Novel in situ gelling vaginal sponges of sildenafil citrate-based cubosomes for uterine targeting

Figures & data

Table 1: Experimental runs, independent variables, and measured responses of the 4² full-factorial design for SIL-loaded cubosomes.

Formulation	P-407^a (% w/w)	PVA^a (% w/w)	EE (%)	Particle size (nm)	Q8h (%)	PDI
C1	5	0	32.15 ± 2.05	446.02 ± 29.81	80.19 ± 3.16	0.21
C2	5	1	35.89 ± 1.13	357.92 ± 20.26	76.36 ± 4.32	0.10
C3	5	2.5	39.78 ± 2.29	308.55 ± 15.57	71.97 ± 3.38	0.15
C4	5	5	45.81 ± 1.75	320.34 ± 19.75	61.82 ± 2.86	0.18
C5	7.5	0	40.57 ± 2.45	252.20 ± 22.36	58.58 ± 3.36	0.13
C6	7.5	1	53.36 ± 2.03	236.60 ± 14.63	45.72 ± 3.06	0.19
C7	7.5	2.5	56.42 ± 3.24	210.04 ± 18.44	43.66 ± 2.35	0.08
C8	7.5	5	57.28 ± 2. 80	227.10 ± 9.11	37.53 ± 1.92	0.11
C9	10	0	43.84 ± 3.48	206.51 ± 10.19	52.88 ± 2.59	0.12
C10	10	1	46.28 ± 1.40	161.54 ± 6.24	48.48 ± 2.74	0.09
C11	10	2.5	65.89 ± 3.85	150.81 ± 6.86	44.08 ± 3.05	0.14
C12	10	5	66.47 ± 2.76	179.50 ± 9.75	31.85 ± 2.87	0.19
C13	15	0	57.34 ± 1.34	249.96 ± 13.10	29.09 ± 1.46	0.15
C14	15	1	59.63 ± 2.54	236.94 ± 18.27	25.74 ± 1.30	0.13
C15	15	2.5	67.82 ± 4.23	229.72 ± 11.39	24.12 ± 1.12	0.10
C16	15	5	72.01 ± 3.33	231.21 ± 17.22	21.03 ± 1.41	0.20

P-407: poloxamer 407; PVA: polyvinyl alcohol.

Concentration of glyceryl monooleate was 5% w/w with respect to the total weight of the dispersion.

Data are mean values (n = 3) ± SD.

^a Concentration with respect to disperse phase.

Figure 1. Response surface plot for the effect of P-407 (X1) and PVA (X2) concentrations on (a) EE%, (b) particle size, (c) Q8h, and (d) desirability of the prepared cubosomal dispersions.

Figure 2. Transmission electron micrograph of the optimized cubosomal formulation (OCF).

Table 2. Morphometric measurement of vaginal tissues in different studied groups.

		Diameter of large blood vessel
Group	Endometrial thickening	Ovarian	Uterine	Vaginal	Vaginal epithelial thickening
Normal control	1013.70 ± 30.14	77.79 ± 6.85	137.50 ± 18.67	36.39 ± 4.80	131.33 ± 9.63
Oral SIL solution	1246.20 ± 30.06^a	185.15 ± 20.582^a	185.64 ± 40.94^a	194.50 ± 10.16^a	166.66 ± 10.17^a
Intravaginal CIS	1381.70 ± 29.61^a^,^b	206.34 ± 19.23^a^,^b	234.20 ± 69.40^a^,^b	198.03 ± 13.16^a^,^b	183.32 ± 5.16^a^,^b
Intravaginal FIS	1105.20 ± 33.32^a^,^Table Footnoteb,Table Footnotec	112.46 ± 10.33^a^,^Table Footnoteb,Table Footnotec	162.32 ± 22.21^a^,^Table Footnoteb,Table Footnotec	142.46 ± 9.43^a^,^Table Footnoteb,Table Footnotec	142.42 ± 7.87^a^,^Table Footnoteb,Table Footnotec

CIS: cubosomal in situ gelling sponges; FIS: free SIL in situ gelling sponges.

Values are means ± SD, with the number of animals =10 for each group.

Using one-way ANOVA followed by Tukey’s post-hoc test.

^a p < .05 versus normal control.

^b p < .05 versus oral SIL solution.

