Cell Membrane Biomimetic Liposomes Based On The Combined Therapy Of Photodynamic Therapy And Hypoxia-Activated Prodrugs In The Preliminary Treatment Of Oral Squamous Cell Carcinoma

Background and Objective:Oral squamous cell carcinoma(OSCC),about ninety percent of malignant tumors occurring in the oral cavity,are currently treated by surgery,chemotherapy,and radiotherapy.It is not improved in the last 20 years about the five-year survival rate of patients with OSCC and conventional treatment has highly toxic side effects.Photodynamic therapy(PDT)uses photosensitizers to convert intracellular oxygen molecules into cytotoxic reactive oxygen species to cause tumor cell death with a minimally invasive,less toxic,highly efficient,and selective emerging treatment strategy.However,the hypoxia bottleneck of PDT limits the therapeutic effect.As one of the hypoxia-targeted chemical agents,hypoxia-activated prodrugs(HAP)are non-toxic under normal conditions but revert to cytotoxic substances under hypoxic conditions,while HAP alone often fails to exert the desired therapeutic effect due to insufficient hypoxia.Photosensitizers represented by dihydro porphyrin e6(Ce6)and hypoxia-activated prodrugs represented by tirapazamine(TPZ)are difficult to effectively act on tumor sites by conventional delivery,which severely limits their use.Nanoliposomes could improve the drug properties,prolong vivo circulation time,and confer its targeting ability by encapsulating the drug.Membrane bionanotechnology is a technique to encapsulate nanoparticles by extracting cell membranes to achieve disguise as autologous components and escape recognition and clearance by the immune system with lower immunogenicity,higher targeting,and better biocompatibility.In the meantime,M1-type macrophage membrane-encapsulated nanoparticles are utilized to enhance drug penetration and accumulation in tumors by homing ability.The purpose of this study is to use liposomes to co-load Ce6 and TPZ efficiently,and then construct a membrane-mimetic liposomal drug delivery system(TPZ/Ce6-Lip@M)to investigate the latent mechanism of action of delivery systematized PDT and HAP for the combined treatment of OSCC by using M1-type macrophage membranes.Methods:1.Firstly,DSPE-PEG-Ce6 was obtained by chemical grafting.Secondly,prepared and screened the inner core liposome TPZ/Ce6-Lip with co-encapsulated TPZ and DSPE-PEG-Ce6 by thin film hydration method,and finally synthesized the prepared inner core liposome TPZ/Ce6-Lip with the extracted macrophage membrane RAW264.7M by co-extrusion method to synthesize the membrane mimetic liposome drug TPZ/Ce6-Lip@M.2.DSPE-PEG-Ce6 was examined the composition through NMR hydrogen spectroscopy and screened the optimal liposome synthesis formulation using particle size and encapsulation rate.Characterized particle size,PDI,zeta potential,and morphology were detected by using a dynamic light scattering method,electrophoretic light scattering method,and transmission electron microscopy for TPZ/Ce6-Lip and membrane mimetic liposome TPZ/Ce6-Lip@M.The fractions were analyzed and the encapsulation rate was measured by UV spectrophotometry.In addition,the physicochemical properties were evaluated by in vitro release assay and placement stability assay,and the expression of membrane-associated proteins was evaluated by western blotting.3.SCC7 cells was as the study subjects and evaluated drug uptake and endosome escape using a lysosomal co-localization assay.The intracellular reactive oxygen species generation after drug administration was detected by using H2DCFDA fluorescent probe.The relationships between intracellular ROS generation and hypoxia after drug administration were evaluated by using reactive oxygen species and hypoxia probe double staining assay.In addition,our group evaluated the relationships between intracellular ROS generation and hypoxia after drug administration using protein blotting.The expression of hypoxia-inducible factor(HIF-1α)was evaluated by western blotting and the abilities of membrane mimetic liposomes to inhibit proliferation and promote apoptosis in OSCC cells were evaluated by CCK8 method and Calcein-AM/PI double-staining assay.Results:1.NMR hydrogen spectroscopy verified the structure of DSPE-PEG-Ce6.The optimal liposome synthesis formulation was successfully screened using particle size and encapsulation rate and used for subsequent experiments;The results of TEM,DLS,PDI,and zeta potential demonstrated that our prepared TPZ/Ce6-Lip@M was suitable in size,homogeneous,and stable.TPZ/Ce6-Lip@M was demonstrated that it has a better encapsulation rate of TPZ and Ce6 by using UV spectrophotometry.In addition,it was demonstrated that the membranemimetic liposome TPZ/Ce6-Lip@M has a better retardation effect in the vitro release experiment.Western blotting demonstrated that TPZ/Ce6-Lip@M camouflage membraneassociated function is well retained.2.Lysosomal co-localization results suggested that TPZ/Ce6-Lip@M was readily and successfully taken up by the OSCC cell’s endosome escape.The ROS fluorescence staining indicated that TPZ/Ce6-Lip@M produced a large amount of toxic ROS after laser irradiation but the cells are hypoxic intensified.CCK8 and Calcein-AM/PI double staining results showed that TPZ/Ce6-Lip@M showed a dose-dependent significant inhibition of OSCC cell proliferation and promotion of OSCC cell apoptosis compared with TPZ and Ce6 alone.TPZ/Ce6-Lip@M showed a dose-dependent significant inhibition of OSCC cell proliferation and promotion of OSCC cell apoptosis.Conclusion:1.TPZ/Ce6-Lip@M,the macrophage membrane mimetic nano liposome,was successfully prepared,owned appropriate size,uniform size,concentrated normal distribution,high encapsulation rate,good stability,and slowly release effect.2.Membrane mimetic liposomes TPZ/Ce6-Lip@M could actively target tumor cells for easy uptake,successful endosome escape,production of large amounts of toxic ROS,and exacerbate hypoxia conditions,creating favorable conditions for TPZ treatment,using hypoxiatargeted chemotherapy and photodynamic therapy combined significantly inhibits OSCC proliferation and promote apoptosis.In conclusion,the macrophage membrane-mimetic nano liposomal drug TPZ/Ce6-Lip@M constructed in this study has excellent physicochemical properties,better drug encapsulation ability,less toxic side effects,good therapeutic selectivity,and significant tumor targeting ability.In addition,cascade incremental the therapeutic effect of OSCC was increasing based on the combination of PDP and HAP.Therefore,this novel membrane-mimetic nano liposomal drug offered a new reliable therapeutic approach for OSCC.