**4.2 Biodistribution**

**Figure 6** shows an overview of the organ distribution patterns of the carriers under study as an approximation of their *in vivo* transport and metabolism processes depending on the route of administration. Perhaps, these behaviors would better correspond to the carried drug since the concentration of the active molecule in the tissues of interest is the measure commonly used to follow the particles in this kind of experiments. Once again, it is the intention to illustrate general behaviors; therefore, the punctual analyses on the particular work conditions used by each research team such as the animal models, the sampling times, and the way as the samples were analyzed are not considered. Thus, caution must be taken to do statements that lead to misinterpretations.

As can be seen in **Figure 6**, after 8 h of oral administration of both lipid and hybrid carriers, the liver, the spleen, and the kidney appear as the organs where lipid and hybrid particles are located. This could be attributed to the important role of the liver in the clearance of the particles and the blood filtration function of spleen within the immune system which might also remove the particles of the bloodstream. On its part, drug concentration in the kidney could mean the normal transit of the carrier because of the systemic circulation and the high irrigation of this organ. Nonetheless, the elimination process of intact carriers would also be happening.

On the other hand, as expected, the brain accumulates substantial amounts of lipid nanoparticles administered via intranasal because of the closeness of this organ to the nasal mucosa and its high blood perfusion. This behavior should be harnessed to improve therapies targeted to the brain as those for the treatment of diseases of the central nervous system. Likewise, particles intended for lung cancer therapies, prepared from a hyaluronan-modified polymer, and administered via

#### **Figure 6.**

quercetin inhibits the mycophenolate metabolism through cytochrome P450, are highlighted. This, together with the slow-release pattern of the particles, improves in a significant way the *in vivo* performance of the hybrid nanoparticles [14].

*General behaviors of plasma concentration reported for polymeric nanoparticles (A and B), lipid nanoparticles*

*administration (B, D, and F) (PTX: paclitaxel; FD: free drug; F68: Pluronic 68; HSA: human serum albumin; KET: ketamine; SH: shellac; ITZ: itraconazole; NEV: nevirapine; AmphB: amphotericin B; P80: polysorbate 80; SA: stearylamine; SL: soy lecithin; P: PLGA; MPA: mycophenolate; PVA: polyvinyl alcohol; QUE: quercetin; cHNP: cholate-modified hybrid nanoparticle; DOX: doxorubicin; DTX: docetaxel; P85: Pluronic 85;*

*(C and D), and hybrid nanoparticles (E and F). Oral administration (A, C, and E); intravenous*

*Nano- and Microencapsulation - Techniques and Applications*

**Figure 5.**

*tf: transferrin).*

**114**

to 10 times higher than free drug. As intended, pegylation of polymeric

Concerning the administration of carriers by the intravenous route, pharmacokinetic advantages were also evidenced compared to the free drug administration. As reported by Bian et al. [22] and Han et al. [27], even if a fraction of the polymeric nanoparticles are quickly removed by the reticuloendothelial system during the first 4 h after the administration, the remaining particles into the systemic circulation allow a sustained drug delivery for more than 20 h achieving AUC0-t values from 2

nanoparticles extends the elimination half-life by 100 h and increases in 84% the AUC regarding the free drug [27]. With respect to lipid carriers, Lahkar et al. [13] evidence a significant increase of their AUC0-t which could remain in the blood circulation four times more than the free drug. Moreover, modifications to the particle surface providing some hydrophilicity with polysorbate 80 result in an MRT eight times higher than that of the free drug. Regarding the hybrid nanoparticles, Zhu et al. [4] provide evidence on their extended drug delivery pattern that is improved as modifications on the particle surface are introduced. Thus, plasma circulation of the particles and their corresponding AUC0-t could be prolonged up to six and seven times, respectively, compared with that for the free drug. Jadon and Sharma [8] illustrate results in the same direction where drug

*General behavior of biodistribution for polymer, lipid, and hybrid nanoparticles after administration by oral, intranasal, intrapulmonary, and intravenous routes. Administration by intravenous, intranasal and oral routes (black-filled symbols) and by intrapulmonary route (crossed symbols) (KET: ketamine; NEV: nevirapine; DTX: docetaxel; DZP: diazepam; AmphB: amphotericin B; MPA: mycophenolate; QUE: quercetin).*

intrapulmonary route, directly locate on lung up to 24 h [59]. This finding confirms the ability of hyaluronan to be recognized by cancer lung receptors allowing the particle concentration in this tissue and consequently, avoiding the waste of active substance in other organs.