^c p < .05 versus intravaginal CIS.

Figure 3. Histopathological characteristics of uterus, ovary and vagina from different studied groups after two weeks of SIL treatment. Hematoxylin and Eosin (H & E) staining. a–d: uterus of female Wistar rats. Magnification ×100. (a) Uterus of normal group showing endometrium lined by simple columnar epithelium (S), normal endometrial gland (G) and blood vessels (arrow). (b) Uterus of rat in oral-treated group showing increased height of endometrial epithelium, activity of endometrial glands (arrow), and increased vascularity of uterus (Bv). (c) Uterus of rat in intravaginal CIS-treated group showing a massive increase in the endometrial epithelium (S), endometrial glands (G) and number of blood vessels as well as the blood flow (Bv). d- uterus of rat in intravaginal FIS-treated group showing the endometrial epithelium (S) and endometrial glands (G) similar to normal group with minor increase in number of blood vessels (arrow). (e–h): uterus of female Wistar rats. Periodic acid Schiff (PAS) staining. Magnification ×100. (e) Uterus of normal group showing moderate reaction in the endometrial glands (arrow). (f) Uterus of rat in oral-treated group showing increased activity of endometrial glands (arrow). Note, increased vascularity of uterus (Bv). (g) Uterus of rat in intravaginal CIS-treated group showing a massive increase in the endometrial vascularity (Bv). Note, strong reaction in the endometrial glands (arrow). (h) Uterus of rat in intravaginal FIS-treated group showing normal endometrial vascularity (Bv) with moderate reaction in the endometrial glands (arrow). (i–l) Ovary of female Wistar rats. Magnification ×100. (i) Ovary of rat in normal group showing normal ovarian tissue with growing follicles (F) and normal ovarian blood vessels (Bv). (j) Ovary of rat in oral-treated group showing increased vascularity of ovarian tissue (Bv) and growing follicles (F). (k) Ovary of rat in intravaginal CIS-treated group showing a massive increase in the ovarian tissue vascularity (Bv) and growing follicles (F). (l) Ovary of rat in intravaginal FIS-treated group showing a slight increase in the ovarian tissue vascularity (Bv) and growing follicles (F). (m–p): Vagina of female Wistar rats. (m) Vagina of normal group showing normal vaginal epithelium (stratified squamous epithelium less keratinized (S)) and normal vaginal blood vessels (arrow). (n) Vagina of oral-treated group showing increased height of vaginal epithelium (S) and increased vascularity of vaginal tissue (arrow). (o)- vagina of intravaginal CIS-treated group showing a massive increase in height of vaginal epithelium (S) and increased vascularity of vaginal tissue (Bv). (p) Vagina of rat in intravaginal FIS-treated group showing increased height of vaginal epithelium (S) and minor changes in vascularity of vaginal tissue (arrow).

Table 3. Mean pharmacokinetic parameters for SIL in rat plasma following administration of oral solution, intravaginal CIS, and intravaginal FIS.

Pharmacokinetic parameter	Mean ± SD
	Oral solution	Intravaginal CIS	Intravaginal FIS
Cmax (ng/mL)	1283.37 ± 219.11	134.30 ± 21.21^a	74.20 ± 12.35^a^,b
tmax (h)	0.88 ± 0.29	8.50 ± 1.00^a	4.55 ± 0.43^a^,b
Kelim (h^−1)	0.1641 ± 0.0326	0.0865 ± 0.0111^a	0.1298 ± 0.0154^a^,b
t1/2 (h)	4.22 ± 0.51	8.02 ± 0.88^a	5.34 ± 0.37^b
AUC0–24 (ng.h/mL)	6556.54 ± 844.29	1544.86 ± 389.48^a	747.76 ± 211.68^a^,b
AUC0–∞ (ng.h/mL)	6655.93 ± 837.19	2012.56 ± 295.57^a	1035.65 ± 223.59^a^,b
Frel (%)	–	30.24 ± 10.32	15.56 ± 5.86^b

CIS: cubosomal in situ gelling sponges; FIS: free SIL in situ gelling sponges.

Values are means ± SD, with the number of animals =10 for each group.

Using one-way ANOVA followed by Tukey’s post-hoc test.

^a p < .05 versus oral SIL solution.

^b p < .05 versus intravaginal CIS.