**2. Influence of nanoparticles preparation parameters on their final properties**

The development of carrier systems involves a lot of study of the variables used in the formulations preparation and their influences on the nanoparticulate properties that reflect on the cellular uptake of nanoparticles, its bioavailability and, consequently, the photodynamic efficiency. The approach to analyze the individual effect and the combinatorial effect (synergistic or antagonistic) of the parameters is usually done by factorial design of experiments, which all levels (experimental domains) of a factor are combined with all levels of the other factors of the experiment [16].

The influence of factors on the characteristics of nanoparticles is intrinsically linked to the production process [16, 20, 21]. Many works have shown the individual effects of some parameters involved in the nanoparticles preparation stage on their properties. However, the influence of a parameter used in a polymeric nanoparticles formulation will not always produce the same response for similar formulations [5].

There are significant challenges to consolidate polymeric nanoformulations in the pharmaceutical market since small changes in the composition of the formulation, for example, the encapsulated drug, can influence the nanoparticles properties, such as the particle size, the surface charge, the residual amount of emulsifier on the surface of the particles, and encapsulation and recovery efficiencies of the nanoparticles [5].

#### **2.1 Size**

The particle size used for the treatment of oncological and non-oncological diseases depends on the administration route and/or the type of diseased tissue. For example, in intravenous administration, the particles must be smaller than 5 μm in order to circulate through the capillaries, however, smaller sizes are necessary for nanoparticles reach the tumor vessels and remain longer in the blood stream [5, 8, 22].

Researches have shown that nanoparticles with sizes smaller than 200 nm have a longer circulation time in the bloodstream due to the reduction of the recognition of the nanoparticle by plasma proteins (opsonin) that signal the reticuloendothelial system to act in the phagocytosis process of the nanoparticles. Remaining longer in the circulatory system, smaller diameter particles could interact more effectively with cell membranes, presenting greater capacity of cellular internalization due to the overexpression of porous in tumor cells membranes, a fact that would result in greater efficiency of nanoparticulate photosensitizers in reducing cell viability through PDT [5, 8, 22].

A highly significant parameter in causing changes in the nanoparticles size was the stirring rate used in the preparation process. The increase of stirring rate leads to smaller sized nanoparticles due to the better dispersion of the organic phase in the aqueous phase, reducing the droplet size of the organic phase and, consequently, the nanoparticle size [20].

Although the stirring rate is considered the main factor responsible for the size reduction of the nanoparticle, in some formulations this parameter is not significant [23]. In the preparation of PLGA-PEG nanoparticles loaded with chloro(5,10,15,20 tetraphenylporphyranato) indium(III) (InTPP – **Figure 2C**), the ethanol percentage in the aqueous phase was the main parameter responsible for size decrease, not the stirring rate [20].

Our group demonstrated that the individual increasing in the ethanol percentage in the aqueous and organic phases contribute to reduce the nanoparticles size. The ethanol present in the aqueous phase causes an increase in viscosity while the addition of ethanol in the organic phase accelerates the separation of phases from

#### *Synergic Influence of Parameters Involved in the Polymeric Nanoparticle Preparation… DOI: http://dx.doi.org/10.5772/intechopen.94176*

the PLGA during the dispersion of the organic phase in the aqueous phase. These effects hinder the coalescence of organic droplets dispersed in the aqueous phase, preventing an increase in the nanoparticles size [16, 20].

Analyzing two preparation methods, the PLGA-PEG nanoparticles loaded with gallium phthalocyanine (GaPc - **Figure 2A**) prepared by the Emulsification-Diffusion Method (EDM) were smaller in size than the nanoparticles prepared by the Emulsification-Evaporation Method (EEM). In the EDM method, the organic solvent is dispersed in the aqueous phase generating droplets that are stabilized by colloidal stabilizing agents, however, the rapid efflux of solvent can cause the formation of aggregates and a population with varying sizes [5].

The aqueous phase temperature is another factor that can positively or negatively influence the nanoparticles size. The increase in temperature reduces the viscosity of the mixture between the organic phase and the aqueous phase, favoring the coalescence of organic solvent drops and consequently increasing the particles size, but the increase in temperature also favors the diffusion of the organic solvent into the aqueous phase, favoring the reduction of particle size. In the EDM method the effect of coalescence is more pronounced, causing the size increase, while in the EEM method the diffusion of the organic solvent to the aqueous phase is more pronounced, decreasing the nanoparticle size [5].

