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[8] Balunas MJ, Douglas Kinghorn A. Drug discovery from medicinal plants. Life Sciences.

[9] Cragg GM, Newman DJ. Natural products: A continue source of novel drug leads.

[10] Malve H. Exploring the ocean for new drug developments marin pharmacology. Journal

[11] Rasheed A, Farhat R. Cominatorial Chemistry: A Review. International Journal of Pharma-

[12] Sharma N, Harikumar SL. Use of genomics and proteomics in pharmaceutical drug discovery and development. International Journal of Pharmacy and Pharmaceutical

[13] Katiyar C. Drug discovery from plant sources: An integrated approach. An International

[14] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimates solubility and permeability in drug discovery and development

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settings. Advanced Drug Delivery Reviews. 2001;**46**:3-26

Sciences. 2013;**3**(15):975-1491

2005;**78**:431-441

44 Molecular Insight of Drug Design

**Chapter 4**

Provisional chapter

**Molecular Classification of Antitubulin Agents with**

DOI: 10.5772/intechopen.73744

Algorithms for classification and taxonomy are proposed based on criteria as information entropy and its production. A set of 59 antitubulin agents with trimethoxyphenyl (TMP), indole, and C=O bridge present inhibition of gastric cancer cell line MNK-45. On the basis of structure-activity relation of TMPs, derivatives are designed that are classified using seven structural parameters of different moieties. A lot of categorization methods are founded on the entropy of information. On using processes on collections of reasonable dimension, an extreme amount of outcomes occur, matching information and suffering a combinatorial increase. Notwithstanding, following the equipartition conjecture, an assortment factor appears among dissimilar alternatives resultant from categorization among pecking order rankings. The entropy of information allows classifying the compounds and agrees with principal component analyses. A table of periodic properties TMPs is obtained. Features denote positions R1–<sup>4</sup> on the benzo and X–R5/6 on the pyridine ring in indole cycle. Inhibitors in the same group are suggested to present similar properties;

those in the same group and period will present maximum resemblance.

Keywords: periodic law, periodic property, periodic table, information entropy,

Experimentally, antitubulin analogues were synthesized/tested for antitubulin activity, revealing ligand-interaction principles with tubulin/related bioactivity [1–13]. Molecular modeling studies of antitubulin agents were performed to aid in the design of better antitubulin inhibitors [14–16]. In computer-aided drug design studies, comparative molecular field analysis

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Classification of Antitubulin Agents with

**Indole Ring Binding at Colchicine-Binding Site**

Indole Ring Binding at Colchicine-Binding Site

Francisco Torrens and Gloria Castellano

Francisco Torrens and Gloria Castellano

http://dx.doi.org/10.5772/intechopen.73744

Abstract

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

equipartition conjecture, anticancer activity

#### **Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site** Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

DOI: 10.5772/intechopen.73744

Francisco Torrens and Gloria Castellano Francisco Torrens and Gloria Castellano

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73744

#### Abstract

Algorithms for classification and taxonomy are proposed based on criteria as information entropy and its production. A set of 59 antitubulin agents with trimethoxyphenyl (TMP), indole, and C=O bridge present inhibition of gastric cancer cell line MNK-45. On the basis of structure-activity relation of TMPs, derivatives are designed that are classified using seven structural parameters of different moieties. A lot of categorization methods are founded on the entropy of information. On using processes on collections of reasonable dimension, an extreme amount of outcomes occur, matching information and suffering a combinatorial increase. Notwithstanding, following the equipartition conjecture, an assortment factor appears among dissimilar alternatives resultant from categorization among pecking order rankings. The entropy of information allows classifying the compounds and agrees with principal component analyses. A table of periodic properties TMPs is obtained. Features denote positions R1–<sup>4</sup> on the benzo and X–R5/6 on the pyridine ring in indole cycle. Inhibitors in the same group are suggested to present similar properties; those in the same group and period will present maximum resemblance.

Keywords: periodic law, periodic property, periodic table, information entropy, equipartition conjecture, anticancer activity

#### 1. Introduction

Experimentally, antitubulin analogues were synthesized/tested for antitubulin activity, revealing ligand-interaction principles with tubulin/related bioactivity [1–13]. Molecular modeling studies of antitubulin agents were performed to aid in the design of better antitubulin inhibitors [14–16]. In computer-aided drug design studies, comparative molecular field analysis

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(CoMFA) combined with docking calculations was applied to protein-ligand-binding complexes [17–21]. A class of antitubulin agents, binding at colchicine (COL) site with an indole ring, was developed and underwent examinations for binding, antitubulin polymerization, and/or anticancer effects. The discovered properties are helpful for better-inhibitor design. Half inhibitory concentrations (IC50) were collected for the inhibition of gastric cancer cell MKN-45, for 59 COL-like compounds with indole and trimethoxyphenyl (TMP) rings (Figure 1), which bind at COL site [22]. The IC50 were measured for 24 compounds and reviewed for others: 71 compounds were collected. Trial CoMFA calculations for all gave a low leave-one-out determination coefficient q<sup>2</sup> ~0.2. Examination of functional groups showed that three ones are much more bulky than the others. Functional groups of eight are much different from others. Compounds were excluded leaving 59 substances in CoMFA calculation. With data, threedimensional (3D)-quantitative structure-activity relationship (SAR) (QSAR) examination was performed with CoMFA [23], combined with docking calculations for compounds to illustrate correlation of functional group variations with anticancer effect. An approach was employed to examine QSAR for a number of other protein-ligand-binding complexes. Functional-group substitutions locate at sites around indole ring, i.e., R1–<sup>6</sup> functional-group sites. Comparative QSAR modeling of 2-phenylindole-3-carbaldehyde derivatives was performed as potential antimitotic agents [24]. The KIT kinase mutants showed unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients [25]. Gene expression profiling of gastric cancer was reported [26]. Natural product COL, obtained from Colchicum autumnale, is a bioactive alkaloid used in the treatment of a number of diseases [27]. It received considerable attention in the basic study of neoplasia by its capacity for interrupting mitosis, ending the process in metaphase [28]. The COL acts as an inhibitor of the polymerization of tubulin (a protein that contains eight Trp units) [29]. It was used as a probe to understand microtubule role in cells because of its big affinity to tubulin, in which structure presents a binding site (colchicine domain) [30, 31]. Tubulin is a target for cancer treatment: a number of drugs were developed to target it [32]. Binding with it, ligands interfere with its polymerization dynamics and exhibit an antitumor effect. In addition to developed drugs (viz. taxol, vibrestine), which bind with it at taxol/vibrestine-binding sites, COL presents a tubulin binding site and showed anticancer effects although with significant toxicity. Developing COL-like compounds with lesser toxicity represented an effort in finding better ligands to target tubulin at COL-binding site [33, 34]. A simple computerized algorithm useful for establishing a relation

between chemical structures [35, 36] was proposed. The preliminary idea results the entropy of information for configuration detection. The entropy of information results was expressed based on a similarity matrix among a pair of chemical entities. Because the entropy of information results feebly discerning for categorization reasons, the more influential concepts of entropy production and equipartition conjecture result were presented in [37]. In previous articles, the classifications by periodic properties of local anesthetics [38–40], inhibitors of human immunodeficiency virus [41–43], and anticancer drugs [44, 45] were analyzed. The goal of the current account is expanding the promises of knowledge of the algorithm and, as compounds are unaffectedly explained by a changeable-dimension prearranged model, learning universal methods in the dispensation of prearranged information. Next goal presents a periodic classification of TMPs. A further objective is to perform a validation of the periodic table (PT) with an

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

http://dx.doi.org/10.5772/intechopen.73744

49

The key problem in classification studies is to define similarity indices when several criteria of comparison are involved. The primary stage in counting resemblance for TMPs records the majority of the significant moieties. The vector of properties i = < i1,i2,…ik,… > should be linked to each TMP i, whose parts match with dissimilar characteristic groups in the molecule, in a pecking order consistent the predictable significance of pharmacological potency. Whether moiety m-th results more important than portion k-th then m < k. The parts ik are values "1" or "0", consistent if an alike portion of rank k is present in TMP i contrasted to the recommendation one. The examination comprises two regions of structure variation in TMP molecules: positions R1–<sup>4</sup> on the benzo and locations X and R5/6 on the pyridine ring in the indole cycle. The TMPs are inhibitory to gastric cancer cell line MKN-45. The structural elements of a TMP molecule can be ranked according to their contribution to MKN-45 inhibition in the order: R1 > R4 > R2 >X>R5 > R3 > R6. Index i<sup>1</sup> = 1 denotes R1 =H(i<sup>1</sup> = 0, otherwise), i<sup>2</sup> = 1 means R4 = H, i<sup>3</sup> = 1 signifies R2 = H, i<sup>4</sup> = 1 stands for X = N, i<sup>5</sup> = 1 indicates R5 = H, i<sup>6</sup> = 1 represents R3 = OMe, and i<sup>7</sup> = 1 implies R6 = CH2–OH. In TMP 42, R1 = R4 = R2 = R5 = H, X = N, R3 = OMe and R6 = CH2–OH; obviously its associated vector is <1,111,111>. The TMP 42 was selected as reference because of its greatest MNK-45 inhibition. Vectors were associated with 59 TMPs with gastric anticancer activities. Vector <1,111,110> is associated with TMP 1 since R1 = R4 = R2 = R5 = R6 = H, X = N and R3 = OMe. Mean by rij (0 ≤ rij ≤ 1) the similarity index of a pair of TMPs linked to vectors i and j, in that order. The relationship of similarity results is typified by a similarity matrix R = [rij]. The similarity index among a pair of TMPs i = < i1,i2,…

external property not used in the development of PT.

2. Computational method

ik… > and j = < j1,j2,…jk… > is described by:

weight (ak)

rij <sup>¼</sup> <sup>X</sup> k

tkð Þ ak

where 0 ≤ ak ≤ 1 and tk = 1 whether ik = jk except tk = 0 whether ik 6¼ jk. The definition allocates a

45 gastric cancer inhibition data reported by Lin et al. were used for the present classification

<sup>k</sup> to whichever feature concerned about the explanation of molecule i or j. The MNK-

<sup>k</sup> ð Þ <sup>k</sup> <sup>¼</sup> <sup>1</sup>; <sup>2</sup>;… (1)

Figure 1. General structure motifs: Trimethoxyphenyl (TMP) ring/indole ring/C=O bridge.

between chemical structures [35, 36] was proposed. The preliminary idea results the entropy of information for configuration detection. The entropy of information results was expressed based on a similarity matrix among a pair of chemical entities. Because the entropy of information results feebly discerning for categorization reasons, the more influential concepts of entropy production and equipartition conjecture result were presented in [37]. In previous articles, the classifications by periodic properties of local anesthetics [38–40], inhibitors of human immunodeficiency virus [41–43], and anticancer drugs [44, 45] were analyzed. The goal of the current account is expanding the promises of knowledge of the algorithm and, as compounds are unaffectedly explained by a changeable-dimension prearranged model, learning universal methods in the dispensation of prearranged information. Next goal presents a periodic classification of TMPs. A further objective is to perform a validation of the periodic table (PT) with an external property not used in the development of PT.

#### 2. Computational method

(CoMFA) combined with docking calculations was applied to protein-ligand-binding complexes [17–21]. A class of antitubulin agents, binding at colchicine (COL) site with an indole ring, was developed and underwent examinations for binding, antitubulin polymerization, and/or anticancer effects. The discovered properties are helpful for better-inhibitor design. Half inhibitory concentrations (IC50) were collected for the inhibition of gastric cancer cell MKN-45, for 59 COL-like compounds with indole and trimethoxyphenyl (TMP) rings (Figure 1), which bind at COL site [22]. The IC50 were measured for 24 compounds and reviewed for others: 71 compounds were collected. Trial CoMFA calculations for all gave a low leave-one-out deter-

more bulky than the others. Functional groups of eight are much different from others. Compounds were excluded leaving 59 substances in CoMFA calculation. With data, threedimensional (3D)-quantitative structure-activity relationship (SAR) (QSAR) examination was performed with CoMFA [23], combined with docking calculations for compounds to illustrate correlation of functional group variations with anticancer effect. An approach was employed to examine QSAR for a number of other protein-ligand-binding complexes. Functional-group substitutions locate at sites around indole ring, i.e., R1–<sup>6</sup> functional-group sites. Comparative QSAR modeling of 2-phenylindole-3-carbaldehyde derivatives was performed as potential antimitotic agents [24]. The KIT kinase mutants showed unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients [25]. Gene expression profiling of gastric cancer was reported [26]. Natural product COL, obtained from Colchicum autumnale, is a bioactive alkaloid used in the treatment of a number of diseases [27]. It received considerable attention in the basic study of neoplasia by its capacity for interrupting mitosis, ending the process in metaphase [28]. The COL acts as an inhibitor of the polymerization of tubulin (a protein that contains eight Trp units) [29]. It was used as a probe to understand microtubule role in cells because of its big affinity to tubulin, in which structure presents a binding site (colchicine domain) [30, 31]. Tubulin is a target for cancer treatment: a number of drugs were developed to target it [32]. Binding with it, ligands interfere with its polymerization dynamics and exhibit an antitumor effect. In addition to developed drugs (viz. taxol, vibrestine), which bind with it at taxol/vibrestine-binding sites, COL presents a tubulin binding site and showed anticancer effects although with significant toxicity. Developing COL-like compounds with lesser toxicity represented an effort in finding better ligands to target tubulin at COL-binding site [33, 34]. A simple computerized algorithm useful for establishing a relation

Figure 1. General structure motifs: Trimethoxyphenyl (TMP) ring/indole ring/C=O bridge.

~0.2. Examination of functional groups showed that three ones are much

mination coefficient q<sup>2</sup>

48 Molecular Insight of Drug Design

The key problem in classification studies is to define similarity indices when several criteria of comparison are involved. The primary stage in counting resemblance for TMPs records the majority of the significant moieties. The vector of properties i = < i1,i2,…ik,… > should be linked to each TMP i, whose parts match with dissimilar characteristic groups in the molecule, in a pecking order consistent the predictable significance of pharmacological potency. Whether moiety m-th results more important than portion k-th then m < k. The parts ik are values "1" or "0", consistent if an alike portion of rank k is present in TMP i contrasted to the recommendation one. The examination comprises two regions of structure variation in TMP molecules: positions R1–<sup>4</sup> on the benzo and locations X and R5/6 on the pyridine ring in the indole cycle. The TMPs are inhibitory to gastric cancer cell line MKN-45. The structural elements of a TMP molecule can be ranked according to their contribution to MKN-45 inhibition in the order: R1 > R4 > R2 >X>R5 > R3 > R6. Index i<sup>1</sup> = 1 denotes R1 =H(i<sup>1</sup> = 0, otherwise), i<sup>2</sup> = 1 means R4 = H, i<sup>3</sup> = 1 signifies R2 = H, i<sup>4</sup> = 1 stands for X = N, i<sup>5</sup> = 1 indicates R5 = H, i<sup>6</sup> = 1 represents R3 = OMe, and i<sup>7</sup> = 1 implies R6 = CH2–OH. In TMP 42, R1 = R4 = R2 = R5 = H, X = N, R3 = OMe and R6 = CH2–OH; obviously its associated vector is <1,111,111>. The TMP 42 was selected as reference because of its greatest MNK-45 inhibition. Vectors were associated with 59 TMPs with gastric anticancer activities. Vector <1,111,110> is associated with TMP 1 since R1 = R4 = R2 = R5 = R6 = H, X = N and R3 = OMe. Mean by rij (0 ≤ rij ≤ 1) the similarity index of a pair of TMPs linked to vectors i and j, in that order. The relationship of similarity results is typified by a similarity matrix R = [rij]. The similarity index among a pair of TMPs i = < i1,i2,… ik… > and j = < j1,j2,…jk… > is described by:

$$r\_{i\rangle} = \sum\_{k} t\_k (a\_k)^k \quad (k = 1, 2, \dots) \tag{1}$$

where 0 ≤ ak ≤ 1 and tk = 1 whether ik = jk except tk = 0 whether ik 6¼ jk. The definition allocates a weight (ak) <sup>k</sup> to whichever feature concerned about the explanation of molecule i or j. The MNK-45 gastric cancer inhibition data reported by Lin et al. were used for the present classification study. The grouping algorithm applies the stabilized similarity matrix obtained via the max-min composition rule o as described by:

$$(\mathbf{RoS})\_{\vec{\imath}\vec{\jmath}} = \max\_{k} \left[ \min\_{k} (r\_{ik}, s\_{\vec{\imath}\vec{\jmath}}) \right] \tag{2}$$

case happening with probability p, the amount of astonishment results is proportional to –ln p. Extending the outcome to a random variable X (that is able to present N achievable values x1, …, xN with probabilities p1, …, pN), the astonishing mean is obtained when finding out the value of X results –Σ pi ln pi. The entropy of information is linked to similarity matrix R results:

rijlnrij �<sup>X</sup>

i, j

cation level b. The entropy of information fulfills the following features. (1) h(R) = 0 whether rij = 0 or rij = 1. (2) h(R) results maximum whether rij = 0.5, i.e., as the ambiguity is maximum. (3)

� � <sup>≤</sup> <sup>h</sup>ð Þ <sup>R</sup> for whichever <sup>b</sup>, i.e., categorization directs to a deficit of entropy. (4)

categorization procedure, each hierarchical tree matches to a reliance of the entropy of information on the classification level, and a plot h–b is obtained. The equipartition conjecture of entropy production of Tondeur and Kvaalen results is suggested as an assortment principle, between dissimilar alternatives coming from categorization between pecking order rankings. Consistent with the conjecture, for a provided custody, the top arrangement of a dendrogram results in which the production of entropy results is mainly dispersed regularly, i.e., neighboring a type of equipartition. It is gone on at this point similarly via information entropy in its place of thermodynamic entropy. Equipartition entails a linear relationship, i.e., a steady production of entropy of information all along the extent of b, so that the equipartition line results are

As the categorization results are disconnected, a mean of stating equipartition is a usual staircase function. The most excellent alternative results decided the one minimizing the

Learning procedures alike the ones met in stochastic methods are the results as applied in [52]. Taking into account a provided classification as good or perfect from practice or experience, which matches to a reference similarity matrix S = [sij] obtained for equivalent weights a<sup>1</sup> = a<sup>2</sup> = … = a and any amount of fabricated features. Then, take into account identical collection of molecules as in the good categorization and the real features. The similarity index rij results calculated with Eq. (1) provided matrix R. The amount of features for R and S can vary. The learning process lies in attempting to get categorization outcomes for R as near as

SS <sup>¼</sup> <sup>X</sup> bi

� � if <sup>b</sup><sup>1</sup> <sup>&</sup>lt; <sup>b</sup>2, i.e., entropy is a monotone function of grouping level <sup>b</sup>. In the

\_

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

<sup>1</sup> � rij � �ln 1 � rij � � (4)

heqp ¼ hmaxb (5)

<sup>h</sup> � <sup>h</sup>eqp � �<sup>2</sup> (6)

b, the similarity matrix at the classifi-

http://dx.doi.org/10.5772/intechopen.73744

51

<sup>h</sup>ð Þ¼� <sup>R</sup> <sup>X</sup>

Mean is obtained by Cb, the collection of divisions and R

h R \_ b

h R \_ b1 � � <sup>≤</sup> <sup>h</sup> <sup>R</sup>

explained by:

addition of the square differences:

\_ b2 i, j

where R = [rij] and S = [sij] that result in matrices of the same kind and (RoS)ij, entry (i,j)-th of matrix RoS [46–49]. On using composition rule max-min iteratively, so that R(n + 1) = R(n) o R, an integer n results fulfilling: R(n) = R(n + 1) = … The resultant matrix R(n) is named as stabilized similarity matrix. The importance of stabilization stretches out in the categorization procedure, and stabilization generates a separation in displaced divisions. From the present on, it results implicitly that the stabilized similarity matrix is applied and named as R(n)=[rij(n)]. The grouping rule is as follows: i and j results allocated in the same division whether rij(n) ≥ b. The grouping of i ( i \_ ) results in the collection of molecules j that fulfills the grouping rule rij(n) ≥ b. The matrix of clusters results in

$$\widehat{\mathbf{R}}\ (n) = \begin{bmatrix} \widehat{r}\_{\stackrel{\frown}{i}\stackrel{\frown}{j}} \end{bmatrix} = \max\_{\mathbf{s},t} \mathbf{x}\_{\mathbf{s},t}(r\_{st}) \quad \left(\mathbf{s} \in \stackrel{\frown}{i}, t \in \stackrel{\frown}{j}\right) \tag{3}$$

where s means whichever indicator of a molecule fitting in class i \_ (likewise for t and j \_ ). Rule (3) denotes discovering the main similarity index among molecules of a pair of divisions. In information theory, information entropy h measures the surprise that the source emitting the sequences can give [50, 51]. We consider the utilization of a qualitative mark assay to decide the attendance of Fe in a sample of water. With no sample in the past, the analyst has to start with the pair of results supposing: 0 (Fe not present) and 1 (Fe there), which are equiprobable with likelihood 1/2. As up to a pair of elements are there in the sample solution (e.g., Fe, Ni or both), there are four achievable results neither from (0, 0) to the two being there (1, 1) via on a par likelihood 1/22 . Which of the four options goes is decided by a pair of assays, each one with a pair of clear conditions. Likewise, with three metals, there are eight options, every one with a likelihood 1/2<sup>3</sup> : three assays are necessary. The following configuration clearly connects uncertainty to information necessary to solve it. The amount of options results stated to the power of 2. The power to which 2 is lifted to provide the amount of occurrences N results in the logarithm to base 2 of that amount. Both information and uncertainty are described in terms of the logarithm to base 2 of the amount of achievable analytical results: log2 N. The initial uncertainty is defined in terms of the probability of the occurrence of every outcome; e.g., the definition is as follows: I = H = log2 N = log2 1/p = �log2 p, where I denotes the information held in the reply provided that there were N options, H, the first uncertainty coming from the necessity of taking into account the N options and p, the likelihood of each result whether or not all N occurrences are evenly probable to occur. The equation can be extended to the case in which the likelihood of each result does not result the same; whether it is identified from historical experiment is proven by some metals that result in more probability than other ones, the expression results are corrected so that the logarithms of the particular likelihood appropriately weighted result in: H = �Σ pi log2 pi, where: Σ pi = 1. Take into account the first case but at present, historical experiment proved that 90% of the samples had no Fe. The amount of uncertainty results is computed as: H = �(0.9 log2 0.9 + 0.1 log2 0.1) = 0.469 bits. For a particular case happening with probability p, the amount of astonishment results is proportional to –ln p. Extending the outcome to a random variable X (that is able to present N achievable values x1, …, xN with probabilities p1, …, pN), the astonishing mean is obtained when finding out the value of X results –Σ pi ln pi. The entropy of information is linked to similarity matrix R results:

study. The grouping algorithm applies the stabilized similarity matrix obtained via the max-min

ð Þ RoS ij ¼ max<sup>k</sup> min<sup>k</sup> rik;skj

where R = [rij] and S = [sij] that result in matrices of the same kind and (RoS)ij, entry (i,j)-th of matrix RoS [46–49]. On using composition rule max-min iteratively, so that R(n + 1) = R(n) o R, an integer n results fulfilling: R(n) = R(n + 1) = … The resultant matrix R(n) is named as stabilized similarity matrix. The importance of stabilization stretches out in the categorization procedure, and stabilization generates a separation in displaced divisions. From the present on, it results implicitly that the stabilized similarity matrix is applied and named as R(n)=[rij(n)]. The grouping rule is as follows: i and j results allocated in the same division whether rij(n) ≥ b. The

) results in the collection of molecules j that fulfills the grouping rule rij(n) ≥ b. The

. Which of the four options goes is decided by a pair of assays, each one with

: three assays are necessary. The following configuration clearly connects uncer-

\_ ; t∈ j � �\_

\_

(likewise for t and j

¼ maxs,tð Þ rst s ∈ i

(3) denotes discovering the main similarity index among molecules of a pair of divisions. In information theory, information entropy h measures the surprise that the source emitting the sequences can give [50, 51]. We consider the utilization of a qualitative mark assay to decide the attendance of Fe in a sample of water. With no sample in the past, the analyst has to start with the pair of results supposing: 0 (Fe not present) and 1 (Fe there), which are equiprobable with likelihood 1/2. As up to a pair of elements are there in the sample solution (e.g., Fe, Ni or both), there are four achievable results neither from (0, 0) to the two being there (1, 1) via on a

a pair of clear conditions. Likewise, with three metals, there are eight options, every one with a

tainty to information necessary to solve it. The amount of options results stated to the power of 2. The power to which 2 is lifted to provide the amount of occurrences N results in the logarithm to base 2 of that amount. Both information and uncertainty are described in terms of the logarithm to base 2 of the amount of achievable analytical results: log2 N. The initial uncertainty is defined in terms of the probability of the occurrence of every outcome; e.g., the definition is as follows: I = H = log2 N = log2 1/p = �log2 p, where I denotes the information held in the reply provided that there were N options, H, the first uncertainty coming from the necessity of taking into account the N options and p, the likelihood of each result whether or not all N occurrences are evenly probable to occur. The equation can be extended to the case in which the likelihood of each result does not result the same; whether it is identified from historical experiment is proven by some metals that result in more probability than other ones, the expression results are corrected so that the logarithms of the particular likelihood appropriately weighted result in: H = �Σ pi log2 pi, where: Σ pi = 1. Take into account the first case but at present, historical experiment proved that 90% of the samples had no Fe. The amount of uncertainty results is computed as: H = �(0.9 log2 0.9 + 0.1 log2 0.1) = 0.469 bits. For a particular

� � � � (2)

(3)

\_ ). Rule

composition rule o as described by:

50 Molecular Insight of Drug Design

grouping of i ( i

par likelihood 1/22

likelihood 1/2<sup>3</sup>

\_

R \_ ð Þ¼ n r \_ i \_ j \_ h i

where s means whichever indicator of a molecule fitting in class i

matrix of clusters results in

$$h(\mathbf{R}) = -\sum\_{i,j} r\_{i\bar{j}} \ln r\_{i\bar{j}} - \sum\_{i,j} (1 - r\_{i\bar{j}}) \ln(1 - r\_{i\bar{j}}) \tag{4}$$

Mean is obtained by Cb, the collection of divisions and R \_ b, the similarity matrix at the classification level b. The entropy of information fulfills the following features. (1) h(R) = 0 whether rij = 0 or rij = 1. (2) h(R) results maximum whether rij = 0.5, i.e., as the ambiguity is maximum. (3) h R \_ b � � <sup>≤</sup> <sup>h</sup>ð Þ <sup>R</sup> for whichever <sup>b</sup>, i.e., categorization directs to a deficit of entropy. (4) h R \_ b1 � � <sup>≤</sup> <sup>h</sup> <sup>R</sup> \_ b2 � � if <sup>b</sup><sup>1</sup> <sup>&</sup>lt; <sup>b</sup>2, i.e., entropy is a monotone function of grouping level <sup>b</sup>. In the categorization procedure, each hierarchical tree matches to a reliance of the entropy of information on the classification level, and a plot h–b is obtained. The equipartition conjecture of entropy production of Tondeur and Kvaalen results is suggested as an assortment principle, between dissimilar alternatives coming from categorization between pecking order rankings. Consistent with the conjecture, for a provided custody, the top arrangement of a dendrogram results in which the production of entropy results is mainly dispersed regularly, i.e., neighboring a type of equipartition. It is gone on at this point similarly via information entropy in its place of thermodynamic entropy. Equipartition entails a linear relationship, i.e., a steady production of entropy of information all along the extent of b, so that the equipartition line results are explained by:

$$h\_{\text{eqp}} = h\_{\text{max}}b \tag{5}$$

As the categorization results are disconnected, a mean of stating equipartition is a usual staircase function. The most excellent alternative results decided the one minimizing the addition of the square differences:

$$SS = \sum\_{b\_i} \left( h - h\_{\text{eqp}} \right)^2 \tag{6}$$

Learning procedures alike the ones met in stochastic methods are the results as applied in [52]. Taking into account a provided classification as good or perfect from practice or experience, which matches to a reference similarity matrix S = [sij] obtained for equivalent weights a<sup>1</sup> = a<sup>2</sup> = … = a and any amount of fabricated features. Then, take into account identical collection of molecules as in the good categorization and the real features. The similarity index rij results calculated with Eq. (1) provided matrix R. The amount of features for R and S can vary. The learning process lies in attempting to get categorization outcomes for R as near as likely to the good categorization. The primary weight a<sup>1</sup> results obtained constant and just the next weights a2, a3,… result exposed to random changes. A novel similarity matrix results via Eq. (1) and the novel weights. The distance among the classifications typified by R and S results is provided by:

$$D = -\sum\_{\vec{\eta}} \left( 1 - r\_{\vec{\eta}} \right) \ln \frac{1 - r\_{\vec{\eta}}}{1 - s\_{\vec{\eta}}} - \sum\_{\vec{\eta}} r\_{\vec{\eta}} \ln \frac{r\_{\vec{\eta}}}{s\_{\vec{\eta}}} \quad \forall 0 \le r\_{\vec{\eta}}, s\_{\vec{\eta}} \le 1 \tag{7}$$

maximum partial correlation r = 1 with whichever TMP, which results an artifact. After the conjecture of equipartition, the intercorrelations are illustrated in the partial correlation diagram, which contains 1382 high (Figure 2, red lines), 109 medium (orange), 161 low (yellow), and 59 zero (black) partial correlations. Six out of 58 high partial correlations of Entry 42 were corrected; e.g., its correlations with Entries 3 and 47 are medium, its correlations with Entries

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53

12, 15, and 43 are low, and its correlation with Entry 46 is zero partial correlation.

