Chitosan-coated PLGA nanoparticles of bevacizumab as novel drug delivery to target retina: optimization, characterization, and in vitro toxicity evaluation

Figures & data

Table 1. Levels of independent and dependent variables used in experiments.

	Level used, actual (coded)
Factor	Low	Medium	High
Independent variables
A = Chitosan conc. (%w/v)	0.25	0.50	0.75
B = PVA concentration (% w/v)	0.50	0.75	1.00
C = PLGA content (mg/mL)	2.00	3.50	5.00
D = Sonication time (min)	5	10	15
Dependent variables
Y1 = Response  1 = Particle size (nm)		Minimize
Y2 = Response  2 = Polydispersity index		Minimize (optimize >0.3)

Table 2. Observed values of responses for Box–Behnken design.

	Factor 1	Factor 2	Factor 3	Factor 4	Response 1	Response 2
Run	A:CS (%w/v)	B:PVA (%w/v)	C:PLGA (mg/mL)	D:SONICATION (min)	Particle size (nm)	PDI
1	0.5	0.5	3.5	5	367 ± 29.41	0.26 ± 0.04
2	0.5	1.00	5	10	371 ± 32.11	0.25 ± 0.06
3	0.5	0.75	3.5	10	239 ± 18.49	0.23 ± 0.08
4	0.25	0.75	3.5	5	283 ± 23.37	0.27 ± 0.11
5	0.75	0.75	3.5	15	309 ± 26.51	0.37 ± 0.09
6	0.25	0.75	2	10	270 ± 19.47	0.20 ± 0.03
7	0.5	0.75	2	5	398 ± 41.52	0.31 ± 0.07
8	0.5	0.5	2	10	420 ± 38.67	0.23 ± 0.05
9	0.5	0.75	3.5	10	226 ± 27.14	0.24 ± 0.12
10	0.5	1.00	3.5	5	331 ± 22.75	0.27 ± 0.03
11	0.75	0.5	3.5	10	367 ± 33.21	0.24 ± 0.06
12	0.5	0.5	5	10	350 ± 28.43	0.26 ± 0.08
13	0.5	0.75	3.5	10	236 ± 16.78	0.23 ± 0.02
14	0.5	0.75	5	15	335 ± 35.11	0.42 ± 0.16
15	0.5	0.75	5	5	392 ± 37.87	0.31 ± 0.18
16	0.75	1.00	3.5	10	303 ± 14.89	0.16 ± 0.01
17	0.75	0.75	3.5	5	364 ± 29.61	0.24 ± 0.09
18	0.5	1.00	3.5	15	243 ± 11.98	0.32 ± 0.13
19	0.25	0.75	3.5	15	210 ± 23.41	0.31 ± 0.04
20	0.25	0.75	5	10	296 ± 27.13	0.26 ± 0.12
21	0.25	0.5	3.5	10	265 ± 23.58	0.17 ± 0.02
22	0.5	0.75	2	15	310 ± 30.17	0.34 ± 0.15
23	0.75	0.75	5	10	365 ± 27.34	0.25 ± 0.09
24	0.75	0.75	2	10	386 ± 34.25	0.24 ± 0.05
25	0.5	0.5	3.5	15	339 ± 28.96	0.36 ± 0.14
26	0.25	1.00	3.5	10	212 ± 14.39	0.21 ± 0.02
27	0.5	1.00	2	10	272 ± 17.23	0.20 ± 0.07

Table 3. Formulation selected by point prediction for optimization.

