Design,Synthesis And In Vitro Anti-Tumor Evaluations Of Tumor Microenvironment-Responsive Polymeric Micelles

Polymeric micelles with core-shell architecture self-assembled from amphiphilic or double hydrophilic copolymers have been widely used as delivery vehicles in biomedicine.The hydrophobic core region serves as a reservoir for hydrophobic drugs,meanwhile the hydrophilic shell region stabilizes the hydrophobic core and effectively enhances the stability of the carrier during the blood circulation,therefore endowing advantages of long circulation and stealth to avoid clearance by the reticulo endothelial system(RES).Furthermore,the hydrophilic shell can be modified by various chemical methods with molecules or groups for active targeting to enhance the therapeutic effect.Benefiting from their unique merits,polymeric micelles have been extensively explored in targeted drug delivery systems for tumors.However,in order to efficiently transport drugs to targeted locations,polymeric micelles must navigate a series of barriers,including both extracellular and intracellular barriers.Therefore,there is an urgent need to develop tumor microenvironment-responsive polymeric micelles to achieve precise targeted drug delivery for specific tumor cells,controllable drug release inside tumor cells,improved therapeutic effects and reduced toxic side effects of chemotherapy.Focusing on the scientific problem of“how to efficiently and accurately achieve the precise as well as controlled synthesis of tumor microenvironment-responsive polymeric micelles”,in the first part of the doctoral thesis,we developed a versatile route toward reduction-sensitive block copolymers based on the reversible addition-fragmentation chain transfer-atom transfer radical polymerization(RAFT-ATRP)double-head agent.Specifically,we designed and prepared well-defined triple-responsive polymer prodrugs with reducible disulfide block junctions,and clarified the versatility of this route.The self-assembly,degradation,in vitro drug release,and cellular uptake of the micelle prodrugs were investigated in detail.Finally we explored the possible decoration of the hydrophilic shell using highly efficient click chemistry.Meanwhile,focusing on the problem of toxic and side effects caused by non-specific recognition induced by exposed folic acid(FA),we further designed and prepared polymeric micelles with a dual-functional sheddable monomethoxypolyethylene glycol(PEG)shell,which can be detached at the weakly acidic pH 6.5 of tumor microenvironment to expose FA targeting ligand for endocytosis regulated by the folate receptor.Whereas,once the carrier enters the tumor cell,the further detachment of hydrophilic segments trigger the destaibilization of the self-assembled micelles for accelerated drug release and efficient chemotherapy.The compare between the stability of benzoic-imine and hydrazone bond,as well as the size of self-assembly,in vitro drug release,in vitro cytotoxicity,and cellular uptake behavior in different physiological environments were investigated.The details are listed as follows,1.RAFT polymerization,removal of RAFT group,subsequent ATRP,and final drug conjugation using the reduction-responsive RAFT-ATRP double-head agent,CPADB-SS-iBuBr,were conducted to prepare the target DHBC-based triple-responsive polymeric prodrug,Alkyne-poly(N-(2-hydroxypropyl)methacrylamide-st-(ethylglycinatemethacrylamide-doxorubicin))-SS-poly(N-isopropylacrylamide)(Alkyne-P(HPMA-st-(EGMA-DOX))-SS-P(NIPPAm)),with both reduction-responsive disulfide link in the backbone and acid-sensitive hydrazone links in the side chain to conjugate doxorubicin(DOX).The thermo-induced association behaviours,stability and destabilization of the self-assembled micelle prodrugs were investigated by Dynamic light scattering(DLS).The results indicate that the prodrug can self-assembled into core-shell micelles with an average diameter of 180 nm in water at the physiological temperature of 37 ~oC,whereas the intracellular simultaneous cleavage of the disulfide links and hydrazone bonds led to significantly promoted degradation of the micelle prodrugs.To validate the acidic-pH-and reducing-triggered drug release,in vitro DOX release behaviors were investigated in four different media,i.e.,pH 7.4,5.5,pH 7.4 with 10 mM GSH and pH 5.5 with 10 mM GSH at 37°C.And we conclude that the acidic