Preparation Of CRGD Peptide Targeting Coated Nano Drug Co- Delivery System And Their Study In Amd Therapy

Age-related macular degeneration?AMD?has become one of the main causes of blindness worldwide.Choroidal neovascularization?CNV?is the leading cause of vision impairment in AMD.Bevacizumab?Bev?,as an endogenous anti-angiogenic drug,is the most commonly used anti-VEGF drug by clinicians and is administered by the vitreous administration.However,Bev is not enough to completely block the development of CNV,and the time of vitreous injections that the human eye can bear is limited.Reasonable combination of drugs may produce significantly better therapeutic effect than single drug treatment.Dexamethasone?Dex?,as an exogenous anti-angiogenic drug,has been widely used in ophthalmology.Vitreous administration is required due to its systemic side effects and the unique barrier structure of the eyeball.However,in view of the fast metabolism of Dex,the vitreous administration is not suitable for frequent operation due to pain and side effects.Therefore,the development of suitable carriers will reduce the drug injection frequency and improve the selectivity of Dex.Therefore,it is of great clinical significance to develop a drug delivery system with longer acting time and better effect.VEGF expression increased significantly in the pathogenesis of AMD,which induced the formation of pathological new blood vessels.The secretion of VEGF mainly comes from retinal pigment epithelial cells?RPE?,so RPE are potential targets for reducing VEGF expression and preventing CNV formation in AMD.In the process of ocular neovascularization,integrin receptor?_V?₃ was over expressed on the surface of RPE,and the cyclic RGD?cRGD?peptide has a particular high affinity with?_V?₃.As a new drug delivery carrier,PLGA nanoparticles have the advantages of high biocompatibility and suitable size for cell uptake.Therefore,the cRGD peptide targeting coated nano drug co-delivery system is a promising treatment for AMD.This research mainly includes the following three parts:1.Establishment of drug in vitro analysis methods and study on Bev stabilityIn this part,the in vitro analysis method of Dex was established firstly.The linear relationship of this method was good in the range of 0²00?g/mL.The specificity,accuracy and precision of this analysis method met the requirements.Then,the in vitro analytical method of Bev was established.The concentration of Bev was measured by BCA protein concentration method.The standard curves were linear over the concentration range of 0⁴00?g/mL and 0¹000?g/mL were drawn.The linear relationship was good,and the accuracy and precision were in line with the requirements.And the isoelectric point of Bev was measured as 7.6.In this part,the stability of Bev in vitro was also investigated through size exclusion chromatography,circular dichroism and fluorescence spectroscopy.Compared with the chemical crosslinking system widely used at present,the electrostatic incubation system can better maintain the structural integrity and the secondary and tertiary structural stability of Bev.2.Preparation and evaluation of polyetherimide?PEI?cationic nano carriers co-loaded with Bev and DexIn this part,PLGA was used as the carrier material of nanoparticles,and the branched PEI was added to regulate the potential of nanoparticles for preparing the DPPNs containing Dex.The aBev/DPPNs were prepared by adsorption of negative Bev to the surface of the positive DPPNs.Meanwhile,the cBev/DPNs were prepared by chemical cross-linking method.The optimal prescription of aBev/DPPNs was determined as follows:Dex was 4 mg,PLGA was 20 mg,PVA aqueous solution concentration was 0.5%?w/v?,PEI concentration was 0.6%?w/v?,and the ultrasonic time was 3 min to obtain the DPPNs.Then,10 mg Bev and 10 mg DPPNs were mixed and dispersed in 4 ml PBS of pH 8.0.The above mixed system was incubated at room temperature for 2 h to obtain the aBev/DPPNs.The particle size,PDI and the potential of the aBev/DPPNs were 217.7±5.3 nm,0.279±0.049 and 0.85±0.37 mV,respectively.The drug loading,encapsulation efficiency and binding efficiency of the aBev/DPPNs were 9.50±0.30%,56.97±1.93%and 85.64±0.28%,respectively.The results of native PAGE showed that