Construction Of Biomimetic Nano Drug Delivery System Based On Composite Membrane And Its Antitumor Therapy

Malignant tumors are diseases that seriously threaten human health,and their expensive treatment costs impose a huge economic burden on the patients' families.Researchers have been working on the development of various cancer treatment methods.Among them,the design of cell membrane-based biomimetic nano drug delivery system has attracted special attention.It has become a hotspot of nano drug research because of its biocompatibility,non-toxicity and low immunogenicity.This topic uses a"take from the body,use into the body" and"immuno deception" ideas to construct a biomimetic nanoreactor with tumor targeting,through the combination of Sonodynamic therapy(SDT),chemotherapy immunotherapy exerting an anti-tumor effect.The nanoreactor has the following advantages:(1)pH sensitivity and ultrasonic response characteristics enable the system to remotely control drug release;(2)Cascade-type acoustic-chemotherapy coordinated immunotherapy can effectively inhibit tumor growth and there are no obvious toxic side effects on normal tissue;(3)The biomimetic properties of the cell membrane can reduce the immunogenicity of the system,thereby avoiding the identification and elimination by the immune system and improving the biocompatibility of the nanoreactor;(4)Targeting the tumor by fused platelet membrane and erythrocyte membrane prolong the cycle time in the body,combined with the EPR effect of the tumor site,the system can significantly improve the delivery efficiency of anti-tumor drugs to the tumor site,and enhance the therapeutic effect of melanoma.1.Preparation and characterization of Lipo-Ce6/TPZ@MH.First,the liposome was prepared by membrane dispersion method,and Ce6 and TPZ were simultaneously loaded into the phospholipid shell and aqueous core of the liposome to obtain Lipo-Ce6/TPZ.Then,the erythrocyte membrane and the platelet membrane were separated from the blood taken from the eyelid of the mouse and were fused to obtain a composite membrane.Finally,the composite membrane was co-extruded with the liposome on a liposome extruder to obtain a novel liposome Lipo-Ce6/TPZ@MH.The optimal conditions for the preparation of liposomes were optimized by particle size and encapsulation efficiency.The results of transmission electron microscopy showed that the liposome was uniform in spherical shape,and the liposome coated the cell membrane showed a typical core-shell structure with a particle size of about 90 nm.The UV-visible absorption spectrum contained both 265 nm and 660 nm.The absorption peak is consistent with the characteristic absorption peaks of Ce6 and TPZ;the in vitro drug release results showed that the great pH response characteristics of the drug delivery system;the stability experiment results showed that the cell membrane encapsulation can increase the stability of the preparation;The colocation results showed the cell membrane was successfully coated on the surface of the liposome;Polyacrylamide gel electrophoresis experiments showed that the obtained biomimetic nanoreactor successfully transferred the protein on the cell membrane to the surface of the liposome.2.The antitumor activity of Lipo-Ce6/TPZ@MH in vitro.The in vitro antitumor effect of Lipo-Ce6/TPZ@MH combined with ultrasound stimulation was evaluated using B16F10 melanoma cells as a model.Cellular uptake experiments showed that platelet membrane can increase the uptake rate of tumor cells to the reactor,but the intrinsic biocompatibility and immune evasion ability of the cell membrane can reduce the uptake rate of macrophage to the envelope reactor;Cell inhibition rate experiment showed the biosafety of the reactor was high.The inhibition rate of Lipo-Ce6/TPZ@MH(drug concentration Ce6:2 ?g/mL,TPZ:0.75?g/mL)under US action was about 58%,which was significantly higher than other groups.This is consistent with the results of apoptosis,which can better explain the obvious anti-tumor effect of the reactor;ROS/hypoxia test results showed that Lipo-Ce6/TPZ@MH combined with ultrasound can produce corresponding ROS and hypoxic environment,which helped TPZ to work;the expression of cell surface immunogenic marker calreticulin(CRT)showed that the expression of CRT in Lipo-Ce6/TPZ@MH group was the highest,indicating that sonodynamic therapy can induce strong immunity.Western blotting experiments showed that the sonodynamic treatment method affected Bcl-2/Bax.In summary,the results of cell experiments showed that sonodynamic therapy,coordinated chemotherapy and immunotherapy can effectively inhibit the proliferation of melanoma,presented significant anti-tumor activity in vitro.3.The antitumor activity of Lipo-Ce6/TPZ@MH in vivo.The C57 black mouse was used as an animal model to investigate the tissue distribution,pharmacodynamics and section staining of Lipo-Ce6/TPZ@MH in vivo.In vivo imaging of BALB/c mice bearing 4T1 cells presented the higher fluorescence signal intensity at the tumor site after 4 h of administration indicated the targeting effect of P-selectin on the surface of the platelet membrane to the CD44 receptor on the surface of tumor cells.The obvious fluorescence signal at the tumor site after 7 days indicated that the erythrocyte membrane can prolong the circulation in the body.The tissue distribution results of the black mice were consistent with the results of in vivo imaging,indicating that the enrichment of the tumor site was significantly increased by the targeting of the platelet membrane and long-circulation of erythrocyte membrane of the Lipo-Ce6/TPZ@MH drug delivery system,and the drug bioavailability was significantly improved.In vivo pharmacodynamics was mainly investigated by relative volume changes of tumors,changes in body weight of black mice,H&E staining of melanoma,TUNEL staining and immunofluorescence staining.The results showed that the body weight did not decrease under the action of each group of preparations,this indicated that the system had great biosafety;the experimental results of immunofluorescence staining indicated that Lipo-Ce6/TPZ@MH induced a strong immune response under ultrasound conditions.In vivo anti-tumor assay results indicated that the Lipo-Ce6/TPZ@MH drug delivery system can be targeted to the tumor site for effective treatment of melanoma.