Feasibility of binary composition in development of nanoethosomal glycolic vesicles of triamcinolone acetonide using Box-behnken design: in vitro and ex vivo characterization

Figures & data

Table 1. Actual experiments with coded factor levels for optimization process and pharmacotechnical properties of EGVs.

		Independent variables(% w/v) [coded value]
Formulationcode	Run	X1	X2	X3	Vesicle size (nm)	Zeta potential (mV)	PDI	% EE	% CDR	Modelfitting
TA16	1	2 [0]	35 [0]	15 [0]	451.8 ± 1.89	−52.0 ± 0.52	0.460 ± 0.11	46.98 ± 0.67	88.83 ± 0.14	First
TA14	2	2 [0]	35 [0]	15 [0]	425.4 ± 1.74	−58.9 ± 0.17	0.430 ± 0.19	52.50 ± 0.64	80.55 ± 0.75	First
TA1	3	1 [−1]	30 [−1]	15 [0]	259.0 ± 1.11	−46.9 ± 0.49	0.466 ± 0.15	46.08 ± 0.27	98.67 ± 0.72	First
TA17	4	2 [0]	35 [0]	15 [0]	461.5 ± 1.54	−72.6 ± 0.67	0.460 ± 1.12	54.58 ± 0.66	86.47 ± 0.54	First
TA9	5	2 [0]	30 [−1]	10 [−1]	285.3 ± 1.76	−61.4 ± 0.45	0.705 ± 0.43	36.71 ± 0.59	89.48 ± 0.43	First
TA10	6	2 [0]	40 [1]	10 [−1]	160.6 ± 1.32	−77.7 ± 1.11	0.252 ± 0.57	86.27 ± 0.97	83.33 ± 0.13	First
TA3	7	1 [−1]	40 [1]	15 [0]	214.0 ± 2.17	−51.4 ± 0.29	0.489 ± 0.32	53.48 ± 1.56	51.69 ± 0.15	First
TA5	8	1 [−1]	35 [0]	10 [−1]	500.0 ± 1.55	−55.1 ± 0.19	1.000 ± 0.23	36.71 ± 1.23	87.85 ± 1.11	First
TA6	9	3 [1]	35 [0]	10 [−1]	520.3 ± 1.35	−41.2 ± 0.33	0.702 ± 0.12	50.67 ± 1.53	56.76 ± 0.56	Zero
TA4	10	3 [1]	40 [1]	15 [0]	280.7 ± 2.21	−46.9 ± 0.78	0.383 ± 0.35	84.30 ± 0.97	80.17 ± 0.23	Zero
TA8	11	3 [1]	35 [0]	20 [1]	421.2 ± 1.62	−41.7 ± 0.17	0.067 ± 0.56	51.92 ± 0.88	88.92 ± 0.79	Zero
TA2	12	3 [1]	30 [−1]	15 [0]	260.3 ± 1.18	−61.9 ± 0.11	0.613 ± 0.33	39.12 ± 0.55	77.04 ± 0.91	Zero
TA12	13	2 [0]	40 [1]	20 [1]	221.8 ± 2.36	−35.0 ± 0.18	0.679 ± 0.72	46.70 ± 1.10	53.48 ± 0.22	First
TA7	14	1 [−1]	35 [0]	20 [1]	159.0 ± 1.90	−35.9 ± 0.62	0.928 ± 0.43	40.01 ± 0.71	87.83 ± 0.63	First
TA11	15	2 [0]	30 [−1]	20 [1]	305.6 ± 1.77	−45.6 ± 0.44	0.630 ± 0.51	63.49 ± 0.68	78.34 ± 0.98	First
TA15	16	2 [0]	35 [0]	15 [0]	458.6 ± 1.26	−50.6 ± 0.87	0.723 ± 0.34	53.80 ± 0.60	84.17 ± 0.81	First
TA13	17	2 [0]	35 [0]	15 [0]	497.9 ± 1.40	−57.4 ± 1.10	0.501 ± 0.66	46.43 ± 0.52	83.48 ± 0.37	First

X1 : Soya lecithin; X2 : Ethanol; X3 : Propylene glycol.

Table 2. Formulation design and characterization for the preparation of triamcinolone acetonide (TA) loaded gels.

