Construction Of Intracellular Drug Delivery System With High Efficiency Based On Poly- β- Malic Acid

Multivalent polymers offer a powerful opportunity to develop theranostic materials on the size scale of proteins that can provide targeting, imaging, and therapeutic functionality. Numerous drug delivery systems have been developed,including liposomes, dendrimers, polymer micelles and nanoconjugate. Even polymeric nanoparticles are nontoxic and biodegradable, they still have some disadvantages. Poly(β-malic acid)(PMLA) is a linear anionic C4-polyester consisting of monomeric units of L-malate. It can be degraded into non-toxic malic acid which was used as an intermediate of tri-carboxylic acid cycle. It is generally safe, nontoxic, biocompatible, and biodegradable. PMLA has lots of pendant carboxyl groups, which allow further conjugation of biological active molecules, such as drugs and targeting modules. In previous study of our group, PMLA has strong negative electricity(-30 m V). It is difficult for PMLA to carry the anti-cancer drugs into tumor cells by endocytosis because of the cell membrane’s strong negative electricity. Therefore, ligands like TF, peptides, folic acid or antibodies which could selectively recognize and bind to the specific receptor over-expressed on tumor cells are used, resulting in increased targeting efficiency and less toxicity. The tumor cells are miscellaneous, while the receptor-ligand system is not universal, which limits its application in drug delivery systems.The cell-penetrating peptides(CPPs) conjugated to the nanocarriers have been used extensively for nano drug delivery. The TAT peptide(AYGRKKRRQRRR) is a well-known non-specific cell penetrating peptides. Derived from human immunodeficiency viruses types 1 and 2(HIV-1and HIV-2), that has been reported to be a potent transcriptional activator of viral gene expression. It is proposed as a potent CPP, capable of transporting different molecules and even 200 nm liposomes into various cell lines. Although the TAT peptide has been widespread used in drug delivery systems, its non-selective penetrating also makes it carry drug into normal cells. This disadvantage limited the use of TAT in drug delivery systems. Various stimuli-responsive strategies to shielding and deshielding the TAT peptide to minimize their side effects and maximize their therapeutic efficacy had been utilized.This study aimed to develop a nano drug delivery system that can highly accumulate encapsulated drugs in tumor tissue and then deliver drugs into tumor cells efficiently. PMLA as the drug carrier, long-chain PEG5000 which was connected with PMLA via hydrazone bond had been used to shield TAT peptide. To make sure TAT can only work after removal of cleavable PEG, TAT was attached to the distal end of a shorter PEG spacer(PEG2000), which could be shielded before arrival at tumor tissue. When the nanoconjugate reached the tumor tissue, the hydrazone bond is broken, TAT played a role in carrying DOX into the cells and killing tumor cells. PMLA will be degraded without side-effect to human body. The minimum concentration of TAT peptide and the preferable concentration of cleavable PEG5000 using with PMLA are not explored systematically before. First, the minimum concentration of TAT peptide which can provide better penetration has been investigated because the more concentration of TAT needs more concentration of PEG5000. After that, the preferable concentration of cleavable PEG5000 which has good effect on protective about TAT has been defined. The TAT peptide has been deshielded under tumor p H circumstances through cleavable PEG and shielded under normal p H in vitro experiment. This strategy could not only decrease the side-effect of drug carriers and anti-cancer drugs at normal tissue, but also facilitate nanoconjugate’s disintegration and the efficiently drug carrying in response to the low p H.