Development and evaluation of luteolin loaded pegylated bilosome: optimization, in vitro characterization, and cytotoxicity study

Figures & data

Table 1. Optimization variables and their responses used for Box–Behnken’s design software.

Factor	Levels used
	Low level (–1)	Middle level (0)	High level (+1)
Formulation variables
A: Surfactant (Span 60, mg)	100	200	300
B: Cholesterol (CHOR, %)	1	3	5
C: Bile salt (mg)	0.15	0.25	0.35
D: PEG-2000 (mg)	1	2.5	4
Responses	Constrains
Y1: vesicle size (VS)	Minimized
Y2: entrapment efficiency (EE)	Maximized

Table 2. Formation composition of apigenin bilosomes (APG-BLs) with actual and predicted value of the vesicle size (Y₁) and entrapment efficiency (Y₂).

Code	A (mg)	B (mg)	C (mg)	D (mg)	Y1 (nm)		Y2 (%)
					Actual	Predicted	Actual	Predicted
LL-PG-BLs 1	30	15	12.5	30	235	238	53	51
LL-PG-BLs 2	60	15	12.5	30	100	101	68	66
LL-PG-BLs 3	30	35	12.5	30	283	280	76	75
LL-PG-BLs 4	60	35	12.5	30	285	284	88	87
LL-PG-BLs 5	45	25	5	20	248	247	69	68
LL-PG-BLs 6	45	25	20	20	266	265	77	74
LL-PG-BLs 7	45	25	5	40	324	326	67	64
LL-PG-BLs 8	45	25	20	40	243	245	76	75
LL-PG-BLs 9	30	25	12.5	20	263	262	65	64
LL-PG-BLs 10	60	25	12.5	20	200	202	80	83
LL-PG-BLs 11	30	25	12.5	40	300	298	66	64
LL-PG-BLs 12	60	25	12.5	40	223	225	75	73
LL-PG-BLs 13	45	15	5	30	205	203	58	56
LL-PG-BLs 14	45	35	5	30	325	327	78	79
LL-PG-BLs 15	45	15	20	30	185	183	66	64
LL-PG-BLs 16	45	35	20	30	281	284	86	88
LL-PG-BLs 17	30	25	5	30	296	295	60	59
LL-PG-BLs 18	60	25	5	30	236	233	76	75
LL-PG-BLs 19	30	25	20	30	268	267	70	72
LL-PG-BLs 20	60	25	20	30	200	197	80	78
LL-PG-BLs 21	45	15	12.5	20	179	179	63	61
LL-PG-BLs 22	45	35	12.5	20	290	288	83	83
LL-PG-BLs 23	45	15	12.5	40	206	205	60	58
LL-PG-BLs 24	45	35	12.5	40	320	321	83	82
LL-PG-BLs *25	45	25	12.5	30	260	256	72	74
LL-PG-BLs *26	45	25	12.5	30	254	256	71	74
LL-PG-BLs *27	45	25	12.5	30	256	256	75	74

A: surfactant (mg); B: cholesterol (%); C: bile salt (mg); D: PEG-2000 (mg); Y₁: vesicle size (nm); Y₂: entrapment efficiency (%).

Figure 1. 3D plots showing the influence of formulation variables. (A) Surfactant (Span-60, mg), (B) cholesterol (CHOR, %), (C) bile salt (mg), and (D) polyethylene glycol-2000 (mg) on the vesicle size.

Figure 2. 3D plots showing the influence of formulation variables. (A) Surfactant (Span 60, mg), (B) cholesterol (CHOR, %), (C) bile salt (mg), and (D) polyethylene glycol-2000 (mg) on the entrapment efficiency (%).

Figure 3. Actual and predicted plots of LL-PG-BLs showing the effect on vesicle size (nm) and entrapment efficiency (%).

Table 3. Model statistical summary of all applied models for vesicle size and entrapment efficiency.

