Establishment Of HCTL Membrane-derived Nanoparticles And Evaluation Of Nanoparticles With Low-dose Irradiation In The Treatment Of Gastric Cancer

Purpose:Recently,cell membrane-derived nanoparticles are becoming more attractive because of their ability to mimic many features of their source cells.The current study reports on a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes.And this dissertation focused on:1.developing human cytotoxic T-lymphocyte membrane-based nanoparticles,evaluating their synergistic antitumor effect when combined with low-dose irradiation;2.developing recombinant protein anti-EGFR-iRGD inserted human cytotoxic T-lymphocyte membrane-based nanoparticles,evaluating their synergistic targeting skills when combined with low-dose irradiation.Methods:PLGA nanoparticles were prepared using a modified nanoprecipitation method.The membrane of human cytotoxic T-lymphocytes were collected using a combination of hypotonic lysing and mechanical membrane disruption,and were further purified through a discontinuous density sucrose gradient.The isolated membranes were washed and extruded through a 200-nm porous polycarbonate membrane to form hCTL membrane-derived vesicles.Then,hCTL membrane-derived vesicles and PLGA nanoparticles were coextruded through an apparatus with 200-nm pores to form Ptx-TPNPs.The conformation studies were carried out using dynamic light scattering,transmission electron microscopy and confocal laser scanning microscopy.To determine the encapsulation efficiency,Ptx encapsulated in the PLGA was measured using HPLC.The in vitro release profile of Ptx from the TPNPs was assessed using HPLC as well.The cellular uptake studies were investigated on human gastric cell line MKN45 and the human THP-1 cell line.In vitro cytotoxic studies were also carried on MKN45 cell line,with low-lose irradiation.In vivo targeting ability,in antitumor ability and in vivo toxicity on a murine subcutaneous tumor model were investigated.Recombinant protein anti-EGFR-iRGD was inserted onto human cytotoxic T-lymphocyte membrane-based nanoparticles.The conformation studies were carried out using dynamic light scattering.The cellular uptake studies were investigated on human gastric cell line MKN45.Western blotting and immunohistochemical analysis were used for quantitative determination of EGFR expression.In vivo targeting ability on a murine subcutaneous tumor model was investigated.Results:The T-lymphocyte membrane coating was verified using dynamic light scattering,transmission electron microscopy and confocal laser scanning microscopy.The encapsulation efficiency and the drug-loading efficiency of Ptx-loaded PLGA were 51.20%and 5.07%,respectively.The average diameter of the final Ptx-loaded TPNPs was 165.9±1.0 nm.TEM imaging revealed a membrane coating around the polymeric core.The release exhibited a biphasic pattern characterized by a fast-initial release during the first 48 h,and then a slow and continuous release subsequently.This new platform reduced nanoparticle phagocytosis by macrophages by 23.99%(p=0.002).There was no significant difference in cytotoxicity between Ptx,Ptx-encapsulated PLGA nanoparticles,and Ptx-loaded TPNPs,whethter they are combined with low-dose irradiation or not.Empty TPNPs displayed no cytotoxicity,even at a high concentration that could encapsulate 100 ng/mL Ptx,indicating their good biocompatibility.In in vivo targeting study,after the induction of low-dose irradiation,the fluorescence signal was clearly visualized and gradually increased at the tumor sites.Systemic administration of Ptx-loaded T-lymphocyte membrane-coated nanoparticles inhibited the growth of human gastric cancer by 56.68%in Balb/c nude mice.Application of low-dose irradiation at the tumor site significantly increased the tumor growth inhibition rate to 88.50%and two mice achieved complete remission.Furthermore,low-dose irradiation could up-regulate the expression of adhesion molecules in tumor vessels,which is important in the process of leukocyte adhesion and might contribute to the localization of T-lymphocyte membrane-encapsulated nanoparticles in tumors.Recombinant protein anti-EGFR-iRGD was inserted onto human cytotoxic T-lymphocyte membrane-based nanoparticles.In the cellular uptake studies,anti-EGFR-iRGD-Ptx-TPNPs were uptaken more than other groups by human gastric cell line MKN45.Western blotting and immunohistochemical analysis showed low-lose irradiation increased the expression of EGFR of MNK45.In vivo targeting study showed good targeting ability of anti-EGFR-iRGD-Ptx-TPNPs.When combined with low-dose irradiation,the targeting ability was enhanced.Conclusions:This new drug-delivery platform retained both the long circulation time and tumor site accumulation ability of human cytotoxic T-lymphocytes while local low-dose irradiation could significantly enhance tumor localization.When recombinant protein anti-EGFR-iRGD was inserted onto human cytotoxic T-lymphocyte membrane-based nanoparticles,the targeting ability was improved significantly.Therefore,anti-EGFR-iRGD-Ptx-TPNPs are promising drug carriers for anti-cancer agents,with low-dose irradiation as a chemoattractant for nanoparticle targeting.They hold potential for future clinical cancer treatment.