**2. Physicochemical fundamentals of the nanoprecipitation technique**

To prepare nanoparticles via the nanoprecipitation technique, two miscible solvents are used, one of them being a good solvent (usually an organic solvent as ethanol, isopropanol, or acetone) and the other one acting as a non-solvent for the material that will form the particle (i.e., polymer, lipid, etc.), e.g., water. In general, as shown in **Figure 1**, the nanoprecipitation procedure requires the preparation of an organic phase and a non-solvent phase, frequently named aqueous phase, both guaranteeing the total solubility of all the starting materials. In this sense, the organic phase could contain polymers or solid and liquid lipids, surfactants of low HLB value, and active molecules dissolved in a solvent or mixture of organic solvents. The solubility in the solvent of the active molecule to be entrapped is one of the factors limiting the drug loading of the particles. On its part, the non-solvent phase mainly includes stabilizing agents solubilized in water, which allows the particle formation and the physical stability of the system [2]. Nonetheless, the preparation of particles without stabilizing agents has been reported. In these cases, for example, isoprenoid chains are linked to the active molecule making it easy to form the nanoparticle because of its amphiphilic nature [49].

Nanoparticles are spontaneously formed when the organic phase is dropped or added in a one-shot to the aqueous phase. Indeed, nanoprecipitation is a robust process and operational conditions used to prepare the particles do not seem to have a marked influence on the obtained particle size and polydispersity index. On the contrary, the variables linked to the used formulation appear as determinants of the characteristics of the nanosized system, mainly the nature and concentration of the starting materials [47]. This might be closely related to the proposed mechanisms to

#### **Figure 1.**

nevirapine [13]), immune suppressants (mycophenolate [14]), anti-inflammatories

antihypertensives (nimodipine [28] and atenolol [29]), vitamins or their precursors (β-carotene [30] and vitamin E [31]), and antioxidants (quercetin [14, 32]). Likewise, although in a much smaller number, hydrophilic active molecules such alendronate [33], N-acetylcysteine [34], and calcein [35], have been investigated. Moreover, natural extracts such as Brazilian red propolis extract [36] and essential

Practical matters as the possibility to use solvents of low toxic potential, the simple procedure, the low energy consumption required, and the feasibility to obtain particles from diverse compositions are also highlighted among the pros of the nanoprecipitation method when carriers at the submicron and nanometric scales are intended [3, 38]. Most of the nanoparticulated drug delivery systems reported as prepared by nanoprecipitation have been developed by using the physicochemical principles governing this technique, primarily those who underpin the precipitation of materials from the mixture of a solvent/non-solvent for the involved material. They include in their majority, polymer, lipid, and hybrid nanoparticles; therefore, this review will be fundamentally focused on them. Nevertheless, some interesting developments of nanoparticles prepared by nanoprecipitation have been reported as well. For example, Arizaga et al. [39] and Villela et al. [40] entrapped magnetic nanoparticles inside polymeric particles, Fan et al. [41] designed spatially controlled release multistage carriers via the complexation of dendrimers with gelatin, and Allen et al. [35] entrapped hydrophobic and hydrophilic active molecules into polymersomes. Likewise, modifications to facilitate the

(clobetasol [15], fluticasone propionate [16], dexamethasone [17, 18], and diclofenac [19]), antimicrobial and antifungal agents (polymyxin B [20], amphotericin B [21], itraconazole [22], and linezolid [23]), antihyperlipidemics

*Nano- and Microencapsulation - Techniques and Applications*

(fenofibrate [24, 25]), anesthetics (tetracaine [26] and ketamine [27]),

oils [37] have also been incorporated into polymeric nanoparticles.

industrial scaling-up of the preparation process have been investigated by

Based on the above, the present chapter revises the generalities of the nanoprecipitation technique such as the physicochemical aspects involved, some of the starting materials used to obtain polymer, lipid, and hybrid nanoparticles, and their characteristics. Then, the pharmacokinetic behaviors, safety evaluations, and

efficacy tests are analyzed. It is our interest to provide readers with a

continuous and scalable processes for the synthesis of the particles.

solvents and stabilizing agents.