and reducing environments resulted in the fastest drug release due to the synergistic effect.Finally,thecytotoxicityandcellularuptakeefficiencyof Alkyne-P(HPMA-st-(EGMA-DOX))-SS-P(NIPPAm)as well as reduction-insensitive P(HPMA-st-(EGMA-DOX))-b-P(NIPPAm)micelle prodrugs were assessed by MTT cell viability assay and FCM analysis,respectively,and the results revealed that the reducible polymer backbone of Alkyne-P(HPMA-st-(EGMA-DOX))-SS-P(NIPPAm)micelle prodrugs contributes to the greater in vitro cytotoxicity and cellular uptake efficiency of this construct.Finally,we explored the possible decoration of the terminus of the PHPMA block using highly efficient click chemistry.2.An amphiphilic statistical copolymer,poly(oligo(ethylene glycol)monomethyl ether methacrylate)-st-poly(2-hydroxyethyl methacrylate-g-lactide)(P(OEGMA300)-st-P(HEMA-g-LA))was first synthesized by ATRP using Alkynyl-bis(hydroxymethyl)propinoicacid-SS-bromoisobutyryl(Alkynyl-bis-MPA-SS-iBuBr)as the initiator.Next,the mPEG shell containing weak acidic-pH-sensitive benzoic-imine and hydrazone bond were linked to P(OEGMA300)-st-P(HEMA-g-LA))via a highly efficient click coupling to produce the target polymer,imine-mPEG-P(OEGMA300)-st-P(HEMA-g-LA)and mPEG-P(OEGMA300)-st-P(HEMA-g-LA),respectively.The stability of benzoic-imine and hydrazone bond at pH 7.4(the typical extracellular pH)was investigated by ~1H NMR analysis,and we conclude that benzoic-imine bond in imine-mPEG-P(OEGMA300)-st-P(HEMA-g-LA)is completely cleaved under physiological conditions,whereas the hydrazone bond in mPEG-P(OEGMA300)-st-P(HEMA-g-LA)provide sufficient stability.The association behaviours of P(OEGMA300)-st-P(HEMA-g-LA)and a series of mPEG-P(OEGMA300)-st-P(HEMA-g-LA)in PB(pH 7.4)solution were investigated by DLS.The results revealed that the mPEG linkage is capable of stabilizing the assembly,and when the degree of HEMA-g-LA is fixed,appropriately reducing the weight fraction of hydrophilic OEGMA will further improve the stability of the micelles.Through the investigate in this chapter,the mPEG shell decorated with hydrazone bond were chosen for the next chapter.3.A panel of statistical copolymers,P(OEGMA300)-st-P(HEMA-g-LA),with three different hydrophilic weight fractions was synthesized by ATRP using Alkynyl-bis-MPA-SS-iBuBr as the initiator.FA was next conjugated via a disulfide link to the terminus of the above statistical copolymer by DCC coupling,and finally,the mPEG stealth containing a tumor acidic pH-cleavable hydrazone bond was linked to FA-P(OEGMA300)-st-P(HEMA-g-LA)via a highly efficient click coupling to produce the target polymer construct,mPEG(FA)-P(OEGMA300)-st-P(HEMA-g-LA).The association behaviours of mPEG(FA)-P(OEGMA300)-st-P(HEMA-g-LA)in PB(pH 7.4)solution were investigated by DLS.The results revealed that P1 micelles with the shortest length of OEGMA units showed the greatest stability among the three micelle constructs.Further,the stability and destabilization of P1 micelles were investigated by DLS,and we conclude that comparing to the typical extracellular pH(7.4),tumor microenvironment pH(6.5),endosomal/lysosomal pH(5.0)and reducing environment(10 mM dithiothreitol(DTT))countributing to the cleavage of the hydrazone bonds and disulfide links led to promoted degradation of the P1 micelles.Furthermore,in vitro DOX release behaviors confirmed acidic-pH-and reducing-triggered the deshielding of mPEG stealth countributing to the destaibilization of drug-loaded P1 micelles for accelerated drug release.The cytotoxicity of drug-loaded P1 micelles were assessed by MTT cell viability assay at pH 7.4 and 5.0.The results revealed that the drug-loaded P1 micelles in acidic environment have greater cytotoxicity.Finally,the cellular uptake efficiency of drug-loaded P1 micelles were assessed by FCM analysis and confocal microscopy at pH 7.4,6.5 and 5.0.We confirm that the deshielding of the mPEG stealth at the weakly acidic pH of tumor site can expose FA targeting ligand that is shielded in the physiological condition for active targeting,and trigger the destaibilization of the self-assembled micelles for accelerated drug release.Meanwhile,reducibley conjugated FA via a disulfide link can further promote the cellular uptake efficiency of the micelles by recovery of Folate receptor(FR)and continuous FR-mediated endocytosis,leading to enhanced therapeutic effect.