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

Figures & data

Figure 1 Chemical structure of pravastatin sodium.

Table 1 Formulation Code for Preparation of Various Polymeric Nanoparticles Compositions

Formulation Code		F1	F2	F3	F4	F5	F6	F7	F8	F9
Code Value	A	−1	−1	−1	0	0	0	+1	+1	+1
	B	−1	0	+1	−1	0	+1	−1	0	+1

Notes: A (drug:polymer) ratio, B (surfactant concentration w/w%), −1 (low level), 0 (Medium level) +1 (high level).

Table 2 Levels of Independent Variables and Their Levels Applied Using the Experimental Design

Factor	Name	Type	Low Actual Level	Medium Actual Level	High Actual Level	Low Coded Level	Medium Coded Level	High Coded Level
A	Drug:polymer ratio	Numeric	1:1	1:10	1:20	−1	0	+1
B	Surfactant concentration (%)	Numeric	1%	1.5%	3%	−1	0	+1

Table 3 Independent & Dependent Variables

Independent Variables	Dependent Variables (Responses)
A = Drug:polymer ratio	Y1 = Particle size (nm)
B = Surfactant concentration (%)	Y2 = Entrapment efficiency (%)
	Y3 = Zeta potential (mv)

Table 4 Coded Independent Variables and Properties of PVS -PLGA-NPs

Formula	Independent Variables		Dependent Variables (Mean ± SD)
	Code of A	Code of B	PS (nm)	EE (%)	ZP (mV)
F1	−1	−1	90 ± 0.125	40 ± 2	−31 ± 1
F2	−1	0	95.673 ± 1.5	39 ± 3.606	−41.167±4.594
F3	−1	+1	102.233 ± 0.321	28 ± 2.646	−37.467 ± 2.892
F4	0	−1	172.8 ± 1.51	38.33 ± 3.055	−9.953 ± 0.081
F5	0	0	168.4 ± 2.506	51.7 ± 5	−28.3 ± 1.18
F6	0	+1	151.53 ± 1.193	37.333 ± 2.517	−33 ± 0.987
F7	+1	−1	171.667± 3.512	27.667 ± 2.517	−32 ± 1
F8	+1	0	179.33 ± 4.509	41 ± 3.606	−31.833 ± 0.709
F9	+1	+1	161.667 ± 0.794	33.667 ± 4.509	−30.9 ± 0.755

Note: Data are expressed as mean ± SD (n=3).

Abbreviations: A, drug:polymer ratio; B, surfactant concentration; PS, particle size; EE%, entrapment efficiency percent; ZP, zeta potential.

Table 5 ANOVA Results for Responses Provided by Design Expert

Responses	Source	F value	P value	Inference	Adjusted R^2	Predicted R^2	Adequate Precision
PS (Y1)	Quadratic	163.25	<0.0001	Significant	0.971	0.954	26
EE% (Y2)	Quadratic	35.13	<0.0001	Significant	0.783	0.653	12.5
ZP (Y3)	Quadratic	13.4	<0.0001	Significant	0.634	0.42	9.125

Abbreviations: PS, particle size; EE%, entrapment efficiency percent; ZP, zeta potential.

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.

Table 6 Adjusted Levels of Independent Variables, Predicted and Observed Responses for Optimized Formula

Optimized Formula Coded Level	Optimized Actual Value	Responses	*Predicted Mean Value	Observed Experimental Value
Drug:polymer ratio [0]	1:10	Y1 PS (nm)	168.311 nm	168.4 ± 2.506 nm
Surfactant concentration [0]	1.5 (%)	Y2 EE (%)	48.9259%	51.7 ± 5%
		Y3 ZP (mv)	−27.719 mv	−28.3 ± 1.18

Note: *Point prediction at confidence of 95% and population of 99%.

Abbreviations: PS, particle size; ZP, zeta potential; EE%, entrapment efficiency percent.

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.

Abbreviations: FT-IR, Fourier-transform infrared spectroscopy; DSC, differential scanning calorimetry; PXRD, powder X-ray diffractometry; PVS, pravastatin sodium; PLGA, poly(d,l-lactide-co-glycolide); BSA, bovine serum albumin.

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).

Abbreviation: PVS, pravastatin sodium.

