*3.3. Characterization of NS*

### 3.3.1. Phase Solubility of NS

The initial solubility of PRX in distilled water was found to be 6.6 μg/mL. The solubility study of NS was performed at different concentrations of stabilizer, i.e., poloxamer 188® and at a different speed. The results showed an increase in the solubility of PRX in the NS formulations (Table 7).

#### 3.3.2. Particle Size and Zeta Potential

The particle size in the NS formulation was found to be decreasing with an increase in the poloxamer 188® concentrations and an increase in the speed as shown in Table 7. The particle size and zeta potential of the optimized formulation are shown in Figure 4.


**Table 7.** Particle size, PI, zeta potential, PDE, and solubility of the NSs.

\* All values are expressed as mean ± SD (*n* = 3).

**Figure 4.** Particle size distribution curve (**A**) and zeta potential peak (**B**) of the optimized formulation NS9.

#### Entrapment efficiency.

The amount of non-capsulated PRX was determined by an indirect method. The entrapment efficiency of NSs is shown in Table 7. The optimized formulation (NS9) exhibited the highest drug entrapment, i.e., 89.76 ± 0.76%.

#### 3.3.3. TEM Study

TEM analysis was used to depict the morphology of suspended PRX nanoparticles, and the resulting TEM micrograph of the optimized NS is shown in Figure 5. TEM analysis revealed that the suspended nanoparticles were roughly spherical or irregular in shape with uniform distribution; these findings were in good agreement with the particle size analysis outcomes of the optimized formulation NS9 suggesting an average particle size of 228 nm.

#### 3.3.4. DSC STUDY

DSC was used to examine the thermal behavior of the drug and nanoparticles. The pure drug shows a sharp endothermic peak, which corresponds to its melting point, which was observed at 203.15 ◦C. However, a significant shift in the melting peak of the pure drug (PRX) in the nanoformulation was observed at 198.05 ◦C, indicating a reduction in crystallinity compared to pure PRX (Figure 6). This indicated the change in the crystalline nature of PRX during the preparation of nanosuspension. The shift also may be due to the presence of stabilizers (Poloxamer 188® at 51.05 ◦C and PVP K30® at 152.15 ◦C) in the formulation when compared with the pure drug. A sharp endothermic peak at 164.05 ◦C represents the melting of mannitol used as a cryoprotectant in the formulation. The conclusions drawn from the DSC analysis were in good agreement with the noted FT-IR outcomes and were further validated using the results of the XRPD analysis.

**Figure 6.** Overlain DSC thermograms of the pure drug (PRX) (A) and freeze-dried formulation (NS9) (B).

#### 3.3.5. XRPD Analysis

To characterize the crystalline nature of PRX within the NS and to evaluate the mode of interaction between PRX and NS, XRPD data of pure PRX and freeze-dried formulation (NS9) samples were acquired (Figure 7). Typical diffraction peaks at 8.62◦, 11.65◦, 12.49◦, 13.28◦, 14.51◦, 15.86◦, 16.70◦, 17.71◦, 18.85◦, 21.74◦, and 27.80◦ were used to identify the crystalline nature of PRX. The pure PRX exhibits an intense crystalline peak between 10◦

and 30◦. However, the peaks in the NS9 formulation were less intense; indicating a decrease in crystallinity. Additionally, the peaks at 9.10◦, 20.22◦, 36.42◦, 40.80◦, and 45.10◦ are the peaks of D-mannitol used in freeze drying which was absent in pure drug (PRX). Similar kinds of results were reported in previous research studies in the literature [25,26].

**Figure 7.** XRPD diffractograms of (**A**) pure drug PRX and (**B**) freeze-dried optimized formulation NS9.

3.3.6. In Vitro Dissolution Study

The in vitro dissolution profile of all the NS formulations (Figure 8) revealed that the PRX was released significantly faster than that of the pure drug. The optimized formulation (NS9) exhibits a greater drug release of approximately 96.07% after 120 min as compared to the other NS formulations and pure PRX (36.78%). According to the dissolution profile, all formulations exhibit burst release, which may be caused by the solubilized drug present in the NS. PRX was more easily dissolved by the formulation when the stabilizer, Poloxamer 188®, was used in higher concentrations and stirred at higher rpm. This improvement can be due to the ability of PVP K30® and stabilizer Poloxamer 188® to form a complex with PRX altering its crystalline nature, which leads to an increase in the solubility and

dissolution rate of the PRX in NS than pure PRX. Dissolution efficiency is the area under the dissolution curve within a given range of time. The amount of drug dissolved and the time of solution in contact with the region of absorption, i.e., the GI tract, directly correlate with drug absorption, which increases drug bioavailability.

