Preparation And In Vitro And In Vivo Evaluation Of An Isoliquiritigenin-loaded Ophthalmic Nanoemulsion For The Treatment Of Corneal Neovascularization

Purpose:Pathological corneal neovascularization（CNV）poses a serious threat to vision.Due to the special anatomy of the eye,clinical treatment options are limited.Isoliquiritigenin（ISL）as a natural flavonoid has been shown to have therapeutic potential for CNV,but its poor water solubility and low permeability limit its therapeutic use.In order to overcome the limitations of these clinical applications,a novel ISL loaded nanoemulsion was designed for the topical treatment of CNV,the physicochemical properties of the developed isoliquiritigenin nanoemulsion（ISL-NE）eye drops were evaluated to study its corneal permeability ex vivo and cytotoxicity,the ocular pharmacokinetics characteristics of the ISL-NE eye drops in rabbits and its anti-angiogenesis effect in vivo,to provide the experimental and theoretical basis for the clinical application of ISL-NE.Methods:The method of assaying ISL in vitro was established,and the maximum absorption peak of ISL was observed by full wavelength scanning of ultraviolet photometer to determine the detection wavelength of high-performance liquid chromatography（HPLC）.The method for the determination of ISL in vitro was determined by the investigation of specificity,standard curve,precision and recovery.In order to prepare ISL-NE,the excipients with higher solubility of ISL were screened by solubility test,and the lipids,surfactant and cosurfactant used in the prescription were determined.The blank nanoemulsion eye drops（B-NE）were prepared by phase inversion composition（PIC）.The pseudo-ternary phase diagram was used to determine the quality range of lipid and the mass ratio of surfactant and cosurfactant（Km）.The optimal formulation of ISL-NE was obtained by optimizing using the central composite design-response surface methodology（CCD-RSM）,which the lipid quality（X₁）and total quality of surfactant and cosurfactant（X₂）were independent variables,particle size（Y₁）and entrapment efficiency（EE）（Y₂）were dependent variables.After the preparation,ISL-NE was characterized by the particle size,polydispersity index（PDI）,potential,pH value,osmotic pressure and morphology.The storage stability if ISL-NE at 4℃and 25℃for 3 months.The cumulative release of ISL from ISL-NE in artificial tear medium containing 1%tween-80 was investigated by dialysis bag dissolution method.The ex vivo corneal permeability of ISL-NE was studied using an Side-Bi-Side diffusion cell.In vitro cytotoxicity test was used to evaluate the cytotoxicity of ISL-NE by studying the effect of ISL-NE at different dilution ratios on the viability of human corneal epithelial cells（HCECs）.Six New Zealand white rabbits were used to evaluate the ocular irritation of ISL-NE according to Draize rule.In the study of Ocular pharmacokinetics（PK）in rabbits,a HPLC method was established for the determination of ISL in cornea,conjunctiva,aqueous humor and tear fluids.The ISL concentration in tear,conjunctiva,cornea and aqueous humor of rabbits after single administration was measured to study the in vivo ocular PK characteristics of ISL-NE,compared with ISL suspension（ISL-Susp）.The main PK parameters of ISL-NE in the eyes of rabbits were calculated by DAS2.0 PK software.The anti-CNV effect of different concentrations of ISL-NE was studied by establishing the model of corneal neovascularization induced by alkali burn in mice,and normal saline used as negative and dexamethasone eye drops used as positive group.The images of CNV were recorded by slit-lamp microscope camera system on day 1,3 and 7,and the areas of corneal neovascularization were calculated by Image J software on day 7.To evaluate the effect of ISL-NE on corneal neovascularization by observing corneal histopathology and the levels of expression of protein factor VEGF-A and MMP-2.Results:The method for the determination of ISL in vitro has been successfully established and validated.Three excipients with high ISL solubility were selected according to the solubility test results.Propylene glycol octanoate（PGO）as lipid,Cremophor EL35（EL-35）as surfactant,polyethylene glycol 400（PEG400）as cosurfactant for the preparation of ISL-NE.By drawing the pseudo-ternary phase diagram,it is determined that the area of nano-emulsion was the largest when Km was 4 and the quality range of lipid was 0.4-1.2 g.The formulation was optimized by CCD-RSM as 0.442 g PGO,0.906 g EL-35 and 0.227 g PEG 400.ISL-NE was prepared by adding water to 20 m L.The ISL-NE prepared by CCD-RSM was spherical in shape.The average particle size was 34.56±0.80 nm,PDI was less than 0.05,potential was-1.95±0.97 m V,pH value was 6.26±0.03,osmotic pressure was 289±1.53 mOsmo L/kg,the EE was99.96±0.02%,the drug content was about 1.98±0.03 mg/m L,and the storage stability was good at 4℃and 25℃.In vitro release study showed that the cumulative release of ISL from ISL-NE was 82.88±2.87%after 12 hours,which showed the characteristics of prolonged drug release time and increased drug release,while ISL from ISL-susp was only 36.05±1.06%.Both of them were in accordance with the first-order kinetic model.The ex vivo corneal permeation of the ISL-NE was greater than that of the ISL-Susp.The ISL-NE did not show toxicity to HCECs and exhibited a good tolerability during rabbit eye treatment.Ocular pharmacokinetic studies revealed that the areas under the concentration-time curves(AUC_0-8h)of the tears,cornea and aqueous humor after a single dose of ISL-NE were 5.76-fold,7.80-fold and 2.13-fold higher than those of ISL-Susp,respectively.Furthermore,the efficacy of ISL-NE treatment（0.2%ISL）was comparable to that of dexamethasone treatment（0.025%）in the inhibition of CNV in mice.Enzyme-linked immunosorbent assay（ELISA）showed that the expressions of corneal vascular endothelial growth factor（VEGF-A）and matrix metalloproteinase（MMP-2）were decreased.Conclusion:The ISL-NE eye drops prepared in this study,as a new ocular delivery system,could enhance the solubility of the insoluble drug ISL,improve the corneal permeability of ISL,and have good physicochemical properties and high ocular bioavailability,the tolerance was good.This may be a promising,safe and effective method for the treatment of CNV,providing new ideas for the treatment of corneal neovascularization,and providing experimental and theoretical basis for clinical application of ISL-NE.