**3.2 General characteristics of the particles**

Regardless of the type of particle, the shape, the particle size, the drug entrapment, and loading, and the zeta potential are among the crucial properties determining their pharmaceutical performance [61].

#### *3.2.1 Shape*

Polymeric and hybrid particles prepared by using the nanoprecipitation technique exhibit spherical shape as it is revealed by techniques of microscopy, mainly scanning electron (SEM), transmission electron (TEM), atomic force (AFM), and field emission scanning microscopies (FESEM). To investigate the shape of lipid nanoparticles in most cases, the same techniques were used, and spherical shapes were also reported. However, lipids might be melted during the sample examination destroying their native characteristics; consequently, controversial results could be obtained. For example, platelet shapes for SLN [62, 63] and structures with the liquid lipid located on the surface of the particles in the form of plates for NLC [64, 65] have been reported by using cryo-TEM and freeze-fracture TEM. Nevertheless, Dong et al. [24] report spherical shape from the analysis of SLN by using Cryo-FESEM.

#### *3.2.2 Particle size*

In general, the mean sizes, usually measured by dynamic light scattering, vary between less than 100 and 300 nm with PDI values below 0.4 (**Figure 3A** and **B**). It seems that polymeric nanocapsules and specially hybrid nanoparticles are the smallest; perhaps, any type of structural arrangement among the lipids and polymers could favor a better consolidation of the particle. With respect to lipid carriers, the platelet shapes as the lipids crystallize inside the particle could explain their high polydispersity [66].

#### *3.2.3 Drug entrapment efficiency*

Regarding the entrapment efficiency (**Figure 3C**), clear differences are identified among the carriers. Thus, polymeric nanocapsules entrap almost the totality of the active molecule in contrast with 40% attained by the SNL. As remarked by Westesen et al. [67], Pardeike et al. [68], and Weber et al. [69], when preparing SLN the solidification and the progressive crystallization of the lipid in more stable forms could lead the expulsion of the active substances whether during the particle formation or its consolidation. This results in eventual instabilities of the particle dispersions and, as evidenced in this case, low entrapment efficiency and loading of active molecules. On the other hand, as shown in **Figure 3D**, the best results of drug loading are reported for hybrid nanoparticles; active molecules could be located both in the polymeric core and the lipid layer of the particles maximizing their loading efficiency.

#### *3.2.4 Physicochemical stability*

Stability of the particle dispersions has been investigated by using refrigerated storage [6, 8, 13, 15, 21], room temperature at 25°C [8, 13, 14, 21, 57], and accelerated conditions varying between 35 and 40°C [8, 9, 11, 20, 27]. Particle size, PDI, and zeta potential are usually followed during the storage time, and the physical integrity of the dispersions is observed for up to 6 months. This good stability is expected for these nanosystems considering their colloidal nature and the absolute zeta potential values which are estimated varying between 15 and 40 mV.

could form part of any of the phases according to their solubility. On the contrary, if a physical mixture of polymer and lipid is used, they are dissolved in the organic phase. Unlike polymeric and lipid particles, acetonitrile is reported as the most used organic solvent for preparing hybrid nanoparticles. Another interesting matter of the recipe to prepare hybrid nanoparticles is the versatile composition of the aqueous phase. In this sense, for example, lecithin and cholesterol can be dissolved in ethanol and then incorporated in the aqueous phase that could contain surfactants such as polysorbate and poloxamer. Likewise, dispersions of surfactants, proteins,

*Starting materials reported as used to prepare the organic and aqueous phases for obtaining polymer, lipid, and hybrid nanoparticles by the nanoprecipitation technique. Number of times reported for each starting material considering a total of 18, 11, and 13 research works for polymer, lipid, and hybrid particles, respectively [PLGA: poly(lactic-co-glycolic acid); PCL: polycaprolactone; PLA: poly(lactic acid); PEG: polyethylene glycol; HPMC: hydroxypropyl methylcellulose; DMSO: dimethyl sulfoxide; CAP: cellulose acetate phthalate; EtOH: ethanol; ACN: acetonitrile; MetOH: methanol; DMF: dimethylformamide; DCM: dichloromethane; THF: tetrahydrofuran; MEK: methyl ethyl ketone; H-b-pBG: hyaluronan poly(γ-benzyl-L-glutamate); PBS: phosphate-buffered saline; SDS: sodium dodecyl sulfate; GMS: glycerol monostearate; TPGS: tocopheryl polyethylene glycol succinate; PVA: polyvinyl alcohol; Tf-PEG-OA: transferrin-poly(ethylene glycol)-oleic acid; Tf: transferrin; HSA: human serum albumin; DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-*

or buffers were tested as the aqueous phase.