**Figure 8.** In vitro drug release profiles of prepared PRX NS formulations.

3.3.7. Mathematical Modeling Studies

The R<sup>2</sup> values from fitting the experimental model's in vitro dissolution data into the various release kinetic models are shown in Table 8. Pure PRX drug release data were extremely well-described by the Higuchi model, whereas the Korsmeyer-Peppas model was found to best fit for NS. The Korsmeyer-Peppas model was the most accurate model for describing the release mechanism for all formulations, while all other models were the least accurate.

**Table 8.** Kinetic profiles of in vitro drug release of optimized PRX NS (NS9).


The highest R<sup>2</sup> value noted for the Korsmeyer-Peppas model (0.9929) established it to be the best-fitting model and the release exponent (*n*) value of NS was below 0.5 which indicated that NS had followed Quasi-Fickian release kinetics [27]. This Fickian diffusion corroborates to the transport of water via the diffusion process which was driven by the concentration gradient, i.e., drug release from high concentration to low concentration.

#### **4. Conclusions and Future Scope**

Nano-sizing is the finest method to improve the solubility and dissolution rate of poorly water-soluble drugs. The anti-solvent precipitation method implied in current research vocation is a cost-effective and simple approach for formulating NS. This research work was aimed at optimizing the NS formulation of PRX by varying concentrations of stabilizers and stirring speed. Optimization was successfully achieved implying 32 full factorial design as a Quality by Design (QbD) approach. The obtained 3D surface response analysis results and plots revealed that increasing the stabilizer concentration and stirring speed had resulted in the reduction in particle size and increase in solubility. Different NS batches have depicted a reduction in the particle size from 443 nm to 228 nm and the solubility has increased from 44 μg/mL to 87 μg/mL over changing the independent variables. From all the noted results, it was concluded that the solubility of PRX in water was enhanced by 14- to 15-fold (87.28 μg/mL) than that of the pure PRX (6.6 μg/mL). The optimized NS formulation (NS9) exhibited a greater in vitro drug release of approximately 96.07% post-120 min with respect to the other NS formulations and pure PRX (36.78%). Moreover, the in vitro drug release data from the PRX NS was fitted into diverse mathematical models to predict the drug release mechanism. The release kinetics outcomes established that release profile best fits into the Korsmeyer-Peppas model suggesting the Fickian diffusion as drug release mechanism. Furthermore, from the DSC and XRPD analysis data, it was evident that the PRX was converted into a less crystalline form when formulated into NS, which could be a valid justification for the enhanced PRX solubility. In a nutshell, from all the noted results, it was evident that the NS formulations had improved the solubility and in vitro dissolution of PRX quite significantly in comparison with the pure drug. Hence, from the in vitro-in vivo correlation (IVIVC) point of view, we conclude that prepared and optimized NS formulation would also lead to an increase in the bioavailability of PRX. However, to validate the in vivo performance and to establish the in vivo pharmacokinetic parameters in detail, a multicentric, randomized in vivo pharmacokinetic study should be undertaken in near future. To conclude, the present research outcomes have clearly indicated that NS formulation approach could be a potential alternative to the conventional methods of solubility as well as bioavailability enhancement and need to be explored much for the diverse promising existing and newly developed BCS Class II drugs.

**Author Contributions:** Conceptualization, S.M.H., R.A.M.O. and U.H.; methodology, S.M.H., R.A.M.O., Y.A. and N.K.K.; software, R.R.D. and M.S.C.; validation, R.R.D. and S.M.H.; formal analysis, R.A.M.O., S.P., G.K. and R.V.; investigation, S.M.H., R.A.M.O. and S.P.; resources, S.P., R.R.D., G.K. and R.V.; data curation, M.S.C., R.A.M.O. and U.H.; writing—original draft preparation, Y.A., S.M.H., R.R.D. and S.P.; writing—review and editing, Y.A, G.K., R.V. and R.A.M.O.; visualization, S.M.H., N.K.K. and R.V.; supervision, M.S.C. and Y.A..; project administration, G.K., R.V., R.A.M.O. and U.H.; funding acquisition, Y.A., U.H., G.K. and R.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research work is funded by Deanship of Scientific Research at King Khalid University, Saudi Arabia, through Large Program under grant number (RGP-2/121/43) 1443.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data in support of the findings of this study will be available from the corresponding author upon request.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for funding this work through Large Program under grant number (RGP-2/121/43) 1443. The authors are also thankful to the Principal and Management of the Annasaheb Dange College of B. Pharmacy, Ashta, Maharashtra, India for providing the obligatory support and facility to complete this research.

**Conflicts of Interest:** The authors declare no conflict of interest.
