Ion-sensitive in situ hydrogels of natamycin bilosomes for enhanced and prolonged ocular pharmacotherapy: in vitro permeability, cytotoxicity and in vivo evaluation

Figures & data

Table 1. Composition and physicochemical properties of natamycin bilosomes (mean ± SD; n = 3).

	Formulation
Component/evaluation parameter	NB 1	NB 2	NB 3	NB 4	NB 5	NB 6
Molar ratio (Span 60:Cholesterol)	2:1	1:1	1:2	2:1	1:1	1:2
Span 60 (mg)	66	50	33	60	45	30
Cholesterol (mg)	30	44	59	27	41	54
ST (mg)	10	10	10	21	21	21
PS (nm)	330.1 ± 21.6	235.0 ± 05.2	287.9 ± 12.1	380.4 ± 27.9	303.3 ± 16.0	348.2 ± 13.1
PDI	0.30 ± 0.10	0.20 ± 0.01	0.23 ± 0.13	0.43 ± 0.23	0.28 ± 0.09	0.31 ± 0.03
ZP (mv)	−45.4 ± 4.5	−47.0 ± 1.5	−51.1 ± 3.3	−67.2 ± 2.6	−69.0 ± 1.5	−66.3 ± 9.1
% EE	78.6 ± 4.1	93.7 ± 4.3	84.1 ± 2.6	70.1 ± 3.6	81.4 ± 2.5	74.1 ± 3.5
Drug content (%)	96.7 ± 1.1	96.2 ± 2.0	96.1 ± 2.5	97.9 ± 4.1	90.1 ± 7.2	95.6 ± 1.2
pH	6.2 ± 0.9	6.4 ± 0.3	6.3 ± 0.1	6.5 ± 0.2	7.1 ± 0.2	6.6 ± 0.3

NB and ST indicates natamycin loaded bilosomes and sodium taurocholate, respectively.

PS, ZP, PDI and % EE represents particle size, zeta potential, polydispersity index and % entrapment efficiency respectively.

Total 250 µM of lipid mixture (including sodium taurocholate) was used in all preparations.

10 ml of each formulation contains 10 mg of natamycin and 225 mg of glycerin.

Table 2. Composition and characterization of NB in situ hydrogel formulations (mean ± SD; n = 3).

	Formulation
Component/Evaluation parameter	NB 2	NBG 1	NBG 2	NBG 3	NBX 1	NBX 2	NBX 3
Molar ratio (Span 60:Cholesterol)	1:1	1:1	1:1	1:1	1:1	1:1	1:1
Span 60 (mg)	50	50	50	50	50	50	50
Cholesterol (mg)	44	44	44	44	44	44	44
ST (mg)	10	10	10	10	10	10	10
G (mg)	--	20	30	40	--	--	--
X (mg)	--	--	--	--	20	30	40
Viscosity (cP) (Formulation)	17.1 ± 2.7	24.7 ± 1.5	37.5 ± 2.4	68.3 ± 3.2	39.5 ± 4.1	87.8 ± 2.5	124.2 ± 4.7
GT (sec)	--	< 5	< 5	< 5	> 20	> 20	15-20
GRT (h)	--	3-4	6-8	>12	<1	1-2	5
Viscosity (cP) (hydrogel)	--	104.5 ± 1.5	514.9 ± 3.8	1285. 6 ± 6.8	128.9 ± 6.5	220.3 ± 5.7	410.6 ± 4.2
Work of Adhesion (g. sec)	--	0.80 ± 0.01	1.39 ± 0.06	1.31 ± 0.07	0.68 ± 0.02	0.71 ± 0.01	1.14 ± 0.10

NB, NBG, NBX, ST, GT and GRT represents natamycin loaded bilosomes, natamycin bilosome in situ gels with gellan gum, natamycin bilosome in situ gels with xanthum gum, sodium taurocholate, gel formation time and intact gel residence time, respectively.

Total 250 µM of lipid mixture (including sodium taurocholate) was used in all preparations.

10 ml of each formulation contains 10 mg of natamycin and 225 mg of glycerin.

Figure 1. Transmission electron micrograph (TEM) depicting the layered morphology of NT bilosomes.

Figure 2. FTIR spectra of (A) sodium taurocholate, (B) cholesterol, (C) span 60, (D) natamycin and (E) NBG 2 formulation.

Figure 3. Cytotoxicity profile of NB 2 and NBG 2 formulations in HCLE cells (mean ± SEM; n = 6).

Table 3. In vitro transcorneal permeability parameters of natamycin from bilosomes and bilosomes in situ gel formulations (mean ± SD; n = 3).

Formulation	Flux (µg/cm^2/min)	Papp×10 ^5 (cm/s)	ER
Control	0.015 ± 0.005	0.14 ± 0.05	–
NB 2	0.12 ± 0.02*^,**	1.92 ± 0.40*^,**	9.0 ± 5.6
NBG 2	0.090 ± 0.005*	1.42 ± 0.08*	6.3 ± 2.8

NB, NBG, P_app and ER represents natamycin loaded bilosomes and natamycin bilosome in situ gels with gellan gum, apparent permeability coefficient and enhancement ratio respectively.

