Development and optimization of curcumin analog nano-bilosomes using 2¹.3¹ full factorial design for anti-tumor profiles improvement in human hepatocellular carcinoma: in-vitro evaluation, in-vivo safety assay

Figures & data

Figure 1. Chemical structure of curcumin (CU) and piperidone (PIP).

Table 1. 2¹.3¹ full factorial design variables and constraints.

Independent variables	Levels
	−1	0	+1	Constrains
X1: phospholipid type	PDC	–	PDS	In range
X2: SC concentration	0.1%	0.3%	0.6%	In range
Dependent variables
R1: EE (%)				Maximize
R2: ZP (mV)				Maximize
R3: PS (nm)				Minimize

PDC: phosphatidyl choline; PDS: phosphatidyl serine; SC: sodium cholate; EE%: entrapment efficiency; ZP: zeta potential; PS: particle size.

Table 2. Independent variables and measured responses for the 2¹.3¹ full factorial experimental design of PIP loaded BLs.

Formulation code	X1 phospholipid type	X2 SC conc. (%)	R1 EE (%)	R2 ZP (mV)	R3 PS (nm)	PDI
F1	PDC	0.1	96.98 ± 1.4	−15.13 ± 2.03	168.17 ± 0.08	0.008 ± 0.023
F2	PDC	0.3	91.04 ± 2.6	−21.20 ± 1.56	200.23 ± 1.05	0.005 ± 0.072
F3	PDC	0.6	87.33 ± 2.45	−28.22 ± 1.14	265.2 ± 0.32	0.002 ± 0.112
F4	PDS	0.1	90.21 ± 1	−27.05 ± 1.08	111.68 ± 1.4	0.006 ± 0.023
F5	PDS	0.3	86.11 ± 0.94	−30.96 ± 1.12	141.99 ± 0.98	0.004 ± 0.091
F6	PDS	0.6	81.08 ± 1.7	−35.11 ± 1.56	206.78 ± 1.2	0.003 ± 0.012

Cholesterol (CH) and pluronic 123 (P 123) were kept constant in all formulae at 0.3 and 0.6%, respectively.

Figure 2. In-vitro release profiles of synthesized PIP from different prepared BLs compared to parent CU suspension and synthesized PIP suspension.

Table 3. In vitro release kinetics for the prepared PIP loaded BLs.

Release model	Formulation code
	CU Susp.	PIP Susp.	F1	F2	F3	F4	F5	F6
Higuchi	R^2 = 0.935	R^2 = 0.952	R^2 = 0.964	R^2 = 0.567	R^2 = 0.663	R^2 = 0.963	R^2 = 0.663	R^2 = 0.583
First order	R^2 = 0.760	R^2 = 0.840	R^2 = 0.868	R^2 = 0.945	R^2 = 0.99	R^2 = 0.90	R^2 = 0.977	R^2 = 0.975
Zero order	R^2 = 0.447	R^2 = 0.690	R^2 = 0.283	R^2 = 0.548	R^2 = 0.689	R^2 = 0.38	R^2 = 0.687	R^2 = 0.671

The bolded and underlined values are the highest values of correlation coefficient (R2) representing the kinetic model that best fit the drug release mode of each formula.

Table 4. Summary of 2¹.3¹ full factorial design results for prepared PIP loaded BLs.

Response	SD	R^2	Adjusted R^2	Predicted R^2	Adequate precision	Model F-value
R1: EE(%)	0.45	0.9957	0.9835	0.9917	55.342	457.84
R2: ZP (mV)	0.10	0.9999	0.9998	0.9994	282.408	11,429.77
R3: PS (nm)	0.22	1.0000	1.0000	0.9999	978.8	13,799.5

SD: standard deviation.

Figure 3. One factor plot of: (a) the effect of X₁ on R₁, (b) the effect of X₂ or R₁, (c) the effect of X₁ on R₂, (d) the effect of X₂ or R_{2, (}e) the effect of X₁ on R₃, (f) the effect of X₂ or R₃.

Figure 4. 3D surface plot of: (a) the effect of X₁ and X₂ on R₁, (b) the effect of X₁ and X₂ or R₂, (c) the effect of X₁ and X₂ on R₃.

Figure 5. TEM micrographs of optimized PIP loaded BLs (F4) with 80,000 Å magnification.

Figure 6. Cytotoxicity assay of CU suspension and PIP loaded BL on Wi-38 cell line.

Figure 7. Anticancer and Selectivity index (SI) of CU suspension and PIP loaded BLs against Huh-7 cell line.

Figure 8. Huh-7 cellular uptake of CU suspension and PIP loaded BLs, (A,B) CUD uptake, (C,D) PIP uptake.

Figure 9. Quantification of the induced ROS in Huh-7 cells, (A) the control untreated cells, (B) LPS-induced cells (positive control), (C) PIP-treated cells, (D) CU-treated cells, (E) PIP-LPS-treated cells, (F) CU-LPS-treated cells, (G) the gatting values of the induced ROS in Huh-7 cells.