**102**

Charcosset et al. [42] and D'Oria et al. [43] who developed procedures based on the use of a membrane contactor. On its part, Valente et al. [44] and Tao et al. [45] propose controllable mixing devices such as microfluidic mixer systems that allow

Reviews published to date dealing with the nanoprecipitation technique provide valuable information from different standpoints. For example, regarding the role of the obtained particles as drug delivery systems and their applications in medicine, Martínez et al. [46] highlighted their ability for carrying either natural products or actives obtained via chemical synthesis. On the other hand, with respect to the study of nanoprecipitation as a physicochemical process, Mora-Huertas et al. [47] revised the influence of both the formulation and the work conditions used to prepare nanoparticles. In this case, data available in scientific reports supplemented with a systematic study of the nanoprecipitation method led to an approximation to the particle formation mechanisms and identify the factors influencing the particle properties. Recently, Saad and Prud'homme [48] deepened on the physicochemical principles of the nanoparticle formation when amphiphilic block copolymers are used as stabilizing agents (named flash nanoprecipitation). They focused on the key variables determining the nucleation and growth phenomena related to the particle formation, particularly the supersaturation condition, the mixing step, and the used

*General view of the preparation of polymer, lipid, and hybrid particles by nanoprecipitation summarizing the work conditions commonly reported (PNC: polymeric nanocapsules; PNS: polymeric nanospheres; SLN: solid lipid nanoparticles; HNP: hybrid nanoparticles; RT: room temperature).*

form the particles by the nanoprecipitation technique. As a basic premise, only specific polymer/solvent/non-solvent ratios, where the polymer is in low concentrations and the solvent is in low proportion with respect to the nonsolvent, lead to particles at the nano- and submicron levels [50]. Thus, on the one hand, the mechanical approach states that when the phases are mixed, the organic phase is successively broken as drops within the aqueous phase due to the interfacial turbulence and thermal inequalities in the system because of the mutual miscibility between the solvent and the non-solvent and their different interfacial tensions (Gibbs-Marangoni effect) [51]. This fragmentation process will occur until the difference in interfacial tensions is minimized and the organic solvent migrates from the drops having a submicron size, which creates a non-solubility condition for the material causing the precipitation of the particles. On the other hand, a mechanism based on the chemical instability of the system has also been proposed ("ouzo effect"). In this case, when the phases are mixed, supersaturation of the molecules forming the particles is caused as the organic solvent migrates toward the aqueous phase, allowing the formation of "protoparticles" that grow following the classical nucleation-and-growth process [48, 52, 53]. It seems that depending on the formulation to prepare the nanoparticles, one of those mechanisms could predominate during the nanoprecipitation, and consequently, the adequate work conditions should be defined for allowing the spontaneous formation of submicron or nanoscale particle sizes exhibiting the smallest polydispersity indexes. Difficulties associated with the standardization of the procedure of nanoprecipitation result in the polymer aggregation yielding wide and asymmetric particle size distributions. For example, polymer aggregates are evidenced because of a concentrated organic phase, high organic phase ratio, low concentration of stabilizing agent, and poor mixing of the phases [47].

active molecules and lipophilic surfactants. On its part, the structure of the nanocapsules is proposed as an oil core surrounded by a polymeric shell. Approximately, 90% of the research works published on the preparation of polymeric nanoparticles via the nanoprecipitation technique are devoted to the obtention of

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

entrapment efficiency of the active molecules [58].

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

for polymeric nanoparticles).

water [16] or phosphate buffer [27, 59].

**3.1 Starting materials**

**105**

With respect to lipid nanoparticles, both solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been investigated, although almost 85% of the research works deal with SLN. Lipids nanoparticles are composed of a lipid matrix that is supposed to be surrounded by stabilizing agents. In the case of SLN, the lipid matrix is exclusively formed by solid lipids, while the lipid matrix of NLC is composed of solid and liquid lipids. It seems that the liquid lipid in NLC favors the

Regarding hybrid nanoparticles, they are made from both polymers chemically modified with lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy polyethylene glycol—DSPE-PEG) or the physical mixture between polymers and lipid components (e.g., PLGA and soy lecithin). Nevertheless, in the latter case, although the qualitative recipe is similar to that for polymeric particles, higher concentrations of solid lipids are used to prepare hybrid particles (i.e., lipid concentrations range between 20 and 50% for hybrid particles and between 1 and 5%

**Figure 2** shows in a comparative way the reported starting materials used to prepare the different types of particles via nanoprecipitation. As can be seen, PLGA, PCL, and PLA are the most used polymers to prepare polymeric nanoparticles and when these polymers are chemically modified with, for example, PEG, stealth polymeric nanoparticles can be obtained [16, 17, 27]. Surfactants of low HLB value, e.g., soy phospholipids, could be added to the organic phase for facilitating the particle formation [19, 31] and, if nanocapsules are intended, castor oil, sesame oil, caprylic capric triglycerides, and caprylic capric triglyceride PEG-4 esters are part of the organic phase. Acetone appears as the preferred organic solvent of the organic phase and the non-solvent is water. Thus, the aqueous phases are solutions of stabilizing agents as poloxamer, polyvinyl alcohol, and polysorbate 80, which prevent the particle aggregation phenomena. Likewise, aqueous phases can only be

To prepare SLN, fatty acids and their glyceryl esters are frequently used as lipids

