Application Of Piperazine Derivatives Modified Polymer Carrier For Intracellular Protein Delivery

Intracellular protein delivery plays a critical role in the development of biotherapeutics and biotechnology.Efficient intracellular protein delivery carriers often need to meet the following factors: stable binding of target proteins,efficient cellular uptake and endosome escape,as well as quick protein release in the cytosol.Cationic polymers have the characteristics of diverse structures,strong designability,simple synthesis,and easy modification,and have developed into one of the most promising intracellular delivery carriers for proteins.However,due to the structural complexity and charge uncertainty of proteins,the application of cationic polymer carriers is often limited by poor binding ability and insufficient stability with cargo proteins.In addition,the low endosome escape efficiency greatly affects the intracellular delivery efficiency of proteins.Piperazine is a six-membered heterocyclic organic compound with two nitrogen atoms located in the para position in its structure.Piperazine molecule is weakly basic.The introduction of piperazine group into drug molecule can improve its pharmacokinetics and pharmacodynamics by adjusting the lipid water partition coefficient and acid base equilibrium constant of the drug.Many piperazine compounds have important pharmacological properties and exhibit broad application prospects in anti-tumor,antibacterial,anti-inflammatory,antioxidant,and other fields.In addition,piperazine compounds have the ability to open tight junctions between cells and are widely used as efficient and low toxicity permeation enhancers in drug delivery.Therefore,introducing piperazine and its derivative structures into cationic polymers is of great significance in solving the problems of carrier protein binding stability and endosome escape,improving the protein intracellular delivery efficiency of the carrier,and enhancing the permeability of the delivery system.Based on this fact,a series of cationic polymer carriers containing piperazine and its derivatives were designed for intracellular protein delivery in this study.The specific research methods and conclusions of this work are as follows:(1)A type of phenylpiperazine-modified cationic dendrimer was designed,and the complex formed with cargo protein exhibit rapid internalization,with the loaded proteins being uniformly released into the cytoplasm as early as half an hour after incubation.The endocytosis inhibition experiment showed that the cellular uptake of the complex can be significantly inhibited by inhibitors related to the endocytosis pathway mediated by caveolin,such as genistein,filipin III,Brefeldin A,and nystatin,indicating that the complex mainly enters cells through the caveolin mediated endocytosis pathway.This endocytosis method has further been proven to be a cellular endocytosis pathway that does not pass through endosomes/lysosomes,effectively avoiding the degradation of protein molecules in lysosomes.Phenylpiperazine modified polymers can effectively deliver various bioactive functional proteins into cells and maintain the biological activity of these cargo proteins.In addition,due to the enhanced permeability of piperazine molecules,the phenylpiperazine polymer also exhibits excellent protein delivery performance in three-dimensional multicellular spheres.(2)Based on the coordination between divalent copper ions and piperazine molecules,a copper-phenylmethylpiperazine coordination polymer was obtained for intracellular protein delivery.It is hoped to combined the protein delivery with chemical kinetics therapy to optimize the efficacy of chemical kinetics therapy in tumor by regulating the tumor microenvironment.On the one hand,the introduction of copper ions enhances the stability of complexes of phenylmethylpiperazine dendrimers and protein,significantly improving the efficiency of intracellular protein delivery.On the other hand,this kind of benzylpiperazine dendrimers containing copper ions can efficiently carry out Fenton-like reaction in tumor cells,produce a large number of hydroxyl radical,and induce tumor cells to ferroptosis and cuproptosis at the same time.In this chapter,we use copper-benzylpiperazine coordination polymer to deliver lactate oxidase to generate hydrogen peroxide in situ in tumors,thereby enhancing the copper ion mediated Fenton-like reaction and improving the chemical kinetics treatment effect on tumor cells.In the treatment of in situ osteosarcoma mice,the copper phenylmethylpiperazine coordination polymer/lactate oxidase complex encapsulated in the tumor cell membrane was injected into the tail vein,effectively achieving the enrichment of the complex at the tumor site and significantly inhibiting tumor growth.(3)A type of in phenylmethylpiperazine-and benzamidine-rich polymer was designed,and its potential role in the treatment of pancreatitis was preliminarily explored.Compared with unmodified cationic polymers or cationic polymers with benzamidine modified only,polymers containing both benzamidine and phenylmethylpiperazine exhibit lower cytotoxicity.Studies on endocytosis mechanisms have shown that these polymers rich in phenylmethylpiperazine and benzamidine enter cells through various endocytosis pathways and exhibited efficient endosome escape through proton sponge effects,and ultimately releaseg cargo proteins into the cytoplasm.Meanwhile,due to the introduction of the pancreatic enzyme inhibitor benzamidine,the prepared polymer can bind to trypsin and inhibit its activity,thus having certain application potential in the treatment of pancreatitis.In summary,this study has obtained a series of efficient and safe intracellular protein delivery carriers based on the modification of polymer with piperazine and its derivatives.By integrating piperazine molecules with other functional components,a series of protein delivery systems that meet different application needs have been obtained.In future research,we will further promote the development of in vivo experiments and therapeutic drugs for related applications.