Formulation code	Optimized composition (A:B:C:D)	Responses	Experimental value	Predicted value	%Error	%EE and %DL
CS-NP1	0.25:0.74:3.50:10.0	R1	211.93 ± 17.26	201.63 ± 10.32	+4.86	59.41 ± 7.13
		R2	0.21 ± 0.16	0.20 ± 0.06	+3.57	5.81 ± 0.56
CS-NP2	0.25:1.00:3.50:10.0	R1	222.28 ± 7.45	212.17 ± 8.26	+4.54	67.28 ± 5.18
		R2	0.19 ± 0.08	0.21 ± 0.11	−10.52	6.19 ± 1.07
CS-NP3	0.25:0.91:4.00:10.0	R1	238.11 ± 16.36	223.34 ± 14.21	+6.20	55.47 ± 6.11
		R2	0.23 ± 0.15	0.22 ± 0.05	+4.35	5.32 ± 0.69
CS-NP4	0.35:0.75:3.50:10.0	R1	207.54 ± 11.21	210.85 ± 6.91	−1.59	48.79 ± 6.23
		R2	0.24 ± 0.05	0.22 ± 0.09	+8.33	4.89 ± 0.91
CS-NP5	0.50:0.75:3.50:10.0	R1	239.61 ± 9.27	233.67 ± 11.45	+2.48	52.34 ± 4.16
		R2	0.21 ± 0.02	0.23 ± 0.05	−9.52	5.02 ± 1.21

Where R₁: particle size; R₂: PDI; EE: entrapment efficiency; DL: drug loading.

Figure 1. (A)–(D) Three-dimensional (3D) response surface plots showing relative effects of different process parameters on the particle size and PDI.

Table 4. Summary of results of regression analysis for responses R₁ and R₂

Model	R^2	Adjusted R^2	Predicted R^2	SD	%CV	Remark
Response 1 (R1)
Linear	0.5086	0.4193	0.3210	47.12
2FI	0.5986	0.3477	0.0918	49.94
Quadratic	0.9965	0.9925	0.9833	5.37	1.71	Suggested
Cubic	0.9988	0.9924	0.9642	5.38
Response 2 (R2)
Linear	0.2429	0.1053	−0.1783	0.058
2FI	0.3313	−0.0867	−1.1696	0.064
Quadratic	0.9968	0.9931	0.9841	5.069E-003	1.91	Suggested
Cubic	0.9985	0.9905	0.8872	5.951E-003

Table 5. Analysis of variance of calculated model for responses.

Result of the analysis of variance	Particlesize (nm)	Polydispersityindex (PDI)
Regression
Sum of squares	99,082.21	0.097
Degree of freedom (df)	14	14
Mean squares	7077.30	6.912E-003
F value	245.87	269.00
P	<0.0001	<0.0001
Lack of fit tests
Sum of squares	252.75	2.417E-004
Degree of freedom (df)	10	10
Mean squares	25.28	2.417E-005
F value	0.55	0.72
P	0.7902	0.7042
R^2	0.9965	0.9968
Correlation of variation (% CV)	1.71	1.91
Residual
Sum of squares	345.42	3.083E-004
Degree of freedom (df)	12	12
Mean squares	28.78	2.569E-005
SD	5.37	5.069E-003

Figure 2. (A) TEM image of chitosan-coated PLGA nanoparticles. (B) Zeta potential of the optimized formulation.

Figure 3. (A) In vitro release study of different NPs. (B) Comparison of drug release profile of CS-coated NPs, PLGA NPs, and drug solution.

Figure 4. Ex vivo transscleral permeation of NPs and drug solution.

Figure 5. HET-CAM test image of formulation treated (A), negative control (B), and positive control (C).

Table 6. Scores obtained in HET-CAM test.

		Scores
		Hemorrhage (A)			Hyperemia (B)			Coagulation (C)
		Time (min)			Time (min)			Time (min)			Mean scores for 5 min
Formulation	Egg	0.5	2	5	0.5	2	5	0.5	2	5	A + B + C/3
Formulation	Egg 1	0	0	0	0	0	0	0	0	0.6	0.53
	Egg 2	0	0	0	0	0	0	0	0	0.5
	Egg 3	0	0	0	0	0	0.5	0	0	0
	Egg 1	0	0	0	0	0	0	0	0	0
Normal saline	Egg 2	0	0	0	0	0	0	0	0	0	0
	Egg 3	0	0	0	0	0	0	0	0	0
	Egg 1	0	1	2	0	0	2	0	2	5
0.1 M NaOH	Egg 2	0	2	4	0	0	1	0	0	6	12
	Egg 3	0	0	3	0	1	2	0	0	5