Thymoquinone-entrapped chitosan-modified nanoparticles: formulation optimization to preclinical bioavailability assessments

Figures & data

Table 1. Various independent and dependent variables used in the Box–Behnken design for the preparation of thymoquinone encapsulated chitosan modified polycaprolactone nanoparticles (THQ-CPLNPs).

Factor	Levels used, actual (coded factor)
Independent variables	Low (‒1)	Medium (0)	High (+1)
X1 = Polycaprolactone (mg; w/v)	35	50	65
X2 = Chitosan (%; w/v)	0.3	0.5	0.7
X3 = PVA (%; w/v)	2	2.5	3
Dependent variables	Goal
Y1 = Particle size (PS; nm)	Minimize
Y2 = Polydispersity index (PDI)	Minimize
Y3 = Entrapment efficiency (EE; %)	Maximize

Table 2. Observed Box–Behnken experimental runs of thymoquinone encapsulated chitosan modified polycaprolactone nanoparticles (THQ-CPLNPs) with their experimental value.

Runs	(A) Polycaprolactone	(B) Chitosan	(C) Polyvinyl alcohol	Y1 Particle size	Y2 (Polydispersity index)	Y3 (Entrapment efficiency)
	mg	%	%	nm		%
F1	35	0.7	2.5	164.12	0.16	76.46
F2	35	0.5	2	159.08	0.19	72.08
F3	50	0.7	2	211.07	0.19	81.49
F4	65	0.5	2	225.14	0.23	87.78
F5	50	0.3	3	176.83	0.16	78.49
F6	65	0.5	3	203.97	0.25	90.54
F7	50	0.7	3	186.28	0.19	87.18
F8	50	0.5	2.5	182.48	0.17	80.59
F9	50	0.5	2.5	184.96	0.18	79.72
F10	65	0.7	2.5	227.15	0.23	92.37
F11	65	0.3	2.5	210.06	0.21	86.89
F12	35	0.5	3	146.51	0.14	74.12
F13	50	0.5	2.5	183.54	0.19	78.67
F14	50	0.3	2	189.02	0.21	78.71
F15	35	0.3	2.5	144.56	0.14	70.47

Figure 1. Effect of independent variables polycaprolactone, chitosan and polyvinyl alcohol on (A). size, (B). PDI, and (C). encapsulation efficiency.

Table 3. Summary of regression analysis for particle size (PS; Y₁), polydispersity index (PDI; Y₂), and entrapment efficiency (EE; Y₃) for fitting data to different models.

Model	R^2	Adjusted R^2	Predicted R^2	SD	Desirability	Remark
Response (Y1)					0.969
Linear	0.9786	0.9727	0.9567	4.27		‒
2F1	0.9850	0.9737	0.9308	4.19		‒
Quadratic	0.9988	0.9967	0.9860	1.47		Suggested
Response (Y2)					0.985
Linear	0.8117	0.7604	0.6103	0.0159		‒
2F1	0.9112	0.8446	0.5840	0.0128		‒
Quadratic	0.9993	0.9981	0.9971	0.0014		Suggested
Response (Y3)					0.977
Linear	0.9763	0.9698	0.9560	1.15		‒
2F1	0.9908	0.9840	0.9801	0.8378		‒
Quadratic	0.9990	0.9973	0.9964	0.3455		Suggested

Table 4. Analysis of variance of response surface quadratic model of each response.

Model	Source	PS	PDI	%EE
Regression analysis
Quadratic	Sum of squares	9336.16	0.0148	612.14
	df	9	9	9
	Mean square	1037.35	0.0016	68.02
	F-Value	477.52	806.29	569.74
	P-value, Prob > F	<0.0001	<0.0001	<0.0001
	Remark	Suggested, significant
Lack of fit tests
Quadratic	Sum of squares	7.77	1.500E-06	0.0616
	df	3	3	3
	Mean square	2.59	5.000E-07	0.0205
	F-Value	1.67	0.1154	0.0768
	P-value, Prob > F	0.39	0.94	0.96
	Remark	Suggested, not significant

Figure 2. Predicted and actual value graph of the (A). particle size; (B). PDI; (C). encapsulation efficiency.

Figure 3. Particle size (A). Zeta potential (B) of optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs)..

Figure 4. Thymoquinone suspension (THQ-S) and optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs). (A) 0.1 N HCl, (B) Phosphate buffer saline.

Table 5. Release kinetic models fitting in terms of linear regression coefficient (R²).

		Plot
Model	Equation	X-axis	Y-axis	R^2
Zero order	${\mathrm{M}}_{\mathrm{t}}={\mathrm{M}}_0+{\mathrm{k}}_0 \mathrm{t}$	Fraction of drug released	time	0.6335
First order	$\ln {\mathrm{M}}_{\mathrm{t}}=\ln {\mathrm{M}}_0+ {\mathrm{k}}_1 \mathrm{t}$	Log % drug remaining	time	0.7965
Korsmeyer–Peppas model	${\mathrm{M}}_{\mathrm{t}}/{\mathrm{M}}_{\mathrm{\infty }}={\mathrm{kt}}^{\mathrm{n}}$	Log fraction of drug released	log time	0.9686
Higuchi matrix model	${\mathrm{M}}_{\mathrm{t}}={\mathrm{M}}_0+{\mathrm{k}}_{\mathrm{H}}{\mathrm{t}}^{1/2}$	Fraction of drug released	$\sqrt{t\mathit{ime}}$	0.8379
Best fitted model		Korsmeyer–Peppas model

Figure 5. Thymoquinone suspension (THQ-S) and optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs). (A) THQ permeated, (B) THQ transported.

Figure 6. Permeation depth image (A). Thymoquinone suspension (THQ-S) (B). Optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs).

Figure 7. Gastric mucosa irritation study image of (A). Normal saline (B). Thymoquinone suspension (THQ-S) (C). Optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs).

Figure 8. Comparative pharmacokinetic study of optimized thymoquinone chitosan-polycaprolactone nanoparticles (THQ-CPLNPs) and thymoquinone suspension (THQ-S).

Table 6. Pharmacokinetic parameters of thymoquinone encapsulated chitosan modified polycaprolactone nanoparticles (THQ-CPLNPs) and THQ suspension (THQ-S).

Parameters	THQ-S	THQ-CPLNPs
${\mathrm{C}}_{\max }$ (µg/mL)	47.53 ± 4.27	164.34 ± 8.74*
${\mathrm{T}}_{\max }$ (h)	4 ± 0.00	2 ± 0.00*
${\mathrm{AUC}}_{0\to 24}$ (µ.h/mL)	400.34 ± 17.38	1670.27 ± 71.95*
${\mathrm{AUC}}_{0\to \infty }$ (µ.h/mL)	430.72 ± 19.54	1836.99 ± 82.17*
${\mathrm{AUMC}}_{0\to 24}$ (µ.h^2/mL)	2766.22 ± 172.78	12807.05 ± 373.68*
${\mathrm{AUMC}}_{0\to \mathrm{\infty }}$ (µ.h^2/mL)	3827.38 ± 211.32	18831.29 ± 423.53^a
MRT (h)	6.91 ± 0.73	7.66 ± 0.57
${\mathrm{t}}_{1/2}$ (h)	7.57 ± 0.67	8.41 ± 0.85
${\mathrm{K}}_{\mathrm{el}}$ (h^‒1)	0.091 ± 0.012	0.082 ± 0.01

^aDenotes highly significant (p < .05) values of optimized TQ-CPLNPs when compared with THQ-S.