Neutrophil Membrane-Mimic Nano-Vehicle For The Treatment Of Non-Small Cell Lung Cancer

Non-small cell lung cancer（NSCLC）is one of the cancers that pose the greatest threat to human health.However,Cisplatin,a first-line drug,has serious systemic toxicity due to its non-specific tissue distribution,which limits its application in clinic.In the meantime,the tumor adaptive immune microenvironment still poses a great threat that compromises the therapeutic efficacy of chemotherapy by promoting the tolerance of tumor cells.Therefore,it is urgent to develop safe and effective tumor targeted drug delivery systems.In recent years,membrane camouflage nanotechnology has been widely recognized.Neutrophils are deeply involved in the occurrence and development of tumors,and have natural tumor targeting and immunomodulatory effects,so they are promising cell membrane sources for the development of cell membrane biomimetic nanosystems.In this study,we constructed a neutrophil membrane-mimic nano-vehicle,Neutrosome（L）,which could achieve tumor targeting while could neutralize inflammatory cytokines,inhibit the infiltration of tumor-associated neutrophils（TANs）,thus regulating tumor immune microenvironment and inhibiting tumor growth.Neutrosome（L）also had satisfactory drug-loading capacity.After carrying cisplatin,Neutrosome（L）could achieve tumor-targeting therapy,which was synergist with tumor immune microenvironment modulation for augmented chemotherapeutic effects and reduced systemetic toxicity.In this study,neutrophils from mouse bone marrow were extracted and purified by Percoll discontinuous density gradient centrifugation.And activated neutrophils were obtained by LPS stimulation in vitro.Neutrophil membrane chimeric liposomes with different weight ratios of phospholipid to membrane protein were constructed by membrane dispersion and ultrasonic fusion after separation of cell membrane by repeated freeze-thaw method.According to the particle size,zeta potential,membrane fusion,etc.,the weight ratio of phospholipid to membrane protein was selected as 1:0.5 for subsequent research,and the non-activated and activated neutrophil membrane biomimetic nano-vehicles were named Neutrosome and Neutrosome（L）respectively.The hydrodynamic size and zeta potential of Neutrosome was 203.6±2.1 nm and-8.2±0.4 m V.The whole protein profile of neutrophil membrane was inherited in the meantime.The results of western-blot showed that the expression level of lymphocyte function-associated molecule-1（LFA-1）,L-selectin,β1 integrin,and other target-related characteristic proteins on the surface of Neutrosome（L）was higher than Neutrosome.After carrying cisplatin,the encapsulation efficiencies of the two biomimetic vehicles were 41.7±1.2%and 41.9±0.2%,respectively,which were similar to those of traditional liposomes.The in vitro release results showed that the neutrophil membrane-mimic nano-vehicles could achieve rapid drug release in response to the acidic p H in the tumor microenvironment（TME）.The affinity and tumor targeting ability of the neutrophil membrane-mimic nano-vehicles were investigated through the cellular and animal experiments.Neutrosome/Neutrosome（L）had a good affinity with A549 cells and could be effectively ingested by A549.The cell uptake was significantly higher than that of the liposome group,thus reached the deep part of tumor sphere.On A549 subcutaneously transplanted tumor nude mice,Neutrosome/Neutrosome（L）could target the tumor site.After delivering cisplatin to the tumor tissue,the drug accumulation in the tumor site increased by 4.8 times and 5.8 times,respectively,compared with free cisplatin.The investigation of the tumor targeting mechanism of the neutrophil membrane-mimic nano-vehicles found that Neutrosome（L）had higher affinity with inflammatory endothelial cells and could effectively respond to the induction of inflammatory factors to pass through the endothelial cell layer in a complete form,thereby tranversing tumor blood vessel and targeting tumor tissue.This may be related to the higher level of targeting-associated characteristic proteins on the surface of Neutrosome（L）.When the antitumor effect was investigated in vitro,the vehicles themselves did not show inhibitory effect on tumor growth.But the affinity between the nano-vehicles and A549 cells enabled cisplatin-loaded Neutrosome（L）（N（L）/C）to significantly reduce the IC₅₀ on A549 cells（9.7μg/m L）.Neutrosome（L）showed a significant tumor inhibited effect on A549 subcutaneous transplanted tumor nude mice,which may be related to Neutrosome（L）being able to neutralize the inflammatory factor TNFαand inhibit the infiltration of neutrophils in tumor tissues.After loading cisplatin,the tumor-inhibited effect of Neutrosome（L）was further enhanced,and the renal toxicity of cisplatin was significantly reduced.The median survival time of tumor-bearing nude mice was extended to 64 days,while that of tumor-bearing nude mice in cisplatin and blank treatment groups was only 9 days and 33 days,respectively.In vitro safety evaluation showed that the hemolysis rate of Neutrosome（L）was less than 5%,and it had no significant cytotoxicity on three normal cell lines of human umbilical vein endothelial cells（HUVEC）,human embryonic kidney cells（HEK293）and normal human liver cells（L02）.After systemic administration,Hematoxylin-eosin staining results showed that the main organs of the mice had no obvious lesions,and all blood indices were normal,indicating that Neutrosome（L）had good biological safety.In conclusion,this study constructed a neutrophil membrane-mimic nano-vehicle,Neutrosome（L）,and carried cisplatin for the treatment of NSCLC.Through tumor targeted drug delivery combined with tumor immune microenvironment regulation,synergistic anti-tumor effects and attenuated toxicity could be achieved,providing a facile strategy to augment chemotherapeutic effects.