Table 7 Kinetic Analysis of Drug Release Data

Formula	pH of the Release Media	Coefficients of Determination (R^2)			Korsmeyer–Peppas		Main Transport Mechanism
		Zero-Order	First-Order	Higuchi Model	R^2	Diffusional Exponent (n)
Free PVS	1.2	0.609	0.584	0.61	0.611	0.015	Fickian
	6.8	0.705	0.726	0.705	0.7016	0.035	First order
	7.4	0.643	0.68	0.642	0.6429	0.017	First order
F5 NPs	1.2	0.879	0.907	0.963	0.957	0.566	Non fickian
	6.8	0.844	0.834	0.856	0.9011	0.45	Fckian
	7.4	0.95	0.958	0.964	0.955	0.619	Non fickian

Table 8 PS, PDI, ZP, EE% of PVS–PLGA-NPs Aqueous Dispersion (F5) Stored at Refrigerated (4 ± 2°C) and at Ambient Condition (25 ± 2°C)

Storage Time	Evaluation Parameters (mean ± SD)
	Refrigeration Condition (4 ± 2°C)				Ambient Condition (25 ± 2°C/60 5% RH)
	Particle Size (nm)	PDI	ZP (mv)	EE%	Particle Size (nm)	PDI	ZP (mv)	EE%
Zero time	168.4 ± 2.506	0.121 ± 0.018	−28.3 ±1.18	51.7 ± 5	168.4 ± 2.506	0.141 ± 0.009	−28.3 ± 1.18	51.7 ± 5
1 week	162.5 ± 2.7	0.12 5 ± 0.047	−24.66 ± 4.6	51.33 ±3.055	160.233 ± 3.775	0.91 ± 0.107*	−22 ± 2	45.7 ± 3.5
2 weeks	160.533 ± 2.1	0.143 ± 0.033	−24 ± 5.2	51.33±2.8	160.767 ± 2	0.204 ± 0.079	−17 ± 1.36*	44.7 ± 4.5
1 month	158.567 ± 5.68	0.154 ± 0.045	−35.3 ± 1.16	50.33 ± 2.8	188.3 ± 5*	0.133 ± 0.049	−16.3 ± 2*	44 ± 4
2 months	184.2 ± 4.2*	0.056 ± 0.053	−20.33 ±0.577*	49.66 ±2.346	680.33 ± 109*	1 ± 0.016*	−5 ± 1.36 *	33 ± 4*
3 months	189.1 ± 8.7*	0.165 ± 0.022	−28.66 ± 0.577	50 ± 2.6	206.1 ± 5.3*	0.23 9 ± 0.107	36.5 ± 1.4*	24.3 ± 4.04*

Notes: Each value represents the mean ± SD. The statistical analysis was performed at p < 0.05 using one-way ANOVA. *Indicates a significant difference vs initial measurement at zero time.

Abbreviations: PS, particle size; PDI, polydispersity index; ZP, zeta potential; EE, entrapment efficiency.

