Efficacy of Concanavalin-A conjugated nanotransfersomal gel of apigenin for enhanced targeted delivery of UV induced skin malignant melanoma

Figures & data

Figure 1. (A) Transmission electron microscope images of apigenin loaded nanotransfersome and (B) Con-A conjugated nanotransfersomal gel, (C) Mean vesicle size of nanotransfersomes, (D) Mean vesicle size of Con-A conjugated nanotransfersomal gel, (E) Zeta potential of nanotransfersomes and (F) Zeta potential of Con-A conjugated nanotransfersomal gel.

Table 1. Zeta potential, vesicles size and PDI of optimized formulations.

Formulations	Zeta potential (mV)	PDI	Vesicles size (nm)
Con-A conjugated nanotransfersomal gel	35.1 ± 0.21	0.197 ± 0.07	179.0 ± 0.32
Marketed product	−23.05	0.207 ± 0.22	196.3 ± 0.19
Nanotransfersomal gel	31.06 ± 0.33	0.199 ± 0.03	158.5 ± 0.25

Figure 2. (A) X-ray diffractogram of (a) Apigenin, (b) Con-A, (c) Phospholipids, (d) physical mixture, (e) Con-A conjugated nanotransfersomal gel. (B) FTIR spectra of (a) Apigenin, (b) Con-A, (c) Con-A conjugated nanotransfersomal gel. (C) DSC Thermogram of (a) Apigenin (b) Con-A conjugated nanotransfersomes (c) Con-A (d) Phospholipids (E) Carbodiimide. (D) In vitro drug release profile of Con-A conjugated nanotransfersomal gel, marketed product and pure drug suspension in skin pH 5.5.

Table 2. In vitro drug release profile of Con-A conjugated nanotransfersomal gel, marketed product and pure drug suspension in skin pH 5.5.

S.No.	Time Interval	% Cumulative drug release from different formulations
		Pure drug suspension	Conjugated nanotransfersomal gel	Marketed product
1.	0	0	0	0
2.	2	12.58 ± 0.65	9.23 ± 1.86	11.65 ± 0.26
3.	4	26.21 ± 1.12	20.71 ± 0.34	23.11 ± 0.45
4.	6	47.3 ± 1.54	28.35 ± 2.07	36.32 ± 0.62
5.	12	66.25 ± 1.28	38.04 ± 1.03	51.45 ± 1.02
6.	24	82.8 ± 0.25	54.46 ± 0.75	67.19 ± 1.34

Table 3. Absorbance data for the interference of additives in the estimation of apigenin.

S. No.	Additives	Absorbance
1.	Control (5µg mL)	0.4383
2.	Phospholipids	0.4290
3.	NHS	0.4225
4.	Con-A lectin	0.4152
5.	EDC	0.4260

Table 4. Release behaviour of apigenin from Con-A conjugated nanotransfersomal gel formulations.

Formulation	First order		Higuchi		Ritger–Peppas
	K	R^2	K	R^2	n	R^2
Con-A conjugated nanotransfersomal Gel	0.086	0.965	0.406	0.992	0.653	0.934
Marketed product	0.64	0.97	0.76	1.06	0.86	1.23
Pure drug suspension	0.69	0.98	0.78	1.07	0.98	1.31

K: Release rate constant; R²: coefficient of determination and n: release exponent.

Table 5. Permeation and % drug retention data for apigenin loaded Con-A conjugated nanotransfersomal gel, marketed and pure drug suspension across abdominal goat skin.

Formulation	Jss (µg/ch^−2/h)	P (Ch/h)	LT (hr)	D^d (Ch^2/h)	% drug retention after 24 h
Pure drug suspension	6.01 ± 0.59	0.249 ± 1.42	3.1	6.12	0.63 ± 0.23
Con-Aconjugated nanotransfersomal gel	8.42 ± 0.73	0.235 ± 1.56	2.2	9.6	0.91 ± 0.14
Marketed product	7.14 ± 0.08	0.224 ± 1.59	2.7	7.3	0.72 ± 0.22

Jss: Transdermal flux, P: Permeability coefficient, LT: lag time, D^d: Diffusion coefficient

Table 6. Effect of apigenin, unloaded and apigenin-loaded Con-A conjugated nanotransfersomal gel on cellular viability (as % of control) of MTT assay on (a) HaCaT cells and (b) A375 cells.

Apigenin concentration (µg/mL)	Cellular viability (% of control)
	Apigenin	Marketed product	Drug loaded Con-A conjugated nanotransfersomal gel
(a) HaCaT cells
50	110.54 ± 3.25	119.22 ± 2.07	103.54 ± 5.20
100	85.11 ± 4.21	135.03 ± 4.24	126.18 ± 6.13
200	72.52 ± 4.08	99.85 ± 8.35	135.24 ± 4.22
400	89.41 ± 4.50	68.64 ± 15.09	48.89 ± 17.42
(b) A375 cells
50	107.07 ± 2.09	112.31 ± 5.82	98.12 ± 2.75
100	96.41 ± 6.34	125.22 ± 3.87	120.96 ± 5.49
200	82.08 ± 10.67	109.21 ± 8.20	128.04 ± 5.08
400	98.42 ± 4.63	85.07 ± 84.32	40.52 ± 7.52

Figure 3. (A) (a) Showing the photomicrograph of the Mice skin of control group, (b) UV-Irradiated Mice skin of control group, (c) UV-Irradiated Mice skin of PDS group, (d) UV-Irradiated Mice skin of marketed formulation (Flonida, Shalak Pharmaceutical India) group, (e) UV- Irradiated Mice skin of Con-A Conjugated NTG group. (B) Showing the effect of all prepared formulations on the level of increase of MPO. (C) CLSM image showing the depth of skin penetration of Rhodamine red (florescence marker) after 24 h into the goat skin surface at 250 µm (a) (PDS), (b) MP, (c) CCNTG. (D) effect of apigenin, unloaded and apigenin-loaded Con-A conjugated nanotransfersomal gel on cellular viability (as % of control) of MTT assay on HaCaT Cells (a) and A375 cells (b).

Table 7. Storage stability studies of the Con-A conjugated nanotransfersomal gel at different time interval and temperature conditions.

Storage condition	Vesicles size (nm)	Entrapment efficiency (%)	PDI	ZP(−mV)
Day 0	179.0 ± 0.32	89.05 ± 0.62	0.197 ± 0.07	35.1 ± 0.21
1 month at 25 °C	179.52 ± 0.12	89.15 ± 0.42	0.196 ± 0.15	35.07 ± 0.11
1 month at 40 °C	179.55 ± 0.28	89.30 ± 0.16	0.198 ± 0.83	35.13 ± 0.06
3 months at 25 °C	179.65 ± 0.27	89.68 ± 0.21	0.198 ± 0.06	34.04 ± 0.86
3 months at 40 °C	179.6 ± 0.06	89.69 ± 0.54	0.196 ± 0.69	34.82 ± 0.53
6 months at 25 °C	179.7 ± 0.57	90.02 ± 0.11	0.198 ± 0.92	34.05 ± 0.12
6 months at 40 °C	179.9 ± 0.74	90.27 ± 0.21	0.198 ± 0.24	34.69 ± 0.85