Investigating the Impact of Optimized Trans-Cinnamic Acid-Loaded PLGA Nanoparticles on Epithelial to Mesenchymal Transition in Breast Cancer

Figures & data

Table 1 The Experimental Range for Independent Variables and CQAs of CIN-PLGA-NPs

Formulation Parameters (Independent Variables)	Unit	Levels
		−1	1
X1: PLGA amount	mg	100	200
X2: Poloxamer concentration	Percent (%)	0.5	1
CQAs (Responses)	Unit	Constraints
Y1: EE%	Percent (%)	Maximize
Y2: PS	nm	Minimize
Y3: ZP	mV	Maximize

Abbreviations: EE%; entrapment efficiency percent; PS; particle size; ZP; zeta potential.

Table 2 Composition and the Observed Responses of the Design

Runs	Formulation Parameters		Responses
	PLGA (mg)	Poloxamer (%)	EE% (%)	PS (nm)	ZP (mV)	PDI
1	200	0.5	78.61±0.80	230.4±7.85	−32.9±0.70	0.093±0.053
2	79.29	0.75	60.13±0.20	144.9±9.63	−19.5±0.99	0.133±0.022
3	100	0.5	67.4±0.07	156.0±10.39	−24.7±1.23	0.06±0.062
4	150	0.75	72.55±0.29	174.3±17.11	−26.3±0.56	0.048±0.043
5	100	1	62.32±1.25	149.5±6.65	−19.1±1.66	0.053±0.027
6	150	0.75	71.2±1.31	168.5±5.31	−25.2±2.2	0.056±0.071
7	220.71	0.75	80.19±1.29	260.3±20.17	−28.5±0.19	0.245±0.089
8	150	0.75	73.25±1.05	171.2±10.16	−26.1±3.1	0.049±0.063
9	200	1	75.55±0.21	204.3±11.15	−25.3±1.88	0.094±0.031
10	150	0.39	77.54±0.06	176.6±19.01	−28.8±1.72	0.108±0.099
11	150	1.1	69.21±0.80	161.2±23.02	−22.8±4.1	0.075±0.045
12	150	0.75	74.66±0.10	165.9±16.78	−27.9±2.2	0.052±0.096
13	150	0.75	74.5±0.09	169.5±6.89	−24.8±3.6	0.047±0.072

Note: Data represented as mean ± SD (n=3).

Abbreviations: EE%, entrapment efficiency percentage; PS, particle size; PDI, polydispersity index; ZP, zeta potential.

Figure 1 Response 3D plots (A, B and C) and Contour plots (D, E and F) for the effect of PLGA amount (X₁) and Poloxamer188 concentration (X₂) on EE%, PS and ZP.

Figure 2 Transmission electron micrographs of CIN-PLGA-NPs with magnification of 20 kx (A and B), 15kx (C) and 8 kx (D).

Figure 3 In-vitro dissolution profile of CIN-PLGA-NPs compared to CIN-Free.

Figure 4 Effect of CIN-Free and CIN-PLGA-NPs on the viability of MDA-MB-231 breast cancer cell lines, as detected by MTT assay.

Figure 5 Effect of CIN-Free and CIN-PLGA-NPs on the migration of MDA-MB-231 cells using scratch wound assay. (A) Representative photomicrographs for the effect CIN-Free and CIN-PLGA-NPs on wound closure in MDA-MB-231 cells at 0, 24 and 48 hr. (B and C) Bar charts showing wound closure (%) in control untreated, CIN-Free-, and CIN-PLGA-NPs-treated cells. Values are presented as mean ± SD. One-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc test was applied for statistical analysis. Significant difference at **P < 0.01 and ***P < 0.001.

Figure 6 Bar charts showing tumor volumes (mm³) in control and treated groups at (A) 15, (B) 18, and (C) 21 days post treatment. (D) Tumor volume (mm³) in EAC-bearing mice of control and treated groups at different time points. (E) Body weight of animals in treated and control groups, at different time points. Values are presented as mean ± SE. One-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc test was applied for statistical analysis. Significant difference compared to control at *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7 Photomicrographs of H&E-stained tumor sections (×100 and ×400) in control group (A and (B), CIN-Free (C and (D), and CIN-PLGA-NPs (E and (F). Black arrows indicate malignant cells; red arrows indicate apoptosis, and yellow arrows indicate necrotic areas.

Figure 8 Anti-caspase-3 immunohistochemical staining (DAB, ×400) for (A) Control untreated, (B) CIN-free-treated, and (C) CIN-PLGA-NPs-treated groups. (D) Bar chart for the percentage of caspase-3 positive expression in tumor sections of different groups. Values are presented as mean ± SD. One-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc test was applied for statistical analysis. Significant difference compared to control at ***P < 0.001. Black arrows indicate positively stained cells observed as brown discoloration in the cytoplasm.

Figure 9 Semi-quantitative estimation of (A) Necrotic and (B) Apoptotic indices in H&E-stained tumor sections. Levels of (C) E-cadherin and (D) N-cadherin levels in tumor sections estimated by ELISA. Values are presented as mean ± SD. One-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc test was applied for statistical analysis. Significant difference compared to control at *P < 0.05, **P < 0.01, ***P < 0.001.