C–b<sup>1</sup> = (1,5–8,10,11,13,16,17,26–28,41,42,44,45,48,58,59),(2,4,9,18,19,49),(3),(12),

Figure 2. Partial correlation diagram: High (red), medium (orange), and low (yellow) correlations.

(14,20–25,29–33,35,50–55),(15,43),(34,36–40,56,57),(46),(47)

The grouping rule in the case with equal weights ak = 0.5 for b<sup>1</sup> = 0.97 allows the classes:

The nine groupings are obtained with associated entropy h–R–b<sup>1</sup> = 39.44. The dendrogram (binary tree) matching with <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > and C–b<sup>1</sup> is calculated [56–58]; it provides a binary taxonomy that separates the same nine classes: from top to bottom, the data bifurcate into groupings 3, 4, 8, 9, 1, 2, 5, 6, and 7 with 1, 1, 1, 1, 20, 6, 19, 2, and 8 TMPs, respectively [59]. The TMPs 42, 26, etc. with the greatest inhibitory activity are grouped into the same class.

The definition was suggested by Kullback to measure the distance between two probability distributions, which is an amount of the distance among matrices R and S [53]. As for each matrix a matching categorization exists, the pair of categorizations result contrasted by distance, which results a non-negative amount that approximates zero as the similarity among R and S rises. The outcome of the procedure results a collection of weights permitting proper categorization. The algorithm was utilized in the production of complicated dendrograms via the entropy of information [54]. Our program MolClas is an easy, dependable, effective, and quick process for molecular categorization, founded on the conjecture of the equipartition of the production of the entropy of information consistent with Eqs. (1)–(7). It reads the amount of features and molecular indices. It permits the optimization of the coefficients. It not obligatorily reads the initial coefficients and the amount of iteration cycles. The correlation matrix results are computed by the algorithm or read from input. Code MolClas permits the alteration of the correlation matrix from [�1, 1] to [0, 1]. The program computes the similarity matrix of the features in symmetric storage mode, computes categorizations, checks whether categorizations result is dissimilar, computes distances among categorizations, computes the similarity matrices of categorizations, works out the entropy of information of categorizations, optimizes coefficients, carries out single/complete-linkage hierarchical cluster analyses, and charts classification plots. It was written not only to analyze the equipartition conjecture of entropy production but also to explore the world of molecular classification. Code MolClas is different from other program MolClass as referred in the literature [55]. While MolClas classifies molecules based on hierarchical dichotomic (Boolean) descriptors, MolClass discovers SARs from molecular patterns (fingerprints) extracted from experimental datasets and needs to interrogate big databases (PubChem, ChEMBL, ChemBank). Code MolClas is available at Internet (torrens@uv. es) and is free for academic use.

#### 3. Calculation results and discussion

Matrix of Pearson correlation coefficients results computed among couples of vector properties <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > for 59 TMPs. Pearson correlations result displayed in the partial correlation diagram, which encloses high (r ≥ 0.75), medium (0.50 ≤ r < 0.75), low (0.25 ≤ r < 0.50), and zero (r < 0.25) partial correlations. Couples of inhibitors with superior partial associations present a vector property alike. Notwithstanding, the outcomes have to be gotten with concern since the TMP with steady vector <1,111,111> (Entry 42) presents zero standard deviation, producing maximum partial correlation r = 1 with whichever TMP, which results an artifact. After the conjecture of equipartition, the intercorrelations are illustrated in the partial correlation diagram, which contains 1382 high (Figure 2, red lines), 109 medium (orange), 161 low (yellow), and 59 zero (black) partial correlations. Six out of 58 high partial correlations of Entry 42 were corrected; e.g., its correlations with Entries 3 and 47 are medium, its correlations with Entries 12, 15, and 43 are low, and its correlation with Entry 46 is zero partial correlation.

The grouping rule in the case with equal weights ak = 0.5 for b<sup>1</sup> = 0.97 allows the classes:

C–b<sup>1</sup> = (1,5–8,10,11,13,16,17,26–28,41,42,44,45,48,58,59),(2,4,9,18,19,49),(3),(12),

(14,20–25,29–33,35,50–55),(15,43),(34,36–40,56,57),(46),(47)

likely to the good categorization. The primary weight a<sup>1</sup> results obtained constant and just the next weights a2, a3,… result exposed to random changes. A novel similarity matrix results via Eq. (1) and the novel weights. The distance among the classifications typified by R and S

> 1 � rij 1 � sij

�<sup>X</sup> ij

The definition was suggested by Kullback to measure the distance between two probability distributions, which is an amount of the distance among matrices R and S [53]. As for each matrix a matching categorization exists, the pair of categorizations result contrasted by distance, which results a non-negative amount that approximates zero as the similarity among R and S rises. The outcome of the procedure results a collection of weights permitting proper categorization. The algorithm was utilized in the production of complicated dendrograms via the entropy of information [54]. Our program MolClas is an easy, dependable, effective, and quick process for molecular categorization, founded on the conjecture of the equipartition of the production of the entropy of information consistent with Eqs. (1)–(7). It reads the amount of features and molecular indices. It permits the optimization of the coefficients. It not obligatorily reads the initial coefficients and the amount of iteration cycles. The correlation matrix results are computed by the algorithm or read from input. Code MolClas permits the alteration of the correlation matrix from [�1, 1] to [0, 1]. The program computes the similarity matrix of the features in symmetric storage mode, computes categorizations, checks whether categorizations result is dissimilar, computes distances among categorizations, computes the similarity matrices of categorizations, works out the entropy of information of categorizations, optimizes coefficients, carries out single/complete-linkage hierarchical cluster analyses, and charts classification plots. It was written not only to analyze the equipartition conjecture of entropy production but also to explore the world of molecular classification. Code MolClas is different from other program MolClass as referred in the literature [55]. While MolClas classifies molecules based on hierarchical dichotomic (Boolean) descriptors, MolClass discovers SARs from molecular patterns (fingerprints) extracted from experimental datasets and needs to interrogate big databases (PubChem, ChEMBL, ChemBank). Code MolClas is available at Internet (torrens@uv.

Matrix of Pearson correlation coefficients results computed among couples of vector properties <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > for 59 TMPs. Pearson correlations result displayed in the partial correlation diagram, which encloses high (r ≥ 0.75), medium (0.50 ≤ r < 0.75), low (0.25 ≤ r < 0.50), and zero (r < 0.25) partial correlations. Couples of inhibitors with superior partial associations present a vector property alike. Notwithstanding, the outcomes have to be gotten with concern since the TMP with steady vector <1,111,111> (Entry 42) presents zero standard deviation, producing

rijln rij sij

∀0 ≤ rij, sij ≤ 1 (7)

results is provided by:

52 Molecular Insight of Drug Design

es) and is free for academic use.

3. Calculation results and discussion

<sup>D</sup> ¼ �<sup>X</sup> ij

1 � rij � �ln

> The nine groupings are obtained with associated entropy h–R–b<sup>1</sup> = 39.44. The dendrogram (binary tree) matching with <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > and C–b<sup>1</sup> is calculated [56–58]; it provides a binary taxonomy that separates the same nine classes: from top to bottom, the data bifurcate into groupings 3, 4, 8, 9, 1, 2, 5, 6, and 7 with 1, 1, 1, 1, 20, 6, 19, 2, and 8 TMPs, respectively [59]. The TMPs 42, 26, etc. with the greatest inhibitory activity are grouped into the same class.

Figure 2. Partial correlation diagram: High (red), medium (orange), and low (yellow) correlations.

The TMPs in the same grouping appear highly correlated in the partial correlation diagram. At level b<sup>2</sup> with b<sup>2</sup> = 0.86, the set of classes results in:

C–b<sup>2</sup> = (1,4–8,10,11,13,14,16–42,44,45,48–59),(2,9),(3,47),(12,15),(43),(46).

Six classes result and entropy decays to h–R–b<sup>2</sup> = 16.18. Dendrogram matching to <i1,i2,i3,i4,i5,i6, i<sup>7</sup> > and C–b<sup>2</sup> divides the same six classes: from top to bottom data bifurcate into classes 5, 6, 1, 2, 3, and 4 with 1, 1, 51, 2, 2, and 2 TMPs, respectively. Again, TMPs with the greatest inhibitory potency belong to the same class. The TMPs in the same class appear highly correlated in the partial correlation diagram and dendrogram. An analysis of set containing 1–59 classes was performed, in agreement with partial correlation diagram and dendrograms. In view of partial correlation diagram and dendrograms, we split data into seven classes: (1,26–28,41,42,45,58,59), (5–8,10,11,13,16,17,44,48), (14,20–25,29–33,35,50–55), (34,36–40,56,57), (2,4,9,18,19,49), (3,47), and (12,15,43,46). Figure 3 displays corresponding tree. Again, TMPs with the greatest activity correspond to the same class.

The illustration of the classification above in a radial tree (Figure 4) shows the same classes, in qualitative agreement with the partial correlation diagram and dendrograms. Once more, TMPs with the greatest potency are included in the same grouping.

Program SplitsTree analyzes cluster analysis (CA) data [60]. Based on split decomposition, it takes a distance matrix and produces a graph that represents the relations between taxa. For ideal data, graph is a tree, whereas less ideal data cause a tree-like network, which is interpreted as possible evidence for different and conflicting data. As split decomposition does not attempt to force data on to a tree, it gives a good indication of how tree-like are given data. Splits graph for 59 TMPs in (Figure 5) shows that most TMP groups collapse: (1,2,4–11,13,16– 19,26–28,41,42,44,45,48,49,58,59), (3,47), (12,15,43), (14,20–25,29–33,35,50–55), and (34,36–40,56,57); classes 1, 2, and 5 coincide. No conflicting relation appears between TMPs. Splits graph is in partial agreement with partial correlation diagram, dendrograms, and radial tree.

Usually in quantitative structure-property relationships (QSPRs), the information archive encloses fewer than 100 molecules and thousands of X-variables. There are a lot of X-variables that nobody is able to find out by inspection configurations, tendencies, groupings, etc. in the molecules. Principal component analysis (PCA) results a method helpful to summarize the knowledge enclosed in the X-matrix and place it comprehensible [61–66]. The PCA acts by decomposing the X-matrix as the product of two matrices P and T. The loading matrix (P), with knowledge concerning the variables, encloses some vectors [principal components (PCs)], in which results are obtained as linear combinations of the first X-variables. In the score matrix (T), with knowledge about the molecules, each molecule result is expressed by projections on to PCs instead of original variables: X = TP' + E. Knowledge not enclosed in the matrices stays as unexplained X-variance in a residual matrix (E). Each PC<sup>i</sup> results a novel coordinate stated as a linear combination of the first characteristics xj: PC<sup>i</sup> = Σjbijxj. The novel coordinates PC<sup>i</sup> result scores or factors whereas the coefficients bij result the loadings. The scores are sorted consistently with the knowledge regarding the entire variability between molecules. The score-score plots present the places of the molecules in the novel coordinate scheme, whereas the loading-loading

Figure 3. Dendrogram of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

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Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site http://dx.doi.org/10.5772/intechopen.73744 55

Figure 3. Dendrogram of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

The TMPs in the same grouping appear highly correlated in the partial correlation diagram. At

Six classes result and entropy decays to h–R–b<sup>2</sup> = 16.18. Dendrogram matching to <i1,i2,i3,i4,i5,i6, i<sup>7</sup> > and C–b<sup>2</sup> divides the same six classes: from top to bottom data bifurcate into classes 5, 6, 1, 2, 3, and 4 with 1, 1, 51, 2, 2, and 2 TMPs, respectively. Again, TMPs with the greatest inhibitory potency belong to the same class. The TMPs in the same class appear highly correlated in the partial correlation diagram and dendrogram. An analysis of set containing 1–59 classes was performed, in agreement with partial correlation diagram and dendrograms. In view of partial correlation diagram and dendrograms, we split data into seven classes: (1,26–28,41,42,45,58,59), (5–8,10,11,13,16,17,44,48), (14,20–25,29–33,35,50–55), (34,36–40,56,57), (2,4,9,18,19,49), (3,47), and (12,15,43,46). Figure 3 displays corresponding tree. Again, TMPs

The illustration of the classification above in a radial tree (Figure 4) shows the same classes, in qualitative agreement with the partial correlation diagram and dendrograms. Once more,

Program SplitsTree analyzes cluster analysis (CA) data [60]. Based on split decomposition, it takes a distance matrix and produces a graph that represents the relations between taxa. For ideal data, graph is a tree, whereas less ideal data cause a tree-like network, which is interpreted as possible evidence for different and conflicting data. As split decomposition does not attempt to force data on to a tree, it gives a good indication of how tree-like are given data. Splits graph for 59 TMPs in (Figure 5) shows that most TMP groups collapse: (1,2,4–11,13,16– 19,26–28,41,42,44,45,48,49,58,59), (3,47), (12,15,43), (14,20–25,29–33,35,50–55), and (34,36–40,56,57); classes 1, 2, and 5 coincide. No conflicting relation appears between TMPs. Splits graph is in

Usually in quantitative structure-property relationships (QSPRs), the information archive encloses fewer than 100 molecules and thousands of X-variables. There are a lot of X-variables that nobody is able to find out by inspection configurations, tendencies, groupings, etc. in the molecules. Principal component analysis (PCA) results a method helpful to summarize the knowledge enclosed in the X-matrix and place it comprehensible [61–66]. The PCA acts by decomposing the X-matrix as the product of two matrices P and T. The loading matrix (P), with knowledge concerning the variables, encloses some vectors [principal components (PCs)], in which results are obtained as linear combinations of the first X-variables. In the score matrix (T), with knowledge about the molecules, each molecule result is expressed by projections on to PCs instead of original variables: X = TP' + E. Knowledge not enclosed in the matrices stays as unexplained X-variance in a residual matrix (E). Each PC<sup>i</sup> results a novel coordinate stated as a linear combination of the first characteristics xj: PC<sup>i</sup> = Σjbijxj. The novel coordinates PC<sup>i</sup> result scores or factors whereas the coefficients bij result the loadings. The scores are sorted consistently with the knowledge regarding the entire variability between molecules. The score-score plots present the places of the molecules in the novel coordinate scheme, whereas the loading-loading

level b<sup>2</sup> with b<sup>2</sup> = 0.86, the set of classes results in:

54 Molecular Insight of Drug Design

with the greatest activity correspond to the same class.

TMPs with the greatest potency are included in the same grouping.

partial agreement with partial correlation diagram, dendrograms, and radial tree.

C–b<sup>2</sup> = (1,4–8,10,11,13,14,16–42,44,45,48–59),(2,9),(3,47),(12,15),(43),(46).

is able to be downgraded to two variables {i4,i5} with a 5% error. For F3, variable i<sup>7</sup> assigns the maximum weight and F<sup>3</sup> is able to be downgraded to two variables {i4,i7} with a 3% error. For F4, variable i<sup>3</sup> consigns the maximum weight; however, F<sup>4</sup> is not able to be downgraded to two variables {i2,i3} devoid of a 15% error. For F5, variable i<sup>1</sup> represents the maximum weight and F<sup>5</sup> is able to be downgraded to two variables {i1,i6} with a 6% error. For F6, variable i<sup>2</sup> explains the maximum weight; notwithstanding, F<sup>6</sup> is not able to be downgraded to two variables {i1,i2} devoid of a 25% error. For F7, variable i<sup>5</sup> accounts for the maximum weight; nevertheless, F<sup>7</sup> is not able to be downgraded to two variables {i5,i6} devoid of a 36% error. In PCA F2–F<sup>1</sup> scores plot (Figure 6), TMPs with the same vector property collapse: (1,26–28,41,45,58,59), (2,9), (4,18,19,49) (5–8,10,11,13,16,17,44,48), (14,20–25,29–33,35,50–55) and (34,36–40,56,57). Seven TMP classes are clearly distinguished: class 1 with 9 compounds (0 < F<sup>1</sup> < F2, right), class 2 with 11 substances (F<sup>1</sup> < F<sup>2</sup> ≈ 0, middle), class 3 with 19 molecules (F<sup>1</sup> > > F2, bottom right), class 4 with 8 organics (0 < F<sup>1</sup> < < F2, top), class 5 (6 units, F<sup>1</sup> < F<sup>2</sup> ≈ 0, middle), class 6 (2 units, F<sup>1</sup> < < F<sup>2</sup> < 0, left) and class 7 (4 units, F<sup>1</sup> < F<sup>2</sup> < 0, bottom). The classification is in agreement with partial

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From PCA factor loadings of TMPs, F2–F<sup>1</sup> loadings plot (Figure 7) depicts the seven properties. In addition, as a complement to the scores plot for the loadings, it is confirmed that TMPs in class 1, located in the right side, present a contribution of R3 = OMe situated in the same side. The TMPs in class 3 in the bottom have more pronounced contribution of X = N in the same location. Two classes of properties are clearly distinguished in the loadings plot: class 1 {R1,R4,

correlation diagram, dendrograms, radial tree, and splits graph.

Figure 5. Splits graph of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

R2,R3} (F<sup>1</sup> > F<sup>2</sup> > 0, right) and class 2 {X,R5,R6} (F<sup>1</sup> < F2, left).

Figure 4. Radial tree of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

plots display the position of the properties that correspond to the molecules in the novel coordinate scheme. The PCs show a pair of features. (1) The PCs result taken out in decreasing sequence of significance: the first PC encloses more knowledge than the second one, the second more than the third one, and so on. (2) Each PC results orthogonal to each other: no correlation exists between information contained in different PCs. A PCA was performed for TMPs. The importance of PCA factors F1–<sup>7</sup> for {i1,i2,i3,i4,i5,i6,i7} was calculated. In particular, the use of the first factor F<sup>1</sup> explains 27% of the variability of data (73% error), the combined application of the first two factors F1/2 accounts for 45% of variance (55% error), the utilization of the first three factors F1–<sup>3</sup> justifies 60% of variability (40% error), etc. Factor loadings of PCA were computed. Profile of PCA F1–F<sup>2</sup> for vector property was calculated. For F1, variable i<sup>6</sup> shows the maximum weight in the profile; notwithstanding, F<sup>1</sup> is not able to be downgraded to two variables {i5,i6} devoid of a 48% error. For F2, variable i<sup>4</sup> presents the maximum weight and F<sup>2</sup> Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site http://dx.doi.org/10.5772/intechopen.73744 57

Figure 5. Splits graph of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

plots display the position of the properties that correspond to the molecules in the novel coordinate scheme. The PCs show a pair of features. (1) The PCs result taken out in decreasing sequence of significance: the first PC encloses more knowledge than the second one, the second more than the third one, and so on. (2) Each PC results orthogonal to each other: no correlation exists between information contained in different PCs. A PCA was performed for TMPs. The importance of PCA factors F1–<sup>7</sup> for {i1,i2,i3,i4,i5,i6,i7} was calculated. In particular, the use of the first factor F<sup>1</sup> explains 27% of the variability of data (73% error), the combined application of the first two factors F1/2 accounts for 45% of variance (55% error), the utilization of the first three factors F1–<sup>3</sup> justifies 60% of variability (40% error), etc. Factor loadings of PCA were computed. Profile of PCA F1–F<sup>2</sup> for vector property was calculated. For F1, variable i<sup>6</sup> shows the maximum weight in the profile; notwithstanding, F<sup>1</sup> is not able to be downgraded to two variables {i5,i6} devoid of a 48% error. For F2, variable i<sup>4</sup> presents the maximum weight and F<sup>2</sup>

Figure 4. Radial tree of TMP ring/indole ring/C=O bridge as MKN-45 inhibitors.

56 Molecular Insight of Drug Design

is able to be downgraded to two variables {i4,i5} with a 5% error. For F3, variable i<sup>7</sup> assigns the maximum weight and F<sup>3</sup> is able to be downgraded to two variables {i4,i7} with a 3% error. For F4, variable i<sup>3</sup> consigns the maximum weight; however, F<sup>4</sup> is not able to be downgraded to two variables {i2,i3} devoid of a 15% error. For F5, variable i<sup>1</sup> represents the maximum weight and F<sup>5</sup> is able to be downgraded to two variables {i1,i6} with a 6% error. For F6, variable i<sup>2</sup> explains the maximum weight; notwithstanding, F<sup>6</sup> is not able to be downgraded to two variables {i1,i2} devoid of a 25% error. For F7, variable i<sup>5</sup> accounts for the maximum weight; nevertheless, F<sup>7</sup> is not able to be downgraded to two variables {i5,i6} devoid of a 36% error. In PCA F2–F<sup>1</sup> scores plot (Figure 6), TMPs with the same vector property collapse: (1,26–28,41,45,58,59), (2,9), (4,18,19,49) (5–8,10,11,13,16,17,44,48), (14,20–25,29–33,35,50–55) and (34,36–40,56,57). Seven TMP classes are clearly distinguished: class 1 with 9 compounds (0 < F<sup>1</sup> < F2, right), class 2 with 11 substances (F<sup>1</sup> < F<sup>2</sup> ≈ 0, middle), class 3 with 19 molecules (F<sup>1</sup> > > F2, bottom right), class 4 with 8 organics (0 < F<sup>1</sup> < < F2, top), class 5 (6 units, F<sup>1</sup> < F<sup>2</sup> ≈ 0, middle), class 6 (2 units, F<sup>1</sup> < < F<sup>2</sup> < 0, left) and class 7 (4 units, F<sup>1</sup> < F<sup>2</sup> < 0, bottom). The classification is in agreement with partial correlation diagram, dendrograms, radial tree, and splits graph.

From PCA factor loadings of TMPs, F2–F<sup>1</sup> loadings plot (Figure 7) depicts the seven properties. In addition, as a complement to the scores plot for the loadings, it is confirmed that TMPs in class 1, located in the right side, present a contribution of R3 = OMe situated in the same side. The TMPs in class 3 in the bottom have more pronounced contribution of X = N in the same location. Two classes of properties are clearly distinguished in the loadings plot: class 1 {R1,R4, R2,R3} (F<sup>1</sup> > F<sup>2</sup> > 0, right) and class 2 {X,R5,R6} (F<sup>1</sup> < F2, left).

properties {R1,R4,R2,R3} (class 1) from {X,R5,R6} (class 2), in agreement with PCA loadings plot and dendrogram. A PCA was performed for the vector properties. The use of only the first factor F<sup>1</sup> explains 51% of variance (49% error), the combined application of the first two factors F1/2 accounts for 71% of variability (29% error), the utilization of the first three factors F1–<sup>3</sup> rationalizes 82% of variance (18% error), etc. In the PCA F2–F<sup>1</sup> scores plot, property R4 appears superimposed on R1. Two groupings of properties are distinguished: class 1 {R1,R4, R2,R3} (F<sup>1</sup> > F2, right) and class 2 {X,R5,R6} (F<sup>1</sup> < F2, left), in agreement with PCA loadings plot, dendrogram and splits graph. Format for PT of TMPs (Table 1) indicates that TMPs are categorized first by i1, then i2, i3, i4, i5, i6, and i7. Vertical groups result described by {i1,i2,i3, i4} and horizontal periods, by {i5,i6,i7}. Periods of eight elements are considered; e.g., group g0011 denotes <i1,i2,i3,i<sup>4</sup> > = <0011>: <0011100> (R1 6¼ H, R4 6¼ H, R2 = H, X = N, R5 = H, R3 6¼ OMe, R6 6¼ CH2–OH), etc. The TMPs in the same column appear close in partial

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

The change of property P (inhibition of gastric cancer cell MKN-45) of vector <i1,i2,i3,i4,i5,i6,

structural parameters {i1,i2,i3,i4,i5,i6,i7}, for TMPs. The property was not used in the development of PT and serves to validate it. Most points appear superimposed, and lines i2/6 on i<sup>1</sup> and i<sup>7</sup> on i4. Results show the order of importance of parameters: i<sup>1</sup> > i<sup>2</sup> > i<sup>3</sup> > i<sup>4</sup> > i<sup>5</sup> > i<sup>6</sup> > i7, in agreement with PT of properties with vertical groups defined by {i1,i2,i3,i4} and horizontal periods by {i5,i6,i7}. The variation property P of vector <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > in base 10 vs. the number of group in PT, for TMPs, reveals minima corresponding to compounds with <i1,i2, i3,i<sup>4</sup> > ca. <0011> (group g0011) and maxima ca. <1111> (group g1111). Periods p010, p100, p110, and p111 represent rows 1–4, respectively. For groups 3 and 6, period p110 is superimposed on p100, and for group 8, all periods coincide. The corresponding function P(i1,i2,i3,i4,i5,i6,i7) indicates a series of cyclic waves obviously controlled by minima or maxima, which propose a periodic performance that evokes the shape of a trigonometric function. For <i1,i2,i3,i4,i5,i6,i7>, maximum results are obviously presented. The space in <i1, i2,i3,i4,i5,i6,i<sup>7</sup> > elements among every couple of successive maxima is eight, which agrees with TMP collections in consecutive periods. The maxima are in similar locations in the curve and are in phase. The typical points in phase have to match with the components in similar group in PT. For maxima <i1,i2,i3,i4,i5,i6,i7>, there is consistency among the two descriptions; notwithstanding, the constancy is not universal. The assessment of the waves presents a pair of dissimilarities: (1) periods are incomplete and (2) periods 2 and 3 are somewhat staircase like. The most characteristic points of the plot are maxima that lie about group g1111. The values of <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > are repeated as the periodic law (PL) states. An empirical function P(p) reproduces different <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > values; a minimum of P(p) presents significance just if it is contrasted with the previous P(p–1) and afterward P(p + 1)

Pminð Þp < P pð Þ � 1

Sequenced relationship (8) has to be done again at determined gaps peer to the dimension of

i<sup>1</sup> + 10<sup>5</sup>

i<sup>2</sup> + 10<sup>4</sup>

i<sup>3</sup> + 10<sup>3</sup>

<sup>P</sup>minð Þ<sup>p</sup> <sup>&</sup>lt; P pð Þ <sup>þ</sup> <sup>1</sup> (8)

i<sup>4</sup> + 10<sup>2</sup>

http://dx.doi.org/10.5772/intechopen.73744

59

i<sup>5</sup> + 10i<sup>6</sup> + i<sup>7</sup> vs.

correlation diagram, dendrograms, radial tree, splits graph, and PCA scores.

i<sup>7</sup> > is expressed in the decimal system P = 10<sup>6</sup>

points, necessitating to satisfy:

the period and is equal to:

Figure 6. Principal component analysis F2–F<sup>1</sup> scores plot for TMP ring/indole ring/C=O bridge.

Figure 7. PCA F2–F<sup>1</sup> loadings plot for TMP ring/indole ring/C=O bridge.

Instead of 59 TMPs in the ℜ<sup>7</sup> space of seven vector properties, we consider seven properties in the ℜ<sup>59</sup> space of 59 TMPs. The dendrogram for vector properties separates properties {R1, R4,R2,R3} (class 1) from {X,R5,R6} (class 2), in agreement with PCA loadings plot. The splits graph for properties indicates no conflicting relation between vector components, separating properties {R1,R4,R2,R3} (class 1) from {X,R5,R6} (class 2), in agreement with PCA loadings plot and dendrogram. A PCA was performed for the vector properties. The use of only the first factor F<sup>1</sup> explains 51% of variance (49% error), the combined application of the first two factors F1/2 accounts for 71% of variability (29% error), the utilization of the first three factors F1–<sup>3</sup> rationalizes 82% of variance (18% error), etc. In the PCA F2–F<sup>1</sup> scores plot, property R4 appears superimposed on R1. Two groupings of properties are distinguished: class 1 {R1,R4, R2,R3} (F<sup>1</sup> > F2, right) and class 2 {X,R5,R6} (F<sup>1</sup> < F2, left), in agreement with PCA loadings plot, dendrogram and splits graph. Format for PT of TMPs (Table 1) indicates that TMPs are categorized first by i1, then i2, i3, i4, i5, i6, and i7. Vertical groups result described by {i1,i2,i3, i4} and horizontal periods, by {i5,i6,i7}. Periods of eight elements are considered; e.g., group g0011 denotes <i1,i2,i3,i<sup>4</sup> > = <0011>: <0011100> (R1 6¼ H, R4 6¼ H, R2 = H, X = N, R5 = H, R3 6¼ OMe, R6 6¼ CH2–OH), etc. The TMPs in the same column appear close in partial correlation diagram, dendrograms, radial tree, splits graph, and PCA scores.