*Nano- and Microencapsulation - Techniques and Applications*

*N-[methoxy(polyethylene glycol)]].*

**Figure 2.**

**106**

**Nanoparticle Drug release study Reference**

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

pH 7.4 PBS with 0.5% w/v polysorbate

PNS Dialysis 14 kDa pH 7.4 PBS Shaker 100 rpm 37 2°C [26] PNS Dialysis 10 kDa pH 7.4 PBS 100 shakes/min 37°C [27]

> 1% wt SDS solution

0.2% wt SDS solution

SLN Dialysis 50 kDa pH 7.4 PBS Shaker 50 rpm 37 2°C [24] SLN Dialysis 14 kDa pH 7.4 PBS Shaker 100 rpm 37 2°C [26]

HNP Dialysis 3.5 kDa pH 7.4 PBS Gentle stirring 37 °C [33]

(0.1 M)

0.1% (v/v) DMF

HNP Dialysis 12 kDa pH 7.4 PBS Shaker 100 rpm 37 2°C [4] HNP Dialysis 3.5 kDa pH 7.4 PBS 100 rpm 37 °C [6]

> 0.5% polysorbate 80

> > 5.5 PBS

HNP Dialysis 12 kDa pH 7.4 PBS nr. 37 °C [14]

water, and HCl 0.1 M solution

**Polymeric nanoparticles**

PNC Directly added at the

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

PNS Franz diffusion cells

PNS Directly added at the

PNS Franz diffusion cells

SLN/NLC Directly added at the

SLN/NLC Directly added at the

SLN Dialysis/USP

**Hybrid nanoparticles**

**109**

**Lipid nanoparticles**

release medium

(sheep nasal mucosa)

release medium/ centrifugation

(dialysis 12–14 kDa)

release medium

release medium/ centrifugation

Apparatus II

HNP Dialysis 12–14 kDa pH 6.8 PBS

HNP Dialysis 10–12 kDa pH 7.4. PBS with

HNP Dialysis 8–14 kDa pH 7.4 PBS with

HNP Dialysis 10 kDa pH 7.4; 6.8 and

HNP Dialysis 100 kDa pH 6.8 PBS,

PNS USP Apparatus I pH 6.8 PBS Mechanical

PNS Dialysis 12–14 kDa pH 7.4 PBS Mechanical

PNS Dialysis pH 7.4 PBS Magnetic

**Method Medium Stirring Operating**

**conditions**

37 2°C [33]

37 1°C [29]

37°C [16]

37°C [17]

32°C [18]

37 2°C [15]

37 2°C [28]

37 2°C [13]

Shaker 90 rpm 37 2°C [12]

150 rpm 37 0.5°C [9]

100 rpm 37°C [56]

100 rpm 37 1°C [8]

nr. nr. [32]

pH 6.8 PBS Magnetic 25 rpm 37 2°C [19]

pH 6.4 PBS 100 rpm 37 0.5°C [57]

100 rpm

100 rpm

Eppendorf thermomixer, gentle stirring

100 rpm

600 rpm

Shaker 60 strokes/min

Shaker 60 strokes/min

100 rpm

pH 7.5 PBS Magnetic

pH 7.4 PBS Mechanical

**Figure 3.**

*General behaviors of particle size (A), polydispersity index (B), drug entrapment efficiency (C), and drug loading (D) for polymeric nanospheres (PNS), polymeric nanocapsules (PNC), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and hybrid nanoparticles (HNP).*

#### *3.2.5 Release behavior*

**Table 1** summarizes the reported work conditions used to carry out the release tests. No matter what type of particles, dialysis is the most used technique to investigate their drug release behaviors, usually at 37°C in PBS media of pH 6.8 or 7.4. Comparisons of drug release data is risked because significant changes in the delivery behaviors are caused by the type of particle and its composition, the nature of the active molecule, and the work conditions associated with the release test, however, worth the risk for gaining a general view.

Thus, even though the mathematical modeling of the drug release data reported for the carriers of interest predicts Higuchi and Korsmeyer-Peppas kinetics, differences in the drug release patterns of polymeric, lipid, and hybrid particles are evidenced (**Figure 4**). In this way, biphasic release behaviors seem to be characteristic when nanoprecipitated polymeric particles, whether nanospheres or nanocapsules, are investigated. In these cases, the equilibrium is reached after 20 or 30 h of begun the study, and drug concentrations varying from 60 to 80% are released. Paclitaxel-loaded PLGA nanoparticles are the exception; in this case, a slow and constant drug release process occurs delivering hardly 40% of the active encapsulated after 60 h. Perhaps, the low entrapment efficiency of this molecule into the carriers makes the diffusion phenomena related to the active molecule delivery (37–70%) difficult.

On its part, the drug release patterns observed when lipid nanoparticles are tested seem to be those where the active molecule has faster delivery (before the *Nanoprecipitation: Applications for Entrapping Active Molecules of Interest in Pharmaceutics DOI: http://dx.doi.org/10.5772/intechopen.93338*


*3.2.5 Release behavior*

**Figure 3.**

delivery (37–70%) difficult.

