Rational Design Of ?-glutamyl Transpeptidase-Responsive Charge-reversal Polymers For Highly Effective Cancer Drug Delivery

Polymer-based nanomedicines have advantages of low adverse effects and thus have wide and urgent clinical needs.However,the clinical benefits of most nanoformulations have been largely limited through decreasing in toxicity,improvement in therapeutic efficacy have been much modest.Poor drug penetration has become major bottleneck for effective nanoparticles(NPs)and biomacromolecular based cancer therapy.The underlying reason is that current nanomedicines can accumulate in tumor but are retained around the blood vessels and incapable of further diffusing away to reach remote tumor cells.Cationic charges on nanomedicines can promote their tumor penetration,but the key is how to make the neutral and stealthy nanomedicine extravasating from the blood vessel quickly turns into positive charged.The clinical relevance of this acid-triggered charge-reversal is limited by the low occurrence of the acidic tumor regions in small human tumors and the inaccessibility of nanocarriers to these weakly acidic regions that are far away from tumor blood vessel network.Therefore,the acidity triggered charge reversal system failed to solve the penentraiton dilemma of nanomedicines.Tumor cells surrounding blood vessels are active and overexpress many enzyme,we thus propose that using these enzymes to cleave off the protection groups to generate some amine groups would make nanomedicine positively charged and thus gain strong penetration ability to distribute in the tumor and thus acquire high therapeutic efficacy.Herein,we demonstrate an enzyme-triggered charge-reversal strategy,taking advantage of the more general enzyme overexpression in tumors,exposing primary amine groups,making the polymer positively charged and highly efficient in tumor accumulation,penetration and cell uptake.Therefore,we synthesized y-glutamyl transpeptidase(GGT)-responsive charge-reversal polymer-PGABEA-CPT,which is zwitterionic and water-soluble,and thus long circulating and stealthy in the blood.But once extravasates into GGT-overexpressing hepatic tumors,the polymer's y-glutamyl moieties are cleaved off by GGT and become positively charged and therefore can penetrate and distribute in the tumor,giving rise to very high therapeutic efficacy.This dissertation can be categorized into three parts,Chapter 2-6 in this thesis mainly constitute the first part.In this part,we designed two monomers GABEA and GAEA,which were enlightened by the structures GSH and L-?-glutamyl-glycine.CPT was chosen to be our model anticancer drug to demonstrate the concept.PGABEA-CPT and PGAEA-CPT conjugates were synthesized by copolymerization of the methacrylate monomer of CPT(MMA-CPT)with GAEAM or GABEAM.The zeta-potentials of PGA(B)EA-CPT conjugates after incubation with GGT for different periods were further studied and the charge-reversal results indicate that GGT can efficiently cleave off the y-glutamyl moieties in PGABEA-CPT to produce the primary amine groups but not in PGAEA-CPT,which are consistent with the hydrolysis results of their monomers.Afterwards,the cell cytoxicity,cellular internalization ability,the subcellular distribution,the endoxytosis mechanism of PGABEA-CPT and PGAEA-CPT conjugates were evaluated.And the results indicatd that GGT played an important role on the cellular behavior of these two conjugates.In Chapter 4,the plasma stability assay,and the pharmacological behaviors of PGABEA-CPT and PGAEA-CPT conjugates,including pharmacokinetics and bidistribution were preliminarily studied.The results suggested that these two conjugates were stable and stealthy in blood circulation owing to their zwitterionic nature,meanwhile,these biodistribution profiles indicate a strong tumor targeting capability of PGABEA-CPT.In Chapter 5,the in vitro and in vivo penetration behaviors of PGABEA-CPT and PGAEA-CPT conjugates were extensively investigated using 3D MCS model and in vivo tumor model.The results indicated that PGABEA-CPT had a superior penetration ability compared with PGAEA-CPT.And the penetration of PGABEA-CPT was mainly through a caveola-mediated endocytosis/exocytosis based transcellular process.In the last Chapter of part 1,the anti-cancer activities of PGABEA-CPT were studied using a series of anti-tumor models.As a result,PGABEA-CPT conjugates show much more potent antitumor activity than non GGT-sensitive ones,leading to complete eradication of small solid tumors(?100 mm3)and dramatic regression of large established tumors with the clinically relevant size(?500 mm3).Furthermore,this conjugate also significantly extends the survival of mice bearing orthotopic pancreatic tumors compared to the first-line drug gemcitabine.In the second part of this thesis,to avoid the high concentration of the conjugate in kidney,we thus synthesized a copolymer PGABEMA-PHEMASN3 8 which self-assembled into 60 nm micelles.The preliminary results indicated that PGABEMA-PHEMASN38 micelles showed higher cell cytotoxicity,better cellular internalization ability and improved antitumor activity towards PEG-PHEMASN38,which means tailoring the size of the delivery system can solve the nephrotoxicity of PGABEA-CPT.Finally,in the third part of this thesis,we developed a GGT responsive charge reversal gene delivery system G-PDEAEA.G-PDEAEA polymer successfully condensed the large DNA macromoleculars into cationic polymer/DN A complexes,and the G-PDEAEA/DNA polyplexes can sterically inhibit the protein absorption and enhance serum tolerance due to the shielding effect of zwitterionic nature of G-PDEAEA,which means G-PDEAEA can be a quite promising non-viral gene delivery carrier.