| Objective:Cataract is the leading cause of blindness,while cataract surgery is the most common and usually successful intraocular surgical procedure.Potentially serious complications of cataract surgery include intraocular inflammation,infection,or posterior capsule opacification(PCO).In clinical practice,antibiotics eye drops often are applied for a week while topical corticosteroids often are used for a month.However,limitations of eye drop solutions,include low bioavailability,low patient compliance and poor eye drop administration technique,which could lead to ineffective treatments.Nd: YAG laser capsulotomy is the only effective treatment for PCO;however,there can be post-laser complications such as inflammation and retina detachment.Therefore,development of a multi-drug delivery system containing antibiotics,steroids and anti-PCO agent with special programmed release profiles and can be injected and form in situ gel in the residual lens capsule at the end of cataract surgery may be an ideal solution.Mixture of Pluronic F127(F127)and Pluronic F68(F68)are widely used to construct temperature sensitive in situ gels.The in situ gels can be injected into the eyeball as solution at room temperature,and turn into non-flowing gel once being warmed up to body temperature.In our previous studies,we constructed nanostructured lipid carriers(NLCs)carrying genistein(Gen)which was shown to inhibit the proliferation,migration and myofibroblast transformation of epithelial cell in the whole process of PCO formation.In this study,the genistein nanostructured lipid carrier(Gen-NLC)was modified by methoxy polyethylene glycol-block-Poly(D,L-lactide)(m PEG-PLA),which could improve surface properties and provide sustainable genistein release.In this study,we aimed to develop a temperature-sensitive in situ hydrogel carrying Gen-NLC,dexamethasone(Dex)and moxifloxacin(Mox),which can be injected into the anterior chamber at the conclusion of cataract surgery,especially in the paediatric population,to reduce inflammation,prevent infection and reduce the incidence rate of posterior capsule opacification.Methods:1.Experimental cells and animalsThe human lens epithelial cell line(SRA01/04)and rabbits was provided by the Liaoning Province Key Laboratory of Lens,the Fourth Affiliated Hospital of China Medical University.2.Experimental methods and indicators(1)Gen-NLC was prepared by homogenization emulsification method.(2)Particle size and Zeta potential of Gen-NLC were measured by Malvern Zetasizer Nano ZS instrument.(3)Encapsulation efficiency of Gen-NLC was determined by Gel microcolumn method.(4)Transmission electron microscopy(TEM)were used to detect the morphology of Gen-NLC.(5)Gen-NLC-Dex-Mox was prepared by a homogenization method.(6)The gel temperature of Gen-NLC-Dex-Mox was measured by test tube inversion method.(7)The rheological properties of Gen-NLC-Dex-Mox at different temperatures were measured by quantitative rheometer.(8)Adhesive ability of Gen-NLC-Dex-Mox hydrogel were detected by TA.XT.Plus Texture Analyser.(9)New Zealand white rabbits were used to build cataract surgery animal models.(10)High performance liquid chromatography(HPLC)method was used to build the in vitro release curve.(11)The UV–vis spectrophotometer was used to measure the transmittance of Gen-NLC-Dex-Mox hydrogel.(12)CCK-8 method was used to detect the effect of Gen-NLC-Dex-Mox on the viability of SRA01 / 04 cells.(13)Wound-healing assay was used to detect the inhibition of Gen-NLC-Dex-Mox on the migration of the SRA01/04 cells.(14)RT-PCR,immunofluorescence assay and western blot were used to investigate the inhibition ability of Gen-NLC-Dex-Mox on the proliferation and Epithelial mesenchymal transition(EMT)of SRA01 / 04 cells.(15)Statistical analysisThe data was processed by SPSS 22.0 statistical software,and the results were expressed as Mean ± SD.P < 0.05 was considered statistically significant.Results:1.The Gen-NLCs constructed with MCT and Compritol ATO888 stayed stabilized more than one month without aggregative sediments and significant increase of zeta potentials.The modified Gen-NLCs constructed by this fomulation had an ideal average size(39.47 ± 0.69 nm),moderate polydispersity index(0.213 ± 0.016),and negative Zeta potentials(-4.32 ± 0.84 m V).The encapsulation efficiency(EE)of the Gen-NLCs was 92.75 ± 2.72%.2.As a result,the formulation containing 26% F127 and 1.5% F68 which showed a gelation temperature of 32.0 ± 0.5°C and a gelation time of 20.67 ± 0.58 s met the requirement best.The final formulated Gen-NLC-Dex-Mox temperature sensitive hydrogel was a light yellow,homogeneous and transparent solution.It presented in liquid form at room temperature,while,it formed a non-flowing gel once being warmed up to body temperature.3.In vitro,moxifloxacin showed a quick release for up to 10 days and 98.76 ± 2.31%of moxifloxacin molecules were released till day 9.Dexamethasone was released from hydrogel in the first week at a high but constant rate,then,the rest released sustainably until day 30(97.14 ± 3.38%)with a tapering down rate.Genistein was released the slowly but persistently with a decreasing rate as well.The cumulative release amount of genistein was 63.35 ± 2.49% at day 40.4.Dexamethasone was released at a constant velocity firstly and sustainably released with a tapping down speed.Genistein showed the slowest but persistent release profile from the beginning of the assay with a cumulative release till the final.5.Gen-NLCs and the multi-drug delivery system could inhibit the proliferation of lens epithelial cells in a dose-dependent manner.However,compared with the results of the groups treated with Gen-NLCs(73.74%-42.09%),the cell viabilities in the hydrogel groups were significantly higher(80.75%-47.78%).6.The wounded area of SRA cells was quantified by Image J software.The LECs in the b FGF group had the highest migration rate(20.75%),while,the hydrogel inhibited the cell migration in a dose dependent manner.7.The results of western blotting and RT-PCR both illustrated decreased expression of α-SMA in lens epithelial cells treated by Gen-NLC-Dex-Mox in a dose-dependent manner.The Gen-NLC-Dex-Mox hydrogel treatment dose-dependently reduced the expression levels of PCNA as well as those of Ki-67.Immunofluorescence illustrated the same trends.Conclusion:1.We remodified the genistein nanostructured carrier formulations.Compared with the data in our previous studies,this novel Gen-NLCs had smaller particle sizes in average,and better stability without compromise the encapsulation efficiency.2.The final formulated Gen-NLC-Dex-Mox temperature sensitive hydrogel was a light yellow,homogeneous and transparent solution.It presented in liquid form at room temperature,while,it formed a non-flowing gel once being warmed up to body temperature.3.Moxifloxacin showed a quick release for up to 10 days.Dexamethasone was released from hydrogel in the first week at a high but constant rate,then,the rest released sustainably until day 30 with a tapering down rate.Genistein was released the slowly but persistently with a decreasing rate as well.Also,the gel was injectable during the cataract surgery.4.The in vitro results of the effects of the in situ hydrogel on lens epithelial cells demonstrated that the drug delivery system could effectively inhibit the proliferation,migration and EMT changes of lens epithelial cells in a dose-dependent manner. |
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