The change of property P (inhibition of gastric cancer cell MKN-45) of vector <i1,i2,i3,i4,i5,i6, i<sup>7</sup> > is expressed in the decimal system P = 10<sup>6</sup> i<sup>1</sup> + 10<sup>5</sup> i<sup>2</sup> + 10<sup>4</sup> i<sup>3</sup> + 10<sup>3</sup> i<sup>4</sup> + 10<sup>2</sup> i<sup>5</sup> + 10i<sup>6</sup> + i<sup>7</sup> vs. structural parameters {i1,i2,i3,i4,i5,i6,i7}, for TMPs. The property was not used in the development of PT and serves to validate it. Most points appear superimposed, and lines i2/6 on i<sup>1</sup> and i<sup>7</sup> on i4. Results show the order of importance of parameters: i<sup>1</sup> > i<sup>2</sup> > i<sup>3</sup> > i<sup>4</sup> > i<sup>5</sup> > i<sup>6</sup> > i7, in agreement with PT of properties with vertical groups defined by {i1,i2,i3,i4} and horizontal periods by {i5,i6,i7}. The variation property P of vector <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > in base 10 vs. the number of group in PT, for TMPs, reveals minima corresponding to compounds with <i1,i2, i3,i<sup>4</sup> > ca. <0011> (group g0011) and maxima ca. <1111> (group g1111). Periods p010, p100, p110, and p111 represent rows 1–4, respectively. For groups 3 and 6, period p110 is superimposed on p100, and for group 8, all periods coincide. The corresponding function P(i1,i2,i3,i4,i5,i6,i7) indicates a series of cyclic waves obviously controlled by minima or maxima, which propose a periodic performance that evokes the shape of a trigonometric function. For <i1,i2,i3,i4,i5,i6,i7>, maximum results are obviously presented. The space in <i1, i2,i3,i4,i5,i6,i<sup>7</sup> > elements among every couple of successive maxima is eight, which agrees with TMP collections in consecutive periods. The maxima are in similar locations in the curve and are in phase. The typical points in phase have to match with the components in similar group in PT. For maxima <i1,i2,i3,i4,i5,i6,i7>, there is consistency among the two descriptions; notwithstanding, the constancy is not universal. The assessment of the waves presents a pair of dissimilarities: (1) periods are incomplete and (2) periods 2 and 3 are somewhat staircase like. The most characteristic points of the plot are maxima that lie about group g1111. The values of <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > are repeated as the periodic law (PL) states. An empirical function P(p) reproduces different <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > values; a minimum of P(p) presents significance just if it is contrasted with the previous P(p–1) and afterward P(p + 1) points, necessitating to satisfy:

$$\begin{aligned} P\_{\min}(p) &< P(p-1) \\ P\_{\min}(p) &< P(p+1) \end{aligned} \tag{8}$$

Sequenced relationship (8) has to be done again at determined gaps peer to the dimension of the period and is equal to:

Instead of 59 TMPs in the ℜ<sup>7</sup> space of seven vector properties, we consider seven properties in the ℜ<sup>59</sup> space of 59 TMPs. The dendrogram for vector properties separates properties {R1, R4,R2,R3} (class 1) from {X,R5,R6} (class 2), in agreement with PCA loadings plot. The splits graph for properties indicates no conflicting relation between vector components, separating

Figure 7. PCA F2–F<sup>1</sup> loadings plot for TMP ring/indole ring/C=O bridge.

Figure 6. Principal component analysis F2–F<sup>1</sup> scores plot for TMP ring/indole ring/C=O bridge.

58 Molecular Insight of Drug Design


Pminð Þ� p P pð Þ � 1 < 0

–H –H –H –S –H –OMe –H –H –H –H –S –H –OMe –Me –H –H –H –S –H –OMe –Pr –H –H –H –O –H –OMe –Et –H –H –H –S –H –OMe –Et

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

g1011 g1101 g1110 g1111

Table 1. Periodic properties for 2-phenylindole-3-carbaldehyde derivatives.

Because relationship (9) is just suitable for minima, additional universal others are wanted for all positions p; D(p) = P(p + 1) – P(p) differences are computed by allocating each value to

In the place of D(p), the values of R(p) = P(p + 1)/P(p) are obtained by assigning R(p) to TMP p; whether PL is universal, components in similar group in equivalent locations in dissimilar

Notwithstanding, the outcomes demonstrate that this is not the case, so PL is not universal but with anomalies. The change of D(p) vs. group number shows that for group 6, periods p100 and p110 collapse. It introduces lack of consistency among <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > Cartesian and PT charts. Whether constancy were exact, every position in each period present similar sign: in general, a tendency exists in the positions to provide D(p) > 0 for the lower groups but not for group 8; however, the latter results should be taken with care because D(p) are calculated using data from the next period. In detail, irregularities exist in which TMPs for successive periods are not always in phase. The change of R(p) vs. group number shows that for groups 3 and 6, periods p100 and p110 collapse, and, for group 8, all periods coincide, confirming the lack of steadiness among

TMP p:

either

either

periodic waves assure:

P pð Þ� <sup>þ</sup> <sup>1</sup> <sup>P</sup>minð Þ<sup>p</sup> <sup>&</sup>gt; <sup>0</sup> (9)

–CH2–C�CH

–H –H –H –N –H –OMe –CO–OH –H –H –H –N –H –OMe –CH2–N(CH3)2

http://dx.doi.org/10.5772/intechopen.73744

61

–H –H –H –N –H –OMe –CH2–OH

D pð Þ¼ P pð Þ� þ 1 P pð Þ (10)

D pð Þ > 0 or D pð Þ < 0 (11)

R pð Þ > 1 or R pð Þ < 1 (12)

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site http://dx.doi.org/10.5772/intechopen.73744 61


Table 1. Periodic properties for 2-phenylindole-3-carbaldehyde derivatives.

$$\begin{aligned} P\_{\min}(p) - P(p-1) &< 0\\ P(p+1) - P\_{\min}(p) &> 0 \end{aligned} \tag{9}$$

Because relationship (9) is just suitable for minima, additional universal others are wanted for all positions p; D(p) = P(p + 1) – P(p) differences are computed by allocating each value to TMP p:

$$D(p) = P(p+1) - P(p) \tag{10}$$

In the place of D(p), the values of R(p) = P(p + 1)/P(p) are obtained by assigning R(p) to TMP p; whether PL is universal, components in similar group in equivalent locations in dissimilar periodic waves assure:

either

g0011 g0101 g0111 g1001

–OMe –H –OMe –N –H –OMe –H

–H –H –OMe –N –H –H –H –H –H –OMe –N –H –H –Me

–H –H –OMe –N –H –OMe –H –H –H –NH2 –N –H –OMe –H –H –H –OH –N –H –OMe –H –H –H –O–CH2–Ph –N –H

–H –H –H –O –H –OMe –H –H –H –H –O –H –OMe –Me –H –H –H –O –H –OMe –Pr

–OMe –H

g1011 g1101 g1110 g1111

–OMe –H –H –N –H –H –H –H –OMe –OMe –N

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H

–OMe –H –H –N –H –OMe –H

–H –H –H

–H –H –H –N –CO–CH=CH2

–H –H –H –N –Me –OMe –H –H –H –H –N –Pr –OMe –H –H –H –H –N –Bu –OMe –H –H –H –H –N –CH2–CH2–N(CH3)2

–H –H –H –N –CH2–CH2–CO–OH

–H –H –H –N –CH2–Ph –OMe –H –H –H –H –N –CH2–Pyr –OMe –H –H –H –H –N –CO–Ph –OMe –H –H –H –H –N –CO–2-Furan

–H –H –H –N –CO–C(CH3)3

–H –H –H –N –CO–O–Ph –OMe –H –H –H –H –N –SO2–Ph –OMe –H –H –H –H –N –Et –OMe –H –H –H –H –N –i-Pr –OMe –H –H –H –H –N –CH2–CO–OH

–H –H –H –N –CO–2-Thiofuran

–H –H –H –N –CO–O–C(CH3)3

–H –H –H –N –CO–N(CH3)2

–H –H –H –N –H –F –H –H –H –H –N –H –OEt –H –H –H –H –N –H –OPr –H –H –H –H –N –H –O-i-Pr –H –H –H –H –N –H –NO2 –H –H –H –H –N –H –Br –H –H –H –H –N –H –O–CH2–O– –H –H –H –H –N –H –NHMe –H –H –H –H –N –H –N(Me)2 –H –H –H –H –N –H –OH –H –H –H –H –N –H –NH2 –H

–H –H –H –N –H –OMe –H –H –H –H –N –H –OMe –Me –H –H –H –N –H –OMe –Et –H –H –H –N –H –OMe –Pr –H –H –H –N –H –OMe –CO–O–CH3

–H –H –H –N –H –OMe

–OMe –OMe –H –N –H –H –H

60 Molecular Insight of Drug Design

–H –OMe –H –N –H

–H –H

$$D(p) > 0 \text{ or } D(p) < 0 \tag{11}$$

either

$$R(p) > 1 \text{ or } R(p) < 1\tag{12}$$

Notwithstanding, the outcomes demonstrate that this is not the case, so PL is not universal but with anomalies. The change of D(p) vs. group number shows that for group 6, periods p100 and p110 collapse. It introduces lack of consistency among <i1,i2,i3,i4,i5,i6,i<sup>7</sup> > Cartesian and PT charts. Whether constancy were exact, every position in each period present similar sign: in general, a tendency exists in the positions to provide D(p) > 0 for the lower groups but not for group 8; however, the latter results should be taken with care because D(p) are calculated using data from the next period. In detail, irregularities exist in which TMPs for successive periods are not always in phase. The change of R(p) vs. group number shows that for groups 3 and 6, periods p100 and p110 collapse, and, for group 8, all periods coincide, confirming the lack of steadiness among Cartesian and PT representations. Whether control were precise or not, every position in every period presents R(p) either smaller or larger than one. A tendency exists in the positions to provide R(p) > 1 for the lower groups but not for group 8; however, the latter should be taken with care because R(p) are calculated from the next period. Confirmed incongruities exist in which TMPs for successive periods are not always in phase.

Author details

Francisco Torrens<sup>1</sup>

References

49:6412-6415

and biological evaluation of 2-(3<sup>0</sup>

\* and Gloria Castellano<sup>2</sup>

1 Institute for Molecular Science, University of Valencia, Valencia, Spain

studies. Journal of Medicinal Chemistry. 2006;49:947-954

tion. Journal of Medicinal Chemistry. 2004;47:6120-6123

nal of Medicinal Chemistry. 2006;49:6656-6659

Journal of Medicinal Chemistry. 2004;47:4247-4257

2 Department of Experimental Sciences and Mathematics, Faculty of Veterinary and Experimental Sciences, Valencia Catholic University Saint Vincent Martyr, Valencia, Spain

[1] DeMartino G, Edler MC, LaRegina G, Coluccia A, Barbera MC, Barrow D, Nicholson RI, Chiosis G, Brancale A, Hamel E, Artico M, Silvestri R. New arylthioindoles: Potent inhibitors of tubulin polymerization. 2. Structure-activity relationships and molecular modeling

Molecular Classification of Antitubulin Agents with Indole Ring Binding at Colchicine-Binding Site

http://dx.doi.org/10.5772/intechopen.73744

63

[2] DeMartino G, LaRegina G, Coluccia A, Edler MC, Barbera MC, Brancale A, Wilcox E, Hamel E, Artico M, Silvestri R. Arylthioindoles, potent inhibitors of tubulin polymeriza-

[3] Chang JY, Hsieh HP, Chang CY, Hsu KS, Chiang YF, Chen CM, Kuo CC, Liou JP. 7-Aroyl-aminoindoline-1-sulfonamides as a novel class of potent antitubulin agents. Jour-

[4] Liou JP, Chang YL, Kuo FM, Chang CW, Tseng HY, Wang CC, Yang YN, Chang JY, Lee SJ, Hsieh HP. Concise synthesis and structure-activity relationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes of potent antitubulin agents.

bretastatin derivatives as potent antimitotic agents. Journal of Medicinal Chemistry. 2006;

[6] Liou JP, Mahindroo N, Chang CW, Guo FM, Lee SWH, Tan UK, Yeh TK, Kuo CC, Chang YW, Lu PH, Tung YS, Lin KT, Chang JY, Hsieh HP. Structure�activity relationship studies of 3-aroylindoles as potent antimitotic agents. ChemMedChem. 2006;1:1106-1118

[7] Rappl C, Barbier P, Bourgarel-Rey V, Gregoire C, Gilli R, Carre M, Combes S, Finet JP, Peyrot V. Interaction of 4-arylcoumarin analogues of combretastatins with microtubule

[8] Romagnoli R, Baraldi PG, Remusat V, Carrion MD, Cara CL, Petri D, Fruttarolo F, Pavani MG, Tabrizi MA, Tolomeo M, Grimaudo S, Balzarini J, Jordan MA, Hamel E. Synthesis


network of HBL100 cells and binding to tubulin. Biochemistry. 2006;45:9210-9218

new class of tubulin inhibitors. Journal of Medicinal Chemistry. 2006;49:6425-6428

,40 ,50 -aminocom-

[5] Chang JY, Yang MF, Chang CY, Chen CM, Kuo CC, Liou JP. 2-amino and 2<sup>0</sup>

\*Address all correspondence to: torrens@uv.es

#### 4. Conclusion


#### Acknowledgements

The authors thank support from Generalitat Valenciana (Project No. PROMETEO/2016/094) and Valencia Catholic University Saint Vincent Martyr (Project No. PRUCV/2015/617).

### Author details

Cartesian and PT representations. Whether control were precise or not, every position in every period presents R(p) either smaller or larger than one. A tendency exists in the positions to provide R(p) > 1 for the lower groups but not for group 8; however, the latter should be taken with care because R(p) are calculated from the next period. Confirmed incongruities exist in

1. Several criteria were selected to reduce analysis to manage quantity of trimethoxyphenyl, indole, carbonyl bridge antitubulins referred to structural parameters related to positions R1–<sup>4</sup> on benzo, R5/6 on pyridine, and heteroatom X in indole. Molecular structural elements were ranked according to inhibitory activity: R1 > R4 > R2 >X>R5 > R3 > R6. In compound 42, R1 = R4 = R2 = R5 = H, X = N, R3 = OMe and R6 = CH3–OH <1,111,111>, which was selected as reference. Many classification algorithms are based on information entropy. For moderate-sized sets, an excessive number of results appear compatible with data and suffer a combinatorial explosion; however, after the equipartition conjecture, one has a selection criterion, according to which the best configuration is that in which entropy production is most uniformly distributed. Method avoids the problem of continuum variables because for compound with constant <1,111,111> vector, null standard deviation causes Pearson correlation coefficient of one. Classification is in agreement with the ana-

2. Code MolClas is an easy, dependable, effective, and quick process for the classification of molecules founded on the conjecture of the equipartition of the production of the entropy of information. The code was developed not just to examine the conjecture of equipartition

3. The periodic law does not convince the category of the laws of physics: (1) antitubulin inhibitory powers do not result done again; maybe their chemical nature; (2) sequence relations are done again with exemptions. The examination compels the declaration: relationships that whichever molecule p presents with its neighbor p + 1 are more or less done again for each period. Periodicity result is not universal; notwithstanding, if a usual order of molecules are agreed, the rule should be phenomenological. The antiproliferative potency did not generate the table of periodic classification and serves to confirm it. The examination of other antitubulin features would give an insight into the achievable gener-

The authors thank support from Generalitat Valenciana (Project No. PROMETEO/2016/094)

and Valencia Catholic University Saint Vincent Martyr (Project No. PRUCV/2015/617).

but, in addition, to discover the world of the classification of molecules.

which TMPs for successive periods are not always in phase.

4. Conclusion

62 Molecular Insight of Drug Design

lyses by principal components.

alization of the periodic table.

Acknowledgements

Francisco Torrens<sup>1</sup> \* and Gloria Castellano<sup>2</sup>

\*Address all correspondence to: torrens@uv.es

1 Institute for Molecular Science, University of Valencia, Valencia, Spain

2 Department of Experimental Sciences and Mathematics, Faculty of Veterinary and Experimental Sciences, Valencia Catholic University Saint Vincent Martyr, Valencia, Spain

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**Chapter 5**

**Provisional chapter**

**Multifunctional Polymeric Enveloped Nanocarriers:**

**Multifunctional Polymeric Enveloped Nanocarriers:** 

Over the past several years, employment of multifunctional polymeric excipients-based nanoparticles for controlled and targeted drug delivery of therapeutic modalities to mucosal membrane-based organelles and systemic circulation has gained enormous interest. Because they promise to resolve numerous key therapeutical issues associated with current clinical practice including low treatment efficacy and significant side effects. Potential controlled and targeted drug delivery systems, therefore, should be able to overcome not only extracellular barriers but also intracellular barriers. Extracellularly, targeted nanocarriers ought to provide extended circulation time, selective binding to the targeted mucosal tissues, long residence time at the site of absorption, and controlled drug release. Intracellularly, the targeted nanocarriers should offer cellular uptake, cellular localization, and endosomal release. Hence, this chapter will provide an overview of the unique chemistry of multifunctional polymeric enveloped diverse nanocarriers such as dendrimers, semiconducting polymer dots, quantum dots, carbon dots, and magnetic

as versatile platform addressing both extracellular and intracellular barriers.

**Keywords:** polymeric nanocarriers, extracellular drug targeting, intracellular drug targeting, carbon dots, polymer dots, quantum dots, magnetic nanoparticles

Targeted drug delivery has been massively investigated because of their potential to overcome hurdles of conventional therapy [1]. Administration of drug selectively at desired site ensures the maximum amount of drug to be available at that locality. Moreover, lesser absorption of

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.74624

**Targeting Extracellular and Intracellular Barriers**

**Targeting Extracellular and Intracellular Barriers**

Maria Hassan Kiani, Masoom Yasinzai and

Maria Hassan Kiani, Masoom Yasinzai and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74624

Gul Shahnaz

Gul Shahnaz

**Abstract**

**1. Introduction**

#### **Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers**

DOI: 10.5772/intechopen.74624

Maria Hassan Kiani, Masoom Yasinzai and Gul Shahnaz Maria Hassan Kiani, Masoom Yasinzai and Gul Shahnaz

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74624

#### **Abstract**

Over the past several years, employment of multifunctional polymeric excipients-based nanoparticles for controlled and targeted drug delivery of therapeutic modalities to mucosal membrane-based organelles and systemic circulation has gained enormous interest. Because they promise to resolve numerous key therapeutical issues associated with current clinical practice including low treatment efficacy and significant side effects. Potential controlled and targeted drug delivery systems, therefore, should be able to overcome not only extracellular barriers but also intracellular barriers. Extracellularly, targeted nanocarriers ought to provide extended circulation time, selective binding to the targeted mucosal tissues, long residence time at the site of absorption, and controlled drug release. Intracellularly, the targeted nanocarriers should offer cellular uptake, cellular localization, and endosomal release. Hence, this chapter will provide an overview of the unique chemistry of multifunctional polymeric enveloped diverse nanocarriers such as dendrimers, semiconducting polymer dots, quantum dots, carbon dots, and magnetic as versatile platform addressing both extracellular and intracellular barriers.

**Keywords:** polymeric nanocarriers, extracellular drug targeting, intracellular drug targeting, carbon dots, polymer dots, quantum dots, magnetic nanoparticles

#### **1. Introduction**

Targeted drug delivery has been massively investigated because of their potential to overcome hurdles of conventional therapy [1]. Administration of drug selectively at desired site ensures the maximum amount of drug to be available at that locality. Moreover, lesser absorption of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

drug systemically minimizes the potential for unwanted effects. To target a drug so that it may avoid its uptake by off target tissues or cell and allow its residence at desired site for longer period of time, it is usually escorted with some targeting agent. These targeting molecules can be receptor-specific ligands, vehicles, or biological molecules [2].

Nanocarriers have revolutionized therapeutic approaches by providing numerous means for drug targeting. Nanocarriers have employed various approaches to target drug to a specific organ, tissue, cell, or organelle. The therapeutic efficacy of these carrier systems is highly dependent on their entry into target sites. It has been studied that passage of particles across endothelial cells requires their size to be less than 100 nm approximately [3]. This limits the greater-sized particles to extravagate into tissues having compromised endothelial arrangement. Nanostructures with hydrophobic outer surface have been observed to undergo phagocytosis after being opsonized [4].

Various polymers with diverse chemical nature and various activities and characteristics have been explored to design nanocarriers for delivery of drug molecules. Among them, biodegradable polymers have been of particular interest. Both natural and synthetic biodegradable polymers have been exploited to functionalize nanoparticles exploiting diverse approaches to deliver drug in an effective manner to its target site [1]. Such functionalized polymeric nanostructures (also known as multiplex nanoparticles) have been widely investigated as an effective carrier for a wide variety of drugs as well as biological molecules such as DNA and proteins [1]. Current research has been precisely focused on the use of biodegradable polymers that have shown great promise in modifying the delivery of drug as well as tissue engineering. Such polymers have shown to provide extended and targeted drug release for days to weeks as well as shown great promise for intracellular transport of drugs [5].

untoward side effects [2]. Macromolecules (proteins, peptides, DNA, etc.) are not allowed by lipophilic biological membranes to enter into cytoplasm. Such molecules may follow active transport involving endocytosis via cell surface receptors [6]. This receptor-mediated endocytosis offers a faster course for drug internalization in contrast to untargeted conjugates. These receptors have been located on surface of cells as well as accompany intracellular

Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers

http://dx.doi.org/10.5772/intechopen.74624

71

The bases on which molecules are sorted to follow either clathrin-dependent or clathrin-independent pay are still not fully understood. However, it has been found that some specialized lipid domains are involved in membrane organization, sorting, and signal transduction in clathrin-independent endocytosis [9]. Ligand-associated drug carriers may bind to specific cell receptors that get assembled into particular areas of plasma membrane termed as coated pits. These regions (diameter 0.1 μm approx.) have been explained as plasma membrane invaginations with fuzzy cytoplasmic coat. This coat is mainly composed of clathrin protein present at cytoplasmic periphery of membrane and serves as major route for cellular internalization [10] as depicted in **Figure 2**. These coated pits allow intracellular vesicle formation in less than 1 min of time that is much faster than other mechanisms of endocytosis. A protein known as adaptin is responsible for polymerization of clathrin in the form of polyhedral lattice scaffold by binding with cell surface receptors. Two other proteins amphiphysin and endophilin get neighboring membrane into close vicinity. Dynamin (a cytosolic GTPase) gathers around the neckline of budding vesicle followed by its scission and intracellular discharge [2]. After intracellular entry of vesicle, an uncoating protein (heat shock protein; hsc70) causes the clathrin coat to shed of. At this stage, endocytosis trafficking of endosomal content decides the fate of therapeutic agent delivered. Endosomes may end up into lysosomes that may lead to degradation of drug or may be safely released into cytoplasm to reach

membranes [8].

desired organelle [6].

**2.1. Clathrin-associated, receptor-mediated endocytosis**

**Figure 1.** Pathways adopted for intracellular drug transport.

Various treatment modalities require intracellular delivery of drug as the causative agent is harboring within cell. This is the case commonly associated with infectious diseases such as tuberculosis, leishmaniasis, and leprosy, where the pathogen invades macrophages. Therefore, complete eradication requires utmost delivery of drug at right concentration to infected cells. Certain other conditions also require delivery of therapeutic agents into cytoplasm where they can target various cellular organelles such as endoplasmic reticulum, mitochondria, nucleus, and lysosomes [6]. This has found particular interest in gene therapy for targeting cellular genome [7] as well as drug targeting for the treatment of cancer and lysosomal storage disease [6]. Intracellular transport has also been widely appreciated for bioimaging and biosensing both *in vitro* and *in vivo*.

#### **2. Intracellular drug targeting**

Entry or transport of drug into the cell has never been easy and thus widely explored [6]. Cells offer several mechanisms to allow ingress of drug carriers prominently comprising endocytosis. Internalization of drug into cells may either obey receptor-dependent endocytosis or receptor-independent pathway as shown in **Figure 1**. Combining small drug molecules with macromolecular carriers restricts their entry into highly perfused tissues, thus averting Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers http://dx.doi.org/10.5772/intechopen.74624 71

**Figure 1.** Pathways adopted for intracellular drug transport.

drug systemically minimizes the potential for unwanted effects. To target a drug so that it may avoid its uptake by off target tissues or cell and allow its residence at desired site for longer period of time, it is usually escorted with some targeting agent. These targeting molecules

Nanocarriers have revolutionized therapeutic approaches by providing numerous means for drug targeting. Nanocarriers have employed various approaches to target drug to a specific organ, tissue, cell, or organelle. The therapeutic efficacy of these carrier systems is highly dependent on their entry into target sites. It has been studied that passage of particles across endothelial cells requires their size to be less than 100 nm approximately [3]. This limits the greater-sized particles to extravagate into tissues having compromised endothelial arrangement. Nanostructures with hydrophobic outer surface have been observed to undergo phago-

Various polymers with diverse chemical nature and various activities and characteristics have been explored to design nanocarriers for delivery of drug molecules. Among them, biodegradable polymers have been of particular interest. Both natural and synthetic biodegradable polymers have been exploited to functionalize nanoparticles exploiting diverse approaches to deliver drug in an effective manner to its target site [1]. Such functionalized polymeric nanostructures (also known as multiplex nanoparticles) have been widely investigated as an effective carrier for a wide variety of drugs as well as biological molecules such as DNA and proteins [1]. Current research has been precisely focused on the use of biodegradable polymers that have shown great promise in modifying the delivery of drug as well as tissue engineering. Such polymers have shown to provide extended and targeted drug release for days

Various treatment modalities require intracellular delivery of drug as the causative agent is harboring within cell. This is the case commonly associated with infectious diseases such as tuberculosis, leishmaniasis, and leprosy, where the pathogen invades macrophages. Therefore, complete eradication requires utmost delivery of drug at right concentration to infected cells. Certain other conditions also require delivery of therapeutic agents into cytoplasm where they can target various cellular organelles such as endoplasmic reticulum, mitochondria, nucleus, and lysosomes [6]. This has found particular interest in gene therapy for targeting cellular genome [7] as well as drug targeting for the treatment of cancer and lysosomal storage disease [6]. Intracellular transport has also been widely appreciated for bioim-

Entry or transport of drug into the cell has never been easy and thus widely explored [6]. Cells offer several mechanisms to allow ingress of drug carriers prominently comprising endocytosis. Internalization of drug into cells may either obey receptor-dependent endocytosis or receptor-independent pathway as shown in **Figure 1**. Combining small drug molecules with macromolecular carriers restricts their entry into highly perfused tissues, thus averting

to weeks as well as shown great promise for intracellular transport of drugs [5].

can be receptor-specific ligands, vehicles, or biological molecules [2].

cytosis after being opsonized [4].

70 Molecular Insight of Drug Design

aging and biosensing both *in vitro* and *in vivo*.

**2. Intracellular drug targeting**

untoward side effects [2]. Macromolecules (proteins, peptides, DNA, etc.) are not allowed by lipophilic biological membranes to enter into cytoplasm. Such molecules may follow active transport involving endocytosis via cell surface receptors [6]. This receptor-mediated endocytosis offers a faster course for drug internalization in contrast to untargeted conjugates. These receptors have been located on surface of cells as well as accompany intracellular membranes [8].

#### **2.1. Clathrin-associated, receptor-mediated endocytosis**

The bases on which molecules are sorted to follow either clathrin-dependent or clathrin-independent pay are still not fully understood. However, it has been found that some specialized lipid domains are involved in membrane organization, sorting, and signal transduction in clathrin-independent endocytosis [9]. Ligand-associated drug carriers may bind to specific cell receptors that get assembled into particular areas of plasma membrane termed as coated pits. These regions (diameter 0.1 μm approx.) have been explained as plasma membrane invaginations with fuzzy cytoplasmic coat. This coat is mainly composed of clathrin protein present at cytoplasmic periphery of membrane and serves as major route for cellular internalization [10] as depicted in **Figure 2**. These coated pits allow intracellular vesicle formation in less than 1 min of time that is much faster than other mechanisms of endocytosis. A protein known as adaptin is responsible for polymerization of clathrin in the form of polyhedral lattice scaffold by binding with cell surface receptors. Two other proteins amphiphysin and endophilin get neighboring membrane into close vicinity. Dynamin (a cytosolic GTPase) gathers around the neckline of budding vesicle followed by its scission and intracellular discharge [2]. After intracellular entry of vesicle, an uncoating protein (heat shock protein; hsc70) causes the clathrin coat to shed of. At this stage, endocytosis trafficking of endosomal content decides the fate of therapeutic agent delivered. Endosomes may end up into lysosomes that may lead to degradation of drug or may be safely released into cytoplasm to reach desired organelle [6].

than 10% of endothelial cell membrane. Only cells expressing caveolin-1 protein develop caveolar invaginations [15]. Ligand after association with plasma membrane moves along it toward caveolar invagination, where being retained for some time internalization occurs via certain unidentified receptor as shown in **Figure 2**. The presence of GTPase dynamin has sug-

Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers

http://dx.doi.org/10.5772/intechopen.74624

73

Folic acid, cholesterol, albumin complexes, and serum lipoproteins are commonly encountered ligands internalized via caveolae-dependent endocytosis. These ligands have been considered as attractive candidates for drug targeting especially to intracellular organelles. Caveolar vesicles after getting internalized fuse with caveosomes following delivery of content at subcellular level bypassing acidic and degradative milieu of lysosomes [15]. Another mechanism suggested for caveolae-dependent internalization is 'potocytosis' that implies diffusion of smaller moieties into cytoplasm after interacting caveolae without membrane internalization [16]. One other associated pathway for cells without caveolae expression is 'lipid rafts'. These flat structures are composed of lipid- and protein-based assemblies that allow

Macropinocytosis employs distinct mechanism to transport molecules inside the cell without any direct coordination with receptors [17]. Macropinocytosis begins with actin polymerization at surface of cell membrane that is regulated by tyrosine kinase, epidermal growth factor, and platelet-derived growth factor receptors. This leads to increased ruffling at membrane surface and subsequent formation of macropinosomes (**Figure 2**). It involves absorption of molecules present in extracellular fluid (ECF) and seems to be a slower process as compared to RME. This process has sometimes shown to accompany receptor-mediated endocytosis; thus, absorption of receptor bound ligand and molecules in ECF may occur through clathrincoated vesicles side by side. After entry of fluid vesicles, they are supposed to follow usual endolysosomal trafficking pathway [18]. Negative charge on membrane surface naturally favors positively charged molecules to reside there and eventually get internalized through fluid phase endocytosis. This phenomenon has been exploited by researches for intracellular

Many targeting approaches utilize such mechanisms that exploit extracellular barriers to ensure efficient delivery of drugs. Nanocarriers following intravenous administration are rapidly recognized by reticuloendothelial system (liver and spleen macrophages), making it difficult for drug to reach its site of action at the minimum effective concentration [20]. This owes to opsonization of particle surface with certain plasma proteins (albumin, apolipoprotein-E, etc.) that make them recognizable by body's immune system and thus are rapidly evacuated from circulation. Therefore, the extent of opsonization will determine the fate of nanoparticles

gested involvement of caveolae in membrane internalization [9].

receptor-specific ligands to anchor on raft domain [15].