Combinatory effects of two parameters can also be significant for nanoparticle size. The binary effect between changing the method from EEM to EDM and increasing the aqueous phase temperature tends to increase the nanoparticles size [5]. Univariate methods do not allow to identify the combinatory effect that could be important for a determinate nanoparticulate property being necessary the use of factorial design.

The ratio between the photosensitizer mass and the polymer mass is also a parameter that can influence the nanoparticle size. In the study of the preparation of PLGA nanoparticles loaded with three porphyrins (Hex-m-bis-HPP, Hex-m-tris-HPP and m-THPP - **Figure 3A**-**C**, respectively) with different amphiphilicities, [16] the effect of the porphyrin/polymer mass ratio on the nanoparticle size was only significant in m-THPP nanoparticles, which the increase in the mass ratio caused a reduction in size. The low polymer concentration reduces the organic phase viscous resistance against the shear force during the emulsification process, favoring the reduction of the organic phase droplets size dispersed in the aqueous phase and, consequently, reducing the nanoparticles size [16].

Different results have also been reported in the literature, not observing any effect of the photosensitizer mass/polymer mass ratio on the PLGA nanoparticles size loaded with bupivacaine [24], while others have reported that an increase in the proportion decreased the nanoparticles size [25, 26].

Another parameter that can also influence the nanoparticles size is the polyvinyl alcohol (PVA) concentration, which is the most used emulsifier in formulations preparation stage. In some cases, the effect of increasing the PVA concentration on the particle size may be very similar to the effect of the stirring rate. It is known that PVA molecules anchor in the aqueous/organic interface formed during emulsification, causing a decrease in interfacial tension and favoring the mechanical and spatial stabilizations of the organic droplets dispersed in the aqueous phase [27]. In addition, polymeric PVA chains not anchored to the nanosphere surface can increase the aqueous phase viscosity. The reduction in interfacial tension and the increase in viscosity caused by PVA favor a decrease in the nanosphere size [20].

Emulsification time is a factor that may or may not be significant on size. In the preparation of PLGA-PEG nanoparticles containing gallium phthalocyanine, this parameter did not significantly influence the nanoparticles size [5], however some studies have already shown that it can increase or decrease the size [28]. As stated

earlier, each parameter behaves in a particular way according to the parameters used in the preparation of nanoparticles. All these influences on size were summarized in **Figure 5**.

Besides the preparation parameters, the physicochemical properties of photosensitizers can also influence nanoparticles size. The polymeric PLGA-PEG nanoparticles loaded with GaPc or InTBPPc had different results for size distribution. The average diameter of the InTBPPc nanoparticles was 1.3 times greater than for nanoparticles with GaPc. According to the optimized structures designed by the molecular modeling program (**Figure 2A** and **4**), it was shown that InTBPPc has a volume of 1555.32 Å3 (**Table 1**), that is 2.6 times greater than GaPc. This result suggests that the molecular size of the encapsulated phthalocyanine may influence the increase in the size distribution and the nanoparticles diameter [29].

The storage of lyophilized samples at certain temperatures is another factor that can influence the particles size. Studies were conducted with the PLGA-PEG nanoparticles loaded with GaPc (**Figure 6**) to evaluate the influence of the formulation storage at different temperatures on the nanoparticles size. The experiments were carried out with a formulation characterized by presenting 88.9% of the nanoparticles with a diameter smaller than 199.9 nm, an important outcome since particles smaller than 200 nm remain longer in the circulatory system [5]. The experiments suggest that the temperature of 20°C is more suitable for storage purposes of the formulations for 4 weeks, due to the results of less variation in the average diameter of the particles. Even considering the statistical variation of the measurements, there are changes in the PLGA-PEG nanoparticles size that may be associated with the aggregation of the particles during the storage period and the difficulty of disintegrating them during the process of redispersion in water. Such average size variations were greater for lower or higher temperatures than 20°C.

