?-Glutamyl-Transferase-Activated Charge-reversal Dendrimer For Camptothecin Delivery

Chemotherapy is a widely used way in clinical treatment of cancer.However,effectiveness of traditional chemotherapeutic drugs is limited by their low selectivity and significant toxicity.And drug-polymer conjugated drug delivery system leads to many advantages,including improving the pharmacokinetics,increasing targeting ability and reducing the toxic side effects.Dendrimer,a kind of drug carrier,has specific advantages in this system for its monodisperse molecular weight and numerous free functional groups on its surface.However,the in vitro and in vivo toxicity of some traditional dendrimers(e.g.PAMAM)limits their utilization in clinical research.Decoration of pH-responsive functional groups on their surface is a way to solve the problem,but pH difference can only be significant in terminal cancer rather than primary cancer,indicated that pH-responsive charge-reversal carrier can hardly work in early cancer treatment.And ?-Glutamyltranspeptidase(GGT)is usually overexpressed in lung cancer,colon cancer and ovarian cancer,whether in terminal cancer or primary cancer.Recently,a kind of GGT sensitive fluorescent probe has been reported,but there's no published research in GGT responsive drug carrier at home or abroad.In this project,we designed and synthesized a kind of PAMAM modified by y-glutamic amido bond(gGlu-PAMAM).Then,anticancer drug camptothecin(CPT)was conjugated on it via disulfide bond.And both gGlu-PAMAM and gGlu-PAMAM-S-S-CPT were characterized by 1HNMR and drug loading rate of gGlu-PAMAM-S-S-CPT was determined by UV-concentration standard curve.While adding this dendrimer into GGT solution,its increasing zeta potential which was from negative to positive proved that the y-glutamic amido groups on surface could be hydrolyzed by enzyme after 33 hours,and it indicated that this nano carrier could reverse its charge in a high level GGT environment.Moreover,in vitro cytotoxicity experiments,although antitumor activity of gGlu-PAMAM-S-S-CPT was inferior to CPT,it expressed significant toxicity in A549 and HT-29 cells line where GGT was highly expressed,compared with gGlu-PAMAM.Via flow cytometry and confocal,we discovered that A549 cells endocytosed less gGlu-PAMAM after treatment with GGT inhibitor azaserine and acivicin,and it revealed that GGT in cells could accelerate the speed of gGlu-PAMAM endocytosed into tumor cells.In blood clearance experiments with ICR mice,we proved that gGlu-PAMAM can be stable in blood circulation.Meanwhile,we found that gGlu-PAMAM mainly distributes in the kidneys and tumors through biodistribution experiments in A549 xenograft nude mice model,which revealed its target ability to tumor.And in the last antitumor experiment in A549 xenograft nude mice model,gGlu-PAMAM-S-S-CPT showed almost the same efficiency in nude mice xenograft model,and it showed no toxicity in vivo.In this project,a kind of GGT responsive charge-reversal drug delivery system for cancer therapy was synthesized for the first time,and its biological characteristics in vitro and in vivo were also studied.And it provides new ideals and experimental evidences for development of other enzyme responsive drug carrier for cancer therapy.Due to time limit,there's only a preliminary evaluation of gGlu-PAMAM-S-S-CPT in anti-tumor experiment in vivo with A549 xenograft nude mice model.And we will conduct more scientific and comprehensive studies for this system in the future.