Solutol HS15 based binary mixed micelles with penetration enhancers for augmented corneal delivery of sertaconazole nitrate: optimization, in vitro, ex vivo and in vivo characterization

Figures & data

Table 1. Independent variables with the respective levels investigated in the 3¹.2² mixed factorial design and the dependent variables with their required constraints, together with the detailed Composition of the prepared STZ-MMs formulations and their characterization.

Factors (independent variables)	Levels
A: Auxiliary surfactant type	L121	B58		F68
B: Penetration enhancer type	Trc		PG
C: Total surfactants :drug ratio	10:1		20:1
Responses (dependent variables)	Constraints
Y1: SM (mg/mL)	Maximize
Y2: PS (nm)	Minimize
Y3: PDI	Minimize
Y4: ZP (mV)	Maximize
Formula	Factors’ levels			SM (mg/mL)	Fs	SI	PS (nm)	PDI	ZP (mV)
	A	B	C
MM-1	L121	Trc	10:1	2.56 ± 0.06	111.20	0.32	26.81 ± 0.29	0.302 ± 0.02	19.50 ± 0.57
MM-2	B58	Trc	10:1	5.17 ± 0.50	224.80	0.65	19.09 ± 0.67	0.315 ± 0.03	33.63 ± 4.07
MM-3	F68	Trc	10:1	2.02 ± 0.10	87.62	0.25	22.44 ± 2.14	0.415 ± 0.03	17.88 ± 0.32
MM-4	L121	PG	10:1	2.51 ± 0.05	109.22	0.31	26.87 ± 1.05	0.334 ± 0.05	15.85 ± 1.77
MM-5	B58	PG	10:1	5.40 ± 0.97	234.89	0.68	19.36 ± 0.10	0.360 ± 0.03	36.89 ± 2.81
MM-6	F68	PG	10:1	1.99 ± 0.23	86.57	0.25	23.51 ± 0.08	0.380 ± 0.01	25.15 ± 0.78
MM-7	L121	Trc	20:1	5.19 ± 0.32	225.60	0.65	21.25 ± 1.22	0.264 ± 0.01	30.25 ± 0.92
MM-8	B58	Trc	20:1	7.79 ± 0.17	338.82	0.97	15.87 ± 0.71	0.249 ± 0.04	28.95 ± 4.38
MM-9	F68	Trc	20:1	3.39 ± 0.13	147.55	0.42	18.61 ± 0.06	0.252 ± 0.00	22.55 ± 0.42
MM-10	L121	PG	20:1	4.60 ± 0.26	200.17	0.58	21.67 ± 0.52	0.217 ± 0.01	18.28 ± 1.38
MM-11	B58	PG	20:1	7.59 ± 0.16	329.91	0.95	16.39 ± 0.04	0.234 ± 0.04	23.33 ± 1.17
MM-12	F68	PG	20:1	3.45 ± 0.14	149.91	0.43	19.20 ± 0.20	0.279 ± 0.05	15.25 ± 0.78

Data are represented as (mean ± SD). S_M: solubility of STZ in MMs; F_s: solubility factor; SI: stability index; PS: particle size; PDI: polydispersity index; ZP: zeta potential.

Table 2. Significance of different model terms appearing in the final model for each response, together with models evaluation.

Component	p-Value
	SM (mg/mL)	PS (nm)	PDI	ZP (mV)
A	<0.0001	<0.0001	0.0062	<0.0001
B	–	0.1209	–	0.0041
C	<0.0001	<0.0001	<0.0001	0.0709
AB	–	–	–	0.0056
AC	0.0119	0.021	–	<0.0001
BC	–	–	–	<0.0001
ABC	–	–	0.0597	–
RMSE	0.33	0.73	0.03	2.15
R^2	0.9782	0.9683	0.8322	0.9448
Adjusted R^2	0.9722	0.9572	0.7855	0.9094
Prediction R^2	0.9613	0.9369	0.7016	0.8379
PRESS	3.48	18.21	0.029	190.04
Adeq Precision	34.505	28.293	12.64	15.189

A: Auxiliary surfactant type; B: Penetration enhancer type; C: Total surfactants: drug ratio; S_M: solubility of STZ in MMs; PS: particle size; PDI: polydispersity index; ZP: zeta potential; RMSE: Root mean square error; R²: Regression coefficient.

