A Study On The Preparation And Pharmocodynamics Of Nanoparticles Targeting For Diabetic Retinopathy

Diabetic retinopathy is the most common complication of diabetic microangiopathy and the major cause of blindness worldwide.The main pathogenesis of diabetic retinopathy may be the activation of multiple molecular pathways mediated by hyperglycemia,which leads to neurodegenerative degeneration and retinal neovascularization in turn.At present,most therapeutic approaches start with the suppression of neovascularization by surgery or drug therapy,but the treatment is still not too perfect to recover the retinal damage that has already occurred.Directly controlling the blood glucose level in the retina may be an effective strategy for preventing the progression of diabetic retinopathy.However,due to the particular structure of the eye,the delivery methods and drug delivery systems of retinal diseases are limited in many aspects such as treatment effects,patient compliance and secondary infections.Purpose:According to the supply mechanism of intraocular glucose transported by the glucose transporter of the blood-retinal barrier,a bio-macromolecules drug targeting for glucose transporter 1 was designed and established.The kind of bio-macromolecules drug-loaded nanoparticles enriched in hyaluronic acid on the surface to target retinal pigment epithelial cells was prepared.Then,the characterization,determination and cell viability evaluation of bio-macromolecules drug-loaded nanoparticles were carried out.After intraocular administration of nanoparticles,the pharmacodynamic effects of nanoparticles on diabetic retinopathy should be investigated.Methods:(1)In order to knock out the glucose transporter 1 gene,combined with the results of screening in vitro and T7 endonuclease 1 digestion,a stable single guide RNA(sgRNA)could be selected to assemble into ribonucleoprotein(RNP)with the clustered regularly interspaced short palindromic repeats-associated protein 9(Cas9)to serve as a biological macromolecular drug.(2)synthesizing polyethylene glycol-polylactic acid-polyglutamic acid and using it combined with polylactic acid-hyaluronic acid as the complex carrier material,using double emulsification/solvent volatilization method to prepare nanoparticles containing bio-macromolecules drug,determining their particle size and Zeta potential.Nanodrop-2000 was used to determine the content of bio-macromolecules drugs.(3)For animal study,eight-week-old C57BL/6 mice were divided into three groups randomly,named normal control group,diabetes model group and the nanoparticle treatment group respectively.The mice were intraperitoneally injected with streptozotocin(STZ)to establish a model of diabetic retinopathy.The mice of nanoparticle treatment group were administrated with nanoparticles carrying the glucose transporter 1 gene drug into the subretinal place,while normal control group and the diabetes model group were injected with the equal volume of PBS respectively.The glucose level of the three groups of retinas was measured,the changes of glucose transporter 1 expressions were measured by Enzyme Linked Immunosorbent Assay(ELISA),the mRNA level of glucose transporter 1 was quantitated by qRT-PCR,and the apoptosis of retinal endothelial cells and ganglion cells were evaluated.The effect on cell viability of nanoparticles was also verified by H&E histopathology in this study.Results:(1)Five sgRNAs with optimal gene editing function were designed and finally selected to be used as gene therapy drugs for diabetic retinopathy.(2)~1H-NMR results showed that triblock polymer carrier materials were successfully synthesized.The nanoparticles carrying gene drugs with the proper properties were prepared,whose particle size is about 106 nm,PDI is 0.2~0.3,the surface charge is negatively charged,the potential value is about-17 mV,and the encapsulation rate can reach 83%.(3)Compared with the normal control group,both the glucose level in the retina and the glucose transporter 1 mRNA expression as well as its protein expression in retinal pigment epithelial cells(RPE)of the diabetes model group were significantly increased(P<0.01);compared with the diabetes model group,the glucose level in the retina of the nanoparticle treatment group,the mRNA and protein expression levels of glucose transporter 1 in RPE decreased significantly(P<0.01).Besides,the number of retinal non-nucleated capillaries was reduced compared with the diabetes model group(P<0.01)while the number of nerves increased(P<0.05).It is showed that the nanoparticles had no toxicity towards retina by H&E histopathology.Conclusion:Based on targeted functionalized nanoparticles combined with sub-retinal drug delivery,targeting the glucose transporter 1 gene of retinal pigment epithelial cells can achieve the possibility of precise control of glucose level in the retina.The mice diabetic retinopathy model studies showed that the bio-macromolecules drug-loaded nanoparticles prepared by us had significant effects on neuroprotection and the suppression of neovascularization.This strategy provides an example of comprehensive treatment of diabetic retinopathy with nanoparticles carrying genome editing tools.