Formulation and In-Vitro Evaluation of Saquinavir Nanoemulsion

Abstract

Nanoemulsions have emerged as promising drug delivery systems due to their ability to enhance the solubility and bioavailability of poorly water-soluble drugs. This study aims to develop and evaluate nanoemulsions of Saquinavir, a protease inhibitor with low aqueous solubility, to improve its dissolution rate and therapeutic efficacy. Nanoemulsions were formulated using Capryol 90 as the oil phase, Tween 80 as the surfactant, and Transcutol P as the co-surfactant. A pseudo-ternary phase diagram was constructed to determine the optimal composition for a stable nanoemulsion. Characterization of the prepared nanoemulsions included particle size analysis, zeta potential, polydispersity index (PDI), pH measurement, thermodynamic stability, and in vitro drug release studies. The optimized formulation exhibited a droplet size of less than 200 nm, a negative zeta potential ensuring stability, and enhanced solubility compared to pure Saquinavir. In vitro dissolution studies revealed a significantly higher drug release from the nanoemulsion compared to the unprocessed drug. Stability studies confirmed the physical and chemical stability of the formulation under accelerated conditions. The findings of this study suggest that Saquinavir nanoemulsions could be a viable approach to improve its bioavailability and therapeutic effectiveness in HIV treatment.

Keywords

Introduction

Saquinavir is a protease inhibitor used in combination antiretroviral therapy for the treatment of HIV/AIDS. However, its clinical efficacy is hindered by low aqueous solubility (~4 mg/L) and poor bioavailability (~4%), necessitating the use of high oral doses, which can lead to side effects and resistance development. Nanoemulsions, submicron-sized dispersions (20-200 nm), have demonstrated significant potential in enhancing drug solubility, permeability, and bioavailability^1-3. Their high surface area-to-volume ratio ensures improved absorption and reduced drug metabolism, making them ideal for lipophilic drugs like Saquinavir. This study focuses on developing and characterizing nanoemulsions for the effective delivery of Saquinavir using lipid-based excipients and surfactants^4-7.

2. MATERIALS AND METHODS

2.1. Materials

Saquinavir (API) from Aurobindho Pharma Limited, Capryol 90, Labrafac PG, Transcutol P , Olive oil Gattefosse products, Tween 80, Tween 60, Span 80, PEG 400, Polyvinyl alcohol, potassium chloride, Methanol are laboratory grade.

2.2. Determination of λmax and Calibration Curve

The maximum absorbance wavelength (λmax) of Saquinavir was determined using a UV-Visible spectrophotometer by scanning the drug solution in methanol within a wavelength range of 200-400 nm. The λmax was found to be 240 nm.

A calibration curve was plotted by preparing standard Saquinavir solutions in methanol at different concentrations (5-50 µg/mL) and measuring absorbance at λmax 240 nm. A linear regression equation was obtained, ensuring the method’s suitability for drug estimation in nanoemulsions^8-10.

2.3. Solubility Studies

To select an appropriate oil, surfactant, and co-surfactant, Saquinavir was added in excess to various excipients, vortexed, and equilibrated for 72 hours. Samples were centrifuged and analyzed using a UV-Visible spectrophotometer at λmax = 240 nm^11-14.

2.3. Construction of Pseudo-Ternary Phase Diagram

Nanoemulsion regions were identified using aqueous titration method, where oil, surfactant, and co-surfactant (Smix) were mixed in different ratios (1:9 to 9:1) and titrated with water. The mixtures were visually assessed for clarity and stability^15-17.

2.4. Formulation of Nanoemulsions

Nanoemulsions were prepared using the aqueous titration method, with Capryol 90 (oil), Tween 80 (surfactant), and Transcutol P (co-surfactant). The oil phase was added dropwise into the aqueous phase under continuous stirring and homogenization^17-20.

2.5. Characterization Studies

2.5.1. Particle Size and Zeta Potential Analysis

The size and surface charge were measured using Dynamic Light Scattering (DLS) and Zetasizer (Malvern ZS 90)^24-26.