**2.4. Macropinocytosis**

delivery of drugs [19].

*in vivo* [21].

**3. Extracellular drug targeting**

**Figure 2.** (A) Clathrin-dependent receptor-mediated endocytosis, I—entry of targeting drug carrier in clathrin-coated pits and binding with ligand-specific receptor; II—dynamin-associated endocytosis; III—formation of clathrin-coated vesicle; IV—shedding of clathrin coat; V—early endosomal sorting and uncoupling of ligand-receptor; VI-A—formation of late endosome; VI-B—formation of transport vesicle; VII—fusion of lysosome with transport vesicle; VIII—formation of endolysosome. (B) Caveolin-dependent, receptor-mediated endocytosis, I—interaction of ligand with receptor; II—movement of receptor ligand complex toward caveolar invagination; III—retention of receptor ligand complex in caveolar invagination; IV—dynamin-associated caveolar endocytosis; V—formation of caveosome; VI-A and VI-B transport of drug carrier to endoplasmic reticulum or Golgi apparatus, respectively. (C) Macropinocytosis, I—movement of drug carrier toward membrane ruffling; II—rearrangement of cytoskeleton, folding of ruffle around drug carrier; IV—internalization; V—formation of macropinosome; VI—early maturation of macropinosome; VII—late endosome; VIII—endolysosome.

#### **2.2. Cell adhesion molecule (CAM)-mediated endocytosis**

Drug targeting has also been investigated using cell adhesion molecules (CAMs). Recently, integrins and cadherins have been found to internalize their ligand into intracellular milieu. Thus, many cell adhesion peptides such as arginylglycylaspartic acid (RGD) [11] and peptides derived from intercellular adhesion molecule 1 (ICAM-1) and lymphocyte functionassociated antigen 1 (LFA-1) sequences that bind to specific integrins have been extensively investigated for targeting tumor and vascular endothelial cells and suppressed progression of autoimmune disorders [12]. CAMs undergo cellular internalization while they are recycled via clathrin-coated pits and thus can be useful in cell-specific drug targeting through specific peptides. Such peptides are usually derived from proteins comprising extracellular matrix, immunoglobulins superfamily, and integrins. Integrins have also been associated with cellular uptake of certain viruses and bacteria through surface interactions at unique regions and initiate transduction pathways [13].

#### **2.3. Caveolin-dependent, receptor-mediated endocytosis**

Another version for cellular internalization of drug carriers is caveolin-mediated endocytosis. This process is sensitive to temperature and also dependent on ATP and sulphydral reagents [14]. Caveolin-1 is a protein associated with flask-shaped invaginations making up greater than 10% of endothelial cell membrane. Only cells expressing caveolin-1 protein develop caveolar invaginations [15]. Ligand after association with plasma membrane moves along it toward caveolar invagination, where being retained for some time internalization occurs via certain unidentified receptor as shown in **Figure 2**. The presence of GTPase dynamin has suggested involvement of caveolae in membrane internalization [9].

Folic acid, cholesterol, albumin complexes, and serum lipoproteins are commonly encountered ligands internalized via caveolae-dependent endocytosis. These ligands have been considered as attractive candidates for drug targeting especially to intracellular organelles. Caveolar vesicles after getting internalized fuse with caveosomes following delivery of content at subcellular level bypassing acidic and degradative milieu of lysosomes [15]. Another mechanism suggested for caveolae-dependent internalization is 'potocytosis' that implies diffusion of smaller moieties into cytoplasm after interacting caveolae without membrane internalization [16]. One other associated pathway for cells without caveolae expression is 'lipid rafts'. These flat structures are composed of lipid- and protein-based assemblies that allow receptor-specific ligands to anchor on raft domain [15].

#### **2.4. Macropinocytosis**

**2.2. Cell adhesion molecule (CAM)-mediated endocytosis**

**2.3. Caveolin-dependent, receptor-mediated endocytosis**

initiate transduction pathways [13].

VIII—endolysosome.

72 Molecular Insight of Drug Design

Drug targeting has also been investigated using cell adhesion molecules (CAMs). Recently, integrins and cadherins have been found to internalize their ligand into intracellular milieu. Thus, many cell adhesion peptides such as arginylglycylaspartic acid (RGD) [11] and peptides derived from intercellular adhesion molecule 1 (ICAM-1) and lymphocyte functionassociated antigen 1 (LFA-1) sequences that bind to specific integrins have been extensively investigated for targeting tumor and vascular endothelial cells and suppressed progression of autoimmune disorders [12]. CAMs undergo cellular internalization while they are recycled via clathrin-coated pits and thus can be useful in cell-specific drug targeting through specific peptides. Such peptides are usually derived from proteins comprising extracellular matrix, immunoglobulins superfamily, and integrins. Integrins have also been associated with cellular uptake of certain viruses and bacteria through surface interactions at unique regions and

**Figure 2.** (A) Clathrin-dependent receptor-mediated endocytosis, I—entry of targeting drug carrier in clathrin-coated pits and binding with ligand-specific receptor; II—dynamin-associated endocytosis; III—formation of clathrin-coated vesicle; IV—shedding of clathrin coat; V—early endosomal sorting and uncoupling of ligand-receptor; VI-A—formation of late endosome; VI-B—formation of transport vesicle; VII—fusion of lysosome with transport vesicle; VIII—formation of endolysosome. (B) Caveolin-dependent, receptor-mediated endocytosis, I—interaction of ligand with receptor; II—movement of receptor ligand complex toward caveolar invagination; III—retention of receptor ligand complex in caveolar invagination; IV—dynamin-associated caveolar endocytosis; V—formation of caveosome; VI-A and VI-B transport of drug carrier to endoplasmic reticulum or Golgi apparatus, respectively. (C) Macropinocytosis, I—movement of drug carrier toward membrane ruffling; II—rearrangement of cytoskeleton, folding of ruffle around drug carrier; IV—internalization; V—formation of macropinosome; VI—early maturation of macropinosome; VII—late endosome;

Another version for cellular internalization of drug carriers is caveolin-mediated endocytosis. This process is sensitive to temperature and also dependent on ATP and sulphydral reagents [14]. Caveolin-1 is a protein associated with flask-shaped invaginations making up greater Macropinocytosis employs distinct mechanism to transport molecules inside the cell without any direct coordination with receptors [17]. Macropinocytosis begins with actin polymerization at surface of cell membrane that is regulated by tyrosine kinase, epidermal growth factor, and platelet-derived growth factor receptors. This leads to increased ruffling at membrane surface and subsequent formation of macropinosomes (**Figure 2**). It involves absorption of molecules present in extracellular fluid (ECF) and seems to be a slower process as compared to RME. This process has sometimes shown to accompany receptor-mediated endocytosis; thus, absorption of receptor bound ligand and molecules in ECF may occur through clathrincoated vesicles side by side. After entry of fluid vesicles, they are supposed to follow usual endolysosomal trafficking pathway [18]. Negative charge on membrane surface naturally favors positively charged molecules to reside there and eventually get internalized through fluid phase endocytosis. This phenomenon has been exploited by researches for intracellular delivery of drugs [19].

#### **3. Extracellular drug targeting**

Many targeting approaches utilize such mechanisms that exploit extracellular barriers to ensure efficient delivery of drugs. Nanocarriers following intravenous administration are rapidly recognized by reticuloendothelial system (liver and spleen macrophages), making it difficult for drug to reach its site of action at the minimum effective concentration [20]. This owes to opsonization of particle surface with certain plasma proteins (albumin, apolipoprotein-E, etc.) that make them recognizable by body's immune system and thus are rapidly evacuated from circulation. Therefore, the extent of opsonization will determine the fate of nanoparticles *in vivo* [21].

Recognition of nanoparticles by RES and their uptake by macrophages can be avoided by modifying surface properties of these carrier systems. One of such modifications is by making surface of nanoparticles to be very hydrophilic. This avoids adsorption of opsonins and ensures nanoparticles to pass unrecognized by RES [22]. Surface of nanoparticles has been made more hydrophilic by increasing the thickness of coating layer of poloxamer and poloxamine. Coating layers up to 10 nm were considered necessary to bypass RES [23].

studied for a wide range of therapeutic applications (antivirals, antimicrobials, antitubercular drugs, biological molecules, and gene therapeutics) by offering enhanced accumulation and reduced toxicity at targeted site. However, reduced stability and trouble in immobilization of vector molecules on exterior of liposomes have led to exploration of other more useful nanocarriers [35]. Nanodots have exhibited great promise in therapeutics owing to diverse physicochemical properties, functionalization opportunities, and contenting stability attributes, which make them excellent candidate for bioimaging along with drug delivery. They have also been found substantially successful in photodynamic therapy in treatment of tumors [36]. Further, our discussion will be focused on multifunctional polymeric nanocarriers including dendrimers, various nanodot structures, and magnetic nanoparticles with reference to their application in intracellular and extracellular targeting for diagnostics and

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Dendrimers are three-dimensional, globular structures consisting of highly branched, repeating, and controllable peripheral functionalities originating from a central core [37]. These structures are assembled in layered fashion from core by repetition of two sequential reaction steps. Origination of a new branching point in layer leads to creation of a new generation (denoted as G). Thus, a regular dendrimer structure is usually composed of three major elements, i.e., a central core, branched units, and surface groups [38]. Such a diverse structure and nanometric size range has pulled them to massive exploration as potential carrier for drug delivery to targeted regions. The chemistry of dendrimers offers several modes for incorporation of drugs. Most commonly, drug is linked either covalently or noncovalently to dendrimers. A drug can be noncovalently introduced into dendrimer by simple encapsulation method that is mostly used to enhance solubilization of lipophilic drugs in aqueous media. Charged drugs such as DNA, RNA, or siRNA can also be incorporated into dendrimers through noncovalent electrostatic interactions. Thus, drug can be conjugated in this manner to both the internal and external regions of dendrimer. Covalent incorporation involves for-

Recent research is focused on development of synthetic carriers for delivery of genetic material with low cytotoxicity, highly efficient delivery, and minimal lysosomal degradation [40]. Gene delivery using polyamidoamine (PAMAM) dendrimers has been studied by Haensler and Szoka. Covalent linking of dendrimer with GALA peptide resulted in improved transfection efficiency [41]. Polypropylene imine (PPI) dendrimers enjoying low generations have also shown capacity as DNA carrier for gene transfection with lower cytotoxic potential [42]. Dendrimers have also been explored for delivery of chemotherapeutic agents. Quintana and coworkers have developed PAMAM dendrimers composed of ethylenediamine core. Methotrexate along with targeting ligand and fluorescein was covalently attached to dendritic surface. Experimental data confirmed highly specific binding with KB cell line with 100% improved cytotoxic response as compared to free drug [43]. In another study, siRNA was incorporated in PPI dendrimer that was stabilized using cross-linker to cage the preformed siRNA-dendrimer nanoparticle. PEG layer was applied over this nanostructure that further utilized luteinizing hormone releasing hormone (LHRH) to guide siRNA-loaded nanoparticles

mation of stable bonds between drug and dendrimers [39].

therapeutics.

**4.1. Dendrimers**

Many drug molecules have shown poor penetration across the blood-brain barrier due to their inherent nature. These molecules have been successfully delivered to the brain when incorporated into nanocarriers [24]. Coated nanoparticles have been studied for delivery of drug into the brain. Polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate-80 have been studied to improve penetration of drug across the blood-brain barrier [25]. In a study, transport of nanoparticles across BBB was investigated. Penetration of nanoparticles across BBB was three to four times increased when charged nanoparticles were coated with dipalmitoyl phosphatidyl choline and cholesterol-based lipid bilayer [26]. Multifunctional nanoparticles have also been investigated for delivery of proteins and peptides. These biological molecules are associated with rapid degradation at acidic pH of GIT and by activity of proteolytic enzymes that owe to their shorter half-life. Moreover, lesser partition coefficient and diffusivity make their movement difficult across biological membranes. These limitations can be conquered using functionalized polymeric nanoparticulate drug delivery systems [27]. Properties of PLGA matrices have been modified through hydrogel systems to deliver proteins and peptides. Bovine serum albumin was loaded in poly vinyl alcohol nanoparticles, which were then incorporated into PLGA microspheres and characterized to release the protein for more than 2 months [28]. Poly(isobutyl cyanoacrylate) nanocapsules have also been investigated for oral delivery of insulin [29].

Sustained release of drugs has also been achieved using various functionalized nanocarriers. Poly DL-lactic acid (PLA) nanoparticles have been used to provide sustained release of savoxepine following intramuscular and intravenous administrations [30]. Damge and his coworkers have successfully developed nanocapsules for sustained delivery of insulin. About 100 U/kg of insulin-loaded nanocapsules were effective to reduce blood glucose level for 6 days by 25% on oral administration to diabetic rats [31]. Colloidal suspension of docetaxel-loaded nanospheres has been prepared using PLA and PLGA to study sustained release of drug after intravenous administration [32]. PEG-grafted polyamidoamine (PAMAM) dendrimers were used to control the release of adriamycin and methotrexate [33]. Mu and Feng have used d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) to prepare controlled release paclitaxel nanoparticles with high encapsulation efficiency [34].

#### **4. Nanocarriers for intracellular and extracellular targeting**

Scientists have extensively explored a wide range of drug carriers at nanoscale extending from highly simple to complex geometries. Other than polymeric nanostructures, several lipids-based structures have been investigated for drug delivery, among which liposomes hold a noticeable status. Owing to their resemblance to lipid bilayer structure, they have been studied for a wide range of therapeutic applications (antivirals, antimicrobials, antitubercular drugs, biological molecules, and gene therapeutics) by offering enhanced accumulation and reduced toxicity at targeted site. However, reduced stability and trouble in immobilization of vector molecules on exterior of liposomes have led to exploration of other more useful nanocarriers [35]. Nanodots have exhibited great promise in therapeutics owing to diverse physicochemical properties, functionalization opportunities, and contenting stability attributes, which make them excellent candidate for bioimaging along with drug delivery. They have also been found substantially successful in photodynamic therapy in treatment of tumors [36]. Further, our discussion will be focused on multifunctional polymeric nanocarriers including dendrimers, various nanodot structures, and magnetic nanoparticles with reference to their application in intracellular and extracellular targeting for diagnostics and therapeutics.

#### **4.1. Dendrimers**

Recognition of nanoparticles by RES and their uptake by macrophages can be avoided by modifying surface properties of these carrier systems. One of such modifications is by making surface of nanoparticles to be very hydrophilic. This avoids adsorption of opsonins and ensures nanoparticles to pass unrecognized by RES [22]. Surface of nanoparticles has been made more hydrophilic by increasing the thickness of coating layer of poloxamer and polox-

Many drug molecules have shown poor penetration across the blood-brain barrier due to their inherent nature. These molecules have been successfully delivered to the brain when incorporated into nanocarriers [24]. Coated nanoparticles have been studied for delivery of drug into the brain. Polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate-80 have been studied to improve penetration of drug across the blood-brain barrier [25]. In a study, transport of nanoparticles across BBB was investigated. Penetration of nanoparticles across BBB was three to four times increased when charged nanoparticles were coated with dipalmitoyl phosphatidyl choline and cholesterol-based lipid bilayer [26]. Multifunctional nanoparticles have also been investigated for delivery of proteins and peptides. These biological molecules are associated with rapid degradation at acidic pH of GIT and by activity of proteolytic enzymes that owe to their shorter half-life. Moreover, lesser partition coefficient and diffusivity make their movement difficult across biological membranes. These limitations can be conquered using functionalized polymeric nanoparticulate drug delivery systems [27]. Properties of PLGA matrices have been modified through hydrogel systems to deliver proteins and peptides. Bovine serum albumin was loaded in poly vinyl alcohol nanoparticles, which were then incorporated into PLGA microspheres and characterized to release the protein for more than 2 months [28]. Poly(isobutyl cyanoacrylate) nanocapsules have also been

Sustained release of drugs has also been achieved using various functionalized nanocarriers. Poly DL-lactic acid (PLA) nanoparticles have been used to provide sustained release of savoxepine following intramuscular and intravenous administrations [30]. Damge and his coworkers have successfully developed nanocapsules for sustained delivery of insulin. About 100 U/kg of insulin-loaded nanocapsules were effective to reduce blood glucose level for 6 days by 25% on oral administration to diabetic rats [31]. Colloidal suspension of docetaxel-loaded nanospheres has been prepared using PLA and PLGA to study sustained release of drug after intravenous administration [32]. PEG-grafted polyamidoamine (PAMAM) dendrimers were used to control the release of adriamycin and methotrexate [33]. Mu and Feng have used d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) to prepare controlled release paclitaxel nanoparticles with high encapsulation efficiency [34].

Scientists have extensively explored a wide range of drug carriers at nanoscale extending from highly simple to complex geometries. Other than polymeric nanostructures, several lipids-based structures have been investigated for drug delivery, among which liposomes hold a noticeable status. Owing to their resemblance to lipid bilayer structure, they have been

**4. Nanocarriers for intracellular and extracellular targeting**

amine. Coating layers up to 10 nm were considered necessary to bypass RES [23].

investigated for oral delivery of insulin [29].

74 Molecular Insight of Drug Design

Dendrimers are three-dimensional, globular structures consisting of highly branched, repeating, and controllable peripheral functionalities originating from a central core [37]. These structures are assembled in layered fashion from core by repetition of two sequential reaction steps. Origination of a new branching point in layer leads to creation of a new generation (denoted as G). Thus, a regular dendrimer structure is usually composed of three major elements, i.e., a central core, branched units, and surface groups [38]. Such a diverse structure and nanometric size range has pulled them to massive exploration as potential carrier for drug delivery to targeted regions. The chemistry of dendrimers offers several modes for incorporation of drugs. Most commonly, drug is linked either covalently or noncovalently to dendrimers. A drug can be noncovalently introduced into dendrimer by simple encapsulation method that is mostly used to enhance solubilization of lipophilic drugs in aqueous media. Charged drugs such as DNA, RNA, or siRNA can also be incorporated into dendrimers through noncovalent electrostatic interactions. Thus, drug can be conjugated in this manner to both the internal and external regions of dendrimer. Covalent incorporation involves formation of stable bonds between drug and dendrimers [39].

Recent research is focused on development of synthetic carriers for delivery of genetic material with low cytotoxicity, highly efficient delivery, and minimal lysosomal degradation [40]. Gene delivery using polyamidoamine (PAMAM) dendrimers has been studied by Haensler and Szoka. Covalent linking of dendrimer with GALA peptide resulted in improved transfection efficiency [41]. Polypropylene imine (PPI) dendrimers enjoying low generations have also shown capacity as DNA carrier for gene transfection with lower cytotoxic potential [42]. Dendrimers have also been explored for delivery of chemotherapeutic agents. Quintana and coworkers have developed PAMAM dendrimers composed of ethylenediamine core. Methotrexate along with targeting ligand and fluorescein was covalently attached to dendritic surface. Experimental data confirmed highly specific binding with KB cell line with 100% improved cytotoxic response as compared to free drug [43]. In another study, siRNA was incorporated in PPI dendrimer that was stabilized using cross-linker to cage the preformed siRNA-dendrimer nanoparticle. PEG layer was applied over this nanostructure that further utilized luteinizing hormone releasing hormone (LHRH) to guide siRNA-loaded nanoparticles to tumor cells. *In-vivo* studies suggested highly specific tumor targeting with improved accumulation of siRNA in cytoplasm of cells and effective gene silencing [44].

decreasing their target specificity and binding affinity [51]. These carriers have widely investigated for their outstanding potential for bioimaging and biosensing both *in vitro* and *in vivo*. Major advantage lies in lesser cytotoxicity as was observed in the case of inorganic nanocrystals or quantum dots (Qdots) that were also observed to be associated with genotoxic and

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Biological applications of fluorescent probes can be controlled by manipulating surface chemistry. Usually, aqueous solubility is the primary requirement for these particles to perform biological functions efficiently. Such modifications have been carried out by incorporating charged molecules in polymer side chains [53]. Current research has much interest in development of various multifunctional Pdots that offer a wide range of biological applications due to easy preparation and diverse chemical dynamics. Conjugated polymers on the basis of varying structure of backbone can be distinguished into four major categories including poly-(fluorine), poly-(p-phenylene vinylene), poly-(p-phenylene ethynylene), and poly-(thiophene). These polymeric backbones can be further functionalized to incorporate desired characteristics [54]. Wang and coworkers have developed water-soluble conjugated polymer (polythiophene) with tyrosin kinase inhibitor (lapatinib)-modified side chains for plasma membrane imaging by targeting intracellular regions of transmembrane proteins [55]. Scientists have prepared conjugated polymer nanoparticles for both drug delivery and cell imaging together via exploiting electrostatic interactions among cationic conjugated polymers and anionic functionalities. Doxorubicin was delivered to target cancer cells by conjugating it with cationic fluorescent PFO and anionic poly-(l-glutamic acid) that allowed to monitor

Apart from cellular imaging, Pdots have also been investigated for delivery of DNA and siRNA. It has been found that nucleic acid carrying a negative charge can be easily incorporated into positively charged semiconductor Pdots. Silva and coworkers have demonstrated the delivery of siRNA using fluorescent CPNs for posttranscriptional gene silencing in plant protoplast without any significant impact on cellular viability in 72 h. They also explained the delivery of siRNA to specifically targeted genes in NTCesA-1 pathway associated with cellulose biosynthesis using CPNs [57]. Moon and associates have developed loosely aggregated CPNs for delivery of siRNA for transfection into HELA cells. siRNA-loaded CPNs caused

Scientists have also investigated the role of conjugated polymers for their antimicrobial potential. Cationic, light-absorbing, conjugated polyelectrolytes were studied for their activity against Gram-positive bacterial spores and Gram-negative bacterial strains. The study suggested that conjugated poly-electrolytes formed surface coating on both bacterial types and caused light-induced bactericidal activity [59]. Electrostatic interactions between negatively charged cell surfaces and oppositely charged markers have also been exploited against microbial and cancerous cells. Cationic CPNs have been designed through electrostatic interaction between positively charged PBF and negatively charged SDPA (disodium salt 3,3′-dithiodipropionic acid). These nanoparticles on photoexcitation by white light sensitized production of reactive oxygen species that effectively killed surrounding tumor and bacterial cells along with fluorescent imaging of cellular uptake of these particles [60]. Electrostatic interactions

epigenetic effects in mammalian cells even at minute concentrations [52].

drug release through 'turn on' fluorescent signal generated by PFO [56].

downregulation of actin b protein with a transfection efficiency of 94% [58].

Dendritic structures have also inspired boron neutron capture therapy for tumor targeting. To kill tumor cells, it is necessary for 10B to reach intracellular concentration of at least 109 atoms/cell. This tumor-specific delivery at desired concentrations has been achieved through use of boronated antibodies. Epidermal growth factor receptors (EGFR) that are overexpressed at surface of glioma cells have been targeted by 10B PAMAM dendrimers. Dendrimers after being covalently linked to epidermal growth factor were effectively internalized by endocytosis with substantial accumulation of 10B in lysosomes of cells *in vitro* [45]. However, *in-vivo* studies demonstrated uptake of boron carriers by the liver and less level of accumulation in C6 glioma cells. To address uptake of boronated dendritic conjugates by the liver, scientists have exploited the steric effect provided by polyethylene oxide (PEO) chains. Such PEO-shielded boronated PAMAM dendrimers showed lesser uptake of conjugate by the liver. However, uptake of PEO-shielded dendritic conjugates by liver was increased with an increase in the number of PEO chains [46].

Exploration of dendrimers in photodynamic therapy (PDT) has also captured great interest. Therapy employs a photosensitizing agent that upon exposure to light of specific wavelength causes irreversible photo-chemical or photo-biological damage to tumor cells. Dendrimers on suitable peripheral functionalization can be promising carrier for photosensitizers. Eighteen ALA (5-aminolevulinic acid) units have been conjugated with dendrimer through amide linkage. These ALA-conjugated dendrimers exhibited increased cellular level of protoporphyrin IX (PIX) and thus showed increased cytotoxicity on exposure of radiations in PAM 212 tumorigenic cell lines [47]. Because of increased tissue permeability to near IR or IR light, the photodynamic system with high absorbance at longer wavelength is extremely attractive. To exploit this feature, aluminum-phthalocyanines polymer conjugates have been designed with the maximum absorption observed at 675 nm [48]. A two-photon approach also has great potential to target deeper tissues with near IR laser. Multivalent character of dendrimers has the capacity to accommodate several two-photon absorbing moieties to porphyrin core. Excitation of chromophores at 780 nm resulted in generation of increased singlet oxygen luminescence [49].

#### **4.2. Semiconductor polymer dots**

Semiconducting polymer dots (Pdots), also described as organic nanodots or conjugated polymeric nanoparticles (CPNs), have emerged as promising fluorescent probes due to their exceptional brightness, high quantum yield, nonblinking, photo-stability, and faster emission rate. Pdots, particularly, refer to small semiconducting polymeric nanoparticles and have shown remarkable conduction properties due to the presence of highly delocalized π-conjugated backbone [50]. Pdots prepared by miniemulsion method usually produced polymeric particles with size ranging from 40 to 500 nm depending on nature of polymer and concentration of surfactant. Reprecipitation method yields Pdots in the range of 5–30 nm. Size usually can be modified depending on biological application; however, most bioimaging and assays require smaller nanoparticle. Brightness and photo-stability of fluorescent Pdots appear to increase with size increment but have also exhibited higher steric hindrance, decreasing their target specificity and binding affinity [51]. These carriers have widely investigated for their outstanding potential for bioimaging and biosensing both *in vitro* and *in vivo*. Major advantage lies in lesser cytotoxicity as was observed in the case of inorganic nanocrystals or quantum dots (Qdots) that were also observed to be associated with genotoxic and epigenetic effects in mammalian cells even at minute concentrations [52].

to tumor cells. *In-vivo* studies suggested highly specific tumor targeting with improved accu-

Dendritic structures have also inspired boron neutron capture therapy for tumor targeting. To kill tumor cells, it is necessary for 10B to reach intracellular concentration of at least

Exploration of dendrimers in photodynamic therapy (PDT) has also captured great interest. Therapy employs a photosensitizing agent that upon exposure to light of specific wavelength causes irreversible photo-chemical or photo-biological damage to tumor cells. Dendrimers on suitable peripheral functionalization can be promising carrier for photosensitizers. Eighteen ALA (5-aminolevulinic acid) units have been conjugated with dendrimer through amide linkage. These ALA-conjugated dendrimers exhibited increased cellular level of protoporphyrin IX (PIX) and thus showed increased cytotoxicity on exposure of radiations in PAM 212 tumorigenic cell lines [47]. Because of increased tissue permeability to near IR or IR light, the photodynamic system with high absorbance at longer wavelength is extremely attractive. To exploit this feature, aluminum-phthalocyanines polymer conjugates have been designed with the maximum absorption observed at 675 nm [48]. A two-photon approach also has great potential to target deeper tissues with near IR laser. Multivalent character of dendrimers has the capacity to accommodate several two-photon absorbing moieties to porphyrin core. Excitation of chromophores at 780 nm resulted in generation of increased singlet oxygen luminescence [49].

Semiconducting polymer dots (Pdots), also described as organic nanodots or conjugated polymeric nanoparticles (CPNs), have emerged as promising fluorescent probes due to their exceptional brightness, high quantum yield, nonblinking, photo-stability, and faster emission rate. Pdots, particularly, refer to small semiconducting polymeric nanoparticles and have shown remarkable conduction properties due to the presence of highly delocalized π-conjugated backbone [50]. Pdots prepared by miniemulsion method usually produced polymeric particles with size ranging from 40 to 500 nm depending on nature of polymer and concentration of surfactant. Reprecipitation method yields Pdots in the range of 5–30 nm. Size usually can be modified depending on biological application; however, most bioimaging and assays require smaller nanoparticle. Brightness and photo-stability of fluorescent Pdots appear to increase with size increment but have also exhibited higher steric hindrance,

 atoms/cell. This tumor-specific delivery at desired concentrations has been achieved through use of boronated antibodies. Epidermal growth factor receptors (EGFR) that are overexpressed at surface of glioma cells have been targeted by 10B PAMAM dendrimers. Dendrimers after being covalently linked to epidermal growth factor were effectively internalized by endocytosis with substantial accumulation of 10B in lysosomes of cells *in vitro* [45]. However, *in-vivo* studies demonstrated uptake of boron carriers by the liver and less level of accumulation in C6 glioma cells. To address uptake of boronated dendritic conjugates by the liver, scientists have exploited the steric effect provided by polyethylene oxide (PEO) chains. Such PEO-shielded boronated PAMAM dendrimers showed lesser uptake of conjugate by the liver. However, uptake of PEO-shielded dendritic conjugates by liver was increased with

mulation of siRNA in cytoplasm of cells and effective gene silencing [44].

an increase in the number of PEO chains [46].

