Design,Synthesis And Bioevaluation Of Enzyme Triggerable Cell Penetrating Peptide(ETCPP)-Based Dendrimers For Targeted Cancer Therapy

Cancer is a major public health problem worldwide and is regarded as the leading cause of population death.Camptothecin(CPT),as a traditional chemotherapeutic agent,shows significant antiproliferative activity against a variety of cancer cells and presents a broad application prospect in cancer treatment.However,CPT suffers from several drawbacks including poor water solubility and low selectivity,which hampers its clinical applications.Over the last couple of years,a large number of novel targeted delivery systems have been developed for chemotherapeutic drugs,among which peptide-drug conjugates(PDCs)have received extensive attention from researchers in both industry and academia.This strategy specifically utilizes the targeting or cellpenetrating properties of short peptides to improve the therapeutic efficacy and reduce toxic and side effects.However,single-chain PDCs are usually short and small with simple spatial structure,thus resulting in poor metabolic stability and low in vivo transport efficiency.Therefore,it is of great importance to develop novel delivery systems for CPT with high specificity,low toxicity and relative stability,and to elucidate their mechanism of action.Herein,based on a cationic penetrating peptide(FKKFFRKLL)discovered in our previous work,we developed a series of enzyme(MMP-2/9)triggerable cell penetrating peptide dendrimers(ETCPPDs)with CPT as the warhead.Experiments on both cellular and molecular levels were carried out to unravel the possible antitumor mechanism of action of these ETCPPD conjugates.Meanwhile,the in vivo pharmacological activities including targeted drug delivery efficiency and tumor suppressing capability were further investigated in HCT-116-bearing mice.The specific experiments are as the following three parts:1.Based on the principle of charge complementation,four generations of ETCPPDs(G0,G1,G2 and G3)with lysine as the aggregation center were synthesized by introducing a "blocking peptide" composed of glutamate and glycine into the FKKFFRKLL skeleton.The disulfide bond was introduced for the connection of ETCPPD and CPT to produce a series of MMP-2/9 sensitive ETCPPD conjugates;2.The ETCPPD conjugates were preliminarily evaluated for their pharmacological activities including biofilm transport efficiency and in vitro antiproliferative potency.Among the newly synthesized compounds,the G3-generation ETCPPD conjugate BL_Oc-SS-CPT exhibited the highest cellular uptake efficiency and the lowest IC50 values against a panel of cancer cells,ranging from 31 to 747 nM.Experiments such as cell cycle,apoptosis,mitochondrial membrane potential and DNA damage and repair further confirmed that BL_Oc-SS-CPT could enhance DNA doublestrand breaks and cell cycle arrest to promote cancer cell apoptosis.Meanwhile,BL_Oc-SS-CPT possessed enhanced metabolic stability with a plasma half-life of 4.2 h;3.In vivo biological properties of BL_Oc-SS-CPT,including tissue distribution,anti-cancer efficacy and toxicity,were studied in HCT-116-bearing BALB/c-nu mice.The pharmacodynamics and histological analysis denmonstrated that BL_Oc-SS-CPT exhibited excellent in vivo tumor targeting capability and antitumor efficacy with benign toxicity profiles,which effectively avoided drug enrichment in normal tissues,thus reducing undesirable toxic and side effects.In conclusion,a series of novel ETCPPDs with CPT as the warhead were designed and synthesized in this thesis.Among them,BL_Oc-SS-CPT has several advantages such as great metabolic stability,more potent in vitro&in vivo anti-cancer activity,and lower toxic and side effects.Thus,the novel ETCPPD-based drug delivery system exemplified by BL_Oc-SS-CPT represents a safe and effective strategy for targeted cancer therapy.