Optimized nano-transfersomal films for enhanced sildenafil citrate transdermal delivery: ex vivo and in vivo evaluation

Figures & data

Table 1 Variables and levels of the central composite design applied for the optimization of SLD nano-transferosomes

Independent variables	Levels (actual, coded)
	−1	0	1	+alpha	−alpha
X1 (drug:lipid molar ratio)	1:4	1:6	1:8	1:9.36	1:2.46
X2 (surfactant HLB)	2.1	3.2	4.3	1.35	5.05
X3 (hydration medium pH)	7.5	8	8.5	7.16	8.84
Response	Goal
Y (vesicle size, nm)	Minimize

Abbreviations: SLD, sildenafil citrate; HLB, hydrophilic lipophilic balance.

Table 2 Vesicle size and entrapment efficiency of SLD nano-transfersomes according to the central composite design

Run	Type	Independent variables			Characterization parameters
		Drug:lipid molar ratio	Surfactant HLB	Hydration pH	Vesicle size, Y (nm)a,b	Entrapment efficiency (%)c
1	F	4	4.3	8.5	2,350±73	94.40±2.33
2	F	4	4.3	7.5	1,309±48	94.74±1.67
3	A	9.36	3.2	8	447±13	94.21±1.33
4	C	6	3.2	8	2,290±87	94.46±2.01
5	A	6	3.2	8.84	659±25	95.40±1.66
6	F	8	4.3	8.5	2,979±118	94.36±1.89
7	F	8	2.1	7.5	1,204±39	94.36±0.87
8	A	6	1.35	8	828±37	94.37±2.21
9	F	4	2.1	8.5	735±31	94.34±1.38
10	F	8	2.1	8.5	2,738±82	94.49±2.77
11	A	6	3.2	7.16	1,952±77	95.46±1.42
12	F	4	2.1	7.5	720±29	94.32±0.84
13	C	6	3.2	8	2,082±84	94.98±1.13
14	A	2.64	3.2	8	1,735±73	94.45±1.82
15	A	6	5.05	8	2,446±97	94.44±1.44
16	F	8	4.3	7.5	456±18	95.31±2.08

Notes:

a Results are presented as mean ± SD, n=5;

b response variable for the central composite design;

c results are presented as mean ± SD, n=3.

Abbreviations: SLD, sildenafil citrate; HLB, hydrophilic lipophilic balance; F, factorial; A, axial; C, center; SD, standard deviation.

Table 3 Regression analysis of the measured response (vesicle size) according to different polynomial models

Model	SD	R^2	Adjusted R^2	PRESS
Linear	500.80	0.3960	0.2450	4.740E+006
Two-factor interaction (2 FI)	556.99	0.4396	0.0660	6.481E+006
Quadratic	343.75	0.8977	0.7443	4.373E+006

Note: R² indicates the multiple correlation coefficient and adjusted R² indicates the adjusted multiple correlation coefficient.

Abbreviations: SD, standard deviation; PRESS, predicted residual sum of square.

Table 4 Analysis of variance (ANOVA) and lack of fit parameters for the vesicle size response according to the quadratic model

Source	Sum of squares	df	Mean square	F-value	P-value
Model	4.274E+006	9	4.749E+005	4.02	0.0423*
X1	26,917.64	1	26,917.64	0.23	0.6500
X2	1.869E+006	1	1.869E+006	15.82	0.0073*
X3	76,569.39	1	76,569.39	0.65	0.4515
X1X2	1.174E+005	1	1.174E+005	0.99	0.3574
X1X3	89,676.13	1	89,676.13	0.76	0.4172
X2X3	10,440.13	1	10,440.13	0.088	0.7763
${\mathrm{X}}_1^2$	1.737E+006	1	1.737E+006	14.70	0.0086*
${\mathrm{X}}_2^2$	2.055E+005	1	2.055E+005	1.74	0.2354
${\mathrm{X}}_3^2$	1.051E+006	1	1.051E+006	8.90	0.0246*
Residual	7.090E+005	6	1.182E+005
Lack of fit	7.040E+005	5	1.408E+005	28.16	0.1421
Pure error	5,000.00	1	5,000.00
Corrected total	4.983E+006	15

Notes:

* Significant terms. X₁, X₂, X₃ show the drug to lipid molar ratio, the surfactant HLB, and the hydration medium pH, respectively.