**4.2. Semiconductor polymer dots**

109

76 Molecular Insight of Drug Design

Biological applications of fluorescent probes can be controlled by manipulating surface chemistry. Usually, aqueous solubility is the primary requirement for these particles to perform biological functions efficiently. Such modifications have been carried out by incorporating charged molecules in polymer side chains [53]. Current research has much interest in development of various multifunctional Pdots that offer a wide range of biological applications due to easy preparation and diverse chemical dynamics. Conjugated polymers on the basis of varying structure of backbone can be distinguished into four major categories including poly-(fluorine), poly-(p-phenylene vinylene), poly-(p-phenylene ethynylene), and poly-(thiophene). These polymeric backbones can be further functionalized to incorporate desired characteristics [54]. Wang and coworkers have developed water-soluble conjugated polymer (polythiophene) with tyrosin kinase inhibitor (lapatinib)-modified side chains for plasma membrane imaging by targeting intracellular regions of transmembrane proteins [55]. Scientists have prepared conjugated polymer nanoparticles for both drug delivery and cell imaging together via exploiting electrostatic interactions among cationic conjugated polymers and anionic functionalities. Doxorubicin was delivered to target cancer cells by conjugating it with cationic fluorescent PFO and anionic poly-(l-glutamic acid) that allowed to monitor drug release through 'turn on' fluorescent signal generated by PFO [56].

Apart from cellular imaging, Pdots have also been investigated for delivery of DNA and siRNA. It has been found that nucleic acid carrying a negative charge can be easily incorporated into positively charged semiconductor Pdots. Silva and coworkers have demonstrated the delivery of siRNA using fluorescent CPNs for posttranscriptional gene silencing in plant protoplast without any significant impact on cellular viability in 72 h. They also explained the delivery of siRNA to specifically targeted genes in NTCesA-1 pathway associated with cellulose biosynthesis using CPNs [57]. Moon and associates have developed loosely aggregated CPNs for delivery of siRNA for transfection into HELA cells. siRNA-loaded CPNs caused downregulation of actin b protein with a transfection efficiency of 94% [58].

Scientists have also investigated the role of conjugated polymers for their antimicrobial potential. Cationic, light-absorbing, conjugated polyelectrolytes were studied for their activity against Gram-positive bacterial spores and Gram-negative bacterial strains. The study suggested that conjugated poly-electrolytes formed surface coating on both bacterial types and caused light-induced bactericidal activity [59]. Electrostatic interactions between negatively charged cell surfaces and oppositely charged markers have also been exploited against microbial and cancerous cells. Cationic CPNs have been designed through electrostatic interaction between positively charged PBF and negatively charged SDPA (disodium salt 3,3′-dithiodipropionic acid). These nanoparticles on photoexcitation by white light sensitized production of reactive oxygen species that effectively killed surrounding tumor and bacterial cells along with fluorescent imaging of cellular uptake of these particles [60]. Electrostatic interactions have also been exploited for delivery of doxorubicin through multifunctional CPNs. CPNs (50 nm approx.) with excellent photo-stability and quantum yield but lower cytotoxicity have been prepared by combination of cationic PFO and anionic poly(l-glutamic acid) followed by conjugation with doxorubicin. This carrier offered targeted release of drug in cancer cells along with concurrent examination of drug release via self-luminescence activity [56].

Scientists have also demonstrated use of CDs for detection of DNA. ssDNA was immobilized on CDs that can get hybridized with required complementary DNA molecule to form dsDNA followed by desorption from CDs and quantification of fluorescence [75]. CDs-dsDNA complex has also been investigated to detect histones. The strong interaction between DNA and histone causing the detachment of DNA from CD that turned on the signal for native fluorescence of CD [76]. CDs conjugated with gold and silver nanostructures have been explored as electro-chemiluminescence (ECL) immuno-sensing devices for detection of prostate-specific antigen (PSA) [77]. CDs have also been conjugated with chlorin e6 (Ce6) photosensitizer for efficient intracellular transport of photosensitizer, longer circulation time, and homing ability in tumor cells. These conjugates revealed excellent stability, biocompatibility with least cytotoxicity and exhibited tremendous bioimaging and homing ability in subcutaneous MGC-803

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Another therapeutic application associated with CDs is gene and drug delivery to targeted cells. pH-responsive, COOH-functionalized CDs caped on surface of mesoporous silica nanoparticles (MSPs) have been studied for intracellular tracking and delivery of doxorubicin with strong luminescence and low cytotoxicity both *in vitro* and *in vivo* [79]. CDs-conjugated mesoporous silica nanoparticles capped with PEG have been investigate for tracking controlled release of doxorubicin through quantifiable fluorescent intensity in HELA cells [80]. In another study, hollow CDs have been prepared from bovine serum albumin for pH-dependent delivery of doxorubicin and its rapid intracellular uptake. Such functionalized hollow CDs have been regarded suitable for bioimaging and targeted drug delivery [81]. Quinoloneconjugated fluorescent CDs have also been explored for *in-vitro* cellular imaging and delivery

Quantum dots (Qdots) are inorganic, semiconductor, fluorescent nanostructures composed of II–IV or III–V group elements. They are crystal structures with size smaller than excitation Bohr radius (few nanometers), and these physical dimensions are controllable by time, temperature, and molecules (ligands) used in their synthesis [83]. Qdots in the range of 2–6 nm are of especial interest due to resemblance of their dimensions with biomolecules and have also shown to display strong dimension-dependent electrical and optical characters. Other distinct features include necessity of few Qdots to generate a detectable signal and minimal photo-bleaching property [84]. The idea of quantum confinement is responsible for unique optical and electronic characteristic of Qdots. Both group II–IV and III–V Qdots have been synthesized with relatively lower quantum yield and greater size difference. Higher quantum yield and better luminescence were observed when CdSe core was capped on surface with

The optical character of Qdots has been associated with the interactions among electrons, holes, and surrounding environment. Qdots undergo absorption of photon when excitation energy surpasses band gap where electrons jump from valence band toward conduction band. The presence of multiple electronic states at elevated energy level offers excitation at relatively lower wavelengths across UV-visible spectra. Emission wavelength can be tuned among the region of blue and near infrared (NIR) wavelength by manipulating size

xenografts in nude mice [78].

of drug to cancer cells [82].

ZnS or CdS (higher band gap) [85].

**4.4. Quantum dots**

#### **4.3. Carbon dots**

Carbon dots (CDs), also known as carbon nanoparticles or carbon quantum dots, are quasispherical fluorescent nanoparticles, gaining excessive attention because of unique optical nature, biocompatibility, low cytotoxicity, and simplistic synthesis [61]. These particles were accidently discovered while electrophoretic purification of single-walled carbon nanotubes (SWCNTs) synthesized using arc discharge process. CDs are usually defined as zero-dimensional particles with size range lying around 10 nm. Various synthetic approaches have been investigated for preparation of CDs with efficient photoluminescence, longer wavelength, and multicolor tunable emission [62]. Several types of carbon materials have been engaged to prepare CDs including graphite, activated carbon, carbon nanotubes (CNTs), and nano-diamond using top-down approach [63]. Bottom-up approach has employed citrate, biomolecules, and polymer-silica nanocomposites to prepare CDs using a variety of reaction conditions [64]. CDs have been addressed as safe and biocompatible substitutes to quantum dots that offer better brightness, photo-stability, and lower cytotoxicity both *in vitro* and *in vivo* [65].

Fluorescent CDs have expressed great potential in the field of biosensing, imaging, and photodynamic therapy as well as gene and drug delivery. CDs can be employed for *in-vitro* and *in-vivo* cell imaging using both one- and two-photon excitations. Yang and coworkers have demonstrated biomolecule surface-modified fluorescent carbon dots for *in-vivo* cell imaging along with good biocompatibility and less cytotoxicity [66]. Fluorescent CDs with surface modified with PEG were also studied for *in-vivo* biocompatibility and cytotoxicity through fluorescence imaging [67]. Luo and coworkers have extensively reviewed the optical imaging of carbon dots both *in vitro* and *in vivo* [65]. Various functionalized CDs have been studied for fluorescent imaging of plasma membrane and cytoplasm of COS7 cells, BGC823 cells, MG-63 cells, A549 cells, and HEPG-2 cells [68]. Scientists have also demonstrated uptake of CDs by endosomes and lysosomes in fluorescent imaging of HELA cells [69]. Besides these investigations, some studies have reported distribution of CDs in entire cell including nucleus [70]. CDs have also been explored for cellular imaging and labeling of *E. coli* [63].

CDs have also been investigated as biosensors in various research studies. Fluorescent carbon dots when conjugated with *N*-(2-aminoethyl)-*N*, *N*, *N*-tris (pyridin-2-yl methyl) ethane-1, 2-diamine have been studied to detect intracellular Cu+2 ions with greater specificity and stability [71]. In another study, fluorescent CDs have been used for detection of metal ions. Scientists have prepared carbon dots from citric acid as a carbon source in the presence of PEI for intracellular imaging and detection of Cu+2 ions [72]. Besides metal ion detection, COOHor OH-functionalized CDs have been used as a receptor to detect change in hydrogen ion concentration. This fluorescent C dot probe has been successfully investigated to detect change in pH of 6–8 range in A549 and LLC-MK2 cells [73]. CDs have been studied for detection of glucose as its transport is associated with certain anomalies such as diabetes and cancer [74].

Scientists have also demonstrated use of CDs for detection of DNA. ssDNA was immobilized on CDs that can get hybridized with required complementary DNA molecule to form dsDNA followed by desorption from CDs and quantification of fluorescence [75]. CDs-dsDNA complex has also been investigated to detect histones. The strong interaction between DNA and histone causing the detachment of DNA from CD that turned on the signal for native fluorescence of CD [76]. CDs conjugated with gold and silver nanostructures have been explored as electro-chemiluminescence (ECL) immuno-sensing devices for detection of prostate-specific antigen (PSA) [77]. CDs have also been conjugated with chlorin e6 (Ce6) photosensitizer for efficient intracellular transport of photosensitizer, longer circulation time, and homing ability in tumor cells. These conjugates revealed excellent stability, biocompatibility with least cytotoxicity and exhibited tremendous bioimaging and homing ability in subcutaneous MGC-803 xenografts in nude mice [78].

Another therapeutic application associated with CDs is gene and drug delivery to targeted cells. pH-responsive, COOH-functionalized CDs caped on surface of mesoporous silica nanoparticles (MSPs) have been studied for intracellular tracking and delivery of doxorubicin with strong luminescence and low cytotoxicity both *in vitro* and *in vivo* [79]. CDs-conjugated mesoporous silica nanoparticles capped with PEG have been investigate for tracking controlled release of doxorubicin through quantifiable fluorescent intensity in HELA cells [80]. In another study, hollow CDs have been prepared from bovine serum albumin for pH-dependent delivery of doxorubicin and its rapid intracellular uptake. Such functionalized hollow CDs have been regarded suitable for bioimaging and targeted drug delivery [81]. Quinoloneconjugated fluorescent CDs have also been explored for *in-vitro* cellular imaging and delivery of drug to cancer cells [82].

#### **4.4. Quantum dots**

have also been exploited for delivery of doxorubicin through multifunctional CPNs. CPNs (50 nm approx.) with excellent photo-stability and quantum yield but lower cytotoxicity have been prepared by combination of cationic PFO and anionic poly(l-glutamic acid) followed by conjugation with doxorubicin. This carrier offered targeted release of drug in cancer cells

Carbon dots (CDs), also known as carbon nanoparticles or carbon quantum dots, are quasispherical fluorescent nanoparticles, gaining excessive attention because of unique optical nature, biocompatibility, low cytotoxicity, and simplistic synthesis [61]. These particles were accidently discovered while electrophoretic purification of single-walled carbon nanotubes (SWCNTs) synthesized using arc discharge process. CDs are usually defined as zero-dimensional particles with size range lying around 10 nm. Various synthetic approaches have been investigated for preparation of CDs with efficient photoluminescence, longer wavelength, and multicolor tunable emission [62]. Several types of carbon materials have been engaged to prepare CDs including graphite, activated carbon, carbon nanotubes (CNTs), and nano-diamond using top-down approach [63]. Bottom-up approach has employed citrate, biomolecules, and polymer-silica nanocomposites to prepare CDs using a variety of reaction conditions [64]. CDs have been addressed as safe and biocompatible substitutes to quantum dots that offer

along with concurrent examination of drug release via self-luminescence activity [56].

better brightness, photo-stability, and lower cytotoxicity both *in vitro* and *in vivo* [65].

CDs have also been explored for cellular imaging and labeling of *E. coli* [63].

Fluorescent CDs have expressed great potential in the field of biosensing, imaging, and photodynamic therapy as well as gene and drug delivery. CDs can be employed for *in-vitro* and *in-vivo* cell imaging using both one- and two-photon excitations. Yang and coworkers have demonstrated biomolecule surface-modified fluorescent carbon dots for *in-vivo* cell imaging along with good biocompatibility and less cytotoxicity [66]. Fluorescent CDs with surface modified with PEG were also studied for *in-vivo* biocompatibility and cytotoxicity through fluorescence imaging [67]. Luo and coworkers have extensively reviewed the optical imaging of carbon dots both *in vitro* and *in vivo* [65]. Various functionalized CDs have been studied for fluorescent imaging of plasma membrane and cytoplasm of COS7 cells, BGC823 cells, MG-63 cells, A549 cells, and HEPG-2 cells [68]. Scientists have also demonstrated uptake of CDs by endosomes and lysosomes in fluorescent imaging of HELA cells [69]. Besides these investigations, some studies have reported distribution of CDs in entire cell including nucleus [70].

CDs have also been investigated as biosensors in various research studies. Fluorescent carbon dots when conjugated with *N*-(2-aminoethyl)-*N*, *N*, *N*-tris (pyridin-2-yl methyl) ethane-1, 2-diamine have been studied to detect intracellular Cu+2 ions with greater specificity and stability [71]. In another study, fluorescent CDs have been used for detection of metal ions. Scientists have prepared carbon dots from citric acid as a carbon source in the presence of PEI for intracellular imaging and detection of Cu+2 ions [72]. Besides metal ion detection, COOHor OH-functionalized CDs have been used as a receptor to detect change in hydrogen ion concentration. This fluorescent C dot probe has been successfully investigated to detect change in pH of 6–8 range in A549 and LLC-MK2 cells [73]. CDs have been studied for detection of glucose as its transport is associated with certain anomalies such as diabetes and cancer [74].

**4.3. Carbon dots**

78 Molecular Insight of Drug Design

Quantum dots (Qdots) are inorganic, semiconductor, fluorescent nanostructures composed of II–IV or III–V group elements. They are crystal structures with size smaller than excitation Bohr radius (few nanometers), and these physical dimensions are controllable by time, temperature, and molecules (ligands) used in their synthesis [83]. Qdots in the range of 2–6 nm are of especial interest due to resemblance of their dimensions with biomolecules and have also shown to display strong dimension-dependent electrical and optical characters. Other distinct features include necessity of few Qdots to generate a detectable signal and minimal photo-bleaching property [84]. The idea of quantum confinement is responsible for unique optical and electronic characteristic of Qdots. Both group II–IV and III–V Qdots have been synthesized with relatively lower quantum yield and greater size difference. Higher quantum yield and better luminescence were observed when CdSe core was capped on surface with ZnS or CdS (higher band gap) [85].

The optical character of Qdots has been associated with the interactions among electrons, holes, and surrounding environment. Qdots undergo absorption of photon when excitation energy surpasses band gap where electrons jump from valence band toward conduction band. The presence of multiple electronic states at elevated energy level offers excitation at relatively lower wavelengths across UV-visible spectra. Emission wavelength can be tuned among the region of blue and near infrared (NIR) wavelength by manipulating size and composition of Qdots. This feature allows simultaneous excitation of multicolor Qdots with single light source that makes them excellent candidate for biological application. Bioconjugation and functionalization of Qdots have increased the spectrum of their activities [36]. Qdots have been widely investigated for *in-vitro* and *in-vivo* imaging at molecular and cellular levels, to study intracellular trafficking as well as tumor targeting [86]. Quantum dots have been studied in immunofluorescence assays for detection of biological molecules and labeling of tissues and cells. NIR fluorescent nanoprobes conjugated with copolymer grafts of poly(l-lysine) and methoxy-polyethylene glycol succinate for *in-vivo* imaging of tumor related lysosomal protease activity. These probes successfully detected small-sized solid tumors with higher NIR signals and to examine specific enzyme activity [87]. Qdots have also explored to study the modifications in erythrocyte membranes caused by plasmodium invasion in malaria via immuno-cytochemical studies [88]. Jaiswal and coworkers have demonstrated multicolor imaging of Qdots-labeled live cells. They explained two approaches for cell labeling; either through intracellular uptake of Qdots by endocytic mechanism or use of antibody-conjugated quantum dots specific to cell surface proteins [89]. Parak and associates have used colloidal Qdots to study metastatic potential of cancer cells due to their photochemical stable nature and to study the mechanism of motility and migration of cancer cells. Uptake of nanocrystals was explained to occur through pinocytosis and phagocytosis [90].

PEG functionalization caused improved circulation time and rapid accumulation in cancer

Multifunctional Polymeric Enveloped Nanocarriers: Targeting Extracellular and Intracellular Barriers

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81

Magnetic nanoparticles (MNPs) are one of other fascinating elements of nanotechnology. Their nanometric dimension, biocompatibility, nontoxicity, and surplus accumulation in targeted cells or tissues justify intensive research in this subject matter. MNPs are mostly composed of ferromagnetic material such as ferrous or ferric oxide core with limited use of cobalt and nickel [98]. Magnetic properties are associated with movement of subatomic particles including electrons, holes, protons, and positive-/negative-charged ions. These materials respond to external stimulus of magnetic field and orient themselves according to magnetic moment. This magnetic behavior has been exploited for both *in-vitro* and *in-vivo* biomedical applications [99]. Magnetic nanoparticles have also been suggested for labeling cells in tissue engineering as they can be easily handled using magnets. Streptavidin-functionalized paramagnetic particles in combination with antibodies have been investigated for magnetic

Magnetic nanoparticles have the ability to cause ablation of tumor cells via generation of heat. AC magnetic field causes the magnetic particles dispersed in target cells or tissues to get heated. This heat is rapidly disseminated to diseased cells, and if 42°C (therapeutic temperature threshold) can be maintained for 30 min, tumor cells get destroyed. However, this thermal ablation may be associated with undesirable concurrent killing of healthy cells [101]. Hase and coworkers have used ferromagnetic heating in combination of hepatic arterial embolization to study heat induction of ferromagnetic implants on VX2 hepatic cancer in rabbits. Results indicated extensive degeneration of tumor cells suggesting a suitable therapeutic strategy for localized hepatic carcinomas with little damage to healthy parenchyma of the

Various functionalized magnetic nanocarriers have been investigated for targeted delivery of therapeutic agents. Magnetic drug carriers were designed either by using a magnetic core with surface coated with polymer or magnetic particles precipitated within porous polymeric composite. Such modifications have been studied to protect magnetic particle from harsh physiological vicinity and also to guide the drug carrier to desired location. Magnetic field-guided uncharged magnetic nanoparticles have been investigated for intracerebral targeting of rat glioma-2 in male (Fisher 344) rats. These magnetic nanoparticles (10–20 nm) exhibited greater uptake in brain tumor cells as compared to larger size (1 μm) magnetic particles [103]. In another study, iron oxide core was coated with oleic acid and subsequent coat of PEG-oleic acid for sustained release of doxorubicin and as MRI contrast. These modified magnetic nanoparticles were further conjugated with antibodies for active targeting of MFC-7 cells. These MNPs showed better MRI contrast with longer circulation time. They exhibited sustained release of drug with enhanced antiproliferative effect [104]. Doxorubicin-loaded monodisperse mesoporous single crystal iron oxide nanoparticles have also been developed as a promising carrier with improved drug loading and

cells [97].

**4.5. Magnetic nanocarriers**

field-guided retroviral infection *in vitro* [100].

liver because of selective heat induction [102].

delivery [105].

Qdots have also been explored to prevent their nonspecific uptake by RES. Molecular markers expressed by blood vessels have been exploited to target nanocarriers toward specific tissues or organs. This strategy has been employed to target lung tumor cells using functionalized Qdots in mice [91]. Surface of quantum dots has been functionalized with COOH, NH2, and streptavidin that was further derivitized using PEG. PEG-conjugated Qdots decreased nonspecific uptake by RES, while COOH- and NH2-functionalized Qdots without PEG showed improved intracellular uptake among various cell types [92]. Qdots have also been explored for gene delivery and gene silencing. Sponge proton-coated Qdot-siRNA has been studied to improve gene silencing efficiency and reduced cytotoxicity in MDA-MB231 cells. These nanocarriers also allowed intracellular tracking and localization of siRNA delivery and transfection [93]. In another study, siRNA transfection was performed using Qdots. siRNA-Qdots exhibited greater photo-stability and tunable optical characteristics. This method was developed to observe the function of T-cadherin in intercellular communication [94].

Chitosan-folate-encapsulated ZnO Qdots have been prepared for delivery of anticancer agent doxorubicin with enhanced and longer photo-stability of Qdots. This nanocarrier showed an initial rapid release followed by controlled liberation of drug [95]. Doxorubicinloaded, immuno-liposome-based quantum dots were modified with HER2scFv for targeted delivery of drug to SKB-3 and MCF-7 cells with overexpressed HER2. These Qdot-IL conjugates exhibited receptor-dependent endocytosis in target cells but not in control MFC-7 cells. They also showed longer circulation of Qdots, and their localization in tumor models was confirmed by florescence imaging [96]. Cadmium telluride-incorporated Qdots with PEI functionalization for tracking of plasmid DNA in mice were designed. After intravenous injection, these structures showed rapid accumulation in the lungs, spleen, and liver. PEG functionalization caused improved circulation time and rapid accumulation in cancer cells [97].

#### **4.5. Magnetic nanocarriers**

and composition of Qdots. This feature allows simultaneous excitation of multicolor Qdots with single light source that makes them excellent candidate for biological application. Bioconjugation and functionalization of Qdots have increased the spectrum of their activities [36]. Qdots have been widely investigated for *in-vitro* and *in-vivo* imaging at molecular and cellular levels, to study intracellular trafficking as well as tumor targeting [86]. Quantum dots have been studied in immunofluorescence assays for detection of biological molecules and labeling of tissues and cells. NIR fluorescent nanoprobes conjugated with copolymer grafts of poly(l-lysine) and methoxy-polyethylene glycol succinate for *in-vivo* imaging of tumor related lysosomal protease activity. These probes successfully detected small-sized solid tumors with higher NIR signals and to examine specific enzyme activity [87]. Qdots have also explored to study the modifications in erythrocyte membranes caused by plasmodium invasion in malaria via immuno-cytochemical studies [88]. Jaiswal and coworkers have demonstrated multicolor imaging of Qdots-labeled live cells. They explained two approaches for cell labeling; either through intracellular uptake of Qdots by endocytic mechanism or use of antibody-conjugated quantum dots specific to cell surface proteins [89]. Parak and associates have used colloidal Qdots to study metastatic potential of cancer cells due to their photochemical stable nature and to study the mechanism of motility and migration of cancer cells. Uptake of nanocrystals was explained to occur

Qdots have also been explored to prevent their nonspecific uptake by RES. Molecular markers expressed by blood vessels have been exploited to target nanocarriers toward specific tissues or organs. This strategy has been employed to target lung tumor cells using functionalized Qdots in mice [91]. Surface of quantum dots has been functionalized with COOH, NH2, and streptavidin that was further derivitized using PEG. PEG-conjugated Qdots decreased nonspecific uptake by RES, while COOH- and NH2-functionalized Qdots without PEG showed improved intracellular uptake among various cell types [92]. Qdots have also been explored for gene delivery and gene silencing. Sponge proton-coated Qdot-siRNA has been studied to improve gene silencing efficiency and reduced cytotoxicity in MDA-MB231 cells. These nanocarriers also allowed intracellular tracking and localization of siRNA delivery and transfection [93]. In another study, siRNA transfection was performed using Qdots. siRNA-Qdots exhibited greater photo-stability and tunable optical characteristics. This method was developed to observe the function of T-cadherin

Chitosan-folate-encapsulated ZnO Qdots have been prepared for delivery of anticancer agent doxorubicin with enhanced and longer photo-stability of Qdots. This nanocarrier showed an initial rapid release followed by controlled liberation of drug [95]. Doxorubicinloaded, immuno-liposome-based quantum dots were modified with HER2scFv for targeted delivery of drug to SKB-3 and MCF-7 cells with overexpressed HER2. These Qdot-IL conjugates exhibited receptor-dependent endocytosis in target cells but not in control MFC-7 cells. They also showed longer circulation of Qdots, and their localization in tumor models was confirmed by florescence imaging [96]. Cadmium telluride-incorporated Qdots with PEI functionalization for tracking of plasmid DNA in mice were designed. After intravenous injection, these structures showed rapid accumulation in the lungs, spleen, and liver.

through pinocytosis and phagocytosis [90].

80 Molecular Insight of Drug Design

in intercellular communication [94].

Magnetic nanoparticles (MNPs) are one of other fascinating elements of nanotechnology. Their nanometric dimension, biocompatibility, nontoxicity, and surplus accumulation in targeted cells or tissues justify intensive research in this subject matter. MNPs are mostly composed of ferromagnetic material such as ferrous or ferric oxide core with limited use of cobalt and nickel [98]. Magnetic properties are associated with movement of subatomic particles including electrons, holes, protons, and positive-/negative-charged ions. These materials respond to external stimulus of magnetic field and orient themselves according to magnetic moment. This magnetic behavior has been exploited for both *in-vitro* and *in-vivo* biomedical applications [99]. Magnetic nanoparticles have also been suggested for labeling cells in tissue engineering as they can be easily handled using magnets. Streptavidin-functionalized paramagnetic particles in combination with antibodies have been investigated for magnetic field-guided retroviral infection *in vitro* [100].

Magnetic nanoparticles have the ability to cause ablation of tumor cells via generation of heat. AC magnetic field causes the magnetic particles dispersed in target cells or tissues to get heated. This heat is rapidly disseminated to diseased cells, and if 42°C (therapeutic temperature threshold) can be maintained for 30 min, tumor cells get destroyed. However, this thermal ablation may be associated with undesirable concurrent killing of healthy cells [101]. Hase and coworkers have used ferromagnetic heating in combination of hepatic arterial embolization to study heat induction of ferromagnetic implants on VX2 hepatic cancer in rabbits. Results indicated extensive degeneration of tumor cells suggesting a suitable therapeutic strategy for localized hepatic carcinomas with little damage to healthy parenchyma of the liver because of selective heat induction [102].

Various functionalized magnetic nanocarriers have been investigated for targeted delivery of therapeutic agents. Magnetic drug carriers were designed either by using a magnetic core with surface coated with polymer or magnetic particles precipitated within porous polymeric composite. Such modifications have been studied to protect magnetic particle from harsh physiological vicinity and also to guide the drug carrier to desired location. Magnetic field-guided uncharged magnetic nanoparticles have been investigated for intracerebral targeting of rat glioma-2 in male (Fisher 344) rats. These magnetic nanoparticles (10–20 nm) exhibited greater uptake in brain tumor cells as compared to larger size (1 μm) magnetic particles [103]. In another study, iron oxide core was coated with oleic acid and subsequent coat of PEG-oleic acid for sustained release of doxorubicin and as MRI contrast. These modified magnetic nanoparticles were further conjugated with antibodies for active targeting of MFC-7 cells. These MNPs showed better MRI contrast with longer circulation time. They exhibited sustained release of drug with enhanced antiproliferative effect [104]. Doxorubicin-loaded monodisperse mesoporous single crystal iron oxide nanoparticles have also been developed as a promising carrier with improved drug loading and delivery [105].

#### **5. Conclusion**

Multifunctional nanocarriers offer a wide spectrum of biological applications exploiting both extracellular and intracellular barriers. These polymeric nanostructures have successfully improved the efficacy and safety of molecules delivered for various diagnostic and therapeutic purposes. Such nanocarriers have propounded unique physicochemical properties that have overall augmented the pharmacokinetic and pharmacodynamics parameters of drugs owing to versatility in their dimensions and surface functionalization. Bioavailability of many drug molecules that was compromised due to uptake by RES has been enhanced by exploiting these nanodevices. They have also offered long circulation time with release of drug molecules in a controlled or sustained manner with substantially fewer adverse effects. Nanocarriers have also shown exceptional promise in cellular imaging and diagnosis. By using various functionalization techniques, fluorescent probes have been directed to target tissues and cells to study site-specific delivery as well as intracellular trafficking of targeted molecules. Thus, they have been exploited to perform dual role of cell imaging along with drug delivery. Nanocarriers have also been successfully employed for gene transfection and gene silencing as well as *invitro* and *in-vivo* detection of biological molecules. Most recent therapeutic strategies under research seem substantially captivated in various dot structures for improved delivery of therapeutic agents, and the same is the case for magnetic nanoparticles that also have offered incredibly assuring results. However, much work is yet to be accomplished to prepare a successful commercial candidate with an ultimate therapeutic spectrum. Critical *in-vivo* cytotoxic behavior of these nanocarriers and untoward effects on normal physiological processes still requires intensive exploration. Some drug-loaded coated nanoparticles have been subjected to preliminary human trials after display of promising outcomes in animal studies but will even so require a long while for appearance in clinical market.

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#### **Conflict of interest**

There is no conflict of interests among authors.

