Screening Of Peptide Drug And The Construction And Application Of Peptide Delivery System Based On Tumor Cell Membrane

Peptides are composed of no more than 50 amino acids,with a molecular weight between small molecules and proteins.Peptides have analogous characters to proteins in structures and functions.For example,many hormones,growth factors,and neurotransmitters are peptide substances.As functional molecules,peptides with the advantages of simple synthesis process,low cost,and high safety have attracted the attention to research and development of peptide drugs.In this thesis,peptide drugs were screened and developed for popular target proteins of tumors and infectious diseases,in order to obtain peptide drugs with clinical potential application.Tumor immunotherapy has become one of the important methods of tumor treatment due to its excellent clinical treatment effect.Tumor immunotherapy includes adoptive cell therapy,tumor vaccines,and immune checkpoints.One of the immune checkpoints,programmed death receptor-1/programmed death molecular ligand-1(PD-1/PD-L1),has achieved great clinical success.Nevertheless,due to the high cost and high immunogenicity of antibodies,we need to find more biomolecules that can replace antibodies.We screened and obtained PD-1 specific binding peptides through bacterial surface display technology,and tested the possibility of blocking the PD-1/PD-L1 pathway.Specifically,we used bacterial surface display technology to obtain 6 peptides that could bind to PD-1 from a random peptide library.Sequence alignments revealed two types of conservative sequences DDCWXD(X stands for:D/E/H)and DGW.By ELISA and flow cytometry tests,the peptide with the best binding effect to PD-1 was named PBP-1.Experiments proved that the binding of the peptide to PD-1 had specificity and the function of blocking the interaction of PD-1/PD-L1.The above results preliminarily showed that PBP-1 peptide could be used as a candidate molecule to antagonize PD-1 and participated in tumor immunotherapy.Since the end of 2019,COVID-19(Corona Virus Disease 2019)has spread rapidly around the world,resulting in an increase in global morbidity and mortality and bringing huge social turmoil and economic losses.As reported,the entry of COVID-19 virus into human cells was mediated by the S(Spike)protein on the surface of the viral cell membrane and the ACE-2(Angiotensin converting enzyme 2)receptor in human cells.Therefore,drugs that block the binding of S protein to the ACE-2 receptor may curb the spread of COVID-19.Based on this mechanism,we used bacterial surface display technology to screen ACE-2 molecules for specific binding peptides.After multiple rounds of screening,12 peptides of specifically binding to ACE-2 were finally obtained,and no conservative sequences of these peptides were found by sequence comparison.Through flow cytometry analysis,the peptide with the best binding effect to ACE-2 was selected and named TAP-1.The fluorescence ELISA method was used to further verify the specificity of TAP-1 binding to ACE-2.Through competition experiments and pseudovirus experiments,it was confirmed that TAP-1 peptide could compete with S protein to bind to ACE-2 in concentration-dependent manner.The above results indicated that TAP-1 peptide had the potential to block COVID-19 from entering cells and provided a new treatment plan for the treatment of COVID-19.Because the peptides have the characteristics of short half-life and easy degradation,which limits its wide clinical application.In order to solve this problem,this research combines nano-delivery technology to design and develop the use of tumor cell membrane nano-drug carrier system to transport peptide drugs.We constructed a peptide delivery system based on tumor cell membranes(SPIO NP@M-P),which was used to deliver the PD-L1 antagonistic peptide TPP1 in the early stage of our research.Experiments proved that H460 cell membrane vesicles had superior homologous targeting ability and mediated the enrichment of nanovesicles at tumor sites.The SPIO NPs(Superparamagnetic Iron Oxide)in the system could be used for Magnetic Resonance Imaging to facilitate the monitoring of the therapeutic effects of peptide drugs on tumors.SPIO NP@M-P with surface modified TPP1 peptide not only still could block the interaction between PD-L1 and PD-1,but also has a longer half-life(60 times that of free TPP1 peptide).The substrate peptide of MMP2 enzyme(Metallomatrix protease 2)in SPIO NP@M-P contributed to the release of TPP1 peptide in the tumor microenvironment.The successful construction of the SPIO NP@M-P peptide delivery system for tumor cell membranes provided a new way for the clinical transformation of peptide drugs.In summary,this study used bacterial surface display technology to screen the binding peptide for two targets of PD-1 and ACE-2.The obtained peptides showed targeting ability and blocking function.In terms of peptide applications,we have successfully constructed a peptide delivery system based on tumor cell membranes(SPIO NP@M-P),which extended the half-life of peptides and endowed peptides with theranostic functions in tumors,making the peptides with the possibility of clinical transformation.