Similar analysis was performed with lyophilized formulations of PLGA-PEG nanoparticulate loaded with InTBPPc (**Figure 7**) for only 12 days at temperatures of 5°C and 35°C. Before the storage process, the formulation was characterized with a population of 98.9% of the particles with an average diameter smaller than 199.9 nm. In the short storage period, the generation of small and large aggregates

#### **Figure 5.**

*Effects of some parameters involved in the nanoparticles preparation stage [(A) stirring rate, (B) PVA concentration, (C) ethanol concentration in the aqueous phase, (D) ethanol concentration in the organic phase, (E) emulsification time, (F) changing the preparation method from EEM to EDM, (G) aqueous phase temperature, (H) photosensitizer mass/polymer mass ratio] over different nanoparticulate properties [(RC) residual chloroform, (EE) entrapment efficiency, (RE) recovery efficiency, (PVAr) residual PVA, size, (ZP) zeta potential].*

*Synergic Influence of Parameters Involved in the Polymeric Nanoparticle Preparation… DOI: http://dx.doi.org/10.5772/intechopen.94176*

**Figure 6.**

*Average diameter of the PLGA-PEG nanoparticle loaded with GaPc after storage for 1–4 weeks in different temperatures.*

**Figure 7.**

*Average diameter of the PLGA-PEG nanoparticle loaded with InTBPPc after storage for 12 days in different temperatures.*

was observed at temperatures of 5°C and 35°C, with the size variation being greater for the temperature of 35°C in the first 8 days. This temperature is above the PLGA-PEG glass transition temperature (Tg at 30°C), [30] a fact that favors particle aggregation. Therefore, it can be concluded that storage temperatures, whether low or high, can influence the formation of aggregates, a fact that could reduce the photosensitizer efficiency during PDT.

## **2.2 Zeta potential**

The zeta potential is a property related to the particles physical stability that can be used to measure the magnitude of the repulsion or attraction. The maximization of the repulsive forces between the nanoparticles, minimizes the interactions responsible for colloidal instability, consequently interfering in the photodynamic efficacy of an encapsulated photosensitizer [5, 20].

Nanoparticles coated with amphiphilic polymers, such as PEG, usually have a higher zeta potential due to the increase in the contact surface and, consequently, to the shielding of the nanoparticle surface charge [31–34]. Therefore, the greater surface area (ratio of surface area/volume) of the nanoparticle, the greater is the residual PVA percentage at the nanoparticle interface and, consequently, the greater is the zeta potential value [5].

Thus, it is expected the parameters that influence the nanoparticles size will also be significant on the zeta potential values. As the stirring rate is one of the main factors in causing the decrease of the nanoparticles size, it will also be expected that this factor is able to affect the zeta potential, increasing its value [20].

GaPc-loaded PLGA-PEG nanoparticles presented higher zeta potential values when prepared by EDM than those prepared by the EEM. This fact corroborates with the results obtained from the nanoparticle prepared by the EEM, which presented greater sizes and smaller values of zeta potential, suggesting that they are more stable from an electrostatic point of view [5].

The increase in the aqueous phase temperature also caused a significant decrease in the absolute value of the zeta potential, because this factor induced an increase in the nanoparticles size which have less residual PVA adsorbed on their surfaces, resulting in a smaller zeta potential [5].

In the preparation of PLGA nanoparticles containing three porphyrins (m-THPP, Hex-m-bisHPP and Hex-m-trisHPP) with different amphiphilicities, each formulation presented a different response of the preparation parameters related to the zeta potential, with results intrinsically linked to particle size. For Hex-m-bisHPPloaded nanoparticles, the increase in the ethanol percentage in the aqueous phase caused an increase in the zeta potential due to the decrease of the nanoparticle size. While for nanoparticles containing m-THPP, the porphyrin/polymer mass ratio was the only significant factor that caused an increase in the zeta potential value since this factor decreased the particle size [16]. The summary of all influences on the zeta potential was indicated in **Figure 5**.

#### **2.3 Entrapment efficiency**

The entrapment efficiency relates the amount of drug that was effectively encapsulated/adsorbed on the nanoparticle. This property depends on the physicochemical properties and the interaction between the photosensitizer, the carrier matrix and the surrounding environment. Studies have shown that higher entrapment efficiency is associated with better photodynamic efficiencies for a short period of light activation [19, 35].