### **Author details**

Maria Hassan Kiani<sup>1</sup> , Masoom Yasinzai2 and Gul Shahnaz<sup>1</sup> \*

\*Address all correspondence to: gshahnaz@qau.edu.pk

1 Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

2 Centre for Interdisciplinary Research in Basic Sciences, International Islamic University, Islamabad, Pakistan

#### **References**

**5. Conclusion**

82 Molecular Insight of Drug Design

**Conflict of interest**

**Author details**

Maria Hassan Kiani<sup>1</sup>

Islamabad, Pakistan

Islamabad, Pakistan

Multifunctional nanocarriers offer a wide spectrum of biological applications exploiting both extracellular and intracellular barriers. These polymeric nanostructures have successfully improved the efficacy and safety of molecules delivered for various diagnostic and therapeutic purposes. Such nanocarriers have propounded unique physicochemical properties that have overall augmented the pharmacokinetic and pharmacodynamics parameters of drugs owing to versatility in their dimensions and surface functionalization. Bioavailability of many drug molecules that was compromised due to uptake by RES has been enhanced by exploiting these nanodevices. They have also offered long circulation time with release of drug molecules in a controlled or sustained manner with substantially fewer adverse effects. Nanocarriers have also shown exceptional promise in cellular imaging and diagnosis. By using various functionalization techniques, fluorescent probes have been directed to target tissues and cells to study site-specific delivery as well as intracellular trafficking of targeted molecules. Thus, they have been exploited to perform dual role of cell imaging along with drug delivery. Nanocarriers have also been successfully employed for gene transfection and gene silencing as well as *invitro* and *in-vivo* detection of biological molecules. Most recent therapeutic strategies under research seem substantially captivated in various dot structures for improved delivery of therapeutic agents, and the same is the case for magnetic nanoparticles that also have offered incredibly assuring results. However, much work is yet to be accomplished to prepare a successful commercial candidate with an ultimate therapeutic spectrum. Critical *in-vivo* cytotoxic behavior of these nanocarriers and untoward effects on normal physiological processes still requires intensive exploration. Some drug-loaded coated nanoparticles have been subjected to preliminary human trials after display of promising outcomes in animal studies but will

even so require a long while for appearance in clinical market.

, Masoom Yasinzai2

\*Address all correspondence to: gshahnaz@qau.edu.pk

and Gul Shahnaz<sup>1</sup>

1 Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University,

2 Centre for Interdisciplinary Research in Basic Sciences, International Islamic University,

\*

There is no conflict of interests among authors.


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**Chapter 6**

**Provisional chapter**

**Multifunctional Nanoparticles for Successful Targeted**

**Multifunctional Nanoparticles for Successful Targeted** 

The blood-brain barrier (BBB) is the major problem for the treatment of brain diseases because we need to be able to deliver drugs from the vascular system into the central nervous system (CNS). There are no drug therapies for a wide range of CNS diseases and these include neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and cerebral ischemia. Therefore, the focus of this chapter is to discuss how nanoparticles (NPs) can be modified to transport different drug molecules for the treatment of brain diseases. In essence, NPs' surface can be functionalized with molecules such as peptides, antibodies and RNA aptamers and these macromolecules can be attached to the receptors present at the BBB endothelial cell surface, which allows the NPs cross the barrier and subsequently deliver pharmaceuticals to the brain for the therapeutic and/or imaging of neurological disorders. In fact, part of the difficulty in finding an effective treatment for these CNS disorders is that there is not yet an efficient delivery method for drug delivery across the BBB. However, over the last several years, researches have started to under-

**Keywords:** blood-brain barrier, multifunctional nanoparticles, Alzheimer's, Parkinson's,

Technological innovations, referred to as nanomedicine, is an exciting field of applications of nanotechnology to the diagnostic, treatment and/or prevention of traumatic injury or disease of the human body. This field holds the promise to deeply revolutionize the medicine to treatment and therapy areas such as imaging, drug delivery, cell therapy, tissue regeneration and development of new nanomedicine products. Due to its great importance, recent global

stand some of the design rules to efficiently deliver NPs to the brain.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.76922

**Drug Delivery across the Blood-Brain Barrier**

**Drug Delivery across the Blood-Brain Barrier**

Débora Braga Vieira and Lionel Fernel Gamarra

Débora Braga Vieira and Lionel Fernel Gamarra

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76922

cerebral ischemia, stroke

**1. Introduction**

**Abstract**

#### **Multifunctional Nanoparticles for Successful Targeted Drug Delivery across the Blood-Brain Barrier Multifunctional Nanoparticles for Successful Targeted Drug Delivery across the Blood-Brain Barrier**

DOI: 10.5772/intechopen.76922

Débora Braga Vieira and Lionel Fernel Gamarra Débora Braga Vieira and Lionel Fernel Gamarra

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76922

#### **Abstract**

The blood-brain barrier (BBB) is the major problem for the treatment of brain diseases because we need to be able to deliver drugs from the vascular system into the central nervous system (CNS). There are no drug therapies for a wide range of CNS diseases and these include neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and cerebral ischemia. Therefore, the focus of this chapter is to discuss how nanoparticles (NPs) can be modified to transport different drug molecules for the treatment of brain diseases. In essence, NPs' surface can be functionalized with molecules such as peptides, antibodies and RNA aptamers and these macromolecules can be attached to the receptors present at the BBB endothelial cell surface, which allows the NPs cross the barrier and subsequently deliver pharmaceuticals to the brain for the therapeutic and/or imaging of neurological disorders. In fact, part of the difficulty in finding an effective treatment for these CNS disorders is that there is not yet an efficient delivery method for drug delivery across the BBB. However, over the last several years, researches have started to understand some of the design rules to efficiently deliver NPs to the brain.

**Keywords:** blood-brain barrier, multifunctional nanoparticles, Alzheimer's, Parkinson's, cerebral ischemia, stroke

#### **1. Introduction**

Technological innovations, referred to as nanomedicine, is an exciting field of applications of nanotechnology to the diagnostic, treatment and/or prevention of traumatic injury or disease of the human body. This field holds the promise to deeply revolutionize the medicine to treatment and therapy areas such as imaging, drug delivery, cell therapy, tissue regeneration and development of new nanomedicine products. Due to its great importance, recent global

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

marketed report expects that the applications of nanotechnology in medicine could reach \$528 billion by 2019 [1]. Indeed, a broad range of nanoparticles (NPs) made of various materials (e.g., polymers, dendrimers, gold, silver, lipids, metals, and virus-like particles), differing in their size, architecture and surface properties, has been initially engineered to improve parameters such as the pharmacokinetics and biodistribution of therapeutic molecules and to reduce drugs' toxicity side effects [2]. Additionally, NPs are also useful tools for body or organ imaging [3]. During the past few decades, NPs have been successfully developed as drug, gene and/or imaging delivery vehicles due to their key properties of enhancing water solubility of poorly water-soluble molecules, extending the plasma circulation time and targeting the site of disease, while avoiding nonspecific toxicity effects [4, 5].

amount of drug or therapeutic macromolecules for a sufficient time; (ii) increase residence time in the blood through the use of soluble polymers such as polyethylene glycol (PEG); (iii) increase their accumulation at the desired site in the body by attaching to NPs, surface macromolecules such as antibodies, RNA aptamers and peptides; (iii) respond to several intrinsic or extrinsic stimuli for "on demand" delivery such as abnormal pH, temperature or magnetic and ultrasound fields and (iv) entrap concomitantly an imaging agent to enable the real-time monitoring of their biodistribution, targeted accumulation and/or therapy

Multifunctional Nanoparticles for Successful Targeted Drug Delivery across the Blood-Brain Barrier

http://dx.doi.org/10.5772/intechopen.76922

93

Despite the exciting advances in the discipline of nanotechnology-based approaches, different challenges arise in their efficacy toward the treatment of neurodegenerative diseases. One of the major obstacles that limit the application of NPs for effective delivery of drugs and diagnostic imaging agents to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB) [14]. As a result, new and innovative invasive and noninvasive NPs formulations have been engineered to provide efficacy in crossing the BBB, mainly by the functionalization of NPs' surface with ligands. Invasive strategies show potential and are being explored for efficient NPs' access to the brain. Some examples of invasive strategies are: convention-enhanced delivery, intracerebral or nasal injection and use of implants. With this in mind, it is important to understand the general concept of BBB, mechanisms of transport in

**2. BBB, general concept and the transport of drugs inside the brain**

composed of fibronectin, type IV collagen, heparin sulfate and laminin [16, 17].

The BBB is a formidable physiological structure that acts as an effective security system for the brain, letting in circulating compounds that this organ needs, but at the same time, these cells have evolved a system of biological pumps and if these pumps recognize molecules that should not be on their way to the brain, they will be pumped right back out into the vascular system [15]. The BBB is primarily composed of brain endothelial cells, which are cells that line microvessels and capillaries in the brain, and these are highly specialized cells that are knitted together very tightly by tight junctions, so there are no gaps between the cells (**Figure 1**). In fact, endothelial cells' tight junctions control the flux of hydrophilic molecules and small lipophilic substances such as water and some gases, respectively, that go through the BBB [15, 16]. Also, the brain endothelial cells are surrounded by a structure known as basal lamina,

Other structures such as neurons, astrocytes, pericytes and extracellular matrix components constitute the neurovascular unit that is also part of the BBB structure (**Figure 1**). Neurons are electrically excitable cells responsible for processing and transmitting information throughout the mind and body. In the nervous system, chemical and electrical signals between neurons occur via synapses, or junctions, that connect these cells [18]. Astrocytes play a key role in providing nutrients to neurons by shuttling them from the blood vessels to neurons themselves; they also help to control the ion concentration in the brain; are part of the repair process that happens after brain injury and help neurons recycle their neurotransmitters [19].

and out of the brain and the BBB alterations in pathology.

efficiency [2, 13].

In fact, NPs have provided remarkable progress in therapy and diagnostic imaging of several diseases. Since 1990, a high number of nanocarrier formulations have been approved by regulatory authorities for clinical use [6, 7]. There are five different applications of nanomedicine products on the market within healthcare – *in vitro* diagnostics; biomaterials; drug delivery; *in vivo* imaging and active implants [7]. Of these products, the type of NPs that exists on the market is diverse and it includes the following: (i) liposomes (e.g., Ambisome®, Albelcet®, DaunoXome®, Depocyt® and Myocet®); (ii) polymer-coated liposomes (e.g., Doxil® and Lipo-Dox®); (iii) polymeric drugs (e.g., Copaxone®); (iv) polymer-protein conjugates (e.g., Oncospar®, PEG-Intron® and Pegasys®); (v) nanoparticle containing paclitaxel (e.g., Abraxane™), (vi) antibodies (e.g., Avastin™ and Herceptin®) and (vii) antibody conjugates (e.g., Mylotarg®); (viii) aptamer conjugates (e.g., Macugen®); (viii) micelles (e.g., Estrasorb®); among others. These formulations are considered the first generation of nanomedicine, already bringing clinical benefits to patients [8].

Moreover, researches are constantly focusing on the development of NPs that can accumulate and deliver their cargo specifically at the diseased site, and these efforts are bringing important advances toward the development of NP-based targeted drug delivery systems. To increase the specificity of NPs to the targeted area, nanocarriers that can either passively or actively target the unhealthy site have been engineered. In passive targeting, the capacity of NPs to accumulate in the angiogenic site of tumors by the enhanced permeability and retention effect is explored [6, 9]. This strategy is achieved by recovering surface of NPs with some sort of coating with several compounds such as poly(ethylene glycol) (PEG) and poly(phosphoester) (PEEP) [10]. By binding PEG or PEEP to the surface of NPs, there occurs a change in the protein corona populations that adhere to the surface of NPs, reducing drastically the opsonization process of the nanocarriers thus preventing recognition by macrophages and monocytes and rapid clearance of NPs from the blood [10, 11]. Also, the accumulation and cellular uptake of NPs could be further enhanced by conjugating the NPs with molecules such as antibodies, peptides and aptamers that are able to bind to overexpressed receptor or antigens on the surface of targeted cells [12].

More recently, various researchers have been developing NPs able to perform two or more functions for the simultaneous or sequential delivery of single or multiple therapeutic active principles to the required targeted site in the body, overcoming multiple physiological barriers [13]. Multifunctional NPs often have the ability to: (i) encapsulate sufficient amount of drug or therapeutic macromolecules for a sufficient time; (ii) increase residence time in the blood through the use of soluble polymers such as polyethylene glycol (PEG); (iii) increase their accumulation at the desired site in the body by attaching to NPs, surface macromolecules such as antibodies, RNA aptamers and peptides; (iii) respond to several intrinsic or extrinsic stimuli for "on demand" delivery such as abnormal pH, temperature or magnetic and ultrasound fields and (iv) entrap concomitantly an imaging agent to enable the real-time monitoring of their biodistribution, targeted accumulation and/or therapy efficiency [2, 13].

marketed report expects that the applications of nanotechnology in medicine could reach \$528 billion by 2019 [1]. Indeed, a broad range of nanoparticles (NPs) made of various materials (e.g., polymers, dendrimers, gold, silver, lipids, metals, and virus-like particles), differing in their size, architecture and surface properties, has been initially engineered to improve parameters such as the pharmacokinetics and biodistribution of therapeutic molecules and to reduce drugs' toxicity side effects [2]. Additionally, NPs are also useful tools for body or organ imaging [3]. During the past few decades, NPs have been successfully developed as drug, gene and/or imaging delivery vehicles due to their key properties of enhancing water solubility of poorly water-soluble molecules, extending the plasma circulation time and tar-

In fact, NPs have provided remarkable progress in therapy and diagnostic imaging of several diseases. Since 1990, a high number of nanocarrier formulations have been approved by regulatory authorities for clinical use [6, 7]. There are five different applications of nanomedicine products on the market within healthcare – *in vitro* diagnostics; biomaterials; drug delivery; *in vivo* imaging and active implants [7]. Of these products, the type of NPs that exists on the market is diverse and it includes the following: (i) liposomes (e.g., Ambisome®, Albelcet®, DaunoXome®, Depocyt® and Myocet®); (ii) polymer-coated liposomes (e.g., Doxil® and Lipo-Dox®); (iii) polymeric drugs (e.g., Copaxone®); (iv) polymer-protein conjugates (e.g., Oncospar®, PEG-Intron® and Pegasys®); (v) nanoparticle containing paclitaxel (e.g., Abraxane™), (vi) antibodies (e.g., Avastin™ and Herceptin®) and (vii) antibody conjugates (e.g., Mylotarg®); (viii) aptamer conjugates (e.g., Macugen®); (viii) micelles (e.g., Estrasorb®); among others. These formulations are considered the first

geting the site of disease, while avoiding nonspecific toxicity effects [4, 5].

generation of nanomedicine, already bringing clinical benefits to patients [8].

surface of targeted cells [12].

92 Molecular Insight of Drug Design

Moreover, researches are constantly focusing on the development of NPs that can accumulate and deliver their cargo specifically at the diseased site, and these efforts are bringing important advances toward the development of NP-based targeted drug delivery systems. To increase the specificity of NPs to the targeted area, nanocarriers that can either passively or actively target the unhealthy site have been engineered. In passive targeting, the capacity of NPs to accumulate in the angiogenic site of tumors by the enhanced permeability and retention effect is explored [6, 9]. This strategy is achieved by recovering surface of NPs with some sort of coating with several compounds such as poly(ethylene glycol) (PEG) and poly(phosphoester) (PEEP) [10]. By binding PEG or PEEP to the surface of NPs, there occurs a change in the protein corona populations that adhere to the surface of NPs, reducing drastically the opsonization process of the nanocarriers thus preventing recognition by macrophages and monocytes and rapid clearance of NPs from the blood [10, 11]. Also, the accumulation and cellular uptake of NPs could be further enhanced by conjugating the NPs with molecules such as antibodies, peptides and aptamers that are able to bind to overexpressed receptor or antigens on the

More recently, various researchers have been developing NPs able to perform two or more functions for the simultaneous or sequential delivery of single or multiple therapeutic active principles to the required targeted site in the body, overcoming multiple physiological barriers [13]. Multifunctional NPs often have the ability to: (i) encapsulate sufficient Despite the exciting advances in the discipline of nanotechnology-based approaches, different challenges arise in their efficacy toward the treatment of neurodegenerative diseases. One of the major obstacles that limit the application of NPs for effective delivery of drugs and diagnostic imaging agents to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB) [14]. As a result, new and innovative invasive and noninvasive NPs formulations have been engineered to provide efficacy in crossing the BBB, mainly by the functionalization of NPs' surface with ligands. Invasive strategies show potential and are being explored for efficient NPs' access to the brain. Some examples of invasive strategies are: convention-enhanced delivery, intracerebral or nasal injection and use of implants. With this in mind, it is important to understand the general concept of BBB, mechanisms of transport in and out of the brain and the BBB alterations in pathology.

#### **2. BBB, general concept and the transport of drugs inside the brain**

The BBB is a formidable physiological structure that acts as an effective security system for the brain, letting in circulating compounds that this organ needs, but at the same time, these cells have evolved a system of biological pumps and if these pumps recognize molecules that should not be on their way to the brain, they will be pumped right back out into the vascular system [15]. The BBB is primarily composed of brain endothelial cells, which are cells that line microvessels and capillaries in the brain, and these are highly specialized cells that are knitted together very tightly by tight junctions, so there are no gaps between the cells (**Figure 1**). In fact, endothelial cells' tight junctions control the flux of hydrophilic molecules and small lipophilic substances such as water and some gases, respectively, that go through the BBB [15, 16]. Also, the brain endothelial cells are surrounded by a structure known as basal lamina, composed of fibronectin, type IV collagen, heparin sulfate and laminin [16, 17].

Other structures such as neurons, astrocytes, pericytes and extracellular matrix components constitute the neurovascular unit that is also part of the BBB structure (**Figure 1**). Neurons are electrically excitable cells responsible for processing and transmitting information throughout the mind and body. In the nervous system, chemical and electrical signals between neurons occur via synapses, or junctions, that connect these cells [18]. Astrocytes play a key role in providing nutrients to neurons by shuttling them from the blood vessels to neurons themselves; they also help to control the ion concentration in the brain; are part of the repair process that happens after brain injury and help neurons recycle their neurotransmitters [19].

**Figure 1.** The Blood-Brain Barrier. Schematic cross-sectional representation of the blood brain barrier (BBB) and other components of vascular unit like neurons, astrocytes, pericytes that are essential for the health function of the CNS. Also, depicted in the picture are the BBB mechanism of passage: (A) Water soluble agents; (B) Lipid solid agents; (C) Protein transport; (D) Receptor-mediated transport, and (E) Adsorptive transcytosis.

Pericytes appear to play a key role in BBB endothelial cells barrier formation [20]. Finally, the extracellular matrix occupies 10–20% of brain volume and has a major role in its normal pathology [21]. Therefore, it is the vascular unit that controls permeability and cerebral blood flow throughout the CNS, ensuring physiological CNS functioning.

In **Figure 1**, a schematic overview of the mechanisms of transport through the BBB is depicted. Molecules that present either a high degree of lipophilicity and molecular weight smaller than 500 Da can penetrate the CNS by simple diffusion (**Figure 1A**). However, in the absence of these characteristics, other circulating molecules can cross the BBB by their interaction with specific transport proteins located at the brain endothelial surface. These proteins are classified into two main categories: (i) carrier-mediated transport and (ii) receptor-mediated transport. Carrier-mediated transport (CTM) systems are responsible for the transport of small-drug molecules or small nutrient molecules including monosaccharaides and amino acids with a molecular mass smaller than 600 Da. These molecules can cross the BBB endothelial cells via active transport mediated by specific proteins (**Figure 2C**). The diffusion of molecules from the blood to the brain may be passive or active. For example, the transport of neutral L-amino acids such as leucine, phenylalanine and tyrosine is mediated by the large neutral amino acid transport (LAT1), whereas cationic amino acid transporter (CAT1) mediates the transport of cationic amino acids such as lysine and histidine. Other examples of transporters of polar substances into the brain include the nucleoside transporter (CNT2), the glucose transporter (GLUT1) and the monocarboxylic acid transporter (MCT1) for nucleoside, glucose and carboxylic acids transport, respectively. Moreover, transporters presented at brain endothelial cells' surface are also able to expel endogenous peptides such as Tyr-Pro-Trp-Gly or a multiplicity of drugs from the CNS to the blood are to be mediated, respectively, by peptide

transport system-1 or P-glycoprotein, via active efflux transport (AET). In fact, if a drug is a substrate of any AET protein, multidrug resistance occurs, and this phenomenon is a great

**Figure 2.** Schematic representation of a drug-loaded, multifunctional, stimuli-responsive NP. The structure of a nanocarrier allows the incorporation of one or multiple therapeutic molecules. These NPs can be found in different sizes and shapes. Increased blood circulation time can be achieved with soluble polymers such as polyethylene glycol (PEG). Nonspecifically target the intended site of action can be achieved by exploring, for example, leaky vessels of tumors. NPs can be actively targeted via the attachment of targeted-specific ligands such as antibody, antibody fragments, aptamers and peptides at their surface. Depending on the kind of application of NPs, various compounds can be added to turn the nanocarrier into a responsive device to a specific stimuli such as temperature, pH or magnetic and ultrasound fields. Imaging or contrast agents such as magnetic resonance imaging (MRI) , near infrared (NIR) and/ or polyethylene terephthalate (PET) compounds can also be incorporated into a single platform to enable imaging and releasing of drugs

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Chemotherapy agents, natural, synthesized or recombinant peptides and proteins, nucleic acids, monoclonal antibodies and other pharmaceutical breakthroughs do not readily cross the BBB (**Figure 2D**). Nonetheless, there are some specific proteins that the brain needs to function correctly, so they can access the brain by attaching to receptors, which are transported across the barrier and subsequently release into the brain. This mechanism of transport is known as receptor-mediated transport (RMT) and the internalization of these relatively large compounds is done via endocytosis (**Figure 2E**). It is the most studied transport mechanism for drug delivery, since receptor-specific ligands such as peptides and antibodies against receptors that are expressed on brain endothelial cells surface can be attached to the surface of nanoparticles or drugs, enabling their accumulation and internalization by cells of vascular side and, consequently, being transported into the brain. In addition, adsorptive-mediated

obstacle for therapeutic drug delivery to the CNS.

from NPs.

Multifunctional Nanoparticles for Successful Targeted Drug Delivery across the Blood-Brain Barrier http://dx.doi.org/10.5772/intechopen.76922 95

**Figure 2.** Schematic representation of a drug-loaded, multifunctional, stimuli-responsive NP. The structure of a nanocarrier allows the incorporation of one or multiple therapeutic molecules. These NPs can be found in different sizes and shapes. Increased blood circulation time can be achieved with soluble polymers such as polyethylene glycol (PEG). Nonspecifically target the intended site of action can be achieved by exploring, for example, leaky vessels of tumors. NPs can be actively targeted via the attachment of targeted-specific ligands such as antibody, antibody fragments, aptamers and peptides at their surface. Depending on the kind of application of NPs, various compounds can be added to turn the nanocarrier into a responsive device to a specific stimuli such as temperature, pH or magnetic and ultrasound fields. Imaging or contrast agents such as magnetic resonance imaging (MRI) , near infrared (NIR) and/ or polyethylene terephthalate (PET) compounds can also be incorporated into a single platform to enable imaging and releasing of drugs from NPs.

Pericytes appear to play a key role in BBB endothelial cells barrier formation [20]. Finally, the extracellular matrix occupies 10–20% of brain volume and has a major role in its normal pathology [21]. Therefore, it is the vascular unit that controls permeability and cerebral blood

**Figure 1.** The Blood-Brain Barrier. Schematic cross-sectional representation of the blood brain barrier (BBB) and other components of vascular unit like neurons, astrocytes, pericytes that are essential for the health function of the CNS. Also, depicted in the picture are the BBB mechanism of passage: (A) Water soluble agents; (B) Lipid solid agents; (C) Protein

In **Figure 1**, a schematic overview of the mechanisms of transport through the BBB is depicted. Molecules that present either a high degree of lipophilicity and molecular weight smaller than 500 Da can penetrate the CNS by simple diffusion (**Figure 1A**). However, in the absence of these characteristics, other circulating molecules can cross the BBB by their interaction with specific transport proteins located at the brain endothelial surface. These proteins are classified into two main categories: (i) carrier-mediated transport and (ii) receptor-mediated transport. Carrier-mediated transport (CTM) systems are responsible for the transport of small-drug molecules or small nutrient molecules including monosaccharaides and amino acids with a molecular mass smaller than 600 Da. These molecules can cross the BBB endothelial cells via active transport mediated by specific proteins (**Figure 2C**). The diffusion of molecules from the blood to the brain may be passive or active. For example, the transport of neutral L-amino acids such as leucine, phenylalanine and tyrosine is mediated by the large neutral amino acid transport (LAT1), whereas cationic amino acid transporter (CAT1) mediates the transport of cationic amino acids such as lysine and histidine. Other examples of transporters of polar substances into the brain include the nucleoside transporter (CNT2), the glucose transporter (GLUT1) and the monocarboxylic acid transporter (MCT1) for nucleoside, glucose and carboxylic acids transport, respectively. Moreover, transporters presented at brain endothelial cells' surface are also able to expel endogenous peptides such as Tyr-Pro-Trp-Gly or a multiplicity of drugs from the CNS to the blood are to be mediated, respectively, by peptide

flow throughout the CNS, ensuring physiological CNS functioning.

transport; (D) Receptor-mediated transport, and (E) Adsorptive transcytosis.

94 Molecular Insight of Drug Design

transport system-1 or P-glycoprotein, via active efflux transport (AET). In fact, if a drug is a substrate of any AET protein, multidrug resistance occurs, and this phenomenon is a great obstacle for therapeutic drug delivery to the CNS.

Chemotherapy agents, natural, synthesized or recombinant peptides and proteins, nucleic acids, monoclonal antibodies and other pharmaceutical breakthroughs do not readily cross the BBB (**Figure 2D**). Nonetheless, there are some specific proteins that the brain needs to function correctly, so they can access the brain by attaching to receptors, which are transported across the barrier and subsequently release into the brain. This mechanism of transport is known as receptor-mediated transport (RMT) and the internalization of these relatively large compounds is done via endocytosis (**Figure 2E**). It is the most studied transport mechanism for drug delivery, since receptor-specific ligands such as peptides and antibodies against receptors that are expressed on brain endothelial cells surface can be attached to the surface of nanoparticles or drugs, enabling their accumulation and internalization by cells of vascular side and, consequently, being transported into the brain. In addition, adsorptive-mediated transport (AMT) is a kind of transport where endocytosis is induced by the binding of cationic substances to the negatively charged plasma membrane of brain endothelial cells interaction. Therefore, due to the electrostatic interaction between the negatively charged membranes, the cationic therapeutic compound takes the AMT to enter the CNS.

the barrier such as aglycemia, hypoxia, among others [32]. For decades, two-dimensional or three-dimensional *in vitro* models of BBB have been developed by cultivating either as a monolayer or in cocultivation with mouse brain microvascular endothelial cells and murine or human endothelial cells with pericytes or astrocytes or glial tissue among others in a way that mimics the barrier under physiological or pathological conditions such as Alzheimer's and Parkinson's diseases or stroke [33–37]. Models of BBB based on stem cells are also reported in the literature [38, 39]. Moreover, by taking permeability measurements on the cultured cells, it is possible to test the physiological relevance of the developed model. In addition, experiments such as gene expression analysis using real-time polymerase chain reaction (PCR), permeability analysis [40, 41] and immunocytochemistry can also be used to validate the BBB model obtained. Although we are still not able to make this platform available both in academic and industry setting, this kind of technology has been showing the importance of considering *in vitro* data together with *in vivo* studies to understand the transport process

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**4. Other barriers that limit effective drug delivery into the brain**

In addition, the BBB is not the only physiological barrier for drug delivery to the brain. If we consider the anatomic aspects of our body, the brain and the spinal cord are completely cushioned and protected by the cerebrospinal fluid (CSF) [42, 43]. This fluid is also responsible for carrying nutrients to and waste products away from the brain. The great majority of CSF is produced within ventricular areas of the brain, as a result of the specialized tissue known as choroid plexus. The choroid plexus is located in each of the four ventricles within the brain area: two lateral ventricles, and the third and fourth ventricles. Here, it is important to clarify that the cells of the choroid plexus do not produce the CSF; instead, this fluid is a filtrate of the blood that is performed by the highly specialized cells of choroid plexus known as cuboidal epithelial cells. Cuboidal epithelial cells are exactly located between the capillary and the ventricle. As all capillaries present within the brain, the capillaries of the choroid plexus have a wall formed by single cells responsible for ready transportation of ions and molecules to and from the choroid plexus capillary. Tight-gap junctions hold the choroid plexus epithelial cells together. These gap junctions prevent substances from entering or leaving the CSF; thus, the choroid plexus acts as a blood-CSF barrier. Lastly, although the CSF originates in the ventricles, this fluid flows through to the ventricles and then surrounds the brain and the

Over the last several years, researches have engineered a variety of NPs that can potentially deliver therapies and/or imaging agents directly into the brain [14, 42, 44–48]. It is really challenging to get these nanoparticles across the BBB to treat a CNS disease in sufficient amount and without causing major side effects on healthy brain cells. NPs are available in many sizes

of NPs into the brain.

spinal cord.

