Angelw: peptide brush polymer, a new tool for protein intracellular delivery

High efficient intracellular transfer of macromolecules is a major challenge in the fields of chemical biology, biotechnology and medicine Compared with the transfected gene, the direct transfected protein can accurately control the protein content and duration, and it is easy to modify and reduce the Miss target effect At present, the methods of protein transfection include protein and cell penetrating peptide However, CPP is easy to be degraded by protease, and it is difficult to escape from the inclusion body The substances with strong positive electricity are cytotoxic, and liposomes need to be optimized according to different proteins Because stem cells play an important role in basic research and cell therapy, but conventional methods are difficult to transfect proteins into stem cells Therefore, there is an urgent need for a general method to transport proteins into stem cells without physical or chemical modifications and without cytotoxicity Recently, Angela P Blum team of Hamilton College, gene W Yeo team of University of California, San Diego and Nathan C gianneschi team have combined to find a brush like CPP polymer that can efficiently deliver proteins to stem cells, providing a new method for efficient protein delivery to stem cells Relevant research results are published in Angela W Chem In Ed (DOI: 10.1002/anie.201904894) Previously, the research team developed an effective method to polymerize peptides into high-density brush like polymer by ring opening metathesis polymerization (ROMP) The polymer can withstand the degradation of protease, and can maintain good cell penetration in the presence of protease So in this new study, the team explored the effects of peptide composition and conformation on the ability of the brush like polymer to transfer proteins into living cells (image source: angel Chem Int ed.) they first took CRE protein which can be edited by gene as the model protein, and HEK293T cell as the model cell (Figure 1a) The researchers first studied the cytotoxicity of various brush like polymers, and found that these polymers had no obvious toxicity to cells at the experimental concentration Next, we used HEK293T cells which can express green fluorescent protein EGFP under the action of Cre recombinase to study the ability of various peptides to deliver CRE protein in cells: the results showed that only the brush like macromolecules containing Tat (a commonly used short peptide for promoting protein phagocytosis) could efficiently deliver CRE protein to cells, while the individual Tat peptide or surface modified Tat could The nanoparticles of peptide had no significant transfection effect (Figure 1b) Next, the author explored the transfection effect of brush like polymer composed of other peptides (including tat, cpp30, cpp44, Arg8, uncharged peptide gsgsg or oligopeg, integrin binding protein irgd, etc.) (Figure 2) The results showed that the transfection ability of the protein in turn was Tat > cpp30 > Arg8 > separate Tat > cpp44 > guanidine > OEG > GS > irgd We also found that RhoB labeled polymer was more efficient in transfection In addition, the best polymerization degree of Tat brush polymer is 5 (image source: angelw Chem Int ed.) finally, the authors evaluated the ability of Tat brush like polymers to deliver proteins to pluripotent stem cells The experimental results show that this polymer can effectively deliver CRE protein to stem cells, and has no effect on the activity of stem cells within the range of concentration (Figure 3) (image source: angelw Chem Int ed.) conclusion: This study shows that Tat modified high-density brush like polymer can effectively deliver functional proteins to stem cells without physical binding of proteins and peptides, and has low toxicity to cells Previous studies have shown that this polymer may enter cells by endocytosis or membrane disruption In the next study, the researchers continue to use these materials to try to deliver other functional proteins to stem cells, or to other cells that are difficult to transfect, so as to expand the application of this new material.