Regarding the intravenous administration, it seems that after 24 h, polymeric nanoparticles mainly locate at the lung, liver, and brain; lipid particles are distributed in blood, liver, kidney, and spleen; and hybrid particles accumulate in the liver. However, it should be noted that regardless of the kind of particle and compared with the oral and intranasal administration, when carriers are administered by intravenous route, drug is found in low levels in all the investigated organs. In addition, after extended periods (e.g., 120 h), particles are more homogeneously distributed among the investigated tissues [27]. This is a natural consequence of the systemic circulation and the irrigation of the different organs. Besides, as previously mentioned, there is a high probability that the concentration of carriers on the liver occurs due to the ability of the hepatic Kupffer cells to phagocyte them. Likewise, the phagocytic activity of the alveolar macrophages could explain why high concentrations of the drug are found in the lung. In addition, to find carriers or active molecules in the stomach might also be possible considering that the pH of this tissue could favor the retention of active molecules exhibiting a basic nature.

It is important to highlight the efficacy of targeted carriers to reach the intended tissues. As it has been evidenced by Jeannot et al. [59], working with polymeric nanoparticles, the functionalization of the polymer with polysaccharide hyaluronan, known for its affinity toward certain cancer cells receptors, allows high concentrations of particles on the lung offering an interesting alternative for the lung cancer treatment. In the same direction, Dehaini et al. [7] demonstrate the ability of docetaxel-loaded hybrid nanoparticles functionalized with folate to reach cancerous tumors.

#### **4.3 Stability in biological fluids**

Knowing if nanoparticles aggregate after their *in vivo* administration is of crucial importance for their application as drug delivery systems. To this end, the colloidal stability of the particulate systems dispersed in biological fluids has been investigated by monitoring variables such as the particle size and the drug encapsulation. Thus, Lazzari et al. [73] demonstrated that polymeric nanospheres prepared by flash nanoprecipitation from PMMA were stable up to 60 h in synthetic saliva, gastric juice, intestinal fluid, and lysosomal fluid while PLA nanoparticles aggregate in gastric juice. Likewise, Dehaini et al. [7] report the aggregation of PLGA nanoparticles in fetal bovine serum (FBS). On the other hand, polymeric nanocapsules coated with brush layers of an oligo ethylene glycol derived methacrylate polymer exhibit major stability in human serum albumin solution, FBS, and human blood plasma, that those non-coated [74]. This evidences the usefulness of designing stealth nanoparticles as a strategy to prevent the particle aggregate formation in blood avoiding their rapid removal from the systemic circulation by the immune system. Regarding SLN, Liu et al. [26] verified their colloidal stability in FBS reporting increases in particle size of approximately 50%, although encapsulation efficiency does not vary. Chaudhari et al. [21] delved into the stability of SLN in simulated gastric fluid confirming that after 2 h, amphotericin B remains encapsulated favoring its stability. With respect to hybrid nanoparticles, contradictory results of aggregation [23] and non-aggregation [7] have been reported when the particle dispersions are mixed with FBS. This can be attributed to the experimental conditions used. In the first case, aggregation is reported after 2 days of storage of the samples at 37°C; in the second one, aggregation was investigated immediately

**Nanoparticle**

**117**

PNS

CD44

Human H322, H358, and A549

8 μg/mL

 30 min at 37 C

 Flow cytometry

Dose-dependent

 binding of NP 30 nm

[59]

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

FITC

and NP 300 nm, was observed in the

three cell lines, with a higher intensity for A549 cells compared with H322 and

H358 cells.

NSCLC cell lines

expression

levels

HNP

HNP

HNP

 Cellular uptake

Human breast

adenocarcinoma

1 μg/mL

12 h

CLSM

The improved cell uptake efficiency of

[8]

HNP is attributed by cytosolic delivery

of the drug.

Qualitative

MDA-MB-231

 cells

study

HNP

 Cellular uptake

Human breast

adenocarcinoma

Equivalent to

24 h

CLSM

HNP exhibit improved cellular uptake

[8]

efficiency (45–48%) compared with

free drug (37–39%).

Quantitative

MDA-MB-231

 cells

10, 20, 30, and

40 μg/mL

 1 μg/mL

2 h

 Differential

contrast microscopy

 interference

High cells after 2 h of incubation with

respect to reference

nanoparticles.

[32]

internalization

 of HNP inside the

[14]

study

HNP

HNP

 Cellular uptake

Caco-2 cells

25 μg/mL

0.5–2 h

 Protein

BCA protein assay kit

Drug

quantification:

HPLC

quantification:

HNP exhibited improved cellular

uptake of quercetin relative to its free

form, showing a

uptake

accumulation.

time-dependent

 Cellular uptake

MCF-7 human breast cancer cell

analysis

 Cellular uptake

 A549 human lung

20 μg/mL

24 h

Flow cytometry

role in the uptake of drugs in *in vitro*

lung cancer cells.

Functionalization

 of HNP plays a key

[6]

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

adenocarcinoma

 cells

 Cellular uptake The

MDA-MB-231

cells; human prostate cancer

PC3 cells

 breast cancer

10, 300, and

24 h

CLSM

High cells at the highest

internalization

 of the HNP in the

[9]

concentrations.

