Design,Synthesis And Biological Evaluation Of New PH-responsive Cell Penetrating Peptides Isopropyl-TH?Butyl-TH And HR-1

Chemotherapy is an important means of cancer treatment,but due to the poor selectivity and biological membrane permeability of conventional chemotherapy drugs,combined with the chemotherapy drugs widely used leading to multi-durg resistance,these reasons have become an important problem faced by cancer treatment.Therefore,improving the selectivity and overcoming the multi-drug resistance have become a key to research and development antitumor drugs.On the one hand,cell penetrating peptides?CPPs?can mediate a variety of substances into the cells,as a result,the method of use CPPs modified traditional antitumor drugs,which can significantly improve the permeability of biomembrane.On the other hand,the biggest difference between tumor tissue and normal tissue is that tumor tissue has more acid extracellular environment than blood and normal tissue,therefore,the pH-sensitive peptides drug delivery systems,which target to acidic extracellular environment of tumor tissue can effectively improve the selectivity of antitumor drugs.The first part of the paper,we design and synthesis two new analogs of acid-sensitive cell-penetrating peptide TH.In previous work,our group had designed and synthesized TH?AGYLLGKINLKKLAKL?Aib?KKIL-NH₂?which is a kind of acid-activated CPPs,and the antitumor activity of TH-CPT conjugate has obvious pH-sensibility.In this study,in order to further improve the sensitivity of TH to weak acid,make it can play a role in a wider pH range.The electron donating groups?Isopropyl and Butyl?was introduced into the imidazole group of histidinein,so that increased the alkalinity of N atom on imidazole ring and enhanced the ability to absorb proton,thereby enhancing the pKa value of histidine.Then these histidine analogs?His?Isopropyl?-OH,His?Butyl?-OH?was introduced into TH,formed two new TH analogs?Isopropyl-TH,Butyl-TH?.The penetrating activity,in vitro cytotoxicity and hemolysis activity of these two TH analogs were evaluated.Then,these two new CPPs were connected to the traditional anti-tumor drug CPT,respectively.In addition,the antitumor activity of the two CPP-CPT conjugates Isopropyl-TH and Butyl-TH were evaluated.As a result,the cell penetrating activity of Isopropyl-TH and Butyl-TH showed good pH-dependent.Especially,the fluorescence intensity of the internalized Butyl-TH was approximately 1.72-fold higher than that TH at pH6.5.More importantly,in vitro cytotoxicity and hemolysis activity of Isopropyl-TH and Butyl-TH significantly decreased,compared with TH.The anti-tumor activity of Isopropyl-TH-CPT at low concentration showed significant pH-selectivity in comparing with TH-CPT.However,the anti-tumor activity and pH-selectivity of Butyl-TH-CPT has been greatly improved.The second part of the paper,we designed and synthesized a series of KR analogues,namely KR-1,KR-2 and KR-3.Activity study showed that the cell penetrating activity of KR-1 was best,and the in vitro cytotoxicity and hemolysis activity of KR-1 was also lower.Therefore,we further focused on low-pH condition in tumor tissues as an appropriate target,utilized histidine as a low pH-responsive trigger,made use of KR-1?KRIVKRIKKFLR-NH₂?as a templat and replaced all of lysines by histidines,formed a novel CPPs HR-1?HRIVHRIHHFLR-NH₂?,which with high pH-sensitivity.In vitro uptake study confirmed that HR-1 exhibited excellent pH selectivity compared to the parent peptide KR-1.Namely,only a small amount of HR-1 was uptaken by Hela cells under normal physiological conditions?pH7.4?,but at low-pH?pH6.0 and pH5.5?,HR-1uptake increased obviously.Furthermore,we also found that HR-1 is almost no toxic effects on Hela cells and almost no damage to red blood cells.In this paper,we designed and synthesized series of pH-responsive CPPs,hoping for the development of appropriate antitumor drug delivery vehicles,having good research significance and application prospects.