Study Of Cell Penetrating Peptides-mediated Intraretinal Nucleic Acid Delivery System

Background: Inherited Retinal Degenerations(IRDs)are a group of ocular diseases caused by inherited genetic mutations that cause vision loss and even blindness worldwide.With the intensive research on nucleic acids and gene drugs in recent years,it has been found that the modification of traditional non-viral vectors(physicochemical properties such as surface charge,particle size and amphipathicity)with CCPs(Cell-penetrating peptides)can not only improve the efficiency of gene vector penetration,but also reduce the cytotoxicity problems caused by high doses of drugs.A number of different strategies have been developed for the modification of membrane penetrating peptides CPPs,including the development of nanocomplexes linked to PEI of different molecular weights or degrees of branchedness,which are thought to have applications in the gene therapy of various genetic diseases,but in fact the therapeutic efficacy of these nanocomplexes in retinal diseases has yet to be improved.Aims: We attempted to construct a membrane penetrating peptide(89W)-mediated non-viral ocular nucleic acid delivery system with the aim of achieving safe and efficient intra-retinal delivery of nucleic acid drugs.This study extends the application of non-viral vectors in ocular gene therapy and lays the foundation for the development of effective targeted therapeutic approaches in the future.Methods: The first part of the article evaluated the membrane penetration effect of the synthesized membrane penetrating peptide 89 W in vivo and in vitro,firstly,the non-toxic and efficient 89 W action concentration and time were initially screened in the cellular internalization assay and cytotoxicity assay of the membrane penetrating peptide,then the internalization effect of 89 W under this condition was verified in other three cell lines,and finally whether 89 W could penetrate the cellular membrane into the retinal layers was demonstrated in vivo in mice The second part of the experiment evaluated the intracellular nucleic acid delivery ability of 89 W and BPEI,respectively,and examined the cytotoxicity of BPEI at different concentrations;the third part designed and prepared89W@BPEI/pGFP complexes with different ratios using green fluorescent protein particle(pGFP),the membrane-penetrating peptide 89 W,and 25 KD of branched polyethyleneimine(BPEI)The nitrogen-to-phosphorus ratio screening and physical property characterization were also performed.In the fourth part,the effects of different ratios of 89 W modification on BPEI plasmid transfection and gene knockdown effects were evaluated in vitro in combination with the experimental conditions and complex properties of the previous screening,and the nucleic acid delivery effects of 89W@BPEI complexes in mouse retina were verified.Results: Firstly,we obtained the non-toxic and efficient membrane penetration conditions of 89 W in vitro,i.e.,89 W did not show cytotoxicity at 89 W concentration of 3M and treatment time of 10 h.We also further demonstrated that 89 W could penetrate the cell membrane into the retina in vivo.We then examined the intracellular nucleic acid delivery ability of 89 W and found that its plasmid transfection ability was poor under non-cytotoxic conditions,while increasing the concentration of BPEI,which has a relatively high transfection efficiency,exhibited significant cytotoxicity.the complementary characteristics of BPEI and 89 W in terms of cytotoxicity and transfection efficiency led us to consider using 89 W modified BPEI(89W@BPEI)to deliver nucleic acid drugs to the retina.Then,by designing the ratio of 89 W to BPEI during plasmid delivery and screening by agarose gel electrophoresis experiments,we obtained the nitrogen-phosphorus ratio at which the charge properties of each level of complexes were critically altered,and the nitrogen-phosphorus ratio at which the charge properties were critically altered in the primary complex BPEI/pGFP was BPEI:pGFP = 1:1,and the secondary complex 89W@BPEI/ pGFP is 89W:BPEI:pGFP=2:1:1,and further physical characterization shows that 89 W increases the particle size of the complex,but BPEI can play a role in reducing the particle size.Finally,combining the membrane-penetrating properties of 89 W and the physical properties of 89W@BPEI,we found that 89 W could effectively improve the plasmid transfection and gene knockdown effect of BPEI in different cell lines,and in vivo experiments also demonstrated that 89W@BPEI could effectively deliver siRNA into the mouse retina.Conclusion: In this paper,a non-toxic and efficient intra-retinal nucleic acid delivery system(89W@BPEI)has been screened in vitro and in vivo,and preliminary studies have shown its potential to deliver nucleic acid drugs for hereditary retinal diseases.