500 μg/mL

 **Assay** **Cellular model**

**Experimental**

**Tracer**

**Interaction**

**—**

**Technique**

 **of analysis**

**molecule**

**cellular model**

**(some work**

**conditions)**

**concentration**

 **conditions**

**General results**


*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*

intrapulmonary route, directly locate on lung up to 24 h [59]. This finding confirms the ability of hyaluronan to be recognized by cancer lung receptors allowing the particle concentration in this tissue and consequently, avoiding the waste of active

*Nano- and Microencapsulation - Techniques and Applications*

Regarding the intravenous administration, it seems that after 24 h, polymeric nanoparticles mainly locate at the lung, liver, and brain; lipid particles are distributed in blood, liver, kidney, and spleen; and hybrid particles accumulate in the liver. However, it should be noted that regardless of the kind of particle and compared with the oral and intranasal administration, when carriers are administered by intravenous route, drug is found in low levels in all the investigated organs. In addition, after extended periods (e.g., 120 h), particles are more homogeneously distributed among the investigated tissues [27]. This is a natural consequence of the systemic circulation and the irrigation of the different organs. Besides, as previously mentioned, there is a high probability that the concentration of carriers on the liver occurs due to the ability of the hepatic Kupffer cells to phagocyte them. Likewise, the phagocytic activity of the alveolar macrophages could explain why high concentrations of the drug are found in the lung. In addition, to find carriers or active molecules in the stomach might also be possible considering that the pH of this tissue could favor the retention of active molecules exhibiting a basic nature.

It is important to highlight the efficacy of targeted carriers to reach the intended tissues. As it has been evidenced by Jeannot et al. [59], working with polymeric

hyaluronan, known for its affinity toward certain cancer cells receptors, allows high concentrations of particles on the lung offering an interesting alternative for the lung cancer treatment. In the same direction, Dehaini et al. [7] demonstrate the ability of docetaxel-loaded hybrid nanoparticles functionalized with folate to reach

Knowing if nanoparticles aggregate after their *in vivo* administration is of crucial importance for their application as drug delivery systems. To this end, the colloidal stability of the particulate systems dispersed in biological fluids has been investigated by monitoring variables such as the particle size and the drug encapsulation. Thus, Lazzari et al. [73] demonstrated that polymeric nanospheres prepared by flash nanoprecipitation from PMMA were stable up to 60 h in synthetic saliva, gastric juice, intestinal fluid, and lysosomal fluid while PLA nanoparticles aggregate

in gastric juice. Likewise, Dehaini et al. [7] report the aggregation of PLGA nanoparticles in fetal bovine serum (FBS). On the other hand, polymeric

nanocapsules coated with brush layers of an oligo ethylene glycol derived methacrylate polymer exhibit major stability in human serum albumin solution, FBS, and human blood plasma, that those non-coated [74]. This evidences the usefulness of designing stealth nanoparticles as a strategy to prevent the particle aggregate formation in blood avoiding their rapid removal from the systemic circulation by the immune system. Regarding SLN, Liu et al. [26] verified their colloidal stability in FBS reporting increases in particle size of approximately 50%, although encapsulation efficiency does not vary. Chaudhari et al. [21] delved into the stability of SLN in simulated gastric fluid confirming that after 2 h, amphotericin B remains encapsulated favoring its stability. With respect to hybrid nanoparticles, contradictory results of aggregation [23] and non-aggregation [7] have been reported when the particle dispersions are mixed with FBS. This can be attributed to the experimental conditions used. In the first case, aggregation is reported after 2 days of storage of the samples at 37°C; in the second one, aggregation was investigated immediately

nanoparticles, the functionalization of the polymer with polysaccharide

substance in other organs.

cancerous tumors.

**116**

**4.3 Stability in biological fluids**


#### **Table 3.**

*Summary of experimental conditions and general results reported in research works on cellular uptake of nanoparticles prepared by the nanoprecipitation technique.* the particle dispersions were diluted. On the other hand, when the stability of hybrid particles was tested in human plasma, interactions of particle and serum proteins were evidenced which increased the carrier size. But what is more interesting is that those interactions seem to be related to the type of stabilizing agent used. As reported by Godara et al. [5], by using PVA or stearylamine as stabilizing agents, particle sizes increased 15% that contrast with an increase of 50% when particles were stabilized with human serum albumin. Maybe, the protein layer covering the particle surfaces promote their interaction with the serum proteins.

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

Regarding cellular uptake, **Table 3** reports the experimental conditions and general results. Indeed, researches on this regard have been mostly carried out for the hybrid nanoparticles by using human cancer cells taken in their majority from the breast. Nevertheless, some research works have also investigated on prostate and lung cancer cells. Other used cell lines include Caco-2 and MC3T3-E1 osteoblasts. In general terms, the analyses by flow cytometry and confocal laser scanning microscopy reveal that the functionalization of the hybrid particles favors the *in vitro* cellular uptake when compared to the free drugs and the pattern of cellular

On the other hand, the ability of nanoparticles to penetrate the different physiological barriers and reside in the target tissues has also been demonstrated. For example, SLN could provide efficient *in vivo* skin permeation [26], polymeric nanoparticles might penetrate mucus also exhibiting mucoadhesive behavior [16], and hybrid nanoparticles would cross the enterocyte walls [32] or reach bone tissue [23].

