In vitro and preclinical assessment of factorial design based nanoethosomes transgel formulation of an opioid analgesic

Figures & data

Table I. Experimental runs obtained from Box–Behnken design and their actual and predicted responses for tramadol nanoethosomal formulations.

	Independent variables			Dependent variables
Code	A (mg)	B (%)	C (min)	Predicted value (Y1 = nm)	Actual value (Y1 = nm)	Predicted value (Y2 = %)	Actual value (Y2 = %)	Predicted value (Y3 = μg/cm^2/h)	Actual value (Y3 = μg/cm^2/h)
1	55	30	6	102.19	102.45 ± 2.56	64.90	65.23 ± 3.87	108.04	106.34 ± 18.76
2	55	45	3	94.96	95.21 ± 1.98	51.48	51.56 ± 6.45	80.39	81.65 ± 27.54
3	70	45	6	119.93	119.87 ± 3.67	58.83	58.57 ± 5.65	45.18	45.56 ± 21.36
4	70	30	3	133.57	133.37 ± 3.12	66.49	66.67 ± 4.28	64.20	62.56 ± 15.37
5	55	15	9	101.90	101.65 ± 2.87	53.61	53.53 ± 3.74	90.71	89.45 ± 16.54
6	40	30	3	85.44	84.98 ± 1.56	61.88	61.78 ± 1.67	63.34	61.65 ± 24.54
7	70	30	9	119.81	120.27 ± 2.12	62.35	62.45 ± 6.12	44.87	46.56 ± 22.45
8	40	15	6	92.50	92.55 ± 2.32	60.50	60.76 ± 5.32	73.98	73.6 ± 20.87
9	70	15	6	132.65	132.45 ± 1.52	68.55	68.53 ± 4.33	80.76	80.34 ± 18.65
10	55	30	6	102.19	101.76 ± 1.28	64.90	64.24 ± 4.67	108.04	108.45 ± 14.56
11	55	45	9	90.51	90.11 ± 1.77	39.29	39.45 ± 2.87	45.29	43.23 ± 18.54
12	40	30	9	83.44	83.64 ± 2.65	53.83	53.65 ± 2.43	62.01	63.65 ± 26.65
13	55	30	6	102.19	102.35 ± 2.34	64.90	65.23 ± 1.87	108.04	109.34 ± 27.43
14	55	30	3	113.21	113.61 ± 4.44	53.62	53.46 ± 2.56	76.28	78.34 ± 21.36
15	40	45	6	75.58	75.78 ± 1.46	53.74	53.76 ± 4.45	68.25	68.67 ± 19.65

A, Phospholipid; B, Ethanol; C, Sonication time; Y₁, Size; Y₂, %EE; Y₃, Flux.

Figure 1. 3D-Response surface plots showing the influence of independent variables (Cholesterol, Span60, and phospholipid) on size (A–B), entrapment efficiency (C–D) and flux (E–F) for TRM ethosomes.

Table II. Summary of regression analysis for responses Y₁ [Size (nm)], Y₂ [Entrapment efficiency (%)], and Y₃ [Flux (μg/cm²/h)] for fitting to different models.

Model	R^2	Adjusted R^2	Predicted R^2	SD	Remark
Response (Y1)
Linear	0.9695	0.9612	0.9357	3.44
2F1	0.9814	0.9675	0.9052	3.15
Quadratic	0.9997	0.9991	0.9960	0.51	Suggested
Response (Y2)
Linear	0.1774	−0.0470	−0.2686	22.48
2F1	0.3130	−0.2022	−0.5850	24.09
Quadratic	0.9958	0.9883	0.9430	2.37	Suggested
Response (Y3)
Linear	0.3572	0.1819	−0.2328	6.96
2F1	0.4092	−0.0340	−1.5418	7.83
Quadratic	0.9989	0.9968	0.9927	0.43	Suggested

Figure 2. Linear correlation graph of actual versus predicted value, (A) particle size, (B) entrapment efficiency, (C) flux.

Figure 3. (A) Size distribution of optimized TRM ethosomes (B) Transmission electron micrography following negative staining (80,000×).

Figure 4. Confocal laser microscopy of Rhodamine loaded (A) liposome formulation (B) nanoethosome formulation (100×).

Figure 5. Histopathological image of (A) Control (B) formalin treated (C) TRM loaded nanoethosomal gel (400×).

Table III. Comparative skin irritation score after application of test formulation and standard irritant.

Rats*	Control		Optimized TRM ethosomal gel (OTEG)		Formalin (0.8%)
	Erythema	Edema	Erythema	Edema	Erythema	Edema
H	0	0	0	1	3	3
B	0	0	1	1	3	4
T	0	0	1	1	3	3
N	0	0	1	0	4	4
HB	0	0	0	1	3	3
Mean ± SD	0	0	0.6 ± 0.54	0.8 ± 0.44	3.3 ± 0.45	3.4 ± 0.51
PII	0		1.4		6.7

PII, Primary Irritancy Index; SD, standard deviation.

*Rats were marked by picric acid solution for identification on Head (H), Back (B), Tail (T), Head-Back (HB), Not marked (N).

†Erythema scale: 0, none; 1, slight; 2, well defined; 3, moderate; and 4, scar formation.

††Edema scale: 0, none; 1, slight; 2, well defined; 3, moderate; and 4, severe.

Table IV. Absorption profile of tramadol in rats after administration different formulations (mean ± SD, n = 6).

Pharmacokinetic parameters	TRM Ethosomal gel (OTEG)	TRM Liposomal gel (TLG)	TRM Oral formulation (TCF)
Cmax (ng/ml)	491.93 ± 43.87	83.41 ± 11.12	676.29 ± 35.63
Tmax (h)	4.0 ± 0.32	6.0 ± 0.91	1.0 ± 0.15
AUC 0-48 (ng/mL/h)	10,047.02 ± 350.54	339.56 ± 24.22	1336.31 ± 207.98
AUC 0–∞ (ng/mL/h)	10,800.16 ± 405.98	367.29 ± 18.56	1414.91 ± 327.59
Half-life (h)	10.60 ± 0.19	14.45 ± 1.35	2.69 ± 0.14
AUMC 0-48 (ng/mL/h^2)	139463.80 ± 603.68	3986.23 ± 29.68	5196.75 ± 439.39
AUMC 0-∞ (ng/mL/h^2)	187121.20 ± 587.67	5041.16 ± 39.43	6445.19 ± 421.48

C_max, peak of maximum plasma concentration; T_max, time of peak concentration; AUC_0-48, area under the concentration time profile curve until last observation; AUC_0–∞, area under the concentration time profile curve from time 0 to infinity; AUMC_0-48, area under first moment curve from 0 to 48; AUMC_0-∞, area under first moment curve from 0 to infinity.

Figure 6. In vivo analgesic activity of optimized TRM loaded nanoethosomal gel and oral solution (Mean ± SD, n = 6).