Table 9 Effect of Optimized Formula (F5) and PVS Solution on Serum Lipid Profile

Parameter	Time (hr)/Group	Before Treatment	After Treatment
		12 hr After Induction of Hyperlipidemia	3 Days	4 Days	5 Days	1 Week
TC (mg/dl) Mean ± SD	G1 G2 G3 G4	65.33 ± 4.095^$#@	66 ± 2.08^$#@	61 ± 6.08^$#@	63 ± 4.33^$#@	67± 4.72^$#@
		451.6 ± 56.6*	844.6 ± 26 *^#@	815 ± 33.4*^#@	732 ± 36*^#@	670.3 ± 54*^#@
		482 ± 75.2*	430.66 ± 67.7*^$	360.33 ± 69.7*^$	285.33 ± 65.8*^$	224 ± 44.6 *^$
		439 ± 53.113*	333 ± 47.4*^$	239.33 ± 38.3*^$	168 ± 27*^$	118.66 ± 12*^$
TG (mg/dl) Mean ± SD	G1 G2 G3 G4	80 ± 7^$#@	76 ± 8.33^$#@	78.33 ± 7.26^$#@	77 ± 5.8^$#@	83 ± 4.4^$#@
		838 ± 114.17*	1124.3 ± 167.7*^#@	1129 ± 86.3*^#@	937 ± 99.6*^#@	827.667 ± 107 *^#@
		887 ± 111*	814.66 ± 104.2*	615.33 ± 60.6*^$	395 ± 56*^$	289 ± 42.6 *^$@
		808 ± 96*	693 ± 90.5*^#	405.33 ± 62.3*^$	262.33 ± 40.4*^$	151 ± 7.2*^$#
HDL (mg/dl) Mean ± SD	G1 G2 G3 G4	45.66 ± 2.7^$#@	45.667 ± 4.72^$#	49.7 ± 3^$#@	52 ± 6^$	46 ± 3^$
		175.3 ± 37.5*	233.6 ± 44.3*	300.3 ± 40.8*^@	274 ± 29*^#@	299 ± 14.5*^#@
		193 ± 54*	185 ± 13*	157.6 ± 58.8*	127.33 ± 53^$	88.33 ± 32.4^$
		165 ± 46.5*	154 ± 21.3*	124.33 ± 26*^$	81.66 ± 15^$	46.66 ± 2.1^$
LDL (mg/dl) Mean ± SD	G1 G2 G3 G4	4 ± 1.1^$#@	6 ± 1.155^$#@	6 ± 1.155^$#	5.33 ± 0.8^$#@	9.333 ± 0.88^$#
		108 ± 4.9*	386 ± 79*^#@	289 ± 21*^#@	269.6 ± 16.2*^#@	206 ± 25.5*^#@
		111.66 ± 7.3*	82.666 ± 34*^$	79.66 ±1.76*^$@	78.66 ± 2.6*^$@	77.66 ± 4.8*^$
		112 ± 14*	40.33 ± 8.5*^$	34 ± 4*^$#	34 ± 4.7*^$#	41.66 ± 12.7^$

Notes: Values are expressed as means ± SEM (n=6 rats); *Significantly different from normal control value; ^$Significantly different from positive control value ; ^#Significantly different from PVS solution and ^@Significantly different from F5 NPs. The statistical analysis was performed at p < 0.05 using Student’s t-test (unpaired).

Abbreviations: G1, normal control; G2, non-treated hyperlipidemic rats (positive control); G3, orally treated hyperlipidemic rats by PVS solution; G4, orally treated hyperlipidemic rat by (F5) PVS–PLGA-NPs; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Figure 8 The serum level of the examined lipid biomarker after the end of treatment period (1 week) with F5 NPs and PVS solution.

Notes: Values were expressed as means ± SEM (n=6 rats) *Significantly different from normal control value; ^$Significantly different from positive control value; ^#Significantly different from PVS value and ^@Significantly different from F5 NPs value. The statistical analysis was performed at p < 0.05 using Student's t-test (unpaired).

Table 10 Effect of F5 NPs and PVS Solution on Serum Liver Function Test, Albumin and Alkaline Phosphatase

Parameter/Group	ALT (U/L)	AST (U/L)	ALP (U/L)	Albumin (g/dl)
Normal control (G1)	28.7 ± 3.4^$#	96.6 ± 3.9^$≠@	254.6 ± 9.7^$#	4.8 ± 0.12^$#
Positive control (G2)	61.6 ± 6.1*^@	222.6 ± 15.3*^#@	407 ± 11.6*^@	3.5 ± 0.179*^#@
PVS solution (G3)	43.6 ± 2.9*	151.3 ± 4.9*^$@	369.3 ± 18.5*	4.1 ± 0.049*^$
F5 nanoparticles (G4)	38 ± 2.6^$	125 ± 6.2*^$#	302 ± 28^$	4.3 ± 0.062^$

Notes: Values are expressed as mean± SEM. (n= 6); *Significantly different from normal control value; ^$Significantly different from positive control value; ^#Significantly different from PVS solution and ^@Significantly different from F5 nanoparticles. The statistical analysis was performed at p < 0.05, using Student’s t-test (unpaired).

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

Figure 9 Effect of F5 NPs and PVS solution on serum liver function.

Notes: Values are expressed as mean± SEM (n= 6); *Significantly different from normal control value; ^$Significantly different from positive controlvalue ^#Significantly different from PVS solution and ^@Significantly different from F5 nanoparticles. The statistical analysis was performed at p < 0.05, using Student's t-test (unpaired).

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

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).