**5. Safety and efficacy of carriers prepared by nanoprecipitation**

nanoparticles reduce the toxicity of the active molecules [8, 57].

One of the promising applications of nanoparticles, including those obtained by

nanoprecipitation, is the therapy against cancer. As shown in **Table 5**, hybrid

A revision of the starting materials used to prepare particles via nanoprecipitation shows that the polymers and lipids present in the different recipes are recognized as safe considering their biocompatibility. Likewise, most organic solvents are classified as with low toxic potential according to ICH [75]. In the cases where acetonitrile, dichloromethane, tetrahydrofuran, dimethylformamide, and even methanol are used as solvents, the obtained particles should meet the specific requirements of limited concentrations of residual solvent because of their inherent toxicity. Traces of organic solvents would remain in the nanoparticle dispersions after the stage of solvent removal during their preparation. For example, up to 2300 ppm of tetrahydrofuran can be detected in lipid nanoparticles, which exceed the limit of 720 ppm established by the ICH [75]. However, as shown in **Table 4**, the safety tests including hematological studies on mice [27], hemolysis assays on human blood [8] or with erythrocytes [5], MTT assay on alveolar epithelial cells [34] or osteoblasts [23], cell viability on cancer cells [9, 11], and histological examination of mice [56], evidence concerns on the safety of that particles, and in general, neither of the particles were prepared via nanoprecipitation. Moreover,

**4.4 Cellular uptake**

**5.1 Safety**

**5.2 Efficacy**

**119**

uptake correlates with the carrier drug loading.

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*

the particle dispersions were diluted. On the other hand, when the stability of hybrid particles was tested in human plasma, interactions of particle and serum proteins were evidenced which increased the carrier size. But what is more interesting is that those interactions seem to be related to the type of stabilizing agent used. As reported by Godara et al. [5], by using PVA or stearylamine as stabilizing agents, particle sizes increased 15% that contrast with an increase of 50% when particles were stabilized with human serum albumin. Maybe, the protein layer covering the particle surfaces promote their interaction with the serum proteins.

#### **4.4 Cellular uptake**

Regarding cellular uptake, **Table 3** reports the experimental conditions and general results. Indeed, researches on this regard have been mostly carried out for the hybrid nanoparticles by using human cancer cells taken in their majority from the breast. Nevertheless, some research works have also investigated on prostate and lung cancer cells. Other used cell lines include Caco-2 and MC3T3-E1 osteoblasts. In general terms, the analyses by flow cytometry and confocal laser scanning microscopy reveal that the functionalization of the hybrid particles favors the *in vitro* cellular uptake when compared to the free drugs and the pattern of cellular uptake correlates with the carrier drug loading.

On the other hand, the ability of nanoparticles to penetrate the different physiological barriers and reside in the target tissues has also been demonstrated. For example, SLN could provide efficient *in vivo* skin permeation [26], polymeric nanoparticles might penetrate mucus also exhibiting mucoadhesive behavior [16], and hybrid nanoparticles would cross the enterocyte walls [32] or reach bone tissue [23].

### **5. Safety and efficacy of carriers prepared by nanoprecipitation**

#### **5.1 Safety**

A revision of the starting materials used to prepare particles via nanoprecipitation shows that the polymers and lipids present in the different recipes are recognized as safe considering their biocompatibility. Likewise, most organic solvents are classified as with low toxic potential according to ICH [75]. In the cases where acetonitrile, dichloromethane, tetrahydrofuran, dimethylformamide, and even methanol are used as solvents, the obtained particles should meet the specific requirements of limited concentrations of residual solvent because of their inherent toxicity. Traces of organic solvents would remain in the nanoparticle dispersions after the stage of solvent removal during their preparation. For example, up to 2300 ppm of tetrahydrofuran can be detected in lipid nanoparticles, which exceed the limit of 720 ppm established by the ICH [75]. However, as shown in **Table 4**, the safety tests including hematological studies on mice [27], hemolysis assays on human blood [8] or with erythrocytes [5], MTT assay on alveolar epithelial cells [34] or osteoblasts [23], cell viability on cancer cells [9, 11], and histological examination of mice [56], evidence concerns on the safety of that particles, and in general, neither of the particles were prepared via nanoprecipitation. Moreover, nanoparticles reduce the toxicity of the active molecules [8, 57].

