RAGE receptor targeted bioconjuguate lipid nanoparticles of diallyl disulfide for improved apoptotic activity in triple negative breast cancer: in vitro studies

Figures & data

Table 1. Independent and dependent variables applied in experimental design.

	Levels
Factor	Low (−1)	Medium (0)	High (+1)
Independent variables
Lipid (X1)	50	75	100
Surfactant (X2)	0.5	1.5	2.5
Co surfactant (X3)	1.0	1.5	2.0
Dependent variables
Particle size (Y1)	Minimize
Entrapment efficiency (Y2)	Maximize

Figure 1. Response surface plots showing the effects of lipid, surfactant and co-surfactant on particle size (a) and entrapment efficiency (b). Perturbation plot showing the effect of lipid, surfactant and co-surfactant on particle size (c) and entrapment efficiency (d).

Table 2. Experimental batches and responses obtained for desired parameters.

Std. Order	Run	Block	Factor 1 A: Lipid (mg)	Factor 2 B: Surfactant (%)	Factor 3 C: Co.surfactant (%)	Response 1: PS (nm)	Response 2: EE (%)
9	1	1	75	0.5	1	216.25	40.27
4	2	1	100	2.5	1.5	148.30	55.89
15	3	1	75	1.5	1.5	174.10	57.32
11	4	1	75	0.5	2	149.50	60.18
17	5	1	75	1.5	1.5	166.40	63.12
1	6	1	50	0.5	1.5	106.62	68.35
6	7	1	100	1.5	1	188.12	59.52
5	8	1	50	1.5	1	140.12	58.70
12	9	1	75	2.5	2	192.36	47.40
3	10	1	50	2.5	1.5	100.37	56.45
2	11	1	100	0.5	1.5	202.62	50.71
10	12	1	75	2.5	1	117.51	46.34
13	13	1	75	1.5	1.5	167.23	54.36
16	14	1	75	1.5	1.5	169.41	60.79
7	15	1	50	1.5	2	117.87	79.10
8	16	1	100	1.5	2	214.85	67.00
14	17	1	75	1.5	1.5	166.40	60.79

Table 3. Analysis of variance (ANOVA) for response surface quadratic model (for PS).

	F value	p Value Prob > F	Remarks
Quadratic model			Significant (p < .05)
A-Lipid	356.3461417	<.0001
B-Surfactant	1610.472914	<.0001
C-Co surfactant	263.3023652	<.0001
AB	2.895045264	.1326
AC	89.17677501	<.0001
BC	92.64144846	<.0001
A^2	769.8215948	<.0001
B^2	173.0944432	<.0001
C^2	105.775055	<.0001
Residual	110.0033404	<.0001
Lack of fit	0.122030829	.9424	Not significant

Table 4. Analysis of variance (ANOVA) for response surface quadratic model (for EE).

	F value	p Value Prob > F
Quadratic model	16.51321	.0006	Significant (p < .05)
A-Lipid	12.82983	.0090
B-Surfactant	2.669539	.1463
C-Co surfactant	35.18557	.0006
AB	8.598451	.0220
AC	4.924361	.0620
BC	10.47904	.0143
A^2	32.2222	.0008
B^2	44.61698	.0003
C^2	0.775362	.0078
Residual
Lack of fit	0.33554	.8018	Not significant

Figure 2. Conjugation reaction between –COOH group of palmitic acid and –NH2 group of RAGE antibody.

Figure 3. FT-IR Spectra of Palmitic acid (a); RAGE antibody (b); and DADS-RAGE-SLN (c).

Figure 4. Particle size distribution of DADS-SLN (a) and DADS-RAGE-SLN (b); SEM (c) and TEM (d) of DADS-RAGE-SLNs.

Figure 5. In vitro release of DADS from DADS-SLN and DADS-RAGE-SLN.

Figure 6. Qualitative (a) and quantitative (b) cell uptake of DADS-SLN and DADS-RAGE-SLN. The values are expressed as mean ± SD.*p < .05 vs DADS-SLN (n = 3).

Figure 7. % cell viability after treatment with DADS, RAGE antibody, DADS-SLN, DADS-RAGE-SLN and Blank-SLN. The values are expressed as mean ± SD.*p < .05 vs DADS-SLN (n = 3).

Figure 8. Apoptosis measurement using Annexin-V/PI assay in control (a); DADS-treated cells (b); DADS-SLN-treated cells (c) and DADS-RAGE-SLN-treated cells (d).

Figure 9. Expression pattern of Bcl2, survivin and caspase-9 in MDA-MB231 cells (a); Quantitative analysis of Bcl2 (b), survivin (c) and caspase-9 (d) by observing relative intensity with β-actin. The values are expressed as mean ± SD. *p < .05 vs control (n = 3).