**5. NPs for brain drug delivery**

This becomes a problem when treating diseases of the brain because we need to be able to deliver drugs from the vascular system into the CNS [22]. Unfortunately, at the moment, there are no drug therapies for a wide range of CNS diseases, and these include neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) and cerebral ischemia (or stroke). Therefore, the objective of this chapter is to discuss how NPs can be modified to transport thousands of different drug molecules for the treatment of brain diseases. In essence, NPs for drug delivery into the brain is a method for transporting drug molecules across the BBB using nanocarriers. NPs, surface can be functionalized with molecules such as peptides, antibodies and RNA aptamers, and these macromolecules can be attached to the receptors present at the BBB endothelial cell surface, which allow the NPs across the barrier and subsequently deliver pharmaceuticals to the brain for the therapeutic and/or imaging of neurological disorders [14]. In fact, part of the difficulty in finding an effective treatment for these CNS disorders is that there is not yet an efficient delivery method for drug delivery across the BBB. However, over the last several years, researches have started to understand some of the design rules for efficient delivery of NPs to the brain.

#### **3.** *In vitro* **approaches to study NPs' transport through the BBB**

As mentioned earlier, the BBB is a selective and dynamic barrier restricting the passage of a huge variety of compounds across this barrier, which is essential for the maintenance of homeostasis and functionality of the CNS. Therefore, the BBB is considered the major obstacle for the use of NPs as delivery systems to brain diseases. As shown in **Figure 2**, endothelial cells of the cerebral microvasculature are associated with perivascular cells form the BBB. The functional interaction between endothelial and perivascular cells and their response to injury have led to the concept of the neurovascular unit [15, 17]. Studying the mechanisms of uptake, transport and cytotoxicity of NPs through the BBB is an extremely challenging task *in vivo* because of the technical limitation to access the interface between the vascular system and the brain, since it is estimated that the brain capillary length is about 650 km [23]. To overcome this problem, *in vitro* BBB models have been built to reproduce as precise as possible the major BBB features, allowing investigation of cellular and molecular mechanisms that occur in the barrier; prediction of the transport of compounds across the BBB and performing highthroughput platform to test NPs transport through the barrier for the effective treatment of brain diseases. For example, it was observed that NPs can reach the capillaries into the brain of rats or mice 30 m after intravenous injection and, up to 5 h after NPs administration, they could go through the barrier, decreasing afterward [24–29]. These studies of NPs across the brain tissue are in line with *in vitro* BBB models data reported [30, 31]. In addition, it was observed that *in vitro* BBB model facilitates the manipulation of some parameters that affects the barrier such as aglycemia, hypoxia, among others [32]. For decades, two-dimensional or three-dimensional *in vitro* models of BBB have been developed by cultivating either as a monolayer or in cocultivation with mouse brain microvascular endothelial cells and murine or human endothelial cells with pericytes or astrocytes or glial tissue among others in a way that mimics the barrier under physiological or pathological conditions such as Alzheimer's and Parkinson's diseases or stroke [33–37]. Models of BBB based on stem cells are also reported in the literature [38, 39]. Moreover, by taking permeability measurements on the cultured cells, it is possible to test the physiological relevance of the developed model. In addition, experiments such as gene expression analysis using real-time polymerase chain reaction (PCR), permeability analysis [40, 41] and immunocytochemistry can also be used to validate the BBB model obtained. Although we are still not able to make this platform available both in academic and industry setting, this kind of technology has been showing the importance of considering *in vitro* data together with *in vivo* studies to understand the transport process of NPs into the brain.

#### **4. Other barriers that limit effective drug delivery into the brain**

In addition, the BBB is not the only physiological barrier for drug delivery to the brain. If we consider the anatomic aspects of our body, the brain and the spinal cord are completely cushioned and protected by the cerebrospinal fluid (CSF) [42, 43]. This fluid is also responsible for carrying nutrients to and waste products away from the brain. The great majority of CSF is produced within ventricular areas of the brain, as a result of the specialized tissue known as choroid plexus. The choroid plexus is located in each of the four ventricles within the brain area: two lateral ventricles, and the third and fourth ventricles. Here, it is important to clarify that the cells of the choroid plexus do not produce the CSF; instead, this fluid is a filtrate of the blood that is performed by the highly specialized cells of choroid plexus known as cuboidal epithelial cells. Cuboidal epithelial cells are exactly located between the capillary and the ventricle. As all capillaries present within the brain, the capillaries of the choroid plexus have a wall formed by single cells responsible for ready transportation of ions and molecules to and from the choroid plexus capillary. Tight-gap junctions hold the choroid plexus epithelial cells together. These gap junctions prevent substances from entering or leaving the CSF; thus, the choroid plexus acts as a blood-CSF barrier. Lastly, although the CSF originates in the ventricles, this fluid flows through to the ventricles and then surrounds the brain and the spinal cord.

#### **5. NPs for brain drug delivery**

transport (AMT) is a kind of transport where endocytosis is induced by the binding of cationic substances to the negatively charged plasma membrane of brain endothelial cells interaction. Therefore, due to the electrostatic interaction between the negatively charged membranes, the

This becomes a problem when treating diseases of the brain because we need to be able to deliver drugs from the vascular system into the CNS [22]. Unfortunately, at the moment, there are no drug therapies for a wide range of CNS diseases, and these include neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) and cerebral ischemia (or stroke). Therefore, the objective of this chapter is to discuss how NPs can be modified to transport thousands of different drug molecules for the treatment of brain diseases. In essence, NPs for drug delivery into the brain is a method for transporting drug molecules across the BBB using nanocarriers. NPs, surface can be functionalized with molecules such as peptides, antibodies and RNA aptamers, and these macromolecules can be attached to the receptors present at the BBB endothelial cell surface, which allow the NPs across the barrier and subsequently deliver pharmaceuticals to the brain for the therapeutic and/or imaging of neurological disorders [14]. In fact, part of the difficulty in finding an effective treatment for these CNS disorders is that there is not yet an efficient delivery method for drug delivery across the BBB. However, over the last several years, researches have started to understand some of the design rules for efficient delivery of NPs

**3.** *In vitro* **approaches to study NPs' transport through the BBB**

As mentioned earlier, the BBB is a selective and dynamic barrier restricting the passage of a huge variety of compounds across this barrier, which is essential for the maintenance of homeostasis and functionality of the CNS. Therefore, the BBB is considered the major obstacle for the use of NPs as delivery systems to brain diseases. As shown in **Figure 2**, endothelial cells of the cerebral microvasculature are associated with perivascular cells form the BBB. The functional interaction between endothelial and perivascular cells and their response to injury have led to the concept of the neurovascular unit [15, 17]. Studying the mechanisms of uptake, transport and cytotoxicity of NPs through the BBB is an extremely challenging task *in vivo* because of the technical limitation to access the interface between the vascular system and the brain, since it is estimated that the brain capillary length is about 650 km [23]. To overcome this problem, *in vitro* BBB models have been built to reproduce as precise as possible the major BBB features, allowing investigation of cellular and molecular mechanisms that occur in the barrier; prediction of the transport of compounds across the BBB and performing highthroughput platform to test NPs transport through the barrier for the effective treatment of brain diseases. For example, it was observed that NPs can reach the capillaries into the brain of rats or mice 30 m after intravenous injection and, up to 5 h after NPs administration, they could go through the barrier, decreasing afterward [24–29]. These studies of NPs across the brain tissue are in line with *in vitro* BBB models data reported [30, 31]. In addition, it was observed that *in vitro* BBB model facilitates the manipulation of some parameters that affects

cationic therapeutic compound takes the AMT to enter the CNS.

to the brain.

96 Molecular Insight of Drug Design

Over the last several years, researches have engineered a variety of NPs that can potentially deliver therapies and/or imaging agents directly into the brain [14, 42, 44–48]. It is really challenging to get these nanoparticles across the BBB to treat a CNS disease in sufficient amount and without causing major side effects on healthy brain cells. NPs are available in many sizes and shapes and they can have a positive, negative or neutral surface charge (**Figures 2**, **3**). Their core can be made of a variety of materials such as biological, synthetic or energy receptive. NPs can also be coated with specialized molecules that allow them to interact with their environment. NPs can also be loaded with therapeutic molecules that are released in a controlled way and, at the same time, retain the drug stability and prevent them from degradation once in the blood. Therefore, for an efficient drug delivery into the CNS, it is very important to engineer NPs with the following properties: (i) small size (NP diameter should be smaller than 100 nm); (ii) biocompatible, biodegradable, nontoxic and noninflammatory; (iii) prolonged circulation time in the body; (iv) stable in the plasma; (v) protect the cargo such as small molecules, peptides [43, 49–51], proteins or nucleic acids from degradation; (vi) targetability to the BBB and (vii) controlled drug release [44].

use of cell penetrating peptides such as SynB vectors, Tat and penetratin that were successfully used to target the BBB [52–55]. In fact, a huge variety of molecules that increased targeting strategies to the BBB such as growth factors (e.g., vascular endothelial growth factor; epidermal growth factor) [56], albumin [57], insulin [58], lactoferrin [59], transferrin [60], angiopep-2 [61] and biotin-binding proteins has been reported [62]. Targetability is usually associated with nanoparticles with prolonged circulation time. This characteristic is achieved by coating the surface of NPs with hydrophilic polymers such a polyethylene glycol (PEG), poly(acryloylmorpholine), poly-N-vinylpyrrolidones, polyvinyl alcohol and poly[N-(2-hydroxypropyl) methacrylamide] [63] (**Figures 2**, **3**). Among these polymers, PEG is still the most useful polymer in obtaining long circulating NP. The attachment of polymers onto the surface of NPs works by preventing NPs interaction with opsonins present at the plasma and, in this way, impeding their capture and subsequent clearance from the body. However, it was observed that the blood clearance phenomenon is accelerated after repetitive administration of clinically used PEGylated NPs due to the induction of production of antibodies (the NPs used in these studies were PEGylated liposomes) [64, 65]. Moreover, PEGylated NPs are particularly useful for neurological disease treatment, since the long-circulating NPs into the brain by diverse mechanisms were observed. Nevertheless, for brain tumors, reliance on the enhanced permeability and retention (EPR) effect for drug delivery strategies faces several

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challenges, since the accumulation of NPs at the tumor site is very low [66].

the reader to Vieira and Gamarra's article [14].

(**Figure 3**).

Due to their ability to carry hydrophilic, hydrophobic and/or lipophilic compounds and high specificity, the use of NPs provides a very efficient platform for drug delivery into the CNS. The most popular nanocarrier studied for brain drug delivery is liposomes and several liposomal formulations are clinically available or tested at different clinical trial phases [14]. Liposomes are spherical concentric vesicles, consisting of at least one lipid bilayer, enclosing an aqueous compartment. This nanocarrier has been employed for therapeutically active compounds delivery soon after its discovery by Bangham in the early 1960s. This NP has been successfully engineered for a variety of brain neurodegenerative disorders and brain tumors. For a detailed overview of liposome-based strategies to drug delivery across the BBB, we refer

One of the breakthroughs of nanoparticles formulations is to target the nanocarriers to deliver their cargo into the brain. The brain endothelial cells contain several targets as discussed earlier that are explored on the studies of nanoparticles for brain delivery. Each of these targets could be specific for a brain disease or brain diseases. For example, transferrin has been described as the BBB-targeting ligand in studies of nanoparticle formulations [67, 68]. Transferrin is a glycoprotein (80 kDa) that binds to the transferrin receptor and is taken across the BBB via Receptor mediated endocytosis (RME). Indeed, these studies demonstrated that transferrin conjugated to liposomes exhibited a significant increase in the concentration of therapeutic molecules delivered by NPs into the brain when compared to the administration of the drug alone. In addition, broad ranges of nanocarriers with different shapes, sizes and surface properties have been developed for the transport of therapeutic or imaging molecules across the BBB. These also include carbon nanotubes [69, 70], micelles [71], dendrimers [72, 73], nanofibers [74, 75], polymer [46, 76], gold [77] and iron oxide nanoparticles [78] NPs

One of the most important and challenging characteristics in engineering NPs is their functionalization. Active targeting of NPs can be achieved by attaching onto their surface, in a highly controlled way, specific molecules such as monoclonal antibody, RNA aptamers, transferrin, lactoferrin and peptides (**Figure 2**). An example of such active NP is the extensive

**Figure 3.** Types of nanoparticles for brain delivery. Enhancing brain drug delivery with the use of several nanocarriers, able to carry the most diverse kind of molecules.

use of cell penetrating peptides such as SynB vectors, Tat and penetratin that were successfully used to target the BBB [52–55]. In fact, a huge variety of molecules that increased targeting strategies to the BBB such as growth factors (e.g., vascular endothelial growth factor; epidermal growth factor) [56], albumin [57], insulin [58], lactoferrin [59], transferrin [60], angiopep-2 [61] and biotin-binding proteins has been reported [62]. Targetability is usually associated with nanoparticles with prolonged circulation time. This characteristic is achieved by coating the surface of NPs with hydrophilic polymers such a polyethylene glycol (PEG), poly(acryloylmorpholine), poly-N-vinylpyrrolidones, polyvinyl alcohol and poly[N-(2-hydroxypropyl) methacrylamide] [63] (**Figures 2**, **3**). Among these polymers, PEG is still the most useful polymer in obtaining long circulating NP. The attachment of polymers onto the surface of NPs works by preventing NPs interaction with opsonins present at the plasma and, in this way, impeding their capture and subsequent clearance from the body. However, it was observed that the blood clearance phenomenon is accelerated after repetitive administration of clinically used PEGylated NPs due to the induction of production of antibodies (the NPs used in these studies were PEGylated liposomes) [64, 65]. Moreover, PEGylated NPs are particularly useful for neurological disease treatment, since the long-circulating NPs into the brain by diverse mechanisms were observed. Nevertheless, for brain tumors, reliance on the enhanced permeability and retention (EPR) effect for drug delivery strategies faces several challenges, since the accumulation of NPs at the tumor site is very low [66].

and shapes and they can have a positive, negative or neutral surface charge (**Figures 2**, **3**). Their core can be made of a variety of materials such as biological, synthetic or energy receptive. NPs can also be coated with specialized molecules that allow them to interact with their environment. NPs can also be loaded with therapeutic molecules that are released in a controlled way and, at the same time, retain the drug stability and prevent them from degradation once in the blood. Therefore, for an efficient drug delivery into the CNS, it is very important to engineer NPs with the following properties: (i) small size (NP diameter should be smaller than 100 nm); (ii) biocompatible, biodegradable, nontoxic and noninflammatory; (iii) prolonged circulation time in the body; (iv) stable in the plasma; (v) protect the cargo such as small molecules, peptides [43, 49–51], proteins or nucleic acids from degradation; (vi) targetability to the BBB and

One of the most important and challenging characteristics in engineering NPs is their functionalization. Active targeting of NPs can be achieved by attaching onto their surface, in a highly controlled way, specific molecules such as monoclonal antibody, RNA aptamers, transferrin, lactoferrin and peptides (**Figure 2**). An example of such active NP is the extensive

**Figure 3.** Types of nanoparticles for brain delivery. Enhancing brain drug delivery with the use of several nanocarriers,

(vii) controlled drug release [44].

98 Molecular Insight of Drug Design

able to carry the most diverse kind of molecules.

Due to their ability to carry hydrophilic, hydrophobic and/or lipophilic compounds and high specificity, the use of NPs provides a very efficient platform for drug delivery into the CNS. The most popular nanocarrier studied for brain drug delivery is liposomes and several liposomal formulations are clinically available or tested at different clinical trial phases [14]. Liposomes are spherical concentric vesicles, consisting of at least one lipid bilayer, enclosing an aqueous compartment. This nanocarrier has been employed for therapeutically active compounds delivery soon after its discovery by Bangham in the early 1960s. This NP has been successfully engineered for a variety of brain neurodegenerative disorders and brain tumors. For a detailed overview of liposome-based strategies to drug delivery across the BBB, we refer the reader to Vieira and Gamarra's article [14].

One of the breakthroughs of nanoparticles formulations is to target the nanocarriers to deliver their cargo into the brain. The brain endothelial cells contain several targets as discussed earlier that are explored on the studies of nanoparticles for brain delivery. Each of these targets could be specific for a brain disease or brain diseases. For example, transferrin has been described as the BBB-targeting ligand in studies of nanoparticle formulations [67, 68]. Transferrin is a glycoprotein (80 kDa) that binds to the transferrin receptor and is taken across the BBB via Receptor mediated endocytosis (RME). Indeed, these studies demonstrated that transferrin conjugated to liposomes exhibited a significant increase in the concentration of therapeutic molecules delivered by NPs into the brain when compared to the administration of the drug alone. In addition, broad ranges of nanocarriers with different shapes, sizes and surface properties have been developed for the transport of therapeutic or imaging molecules across the BBB. These also include carbon nanotubes [69, 70], micelles [71], dendrimers [72, 73], nanofibers [74, 75], polymer [46, 76], gold [77] and iron oxide nanoparticles [78] NPs (**Figure 3**).

Although nanotechnology-based strategies to get into the brain have shown progress in animal models, the translation of passive- and active-targeting delivery strategies into clinical studies is still questionable. This might be due to the random nature of receptor-ligand interactions and/or ineffective release of drug from the nanocarrier at the targeted site [79]. Therefore, the development of multifunctional nanoparticles is becoming possible due to the engineering of stimuli-responsive systems that are able to control the release of their cargo and drug distribution in response to specific stimuli such as magnetic field, light, changes in pH, variations in temperature, among others (**Figure 2**).

### **6. NPs in context of brain neurological diseases**

Getting NPs into CNS is not an easy task. As discussed earlier, the BBB is the main structure responsible for brain protection and homeostasis. In addition, it is important to mention that in neurological diseases, several impairments of this structure occur, leading to the perpetuation of the inflammatory cycle that damages neuronal cells and neurodegeneration [80] Moreover, the BBB breakdown can occur, which is clearly a consequence of an ischemic stroke that occurred [81] due to an obstruction within a blood vessel that supplies the brain with oxygen and several nutrients, leading to brain cell death.

In other cases, especially in chronic neurological diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), it remains unclear how these diseases promote the BBB impairment [32]. Importantly, these modifications in the BBB structure should be taken into consideration when you are planning the engineering of effective multifunctional NPs for brain delivery. In this context, NPs have been designed to cross the BBB and this new technology has some applications so far in the treatment of Alzheimer's and Parkinson's diseases, stroke and brain tumors, which are discussed in the following section.

#### **6.1. Stroke**

Stroke is a serious disease that occurs when some or all of the blood supply to a part of the brain is restricted or cut off and, therefore, this can lead to disability, brain injury or death. Thus, the loss of oxygen and nutrients provided by the blood causing the loss of brain function is a stroke also known as cerebral ischemia. There are two ways to disturb the blood supply to the brain (**Figure 4**). The most common type occurs when there is a stoppage of blood flow to a part of the brain due to a blood clot. This cause of strokes accounts for 85% of all cases and it is known as ischemic stroke. The second cause of stroke, that is not as common as the ischemic stroke, but still very serious, happens when one of the blood vessels that is a part of the cerebral circulation supplying the brain ruptures. This kind of stroke is called a hemorrhagic stroke. In addition, "hemorrhagic" refers to a sudden torrential bleeding outburst. However, regardless of whether it is an ischemic stroke or a hemorrhagic stroke, the brain cells start to malfunction after some minutes due to the lack of oxygen and nutrients owing to improper blood flow or improper blood supply.

There is also a related condition called the transient ischemic attack (TIA), also known as a mini-stroke. It is essentially a temporary interruption of blood flow to a part of the brain often lasting between 30 m and several hours. Therefore, the symptoms of TIA and stroke are similar. However, the difference is that a TIA does not actually destroy brain cells and it does not cause permanent disability. Nevertheless, it is often a warning signal that an individual is at risk of having a stroke in the near future. One of the key differences is that a TIA will resolve, whereas, if an individual has a stroke, he/she may not gain normal functioning again

**Figure 4.** Schematic representation of the main event causing ischemic stroke, Alzheimer's and Parkinson's disease. (Adapted from Alvarim et al. [82]) ischemic stroke is caused by the interruption of the blood flow, depriving the brain of oxygen and some nutrients. For Alzheimer's disease, the main characteristic of this disease is the presence of neurofibrillary tangles and amyloid plaques in the brain, leading to shrinkage of some structures of the brain such as the hippocampus and the enlargement of the brain ventricles, resulting in neurodegeneration. In Parkinson's disease,

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for weeks or months, or maybe even for the rest of his/her life.

substantia nigra dopamine neurons are lost.

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Although nanotechnology-based strategies to get into the brain have shown progress in animal models, the translation of passive- and active-targeting delivery strategies into clinical studies is still questionable. This might be due to the random nature of receptor-ligand interactions and/or ineffective release of drug from the nanocarrier at the targeted site [79]. Therefore, the development of multifunctional nanoparticles is becoming possible due to the engineering of stimuli-responsive systems that are able to control the release of their cargo and drug distribution in response to specific stimuli such as magnetic field, light, changes in

Getting NPs into CNS is not an easy task. As discussed earlier, the BBB is the main structure responsible for brain protection and homeostasis. In addition, it is important to mention that in neurological diseases, several impairments of this structure occur, leading to the perpetuation of the inflammatory cycle that damages neuronal cells and neurodegeneration [80] Moreover, the BBB breakdown can occur, which is clearly a consequence of an ischemic stroke that occurred [81] due to an obstruction within a blood vessel that supplies the brain

In other cases, especially in chronic neurological diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), it remains unclear how these diseases promote the BBB impairment [32]. Importantly, these modifications in the BBB structure should be taken into consideration when you are planning the engineering of effective multifunctional NPs for brain delivery. In this context, NPs have been designed to cross the BBB and this new technology has some applications so far in the treatment of Alzheimer's and Parkinson's diseases, stroke

Stroke is a serious disease that occurs when some or all of the blood supply to a part of the brain is restricted or cut off and, therefore, this can lead to disability, brain injury or death. Thus, the loss of oxygen and nutrients provided by the blood causing the loss of brain function is a stroke also known as cerebral ischemia. There are two ways to disturb the blood supply to the brain (**Figure 4**). The most common type occurs when there is a stoppage of blood flow to a part of the brain due to a blood clot. This cause of strokes accounts for 85% of all cases and it is known as ischemic stroke. The second cause of stroke, that is not as common as the ischemic stroke, but still very serious, happens when one of the blood vessels that is a part of the cerebral circulation supplying the brain ruptures. This kind of stroke is called a hemorrhagic stroke. In addition, "hemorrhagic" refers to a sudden torrential bleeding outburst. However, regardless of whether it is an ischemic stroke or a hemorrhagic stroke, the brain cells start to malfunction after some minutes due to the lack of oxygen and nutrients owing to improper blood flow or improper blood supply.

pH, variations in temperature, among others (**Figure 2**).

100 Molecular Insight of Drug Design

**6. NPs in context of brain neurological diseases**

with oxygen and several nutrients, leading to brain cell death.

and brain tumors, which are discussed in the following section.

**6.1. Stroke**

**Figure 4.** Schematic representation of the main event causing ischemic stroke, Alzheimer's and Parkinson's disease. (Adapted from Alvarim et al. [82]) ischemic stroke is caused by the interruption of the blood flow, depriving the brain of oxygen and some nutrients. For Alzheimer's disease, the main characteristic of this disease is the presence of neurofibrillary tangles and amyloid plaques in the brain, leading to shrinkage of some structures of the brain such as the hippocampus and the enlargement of the brain ventricles, resulting in neurodegeneration. In Parkinson's disease, substantia nigra dopamine neurons are lost.

There is also a related condition called the transient ischemic attack (TIA), also known as a mini-stroke. It is essentially a temporary interruption of blood flow to a part of the brain often lasting between 30 m and several hours. Therefore, the symptoms of TIA and stroke are similar. However, the difference is that a TIA does not actually destroy brain cells and it does not cause permanent disability. Nevertheless, it is often a warning signal that an individual is at risk of having a stroke in the near future. One of the key differences is that a TIA will resolve, whereas, if an individual has a stroke, he/she may not gain normal functioning again for weeks or months, or maybe even for the rest of his/her life.

Nevertheless, the cellular and molecular mechanisms of a stroke episode have been very well known due to the development of several experimental animal models of ischemic stroke [83]. From the studies with these animal models, it was observed that during ischemic stroke, first occurs the opening of the BBB for a short time period. Then, occurs a refractory interval, followed by a reopening of the BBB, but this time for a long period [84] The reopening of the BBB is the step responsible for the activation of the endothelium, leukocyte recruitment, reactive oxygen species (ROS) and cytokine productions and edema formation [85], leading to an inflammatory response and the BBB breakdown and cell death upon stroke [86]. Moreover, dysregulation of tight-junction proteins is also observed during ischemic stroke, due to their degradation by matrix metalloproteinases, which are involved in the process of BBB extracellular degradation, leading to an increase in the permeability of the brain structure [32].

the lipid-squalene and, then, yielded negatively charged NPs, showing promising results [89]. *In vivo* experiments showed that this formulation was able to extend adenosine circulation time in the plasma, interact with the neurovascular unit, enhance animal neurological deficit

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It has been estimated that AD, only in the USA, affects over 5.5 million people. Moreover, Alzheimer's AD is the most common cause of dementia [90]. Dementia is a serious brain disease that has as major symptoms deterioration in memory, behavior and thinking. In 2015, dementia affected 47.5 million people worldwide. Most were over the age of 60. The United States data related to AD account that this disease was the sixth highest cause of death in 2005. It was also observed that 1 in 3 seniors who died had AD or other kinds of dementia. It is also expected that the number of people with Alzheimer's will grow as the population of those over the age of 65 rises. In 2015, Alzheimer's disease and other dementia cost the nation \$226

AD was named after the German physician Dr. Alois Alzheimer who presented a case history before an important medical meeting. In 1901, he was closely following a 51-year-old woman patient with a mental disorder, the manifestations of which were language problems and memory loss. After her death, Dr. Alzheimer took a serious examination of her brain and found the presence of plaques and tangles that today characterize AD [91]. This disease accounts for about 60–80% of the dementia cases. In most cases, Alzheimer's clinical manifestations first appear after the age of 65. However, Alzheimer's disease is not considered normal aging although the greatest risk factor of developing the disease is increased age. This is actually the greatest known risk factor for developing AD. However, as mentioned earlier, Alzheimer's is not a normal part of aging. It was observed that a greater proportion of patients over 85 years have AD compared to those over 65 years as AD is more likely to affect older individuals. Dominant genes that are transmitted through generations cause less than 5% of Alzheimer's. However, family risk is the second biggest factor for the development of AD after a certain age. In these families, individuals usually present symptoms of Alzheimer's before the age of 65 and these symptoms sometimes appear in their 30s. This form of AD that is hereditary and marked by Alzheimer's symptoms at an early age is called early-onset familial Alzheimer's disease (EOFAD). To date, mutations in presenilin (PS1 on chromosome 14 and PS2 on chromosome 1) and the amyloid precursor protein gene (APP) on chromosome 21 have been associated with EOFAD. All these three gene mutations (PS1/PS2/APP) affect the pathway in amyloid precursor protein synthesis, which leads to the increase of production of Aβ, creating plaques in the brain [92]. Additionally, there are certain genes such as apoE gene on chromosome 19 that increase the susceptibility to AD. There are three forms of the apoE gene: APOE2, APOE3 and APOE4, the last one being the one associated with a high risk for developing AD. Actually, an individual with two copies of this gene is at three to eight more

The human brain contains about 100 billion neurons that communicate to one another via synapses, when a burst of chemicals called neurotransmitters are released [93]. The neurotransmitters are synthesized into the synaptic gap. Then, neurotransmitters move across

scores and decrease the size of the infarcted area [89].

billion and by 2050 this may rise to \$1.1 trillion [90].

risk than people with one copy of this gene.

**6.2. Alzheimer's disease**

Therefore, besides the BBB itself being an excellent target for itself for treating ischemic stroke, the design of effective drug delivery systems has also to take into consideration the cellular and molecular mechanisms described earlier. One of the strategies described in the literature to overcome neuronal tissue damage after a stroke event is the use of multifunctional NPs to deliver neuroprotective drugs into the brain, since the majority of neuroprotective drugs do not cross the BBB in their free form. For example, the inactive caspase-3 activation in the brain cells likely decreases the probability of brain cell damage after a stroke event. Although it was shown that some peptide inhibitors of caspase are effective compounds in promoting neuroprotection, they cannot readily cross the BBB. For this purpose, a positively charged NP of chitosan conjugated with transferrin receptor was designed to deliver the relatively specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK) across the BBB [36]. By clearly reducing the caspase 3-activity *in vivo*, this formulation was also readily transported across the BBB (less than 1 h) and it decreased neurological deficits and the infarcted area, proving to be a very promising formulation [27].

In the same way, several other compounds have been described to provide neuroprotection and to prevent neurodegeneration. One of these compounds is the Tanshinone IIA that has demonstrated neuroprotective effects against ischemic injury [87]. However, its use in the treatment of this disease is limited due to the compound's low solubility in aqueous medium, short-half circulation in the plasma and inability to cross the BBB. To overcome these limitations, Tanshinone IIA was successfully conjugated to PEGylated-cationic bovine serum albumin. These nanoparticles were able to cross the BBB *in vivo*, as a significant decreasing in the infarcted volume was observed. In addition, a reduction in the neutrophil infiltration and neuronal apoptosis was observed [88]. The authors also explored the molecular mechanisms by which this formulation conferred neuroprotection. It seems that the mechanism of action of this formulation in the brain is related to the down-regulation of pro-inflammatory cytokines (mainly IL-8 and TNF-α), to the up-regulation of anti-inflammatory cytokines (mainly transforming growth factor-β1 and IL-10) and to the reduction or inhibition of mRNA and proteins (mainly GFAP, MMP-9, COX-2, p38MAPK, ERK1/2 and JNK) [88].