#### **5.2 Efficacy**

One of the promising applications of nanoparticles, including those obtained by nanoprecipitation, is the therapy against cancer. As shown in **Table 5**, hybrid

**Nanoparticle**

**118**

HNP

into osteoblasts

> HNP

*PNS: polymeric nanospheres;*

*scanning microscope;*

**Table 3.**

*Summary of* 

*experimental*

 *conditions and general results reported in research works on cellular uptake of* 

 *MRSA:* 

 *HNP: hybrid*  *methicillin-resistant*

*nanoparticles;*

 *NP: nanoparticle;*

Staphylococcus

 aureus*; nr.: non-reported*

 *MTX:* 

*methotrexate;*

 *FITC: fluorescein* 

 *data.*

*isothiocyanate;*

*nanoparticles*

 *prepared by the* 

*nanoprecipitation*

 *technique.*

 *HPLC:* 

*high-performance*

 *liquid* 

*chromatography;*

 *CLSM: confocal laser*

Cellular

Prostate cancer cells (PC3-

100

300

3 min at 37°C

CLSM

Cellular uptake ability depends on

particle

concentration.

[11]

*Nano- and Microencapsulation - Techniques and Applications*

FITC

μg/mL

internalization

MM2) and human breast cancer

cells

(MDA-MB-231)

Internalization

MC3T3-E1 osteoblasts

 2, 4, and

6 h at 37°C

CLSM

HNP were more effective in reducing

[23]

the intracellular

 MRSA counts than the

free linezolid.

8 μg/mL

 **Assay** **Cellular model**

**Experimental**

**Tracer**

**Interaction**

**—**

**Technique**

 **of analysis**

**molecule**

**cellular model**

**(some work**

**conditions)**

**concentration**

 **conditions**

**General results**


**Nanoparticle**

**121**

**Cellular/animal**

HNP

MDA-MB-231

human prostate cancer PC3 cells;

colon cancer HT29 cells

> HNP

HNP

HNP HNP HNP HNP *PNS: polymeric nanospheres;*

*operation and* 

*lactide-co-glycolide);*

**Table 4.**

*Summary of* 

*experimental*

 *conditions and general results reported in research works on safety testing of* 

 *ALT: alanine* 

*Development;*

 *KB: kokum butter; H2DCFDA:*

*transaminase;*

 *AST: aspartate* 

 *SLN: solid lipid* 

*nanoparticles;*

 *HNP: hybrid* 

*transaminase;*

 *DTX: docetaxel; nr.: non-reported*

*nanoparticles;*

*6-carboxy-2*0*,7*0*-dichlorodihydrofluorescein*

 *diacetate; NHK: normal human* 

 *data.* *nanoparticles*

 *prepared by the* 

*nanoprecipitation*

 *technique.*

 *MTT:* 

*3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium*

 *bromide; OECD: Organisation*

*keratinocytes;*

 *FITC: fluorescein* 

 *for Economic Co-*

*isothiocyanate;*

 *PLGA: poly(D,L-*

Prostate cancer cells (PC3-MM2)

In vitro

25, 50, 100, 150,

48 h

Measure the

No cytotoxic effects were observed.

 [11]

luminescence

cytotoxicity

200, and 300 μg/

studies

mL

and human breast cancer cells

(MDA-MB-231)

Erythrocytes

Hemolysis

0.7 mg/mL

1 h

Spectrophotometric

 The average percentage hemolysis rate of

nanoparticles

 was found between 7 and

16%.

assay

MC3T3-E1 osteoblasts

 MTT assay

 0.5–40 μg/mL

 16 h

 Microplate reader

 All groups showed minimum cytotoxicity

against osteoblasts.

[23]

[5]

Sprague-Dawley

 rats

In vivo

25 mg/kg

 30 days

Spectrophotometric

biomarkers

insignificant

 as compared to control.

 (ALT and AST) was

Concentration

 of

hepatotoxicity

[14]

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

toxicity

 Whole human blood (from a healthy

person)

BALB/c female mice

 Side effects on

3 mg/kg

 21 days

 Histological

No significant toxicity to the heart, liver,

[56]

examination

spleen, lung, or kidney.

vital organs

Hemolysis

1 μL of suitably

0.5 h

Spectrophotometric

 The hemolysis of nanoparticle

was lesser than free drug.

 formulation

[8]

assay

diluted free DTX

and HNP

 breast cancer cells;

Cell viability

 50 to 300 μg/mL

 Over night

Fluorescence

No cytotoxic effects were observed for the

intensity

particles tested.