**108**

**Table 1** summarizes the reported work conditions used to carry out the release

Thus, even though the mathematical modeling of the drug release data reported for the carriers of interest predicts Higuchi and Korsmeyer-Peppas kinetics, differences in the drug release patterns of polymeric, lipid, and hybrid particles are evidenced (**Figure 4**). In this way, biphasic release behaviors seem to be character-

nanocapsules, are investigated. In these cases, the equilibrium is reached after 20 or 30 h of begun the study, and drug concentrations varying from 60 to 80% are released. Paclitaxel-loaded PLGA nanoparticles are the exception; in this case, a slow and constant drug release process occurs delivering hardly 40% of the active encapsulated after 60 h. Perhaps, the low entrapment efficiency of this molecule into the carriers makes the diffusion phenomena related to the active molecule

On its part, the drug release patterns observed when lipid nanoparticles are tested seem to be those where the active molecule has faster delivery (before the

tests. No matter what type of particles, dialysis is the most used technique to investigate their drug release behaviors, usually at 37°C in PBS media of pH 6.8 or 7.4. Comparisons of drug release data is risked because significant changes in the delivery behaviors are caused by the type of particle and its composition, the nature of the active molecule, and the work conditions associated with the release test,

*General behaviors of particle size (A), polydispersity index (B), drug entrapment efficiency (C), and drug loading (D) for polymeric nanospheres (PNS), polymeric nanocapsules (PNC), solid lipid nanoparticles*

istic when nanoprecipitated polymeric particles, whether nanospheres or

however, worth the risk for gaining a general view.

*(SLN), nanostructured lipid carriers (NLC), and hybrid nanoparticles (HNP).*

*Nano- and Microencapsulation - Techniques and Applications*


appear to be more efficient than SLN during the release process. The highest amounts of active molecule that could be encapsulated because of the oil component in the particle structure might have influence. Once again, there are exceptions

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

to the general behavior. In this way, slow-release processes as in the case of

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

availability behaviors, including the pharmacokinetic parameters and the

paramount importance to investigate their applicability in pharmaceutics.

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 bio-

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

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

and that clobetasol propionate was the starting material to prepare the

the particle would difficult its delivery process.

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

to facilitate the drug delivery [9].

urine [70].

**111**

clobetasol and fenofibrate, lead to less than 40% of active molecule released even at 100 h. It is important to keep in mind that fenofibrate has a high logP value (5.2)

nanoparticles. Thus, a high affinity of the active molecules for the lipid matrix of

Hybrid nanoparticles, irrespective of whether the particles are obtained from the mixture of polymers and lipids (**Figure 4E**) or by using chemically modified polymers with lipids (**Figure 4F**), characterize by a very slow release of the active molecule where, for example, some carriers deliver above 90% of the drug after 50 h of started the test. It should be noted that in this case, the data are reported twice the set time for the other carriers. For some active molecules such as methotrexate, N-acetylcysteine, psoralen, quercetin, and paclitaxel, the prolonged drug release could be related to the uniform distribution presumed for the drug into the matrix and the core-shell structure of the particle, which difficult the diffusion of the drug toward the release medium [9]. Likewise, the hydrolysis and erosion processes of the polymeric core could be hindered by the lipid layer surrounding the polymeric core [34] or, perhaps, the hydrophobic interactions of the active molecule with the polymer might result of relevance for the drug release [5]. These effects offset, for example, the favorable solubility gained because of the precipitation of amorphous active during the preparation of the particles, which is expected

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

#### **Table 1.**

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

#### **Figure 4.**

*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).*

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

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

appear to be more efficient than SLN during the release process. The highest amounts of active molecule that could be encapsulated because of the oil component in the particle structure might have influence. Once again, there are exceptions to the general behavior. In this way, slow-release processes as in the case of clobetasol and fenofibrate, lead to less than 40% of active molecule released even at 100 h. It is important to keep in mind that fenofibrate has a high logP value (5.2) and that clobetasol propionate was the starting material to prepare the nanoparticles. Thus, a high affinity of the active molecules for the lipid matrix of the particle would difficult its delivery process.

Hybrid nanoparticles, irrespective of whether the particles are obtained from the mixture of polymers and lipids (**Figure 4E**) or by using chemically modified polymers with lipids (**Figure 4F**), characterize by a very slow release of the active molecule where, for example, some carriers deliver above 90% of the drug after 50 h of started the test. It should be noted that in this case, the data are reported twice the set time for the other carriers. For some active molecules such as methotrexate, N-acetylcysteine, psoralen, quercetin, and paclitaxel, the prolonged drug release could be related to the uniform distribution presumed for the drug into the matrix and the core-shell structure of the particle, which difficult the diffusion of the drug toward the release medium [9]. Likewise, the hydrolysis and erosion processes of the polymeric core could be hindered by the lipid layer surrounding the polymeric core [34] or, perhaps, the hydrophobic interactions of the active molecule with the polymer might result of relevance for the drug release [5]. These effects offset, for example, the favorable solubility gained because of the precipitation of amorphous active during the preparation of the particles, which is expected to facilitate the drug delivery [9].
