Preparation Of Co-deliveried Solid Lipid Nanoparticles Modified By NGR Peptide Target And Investgation On The Therapeutic Effects For AMD

Age-related macular degeneration(Age-related macular degeneration,AMD)is a chronic eye disease and the leading cause of irreversible blindness among middle-aged and elderly people worldwide.The pathogenesis of AMD is complicated,and studies demonstrated that AMD is mainly related to choroidal neovascularization(CNV)and oxidative damage of retinal cells.Quercetin(Que),potent with anti-tumor and antiinflammatory effects,low toxicity and side effects,is considered as a novel therapeutic agent for oxidative damage related diseases.Studies showed that intravitreal injection of microRNA-150(mR150)significantly down-regulate the expression of CXCR4,DLL4,FZD4 and other angiogenesis target genes,thereby inhibiting CNV.Therefore,it is hypothesized that combined application of Que and mR150 might improve the therapeutic efficacy of AMD based on multi-target treatment strategies.CNV is one of the most obvious features in AMD.CD13 is highly expressed in abnormally growing new blood vessels,and NGR peptides can specifically target CD13 receptors.Therefore,cationic solid lipid nanoparticles modified with NGR peptides might improve the active targeting ability of nanoparticles in lesions.In this study,solid lipid nanoparticles with good biocompatibility were selected as drug carriers.The cationic solid lipid nanoparticles carried with quercetin and microRNA-150 and modified by NGR peptide was developed.Injection of Que/mR150-NSLNs will reduce the frequency of drug administration,improve the drugs targeting and enhance the efficacy of drugs,which might provide ideas for the treatment of vascular proliferationrelated diseases such as AMD.ObjectiveCo-delivery of microRNA-150 and quercetin in solid lipid nanoparticles to improve the inherent defects of microRNA-150 and quercetin.To investigate on the preparation process,and evaluate its in vitro release and cellular uptake ability,efficacy and safety of ocular administration.MethodFirst,thin-film dispersion method was used to prepare quercetin-encapsulated cationic solid lipid nanoparticles(Que-SLNs),and the preparation process was optimized based on the particle size,PDI and encapsulation rate.Electrostatic adsorption method was used to co-load mR150 in nanoparticles(Que/mR150-SLNs),and the adsorption efficiency of the mi RNA by the nanoparticles was examined by agarose gel electrophoresis experiment.The effect of Que/mR150 SLNs on the proliferation of HUVEC of human umbilical vein endothelial cells was measured by MTT method,and fluorescence labeling was used to observe their uptake in HUVEC.Screening of the optimal content of DSPE-PEG2000-NGR in the nanosystem,and further preparing nanoparticles modified with NGR peptides(Que/mR150-NSLNs),and investigating the targeting ability and time of NGR peptide-modified solid lipid nanoparticles in HUVEC cells.Laser photocoagulation was used to establish a mouse CNV model and its location in the choroid was observed.The ability for inhibition of CNV in mice and toxicity were investigated.At the same time,qPCR was used to determine the expression of HIF-1α and CXCR4 mRNA in the eyes of mice after administration.ResultsAfter process optimization,the drug-loading efficacy,particle size distribution and stability of cationic nano Que-SLNs is acceptable.The appearance of the cationic nanoQue-SLNs was spherical,and it could be kept stable for two months.The quercetin encapsulation rate was 85.25±1.29%,the drug load capacity was 1.67±0.02%,the average particle size was 110.00 ± 2.10 nm,and the Zeta potential is 53.2±5.12 m V;In vitro drug release results showed that the release of quercetin in the nanoparticles was slow,and the cumulative release amount within 48 h was about 80.69 ± 1.29%;When the mass ratio of dioctadecyl dimethyl ammonium bromide to mR150(DDAB/RNA)of different cationic materials was 6:1,the cationic solid lipid nanoparticles basically encapsulated mR150 completely with little effect on its particle size and potential.MTT experiments showed that blank nanometer mass concentration of 50-150 mg/L had no significant proliferation toxicity on HUVEC cells.Cell uptake experiments showed that Cy5 and coumarin-6 dual fluorescently labeled and co-loaded nanometers could effectively enter HUVEC cells.The material DSPE-PEG2000-NGR was synthesized,and the structure was confirmed by 1H-NMR,and it was determined that the percentage of 5% NGR has little effect on the nanosystem,and the uptake efficiency in the cell is the best.Intravitreal administration of mice after 14 days,the NGR peptide-modified co-drug-loaded nanoparticles had the strongest inhibitory effect on CNV.The rate reached 79.10%(P<0.001).Pathological sections of the retina and choroid showed that the area of new blood vessels in the Que/mR150-NSLNs group was significantly smaller,which significantly repaired the damage caused by laser photocoagulation and inhibited the growth of CNV.Fundus localization experiments showed that Que/mR150-NSLNs can increase the accumulation of Que and mR150 in fundus lesions.qPCR measurement found that after vitreous injection of nanoparticles,HIF-1α and CXCR4 in the eyeball were significantly down-regulated.pathological tissue sections obsercation for mouse eyeballs showed that Que/mR150-NSLNs had no obvious damage to the retinal tissue.ConclusionThe preparation process of Que/mR150-NSLNs solid lipid nanoparticles is stable and reliable,with good reproducibility,storage stability,good biological safety,good targeting in vivo and in vitro,and can effectively inhibit CNV in mice.This novel targeting system might provide new ideas for the treatment of AMD and other vascular proliferation-related diseases.