[9]

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

 **model**

**Experimental**

 **conditions**

**Assay**

**Drug**

**Time of**

**Technique**

 **of**

**interaction**

**cellular model**

**(h)**

**—**

**analysis**

**concentration**

**or dosage used**

 **for toxicity testing**

**General results**

**Reference**

#### *Nano- and Microencapsulation - Techniques and Applications*


 **4.**

*Summary of* 

*experimental*

 *conditions and general results reported in research works on safety testing of* 

*nanoparticles*

 *prepared by the* 

*nanoprecipitation*

 *technique.*

#### *Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*

**Nanoparticle**

**120**

**Cellular/animal**

**Polymeric** 

PNS

PNS PNS PNS **Lipid** 

SLN SLN SLN/PNS

**Hybrid** 

HNP

 Normal L929 alveolar epithelial cells

 MTT assay

 0.1–30 mg/mL

 48 h

Spectrophotometric

 No significant cytotoxicity

 against normal

[34]

alveolar cells.

**nanoparticles**

BALB/c 3T3

MTT assay

 50–200 μM

 24 h

 Microplate reader

 A moderate effect on cell viability, no

[26]

obvious changes were found.

Wistar rats

Sprague-Dawley

 rats

 Renal toxicity

3.6 mg/kg

72 h

UV-visible

Freeze-dried

as a safe oral alternative.

nanoparticles

 are considered

[21]

[13]

assessment

(OECD)

5–2000 mg/kg

 14 days

 LD50 by Karber

None of the animals showed any sign of

toxicity. The lethal dose (LD50) of KB is

higher than 2000 mg/kg.

method

guidelines, 423

**nanoparticles**

Male C57BL/6 J mice

Normal human

keratinocytes

(H2DCFDA)

0.5 mg/mL

1 h

 FITC

fluorescence

 The

nanoparticles

 have no oxidative stress

[18]

induction potential.

assay

Hematological

1 mg/kg

5 days

 Hematology

All

hematological

 parameters

mice.

 assessed at

[27]

analyzer

study remained in the normal range for

studies

Normal human

keratinocytes

 MTT assay

 0.05 and 0.5 mg/

48 h

 Microplate reader

 No cytotoxic effect was detected after

exposure of the NHK for 24 and 48 h to the

nanoparticles.

mL

 Vero cell line (green monkey kidney

epithelial cells)

MTT assay

 3.12–100 μg/mL

 24 h

 ELISA microplate

Nanoparticles

 reduce cytotoxicity

active molecule.

 of the

[57]

[18]

*Nano- and Microencapsulation - Techniques and Applications*

reader

**nanoparticles**

 **model**

**Experimental**

 **conditions**

**Assay**

**Drug**

**Time of**

**Technique**

 **of**

**interaction**

**cellular model**

**(h)**

**—**

**analysis**

**concentration**

**or dosage used**

 **for toxicity testing**

**General results**


**Nanoparticle**

**123**

**Assay**

SLN/PNS

Franz diffusion

Hairless abdominal full-

Corresponding

 to

72 h

Spectrophotometry

 UV

 SLN provides an efficient *in vitro*

permeation

performance

PNS exhibited prolonged

antinociceptive

efficiency until 3.5 h of study. The analgesic effects are maintained

for a longer period. Greater than

50% pain control was still found at 6

and 4 h for PNS and SLN,

respectively.

 effect showing

 up to 72 h.

 and sustained

[26]

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

thickness skins of

5 mg of tetracaine

Sprague-Dawley

 rats

cell

Tail-flick test Paw pressure test

> SLN (polymyxin

SLN

B)

**Hybrid** 

HNP (docetaxel)

 Targeting studies

*In vivo* tumor

Female nude mice

4 mg/kg

 35 days

 Tumor width, length, and

size

64 days after tumor challenge. As an

indicator of global health, body weights were monitored over the

course of the study.

Half of the mice were still alive at

> treatment efficacy

 KB cells

0.25 mg/mL

 30 min

 Flow cytometry

 The targeted hybrid

were found much deeper within the

tumor and further away from the

vasculature.

nanoparticles

[7]

**nanoparticles**

(amphotericin

*In vitro*

antifungal

*C. albicans*

0.5–250 μg/mL

 48 h

 Change in original blue color

of resazurin to pink.

efficacy

 B)

Antimicrobial

Strain of *E. coli*

6.6 μg/mL

 2–18 h

Turbidimetry

 Activity (% inhibition of growth) by

[20]

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

the plain drug and SLN were 52.7

and 56.7%,

Minimum inhibitory value of 7.812 μg/mL attributed to

controlled release of drug from the

nanoparticulate

 matrix.

concentration

[21]

respectively.

evaluation

Sprague-Dawley

 rats

 0.25 mg/mL

 8 h

 Response threshold (amount

of pressure supported by the

animal before removing the

paw).

Sprague-Dawley

 rats

 0.25 mg/mL

 4 h

 Response threshold (seconds

to withdraw the tail before

the thermal stimulus).