* indicates the level of significance at p < .05 between NB2/NBG 2 and control.

**represents the level of significance at p < .05 with respect to NBG 2.

Table 4. The amount of natamycin in the ocular tissues of rabbits at the end of 2 h and 6 h post-topical administration with control, NB 2 and NBG 2 formulations (mean ± SEM; n = 4).

			NT levels in ocular tissues (ng/g or ng/mL)
Formulations	Dose (µg)	Time point (h)	Cornea	AH	IC	RC	Sclera	VH
Control	2500	2	4390.4 ± 894.2	100.6 ± 17.9	225.3 ± 70.1	ND	2691.3 ± 923.4	ND
NB 2	50		333.6 ± 59.4	28.1 ± 8.2	369.4 ± 64.6	ND	416.2 ± 42.4	ND
NBG 2	50		238.1 ± 10.2	17.8 ± 1.5	264.5 ± 16.2	ND	232.1 ± 33.5	ND
Control	2500	6	290.1 ± 56.9	ND	490.2 ± 60.6	ND	624.7 ± 75.3	ND
NB 2	50		ND	ND	ND	ND	66.4 ± 14.4	ND
NBG 2	50		212.3 ± 44.2	13.0 ± 1.4	233 ± 4	158.4 ± 18.3	146.1 ± 20.8	ND

AH, IC, RC and VH indicates aqueous humor, iris ciliary body, retina choroid and vitreous humor, respectively.

NT, NB and NBG represents natamycin, natamycin bilosomes and natamycin bilosome in situ gel formulation, respectively.

Figure 4. Histology images of rabbit corneas exposed to (A) DPBS, (B) control, (C) natamycin bilosomes (NB 2) and (D) natamycin bilosomes in situ gel (NBG 2) formulations. 1 and 2 indicates the 10 × and 100 × magnifications respectively. EP: epithelial layer; BM: Bowman's membrane; SL: stromal layer; DM: Descemet's membrane.

Figure 5. Mean dose normalized natamycin levels in rabbit ocular tissues from control, optimized natamycin bilosomes (NB 2) and bilosomes in situ gel (NBG 2) formulations, at (A) 2 h and (B) 6 h time point (mean ± SEM; n = 4). *– significantly different at p < .05. ND-not detected; Cr-Cornea; AH-Aqueous Humor; IC-Iris Ciliary Body; SC-Sclera; RC-Retina Choroid; VH-Vitreous Humor.

Table 5. Physical stability of optimized natamycin bilosomes and bilosome in situ gel formulations stored at 4 °C and 25 °C for 60 days (mean ± SD; n = 3).

Formulation	Stored at	Time (days)	PS (nm)	ZP (mv)	PDI	% EE	Drug content (%)
NB 2	Initial		235.0 ± 5.3	−47.0 ± 1.6	0.20 ± 0.01	93.8 ± 4.3	99.2 ± 2.0
	4°C	30	239.3 ± 4.4	−44.6 ± 3.9	0.21 ± 0.01	91.5 ± 1.2	98.4 ± 1.0
		60	243.6 ± 5.9	−45.7 ± 2.0	0.21 ± 0.01	91.4 ± 4.6	98.5 ± 1.3
	25°C	30	240.7 ± 3.7	−44.6 ± 3.2	0.21 ± 0.02	92.4 ± 2.2	98.4 ± 1.2
		60	264.2 ± 10.9*	−44.2 ± 4.7	0.28 ± 0.04*	81.9 ± 1.9*	97.9 ± 2.0
NBG 2	Initial		234.3 ± 6.1	−48.0 ± 1.5	0.22 ± 0.07	90.5 ± 1.2	98.9 ± 1.7
	4°C	30	236.7 ± 6.9	−46.8 ± 0.4	0.21 ± 0.02	90.5 ± 0.3	98.4 ± 1.5
		60	244.4 ± 8.4	−46.7 ± 0.9	0.22 ± 0.01	90.4 ± 1.2	98.3 ± 1.9
	25°C	30	240.4 ± 3.4	−47.5 ± 4.2	0.22 ± 0.01	89.7 ± 2.3	98.0 ± 1.9
		60	245.4 ± 10.4	−46.7 ± 6.2	0.29 ± 0.16	81.9 ± 1.2*	97.9 ± 0.5

NB, NBG, PS, ZP, PDI and % EE represents natamycin bilosomes, natamycin bilosome in situ gel formulation, particle size, zeta potential, polydispersity index and % entrapment efficiency respectively.

*indicates significant difference (p < .05) between the NB 2 or NBG 2 at 25 °C for 60 days and fresh NB 2 or NBG 2 at initial conditions (day 0 at room temperature).