**4.** *In vivo* **performance of carriers prepared by nanoprecipitation**

Drug delivery systems such as the polymeric, lipid, and hybrid nanoparticles have been promoted for use in therapeutics as an interesting approach to facilitate uptake of drugs at the desired site of action, particularly when free drugs might give rise to significant off-site toxicities or characterize by poor bioavailability because of their molecular and physicochemical properties. Accordingly, knowing the bioavailability behaviors, including the pharmacokinetic parameters and the biodistribution of the carriers obtained via the nanoprecipitation technique, as well as the stability of the carriers in biological fluids and their cellular uptake, result of paramount importance to investigate their applicability in pharmaceutics.

Considering that submicron sizes for most particles prepared by nanoprecipitation range between 200 and 300 nm, which are larger than pores between endothelial cells, it is expected that, in the absence of specific affinity for receptors, their distribution is limited to the vascular space. Nevertheless, for example, larger endothelial pores such as the fenestrations in the liver and the spleen might lead to the uptake of the particles by these tissues via bulk fluid flow. Once in the bloodstream, particles are coated with a layer of plasma proteins (opsonization or protein corona formation) facilitating their elimination by immune cells. Besides, dynamic interactions between nanoparticles and blood cells, e.g., erythrocytes, platelets, and leukocytes, could occur. Then, the carriers are entrapped in the microvasculature and clearing compartments of the reticuloendothelial system like the liver, the spleen, the bone marrow, and the lung, via phagocytic uptake by cells accessible from the vascular space such us the hepatic Kupffer cells. This allows the elimination of the particles from the organism via the bile ducts into the feces or in the urine [70].

first 20 h) and, at a rate, higher than 80%. Nimodipine reached delivered concentrations near 100% at 10 h, and other molecules such as tetracaine and nevirapine exhibit biphasic behaviors reaching drug deliveries higher than 80% at 25 h. NLC

*Drug release behaviors for polymeric nanospheres (A), polymeric nanocapsules (B), solid lipid nanoparticles (C), nanostructured lipid carriers (D), hybrid nanoparticles obtained from the mixture of polymers and lipids (E), and hybrid nanoparticles obtained from chemically modified polymers with lipids (F) (PTX: paclitaxel; PVA: polyvinyl alcohol; ATE: atenolol; F: formulation; DEX: dexamethasone; CAP: cellulose acetate phthalate; HSA: human serum albumin; DZP: diazepam; TETR: tetracaine; F68: Pluronic 68; FLU: fluticasone propionate; KET: ketamine; SH: shellac; DOX: doxorubicin; P85: Pluronic 85; DICLO: diclofenac; MGL: Miglyol 810; LAB: labrafac; FEN: fenofibrate, NEV: nevirapine; NIM: nimodipine; P80: polysorbate 80; CLOB: clobetasol propionate; CCT: caprylic/capric triglycerides; EFA: efavirenz; SA: stearylamine, SL: soy lecithin; Lec: lecithin; METH: methotrexate; PSO: psoralen; TPGS: tocopheryl polyethylene glycol succinate; QUE: quercetin; Tf: transferrin; SOR: sorafenib; LIN: linezolid; DTX: docetaxel; MPA: mycophenolate; TL: triptolide).*

**Nanoparticle Drug release study Reference**

HNP Dialysis 10 kDa pH 7.4 PBS nr. 37 °C [23]

FBS (10%)

*PNS: polymeric nanospheres; PNC: polymeric nanocapsules; SLN: solid lipid nanoparticles; NLC: nanostructured lipid carriers; HNP: hybrid nanoparticles; SDS: sodium dodecyl sulfate; DMF: dimethylformamide; PBS: phosphate*

*Summary of the work conditions used to investigate the drug release behavior of nanoparticles prepared by the*

HNP Dialysis 12 kDa pH 7.4 PBS Magnetic

HNP Dialysis 10–12 kDa PBS with pH 7.4

*Nano- and Microencapsulation - Techniques and Applications*

*buffer solution; FBS: fetal bovine serum; nr.: non-reported data.*

**Table 1.**

**Figure 4.**

**110**

*nanoprecipitation technique.*

**Method Medium Stirring Operating**

200 rpm

**conditions**

100 rpm 37°C [11]

