Pivotal role of Acitretin nanovesicular gel for effective treatment of psoriasis: ex vivo–in vivo evaluation study

Figures & data

Table 1 Composition of Act-loaded niosomal formulations

Formulation code	Span 60:CH (molar ratio)
F1	1:1
F2	1.5:1
F3	2:1
F4	3:1
F5	1:1.5
F6	1:2
F7	1:3

Note: Each formulation contains 15 mg Act.

Abbreviations: Act, Acitretin; CH, cholesterol.

Table 2 Characterization of Act-loaded niosomal formulations

Formulation code	PS (nm)	PDI	ZP (mV)	EE (%)
F1	369.73±45.45	0.095±0.03	−36.33±1.80	90.32±3.80
F2	505.30±73.98	0.141±0.03	−37.83±0.31	86.64±2.58
F3	607.83±69.26	0.226±0.06	−39.17±0.58	82.17±4.02
F4	784.43±18.01	0.467±0.12	−41.20±0.36	78.50±5.53
F5	279.67±20.13	0.179±0.04	−20.77±0.81	80.32±3.19
F6	265.93±28.38	0.225±0.05	−21.50±1.14	77.30±2.45
F7	221.60±24.24	0.212±0.06	−27.23±1.16	68.63±2.79

Note: Each value represents the mean ± SD (n=3).

Abbreviations: Act, Acitretin; EE, entrapment efficiency; PDI, polydispersity index; PS, particle size; ZP, zeta potential.

Figure 1 TEM image, size, and zeta potential distribution curves of Act-loaded niosome (F1).

Note: (A) TEM image, (B) size distribution curve, and (C) zeta potential distribution curve.

Abbreviations: Act, Acitretin; TEM, transmission electron microscope.

Figure 2 DSC thermograms.

Note: (A) Act, (B) Span 60, (C) CH, (D) physical mixture, (E) plain niosome, and (F) Act-loaded niosome (F1).

Abbreviations: Act, Acitretin; CH, cholesterol; DSC, differential scanning calorimetry.

Figure 3 PXRD patterns.

Note: (A) Act, (B) Span 60, (C) CH, (D) physical mixture, (E) plain niosome, and (F) Act-loaded niosome (F1).

Abbreviations: Act, Acitretin; CH, cholesterol; PXRD, powder X-ray diffractometry.

Figure 4 Ex vivo skin permeation profiles of Act from different niosomal formulations.

Note: Each point represents the mean ± SD (n=3).

Abbreviation: Act, Acitretin.

Table 3 Ex vivo skin permeation parameters of Act from different niosomal formulations across the excised rat skin after 30 h

Formulation code	Q30 h (µg/cm^2)	Jss (µg/cm^2/h)	Kp (cm/h) ×10^−3	ERflux
Act in propylene glycol (control)	63.19±5.48	1.88±0.17	1.09±0.00	–
F1	225.40±6.78*,**	7.07±0.15*,**	4.11±0.00*,**	3.75±0.08**
F2	171.10±6.08*	6.11±0.15*	3.55±0.00*	3.25±0.08
F3	143.72±5.39*	4.44±0.16*	2.58±0.00*	2.36±0.08
F4	108.48±4.70*	3.54±0.15*	2.06±0.00*	1.89±0.08
F5	93.99±6.62*	2.95±0.22*	1.71±0.00*	1.57±0.12
F6	105.63±8.51*	3.55±0.23*	2.07±0.00*	1.89±0.12
F7	136.42±7.90*	4.20±0.23*	2.44±0.00*	2.23±0.12

Notes: Each value represents the mean ± SD (n=3).

* p<0.05 vs control;

** p<0.05 F1 vs other formulations.

Abbreviations: Act, Acitretin; ER, enhancement ratio; J_ss, steady state flux; K_p, permeability coefficient; Q_{30 h}, cumulative amount that permeated after 30 h.

Table 4 Stability study data of Act-loaded niosomal formulation (F1) after storage at refrigerated and room temperatures

Parameters	Refrigerated temperature (4±1°C)				Room temperature (25±1°C)
	Initial	1 month	2 months	3 months	1 month	2 months	3 months
PS (nm)	369.73±45.45	421.49±24.39*,≠	443.66±32.87*,#	498.06±45.03*,^	475.37±38.76*	528.19±26.04*	683.83±43.89*
PDI	0.095±0.03	0.340±0.10	0.346±0.11	0.306±0.11	0.365±0.11	0.443±0.07*	0.409±0.15*
EE (%)	90.32±3.80	87.40±2.32	85.00±3.96	82.88±4.33^	82.33±3.95	77.60±4.87*	71.30±6.31*
Drug retention (%)	100.00±0.00	96.77±2.56	94.11±4.39	91.77±4.79^	91.16±4.38	84.63±3.91*	78.95±6.96*

Notes: Each value represents the mean ± SD (n=3).

* p<0.05 vs initial measurements;

≠ p<0.05 refrigerated temperature vs room temperature after 1-month storage;

# p<0.05 refrigerated temperature vs room temperature after 2-month storage;

^ p<0.05 refrigerated temperature vs room temperature after 3-month storage.