^* p Values for significant terms are shown in bold face.

Figure 1. (I) line plots of the significant effects of type of auxiliary surfactant, total surfactants: drug ratio and type of penetration enhancer on (A) S_M, (B) PS, (C) PDI, and (D) ZP. (II) DSC thermograms of STZ, HS15, B58, MM-8 physical mix., plain MM-8, STZ-MM-8, MM-11 physical mix., plain MM-11, and STZ-MM-11.

Table 3. Predicted values of different responses for optimized formulations (MM-8 and MM-11) compared to the actual values together with the prediction intervals and percent deviations.

Formula	Validity parameters	Y1: SM (mg/mL)	Y2: PS (nm)	Y3: PDI	Y4: ZP (mV)
MM-8	Predicted value	7.69	15.88	0.247	29.38
	95% prediction interval	6.92–8.46	14.12–17.64	0.18–0.32	23.89–34.86
	Actual values	7.79	15.87	0.249	28.95
	% Deviation	1.29	0.08	0.81	1.45
MM-11	Predicted value	7.69	16.37	0.230	22.90
	95% prediction interval	6.92–8.46	14.61–18.13	0.16–0.3	17.41–28.38
	Actual values	7.59	16.39	0.234	23.33
	% Deviation	1.31	0.11	1.87	1.89

SM: solubility of STZ in MMs; PS: particle size; PDI: polydispersity index; ZP: zeta potential.

Figure 2. (I) Mucoadhesion results: (A) PS measurements, (B) ZP measurements and (C) Turbidimetric measurements. (II) Evolution of the absorbance of optimized formulations (MM-8 and MM-11) after 100-folds dilution in STF. (III) The ex-vivo permeation profile of STZ from optimized formulations (MM-8 and MM-11) compared to the STZ-suspension.

Table 4. Effect of storage on the physicochemical properties of optimized STZ-MMs formulations (MM-8 and MM-11).

Formula	Storage period	SM (mg/mL)	PS (nm)	PDI	ZP (mV)
MM-8	Fresh	7.79 ± 0.17	15.87 ± 0.71	0.249 ± 0.04	28.95 ± 4.38
	1 Month	7.72 ± 0.28	15.99 ± 0.17	0.250 ± 0.01	28.12 ± 2.93
	2 Months	7.69 ± 0.15	16.02 ± 0.01	0.249 ± 0.05	28.80 ± 1.99
	3 Months	7.64 ± 0.25	16.02 ± 0.33	0.247 ± 0.02	25.10 ± 4.10
	6 Months	7.61 ± 0.37	16.04 ± 0.23	0.249 ± 0.04	25.21 ± 0.76
MM-11	Fresh	7.59 ± 0.16	16.39 ± 0.04	0.234 ± 0.04	23.33 ± 1.17
	1 Month	7.53 ± 0.08	16.38 ± 0.08	0.239 ± 0.07	25.20 ± 1.85
	2 Months	7.56 ± 0.14	16.47 ± 0.45	0.242 ± 0.03	25.00 ± 2.83
	3 Months	7.51 ± 0.12	16.52 ± 0.33	0.247 ± 0.06	26.20 ± 4.53
	6 Months	7.25 ± 0.31	16.83 ± 0.39	0.271 ± 0.04	25.70 ± 0.42

S_M: solubility of STZ in MMs; PS: particle size; PDI: polydispersity index; ZP: zeta potential.

Figure 3. (I) Transmission electron micrographs of optimized formulations (MM-8 and MM-11). (II) Photomicrographs of the rabbits’ corneas after instillation of; (A) normal saline solution (negative control), (B) Isopropyl alcohol (positive control), (C) MM-8 formulation and (D) MM-11 formulation. (III) Confocal laser scanning micrographs of the rabbits’ corneas after instillation of (A) 0.1% RhB aqueous solution, (B) RhB loaded MM-8, and (C) RhB loaded MM-11.