37 2°C [5]

To provide a therapeutic response, nanoparticles must overcome these physiological clearance mechanisms and distributional barriers. The objective is to guarantee a high mean residence time for the carriers in the systemic circulation while their drug release delivery is modulated. Some alternatives in this way include the development of particles exhibiting sizes less than 100 nm or a positive surface charge. Stealth particles by using nonionic polymers or mimic the outer surface of blood cells by locating mixtures of phospholipids, cholesterol, sphingomyelin, and ganglioside molecules on the particle surface have also been proposed, and the modification of the particle surface with specific ligands appears as the best strategy for the target delivery of active substances up to now [61, 70].

Regarding the carriers prepared by nanoprecipitation, among the reported developments of particles that could theoretically allow them a better *in vivo* performance are: (i) particle sizes lesser than 100 nm for polymeric nanospheres [25, 26, 34], solid lipid nanoparticles [71], and hybrid nanoparticles [7, 34, 56], (ii) positively charged polymeric nanospheres by using chitosan [72] and Eudragit® RL 100 [18] as polymers or positively charged hybrid nanoparticles prepared from lipids as the stearylamine [5], (iii) stealth polymeric nanospheres [17, 27] and stealth hybrid particles [4–8], and (iv) targeted cancer hybrid particles [7, 59].

#### **4.1 Pharmacokinetic parameters**

An approach to the pharmacokinetic aspects of the particles prepared via nanoprecipitation is made from the reported studies where carrier dispersions were administered by the intravenous, oral, and intranasal routes to animal models as Sprague-Dawley rats, Wistar rats, and BALB/c mice (**Table 2**). First, the slowrelease patterns previously discussed appear to be maintained in the *in vivo* behavior, i.e., nanoparticles extend in some way the drug delivery regardless of the administration route and the carrier properties. Thus, mean residence times (MRT) in the systemic circulation between 1.2 and 20 folds higher than that for the free drug and elimination half-lives between 5 and 10 folds higher than free drug are achieved. Likewise, larger values of area under curve (AUC) are reported which, provided that the amount of drug that is released allows the therapeutic dose required, are attractive for treating chronic diseases where less frequent dosing regimens are convenient.

A general view depending on the administration route (**Figure 5**, where solid and dashed lines correspond to carriers and free-drug plasma profiles, respectively) shows that polymeric particles orally administered increase the Tmax, Cmax, and AUC0-t values compared with free drugs administered in suspension or, as in the case of lipid nanoparticles, with an intravenously administered solution of the drug. The slow drug release behavior characteristic of lipid particles, where Tmax is abruptly reached after 20 h of administration is interesting. On the other hand, although Tmax, Cmax and, AUC are increased when using hybrid particles, it must be noted that drug could be rapidly or slowly delivered to the serum which might be related to the location of the active molecule into the particle. For example, if the active molecule is located at the lipid shell surrounding the polymeric core, the drug might be easily released; on the contrary, if the active molecule locates at the polymeric core, more extended drug release behaviors could be obtained. Zhu et al. [4] and Godara et al. [5] demonstrate the usefulness of the lipid layer covering the polymeric core in the hybrid particles to prolong the circulation time of the particles. Probably, the lipid shell restricts the plasma protein adsorption reducing the opsonization phenomena. Moreover, the modifications of the particle with cholate enhance the drug absorption by the oral route. Likewise, developments as that of mycophenolate particles containing quercetin, where the antioxidant activity of

**Active molecule**

**113**

**carrier**

**Polymeric** 

PNS PNS PNS

PNS PNS **Lipid** 

SLN (P80)

SLN SLN **Hybrid** 

HNP HNP (P85) HNP (Tf-P85)

HNP HNP HNP HNP HNP *PNS: polymeric nanospheres;* *half-life; Cmax: maximum concentration;*

