Tailoring of Retinyl Palmitate-Based Ethosomal Hydrogel as a Novel Nanoplatform for Acne Vulgaris Management: Fabrication, Optimization, and Clinical Evaluation Employing a Split-Face Comparative Study

Figures & data

Table 1 Composition of the Independent Variables with Their Respective Levels and Their Dependent Variables

Variables	Levels
Independent variables (Factors)	Low(−1)					Medium(0)			High(+1)
A = PC concentration % (w/v)	1%					2%			3%
B = Ethanol concentration % (v/v)	10%					20%			30%
C = PG concentration % (v/v)	5%					10%			15%
Formula no.	Independent variables			Dependent variables							PDI
	A	B	C	Y1: particle size (nm)	Y2: zeta potential(mV)		Y3:EE%	Y4: % drug released over 24h		Y5: % drug permeated over 24h
F1	+1	+1	0	354.3±9.36	−77.5±3.99		98.86±5.01	83.69±8.29		74.26±5.92	0.46
F2	0	−1	−1	340±4.35	−61.1±4.32		89.23±4.88	67.2±6.38		55.16±5.28	0.43
F3	0	0	0	271.4±6.48	−59.5±4.28		93.81±3.74	76.73±4.41		73.5±6.99	0.35
F4	0	−1	+1	311±8.27	−54.5±7.84		92.76±6.55	73.23±11.01		61.38±3.09	0.30
F5	−1	+1	0	174.5±5.36	−56.1±2.09		91.24±7.98	97.9±3.92		85.18±5.61	0.26
F6	+1	−1	0	404±10.43	−66.3±5.82		95.84±4.72	62.48±8.54		51.53±4.33	0.36
F7	+1	0	+1	367.6±13.49	−72.6±9.75		98.27±3.91	75.4±4.09		63.28±4.72	0.37
F8	−1	−1	0	310.1±3.52	−51±8.35		82.01±6.02	79.87±2.74		68.38±9.51	0.39
F9	+1	0	−1	386.4±5.33	−74.2±3.17		97.97±7.53	70.43±10.61		58.83±6.42	0.34
F10	0	0	0	283.5±6.63	−62±5.29		95.09±11.23	78.83±6.81		68.87±3.99	0.46
F11	0	+1	+1	237.4±3.81	−67±10.56		96.39±2.84	93.87±8.49		83.28±5.91	0.33
F12	0	+1	−1	261.3±2.69	−72.6±3.88		96.03±3.52	87.9±7.90		76.94±8.53	0.36
F13	−1	0	−1	228.4±11.51	−52.3±3.11		84.12±9.19	82.24±14.73		72.38±7.28	0.32
F14	−1	0	+1	199.4±3.33	−58.2±4.02		87.13±4.92	89.24±5.44		78.53±4.74	0.26
F15	0	0	0	293.6±9.46	−60.6±6.53		93.91±3.06	75.49±5.38		71.32±5.26	0.21
F optimized	1.33%	30%	15%	195.8±5.45	−62.1±2.85		92.63±4.33	96.63±6.81		85.98±4.79	0.37

Note: Data are mean values (n = 3) ± SD.

Abbreviations: PC, Phosphatidyl choline; PG, Propylene glycol; EE%, entrapment efficiency percent; PDI, polydispersity index.

Table 2 Formulation Parameters for RP Ethosomal Hydrogel

Independent Variables	Levels				Dependent Variables				Constraint
	0		1
Polymer type (X1)	Carbopol 971P		Chitosan		% RP released from ethosomal hydrogel				Maximum
Polymer conc. (X2)	1		2		% RP permeated from ethosomal hydrogel				Maximum
Surfactant (DMSO) conc. (X3)	1		2
Formulation	G1	G2	G3	G4	G5	G6	G7	G8		Goptimized
Polymer type (X1)	0	0	0	0	1	1	1	1		0
Polymer conc. (X2)	1	1	2	2	1	1	2	2		1
Surfactant conc. (X3)	1	2	1	2	1	2	1	2		2
% drug released (Y1)	84.83± 2.33	89.39± 3.84	72.61± 7.63	77.99± 4.92	63.97± 9.16	65.94± 3.94	53.54± 4.81	59.16± 4.02		86.75± 5.82
% drug permeated (Y2)	72.96± 8.83	75.47± 3.80	58.97± 1.42	65.64± 0.34	42.58± 3.92	49.99± 4.55	38.25± 5.12	41.36± 3.68		75.89± 3.88
Viscosity (cP)	5448.64	4768.96	6405.12	6029.28	1351.84	1272.32	2067.52	1431		4798.34
Farrow’s constant (N)	5.281	3.507	4.478	3.42	2.272	1.5	1.779	3.453		3.550
Area of hysteresis loop (Dyne/cm^2.sec)	1527.36	2035.71	667.96	1953.12	683.87	858.81	763.39	1304.05		2835.288