Hybrid nanoparticles were designed to integrate the favorable characteristics of both polymeric and lipid systems and overcome their drawbacks [34]. These systems are proposed as an inner polymeric core surrounded by a lipid shell [60]. To obtain it, as is the rule in nanoprecipitation, organic and aqueous phases are designed so that solubility of the starting materials is guaranteed. As one of the strategies to prepare hybrid particles is employing polymers chemically modified with lipids (e.g., DSPE-PEG-NH2), they behave as amphiphilic compounds that

(e.g., glyceryl monostearate, tristearate, behenate, and dilaurate); they are dispersed at a molecular level in organic solvents such as acetone and ethanol for obtaining the organic phase. As in the case of polymeric nanoparticles, phospholipids can be used to favor the particle formation and, to make NLC, liquid lipids as caprylic capric triglycerides are also dissolved in the organic phase. With respect to the non-solvent phase, aqueous solutions of stabilizing agents of varied nature are reported. Among them, surfactants as those mentioned for polymeric nanoparticles, proteins such as sodium caseinate and lactoferrin, and osmotic active compounds

such as glucose and magnesium sulfate have been investigated.

nanospheres.

It is worth clarifying that in-depth studies on how particles are formed via the nanoprecipitation technique and the operating variables determining their characteristics have been carried out by using polymeric systems. Regarding lipid nanoparticles, only systematic studies have been reported to aid in understanding the variables that influence the preparation of the carriers; among them, the contributions of Martínez-Acevedo et al. [54] on the influence of the used recipe and Noriega-Pelaez [55] on the study of the particle preparation process are highlighted. Concerning the hybrid particles, research works to date have focused primarily on the impact of the starting materials on the particle characteristics [5, 9, 12, 23, 56].

Once the nanocarriers are formed, the particle dispersions are further processed to purified and concentrate them. To this end, rotary evaporation [14, 18, 22–24, 31, 55] and centrifugation [5, 6, 12, 13, 15, 16, 23, 25, 27–29, 36, 56, 57] are the most used methods; however, filtration [4, 6, 16, 18, 24] and dialysis [7–9, 21, 27, 34] have also been reported. Likewise, lyophilization is the preferred technique to stabilize the nanoparticles, although the storage to low temperatures has been used to preserve the aqueous dispersions [5, 8, 9, 13, 21, 29].

## **3. Starting materials and general characteristics of particles prepared by nanoprecipitation**

As mentioned above, although different types of carriers intended for pharmaceutical applications can be prepared via nanoprecipitation, only polymer, lipid, and hybrid particles were chosen to be analyzed in detail because of the amount of reported research works to date. Polymeric nanoparticles are classified as polymeric nanospheres (PNS) and polymeric nanocapsules (PNC). The first ones correspond to a solid matrix conformed by the used polymers and other components, e.g.,

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

active molecules and lipophilic surfactants. On its part, the structure of the nanocapsules is proposed as an oil core surrounded by a polymeric shell. Approximately, 90% of the research works published on the preparation of polymeric nanoparticles via the nanoprecipitation technique are devoted to the obtention of nanospheres.

With respect to lipid nanoparticles, both solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been investigated, although almost 85% of the research works deal with SLN. Lipids nanoparticles are composed of a lipid matrix that is supposed to be surrounded by stabilizing agents. In the case of SLN, the lipid matrix is exclusively formed by solid lipids, while the lipid matrix of NLC is composed of solid and liquid lipids. It seems that the liquid lipid in NLC favors the entrapment efficiency of the active molecules [58].

Regarding hybrid nanoparticles, they are made from both polymers chemically modified with lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy polyethylene glycol—DSPE-PEG) or the physical mixture between polymers and lipid components (e.g., PLGA and soy lecithin). Nevertheless, in the latter case, although the qualitative recipe is similar to that for polymeric particles, higher concentrations of solid lipids are used to prepare hybrid particles (i.e., lipid concentrations range between 20 and 50% for hybrid particles and between 1 and 5% for polymeric nanoparticles).