(tetracaine)

**Cellular/animal**

 **model Drug** 

**Experimental**

 **conditions**

 **for efficacy testing**

**concentration**

 **Time of**

**Technique**

 **of analysis**

**interaction**

**—cellular**

**model**

**General results**


#### *Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*

**Nanoparticle**

**122**

**Assay**

**Polymeric**  PNS (Brazilian red

Antioxidant

—

80 μg/mL

 30 min

Spectrophotometry

 UV

 The PNS displayed good antioxidant

[36]

activity with inhibition values

higher than 75%.

activity by using

DPPH method

Antileishmanial

L. (V.) braziliensis

5–100 μg/mL

 24 h

 Inverted microscopy

30 and 40% of EEP maintained

antileishmanial

 activity like the EEP

in its original form.

Nanoparticles

 containing between

*Nano- and Microencapsulation - Techniques and Applications*

culture

*in vitro* assay

> PNS (fluticasone

Mucus mobility

Human

cervicovaginal

0.2–0.5 μL

30 min

 Fluorescent

 microscopy

 Particles exhibit rapid mucus

penetration

 and behavior.

mucoadhesive

[16]

(nanosuspension)

mucus

by multiple particle tracking

Antiinflammatory

Lewi rats

0.1 mg of FP/kg

 24 h

 Total and differential

counts on an automated cell

counter.

HPLC/MS

Upon deposition onto respiratory

tissue, solution

encapsulated

removed through absorption into

systemic circulation compared with

nanoparticles.

 drugs are rapidly

formulations

 or non-

 cell

Inhibition of fluid (BAL) lavage neutrophils

between 50 and 70% in 24 h.

bronchoalveolar

 lavage

action

Duration of

CF-1 mouse

 10 μg of FP per

24 h

animal

residence in

mouse lung

> **Lipid**

L-βCD-C10

Complement

Polyclonal anti C3

Topical

 75 min

Immuno-electrophoretic

low level of

L-βCD-C10/DOPE-PEG

 shows a

[71]

complement

activation.

 C3

antibody

protein C3

activation

L-βCD-C10/DOPE-

PEG

L-βCD-C10/

stabilizer (nr.)

**nanoparticles**

propionate)

propolis extract)

**nanoparticles**

**Cellular/animal**

 **model Drug** 

**Experimental**

 **conditions**

 **for efficacy testing**

**concentration**

 **Time of**

**Technique**

 **of analysis**

**interaction**

**—cellular**

**model**

**General results**


**Nanoparticle**

**125**

**Assay**

HNP (psoralen) HNP (docetaxel)

(MTT assay) Annexin V-FITC/

MDA-MB-231

 cells

 10 μg/mL

 24 h

Flow cytometry

> propidium iodide

apoptosis assay

Antitumor

BALB/c female mice

 10 mg/kg

 3 weeks

 Tumor width, length and

size

The repeated dosing of HNP exhibit

less mortality (33%) than with free

drug.

efficiency

HNP

Annexin V

MCF-7

10, 20, 40, and

6 h

CLSM

Apoptosis indices of MPA-NP and

[14]

QC-NP are higher compared to

respective free drugs. Moreover, the

apoptosis index is significantly

higher when

NP + QC-NP is used.

Significantly

inhibition was observed in MPA-NP

than free MPA.

 higher enzyme

combination

 MPA-

60 μg/mL

apoptosis assay

Inosine-50

monophosphate

dehydrogenase

(IMPDH) assay


MCF-7

1 μM MPA NP

 24 h

IMPDH assay kit

(mycophenolate;

quercetin)

Cytotoxicity

Human breast

0.05, 0.1, 1, 10, and

24, 48, and

ELISA plate reader

be attributed to

cytosolic delivery of the drug which

is dose dependent. Injured cells (including early apoptosis, late apoptosis, and

necrotic cells) with HNP are greater

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

(87%) as compared with free drug

(51%).

lipid-mediated

[8]

Cytotoxicity

 activity of HNP could

> 72 h

20 μg/mL

adenocarcinoma

MB-231 cells

 MDA-

 Antitumor

MCF-7 cells

3 mg/kg

 21 days

 Changes in the tumor

HNP show more efficient antitumor

[56]

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

effects respect to other

formulations.

volume and final tumor

weight

efficacy

**Cellular/animal**

 **model Drug** 

**Experimental**

 **conditions**

 **for efficacy testing**

**concentration**

 **Time of**

**Technique**

 **of analysis**

**interaction**

**—cellular**

**model**

**General results**


#### *Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*

**Nanoparticle**

**124**

**Assay**

HNP

Antiproliferation

MDB-MB-231

 breast

5, 10, 20, 50, 100,

150, and 200 μg/mL

assay

cancer and PC3 prostate

cancer cells

(methotrexate)

HNP

(doxorubicin)

Cytotoxicity

HL-60 cells and HL-60/

0.25, 0.5, 1, 2, 5, 10,

70 h

 Microplate reader

 The cytotoxicity

was superior to DOX solution. The

IC50 of HNP was lower than DOX

solution.

 activity of HNP

[4]

*Nano- and Microencapsulation - Techniques and Applications*

and 20 μg/mL

evaluation

Tumor growth

Male BALB/c mice

 20 mg/kg

 18 days

 Xenograft model

 The tumor growth inhibition was

(68.9–89.6%).

the mice in any of HNP treatments

groups showed no obvious decrease

in comparison

 with untreated

groups.