The diffusion process of the photosensitizer from the organic phase to the aqueous phase has significantly influenced the substance entrapment efficiency during the nanoparticle preparation process. Results have shown that the individual increase in the PVA concentration and the ethanol concentration in the aqueous phase tend to increase the photosensitizer encapsulation. The aqueous phase viscosity increases with the PVA and ethanol concentrations, which favors the formation of smaller sizes nanoparticles, having a specific surface area (area/volume) that

#### *Synergic Influence of Parameters Involved in the Polymeric Nanoparticle Preparation… DOI: http://dx.doi.org/10.5772/intechopen.94176*

allows a greater number of PVA molecules at the interface of the organic/aqueous phase. This hinders the diffusion of photosensitizers from the organic phase to the aqueous phase, favoring an increase in nanoparticle encapsulation. On the other hand, the combinatory effect caused by the simultaneous increase in the concentration of PVA and ethanol in the aqueous phase decreases the entrapment efficiency of InTPP in PLGA nanoparticles, as experiments showed that PVA and ethanol favor the solubilization of InTPP in aqueous medium [20].

When the method is changed from EEM to EDM, the entrapment efficiency decreases since the EDM method favors the formation of smaller diameter nanoparticles, facilitating the organic solvent diffusion into the aqueous phase and decreasing the entrapment efficiency of the photosensitizer in nanoparticles [5].

The increase of the aqueous phase temperature combine with the change in the preparation method also influences the photosensitizer entrapment efficiency. In the EEM method, the increase in the aqueous phase temperature causes the more effectively evaporation of the organic solvent, leading to fast polymer coacervation and, consequently, the organic/aqueous interface solidification. This increases the photosensitizer entrapment efficiency in the PLGA-PEG nanoparticles prepared by EEM. In the EDM method, the same increase in the aqueous phase temperature favors the solvent diffusion from the organic phase to the aqueous phase that carries the photosensitizer out of the nanoparticle, decreasing the entrapment efficiency [5]. All effects of parameters on entrapment efficiency were registered in **Figure 5**.

In addition to the parameters used in the nanoparticle preparation, the physicochemical properties of the photosensitizer may interfere on the entrapment efficiency. As an example, molecules of greater polarity tend to diffuse more easily from the organic phase to the aqueous phase, decreasing the entrapment efficiency [16]. The theoretical calculations compared to experimental results have suggested that photosensitizers with higher volume tend to be less efficiently encapsulated by nanoparticles. This was observed for InTBPPc molecules and also for Hex-m-TrisHPP molecules (**Table 1**). Molecules that have close volume values have shown similar entrapment efficiency as GaPc and InPc (**Figure 2A**, **B**, respectively).

#### **2.4 Recovery efficiency**

The recovery efficiency calculates the percentage of nanoparticle that has been produced and recovered. It is a property that has economic importance and has great value for the pharmaceutical industries, since they aim to reduce the production costs of the nanoparticulate formulation.

The nanoparticles size influences directly the recovery efficiency. Smaller nanoparticles are expected to be less recovered during the washing step than larger nanoparticles since the sedimentation rate of the particles in a centrifugal field is proportional to the square particle diameter. Thus, the parameters that influence the nanoparticles size tend to influence the recovery efficiency [20].

Parameters that cause a reduction in size, such as stirring rate, the EDM preparation method, the ethanol concentration in the aqueous or organic phase, as well as the emulsification time can favor the decrease of the recovery efficiency [5, 20].

Synergistic effects can be significant for recovery efficiency. For example, increasing the aqueous phase temperature together with the change in the preparation method, or increasing the emulsification time together with the change from the EEM method to EDM, can increase the recovery efficiency [5]. All effects of the parameters used to prepare of nanoparticles on the recovery efficiency are shown in **Figure 5**.