#### **3.1 Starting materials**

form the particles by the nanoprecipitation technique. As a basic premise, only specific polymer/solvent/non-solvent ratios, where the polymer is in low concentrations and the solvent is in low proportion with respect to the nonsolvent, lead to particles at the nano- and submicron levels [50]. Thus, on the one hand, the mechanical approach states that when the phases are mixed, the organic phase is successively broken as drops within the aqueous phase due to the interfacial turbulence and thermal inequalities in the system because of the mutual miscibility between the solvent and the non-solvent and their different interfacial tensions (Gibbs-Marangoni effect) [51]. This fragmentation process will occur until the difference in interfacial tensions is minimized and the organic solvent migrates from the drops having a submicron size, which creates a non-solubility condition for the material causing the precipitation of the particles. On the other hand, a mechanism based on the chemical instability of the system has also been proposed ("ouzo effect"). In this case, when the phases are mixed, supersaturation of the molecules forming the particles is caused as the organic solvent migrates toward the aqueous phase, allowing the formation of "protoparticles" that grow following the classical nucleation-and-growth process [48, 52, 53]. It seems that depending on the formulation to prepare the nanoparticles, one of those mechanisms could predominate during the nanoprecipitation, and consequently, the adequate work conditions should be defined for allowing the spontaneous formation of submicron or nanoscale particle sizes exhibiting the smallest polydispersity indexes. Difficulties associated with the standardization of the procedure of nanoprecipitation result in the polymer aggregation yielding wide and asymmetric particle size distributions. For example, polymer aggregates are evidenced because of a concentrated organic phase, high organic phase ratio, low concentration of stabilizing agent, and poor

*Nano- and Microencapsulation - Techniques and Applications*

It is worth clarifying that in-depth studies on how particles are formed via the nanoprecipitation technique and the operating variables determining their characteristics have been carried out by using polymeric systems. Regarding lipid nanoparticles, only systematic studies have been reported to aid in understanding the variables that influence the preparation of the carriers; among them, the contributions of Martínez-Acevedo et al. [54] on the influence of the used recipe and Noriega-Pelaez [55] on the study of the particle preparation process are highlighted. Concerning the hybrid particles, research works to date have focused primarily on the impact of the starting materials on the particle characteristics [5, 9, 12, 23, 56]. Once the nanocarriers are formed, the particle dispersions are further processed to purified and concentrate them. To this end, rotary evaporation [14, 18, 22–24, 31, 55] and centrifugation [5, 6, 12, 13, 15, 16, 23, 25, 27–29, 36, 56, 57] are the most used methods; however, filtration [4, 6, 16, 18, 24] and dialysis [7–9, 21, 27, 34] have also been reported. Likewise, lyophilization is the preferred technique to stabilize the nanoparticles, although the storage to low temperatures has been used to preserve

**3. Starting materials and general characteristics of particles prepared by**

As mentioned above, although different types of carriers intended for pharmaceutical applications can be prepared via nanoprecipitation, only polymer, lipid, and hybrid particles were chosen to be analyzed in detail because of the amount of reported research works to date. Polymeric nanoparticles are classified as polymeric nanospheres (PNS) and polymeric nanocapsules (PNC). The first ones correspond to a solid matrix conformed by the used polymers and other components, e.g.,

mixing of the phases [47].

the aqueous dispersions [5, 8, 9, 13, 21, 29].

**nanoprecipitation**

**104**

**Figure 2** shows in a comparative way the reported starting materials used to prepare the different types of particles via nanoprecipitation. As can be seen, PLGA, PCL, and PLA are the most used polymers to prepare polymeric nanoparticles and when these polymers are chemically modified with, for example, PEG, stealth polymeric nanoparticles can be obtained [16, 17, 27]. Surfactants of low HLB value, e.g., soy phospholipids, could be added to the organic phase for facilitating the particle formation [19, 31] and, if nanocapsules are intended, castor oil, sesame oil, caprylic capric triglycerides, and caprylic capric triglyceride PEG-4 esters are part of the organic phase. Acetone appears as the preferred organic solvent of the organic phase and the non-solvent is water. Thus, the aqueous phases are solutions of stabilizing agents as poloxamer, polyvinyl alcohol, and polysorbate 80, which prevent the particle aggregation phenomena. Likewise, aqueous phases can only be water [16] or phosphate buffer [27, 59].

To prepare SLN, fatty acids and their glyceryl esters are frequently used as lipids (e.g., glyceryl monostearate, tristearate, behenate, and dilaurate); they are dispersed at a molecular level in organic solvents such as acetone and ethanol for obtaining the organic phase. As in the case of polymeric nanoparticles, phospholipids can be used to favor the particle formation and, to make NLC, liquid lipids as caprylic capric triglycerides are also dissolved in the organic phase. With respect to the non-solvent phase, aqueous solutions of stabilizing agents of varied nature are reported. Among them, surfactants as those mentioned for polymeric nanoparticles, proteins such as sodium caseinate and lactoferrin, and osmotic active compounds such as glucose and magnesium sulfate have been investigated.

Hybrid nanoparticles were designed to integrate the favorable characteristics of both polymeric and lipid systems and overcome their drawbacks [34]. These systems are proposed as an inner polymeric core surrounded by a lipid shell [60]. To obtain it, as is the rule in nanoprecipitation, organic and aqueous phases are designed so that solubility of the starting materials is guaranteed. As one of the strategies to prepare hybrid particles is employing polymers chemically modified with lipids (e.g., DSPE-PEG-NH2), they behave as amphiphilic compounds that
