Negative Charge Biased Zwitterionic Polypeptide Nanodrug Vehicle For Tumor Targeting

In the past few decades,the development of nanomedicine has partially solved many obstacles from the initial injection site of nanodrug carriers to tumor targets,for enhancing the efficacy of a chemotherapeutic drug in cancer treatment.The welldesigned nanodrug carrier must have excellent blood compatibility,specific tumor targeting,and rapid drug release.These characteristics improve the therapeutic efficacy of nanodrugs in tumor treatment.Polyethylene glycol（PEG）and methacrylate（or acrylate）-based zwitterionic polymers are the most effective materials to improve the blood compatibility of nanodrug carriers and play an important role in the research of nanodrug carriers.However,their non-enzymatic degradation and metabolism will not only reduce the release rate of drugs but also cause excessive accumulation in tumors and healthy organs after off-target delivery,resulting in systemic toxicity.This leads to the problems of a narrow drug treatment window and poor efficacy.Based on the characteristics of enzymatic degradation and physiological metabolism of zwitterionic peptides,the albumin-mimicking nanodrug carrier based on zwitterionic peptides is expected to obtain higher safety and the characteristics of peptides,such as secondary structure conversion and special enzymatic digestion in the tumor microenvironment,for further improving the efficacy of the chemotherapeutic drug in tumor treatment,which is expected to become a better choice for intelligent nanodrug carriers.Therefore,aiming to mimick the surface characteristics of albumin,this work investigated the negatively biased zwitterionic polypeptide nanocarriers of doxorubicin（Dox）,including:1)P（EK-D-C）polypeptides were prepared by copolymerization of side-chain protected glutamyl lysine dimer（EK dimer）with different proportions of side-chain protected cysteine（C）and a small amount of side-chain protected aspartic acid（D）.The hydrophobic anticancer drug Dox was encapsulated by the P（EK-D-C）polypeptides after clicking 11-maleimidoundecanoic acid（M）on sulfhydryl groups of cysteine residues.The results showed that the slightly negatively charged nanodrug carrier having the EK:D:C ratio of 4:0.6:1[P（EK4-D0.6-C1M）/Dox]has a stable particle size below 100nm diameter,which showed excellent blood compatibility,trypsin degradation ability and selective inhibition of tumor cells in a slightly acidic tumor microenvironmental pH by mimicking the charge ratio on the surface of albumin.2)Dox molecules were encapsulated by the P（EK-D-C）polypeptides after the clicked 11-maleimidoundecanoic acid（M）was replaced by conjugated n-mercaptoalkanoic acids with different chain lengths through reductive disulfide bonds on cysteine residues.The results showed that 12-mercaptododecanoic acid（MA12）modified polypeptide[P(EK₆-D_0.9-C₁MAl2)]with the EK:D:C ratio of 6:0.9:1 had the best nanodrug stability,drug loading content（DLC）（16.2%）and drug loading efficiency（DLE）（90%）,and maintained blood compatibility,trypsin degradation ability,and selective inhibition of tumor cells in slightly acidic tumor microenvironmental pH,Moreover,it further showed the drug’s release ability in response to the high concentration of reductive glutathione（GSH）in tumor cells,which increases the release rate of Dox in tumor cells.The study of tumor inhibition through intravenous administration showed that P(EK₆-D_0.9-C₁MA12)/Dox nanodrug had excellent MCF-7 tumor inhibition capability with negligible systemic toxicity.3)Dox-encapsulated nanodrugs by negative biased zwitterionic polypeptides copolymerization with extra phenylalanine（F）were prepared and studied.The results showed that both P（EK6-D0.9-F1.2C1M）/Dox,the one with clicked 11maleimidoundecanoic acid（M）,and P(EK₈-D_1.2-F_3.2-C₁)/Dox,the one only containing hydrophobic phenylalanine,had good stability,selective inhibition of MCF-7 tumor cells in slightly acidic tumor microenvironmental pH,as well as being digested by overexpressed cathepsin B in the tumor microenvironment to accelerate the release of Dox molecules.This shows that the enzyme digestion of the F contained zwitterionic polypeptides by cathepsin B can be realized by mixing an appropriate amount of phenylalanine into the zwitterionic polypeptide,which plays a foundation for further exploring the multifunctional zwitterionic polypeptide nanodrug carrier for tumor tissue targeting.In short,combining all these results,it is shown that the zwitterionic polypeptidebased nanodrug carrier through albumin-mimicking can not only realize the multifunctional design for tumor-targeting delivery,which improves the efficacy of the chemotherapeutic drug in cancer treatment but also further reduces the toxification of healthy organs caused by the accumulation of off-targeting nanodrugs through metabolizing the carrier materials.