Preparation Of Dynamic Amphiphilic Polymer And Its Application In The Field Of Protein Intracellular Delivery

Protein is a very important biological macromolecule in life activities and plays an important role in physiological processes such as enzyme catalysis,gene transcription regulation,signal transduction and so on.Over the past decades,protein drugs such as polyclonal antibodies,vaccines,hormones have gradually emerged in clinic and occupied an extremely important position in the field of medicine.However,due to the large molecular weight and complex charged nature of proteins,proteins are unable to cross the cell membrane itselfs,let alone enter the cell to play its special physiological function.For many proteins which can work in the cell but cannot cross the cell membrane,it is very urgent to develop methods that can efficiently deliver proteins.Current protein delivery methods still have many shortcomings that need to be solved urgently,such as common physical methods including electric pulse,physical extrusion,microneedles and so on,which are easy to cause irreversible damage to cells;although protein modifications have improved the transmembrane performance after chemical or genetic engineering,they are prone to cause irreversible changes in protein structure,thus losing the original biological activity;Polymer carrier is widely used in the delivery of biological macromolecules because of its easy modification and high efficiency,but high molecular weight is often accompanied by high toxicity.In order to solve these problems,we designed a new type of polymer material: introducing hydrophobic alkyl chain through the recognition between phenylboronic acid which grafted to a-polylysine and phenolic hydroxyl end which attaced to alkyl chain to form borate ester bond,after polymers entering the cell,the acidic environment will make the borate ester bond broken,thus constructing a dynamic amphiphilic polymer.Amino groups on polylysine can interact with carboxyl groups in proteins by electrostatic interaction,and the introduced hydrophobic alkyl chains can insert into the hydrophobic regions of proteins,thus better binding to proteins;meanwhile,the "detachable" alkyl chains reduce the toxic side effects to cells during transfection and have better biocompatibility.Based on the above experimental theory,we synthesized a-polylysine containing 15 phenylboronic acids,after binding to different lengths of phenolic hydroxyl-terminal alkyl chains,polymers were combined with bovine serum protein(BSA)linked with fluorescein.Then we screened out the ratio of phenylboronic acid to phenolic hydroxyl group and alkyl chain length at the highest delivery efficiency.The results showed that the alkyl chains of C10 and C12 most significantly promoted the endocytosis of the complex when the ratio of phenylboronic acid to phenolic hydroxyl was 1:1.Next,we further explored the mechanism of protein delivery by such dynamic amphiphilic polymers,including the binding efficiency of phenylboronic acid to phenolic hydroxyl groups,the effect of alkyl chains of different lengths on protein loading capacity and cell entry behavior of complexes under physiological conditions.Through NMR characterization,the binding efficiency of phenylboronic acid and phenolic hydroxyl groups was about 100% at different feed ratios;we further found that the longer the introduced alkyl chain,the stronger hydrophobic force the alkyl chain provide,the more the material entered the cel,and the higher the stability of the complex under physiological conditions;However,due to the existence of steric hindrance effect,the longer the alkyl chain,the lower the protein mass loaded.Under the influence of multiple factors,the efficiency of protein transfection by the complex under physiological conditions showed a trend of rising first and then declining,thus showing the highest efficiency when the alkyl chain was C10 and C12.Next,we selected a variety of proteins for intracellular delivery using polymers that introduced alkyl chains C10 and C12.By means of flow cytometry,laser confocal,toxicity detection,intracellular and extracel ular enzyme activity determination,we found that: Efficient delivery can be observed for BSA and egg ovalbumin;Fluorescent phycoerythrin(R-PE)has a very high intracellular fluorescence intensity;for proteins with biological activity such as beta-Gal,Saporin,and alpha-chymotrypsin,it can maintain high efficiency of enzyme activity or its toxic effect while efficiently delivering to the cels;there is also a special class of oxidoreductases,peroxide dismutase(SOD)and catalase,which play a role in oxidative stress.Both can exert their special ROS-lowering effect.Based on the above studies,this kind of dynamic amphiphilic polymer can deliver a variety of proteins with low efficiency and has certain universality.At the same time,our exploration of its mechanism provides researchers with a new polymer design strategy.And this material has great potential in clinical treatment,which provides the possibility to develop more protein drugs for clinical treatment...