Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages

Figures & data

Table 1 Levels and Code of Variables Chosen for Box–Behnken Design

Factors	Code	Levels and Range
		−1	0	1
A (lipid-to-cholesterol molar ratio)	X1	4:1	6:1	8:1
B (addition of DSPE-PEG2000 (lipid molar ratio (%))	X2	1%	5%	9%
C (lipid to drug (w/w))	X3	5:1	10:1	15:1

Table 2 The Design and Results of Box–Behnken Experiments

No.	X1: Lipid-to-Cholesterol Molar Ratio	X2: Doses of DSPE-PEG2000	X3: Lipid-to-Drug Weight Ratio	Response: EE (%)
				Predicted EE	Practical EE
1	−1	0	−1	90.86	86.31
2	1	−1	0	82.34	82.41
3	0	−1	−1	89.93	88.57
4	0	1	−1	92.86	94.56
5	−1	1	0	88.03	90.79
6	1	0	1	92.86	80.01
7	0	−1	1	88.05	80.05
8	0	0	0	93.60	91.55
9	−1	−1	0	87.41	90.48
10	0	0	0	92.86	92.02
11	0	1	1	92.86	87.51
12	0	0	0	89.19	92.26
13	−1	0	1	81.21	89.80
14	0	0	0	92.86	94.93
15	0	0	0	92.84	93.51
16	1	1	0	93.39	93.05
17	1	0	−1	78.91	92.23

Table 3 ANOVA for Response Surface Quadratic Model

Source	Sum of Squares	df	Mean Square	F Value	P Value
Model	342.02	9	38.00	18.24	0.0005 Significant
A	11.72	1	11.72	5.63	0.0495
B	74.37	1	74.37	35.70	0.0006
C	73.81	1	73.81	35.44	0.0006
AB	26.63	1	26.63	12.79	0.0090
AC	61.73	1	61.73	29.63	0.0010
BC	0.54	1	0.54	0.26	0.6252
A^2	19.03	1	19.03	9.14	0.0193
B^2	10.05	1	10.05	4.82	0.0641
C^2	55.81	1	55.81	26.79	0.0013
Residual	14.58	7	2.08
Lack of fit	7.12	3	2.37	1.27	0.3971 not significant
Pure error	7.46	4	1.87
Cor total	356.60	16

Notes: R²=0.9591, R_Adj²=0.9065, R_Pred²=0.6480.

Figure 1 2-D contour plots and 3-D response surface plots showing the effects of various parameters on EE. (A and B) The effects of X₁ and X₂ on EE. (C and D) The effects of X₁ and X₃ on EE. (E and F) The effects of X₂ and X₃ on EE.

Abbreviations: X₁, lipid-to-cholesterol molar ratio; X₂, doses of DSPE-PEG₂₀₀₀; X₃, lipid-to-drug weight ratio; EE, encapsulation efficiency.

Figure 2 Particle size and morphology of the optimized pRL. (A) Morphology of the pRL observed by TEM. (B) Particle size of the optimized pRL was measured by DLS.

Abbreviations: pRL, PEGylation nano-RGP; DLS, dynamic light scattering; TEM, transmission electron microscopy.

Table 4 The Effects of Different Lyoprotectants on Formability and Redispersibility of the Lyophilized pRL

Cryoprotectant	Formability	Redispersibility
Lactose	+++	++++
Sucrose	++++	++++
Mannitose	++	+
Mycose	++++	++++
Sorbitol	+	++
Glucose	+	+++
Xylitol	+	+++

Abbreviation: pRL, PEGylation nano-RGP.

Table 5 Comparision of the EE and the Retention Rate Under Different Lipid-to-Cryoprotectant Ratios

	Lipid: Cryoprotectant	EE After Lyophilization (%)	Retention Rate (%)
Lactose	1:1	91.67±3.02	97.41±2.90
	2:1	92.49±1.15	99.08±1.31
	3:1	86.00±3.82	92.43±4.10
	4:1	88.82±4.16	95.46±4.47
Mycose	1:1	91.26±6.68	95.67±6.53
	2:1	89.19±5.10	95.86±5.49
	3:1	80.87±5.87	86.92±6.31
	4:1	79.73±10.48	85.68±1.26
Sucrose	1:1	59.10±10.82	63.51±1.63
	2:1	60.43±8.34	64.94±8.96
	3:1	73.51±1.17	78.99±1.26
	4:1	70.23±1.19	75.47±1.28

Abbreviation: EE, encapsulation efficiency.

Figure 3 (A) Cell viability of RAW264.7 in the stimulation of pRL, RL, BL and RGP at 450nm. LPS was the positive control and the BC was the negative control. (B-E) The effects of pRL on the secretion of IL-6, IL-12, IL-1β and TNF-α. (**P<0.01).

Abbreviations: pRL, PEGylation nano-RGP; RL, nano-RGP without PEGylation; BL, blank nano-vehicle; RGP, Rehmannia glutinosa polysaccharide; LPS, lipopolysaccharide; BC, blank control.

Figure 4 Cellular uptake of RAW264.7 cell lines. (A) The intracellular uptake and subcellular distribution of DiD-labeled pRL (50 μg/mL) were investigated after 4hr incubation under the confocal (Green: LysoTracker) (upper line: the scale bar is 50 μm. Lower line: the scale bar is 20 μm). (B) The intracellular uptake of DiD-labeled pRL was quantified by flow cytometry. (C) The quantification data of the flow cytometry results (***P<0.001).

Abbreviation: pRL, PEGylation nano-RGP.

Figure 5 pRL internalized cell via the macropinocytosis- and caveolae-mediated endocytotic pathway. The changes in cell viability by cytochalasin D (A), genistein (B) and chlorpromazine (C) were measured by CCK-8 method (*P<0.05, ***P<0.001).

Abbreviation: pRL, PEGylation nano-RGP.