Implementing Spanlastics for Improving the Ocular Delivery of Clotrimazole: In vitro Characterization, Ex vivo Permeability, Microbiological Assessment and In vivo Safety Study

Figures & data

Table 1 The Independent Variables Levels Used to Formulate CLT Loaded SPs Utilizing (3²) Complete Factorial Design

Factors (Independent Variables)	Levels
X1: Span 60: EA ratio (w/w)	80: 20	70: 30	60: 40
X2: Type of EA	Tween 80	Pluronic F127	Kolliphor RH40
Responses (Dependent Variables)	Desirability Constraints
Y1: EE%	Maximize
Y2: VS (nm)	Minimize
Y3: PDI	Minimize
Y4: ZP (mV)	Maximize (as absolute value)

Abbreviations: CLT, clotrimazole; SPs, spanlastics; EA, edge activator; EE%, encapsulation efficiency percent; VS, vesicle size; PDI, polydispersity index; ZP, zeta potential.

Table 2 Measured Responses of CLT Formulations of the Experimental Complete 3² Factorial Design

Formulations	X1 Span 60: EA Ratio	X2 Type of EA	Y1 EE%	Y2 VS (nm)	Y3 PDI	Y4 ZP (mV)
S1	80: 20	Tween 80	66.54 ± 7.57	206.20 ± 4.95	0.39 ± 0.00	−29.60 ± 0.99
S2	80: 20	Pluronic F127	67.38 ± 0.00	366.70 ±102.00	0.56 ± 0.10	−22.10 ± 0.49
S3	80: 20	Kolliphor RH40	40.96 ± 24.25	356.10 ± 8.84	0.59 ± 0.09	−29.40 ± 0.49
S4	70: 30	Tween 80	21.91 ± 8.76	162.10 ± 0.14	0.36 ± 0.03	−29.00 ± 6.51
S5	70: 30	Pluronic F127	33.34 ± 4.38	227.10 ± 2.69	0.36 ± 0.05	−28.20 ± 1.41
S6	70: 30	Kolliphor RH40	97.74 ± 1.51	411.30 ± 13.00	0.81 ± 0.05	−26.10 ± 2.83
S7	60: 40	Tween 80	94.52 ± 0.00	309.50 ± 5.23	0.44 ± 0.01	−25.40 ± 0.21
S8	60: 40	Pluronic F127	64.53 ± 4.38	179.00 ± 3.96	0.33 ± 0.04	−27.20 ± 0.64
S9	60: 40	Kolliphor RH40	80.12 ± 5.90	245.40 ± 1.13	0.47 ± 0.00	−21.20 ± 0.57

Notes: All the prepared SPs contained 10 mL CLT nanosuspension (equivalent to 20 mg drug). Data are presented as mean ± SD (n=3).

Abbreviations: CLT, clotrimazole; EA, edge activator; EE%, encapsulation efficiency percent; VS, vesicle size; PDI, polydispersity index; ZP, zeta potential; SPs, spanlastics.

Table 3 The Statistics Summary of Complete Factorial Design (3²) Used for Optimization of SPs Formulations

Responses	R^2	Adjusted R^2	Predicted R^2	Adequate Precision	Significant Factors
Y1: EE%	0.93	0.87	0.73	11.39	X1, X2
Y2: VS (nm)	0.92	0.85	0.69	10.12	X1, X2
Y3: PDI	0.94	0.89	0.76	13.23	X1, X2
Y4: ZP (mV)	0.74	0.50	−0.05	4.82	–

Abbreviations: SPs, Spanlastics; EE%, encapsulation efficiency percent; VS, vesicle size; PDI, polydispersity index; ZP, zeta potential.

Figure 1 Linear correlation plots presenting the effect of (A) Span 60: EA ratio and (B) EA type on the following parameters: EE%, VS, PDI and ZP.

Abbreviations: EA, edge activator; EE%, encapsulation efficiency percent; VS, vesicle size; PDI, polydispersity index; ZP, zeta potential.

Figure 2 The optimum SPs formulation (S1) transmission electron micrograph.

Abbreviation: SPs, Spanlastics.

Table 4 Effect of Storage on Physical Properties of the Optimum Formulation S1

Parameter	S1 Fresh	S1 After 45 Days at 4°C	S1 After 45 Days at 25°C	S1 After 90 Days at 4°C	S1 After 90 Days at 25°C
EE%	66.54 ± 7.57	66.68 ± 1.90	68.82 ± 1.18	66.67 ± 2.20	67.60 ± 3.68
VS (nm)	206.20 ± 4.95	210.30 ± 7.42	207.40 ± 3.96	219.00 ± 2.19	211.50 ± 0.78
PDI	0.39 ± 0.00	0.47 ± 0.03	0.46 ± 0.02	0.42 ± 0.02	0.40 ± 0.03
ZP (mV)	−29.60 ± 0.99	−30.60 ± 0.07	−28.90 ± 1.34	−31.00 ± 0.14	−30.00 ± 1.13

Note: Data are presented as mean ± SD (n = 3).

Abbreviations: EE%, encapsulation efficiency percent; VS, vesicle size; PDI, polydispersity index; ZP, zeta potential.

Figure 3 In vitro release study of CLT formulations.

Abbreviation: CLT, clotrimazole.

Table 5 Kinetics of CLT Release from S1 According to Different Kinetic Models

Formulation	Zero Order	First Order	Second Order	Third Order	Diffusion
	Correlation Coefficient (R^2) *
S1	0.98 ± 0.00	0.77 ± 0.03	0.44 ± 0.03	0.34 ± 0.04	0.97 ± 0.03

Note: *Mean ± SD (n=3).

Abbreviation: CLT, clotrimazole.

Figure 4 Ex vivo corneal permeability of CLT formulations.

Abbreviation: CLT, clotrimazole.

Figure 5 Microbiological assay of (A) CLT suspension, (B) Non-medicated S1 and (C) S1 for the treatment of Candida albicans infection. Data are presented as mean ± SD, (n=3).

Abbreviation: CLT, clotrimazole.

Figure 6 Photomicrographs presenting histopathological sections (stained by hematoxylin and eosin) of normal untreated rabbit eye (group 1), rabbit eye treated with CLT suspension (group 2) and rabbit eye treated with S1 (group 3). (A) Shows histological structure of the cornea, (B) Shows histological structure of the iris and (C) Shows histological structure of the sclera, retina and choroid.

Abbreviation: CLT, clotrimazole.