Polyvinyl Alcohol (PVA)-Based Nanoniosome for Enhanced in vitro Delivery and Anticancer Activity of Thymol

Figures & data

Table 1 Optimization of Nio-TH Using Box–Behnken Method

Run	Variables			Responses
	Lipid, µmol	Hydration Time, Min	Vitamin E: Span 60, Molar Ratio	Average Size (nm)	PDI	EE (%)
1	0	−1	1	232.5	0.251	64.49
2	−1	0	1	220.5	0.305	70.25
3	−1	0	−1	249.3	0.325	63.52
4	0	0	0	162.9	0.178	67.54
5	1	0	−1	322.1	0.295	68.41
6	1	1	0	195.4	0.243	72.91
7	0	0	0	170.1	0.194	66.04
8	−1	−1	0	265.9	0.291	66.31
9	1	0	1	275.4	0.342	77.43
10	0	−1	−1	367.9	0.334	59.75
11	−1	1	0	191.1	0.234	63.75
12	0	1	1	162.7	0.223	74.59
13	0	0	0	156.4	0.165	64.45
14	0	1	−1	208.6	0.29	54.39
15	1	−1	0	329.7	0.369	81.95

Figure 1 (A–C) Box–Behnken method for size (A), PDI (B), and EE (C) as a function of the lipid content and molar ratio of Vitamin (E) Span 60; (D–G) The optimized responses obtained by Box–Behnken design method under the optimum conditions: (D) Vesicle size, (E) PDI, (F) EE (%), and (G) Zeta potential. Data are represented as mean ± SD and n=3; ***p<0.001, **p<0.01, *p<0.05.

Table 2 ANOVA Statistical Analysis for the Quadratic Polynomial Model for Particle Size, PDI, and Entrapment Efficiency

Source	F-value	p-value
Particle size (nm)
Model	16.84	0.0031	Significant
A	12.06	0.0178
B	60.39	0.0006
C	20.74	0.0061
AB	2.23	0.1958
AC	0.20	0.6723
BC	5.04	0.0748
A^2	26.24	0.0037
B^2	7.95	0.0372
C^2	23.74	0.0046
PDI
Model	5.33	0.0281	Significant
A	0.98	0.3598
B	7.23	0.0361
C	1.68	0.2422
AB	1.06	0.3431
AC	1.00	0.3562
BC	17.87	0.0055
A^2	3.26	0.1210
B^2	13.46	0.0105
C^2	5.33	0.0281
Entrapment efficacy (%)
Model	4.91	0.0473	Significant
A	12.32	0.0171
B	0.43	0.5425
C	15.01	0.0117
AB	0.76	0.4229
AC	0.095	0.7703
BC	4.33	0.0919
A^2	9.35	0.0282
B^2	0.13	0.7361
C^2	1.09	0.3446

Table 3 Predicted Models of Nio-TH

Models
Particle Size (nm) = +163.13+24.47 * A-54.77* B-32.10 * C-14.88* A * B-4.48* A * C+22.37* B * C+53.15 * A^2+ 29.25 * B^2+ 50.55 * C^2
PDI = +0.18+0.012 * A- 0.032* B- 0.015* C-0.017* A * B +0.017* A * C +0.074 * A^2+ 0.031 * B^2+ 0.064 * C^2
Entrapment Efficiency (%) = +66.01+ 4.61 * A-0.86* B+5.09* C-1.62* A * B +0.57* A * C +3.87* B * C+ 5.91 * A^2- 0.69 * B^2-2.02 * C^2

Table 4 Summary of the Results of Regression Analysis for the Various Parameters (Size, PDI, and Entrapment Efficiency) Employed for Fitting to the Quadratic Model

Response	R-Square	Adjusted R- Square	Adequate Precision	Lack of Fit
Particle size	0.9681	0.9106	11.614	0.0701
PDI	0.8766	0.7121	6.398	0.1213
Entrapment efficacy (%)	0.8983	0.7153	8.796	0.1020

Table 5 Desirability Criteria and Predicted Values for the Variables

Lipid, µmol	Hydration Time, Min	Vitamin E: Span 60, Molar Ratio	Desirability
282	43.70	40:60	0.707

Figure 2 (A) Particle size distribution of Vehicle (Nio/PVA), Nio-TH, and Nio-TH/PVA by Dynamic Light Scattering (DLS). (B and C) Morphological analysis by SEM; (B) optimum Nio-TH, (C) Nio-TH/PVA. (D and E) Morphological analysis by TEM. (D) optimum Nio-TH, (E) Nio-TH/PVA.

Figure 3 (A) In vitro release of TH from Nio-TH and Nio-TH/PVA at pH 7.4 and pH 5.4; (B and C) Size stability evaluation of Nio-TH (B), and Nio-TH/PVA (C); (D and E) PDI stability evaluation of Nio-TH (D), and Nio-TH/PVA (E); (F and G) EE (%) stability evaluation of Nio-TH (F), and Nio-TH/PVA (G) after two months of storage at 4 ± 2°C and 25 ± 2 °C. Data are represented as mean ± SD and n=3; ***P<0.001, **P<0.01, *P<0.05.