*coglycolic acid), SH: shellac, nr.: non-reported*

**Table 2.**

*Summary of the* 

*pharmacokinetic*

 *parameters*

 *reported in research works on* 

*nanoparticles*

 *prepared by the* 

*nanoprecipitation*

 *technique.*

 *SLN: solid lipid nanoparticles;*

 *AUC 0-t: area under the curve of a plasma concentration*

 *data.*

 *NLC:* 

*nanostructured*

 *lipid carriers; HNP: hybrid nanoparticles;*

 *versus time profile; MRT: mean residence time; P80: polysorbate 80; PEG: poly(ethylene*

Docetaxel Mycophenolate

Mycophenolate

 + quercetin

Quercetin Paclitaxel

Oral

Oral

Wistar rats

Sprague-Dawley

 rats

 25 mg/kg

10 mg/kg

 *Tf: transferrin; P85: Pluronic 85; Tmax: time taken to reach peak plasma concentration;*

 6.0

 6.8–7.6 μg/mL

 nr.

 nr.

 *glycol), PLGA: poly (D,L-lactic-*

 nr.

 [5]

 *t 1/2:*

 1.0

 8.8 μg/mL

 33.3 μg h/mL

 nr.

 3.4

 [32]

Parenteral

Oral

Sprague-Dawley

 rats 25 mg/kg equivalent

to MPA and QC

 BALB/c female mice

 10 mg/kg

 nr. nr.

nr.

 1.2 μg/mL

 35.9 μg h/mL

 46.0

 28.4

 1.2 μg/mL

 27.4 μg h/mL

 34.0

 24.1

 [14]

 8.0 μg/mL

 198.5 μg h/mL

 34.9

 25.7

 [8]

Doxorubicin

 Parenteral

Male Sprague-

20 mg/kg

nr.

nr.

nr.

 19.9 μg/mL

 107.1 μg h/mL

 11.43

 8.0

 17.8 μg/mL

 75.4 μg h/mL

 10.82

 7.1

 17.5 μg/mL

 62.9 μg h/mL

 9.5

 6.4

 [4]

> Dawley rats

**nanoparticles**

Amphotericin

 B

Oral

Sprague-Dawley

 rats

 3.6 mg/kg

 24.0

 1.1 μg/mL

 27.9 μg h/mL

 nr.

 15.9

 [21]

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

Nevirapine

Parenteral

 Wistar rats

20 mg/kg

4.0

4.0

 5.8 μg/g

 1.1 μg h/g

 8.6

 7.2

 9.3 μg/g

 2.9 μg h/g

 17.4

 27.6

 [13]

**nanoparticles**

Paclitaxel Diazepam

Oral Intranasal

Sprague-Dawley

 rats

 0.2–0.25 mg/kg

 2.0

 2.4%/g

 13.9% h/g

 nr.

 nr.

 [57]

Wistar rats

10 mg/kg

 6.0

 3.6–4.2 μg/mL

 nr.

 nr.

 nr.

 [5]

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

(PEG-PLGA:SH)

(PEG-PLGA)

Itraconazole

Ketamine

Parenteral

Parenteral

 Male C57BL/6 J mice

Sprague-Dawley

 rats

 5 mg/kg

 1 mg/kg

 nr. nr.

 19.6 μg/mL

 86.8 μg h/mL

 nr.

 79.7

 20.1 μg/mL

 88.6 μg h/mL

 nr.

 103.1

 [27]

 7.7

 nr.

 1.2 μg h/mL

 12.4

 nr.

 [22]

**nanoparticles**

**—**

**Active ingredient**

**Route of**

**Animal model**

 **Equivalent dose of**

**Tmax**

**Cmax**

**AUC0-t**

**MRT**

**t 1/2**

**Reference**

**(h)**

**(h)**

**(h)**

**active molecule**

**administration**


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

**Table** 

To provide a therapeutic response, nanoparticles must overcome these physiological clearance mechanisms and distributional barriers. The objective is to guarantee a high mean residence time for the carriers in the systemic circulation while their drug release delivery is modulated. Some alternatives in this way include the development of particles exhibiting sizes less than 100 nm or a positive surface charge. Stealth particles by using nonionic polymers or mimic the outer surface of blood cells by locating mixtures of phospholipids, cholesterol, sphingomyelin, and ganglioside molecules on the particle surface have also been proposed, and the modification of the particle surface with specific ligands appears as the best strategy

