Experimental Study Of Penetrating Peptide Loaded Anti VEGF Eye Drops In The Treatment Of Choroidal Neovascularization

Age-related macular degeneration is one of the main threats of old people’s central vision Irreversible loss.The hemorrhage and leakage caused by the abnormal growth of choroidal neovascularization lead to the damage of retinal pigment epithelium and neurosensory retina,which is the main cause of the irreversible loss of central vision.Intravitreal injection of anti-vascular endothelial growth factor(VEGF),as the mainstream therapy,can improve patients’ visual acuity,but long-term and repeated invasive treatment will increase the risk of post-injection complications and adverse drug reactions.This topic aims to use the new type non-viral carrier cell penetrating peptides as the carrier,modified fluorescent groups fluorescein isothiocyanate and load anti-VEGF drugs ranibizumab and conbercept,synthetic product particles respectively 5-FITC-CPP-RBZ(5-FCR)and 5-FITC-CPP-CBC(5-FCC),using noninvasive eye drops way to treat laser induced mice choroid neovascularization,through the viral drug carrier platform construction,providing a new treatment model for wet macular degeneration.Methods:The finished penetrating peptide particles loaded with ranibizumab and conbercept synthesized respectively.The hydrated particle size and surface potential of the material were measured by Nano-ZS particle size analyzer and Zeta potential analyzer.5-FITC-CPP was added into human retinal pigment epithelial cells(ARPE-19)cultured in vitro.Morphology was observed by optical microscopy and cytotoxicity of the finished granules was detected by CCK-8 kit.According to experimental groups,C57BL/6 mice were treated with 5-FCR and 5-FCC eye drops respectively,and the corneal morphology of mice was observed by fluorescein sodium staining.HE staining was used to observe the changes of tissue structure and morphology of each organ(heart,liver,spleen,lung,kidney)and eyeball(cornea,retina).CNV model of C57BL/6 mice was established by laser photocoagulation,and use PBS,5-FITC-CPP(5mg CPP per ml),5-FCR(5mg/ml CPP and 0.072mg,0.36mg and 1.8mg RBZ per ml),5-FCC(5mg CPP per ml and 0.072mg,0.36mg and 1.8mg CBC)by dropping on eyeballs surface and intravitreal injection of RBZ and CBC were respectively applied.Fluorescent fundus angiography was performed at 7d,14d and 28d after intervention to observe the changes of CNV lesions and fundus conditions.FITC-Dextran and IB4 double fluorescence staining and HE staining were used to observe the changes of lesion size on 28d after intervention.The expression and localization of CD31 and VEGF,two angiogenesis related indicators,were detected by tissue immunofluorescence method.Results:5-FCR and 5-FCC were successfully synthesized.The results of hydration particle size showed that due to the combination of RBZ and CBC,the particle size of 5-FCR and 5-FCC increased compared with that of 5-FITC-CPP without drug loading.The Zeta potential of 5-FCR and 5-FCC was lower than that of 5-FITC-CPP due to the successful binding of protein drugs.The results of light microscopy and CCK-8 showed that the morphology and survival rate of cells cultured with different concentrations of 5-FITC-CPP(lug/mL-100ug/ml)for 24h had no significant changes compared with the control group.After intervention for 28 days,the corneal fluorescence staining showed no obvious damage on the corneal surface,and HE staining showed no obvious abnormalities in the structure and morphology of each organ and eyeball.CNV model of C57BL/6 mice was successfully established by laser.After intervention for 7 days,there was no significant difference in the fluorescence leakage signals of each group.After 14d and 28d intervention,the fluorescence signal intensity of CNV foci leakage in the PBS group and 5-FITC-CPP group was high,while the fluorescence signal intensity of CNV foci leakage in the 5-FCR and 5-FCC groups was decreased.Dual fluorescence staining was performed on the choroidal retinal lamination of mice after intervention for 28 days using FITC-Dextran and IB4,and the lesion area was significantly reduced in the treatment group.HE staining showed the same results.After treatment,the retinal layers were arranged neatly compared with the control group,and the area of CD31 and VEGF positive expression was significantly reduced.Conclusions:In this paper,the finished particles 5-FITC-CPP-RBZ and 5-FITC-CPP-CPC have been successfully prepared,and the materials have good biocompatibility and low toxicity.There was no significant effect on the structure and function of the organs and eyeball in experimental animals.Cell-penetrating peptides can successfully carry drugs to the lesions and inhibit the further development of laser-induced CNV lesions,which provides a new therapeutic strategy for wet macular degeneration.Figure 26 table 4 reference 62...