Note: Data are mean values (n = 3) ± SD.

Abbreviations: DMSO, Dimethyl Sulfoxide; RP, Retinyl Palmitate.

Table 3 Summarize Statistics Model of Box–Behnken Design Used for Optimization of Ethosomal RP with Their Regression Equation

Dependent Variables	R^2	Adjusted R^2	Predicted R^2	P value
Y1: particle size(nm)	0.9341	0.9161	0.8692	0.0001
Y2: ZP (mV)	0.9032	0.8768	0.8127	0.0001
Y3: % EE	0.9918	0.9772	0.9070	0.0001
Y4: % drug released over 24 hr	0.9658	0.9564	0.9462	0.0001
Y5: % drug permeated over 24 hr	0.9715	0.9638	0.9532	0.0001
Regression equation of the fitted model
Y1= 294.86 + 74.9875A - 42.2 B - 12.5875C
Y2= 63.0333 + 9.125A + 5.0375 B - 0.9875 C
Y3= 94.27 + 5.805 A+ 2.835B + 0.9C- 1.5525AB - 0.6775AC - 0.7925 BC- 2.00625 A^2–0.27625 B^2 –0.39125 C^2
Y4= 79.6333–7.15625A + 10.0725B + 2.99625 C
Y5= 69.5213–7.07125A + 10.4013B + 2.895 C

Note: Data are mean values (n = 3) ± SD.

Abbreviation: EE%, entrapment efficiency percent.

Figure 1 Response surface plot for the effect of independent variables on (A) particle size, (B) ZP, (C) EE%, (D) % drug released over 24 hr, (E) % drug permeated over 24 hr and (F) desirability of the optimized ethosomal RP formula.

Table 4 Ex vivo Permeation Parameters of Ethosomal RP Formulations

Formulation	Lag Time (min)	Jss (µg/cm^2 h)	Kp (cm/h)	EI
F1	25.4961	19.21	0.01921	1.24
F2	39.60187	25.62	0.02562	1.66
F3	25.13599	22.87	0.02287	1.48
F4	56.02113	17.04	0.01704	1.1
F5	15.37897	24.54	0.02454	1.59
F6	16.5859	21.13	0.02113	1.37
F7	35.55245	21.45	0.02145	1.39
F8	55.94397	16.42	0.01642	1.06
F9	25.40839	27.18	0.02718	1.76
F10	52.13483	24.03	0.02403	1.56
F11	62.58813	21.56	0.02156	1.4
F12	44.97833	20.77	0.02077	1.34
F13	36.67233	17.67	0.01767	1.14
F14	22.37778	21.6	0.0216	1.4
F15	51.42857	18.97	0.01897	1.23
RP suspension	73.12621	15.45	0.01545	–

Table 5 Predicted and Experimental Values of the Optimal Ethosomal RP Formulations

Solution	PC Conc.	Ethanol Conc.	PG Conc.	Y1	Y2	Y3	Y4	Y5	Desirability
Predicted	1.33	30	15	190.01	60.99	93.26	97.48	87.54	0.886
Experimental	1.33	30	15	195.80	62.10	92.63	96.63	85.98	0.886
Bais %	–	–	–	2.95	1.78	0.68	0.87	1.81	–

Note: Bias % = [(predicted value-experimental value)/experimental value]×100.

Figure 2 Contour plot for the effect of independent variables on (A) particle size, (B) ZP, (C) EE%, (D) % drug released over 24 hr, (E) % drug permeated over 24 hr and (F) desirability of the optimized ethosomal RP formula.

Figure 3 Transmission electron micrograph of the optimized ethosomal RP formulation.

