Tailored nanostructured platforms for boosting transcorneal permeation: Box–Behnken statistical optimization, comprehensive in vitro, ex vivo and in vivo characterization

Figures & data

Table 1 Experimental runs, independent variables and measured responses of the Box–Behnken model for tenoxicam nanostructured formulations

Formulae	GTP % (w/v)	PF127% (w/v)	GG % (w/v)	PS (nm)	ZP (mV)	EE (%)	GT (°C)
NF1	0.25	4	0.3	631.6±22.7	−14.4±1.2	94.4±3.4	45±3.9
NF2	0.25	7	0.1	1,020.1±45.2	−10.5±0.8	89.7±2.7	44±1.4
NF3	0.25	7	0.5	107.8±3.3	−21.1±1.5	92.7±4.0	34±1.3
NF4	0.25	10	0.3	26.3±1.0	−21.5±0.7	90.5±6.1	31±0.9
NF5	0.625	4	0.1	937.5±31.7	−8.6±0.5	94.8±6.9	44±1.1
NF6	0.625	4	0.5	968.7±58.9	−29.2±2.0	95.7±3.3	36±2.8
NF7	0.625	7	0.3	799.9±10.3	−15.6±0.7	91.4±1.9	38±3.1
NF8	0.625	10	0.1	1,160.4±24.2	−9.7±0.3	91.2±4.8	46±0.8
NF9	0.625	10	0.5	900.2±40.8	−13.1±0.9	91.5±7.2	38±1.0
NF10	1	4	0.3	933.4±36.9	−18.5±0.2	93.4±2.0	37±2.7
NF11	1	7	0.1	1,035.4±25.0	−9.9±1.1	91.2±6.1	46±1.7
NF12	1	7	0.5	1,640.5±57.1	−25.0±0.1	87.9±3.7	32±3.9
NF13	1	10	0.3	1,968.0±14.6	−15.6±1.4	90.0±4.3	45±2.6

Note: Data are presented as mean ± SD.

Abbreviations: EE, entrapment efficiency; GG, gellan gum; GT, gelation temperature; GTP, glyceryl tripalmitate; PF, Pluronic F; PS, particle size; ZP, zeta potential.

Figure 1 Response surface plots for the effects of the independent variables on the PS (A), ZP (B), EE (C) and GT (D) of the prepared nanostructured formulations.

Abbreviations: conc, concentration; EE, entrapment efficiency; GT, gelation temperature; GTP, glyceryl tripalmitate; PS, particle size; ZP, zeta potential.

Table 2 Physical characteristics of the optimized formulations

Formulae	PS (nm)	ZP (mV)	EE (%)	GT (°C)
PRE-I	112.0±8.2	−24.6±3.8	89.0±7.4	–
OIG	93.6±5.9	−25.8±1.6	93.3±5.7	32±0.6

Note: Data presented as mean ± SD.

Abbreviations: EE, entrapment efficiency; GT, gelation temperature; OIG, optimized in situ gel; PRE-I, pre-insert; PS, particle size; ZP, zeta potential.

Figure 2 Desirability values for the development of OIG (A and B) and PRE-I (C and D) formulations.

Abbreviations: conc, concentration; GTP, glyceryl tripalmitate; OIG, optimized in situ gel; PRE-I, pre-insert.

Figure 3 Characteristics of the lyophilized formulations, compared to the original nanostructured formulation (PRE-I).

Abbreviations: EE, entrapment efficiency; PRE-I, pre-insert; PS, particle size; ZP, zeta potential.

Figure 4 Transmission electron micrographs of the OIG formula at different magnifications.

Abbreviation: OIG, optimized in situ gel.

Figure 5 Scanning electron micrographs of the selected lyophilized formula (L3) at magnifications of 60× (A), 140× (B) and 800× (C).

Figure 6 Rheological characteristics of the optimized formulations, including the changes in shear stress (A) or viscosity (B) with increasing rate of shear.

Abbreviations: OIG, optimized in situ gel; L3, selected lyophilized formula.

Figure 7 In vitro drug release from the optimized formulations, compared to the drug solution.

Abbreviations: OIG, optimized in situ gel; L3, selected lyophilized formula.

Figure 8 Ex vivo drug permeation from the optimized formulations, compared to the drug solution.

Abbreviations: OIG, optimized in situ gel; P₂, the diffusion; J, flux; P₁, partition; K_P, permeability; T_L, the lag time; L3, selected lyophilized formula.

Figure 9 Histopathologic evaluation of the rabbit corneas after instillation of normal saline solution (A), OIG (B) and L3 (C) formulations.

Abbreviations: OIG, optimized in situ gel; L3, selected lyophilized formula.

Figure 10 Confocal laser scanning micrographs of the rabbit corneas after instillation of RhB loaded in an aqueous solution (A), OIG (B) and L3 (C) formulations.

Abbreviations: OIG, optimized in situ gel; RhB, Rhodamine B; L3, selected lyophilized formula.