Construction And Mechanism Of Biomimetic Liposomes Camouflaged By Colorectal Cancer Cell Membranes

Colorectal cancer（CRC）is one of the most common cancers at home and abroad,and 5-fluorouracil（5-Fu）,as one of the main chemotherapeutic agents in the first-line treatment of CRC,has the disadvantages of lack of specificity in in vivo distribution and induction of drug resistance,which makes its efficacy and safety unsatisfactory.5-Fu induces drug resistance by inducing autophagy in tumor cells,therefore,the use of autophagy inhibitors can theoretically overcome 5-Fu resistance,thus improving the efficacy.Our group synthesized acid ester of hydroxychloroquine and linolenic acid（AHQ）,a precursor drug of autophagy inhibitor hydroxychloroquine（HCQ）,independently in the early stage,which improved the uptake of the drug by tumor cells.AHQ showed the best natural targeting to CRC cells（HT-29）,while the results of in vitro and in vivo studies of AHQ in combination with 5-Fu showed synergistic anti-CRC effects.However,the poor water solubility and short half-life(t_1/2)of AHQ prevented long-lasting anti-tumor therapy.Cancer cell membrane（CCM）-based bionanosystems show strong tumor homing targeting ability and good biocompatibility,and liposomes（LPs）can improve the water solubility of hydrophobic drugs,thus enhancing antitumor efficacy and safety.In this study,AHQ was loaded into a bionic nano-drug delivery system system（CCM-AHQ-LPs）formed by the fusion of CCM and LPs to improve the solubility of AHQ,prolong its circulation time,and achieve targeted drug delivery and optimal anti-tumor activity in tumor cells.Methods:1.A method for the determination of AHQ was established based on high performance liquid chromatography technique.AHQ-LPs were prepared by thin film hydration method,and the optimal preparation process was screened by single-factor examination,and the formulation of AHQ-LPs was systematically evaluated.2.HT-29 cell membrane was extracted by the kit,and the CCM protein concentration and integrity were quantified by BCA and SDS-PAGE.3.CCM was coated on AHQ-LPs by ultrasonication to prepare CCM-AHQ-LPs,and they were evaluated systematically.4.Coumarin-6 was used to label LPs with fluorescence,and confocal laser scanning microscopy（CLSM）was used to investigate the colocalization of CCM and LPs,the uptake of CCM-LPs by HT-29 cells,the mechanism of endocytosis and the lysosome of CCM-LPs escape ability.5.The safety of CCM-LPs and the effect of 5-Fu combined with AHQ,AHQ-LPs and CCM-AHQ-LPs on the viability of HT-29 cells were investigated by CCK-8 method.6.To examine the effect of 5-Fu combined with AHQ,AHQ-LPs,and CCM-AHQ-LPs respectively on the proliferation and migration of HT-29 cells by plate cloning and wound healing assay.7.To investigate the effect of 5-Fu combined with AHQ,AHQ-LPs,CCM-AHQ-LPs on HT-29 cell cycle and apoptosis by flow cytometry,respectively.8.The fluorescent probe DCFH-DA was used to detect the effect of 5-Fu combined with AHQ,AHQ-LPs and CCM-AHQ-LPs on the production of reactive oxygen species in HT-29 cells,respectively.Results:1.AHQ-LPs（size:108.47±0.42nm,Zeta potential:21.2±2.04m V,AHQ encapsulation efficiency:80.42±1.01%）and CCM-AHQ-LPs（size:124.97±7.88nm,Zeta potential:-21.97±0.21m V,AHQ encapsulation efficiency:87.55±1.58%）were successfully prepared.AHQ-LPs and CCM-AHQ-LPs showed good in vitro release properties.The storage stability of AHQ-LPs and CCM-AHQ-LPs within 5 days was good.In vitro hemolysis assay showed that neither AHQ-LPs nor CCM-AHQ-LPs could induce hemolysis of erythrocytes.2.The results of CCM integrity assay showed that CCM and CCM-LPs could retain most of the proteins on HT-29 cells,and the results of CCM co-localization assay with LPs showed the successful encapsulation of CCM on LPs.3.The results of CCK-8 assay verified the biosafety of CCM-LPs.When combined with 5-Fu,AHQ,AHQ-LPs and CCM-AHQ-LPs showed dose-and time-dependent inhibitory effect on HT-29 cells,and CCM-AHQ-LPs showed the strongest inhibitory effect on HT-29 cells.4.Cellular uptake results showed that HT-29 cells exhibited the highest level of uptake of HT-29-derived CCM-LPs compared to HCT116 and MCF-7 cells.both LPs and CCM-LPs entered the cells through caveolae-mediated endocytosis.CCM-LPs have lysosomal escape ability.5.The results of plate cloning and wound healing assay showed that the group of 5-Fu in combination with CCM-AHQ-LPs showed the best inhibitory effect on the proliferation and migration ability of HT-29 cells.6.When 5-Fu was combined with AHQ-LPs and CCM-AHQ-LPs,it could lead G0/G1 phase and S phase arrest in HT-29 cells,while the free drug group（5-Fu+AHQ）could lead G0/G1 phase arrest in HT-29 cells.5-Fu combined with AHQ induced an increased rate of late apoptosis in HT-29 cells.7.The results of oxidative stress showed that,compared with the free drug group（5-Fu+AHQ）,the 5-Fu combined with CCM-AHQ-LPs group had the strongest ability to promote the production of reactive oxygen species in HT-29 cells.Conclusions:In this experiment,CCM-AHQ-LPs with good pharmacological properties and tumor homing targeting were successfully constructed,and showed the best anti-CRC activity in combination with 5-Fu.