Table 6 The Kinetic Release Models and the Parameters Obtained for Optimum Niosomal Formulation

Release Model	Equation	R2
		Free TH (pH=7.4–37°C)	Nio- TH (pH=7.4–37°C)	Nio- TH (pH=5.4–37°C)	Nio-TH/PVA (pH=7.4–37°C)	Nio-TH/PVA (pH=5.4–37°C)
Zero-Order	Ct=C0+K0t	R^2=0.6552	R^2=0.7890	R^2=0.8595	R^2=0.8193	R^2=0.8158
First-Order	LogC=LogC0+Kt/2.303	R^2=0.9535	R^2=0.8319	R^2=0.9398	R^2=0.8503	R^2=0.8504
Higuchi		R^2=0.8217	R^2=0.9216	R^2=0.9622	R^2=0.9405	R^2=0.9362
Korsmeyer-Peppas	Mt/Mꝏ=Ktn	R^2=0.8885	R^2=0.9389	R^2=0.9580	R^2=0.9411	R^2=0.9713
		n=0.4199	n=0.5076	n=0.5161	n=0.5999	n=0.4883

Table 7 IC50 Levels in the Three Cancerous Cell Lines Treated with TH, Nio-TH and Nio-TH/PVA. Data are Represented as Mean ± SD and n=3

Cell Line	TH	Nio-TH	Nio-TH/PVA
MCF-7	55.17±1.89	37.14±1.76	27.17±1.35
AGS	92.46±2.32	63.20±1.93	39.74±1.55
HepG2	170.25±1.85	122.50±2.64	74.45±2.35

Figure 4 Different concentrations of TH, Nio-TH, and Nio-TH/PVA (0, 10, 20, 40, 80 and 160 μg/mL) were added to the cultured MCF-7, AGS, HepG2 and HFF cells seeded onto 96-well (2×10⁵ cell/well) (A) The effect of different dilutions of Nio/PVA on cell viability in MCF-7, AGS, HepG2 and HFF cell lines. (B) Cytotoxicity evaluation of TH, Nio-TH and Nio-TH/PVA on HFF cell line. The impact of TH, Nio-TH, and Nio-TH/PVA on cell viability in (C) MCF-7, (D) AGS, (E) HepG2 cancerous cell lines. (F) Bar plot and (G) Heat-map of the IC50 levels in the three cancerous cell lines treated with nanodrug samples. Data are represented as mean ± SD and n=3; The p-values are *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5 MCF-7, AGS, and HepG2 cancer cells (1×10⁸ cells/well in 96-well plate) were treated with IC50 concentration of Vehicle, TH, Nio-TH, and Nio-TH/PVA. The expression levels and its heat-map for Caspase-3 (A and B), Caspase-9 (C and D), MMP-2 (E and F), MMP-9 (G and H), CyclinD (I and J), CyclinE (K and L) genes in MCF-7, AGS, and HepG2 cells after treatment with various samples. The IC50 was used for each sample. Data are represented as mean ± SD and n=3; ***p<0.001, **p<0.01, *p<0.05.

Figure 6 Flowcytometric analysis of all samples in (A) MCF-7, (B) AGS, and (C) HepG2 cell lines. Cell suspension was adjusted to 5×10⁵ cells/mL and were seeded onto 6-well and treated with IC50 concentration of various formulations. Analysis of the apoptosis rate in (D) MCF-7, (E) AGS, and (F) HepG2 cancer cell lines after incubation with different samples. Q1 (necrotic cells), Q2 (late apoptosis), Q3 (early apoptosis), and Q4 (alive cells). (G) Apoptosis rate in MCF-7, AGS, and HepG2 cell lines treated with all samples. (H) Heat-map of apoptosis analysis of all samples in MCF-7, AGS, and HepG2 cell lines. Data are represented as mean ± SD and n=3; ***p<0.001, **p<0.01, *p<0.05.

Figure 7 Cells were seeded at a density of 1×10⁶ cells per well in complete medium in 6-well plates All of these experiments were conducted using IC50 concentration. Histograms showing DNA content of cell cycle progression of all samples in (A) MCF-7, (B) AGS, and (C) HepG2 cell lines. Cell cycle distribution of (D) MCF-7, (E) AGS, (F), HepG2 cell lines treated with all samples. The SubG1 percentage in all the cancerous cell lines treated with all samples, shown as (G) A bar plot and (H) A heat-map. Data are represented as mean ± SD and n=3; ***p<0.001, **p<0.01, *p<0.05.

Figure 8 (A) Bar plot and (B) heat-map showing activity levels of Caspase-3 of all samples in MCF-7, AGS, and HepG2 cell lines that were treated with IC50 concentration. (C) Bar plot and (D) heat-map showing activity levels of Caspase-9 of all samples in MCF-7, AGS, and HepG2 cell lines. (E) Bar plot and (F) heat-map showing DCF fluorescence of all samples in MCF-7, AGS, and HepG2 cell lines. Data are represented as mean ± SD and n=3; ***p<0.001, **p<0.01, *p<0.05.

Figure 9 Qualitative Migration analysis of all samples in (A) MCF-7, (B) AGS, and (C) HepG2 cell lines exposed to IC50 concentration of vehicle, TH, Nio-TH, and Nio-TH/PVA. (D) Migration rate of the three cancerous cell lines in all samples presented as bar plots. (E) Heat-map of migration analysis of all samples in MCF-7, AGS, and HepG2 cell lines. Data are represented as mean ± SD and n=3; ***p<0.001.

Figure 10 DAPI staining determining the number of nuclei of all samples in (A) MCF-7, (B) AGS, and (C) HepG2 cell lines. MCF-7, AGS, and HepG2 cells were plated onto a 24-well plate and exposed to the IC50 concentration of formulations for 48 hrs.