Figure 4 Effect of storage at 4°C and 25°C for 3 months on particle size, ZP and EE % of the optimized Ethosomal RP formula.

Figure 5 Rheograms of RP hydrogel formulations; G1-G8.

Figure 6 Flow curves of RP hydrogel formulations; G1-G8.

Figure 7 Response surface plot for the effect of independent variables on (A) % drug released over 24 h, (B) % drug permeated over 24 h, (C) desirability of the optimized RP ethosomal hydrogel.

Figure 8 % Drug permeated from control gel and optimized RP ethosomal hydrogel.

Figure 9 Light photomicrographs showing histopathological sections of (A) rat skin of the control group, (B) rat skin of tretinoin cream treated group, and (C) rat skin of ethosomal RP hydrogel treated group. (H&E stain; Bar= 100μm).

Table 6 Mean Acne Lesion Count Before and After Treatment

Lesion Count	Right Side (Conventional Tretinoin)		Left Side (Ethosomal RP Hydrogel)		P-value^b
	Mean ± SD	P-value^a	Mean ± SD	P-value^a
Non-inflammatory lesions
Baseline	9.80 ± 5.27		9.35 ± 4.99		0.529
After 2 weeks	7.79 ± 4.93	0.016	6.37 ± 3.39	0.002
After 4 weeks	6.35 ± 3.51	< 0.001	4.70 ± 2.342	0.001
After 6 weeks	6.45 ± 3.75	< 0.001	4.65 ± 2.56	< 0.001	0.008
Final % reduction	34.32 ± 0.2		46.69 ± 0.24		0.071
Inflammatory lesions
Baseline	9.3 ± 6.47		9.2 ± 5.88		0.706
After 2 weeks	7.42 ± 4.86	0.006	6.11 ± 3.59	0.007
After 4 weeks	6 ± 4.32	0.005	4.55 ± 3	0.001
After 6 weeks	6.2 ± 4.1	0.001	4.6 ± 2.81	0.001	0.083
Final % reduction	28.35 ± 0.32		39.32 ± 0.37		0.432
Total lesions
Baseline	19.1 ± 8.39		18.55 ± 8.42		0.587
After 2 weeks	15.21 ± 6.96	0.005	12.47± 4.91	0.004
After 4 weeks	12.35 ± 5.57	< 0.001	9.25 ± 3.99	0.001
After 6 weeks	12.65 ± 5.62	< 0.001	9.25 ±3.89	< 0.001	0.016
Final % reduction	28.49 ± 0.21		39.95 ± 0.24		0.179

Notes: ^aComparison vs baseline within the same treatment side. ^bComparison of the two treated sides. Significance is considered at p < 0.05.

Table 7 Local Adverse Effects in the Two Treatment Sides

Side Effect	Right Side (Conventional Tretinoin)	Left Side (Ethosomal RP)	p-value
Erythema, n (%)
No	0 (0)	7 (35)	< 0.001
Mild	3 (15)	12 (60)
Moderate	15 (75)	1 (5)
Severe	2 (10)	0 (0)
Peeling, n (%)
No	0 (0)	7 (35)	< 0.001
Mild	7 (35)	12 (60)
Moderate	11 (55)	1 (5)
Severe	2 (10)	0 (0)
Burning, n (%)
No	0 (0)	3 (15)	< 0.001
Mild	5 (25)	15 (75)
Moderate	11 (55)	2 (10)
Severe	4 (20)	0 (0)
Pruritus, n (%)
No	1 (5)	4 (20)	< 0.001
Mild	1 (5)	15 (75)
Moderate	12 (60)	1 (5)
Severe	6 (30)	0 (0)

Figure 10 Right side of face before (A) and after topical tretinoin (B). Left side of the face before (C) and after ethosomal RP hydrogel (D). Peeling and dryness noted after topical tretinoin.

Figure 11 Right side of face before (A) and after topical tretinoin (B). Left side of the face before (C) and after ethosomal RP hydrogel (D). Moderate erythema noted after topical tretinoin.

Figure 12 Right side of face before (A) and after topical tretinoin (B). Left side of the face before (C) and after ethosomal RP hydrogel (D).

Figure 13 Right side of face before (A) and after topical tretinoin (B). Left side of the face before (C) and after ethosomal RP hydrogel (D).