Abbreviations: Act, Acitretin; EE, entrapment efficiency; PDI, polydispersity index; PS, particle size.

Table 5 Evaluation parameters of different gel formulations

Parameters	Act gel	Act niosomal gel
Appearance	Translucent homogeneous gel	Opaque homogeneous gel
Color	Yellow	Yellowish white
pH*	6.23±0.23	6.18±0.16
Viscosity (cP)*	1,164.84±8.65	1,386.72±9.85
Drug content (%)*	98.78±0.73	97.67±0.64

Note:

* Each value represents the mean ± SD (n=3).

Abbreviation: Act, Acitretin.

Table 6 Ex vivo skin permeation parameters of Act from different gel formulations across the excised rat skin after 30 h

Formulation	Q30 h (µg/cm^2)	Jss (µg/cm^2/h)	Kp (cm/h) ×10^−3	ERflux
Act gel	35.66±4.61	1.13±0.14	0.65±0.00	–
Act niosomal gel	112.81±9.87*	3.73±0.25*	2.17±0.00*	3.31±0.22

Notes: Each value represents the mean ± SD (n=3).

* p<0.05 vs Act gel using Student’s t-test (unpaired t-test).

Abbreviations: Act, Acitretin; ER, enhancement ratio; J_ss, steady state flux; K_p, permeability coefficient; Q_{30 h}, cumulative amount that permeated after 30 h.

Figure 5 Ex vivo skin permeation profiles of Act from different gel formulations.

Note: Each point represents the mean ± SD (n=3).

Abbreviation: Act, Acitretin.

Figure 6 Ex vivo skin deposition profiles of Act from different gel formulations.

Notes: Each value represents the mean ± SD (n=3). *p<0.05 vs Act gel using Student’s t-test (unpaired t-test).

Abbreviations: Act, Acitretin; SC, stratum corneum; VED, viable epidermis/dermis.

Table 7 Kinetic analysis of the release data of Act from niosomal dispersion and different gel formulations

Formulation	Zero order	First order	Higuchi model	Release mechanism	Korsmeyer–Peppas		Drug release mechanism
	Correlation coefficient (r^2)				Diffusional exponent
					(r^2)	(n)
Act niosomal dispersion (F1)	0.976	0.983	0.990	Diffusion	0.985	0.725	Non-Fickian
Act gel	0.978	0.979	0.990	Diffusion	0.991	0.706	Non-Fickian
Act niosomal gel	0.979	0.982	0.985	Diffusion	0.991	0.904	Non-Fickian

Abbreviation: Act, Acitretin.

Figure 7 Ex vivo cytotoxicity studies performed by MTT assay.

Notes: (A) L929 cell line. (B) HaCaT cell line. Each value represents the mean ± SD (n=3). *p<0.05 vs plain niosome corresponding to the same concentration, **p<0.05 vs Act corresponding to the same concentration.

Abbreviations: Act, Acitretin; HaCaT, human epidermal keratinocytic cells; L929, mouse dermal fibroblast cells.

Figure 8 Monitoring progression of irritation before and after (7 days) topical application of different gel formulations.

Abbreviation: Act, Acitretin.

Figure 9 Primary irritation index after topical application of different gel formulations for 7 days (n=6).

Abbreviation: Act, Acitretin.

Figure 10 Photomicrographs of histopathologic examination of skin irritation after topical application of different gel formulations for 7 days.

Notes: (A) Control rat skin, (B) plain gel, (C) zarotex gel, (D) Act gel, and (E) Act niosomal gel. H&E (100×). Photomicrograph of skin treated with zarotex gel showed necrosis of the epidermis with crust formation of the overlying superficial layer (arrow) and dilated blood capillaries in the underlying dermis (arrowhead). Photomicrograph of skin treated with Act gel exhibited degenerative changes of the prickle cell layer and status spongiosus formation (arrow) with normal dermis and skin appendages.

Abbreviation: Act, Acitretin.

Table 8 Effect of different gel formulations on the degree of orthokeratosis, drug activity, and reduction in epidermal thickness in mouse tail model after topical application for 4 weeks

Groups	Orthokeratosis (%)	Drug activity (%)	Reduction in epidermal thickness (%)
Control (normal tail)	11.20±2.10	–	–
Plain gel	12.31±2.37	1.25	9.25±2.09
Zarotex gel (0.1%; commercial product)	34.13±3.76*	25.82	22.64±3.08
Act gel	41.42±3.46*	34.03	59.49±3.48
Act niosomal gel	68.51±3.22*,**	64.54	73.60±2.32**

Notes: Each value represents the mean ± SE (n=6).

* p<0.05 vs control;

** p<0.05 Act niosomal gel vs other gel formulations.

Abbreviation: Act, Acitretin.

Figure 11 Photomicrographs of histopathologic examination of mouse tail skin after topical application of different gel formulations for 4 weeks.

Notes: (A) Control tail skin, (B) plain gel, (C) zarotex gel, (D) Act gel, and (E) Act niosomal gel. H&E (100×).

Abbreviations: Act, Acitretin; C, blood capillaries; D, dermis; EP, epidermis; M, Munro abscess; R, rete ridges; SC, stratum corneum.

Figure S1 Schematic diagram of the skin permeation measurement apparatus.