Response Surface Methodology for Statistical Optimization of Chitosan/Alginate Nanoparticles as a Vehicle for Recombinant Human Bone Morphogenetic Protein-2 Delivery

Figures & data

Table 1 The Data from Experimental Design of Independent Variables for Optimization of Cs/Alg/B NPs

Variables and Their Levels in the Experiment Design
Independent Variables	Symbol	Levels						Dependent Variables						Units		Constraints
		−1			0	1
pH	A	4.8			5.15	5.5		Y1=Particle Size (PS) Y2=Loading efficacy						nm %		Minimize Maximize
Cs/Alg Mw ratio	B	0.5			1. 75	3		Y3=Polydispersity index (PDI) Y4=ζ-potential						- mv		Less than 0.5 Maximize
Stirring rate (rpm)	C	900			1100	1300		Y5=Cumulative Release (CR) Y6=Morphological degradation time (MDT)						% Day		Maximize Maximize
Experimental design matrix and results of Box–Behnken design
	Independent variables						Dependent variables
Run	A		B	C			Size (nm)	LE (%)			CR (%)	ζ-potential (mV)		PDI				MDT
1	0		0	0			525	25			15	34.3		0.62				4
2	1		0	1			611	19			10	34		0.7				4
3	0		0	0			522	27			17	35		0.65				4
4	1		1	0			898	22			12	23		0.8				2
5	0		−1	−1			189	76			66	22		0.4				14
6	0		−1	1			256	70			60	23		0.55				12
7	−1		0	1			557	22			15	38		0.7				3
8	0		1	−1			802	20			10	23		0.72				2
9	1		−1	0			275	72			60	23		0.6				13
10	0		0	0			523	25			16	37		0.67				4
11	0		1	1			900	19			15	39		0.86				3
12	−1		−1	0			235	70			61	24		0.5				13
13	0		0	0			520	29			19	37		0.61				5
14	0		0	0			519	27			18	35		0.56				4
15	−1		0	−1			501	25			18	33		0.63				4
16	−1		1	0			811	20			15	23		0.76				3
17	0		−1	−1			598	17			11	33		0.68				4
Analysis of variance for Box–Behnken design refined models
Dependent Variables	Source of Variations						Mean Square		F-value	P-value > F			R^2		Adjusted R^2		Predicted R^2
Particle size (PS)	Model Lack of fit						85155.65		534.47	‹0.0001			0.9985		0.9967		0.9772
Polydispersity index (PDI)	Model Lack of fit						0.020		8.46	0.0051			0.9158		0.8075		0.1676
ζ-potential	Model Lack of fit						70.90		45.96	‹0.0001			0.9834		16.074		0.8742
Morphological degradation time (MDT)	Model Lack of fit						31.53		196.77	‹0.0001			0.9954		0.9676		0.9896
Cumulative release (CR)	Model Lack of fit						765.09		212.10	‹0.0001			0.9963		0.9624		0.9916
Loading efficacy (LE)	Model Lack of fit						836.91		117.28	‹0.0001			0.9934		0.9159		0.9849

Figure 1 SEM images of morphological degradation of Cs/Alg/B NPs. (A) The initial stage of degradation on the 7th day of preparation. (B) End-stage of morphological degradation after the 14th day of preparation. For each run MDT is included in optimization as a quantitative factor. The scale bar is 300 nm. More images are shown in Supplementary Figure 5.

Figure 2 3D Response surface plots (A−C) and contour plots (D−F) for the mean particle size of Cs/Alg/B NPs. 3D and contour graphs are the preservative relative relationship between two variables on the size of Cs/Alg/B NPs. Contour plots show a three-dimensional surface on a two-dimensional plane. It graphs two predictor variables, X and Y, on the y-axis and a response variable, Z, as contours. Cs/Alg/B NPs, rhBMP-2-loaded chitosan/alginate nanoparticles.

Figure 3 3D Response surface plots (A−C) and contour plots (D−F) for the mean LE of Cs/Alg/B NPs. 3D and contour graphs are the preservative relative relationship between two variables on the size of Cs/Alg/B NPs LE (%). Contour plots show a three-dimensional surface on a two-dimensional plane. It graphs two predictor variables, X and Y, on the y-axis and a response variable, Z, as contours. LE, loading efficacy (%); Cs/Alg/B NPs, rhBMP-2-loaded chitosan/alginate nanoparticles.

Figure 4 SEM micrographs of Cs/Alg/B NPs prepared with the optimum formulation of Cs/Alg/B NPs with variables as; Cs/Alg Mw ratio=1, stirring rate=5, pH=5.3 at preparation day. The scale bar is 1 µm.

Figure 5 Cytotoxicity effects of chitosan, alginate, and Cs/Alg/B NPs on NIH 3T3cells at 24 and 48 hours. The NIH 3T3 cells were cultured in 96-well plates and were exposed to 10 µg chitosan, alginate, Cs/Alg/B NPs (26 µg/mL), and negative control (RPMI 1640). Positive control (untreated cell). Cell viability calculated as; viability (%)=Mean OD/Control OD×100%. All data are presented as the mean of three different measurements±SD, asterisks (*) represent means the significant difference from the control group by the Bonferroni posttest (P<0.05) and asterisks (****) P<0.0001. OD, optical density; SD, standard deviation.; Cs/Alg/B NPs(rhBMP-2 NPs), rhBMP-2-loaded chitosan/alginate nanoparticles.