Formulation and Evaluation of Pravastatin Sodium-Loaded PLGA Nanoparticles: In vitro-in vivo Studies Assessment

Abstract

Purpose: Pravastatin sodium (PVS) is a hypolipidemic drug which suffers from extensive first-pass metabolism and short half-life. Poly(d,l-lactide-co-glycolide) (PLGA) is considered a promising carrier to improve its hypolipidemic and hepatoprotective activities.

Methods: PVS-loaded PLGA nanoparticles (PVS-PLGA-NPs) were prepared by double emulsion method using a full 3² factorial design. The in vitro release and the physical stability studies of the optimized PVS-PLGA-NPs (F5) were performed. Finally, both hypolipidemic and hepatoprotective activities of the optimized F5 NPs were studied and compared to PVS solution.

Results: All the studied physical parameters of the prepared NPs were found in the accepted range. The particle size (PS) ranged from 90 ± 0.125 nm to 179.33 ± 4.509 nm, the poly dispersity index (PDI) ranged from 0.121 ± 0.018 to 0.158 ± 0.014. The optimized NPs (F5) have the highest entrapment efficiency (EE%) (51.7 ± 5%), reasonable PS (168.4 ± 2.506 nm) as well as reasonable zeta potential (ZP) (-28.3 ± 1.18mv). Solid-state characterization indicated that PVS is well entrapped into NPs. All NPs have distinct spherical shape with smooth surface. The prepared NPs showed a controlled release profile. F5 showed good stability at 4 ± 2°C during the whole storage period of 3 months. In vivo study and histopathological examination indicated that F5 NPs showed significant increase in PVS hypolipidemic as well as hepatoprotective activity compared to PVS solution.

Conclusion: The PVS-PLGA-NPs could be considered a promising model to evade the first-pass effect and showed improvement in the hypolipidemic and hepatoprotective activities compared to PVS solution.

Keywords: PLGA; hypolipidemic and hepatoprotective activity; nanoparticles; pravastatin sodium.

Figure 1

Chemical structure of pravastatin sodium.

Figure 2

Particle size distribution graph (A) and zeta potential distribution graph (B).

Figure 3

Continued.

Figure 3

Contour plots (A, C and E) and three-dimensional surface plots (B, D and F) representing the effect of interaction between drug:polymer ratio (A) and surfactant concentration (B) on particle size, entrapment efficiency percent and zeta potential, respectively.

Figure 4

Transmission electron microscope (TEM) of F5 NPs.

Figure 5

Scanning electron microscope (SEM) of F5 NPs.

Figure 6

Solid characterizations of the optimized formula (F5), FTIR spectra (A), DSC thermograms (B) and PXRD patterns (C) of (I) PVS, (II) PLGA, (III) BSA, (IV) physical mixture, (V) optimized F5 and (VI) plain F5.

Figure 7

In vitro release profile of free PVS and the optimized formula F5 in 0.1 N HCL pH 1.2 (A), phosphate buffer pH 6.8 (B) and phosphate buffer pH 7.4 (C).

Figure 8

The serum level of the examined lipid biomarker after the end of treatment period (1 week) with F5 NPs and PVS solution.

Figure 9

Effect of F5 NPs and PVS solution on serum liver function.

Figure 10

Microscopic pictures of H&E-stained hepatic sections showing normal organization of hepatic cords H around central veins (CV) with normal portal areas (PA) and sinusoids (S) in the negative control group (A1 and A2). Hepatic sections from positive control group (B1 and B2) showing congested central veins (CV, red arrow), fat vacuoles (black arrows) in hepatocytes (H), dilated sinusoids (S), mononuclear cells infiltration in portal areas (PA, yellow arrows), bile duct dilation (arrowheads). Hepatic sections from PVS-treated group (C1 and C2) showing fewer fat vacuoles in hepatocytes (black arrows), dilated sinusoids (S), fewer mononuclear cells infiltration in portal areas (PA, yellow arrow), mild bile duct dilation (arrowheads). Hepatic sections from F5 NPs-treated group (D1 and D2) showing restored normal organization of hepatic cords (H) around central veins (CV) with normal portal areas (PA) and sinusoids (S). Low magnification ×: 100 bar 100 and high magnification ×: 400 bar 50.

Figure 11

Microscopic pictures of H&E stained longitudinally sectioned skeletal muscles showing normal organization of striated muscle fibers with peripherally located nuclei in negative control group (A1). The longitudinally sectioned skeletal muscles from the positive control group (B1) showing hyaline degeneration (black arrow) in some sections, marked lipid infiltration in muscle fiber and marked mononuclear cells infiltration in interstitial tissue (yellow arrow). The longitudinally sectioned skeletal muscles in the free PVS-treated group (C1), showing moderate lipid infiltration in muscle fibers (blue arrow) and in the F5 nanoparticles-treated group (D1), showing lower lipid infiltration in muscle fibers (blue arrow). The crossly sectioned skeletal muscles showing normal muscle fibers with peripherally located nuclei in negative control group (A2). The crossly sectioned skeletal muscles from positive control group (B2) showing marked lipid infiltration, mild mononuclear cells infiltration (yellow arrows) in muscle fibers and hyaline degeneration in some sections with mild mononuclear cells infiltration. The cross-sectioned skeletal muscles from the free PVS-treated group (C2) showing moderate lipid infiltration in muscle fibers (blue arrow) and in the F5 NPs-treated group (D2) showing very mild lipid infiltration in muscle fibers (blue arrow).