	Formulation code
Ingredients (%w/v)	CG	RG	EG	HEG	RGP	EGP
TA	0.1	–	–	0.1	–	–
Carbopol 934P	1	1	1	1	1	1
TA10	–	–	0.1	–	–	0.1
RE	–	0.1	–	–	0.1	–
Ethanol (95% v/v)	–	–	–	30	–	–
PEG 600	–	–	–		5	5
Triethanolamine	1	1	1	1	1	1
Distilled water	100	100	100	100	100	100
Evaluation parameters
Ph	6.82 ± 1.02	6.79 ± 1.11	6.84 ± 1.14	6.72 ± 1.04	6.74 ± 1.32	6.85 ± 1.15
Viscosity (cp)	1195 ± 5.03	1378 ± 7.00	1484 ± 7.94	1452 ± 7.04	1398 ± 7.64	1086 ± 5.57
Percent drug content	27.36 ± 0.03	71.83 ± 0.05	84.60 ± 0.02	64.61 ± 0.04	74.81 ± 0.04	86.06 ± 0.06
Cumulative drug permeated (%)	25.92 ± 1.08	70.83 ± 1.06	82.42 ± 0.89	63.17 ± 1.21	72.63 ± 0.90	83.76 ± 0.72
Kinetic model	First	Zero	Zero	First	First	Zero
Flux (μg/cm^2/min)	0.441 ± 0.99	5.00 ± 1.11	6.67 ± 0.94	4.76 ± 1.04	6.67 ± 0.89	9.52 ± 0.66
Permeation coefficient (cm/min)x 10^−3	0.07 ± 1.03	0.80 ± 1.19	1.06 ± 1.10	0.76 ± 0.98	1.06 ± 1.01	1.52 ± 1.07
ER	0.70	8.00	10.59	7.56	10.59	15.11
f2	100	32	21	28	22	22
Lag time (min)	48	30	30	36	30	36

CG : Control Gel; RG : Reference Ethosomal gel; EG : Ethosomal glycolic gel; HEG : Hydroethanolic gel; RGP/EGP: Corresponding gels with penetration enhancer.

Figure 1. (A) In vitro release profiles of triamcinolone acetonide in phosphate buffer, pH 6.8 in 8 h using dialysis membrane; (B) In vitro cumulative permeability profiles of different gel formulations in phosphate buffer, pH 6.8.

Figure 2. Transmission electron micrographs of (A) Reference ethosomes, RE; (B) Ethosomal glycolic vesicles, TA10.

Figure 3. Skin histological micrographs (A) Control skin; (B) EGVs treated skin.

Table 3. Comparative stability investigation of optimized ethosomal glycolic vesicles (TA10) and reference ethosomes (RE).

		Parameters
Formulation Code	Storage time (months)	Vesicle size (nm)	Zeta potential (mV)	% EE
TA10	0	160.6 ± 0.32	−77.7 ± 1.11	86.27 ± 0.97
	6	198.1 ± 0.19	61.3 ± 0.99	78.88 ± 0.48
RE	0	257.0 ± 0.55	−40.3 ± 0.59	72.16 ± 1.23
	6	313.9 ± 1.01	29.1 ± 0.87	61.08 ± 1.50

% EE: Percent Entrapment Efficiency; AD: Atopic dermatitis; BCS: Biopharmaceutics Classification System; CG: Control Gel; CLSM: Confocal Laser Scanning Microscopy; DRS: Diffuse Reflectance Spectroscopy; EG: TA10 loaded ethosomal gel; EGP: TA10 loaded ethosomal gel with penetration enhancer; EGVs: Ethanolic Glycolic Vesicles; FTIR: Fourier Transform Infrared; HEG: Hydroethanolic gel; PDI: Polydispersity Index; RE: Reference Ethosomes; RG: Reference ethosomal Gel; RGP: Reference ethosomal Gel with Penetration enhancer; TA: Triamcinolone acetonide.

Figure 4. Formulation optimization via Design-Expert software showing (A) Desirability ramp for optimization of vesicles; (B) Bar graphs; (C) Contour plot for optimization; (D) Overlay plot from graphical optimization technique.

Figure 5. Confocal photomicrograph of (A) Control gel; (B) Reference ethosomal gel; (C) ethosomal glycolic vesicular gel through confocal laser scanning microscope at 100×.