Source	Std. Dev.	R^2	Adjusted R^2	Predicted R^2	PRESS	Remark
Vesicle size (Y1)
Linear	23.32	0.8259	0.7943	0.72679	18786.54
2FI	16.53	0.9363	0.8966	0.79256	14264.05
Quadratic	2.92	0.9985	0.9968	0.99239	523.6022	Suggested
Entrapment efficiency (Y2)
Linear	1.46	0.9776	0.9734	0.9679	66.70
2FI	1.17	0.9895	0.9830	0.9800	41.53
Quadratic	0.51	0.9984	0.9967	0.9916	17.34	Suggested

Table 4. ANOVA evaluation of best fitted quadratic model for vesicle size and entrapment efficiency.

Source	Vesicle size (Y1)				Entrapment efficiency (Y2)
	Sum of squares	df	F Value	p Value Prob>F	Sum of squares	df	F Value	p Value Prob>F
Model	68658.83	14	575.39	<.0001	2076.57	14	568.11	<.0001
A: surfactant (Span 60)	13240.37	1	1553.45	<.0001	511.97	1	1960.92	<.0001
B: CHOR	37985.85	1	4456.76	<.0001	1337.37	1	5122.34	<.0001
C: bile salt	3005.04	1	352.57	<.0001	173.72	1	665.39	<.0001
D: PEG-2000	2562.84	1	300.69	<.0001	9.97	1	38.21	<.0001
AB	4944.13	1	580.08	<.0001	4.23	1	16.18	.0017
AC	16	1	1.87	.1957	9.42	1	36.07	<.0001
AD	48.81	1	5.73	.0339	9.02	1	34.54	<.0001
BC	135.21	1	15.86	.0018	0.14	1	0.54	.4751
BD	16.12	1	1.89	.1941	1.58	1	6.05	.0300
CD	2432.46	1	285.39	<.0001	0.52	1	2.01	.1814
A^2	1382.57	1	162.21	<.0001	16.2	1	62.13	<.0001
B^2	1130.33	1	132.61	<.0001	6.07	1	23.26	.0004
C^2	330.39	1	38.76	<.0001	6.08	1	23.31	.0004
D^2	236.08	1	27.69	.0002	4.39	1	16.85	.0015
Residual	102.27	12	–	–	3.13	12	–	–
Lack of fit	83.61	10	0.8958	.6349	2.93	10	2.95	.2791
Pure error	18.66	2	–	–	0.19	2	–	–
Cor total	68761.11	26	–	–	2079.70	26	–	–

Figure 4. Showing the (A) particle size distribution graph and (B) TEM image of the optimized LL-PG-BLs-opt.

Figure 5. DSC curve: (A) pure luteolin, (B) bile salt, (C) polyethylene glycol 2000, (D) cholesterol, and (E) LL-PG-BLs-opt.

Figure 6. XRD-spectra: (A) pure luteolin, (B) cholesterol, (C) bile salt, (D) polyethylene glycol 2000, and (E) LL-PG-BLs-opt.

Figure 7. In vitro release profile of LL-dispersion, LL-BLs, and LL-PG-BLs-opt. Value represented as mean ± SD (n= 3).

Figure 8. In vitro antioxidant activity of LL-dispersion, LL-BL-opt, and PG-LL-BL-opt. Data are the mean of three independent experiments and presented as mean ± SD, *significantly different from LL-dispersion (p<.05), ^#significantly different from LL-BL-opt (p<.05).

Figure 9. Cytotoxicity of LL incorporated in various formulations using MDA-MB-231 cell line at 24 h (A) and 48 h (B). Data shown are mean of three experiments and presented as mean ± SD. Tukey–Kramer’s multiple comparison test was used to evaluate the statistically significant difference between exposed different concentration and control. Difference was considered significant if p<.05. ***p<.001 when compared with control; **p<.001 when compared with the same concentration groups of pure LL.

Figure 10. Cytotoxicity of LL incorporated in various formulation using MCF7 cell line at 24 h (A) and 48 h (B). Data shown are mean of three experiments and presented as mean ± SD. Tukey–Kramer’s multiple comparison test was used to evaluate the statistically significant difference between exposed different concentration and control. Difference was considered significant if p<.05. ***p<.001 when compared with control; **p<.001 when compared with the same concentration groups of pure LL.

Figure 11. Stability study of optimized formulation (LL-PG-BL-opt) conducted at 4 °C and 25 °C. Experiment performed in triplicate and data shown as mean of three experiments (mean ± SD).