Study On Stiffness Of Cell Membrane Camouflaged Nanocarriers Influences Their Systemic Circulation And Cellular Uptake

Nanoparticle drug delivery systems have been used more and more in the treatment of tumors because of their long circulation effect and tumor-targeting ability.After PEG modification,liposomes can significantly reduce the phagocytosis of macrophages and accumulate to tumor sites through the Enhanced Permeability and Retention effect?EPR?.However,due to the accelerated Blood Clearance phenomenon,ABC phenomenon exists that after repeated injections of PEGylated liposomes,the immune system will be activated and the liposomes will be cleared,resulting in a significant reduction in the anti-tumor effect of liposomes.The bionic nanoparticle carrier of the erythrocyte membrane is to coat the purified erythrocyte membrane on the core surface of inorganic or organic nanoparticles.The core-shell nanocarrier can significantly reduce the recognition of reticuloendothelial system,avoid the ABC effect,and prolong the carrier cycle time.Healthy red blood cells have excellent elasticity,allowing them to move freely through narrow capillaries.However,after the red blood cell senescence,cytoskeletal composition changes,cytoplasmic thickening of the cell body becomes hard,which is easily recognized by the in vivo immunoglobulin,thereby activating the reticuloendothelial system and being cleared.Based on the changes in the hardness of red blood cells during the aging process,it is speculated that the hardness is also a key factor affecting the in vivo behavior of the bionic nanocarrier of the erythrocyte membrane.In order to verify this hypothesis,this study isolated the erythrocyte membrane and constructed a bionic nanocarrier of erythrocyte membrane that simulated the hardness of diseased erythrocytes,healthy erythrocytes and aging erythrocytes respectively.The three vectors were flexible erythrocyte membrane nanovesicles?RBC-SNVs?,elastic erythrocyte membrane core-shell nanoparticles?RBC-ENPs?,and rigid erythrocyte membrane core-shell nanoparticles?RBC-HNPs?,and their effects on drug delivery behavior in vivo were investigated.The characterization results of the bionic erythrocyte membrane nanocarriers showed that the particle size of the three carriers was about 170 nm and the dispersion was good.Under transmission electron microscopy?TEM?,the structure of RBC-SNVs was vesicular,while RBC-ENPs and RBC-HNPs were core-shell characteristics.SDS-PAGE and Western Blot experiments confirmed that the bionic erythrocyte membrane carrier inherited the surface membrane protein of erythrocyte well.The assembly and coating directions of the erythrocyte membrane were consistent with those of the natural erythrocyte membrane by Dot Blot.According to the scanning results of the Atomic Force Microscope?AFM?,the hardness values of the bionic carriers of red blood cells in each group were calculated.It was found that RBC-ENPs had a more similar hardness with natural healthy red blood cells,both of which were about 10 N/m.The bionic carriers of the erythrocyte membrane in each group had good stability and biological safety in vitro.The results of in vivo pharmacokinetic experiments and in vivo distribution experiments confirmed that the fluorescence intensity of RBC-ENPs in the liver of the main reticuloendothelial system was 39.4%and 58.6%lower respectively than that of RBC-SNVs and RBC-HNPs,and the fluorescence uptake in Kupffer cells was 22.5%and 29.3%lower respectively,with longer in vivo circulation time and elimination half-life.In addition,the mechanism of the difference between erythrocyte carrier and PEG long-circulating liposomes in vivo circulation was studied from the perspective of protein crown.By LC-MS/MS experiment analysis of protein adsorption type and quantity,the results showed that red cell membrane carrier and PEG long circulation liposomes are a variety of plasma protein adsorption,but plasma protein adsorption quantity have obvious difference,RBC-ENPs compared to the amount of plasma protein adsorption RBC-SNVs and RBC-HNPs reduced by 26.9%and 36.2%respectively,adsorption of immunoglobulin IgG and IgM has significantly reduced,it can effectively avoid the identification and macrophage,long cycle so as to achieve a better effect.After the bionic erythrocyte carrier was loaded with doxorubicin?Dox?,the cell uptake experiment was carried out on breast cancer tumor cells.The results showed that Dox@RBC-SNVs,Dox@RBC-ENPs and Dox@RBC-HNPs showed no significant difference in the uptake of tumor cells,but all showed a significant increase compared with Dox@PEG-Lipo.The in vivo distribution experiment of the preparation was conducted in 4T1 tumor-bearing mice,and the results showed that the fluorescence value of RBC-ENPs in the tumor site was significantly increased by51.8%and 93.7%compared with RBC-SNVs and RBC-HNPs,indicating that RBC-ENPs could significantly reduce the immune activation ability in vivo and prolong the circulation time in vivo,and significantly increase the accumulation of tumor site through EPR effect.The results of the MTT experiment showed that the IC₅₀0 value was also reduced by 86.3%,87.90%and 84.2%respectively,indicating that in the absence of plasma protein adsorption,the bionic erythrocyte nanocarrier significantly increased the anti-tumor effect in vitro due to the increased uptake of4T1 tumor cells.The anti-tumor test showed that the tumor inhibition rate of Dox@RBC-ENPs reached 89.5%,which increased the survival rate of the mice,and had no visible toxic and side effects on the organs and organs of the animals.To sum up,the red cell membrane bionic carrier RBC-ENPs from two aspects of carrier surface composition and hardness of the physical properties of better simulate the natural state of healthy red blood cells,and can in the body has the best long cycle effect,significantly reduce macrophage activation and recognition,in the animal in vivo antitumor experiments also have good tumor inhibition effect,or even better than a gleam of antitumor nano drugs Doxil.Through the design of a new bionic delivery carrier,the problems of low delivery efficiency and poor efficacy of nanocarriers were solved.It provides a new idea and method for the design and preparation of a new generation of bionic delivery carriers and lays a research foundation for the clinical transformation of anti-tumor drug nanometer carriers in the treatment of breast cancer.