Stimuli-responsive Polypeptide Micelles For Incellular Anti-cancer Drug Delivery

Recently,polymeric micelle has gained tremendous interests for scientists and medical workers due to its high delivery efficiency and less side effects.However,there are still challenges on the study of polymeric micelles as following:1)poor in vivo circulation,2)the in vivo stability of nanoparticles,3)poor biocompatibility of polymer,and 4)non-responsiveness in the cellular microenvironment.Therefore,to solve above problems,a novel polymeric drug delivery system with controllable drug release,improved bio-stability,and good biocompatibility should be established.According to previous studies,polyamino acid is generally used as a hydrophilic shell to stabilize nanoparticles,which not only has good biocompatibility,but also can be degraded in vivo.In this experiment,the chemical structure,nanoscale morphology and drug release properties of polymeric micelle were studied.Main contents of the experiment are listed as follows:?1?A novel stepwise pH-sensitive and biodegradable polypeptide hybrid terpolymer,poly?lysine-co-N,N-Bis?acryloyl?cystamine-co-dimethylmaleic anhydride??PLBD?was designed and synthesized to achieve efficiently cellular internalization and significantly promote the delivery of doxorubicin?DOX?.The PLBD could self-assemble into spherical nano-micelles?NMs?with narrow distributions and elicited stepwise responses to extracellular and intracellular pH condition.The as prepared NMs showed a negative surface charge under physiological conditions,which could protect the NMs from the attack of plasma proteins and recognition by the reticuloendothelial system,exhibiting advantageous stability during blood circulation.By the first-step pH response,the surface charge of the NMs transferred from negative to positive in acidic medium to enhance cellular uptake under the slightly acidic tumor extracellular environment.After internalization into tumor cells,the second-step pH response resulted in a disassembly of the NMs due to the further protonation of the polymer back bone in the endo/lysosome acid environment of the cancer cell.Additionally,a rapid drug release was triggered in response to the intracellular reductive environment of tumor cells via the destruction of disulfide-linked polymer chains to enhance the nuclear delivery of the DOX.In vitro cell assays showed that the blank NMs showed negligible systemic toxicity against normal cells while the DOX-loaded NMs significantly inhibited growth of the cancer cells,suggesting that the developed stepwise pH-sensitive and biodegradable PLBD-based NMs would be a smart and promising drug delivery candidate for anti-cancer therapy.?2?The synthesis of a charge-reversed cross-linked polymeric micelle.Firstly,?-Benzyl-L-glutamate N-carboxyanhydride?Bz-L-Glu NCA?was prepared utilizing triphosgene and?-Benzyl-L-glutamate.Allylamine was used to initiate the ring opening polymerization?ROP?of Bz-L-GluNCA to prepare allyl-terminated poly??-Benzyl-L-glutamate N-carboxyanhydride??PBLG?in a dry Schlenk flask.?-Benzyloxycarbonyl-L-lysine N-carboxyanhydride?Zlys-NCA?was prepared utilizing triphosgene and?₆-Carbobenzoxy-L-Lysine.Allylamine was used to initiate the ring opening polymerization?ROP?of Zlys-NCA to prepare allyl-terminated PZLL in a dry Schlenk flask.Secondly,cystamine hydrochloride and acryloyl chloride were used to synthesize BACy.The cross-linking reaction of PZLL and PBLG occurs under the initiation of azobisisobutyronitrile?AIBN?.Then,a hydrolysis was induced to prepared the charge-reversed cross-linked polymer.The cross-linked polymeric micelle has good stability due to chemo-stability of the micelles structure.The experimental results showed that the cross-linked polymeric micelle exhibited a spherical structure.And the critical micelle concentration of the cross-linked polymeric micelle was relatively low,indicating that the cross-linked polymeric micelle can provide excellent colloidal stability in very dilute solutions.The particle size distribution at different time points indicates that the cross-linked polymeric micelle can be degraded in the presence of the reducing agent.The drug release results showed that the cumulative release of cross-linked polymeric micelle in phosphate buffer was less than that in the buffer containing GSH,and it was also found that the release rate in the acetic acid buffer?pH=5.0?was higher than that in the phosphate buffer?pH=7.4?.Protein absorption and cytotoxicity experiments showed that the cross-linked polymeric micelle have good biocompatibility,and efficient cancer cell killing effect.In brief,the experimental results showed that the as prepared cross-linked polymeric micelle exhibited dual response characteristics of reduction and pH responsiveness,which can achieve long-term in vivo circulation and high cell uptake.?3?The synthesis of a multiple-stimuli-responsive cross-linked polymeric micelle,the chemical structure,morphology,and size of the cross-linked polymeric micelle were characterized.The experimental results indicated that the hydrodynamic size of the polymeric micelle were about 100 nm,and the as prepared polymeric micelle could be degraded and swelled in presence of reducing glutathione?GSH?.The low critical solution temperature?LCST?of the polymeric micelle was around39.4?.According to the experimental results,the polymeric micelle will shrink at temperature above the LCST.Subsequently,the accumulative drug release rate was up to 91.78%under acidic?pH 5.0?,reductive?GSH 10 mmol/L?and high temperature?42.0??conditions mimicking the tumor microenvironment,while a relatively low release rate of 1.12%was observed without stimulation.The drug-conjugated cross-linked polymeric micelle showed a strong cell uptake behavior.In the cytotoxicity assay,the polymeric micelle exhibited effective anti-cancer activity and excellent biocompatibility.In brief,the experimental results showed that the as prepared cross-linked polymeric micelle exhibits multiple stimuli-responsiveness,which holds great promise for anti-cancer drug delivery.