Construction Of A GSH-Responsive Hyperbranched Poly(Amido Amine) As The Drugs And Gene Vectors And Its Application In Tumor Therapy

Nano-delivery systems for drugs and genes developed in the past decades have many defects in stability,loading capacity,targeting,and controlled release etc.Recently,stimuli-responsive carriers on the basis of tumor microenvironment with enhanced tumor permeability,active targeting and controllable release kinetics have provided a new strategy to address the as-mentioned problems.Therefore,it is desirable to rationally design nanoparticles with controllable properties to overcome the multiple obstacles during transportation,thus improving the efficacy of the encapsulated drugs.Studies reported previously have shown that the expression level of glutathione?GSH?in tumor cells??2-10 m M?exhibits an exponential increase compared to the normal cells(?2-10×10^-3 m M).Based on this,we designed a GSH-responsive hyperbranched poly?amido amine??HPAA?,whose polymer skeleton contains a large amount of disulfide bonds,which can not only be reduced to mercapto moieties under high GSH concentration of tumor cells,thereby achieving the rapid release of encapsulated drugs or genes in the tumor sites;but also exhaust GSH in tumor cells,so as to enhance the sensitivity of tumor cells to chemotherapeutic drugs.In the first chapter,HPAA was used as the basic backbone,which was grafted with amphiphilic methoxy ether polyethylene glycol-poly?D,L-lactide-co-glycolide?block copolymer?m PEG-PLGA?.Then the prepared m PEG-PLGA-HPAA could encapsulate the hydrophobic paclitaxel?PTX?to achieve the self-sensitizing chemotherapy on triple-negative breast cancer cells.In addition,due to the large number of amino moieties on the surface of HPAA,it can be used as highly-efficient vehicles for genes and be functionalized by simple chemical reactions.In chapter 2,HPAA was modified with the rabies virus glycoprotein peptide?RVG?followed by loading small interfere ribonucleic acid?si RNA?to form HPAA-RVG/si RNA.The prepared gene delivery carriers are expected to provide new mean for neuroblastoma cell gene therapy.The overall research is mainly divided into the following two parts:1.A chemotherapeutic self-sensibilized hyperbranched poly?amido amine?carrier to deliver paclitaxel for the enhanced chemotherapy on triple-negative breast cancerOwing to the lack of valid targets of triple-negative breast cancer,it can only be treated by traditional chemotherapy in clinical.However,traditional chemotherapy drugs may cause serious toxic side effects during treatment due to their poor water solubility,low selectivity and low bioavailability.Thus,to enhance the chemotherapy effect on triple-negative breast cancer cell?MDA-MB-231?and reduce toxic side effect caused during therapy,we grafted amphiphilic monomethyl ether polyethylene glycol-poly?D,L-lactide-co-glycolide?block copolymer?m PEG-PLGA?onto HPAA through amide reaction,and further loaded paclitaxel?PTX?to obtain m PEG-PLGA-HPAA/PTX nanocomposite micelles.Because of the large amount of disulfide bond in the nanocomposite micelles,m PEG-PLGA-HPAA/PTX can degrade under the high concentration of GSH of the tumor microenvironment,leading to the rapidly release PTX and the reduce of GSH needed for tumor growth,thus inhibiting tumor cell proliferation and reducing the tolerance of tumor cells to PTX.The chemical structure and morphology of m PEG-PLGA-HPAA were characterized by NMR and TEM,and the in vitro GSH-responsive performance were characterized by DLS,GPC and HPLC;The inhibitory effect of drug-loaded micelles on tumor cells were characterized by cytotoxicity and apoptosis experiments.The experimental results shown that the m PEG-PLGA-HPAA/PTX nanocomposite micelles could degrade exposed to the GSH,showing a GSH-controlled PTX release;Compared with PTX,the drug-loaded micelles significantly enhanced the efficacy of PTX by inhibiting cells proliferation and inducing cells apoptosis,performing the self-sensitization effect of chemotherapy.The biocompatibility of the prepared carrier was evaluated through cell experiments in vitro,hematological parameters and histological analysis in vivo,which revealed that the m PEG-PLGA-HPAA has good biological safety.The self-sensitizing chemotherapeutic drug carrier is expected to provide a new method for tumor chemotherapy.2.Hyperbranched poly?amido amine?gene carrier grafted with rabies virus glycoprotein peptide for targeted therapy of neuroblastoma cellsNeuroblastoma is a common extra-cranial nerve tumor,which mostly occurs in children under14 years old.Due to the poor tolerance of children to radiotherapy and chemotherapy,emerging gene therapy has become a relatively mild and effective method for this cancer.However,gene therapy often has the problem of mutual restriction between high transfection efficiency and low toxicity.To improve the safety and effectiveness of gene therapy,we used the GSH-responsive cationic polymer HPAA as the backbone to link rabies virus glycoprotein?RVG?via the bridge of maleimide-polyethylene glycol-succinyl ester?MAL-PEG-NHS?followed by loading si RNA to form HPAA-RVG/si RNA complex for targeted gene therapy on neuroblastoma?SK-N-SH?cells.The chemical structure and physical properties of HPAA-RVG were verified by NMR and HPLC;The capacity of HPAA-RVG to bind si RNA was verified by DLS,agarose gel electrophoresis and TEM;The targeting effect of RVG was proved by endocytosis and intracellular colocalization.The experimental results showed that HPAA-RVG could load si RNA efficiently,and perform highest endocytosis efficiency in SK-N-SH cells,showing a specific targeting effect.Cell transfection experiment confirmed that compared to PEI,HPAA-RVG/si RNA could significantly improve the transfection efficiency of SK-N-SH cells in the presence of serum.Through in vivo imaging experiments,HPAA-RVG/si RNA was proved with the ability to cross the blood-brain barrier.Moreover,the biocompatibility of the prepared HPAA-RVG/si RNA was evaluated by cytotoxic and blood compatibility experiments,which revealed that the carrier material has low cytotoxicity and good blood compatibility.The prepared HPAA-RVG/si RNA with good targeting ability as well as the capacity to penetrate the blood-brain barrier is expected to provide a new strategy for precise gene therapy of neuroblastoma.