A Cosmeceutical Topical Water-in-Oil Nanoemulsion of Natural Bioactives: Design of Experiment, in vitro Characterization, and in vivo Skin Performance Against UVB Irradiation-Induced Skin Damages

Figures & data

Figure 1 Rats’ dorsal skin photographs of (A) normal control group (unexposed to UVB-irradiation) and (B) positive control group (exposed to UVB-irradiation, where skin alterations were visible, as the skin was erythematous and wrinkled, as well as, being hyperpigmented in certain areas).

Table 1 The Composition of the Experimental Runs According to the D-Optimal Statistical Design and Their Responses

Runs	A Oils (%)	B S/Cos (%)	C Water (%)	Y1* GS (nm)	Y2* PDI	Y3* %T	Y4* Viscosity (cp)
1	28.49	53.20	18.31	26.42±0.37	0.42±0.08	98.76±0.08	342.83±3.55
2	24.36	51.23	24.41	29.52±0.10	0.41±0.02	86.12±0.72	495.83±5.78
3	23.20	62.03	14.77	25.87±1.31	0.43±0.07	98.36±0.57	285.50±2.04
4	31.38	58.62	10.00	34.12±1.42	0.35±0.05	92.56±1.54	320.20±3.44
5	40.55	49.45	10.00	73.01±3.03	0.59±0.07	75.25±0.51	336.30±2.46
6	45.00	45.00	10.00	68.64±2.18	0.29±0.05	61.93±4.28	324.17±5.92
7	38.46	45.00	16.54	58.22±3.58	0.42±0.06	79.05±2.32	354.77±6.23
8	45.00	45.00	10.00	63.96±3.10	0.25±0.02	65.21±3.23	333.03±2.59
9	20.00	57.26	22.74	18.75±1.86	0.68±0.02	99.11±0.30	433.90±1.47
10	20.00	70.00	10.00	151.00±28.82	0.58±0.01	72.03±0.65	312.40±5.20
11	20.00	70.00	10.00	147.00±33.93	0.56±0.01	68.85±0.06	340.87±2.95
12	29.44	45.56	25.00	29.89±1.78	0.84±0.01	85.29±2.10	418.97±2.50
13	29.44	45.56	25.00	28.37±2.26	0.80±0.01	80.86±1.82	456.63±3.43
14	20.00	57.26	22.74	15.20±1.18	0.65±0.00	99.63±0.60	470.23±19.20
15	38.46	45.00	16.54	46.15±3.02	0.47±0.04	87.39±2.56	321.77±4.46
16	35.32	51.81	12.87	59.49±3.37	0.31±0.03	77.85±1.35	382.80±3.46

Notes: *All values are represented as mean ± standard deviation of three measurements for three different batches (n = 3). Collagen (10 mg) was incorporated in all formulations with a final concentration of 1% w/w.

Abbreviations: S/Cos, surfactant/cosurfactant; GS, globule size; PDI, polydispersity index; %T, percent transmittance.

Figure 2 Pseudo-ternary diagram of vitamin E and safflower oil (1:3), Span 80/kolliphor EL/ Arlasolve (1:1:1), and water. The defined percentages of the oils (20–45%), the surfactant/ cosurfactant (S/Cos) (45–70%), and water (10–25%) are represented by the black triangle.

Table 2 Summary of Model Statistics for the Measured Responses

Response	Model	SD	R^2	Adjusted R^2	Predicted R^2	PRESS
Y1: GS	Linear	29.67	0.5488	0.4794	0.2536	18,933.41
	Quadratic	16.80	0.8887	0.8331	0.7157	7212.14
	Cubic	4.07	0.9961	0.9902	0.9823	449.19
Y2: PDI	Linear	0.14	0.4569	0.3734	0.1654	0.39
	Quadratic	0.13	0.6364	0.4546	0.2286	0.36
	Cubic	0.026	0.9915	0.9787	0.7273	0.13
Y3: %T	Linear	10.02	0.4405	0.3544	0.1000	2100.27
	Quadratic	5.39	0.8755	0.8133	0.6779	751.64
	Cubic	3.26	0.9727	0.9318	0.3152	3069.33
Y4: Viscosity	Linear	38.29	0.6911	0.6436	0.5711	26,470.12
	Quadratic	32.74	0.8264	0.7395	0.6138	23,830.24
	Cubic	21.63	0.9545	0.8863	1.6081	160,900

Note: The statistic values of the selected model for each response are underlined.