As a last example, adenosine is also a powerful molecule that has demonstrated neuroprotection to the brain after an ischemic stroke event. This molecule also presents as limitations short-life time in the plasma and inability to cross the BBB. Here, adenosine was conjugated to the lipid-squalene and, then, yielded negatively charged NPs, showing promising results [89]. *In vivo* experiments showed that this formulation was able to extend adenosine circulation time in the plasma, interact with the neurovascular unit, enhance animal neurological deficit scores and decrease the size of the infarcted area [89].

#### **6.2. Alzheimer's disease**

Nevertheless, the cellular and molecular mechanisms of a stroke episode have been very well known due to the development of several experimental animal models of ischemic stroke [83]. From the studies with these animal models, it was observed that during ischemic stroke, first occurs the opening of the BBB for a short time period. Then, occurs a refractory interval, followed by a reopening of the BBB, but this time for a long period [84] The reopening of the BBB is the step responsible for the activation of the endothelium, leukocyte recruitment, reactive oxygen species (ROS) and cytokine productions and edema formation [85], leading to an inflammatory response and the BBB breakdown and cell death upon stroke [86]. Moreover, dysregulation of tight-junction proteins is also observed during ischemic stroke, due to their degradation by matrix metalloproteinases, which are involved in the process of BBB extracellular degradation, leading to an increase in the permeability of

Therefore, besides the BBB itself being an excellent target for itself for treating ischemic stroke, the design of effective drug delivery systems has also to take into consideration the cellular and molecular mechanisms described earlier. One of the strategies described in the literature to overcome neuronal tissue damage after a stroke event is the use of multifunctional NPs to deliver neuroprotective drugs into the brain, since the majority of neuroprotective drugs do not cross the BBB in their free form. For example, the inactive caspase-3 activation in the brain cells likely decreases the probability of brain cell damage after a stroke event. Although it was shown that some peptide inhibitors of caspase are effective compounds in promoting neuroprotection, they cannot readily cross the BBB. For this purpose, a positively charged NP of chitosan conjugated with transferrin receptor was designed to deliver the relatively specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK) across the BBB [36]. By clearly reducing the caspase 3-activity *in vivo*, this formulation was also readily transported across the BBB (less than 1 h) and it decreased neurological deficits and the infarcted area, proving to be a very promising formulation [27].

In the same way, several other compounds have been described to provide neuroprotection and to prevent neurodegeneration. One of these compounds is the Tanshinone IIA that has demonstrated neuroprotective effects against ischemic injury [87]. However, its use in the treatment of this disease is limited due to the compound's low solubility in aqueous medium, short-half circulation in the plasma and inability to cross the BBB. To overcome these limitations, Tanshinone IIA was successfully conjugated to PEGylated-cationic bovine serum albumin. These nanoparticles were able to cross the BBB *in vivo*, as a significant decreasing in the infarcted volume was observed. In addition, a reduction in the neutrophil infiltration and neuronal apoptosis was observed [88]. The authors also explored the molecular mechanisms by which this formulation conferred neuroprotection. It seems that the mechanism of action of this formulation in the brain is related to the down-regulation of pro-inflammatory cytokines (mainly IL-8 and TNF-α), to the up-regulation of anti-inflammatory cytokines (mainly transforming growth factor-β1 and IL-10) and to the reduction or inhibition of mRNA and

As a last example, adenosine is also a powerful molecule that has demonstrated neuroprotection to the brain after an ischemic stroke event. This molecule also presents as limitations short-life time in the plasma and inability to cross the BBB. Here, adenosine was conjugated to

proteins (mainly GFAP, MMP-9, COX-2, p38MAPK, ERK1/2 and JNK) [88].

the brain structure [32].

102 Molecular Insight of Drug Design

It has been estimated that AD, only in the USA, affects over 5.5 million people. Moreover, Alzheimer's AD is the most common cause of dementia [90]. Dementia is a serious brain disease that has as major symptoms deterioration in memory, behavior and thinking. In 2015, dementia affected 47.5 million people worldwide. Most were over the age of 60. The United States data related to AD account that this disease was the sixth highest cause of death in 2005. It was also observed that 1 in 3 seniors who died had AD or other kinds of dementia. It is also expected that the number of people with Alzheimer's will grow as the population of those over the age of 65 rises. In 2015, Alzheimer's disease and other dementia cost the nation \$226 billion and by 2050 this may rise to \$1.1 trillion [90].

AD was named after the German physician Dr. Alois Alzheimer who presented a case history before an important medical meeting. In 1901, he was closely following a 51-year-old woman patient with a mental disorder, the manifestations of which were language problems and memory loss. After her death, Dr. Alzheimer took a serious examination of her brain and found the presence of plaques and tangles that today characterize AD [91]. This disease accounts for about 60–80% of the dementia cases. In most cases, Alzheimer's clinical manifestations first appear after the age of 65. However, Alzheimer's disease is not considered normal aging although the greatest risk factor of developing the disease is increased age. This is actually the greatest known risk factor for developing AD. However, as mentioned earlier, Alzheimer's is not a normal part of aging. It was observed that a greater proportion of patients over 85 years have AD compared to those over 65 years as AD is more likely to affect older individuals. Dominant genes that are transmitted through generations cause less than 5% of Alzheimer's. However, family risk is the second biggest factor for the development of AD after a certain age. In these families, individuals usually present symptoms of Alzheimer's before the age of 65 and these symptoms sometimes appear in their 30s. This form of AD that is hereditary and marked by Alzheimer's symptoms at an early age is called early-onset familial Alzheimer's disease (EOFAD). To date, mutations in presenilin (PS1 on chromosome 14 and PS2 on chromosome 1) and the amyloid precursor protein gene (APP) on chromosome 21 have been associated with EOFAD. All these three gene mutations (PS1/PS2/APP) affect the pathway in amyloid precursor protein synthesis, which leads to the increase of production of Aβ, creating plaques in the brain [92]. Additionally, there are certain genes such as apoE gene on chromosome 19 that increase the susceptibility to AD. There are three forms of the apoE gene: APOE2, APOE3 and APOE4, the last one being the one associated with a high risk for developing AD. Actually, an individual with two copies of this gene is at three to eight more risk than people with one copy of this gene.

The human brain contains about 100 billion neurons that communicate to one another via synapses, when a burst of chemicals called neurotransmitters are released [93]. The neurotransmitters are synthesized into the synaptic gap. Then, neurotransmitters move across these synaptic gaps between neurons and bind to receptor sites on the dendrites of the next neuron. Unfortunately, neurons are the type of cells affected by AD. To date, scientists still do not know exactly the causes of AD and how this process begins. However, according to recent studies, it appears to be likely that astrocytes' activation contributes to the neuroinflammatory component responsible for the damage of neurons decades before the issue becomes obvious [94].

serum. For example, PEG-PLA NPs were able to protect the neuroprotective peptide NAPVSIPQ from degradation. However, just NPs modified with B6 peptide (similar to transferrin) were able to cross the blood-brain barrier in mice and successfully deliver the neuroprotective peptide into the brain [102]. Moreover, it was observed that the treatment with this formulation improved cholinergic function and ameliorated spatial learning of AD mouse model [102]. In the same way, the nerve growth factor (NGF) has also been explored as a good drug for treating AD, although it is not able to cross the BBB. For this purpose, NGF was encapsulated into PBCA NPs, decorated with polysorbate 80 [103]. These NPs presented very promising results, since they were able to reach the mice brain parenchyma in less than 1 h after administration and these nanocarriers also proved to be able to improve recognition and memory of mice and to reduce by almost 40% the PD symptoms such as rigidity, tremor and oligokinesia in animal models [103]. Coenzyme Q10, a powerful antioxidant macromolecule, has also been explored at AD therapy. In this way, this coenzyme was encapsulated within PLGA NPs decorated with trimethylates chitosan. The results showed that these nanoparticles were able to cross the BBB and accumulate in the choroid plexus, ventricles and cortex. Moreover, the authors also observed an improvement in the cognitive and spatial memory performance of AD mice

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105

models and a significant reduction of senile plaques and levels of ROS [104].

brain, especially in an area known as basal ganglia [105].

After Alzheimer's, PD is the second most common disease in terms of neurodegenerative diseases. As the aging population increases, the number of people with this disease is expected to rise. It affects 0.3% and 1% of the population worldwide over the age of 40 years and 65 years, respectively. Pathologically, PD is characterized by progressive loss of muscle control, which leads to tremor of hands, bradykinesia, rigidity and postural instability [105] Motor impairment in PD can also cause hypomimia, which is the decreased degree of facial expression. Dysphagia and hypophonia, which are disruption of the swallowing process and lack of coordination in the vocal musculature, are also common features in PD. Other symptoms also include ophthalmologic complaints such as blurred vision and gate. It is important to mention that all these signs and symptoms are a result of affected areas that occur within the

Thus, PD is a result of problems that occur within the basal ganglia. The basal ganglia is a collection of nuclei located deep beneath the cerebral cortex and it is responsible for the correct execution of voluntary muscle movements and learned movement patterns. The components of basal ganglia are caudate nucleus and putamen (dorsal striatum); nucleus accumbens and olfactory tubercule (ventral striatum), ventral pallidum, globus pallidus, subthalamic nucleus and substantia nigra. In PD, the basal ganglia is disrupted, causing degeneration of dopaminergic neurons located at the substantia nigra. Essentially, it is considered a disease of the basal ganglia because what happens is that when the cerebral cortex wants to initiate a movement, the basal ganglia receives these signals and sends it back the motor cortex via the thalamus. Through various pathways, the substantia nigra is connected with nuclei in basal ganglia. The basal ganglia plays an essential role in integrating multiple input signals to modulate the output of the motor cortex. Inhibitory or excitatory connections can occur in this process. Thus, the loss of dopamine from substantia nigra in this process underlies the symptoms described earlier [105].

**6.3. Parkinson's disease**

Abnormal structures called β-amyloid plaques and neurofibrillary tangles are classical biological hallmarks of the disease [91]. The formation of extracellular β-amyloid plaques occurs when amyloid precursor protein in the neuron cell membrane is cleaved at different positions, releasing small fragments called amyloid β (Aβ) that are highly toxic to the neurons and also interfere with the function of the brain cells [95]. Neurofibrillary tangles, on the other hand, are aggregates of hyperphosphorylate of a microtubule-associated protein known as tau. Tau protein, which in normal cells is responsible for helping nerve cells transport nutrients and maintain their proper shape, is altered in AD and, as a consequence, the transport of nutrients and other essential supplies into the neuron is affected, causing its death. At the same time, the health neurons start working less effectively. After some time, these neurons start losing their capacity to function and communicate to one another and, eventually, they die. Then, the harm may spread to structures in the brain such as hippocampus and entorhinal cortex, which are crucial areas of the brain responsible for forming new memories, thus causing memory loss. As neurons continue to die, affected areas of the brain begin to shrinks and brain functions are lost (**Figure 4**).

The BBB impairment in AD has been controversial [96]. However, several studies carried out in AD patients or AD animal models have been suggesting that the cause of cerebrovascular alterations in the BBB of the diseased brains is the accumulation of Aβ peptide [97–99]. Nevertheless, there are also studies suggesting that the BBB impairment is the cause of neurodegeneration, since the dysfunction of the brain structure in AD animal models was observed before Aβ aggregates were accumulated [100]. In any case, both hypotheses for the dysfunction of the BBB consider as a secondary event the tauopathies. However, a study reported that the tau filaments alone are able to start the disruption of the BBB and when it was deregulated, the BBB integrity was recovered [101].

Currently, there are no drug treatments that can cure AD. For this reason, approaches for treating AD are focused more on therapeutic interventions that alleviate symptoms, slow down or delay the progression of the disease, improving the patient's quality of life. To date, there are two types of medications for Alzheimer's treatment: acetylcholinesterase inhibitors (Aricept, Reminyl, Exelon and Cognex) and N-Methyl-D-aspartate (NMDA) receptor antagonist (Namenda). Nevertheless, the administration of these therapeutic molecules is associated with severe side effects. Thus, it would be desirable to develop drugs that can efficiently deliver these drugs into the brain. Moreover, there are also several studies showing that neuroprotective peptides might be an excellent compound for AD therapy, since they have shown to be able to break down and degrade Aβ plaques.

Multifunctional NPs are a good option to carry these peptides, since nanocarriers can protect them from degrading into the plasma by proteolytic enzymes and increase their stability in the serum. For example, PEG-PLA NPs were able to protect the neuroprotective peptide NAPVSIPQ from degradation. However, just NPs modified with B6 peptide (similar to transferrin) were able to cross the blood-brain barrier in mice and successfully deliver the neuroprotective peptide into the brain [102]. Moreover, it was observed that the treatment with this formulation improved cholinergic function and ameliorated spatial learning of AD mouse model [102]. In the same way, the nerve growth factor (NGF) has also been explored as a good drug for treating AD, although it is not able to cross the BBB. For this purpose, NGF was encapsulated into PBCA NPs, decorated with polysorbate 80 [103]. These NPs presented very promising results, since they were able to reach the mice brain parenchyma in less than 1 h after administration and these nanocarriers also proved to be able to improve recognition and memory of mice and to reduce by almost 40% the PD symptoms such as rigidity, tremor and oligokinesia in animal models [103]. Coenzyme Q10, a powerful antioxidant macromolecule, has also been explored at AD therapy. In this way, this coenzyme was encapsulated within PLGA NPs decorated with trimethylates chitosan. The results showed that these nanoparticles were able to cross the BBB and accumulate in the choroid plexus, ventricles and cortex. Moreover, the authors also observed an improvement in the cognitive and spatial memory performance of AD mice models and a significant reduction of senile plaques and levels of ROS [104].

#### **6.3. Parkinson's disease**

these synaptic gaps between neurons and bind to receptor sites on the dendrites of the next neuron. Unfortunately, neurons are the type of cells affected by AD. To date, scientists still do not know exactly the causes of AD and how this process begins. However, according to recent studies, it appears to be likely that astrocytes' activation contributes to the neuroinflammatory component responsible for the damage of neurons decades before the issue becomes

Abnormal structures called β-amyloid plaques and neurofibrillary tangles are classical biological hallmarks of the disease [91]. The formation of extracellular β-amyloid plaques occurs when amyloid precursor protein in the neuron cell membrane is cleaved at different positions, releasing small fragments called amyloid β (Aβ) that are highly toxic to the neurons and also interfere with the function of the brain cells [95]. Neurofibrillary tangles, on the other hand, are aggregates of hyperphosphorylate of a microtubule-associated protein known as tau. Tau protein, which in normal cells is responsible for helping nerve cells transport nutrients and maintain their proper shape, is altered in AD and, as a consequence, the transport of nutrients and other essential supplies into the neuron is affected, causing its death. At the same time, the health neurons start working less effectively. After some time, these neurons start losing their capacity to function and communicate to one another and, eventually, they die. Then, the harm may spread to structures in the brain such as hippocampus and entorhinal cortex, which are crucial areas of the brain responsible for forming new memories, thus causing memory loss. As neurons continue to die, affected areas of the brain begin to shrinks and

The BBB impairment in AD has been controversial [96]. However, several studies carried out in AD patients or AD animal models have been suggesting that the cause of cerebrovascular alterations in the BBB of the diseased brains is the accumulation of Aβ peptide [97–99]. Nevertheless, there are also studies suggesting that the BBB impairment is the cause of neurodegeneration, since the dysfunction of the brain structure in AD animal models was observed before Aβ aggregates were accumulated [100]. In any case, both hypotheses for the dysfunction of the BBB consider as a secondary event the tauopathies. However, a study reported that the tau filaments alone are able to start the disruption of the BBB and when it was deregulated,

Currently, there are no drug treatments that can cure AD. For this reason, approaches for treating AD are focused more on therapeutic interventions that alleviate symptoms, slow down or delay the progression of the disease, improving the patient's quality of life. To date, there are two types of medications for Alzheimer's treatment: acetylcholinesterase inhibitors (Aricept, Reminyl, Exelon and Cognex) and N-Methyl-D-aspartate (NMDA) receptor antagonist (Namenda). Nevertheless, the administration of these therapeutic molecules is associated with severe side effects. Thus, it would be desirable to develop drugs that can efficiently deliver these drugs into the brain. Moreover, there are also several studies showing that neuroprotective peptides might be an excellent compound for AD therapy, since they have

Multifunctional NPs are a good option to carry these peptides, since nanocarriers can protect them from degrading into the plasma by proteolytic enzymes and increase their stability in the

obvious [94].

104 Molecular Insight of Drug Design

brain functions are lost (**Figure 4**).

the BBB integrity was recovered [101].

shown to be able to break down and degrade Aβ plaques.

After Alzheimer's, PD is the second most common disease in terms of neurodegenerative diseases. As the aging population increases, the number of people with this disease is expected to rise. It affects 0.3% and 1% of the population worldwide over the age of 40 years and 65 years, respectively. Pathologically, PD is characterized by progressive loss of muscle control, which leads to tremor of hands, bradykinesia, rigidity and postural instability [105] Motor impairment in PD can also cause hypomimia, which is the decreased degree of facial expression. Dysphagia and hypophonia, which are disruption of the swallowing process and lack of coordination in the vocal musculature, are also common features in PD. Other symptoms also include ophthalmologic complaints such as blurred vision and gate. It is important to mention that all these signs and symptoms are a result of affected areas that occur within the brain, especially in an area known as basal ganglia [105].

Thus, PD is a result of problems that occur within the basal ganglia. The basal ganglia is a collection of nuclei located deep beneath the cerebral cortex and it is responsible for the correct execution of voluntary muscle movements and learned movement patterns. The components of basal ganglia are caudate nucleus and putamen (dorsal striatum); nucleus accumbens and olfactory tubercule (ventral striatum), ventral pallidum, globus pallidus, subthalamic nucleus and substantia nigra. In PD, the basal ganglia is disrupted, causing degeneration of dopaminergic neurons located at the substantia nigra. Essentially, it is considered a disease of the basal ganglia because what happens is that when the cerebral cortex wants to initiate a movement, the basal ganglia receives these signals and sends it back the motor cortex via the thalamus. Through various pathways, the substantia nigra is connected with nuclei in basal ganglia. The basal ganglia plays an essential role in integrating multiple input signals to modulate the output of the motor cortex. Inhibitory or excitatory connections can occur in this process. Thus, the loss of dopamine from substantia nigra in this process underlies the symptoms described earlier [105].

At the beginning, researches believed that the BBB did not suffer any kind of alteration during the disease development [106]. Surprisingly, tracking compounds such as [11C]-verapamil and benserazide in the brain of PD patients or PD animal models, it was observed that the concentration of these compounds in the brain was increased, what does not happen in the brain of health patients or animals since these drugs are not able to cross the BBB [107]. In addition to this, a good correlation between the albumin ratio and progressive BB integrity loss in the brain of patients with PD was observed [107]. Moreover, other signals of the BBB impairment such as vascular alterations and blood flow deficiencies were reported [108]. Most important, the increased expression of vascular endothelial growth factor (VEGF) was directly correlated with the high amount of blood vessels presented in the damaged dopaminergic neurons in the brain of monkeys [109]. Later, it was observed that the injection of VEGF into the substantia nigra in the brain of rats disrupted the BBB, leading to a strong inflammation response and loss of dopaminergic neurons [110]. Lastly, alpha-synuclein aggregates are the central hallmark of PD and their accumulation seems to be correlated with the downregulation of the P-glycoprotein (**Figure 4**) [111]. Moreover, higher concentration of some metals like iron was found in the brain of PD patients and PD animal models because of the higher levels of lactoferrin receptor in the substantia nigra dopaminergic neurons of the diseased brain [32].

combine several of these characteristics, known as multifunctional NPs. Long-circulating and target-specific NPs capable of prolonged circulation time in the blood and targeted delivery of drug to the brain and *in vivo* imaging represent one example of a multifunctional nanocarrier [44]. Moreover, we hope this chapter was a bridge between nanotechnology and central nervous systems disorders, since multifunctional NPs have a great potential in the treatment of neurological disorders in the near future [14, 44, 120]. However, as discussed, the BBB is one of the major obstacles to the delivery of drugs into the brain and, consequently, for the treatment of neurological diseases [48, 121]. The BBB is composed of very tightly connected endothelial cells and a variety of transporters [15, 17, 120, 122]. This results in a highly selective permeability barrier that separates the circulating blood from the cerebral parenchyma, thus limiting the entry of drugs into the brain. As discussed earlier, several multifunctional NPs for delivering therapeutic and/or imaging molecules into the brain have been developed [44, 47, 123]. Thus, this part of the chapter was organized in a way to carry the reader through the fundamentals of common neurological diseases such as Alzheimer's, Parkinson's and cerebral ischemia and their potential treatments with these kinds of NPs [44, 68, 88, 102, 124–139]. The purpose was to analyze some of the major scientific data indexed in PubMed, Web of Science and Scopus to explore different approaches engineered to transport and deliver imaging or therapeutic molecules to the brain by using multifunctional NPs technology. In this way, our gathered data on different strategies for the delivery of drugs across the BBB using multifunctional NPs were reviewed, discussed and grouped in self-explanatory figures. Results of our analysis from some research articles on our search showed that several strategies have been used to deliver several therapeutic compounds to the brain by these NPs. Functionalization of the surface of these NPs by covalent ligation of macromolecules such as antibodies, RNA aptamers as well peptides is an effective method for receptor targeting nanocarriers, which allows their BBB-penetration and the efficient delivery of their cargo specifically to the disease site. Additionally, methods for the development of multifunctional NPs that can respond to external stimuli were employed, concluding that the development of multifunctional NPs for treating neurological disorders still is at its infancy, although these systems have a huge chance to revolutionize the ways that brain diseases are treated.

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**Author details**

**References**

1038/nnano.2015.341

Débora Braga Vieira and Lionel Fernel Gamarra\*

Hospital Israelita Albert Einstein, São Paulo, Brazil

\*Address all correspondence to: lionelgamarra7@gmail.com

[1] Kendall M, Lynch I. Long-term monitoring for nanomedicine implants and drugs. Nature Nanotechnology. 2016;**11**(3):206-210. Available at: http://www.nature.com/doifinder/10.

Currently, there is no cure for PD. However, there are drugs that work to decrease and relieve the symptoms of PD and maintain the quality of life of the patient. The most effective treatment for PD is the use of the drug levodopa—also called L-dopa—or dopamine that is both able to restore or increase the concentration of dopamine in the basal ganglia. But here, we want to discuss an article that explored the increased expression of lactoferrin in some region of the brain in PD patients [112]. In this way, a PAMAM and PEG NPs were developed, coated with lactoferrin to the delivery of a plasmid of human glial cell line-derived neurotrophic factor plasmid (GDNF), since GDNF is a promising factor in treating PD, but as all plasmids are unable to cross the BBB. These multifunctional nanoparticles were able to not only cross the brain barrier but also effectively deliver the plasmid into the brain, since a neuroprotective effect on dopaminergic neurons and improvement of locomotor activities in AD animal models was observed [113]. Another example is the encapsulation of urocortin (hormone-related peptide) in PEGylated-PLGA NPs covered by lactoferrin. From the results presented, this formulation was able to quickly cross the BBB and to promote protection to the dopaminergic neurons and improve locomotor functional deficits [114].

#### **7. Conclusions**

Nanotechnology in the field of medicine has brought a variety of new ways to treat and/or detect diseases [13, 115, 116]. Currently, engineered pharmaceutical NPs demonstrated abilities such as long blood circulation time in the body fluids for their accumulation at disease sites with leaky vasculature [117]; specific targeted drug delivery to the pathological area due to the surface functionalization of NPs with ligands such as antibodies [118]; contrast properties due to their unique capacity of carrying contrast agents allowing their tracking *in vivo* [119]; drug delivery from the particles responsive to a specific stimuli [79] and others. The tremendous advances in nanomedicine during the past decade have significantly advanced on the engineering of nanoparticles that combine several of these characteristics, known as multifunctional NPs. Long-circulating and target-specific NPs capable of prolonged circulation time in the blood and targeted delivery of drug to the brain and *in vivo* imaging represent one example of a multifunctional nanocarrier [44].

Moreover, we hope this chapter was a bridge between nanotechnology and central nervous systems disorders, since multifunctional NPs have a great potential in the treatment of neurological disorders in the near future [14, 44, 120]. However, as discussed, the BBB is one of the major obstacles to the delivery of drugs into the brain and, consequently, for the treatment of neurological diseases [48, 121]. The BBB is composed of very tightly connected endothelial cells and a variety of transporters [15, 17, 120, 122]. This results in a highly selective permeability barrier that separates the circulating blood from the cerebral parenchyma, thus limiting the entry of drugs into the brain. As discussed earlier, several multifunctional NPs for delivering therapeutic and/or imaging molecules into the brain have been developed [44, 47, 123]. Thus, this part of the chapter was organized in a way to carry the reader through the fundamentals of common neurological diseases such as Alzheimer's, Parkinson's and cerebral ischemia and their potential treatments with these kinds of NPs [44, 68, 88, 102, 124–139]. The purpose was to analyze some of the major scientific data indexed in PubMed, Web of Science and Scopus to explore different approaches engineered to transport and deliver imaging or therapeutic molecules to the brain by using multifunctional NPs technology. In this way, our gathered data on different strategies for the delivery of drugs across the BBB using multifunctional NPs were reviewed, discussed and grouped in self-explanatory figures. Results of our analysis from some research articles on our search showed that several strategies have been used to deliver several therapeutic compounds to the brain by these NPs. Functionalization of the surface of these NPs by covalent ligation of macromolecules such as antibodies, RNA aptamers as well peptides is an effective method for receptor targeting nanocarriers, which allows their BBB-penetration and the efficient delivery of their cargo specifically to the disease site. Additionally, methods for the development of multifunctional NPs that can respond to external stimuli were employed, concluding that the development of multifunctional NPs for treating neurological disorders still is at its infancy, although these systems have a huge chance to revolutionize the ways that brain diseases are treated.

#### **Author details**

At the beginning, researches believed that the BBB did not suffer any kind of alteration during the disease development [106]. Surprisingly, tracking compounds such as [11C]-verapamil and benserazide in the brain of PD patients or PD animal models, it was observed that the concentration of these compounds in the brain was increased, what does not happen in the brain of health patients or animals since these drugs are not able to cross the BBB [107]. In addition to this, a good correlation between the albumin ratio and progressive BB integrity loss in the brain of patients with PD was observed [107]. Moreover, other signals of the BBB impairment such as vascular alterations and blood flow deficiencies were reported [108]. Most important, the increased expression of vascular endothelial growth factor (VEGF) was directly correlated with the high amount of blood vessels presented in the damaged dopaminergic neurons in the brain of monkeys [109]. Later, it was observed that the injection of VEGF into the substantia nigra in the brain of rats disrupted the BBB, leading to a strong inflammation response and loss of dopaminergic neurons [110]. Lastly, alpha-synuclein aggregates are the central hallmark of PD and their accumulation seems to be correlated with the downregulation of the P-glycoprotein (**Figure 4**) [111]. Moreover, higher concentration of some metals like iron was found in the brain of PD patients and PD animal models because of the higher levels of lactoferrin receptor in the substantia nigra dopaminergic neurons of the diseased brain [32]. Currently, there is no cure for PD. However, there are drugs that work to decrease and relieve the symptoms of PD and maintain the quality of life of the patient. The most effective treatment for PD is the use of the drug levodopa—also called L-dopa—or dopamine that is both able to restore or increase the concentration of dopamine in the basal ganglia. But here, we want to discuss an article that explored the increased expression of lactoferrin in some region of the brain in PD patients [112]. In this way, a PAMAM and PEG NPs were developed, coated with lactoferrin to the delivery of a plasmid of human glial cell line-derived neurotrophic factor plasmid (GDNF), since GDNF is a promising factor in treating PD, but as all plasmids are unable to cross the BBB. These multifunctional nanoparticles were able to not only cross the brain barrier but also effectively deliver the plasmid into the brain, since a neuroprotective effect on dopaminergic neurons and improvement of locomotor activities in AD animal models was observed [113]. Another example is the encapsulation of urocortin (hormone-related peptide) in PEGylated-PLGA NPs covered by lactoferrin. From the results presented, this formulation was able to quickly cross the BBB and to promote protection to the dopaminergic

neurons and improve locomotor functional deficits [114].

Nanotechnology in the field of medicine has brought a variety of new ways to treat and/or detect diseases [13, 115, 116]. Currently, engineered pharmaceutical NPs demonstrated abilities such as long blood circulation time in the body fluids for their accumulation at disease sites with leaky vasculature [117]; specific targeted drug delivery to the pathological area due to the surface functionalization of NPs with ligands such as antibodies [118]; contrast properties due to their unique capacity of carrying contrast agents allowing their tracking *in vivo* [119]; drug delivery from the particles responsive to a specific stimuli [79] and others. The tremendous advances in nanomedicine during the past decade have significantly advanced on the engineering of nanoparticles that

**7. Conclusions**

106 Molecular Insight of Drug Design

Débora Braga Vieira and Lionel Fernel Gamarra\* \*Address all correspondence to: lionelgamarra7@gmail.com Hospital Israelita Albert Einstein, São Paulo, Brazil

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## *Edited by Arli Aditya Parikesit*

The approaches in drug design are mainly comprised of these three multidisciplinary sciences. First, Bioinformatics has successfully gather biological data in form of biomolecular sequences, in order to construct knowledge on drug and vaccine design. It is of considerable importance for drug designers to comprehend the utilization of bioinformatics tools for resolving their research questions. Second, Nanotechnology has made possible the design and delivery of the nano-based drug. Third, Pharmaceutical Chemistry made it possible to investigate the adsorption, distribution, metabolism, and toxicology of the drug candidates in a fine-grained resolution.

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Molecular Insight of Drug Design

Molecular Insight of

Drug Design