Abbreviations: df, degrees of freedom; HLB, hydrophilic lipophilic balance.

Figure 1 Residuals plot for the observed and predicted vesicle size of SLD nano-transfersomes prepared according to central composite design.

Abbreviation: SLD, sildenafil citrate.

Figure 2 Three-dimensional surface plots (A) and corresponding contour plots (B) showing the effects of the independent variables on the vesicle size (Y) response.

Notes: Independent variables are drug: PL molar ratio (X₁), surfactant HLB (X₂), and hydration medium pH (X₃). Two variables are considered at a time and the third one is kept constant at its mid value.
Abbreviations: HLB, hydrophilic lipophilic balance; PL, phospholipid.

Figure 3 TEM photomicrograph of optimized SLD nano-transfersomes (×10,000).

Abbreviations: TEM, transmission electron microscopy; SLD, sildenafil citrate.

Table 5 Ex vivo permeation parameters of SLD optimized nano-transfersomal and control transdermal films

Formulation	Dmax (µg) ± SD	Jss (µg/cm^2 h)	Pc (cm/h) ×10^−3	D ×10^−3
Control SLD films	152.87±2.09	0.21010805	0.440216	0.284859
Optimized SLD nano-transfersomal films	235.89±3.76	0.22114828	0.552871	0.44891

Note: J_ss indicates the steady-state flux.

Abbreviations: SLD, sildenafil citrate; D_max, maximum amount of drug permeated; P_c, permeability coefficient; D, diffusion coefficient; SD, standard deviation.

Figure 4 Mean cumulative percent SLD permeated across excised rat abdominal skin from SLD optimized nano-transfersomal films compared to control films.

Abbreviations: SLD, sildenafil citrate; SD, standard deviation; h, hour.

Table 6 Kinetic analysis of permeation data of SLD optimized nano-transfersomal and control transdermal films

Formulation	Correlation coefficient (R)			n	Release mechanism
	Zero	First	Higuchi
Control SLD films	0.700865	0.72011	0.822014	0.204216	Fickian diffusion
Optimized SLD nano-transfersomal films	0.862806	0.8888	0.957981	0.652354	Anomalous transport

Abbreviation: SLD, sildenafil citrate.

Table 7 In vivo mean pharmacokinetics parameters of optimized SLD nano-transfersomal transdermal films compared to control SLD transdermal films and SLD oral suspension

PK parameter	SLD suspension	Control SLD films	Optimized SLD nano-transfersomal films
K (h^−1)a	0.128±0.024	0.075±0.024	0.086±0.01
Tmax (h)b	1.00	4.00	15.00
Cmax (ng/mL)a	128.81±19.8	79.91±15.16	114.83±12.82
AUC0–36 (ng h/mL)a	691.23±128.7	942.55±214.8	2,103.77±247.56
AUC0–∞ (ng h/mL)a	699.17±132.7	1,023.54±243.59	2,326.65±300.91

Notes:

a Results are presented as mean ± SD (n=12).

b Results are presented as median (n=12). K is the elimination half-life, T_max is the time to maximum plasma concentration, and C_max is the maximum plasma concentration.

Abbreviations: PK, pharmacokinetics; SLD, sildenafil citrate; AUC, area under the curve; SD, standard deviation.

Figure 5 Mean SLD plasma concentrations following the oral administration of SLD suspension and the transdermal application of SLD optimized nano-transfersomal and control films.

Abbreviations: SLD, sildenafil citrate; SD, standard deviation; h, hour.