Optimal Design Of Novel Functionalized Nano Drug Delivery System Based On Polymalic Acid For Efficient Tumor Endocytosis With Enhanced Anticancer Activity

In recent years,nanoparticles(NPs)have been widely studied to improve selective delivery of drugs to the tumor site.NPs achieve this objective thanks to their ability to accumulate in the solid tumor by either passive(NPs size < 200 nm)or active targeting mechanisms and specifically trigger internalization inside tumor cells.Passive targeting relies on a phenomenon discovered in 1986,known as Enhanced Permeability and Retention(EPR)effect.This effect is based on abnormal features of blood vessels and the less lymphatic drainage in solid tumors,resulting in prolonged retention of NPs inside the tumor mass and increased concentrations up to 10–50 fold with respect to normal tissues.Tumor microenvironment(TME)has been broadly exploited recently,in which the cellular metabolism,biosynthetic intermediates and physical environment are prominently different from those in normal tissues.The idea of stimuli-responsive NPs comes from these abnormalities.A number of environment sensitive nanoscale polymer drug carriers are developed for drug delivery with the ability to enhance the therapeutic efficacy of antitumor drugs both in vitro and in vivo.Asparaginyl endopeptidase(AEP,legumain)is a highly conserved lysosomal/vascuolar cysteine protease,which was originally identified in legumes.It has restricted substrate specificity cleaving peptides on the C-terminal side of asparagines.The selection of legumain as a target for tumor therapy is based on the fact that the gene encoding of this asparaginyl endopeptidase is found to be highly upregulated in many murine and human tumor tissues but absent or present only at very low levels in all normal tissues from which such tumors arise.The function of legumain is to recognize a specific sequence(a stretch of peptide,AANL)in proteins and cleave to the proteins to either activate or destroy them.On the basis of these changes in its specific expression pattern,legumain represents a good candidate molecule for the design of an anticancer drug carrier.Several different delivery systems based on legumain have been created and used effectively as carriers for anticancer agents.However,legumain responsive drug delivery systems using other promising materials are rarely reported.In order to compose proper NPs,Various biocompatible and biodegradable polymers have been evaluated as potential drug carriers.Among them,PMLA being non-toxic,non-immunogenic,biocompatible and biodegradable,it also has carboxylic acid lateral functional groups,which can be used to chemically bind various molecules of interest such as drugs,targeting moieties,etc.However,the strong negative charge of carboxyl groups in PMLA decreases the compatibility with the negatively charged cell membrane,leading to the low cellular uptake,and limits its application as the drug carrier.Although the targeting ligands and a variety of stimuli-responsive moieties of nanomedicines significantly increased the drug accumulation in the tumor area,insufficient cellular internalization of nanomedicines can be another significant barrier,especially for macromolecular drugs such as proteins or RNA.To improve the cellular uptake,other delivery pattern(cell penetrating peptides,such as TAT)was used to modify the NPs since they were shown to be efficient in aiding translocation across the plasma membrane.Nevertheless,the application of TAT in DDSs has been limited due to the non-specificity of TAT.Herein,an enzyme-and pH-sensitive PMLA-based nanoparticle was prepared for effective drug delivery,enhanced cellular uptake,and intracellular drug release.Covalent conjugation of pH-sensitive cis-aconitic anhydride-modified doxorubicin(CAD)with PMLA afforded the amphiphilic co-polymers that readily formed the polymeric nanoparticle.The legumain-cleavable tetrapeptide(AANL)was used as a sensitive linker between PMLA and its long-chain PEG block to serve as a steric shield for the nanoparticle and surface-attached cell-penetrating function(TAT)in the blood.In the blood(at normal pH),the PEGylated nanoparticle has been used to hide the TAT,which prolongs its circulation time and minimizes the rapid clearance of the nanoparticle from the body.When in the tumor microenvironment,the peptide is expected to be cleaved by the highly expressed extracellular legumain,leading to the exposure of TAT and enhanced intracellular penetration.After endocytosis,the nanoparticles could rapidly release the loaded drugs at the endosomal/lysosomal pH,to facilitate the drug action.To the best of our knowledge,this is the first report on a nanoparticle based on biodegradable branched PMLA copolymer-drug conjugate as a legumain-sensitive NDDS for cancer therapy.