Regarding the carriers prepared by nanoprecipitation, among the reported developments of particles that could theoretically allow them a better *in vivo* performance are: (i) particle sizes lesser than 100 nm for polymeric nanospheres [25, 26, 34], solid lipid nanoparticles [71], and hybrid nanoparticles [7, 34, 56], (ii) positively charged polymeric nanospheres by using chitosan [72] and Eudragit® RL 100 [18] as polymers or positively charged hybrid nanoparticles prepared from lipids as the stearylamine [5], (iii) stealth polymeric nanospheres [17, 27] and stealth hybrid particles [4–8], and (iv) targeted cancer hybrid particles [7, 59].

An approach to the pharmacokinetic aspects of the particles prepared via nanoprecipitation is made from the reported studies where carrier dispersions were administered by the intravenous, oral, and intranasal routes to animal models as Sprague-Dawley rats, Wistar rats, and BALB/c mice (**Table 2**). First, the slowrelease patterns previously discussed appear to be maintained in the *in vivo* behavior, i.e., nanoparticles extend in some way the drug delivery regardless of the administration route and the carrier properties. Thus, mean residence times (MRT) in the systemic circulation between 1.2 and 20 folds higher than that for the free drug and elimination half-lives between 5 and 10 folds higher than free drug are achieved. Likewise, larger values of area under curve (AUC) are reported which, provided that the amount of drug that is released allows the therapeutic dose required, are attractive for treating chronic diseases where less frequent dosing

A general view depending on the administration route (**Figure 5**, where solid and dashed lines correspond to carriers and free-drug plasma profiles, respectively) shows that polymeric particles orally administered increase the Tmax, Cmax, and AUC0-t values compared with free drugs administered in suspension or, as in the case of lipid nanoparticles, with an intravenously administered solution of the drug. The slow drug release behavior characteristic of lipid particles, where Tmax is abruptly reached after 20 h of administration is interesting. On the other hand, although Tmax, Cmax and, AUC are increased when using hybrid particles, it must be noted that drug could be rapidly or slowly delivered to the serum which might be related to the location of the active molecule into the particle. For example, if the active molecule is located at the lipid shell surrounding the polymeric core, the drug might be easily released; on the contrary, if the active molecule locates at the polymeric core, more extended drug release behaviors could be obtained. Zhu et al. [4] and Godara et al. [5] demonstrate the usefulness of the lipid layer covering the polymeric core in the hybrid particles to prolong the circulation time of the particles. Probably, the lipid shell restricts the plasma protein adsorption reducing the opsonization phenomena. Moreover, the modifications of the particle with cholate enhance the drug absorption by the oral route. Likewise, developments as that of mycophenolate particles containing quercetin, where the antioxidant activity of

for the target delivery of active substances up to now [61, 70].

*Nano- and Microencapsulation - Techniques and Applications*

**4.1 Pharmacokinetic parameters**

regimens are convenient.

**112**

**<sup>2.</sup>** *Summary of the pharmacokinetic parameters reported in research works on nanoparticles prepared by the nanoprecipitation technique.*

delivery from the hybrid particles continues to be detected 72 h after the adminis-

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

**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

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

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,

*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:*

prepared from a hyaluronan-modified polymer, and administered via

tration with AUC0-t values around 3.6 times higher than free drug.

**4.2 Biodistribution**

happening.

**Figure 6.**

*quercetin).*

**115**

statements that lead to misinterpretations.

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

#### **Figure 5.**

*General behaviors of plasma concentration reported for polymeric nanoparticles (A and B), lipid nanoparticles (C and D), and hybrid nanoparticles (E and F). Oral administration (A, C, and E); intravenous 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; tf: transferrin).*

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].

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

delivery from the hybrid particles continues to be detected 72 h after the administration with AUC0-t values around 3.6 times higher than free drug.