#### **2.5 Residual polyvinyl alcohol (PVA)**

PVA is the emulsifier most commonly used in the polymeric nanoparticles preparation. Even with the washing steps during the process, aiming to reduce the excess of PVA, an amount of these molecules remains adsorbed to the nanoparticle polymeric matrix due to the orientation of the PVA hydrophobic part in the organic phase, keeping molecules attach on the surface of the particle after the coacervation process [31]. This residual PVA on the particles surface can interfere on the nanoparticles physicochemical and biological properties, such as the size, release profiles of encapsulated drugs and intracellular uptake of the nanoparticles.

PVA tends to be adsorbed on the nanoparticle surface through the hydrophobic part of vinyl acetate, which tends to anchor the polymer in the aqueous/organic interface formed during the emulsification process. Smaller sized particles have a greater specific surface area, so it requires a greater amount of PVA to stabilize the emulsion droplets. Thus, these nanoparticles retain a greater amount of PVA adsorbed on its surface. Therefore, parameters that influenced the particle size, tend to affect the percentage of residual PVA [5, 16, 20, 22].

As the ethanol in the aqueous phase and the stirring rate favor the preparation of smaller nanoparticles, it is expected a higher amount of residual PVA on the small nanoparticle surface. However, the relation between the nanoparticles size and residual PVA is not immutable. An example is the PLGA-PEG nanoparticles containing gallium phthalocyanine [20]. It was reported that the aqueous phase temperature increased the nanoparticles size and the residual PVA while the change in the preparation method from EEM to EDM decreased the nanoparticles size and the residual PVA. Therefore, a different situation that it was expected. Probably, the presence of PEG linked to PLGA hindered the interactions of PVA molecules with the organic/aqueous interface [20].

Residual PVA can also be influenced by synergistic effects. For example, changing the preparation method from EEM to EDM, associated with an increase in the aqueous phase temperature can cause an increase in the residual PVA. However, the increase of the emulsification time together with the change of the preparation method can reduce the residual PVA [5]. All influences of the parameters used in the preparation of polymeric nanoparticle were summarized in the **Figure 5**.

It should be noted that the residual PVA values can still vary according to the number of washing steps and the method used to wash the nanoparticle suspension [5, 22].

#### **2.6 Residual chloroform**

The organic solvent can be retained by nanoparticles during the preparation of the nanoparticulate formulation, becoming a residual organic impurity. Therefore, the quantification of solvent residual is necessary to eliminate toxicological risks for patients. According to the American Pharmacopeia, the residual chloroform limit is 60 ppm for pharmaceutical formulations. Thus, it is very important to evaluate the influence of the factors involved in the nanoparticle preparation on the residual chloroform concentration [20].

The percentage of residual chloroform, as described for other properties, is also related to the nanoparticle size. Thus, there is a tendency to reduce residual chloroform linked to the reduction in the nanoparticles size.

In the preparation of PLGA-PEG nanoparticles containing chloro(5,10,15,20 tetraphenylporphyranato) indium(III) (InTPP - **Figure 2C**), the influence of four parameters on the residual chloroform percentage was studied: PVA concentration, stirring rate, ethanol percentage in the aqueous phase and in the organic phase [20]. *Synergic Influence of Parameters Involved in the Polymeric Nanoparticle Preparation… DOI: http://dx.doi.org/10.5772/intechopen.94176*

The stirring rate and the ethanol percentage in the aqueous phase were the factors that significantly influenced the residual solvent, favoring the decrease of residual chloroform. The increase in the stirring rate favors the organic phase dispersion into the aqueous phase, generating small organic droplets that favor the fast solvent diffusion into the aqueous phase. On the other hand, ethanol in the aqueous phase hinders the coalescence of organic droplets dispersed in the aqueous phase, favoring the formation of smaller diameter particles [20].

Although the individual effects of ethanol in the aqueous phase and the increase in stirring rate cause a decrease in residual chloroform, the synergistic effect of the simultaneous increase of these factors caused an increase in residual chloroform. Both factors favored the decrease in the nanoparticles size. Small size nanoparticles tend to present a higher residual PVA percentage on the particles surface, which makes difficult the solvent diffusion from organic droplets into the aqueous phase. This diffusion becomes even more difficult after the solidification of the polymeric matrix surface layer during the process of evaporation of the organic solvent, favoring the residual increase of chloroform in the nanoparticles [20]. All influences on residual chloroform have been reported in **Figure 5**.