Abbreviations: SD, Standard deviation; GS, globule size; PDI, Polydispersity index; %T, Percent transmittance, PRESS, Predicted residual error sum of squares.

Figure 3 3D surface plots demonstrating the effect of the independent variables; the percentage of oils (A), the percentage of S/Cos (B), and the percentage of water (C) on (I) globule size in nm (GS), (II) Polydispersity index (PDI), (III) Percent transmittance (%), and (IV) Viscosity in cp.

Table 3 The Observed and the Predicted Values of the Optimized Collagen-Loaded NE

Factor	Optimized Level (D=0.808)
A: Oils (%)	25
B: S/Cos (%)	54.635
C: Water (%)	20.365
Response	Expected	Observed^#	Residual*
Y1: GS (nm)	15.20	19.41±4.17	−4.21
Y2: PDI	0.51	0.50 ± 0.11	−0.01
Y3: %T	99.63	99.98±0.01	−0.35
Y4: Viscosity (cp)	407.89	402.00±5.02	4.11

Notes: *Residual = expected value – observed value. ^#Mean ± Standard deviation (n = 3).

Abbreviations: D, desirability value; S/Cos, surfactant/cosurfactant; GS, globule size; PDI, polydispersity index; %T, percent transmittance.

Figure 4 TEM micrographs of (A) collagen-loaded NE, and (B) Vit C-loaded NE.

Table 4 Characteristics of the Optimized Collagen-Loaded NE and Vit C-Loaded NE Before and After Storage at (25±2 °C/60±5% RH) for 6 Months

Formulations	GS (nm)	PDI	T (%)	Viscosity (cp)
Collagen-loaded NE before storage	19.41±4.17	0.50 ± 0.01	99.98±0.01	402.00±5.02
Collagen-loaded NE after storage	19.95 ± 3.37	0.51± 0.1	99.98± 0.12	411.00±9.31
Vit C-loaded NE before storage	26.15 ± 1.02	0.49±0.04	98.34±0.21	357.00±11.31
Vit C-loaded NE after storage	25.82 ± 2.35	0.52±0.03	98.57±0.11	365.00±8.78

Abbreviations: GS, globule size; PDI, polydispersity index; %T, percent transmittance.

Figure 5 Surviving fraction after applying the different treatments (Vit E, Vit C, collagen, plain NE lacking Vit E, Plain NE, Vit C-loaded NE, collagen-loaded NE, and a mixture of collagen and Vit C-loaded NEs) for 48 h on (A) HFB4 cells, (B) BHK cells, (C) HFB4 cells after applying the treatments at the highest concentration (50 µL/mL), and (D) BHK cells after applying the treatments at the highest concentration (50 µL/mL). The actual data represent the mean ± SD of three separate experiments performed in duplicates. Results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison post-hoc test. ^aSignificant difference from Vit E, ^bSignificant difference from Vit C, ^cSignificant difference from collagen, ^dSignificant difference from Plain NE lacking Vit E, ^eSignificant difference from Plain NE, ^fSignificant difference from Vit C-loaded NE, and ^gSignificant difference from collagen-loaded NE (p<0.05).

Figure 6 Micrographs of the stained healed monolayer of HFB4 and BHK cells. (A) Control at 0 h, (B) control after 48 h, (C) Vit E, (D) Vit C, (E) collagen, (F) plain NE lacking Vit E, (G) plain NE, (H) Vit C-loaded NE, (I) collagen-loaded NE, and (J) A mixture of collagen- and Vit C-loaded NEs.

Figure 7 CLSM micrographs of (A) Rhodamine B dye aqueous solution and (B) Rhodamine B dye-loaded NE.

Figure 8 Microscopic examination of skin samples from (A) control group revealing normal histology of skin, (B) positive control showing histopathological alterations mainly in the epidermis. Hyperkeratosis and acanthosis (black arrows) are manifested by thickening in the epidermal layer (red arrow). The epidermis contained numerous shrunken cells known as sunburn cells near the basal cell layer (3 black arrows), (C) Vit C-loaded NE showing apparently normal skin with mild inflammatory cells infiltration, (D) collagen-loaded NE showing apparently normal skin, and (E) a mixture of collagen and Vit C-loaded NEs revealing skin free from any detectable alterations.