the adsorption method retained the structural integrity of Bev.SEM results showed that the nano carriers were well dispersed and spherical.Stability experiments showed the aBev/DPPNs were stable in PBS at 37?for 72h.Under the 37?vitreous humor conditions,the particle size of the aBev/DPPNs decreased from 207.7±3.8 nm to 194.0±12.8 nm within 72 h,several proteins in vitreous humor and a small amount of Bev shedding had a slight effect on particle size.In vitro release experiments showed that more complete release of Dex and Bev in the aBev/DPPNs could make them play a more effective role.Then we carried out a series of anti-angiogenic experiments in vitro.In Human umbilical vein endothelial cells?HUVEC?apoptosis assay,migration assay,invasion assay and tube formation assay.The aBev/DPPNs showed significant inhibition on HUVEC angiogenesis in vitro,which indicated that the aBev/DPPNs had significant inhibition on all stages of angiogenesis in vitro?p<0.001?.In chick embryo chorioallantoic membrane?CAM?assays experiments,the percentages of vessels in Dex,Bev,cBev/DPNs and aBev/DPPNs groups were 43.10±6.85%,42.98±6.57%,7.93±0.74%and 4.43±1.55%,respectively.Therefore,the aBev/DPPNs showed a strong inhibition effect on angiogenesis in vivo CAM model?p<0.001?.3.Preparation and evaluation of cRGD peptide-modified PEI cationic nanoccarrier with co-loading Bev and Dex and its application in AMDIn this part,we prepared PEI cationic nano carriers of Bev and Dex with cRGD peptide as the target.In the prescription optimization experiment,based on the preparation method of the aBev/DPPNs,we determined that the quality of PLGA and cRGD-PEG-PLGA in the optimal prescription were 18 mg and 2 mg,respectively.The particle size,PEI and potential of the aBev/cRGD-DPPNs was 213.8±1.5 nm,0.153±0.036 and 0.30±1.61 mV.The drug loading,encapsulation efficiency and binding efficiency of the aBev/cRGD-DPPNs were 9.35±0.41%,56.07±2.46%and 83.15±1.66%,respectively.SEM results showed that the nano carriers were well dispersed and spherical.Stability experiments showed the aBev/cRGD-DPPNs were stable in PBS at 37?for 72 h.Under the 37?vitreous humor conditions,a small amount of Bev was shed and released within 72 h.The cumulative release of Dex and Bev within 168 h of the aBev/cRGD-DPPNs was 66.5±4.2%and 56.3±4.8%,respectively.CLSM assay and flow cytometry were used to observe the uptake of nanoparticles in Adult retinal pigment epithelial cells?ARPE-19?and Human embryonic kidney cells?293T?.Rhod B and FITC were used to mark the nanoparticles.The results showed that the aBev/cRGD-DPPNs had the highest fluorescence intensity in ARPE-19,indicating that aBev/cRGD-DPPN had the highest uptake efficiency.However,it was rarely taken in 293T,further demonstrated the selectivity of the aBev/cRGD-DPPN.In the experiments of HUVEC cell apoptosis,cell migration,cell invasion and cell tubule formation,the aBev/cRGD-DPPNs showed significant inhibition of angiogenesis in vitro of HUVEC,indicating that the aBev/cRGD-DPPNs had significant inhibition of angiogenesis in vitro at all stages?p<0.001?.In the in vivo anti-angiogenesis experiment,this study firstly established the CNV model of chinchillaf rabbit through laser-induced.It was proved that the aBev/cRGD-DPPNs most significantly inhibited the fluorescence leakage of CNV in the fundus through fluorescein fundus angiography?FFA?experiment.In histopathological experiments,the aBev/cRGD-DPPNs group showed the most significant reduction in CNV structure.In the immunohistochemical experiment,the positive integrated optical density?IOD?of VEGF expression in the aBev/cRGD-DPPNs group was the lowest.In the Enzyme-linked immunosorbent assay?ELISA?experiment,the RPE-choroid tissues in the aBev/cRGD-DPPNs group contained the least VEGF amount.Therefore,the aBev/cRGD-DPPNs showed stronger ability to reduce VEGF and inhibit CNV in rabbit model.In addition,intraocular pressure tests demonstrated the biosafety of nanoparticles.In conclusion,the drug co-delivery nano carrier modified with cRGD peptide in this study can effectively deliver drugs to the site of AMD and exert effects,and the combination of Dex and Bev can better inhibit CNV.This study presents an ideal carrier for the treatment of AMD.