 The body weight of

inhibition

HNP (paclitaxel)

(MTT assay)

Synergistic

*In vivo*

antitumor

BALB/c-nude

 mice

 5 mg/kg of paclitaxel

18 days

 Tumor width, length, and

size

and 3 mg/kg of

triptolide

efficacy

 effects

 A549 human lung

0.5–10 mg/mL

 48 h

 The results of cytotoxicity

were evaluated via the

Combination

 Index

adenocarcinoma

 cells

Cytotoxicity

A549 human lung

0.5–10 mg/mL

 48 h

 Microplate reader

 Drugs loaded HNP exhibited

marked cytotoxicity

dose-dependent

higher cytotoxicity their free drug The *in viv*o and *in vitro* results show

synergetic effect of the two drugs

incorporated

 in HNP against the

lung cancer.

The inhibition of the *in vivo* tumor

growth was lesser than that of the

control group.

 way and showed

 compared with

counterparts.

 on cells in a

[6]

adenocarcinoma

 cells

DOX. MTT assay

**Cellular/animal**

 **model Drug** 

**Experimental**

 **conditions**

 **for efficacy testing**

**concentration**

 **Time of**

**Technique**

 **of analysis**

**interaction**

**—cellular**

**model**

72 h

 ATP-based

 cell viability kit

 MTX

preserve its anticancer activity.

encapsulated

 in the HNP

[9]

**General results**


**Nanoparticle**

**127**

**Assay**

HNP (linezolid)

 Minimum

Strains of

USA300-0114,

500 μg/mL stock

24 h

 Broth dilution method

The MIC50 and MIC90 values of

free linezolid were 40–50% of the values of HNP.

approximately

*DOI: http://dx.doi.org/10.5772/intechopen.93338*

[23]

Microplate reader

solution in TSB, and

serially diluted for

the assay

inhibitory

CDC-587, and RP-62A

concentration

(MIC)

Biofilm

*S. aureus*

32, 64, 128, 164, and

12 h

 Microplate reader

 HNP were more effective than free

linezolid for eradicating

 the MRSA

biofilm.

256 μg/mL

microplate assay

Biofilm

*S. aureus*

32, 64, 128, 164, and

12 h

CLSM

Extensive retention of the

nanoparticles

after multiple buffer washing.

*Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics*

 in the biofilms even

256 μg/mL

microplate assay

Drug's levels in

Sprague-Dawley

 rats

 32, 64, 128, 164, and

24 h

HPLC

Bone linezolid levels from HNP increase to over four-folds those of

the free drug.

256 μg/mL

animals' bones

> HNP (sorafenib)

*PNS: polymeric nanospheres;*

*mycophenolate;*

*luciferase reporter phage; CFU:* 

*DPPH:* 

*liquid*  **Table 5.** *Summary of* 

*experimental*

 *conditions and general results reported in research works on efficacy testing of* 

*chromatography;*

 *CLSM: confocal laser scanning microscopy,*

 *nr.: non-reported*

 *data.*

*2,2-diphenyl-1-picryhydrazyl;*

 *Nv: nevirapine; QC: quercetin; MIC: minimum inhibitory* 

*colony-forming*

 *EEP: ethanolic extract of propolis; NSCLCs: non-small cell lung cancers; NMRI: Naval Medical Research Institute; MS: mass spectrometer;*

 *units; MTT:* 

 *SLN: solid lipid* 

*nanoparticles;*

 *NLC:* 

*nanostructured*

 *lipid carriers; HNP: hybrid* 

*concentration;*

 *MTS:*  *3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium*

 *bromide; DOX: doxorubicin;*

*nanoparticles*

 *prepared by the* 

*nanoprecipitation*

 *technique.*

*nanoparticles;*

 *CD: cyclodextrin;*

 *IC50:*  *3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;*

 *MTX:* 

*methotrexate;*

 *HL-60: human leukemia cell line;*

 *HPLC:* 

*high-performance*

*half-maximal*

 *inhibitory* 

*concentration;*

 *MPA:*

 *LRP:*

 Cell growth

Prostate cancer cells

5, 10, 20, 50, 100,

72 h

Cell viability kit

 The inhibition of the tumor cell

[11]

growth was found to be time- and

dose dependent for drug solution as

well as HNP.

150, and 200 μg/mL

(PC3-MM2)

breast cancer cells

(MDA-MB-231)

 and human

inhibition assay

**Cellular/animal**

 **model Drug** 

**Experimental**

 **conditions**

 **for efficacy testing**

**concentration**

 **Time of**

**Technique**

 **of analysis**

**interaction**

**—cellular**

**model**

**General results**