2.5.2. Thermodynamic Stability

Nanoemulsions were subjected to centrifugation (3500 rpm for 30 min) and freeze-thaw cycles (-20°C to 25°C) to assess their physical stability^24-26.

2.5.3. In Vitro Drug Release Studies

Drug release was evaluated using a USP dissolution apparatus (Type I, basket method) in simulated gastric fluid (pH 1.2) at 37°C ± 0.5°C. Aliquots were analyzed at λmax = 240 nm^24-26.

2.5.4. Accelerated Stability Studies

Nanoemulsions were stored at 40°C ± 2°C, 75% RH for three months and analyzed for droplet size, zeta potential, and drug content^21-24.

3. RESULTS AND DISCUSSION

The UV-Visible spectroscopic analysis of Saquinavir confirmed a maximum absorption wavelength (λmax) of 240 nm. This wavelength was selected for all subsequent quantitative estimations of the drug in nanoemulsion formulations. The calibration curve was established by preparing a series of standard solutions of Saquinavir in methanol with concentrations ranging from 5 to 50 µg/mL. The absorbance of these solutions was measured at λmax = 240 nm (fig-1), and a linear regression equation was derived. The plot of absorbance versus concentration exhibited a strong linear correlation with an R² value of 0.998 (fig-2), confirming the accuracy, precision, and reliability of the analytical method for drug quantification.

Fig-1: Scan Spectrum of Saquinavir

Fig-2: Calibration curve of Saquinavir (n=3)

Fig-6: Solubility of Saquinavir in Oils

Fig-7: Solubility of Saquinavir in Surfactants

Fig-8: Solubility of Saquinavir in Co-surfactant

Fig-15: Droplet size of CT4T1(2:8)

Fig-16-: Zeta potential of CT4T1(2:8)

Fig-19: Drug release studies for Saquinavir Nanoemulsions

The drug release continued to increase at later time points, with differences in release rates and extent observed between the formulations. By the end of the 6-hour study period, CT3T1(2:8) had released 97.9% of the drug with a standard deviation of ±3.38%, CT4T1(2:8) had released 99.1% with a standard deviation of ±3.18% and CT4T1(3:7) had released 99.3% with a standard deviation of ±3.23%. Based on the results, CT4T1(2:8) is chosen for the accelerated stability studies.

To assess the stability of the nanoemulsion, accelerated stability studies were conducted under ICH guidelines (40°C ± 2°C, 75% relative humidity) for three months. The formulation was periodically analyzed for changes in droplet size, zeta potential, and drug content. The results indicated that the nanoemulsion maintained its physicochemical properties with no significant changes in particle size, charge, or drug content, confirming its high stability under stress conditions. Additionally, thermodynamic stability tests, including centrifugation and freeze-thaw cycling, showed no signs of phase separation, flocculation, or precipitation, further demonstrating the robust stability of the nanoemulsion formulation.

In summary, the optimized Saquinavir nanoemulsion exhibited superior solubility, rapid drug release, and excellent stability compared to the pure drug. The small droplet size, low PDI, and stable zeta potential suggest that nanoemulsion technology can significantly improve the bioavailability of Saquinavir, potentially enhancing its therapeutic efficacy in HIV treatment. These findings indicate that nanoemulsions could serve as a viable approach to overcome the solubility and bioavailability limitations associated with Saquinavir, thereby improving patient outcomes in antiretroviral therapy.

CONCLUSION

This study successfully developed Saquinavir-loaded nanoemulsions, demonstrating improved solubility, enhanced drug release, and stability. The CT4T1(2:8)-optimized formulation (Capryol 90, Tween 80, and Transcutol P) exhibited a particle size of 98.24 nm with 0.231 PDI, a stable zeta potential of -31.1 mV, and a significant enhancement in dissolution rate. These findings suggest that nanoemulsions are a viable strategy for improving the bioavailability of Saquinavir in HIV therapy.

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