The Modulation Of Secondary Structure Of PH-responsive Polypeptides And Its Application On Selective Tumor Therapy

Polypeptides,which have the same amine backbone as proteins,can mimic the secondary structure and biological function of proteins.Biological activity of polypeptides is significantly affected by their secondary structure,which can be effectively regulated.A large number of studies have demonstrated that there are various acidic environments in tumor tissues and phagocytic lysozymes.It is challenging to accurately control biological activity of polypeptides by regulating their secondary structure under these acidic environments.In this study,we developed two kinds of pH-responsive polypeptides based on ionizable tertiary amines and acid-sensitive amide bonds,studied their secondary structure and biological activity in various acidic microenvironments,and studied their applications on selective tumor therapy,mainly including the following three aspects of work:（1）pH-responsive PEG-peptide conjugates were prepared by introducing ionizable tertiary amines into the polylysine side chain through the reaction of amino group and alcohol.We synthesized a series of pH-responsive polypeptides with different PEG molecular weights,tertiary amine types,degree of polymerization and proportion of copolymerized hydrophobic leucine.The study showed that the smaller the molecular weight of PEG,the closer the tertiary amine to the main chain,the larger the degree of polymerization,the stronger the hydrophobicity of tertiary amine,the larger the proportion of leucine copolymerized,the lower the p K_a of polypeptide.The polypeptides exhibited a pH responsive helical to non-helical transition when the tertiary amines are relatively hydrophilic,such as N-（2-hydroxyethyl）-pyrrolidine（C5）,Diethylaminoethanol（DE）,N-hydroxyethyl piperidine（C6）and N-（2-Hydroxyethyl）-hexamethyleneimine（C7）.At high pH,the polypeptides show a helical structure when most tertiary amines are protonated.With the decrease of pH,the protonation of tertiary amine enables polypeptide to transform into a non-helical structure.The secondary structural transformation can be achieved in the pH range of 6.0 to 7.2 by regulating the types of polypeptide tertiary amines and the proportions of copolymer amino acids.Polypeptides showed helical structure in the pH range of 2.0 to 7.4 when the tertiary amines are relatively hydrophobic or containg benzyl structure,such as N,N-dibutylethanolamine（DB）,N-benzyl-N-methylethanolamine（MP）,（S）-3-dimethylamino-3-phenylpropanol（DMP）,（1-benzyl-2-pyrrolidinyl）-methanol（C5P）,（S）-1-benzyl-3-pyrrolidinol（C5P2）,or with a high proportion of hydrophilic amino acids.（2）We further screened the above-mentioned pH-responsive polypeptides in search of polypeptides that are low toxicity in normal tissues（pH 7.4）and highly efficient in killing tumor cells in tumor tissues（pH 6.8）.Highly selective anti-tumor polypeptide PPL₈₆-DB was screened by hemolysis assay at pH 7.4 and tumor cell killing assay at pH 6.8.Subsequently,the assembly behavior of PPL₈₆-DB was studied in detail.It was found that the polypeptide was assembled into a hollow vesicle structure with a particle size of 120 nm in neutral environment,which could support hydrophilic or hydrophobic molecules.With the decrease of pH,the polypeptide assembly shifted to micellar structure and could achieve responsive release.Further study showed that PPL₈₆-DB could induce PS lipid aggregation which affected secondary structure of PPL₈₆-DB under acidic microenvironment.At the cellular level,PPL₈₆-DB was observed to disrupt cell membranes leading to leakage of contents,resulting in cell death,and the emergence of filamentous assemblages.PPL₈₆-DB has been demonstrated to have an excellent inhibitory effect on tumor in vivo treatment experiment.（3）In addition,we have developed pH-responsive polypeptides by using maleic anhydride derivatives reacting with primary amines to form acid-sensitive amide bonds.The modification of maleic anhydride introduces both amide bond and negative charge into the side chain of polypeptide.The hydrogen bond interaction and charge interaction in the side chain can destroy the helical structure of polypeptide.Half of the PBut LG-CA side chain was modified with methyl maleic anhydride（MMA）,1-cyclohexene-1,2-dicarboxylic anhydride（CDA）and 2,3-dimethyl maleic anhydride（DMMA）to prepare pH-sensitive non-helical to helical convertible polypeptides activated under lysosome acidity,incline acidity and tumor acidity,respectively.In a specific acidic environment,the side chain amide bond breaks,the carboxyl group（negative charge）leaves,and the side chain hydrogen bond interaction and charge interaction disappear,allowing the polypeptide to restore the helical structure and activate its membranolytic activity.The polypeptide maintained low helicity and low hemolysis activity in body fluid neutral environment（pH 7.4）,but activated membranolytic activity at pH that are specific to tumor microenvironment and tumor endocytotic pathway.In this study,we develop two kinds of pH-responsive polypeptides based on ionizable tertiary amines and acid-sensitive amide bonds,which can achieve the regulation of secondary structure and biological activity under different acidities,allowing selectively inhibiting tumor under tumor acidity.This study provides a new idea for the study of secondary structure regulation and activity regulation of pH-responsive polypeptides and a new strategy for selective tumor therapy.