Construction Of Stimulus-responsive Hybrid-core Polymeric Nanoparticles Based On Monomethoxy(Polyethylene Glycol)-b-P(D,L-lactic-co-glycolic Acid)-b-P(L-glutamic Acid)

In the biomedical filed,existing studies related to amphililic polymer mainly focus on the biodegradable and biocompatible PEGylated poly(lactide-co-glycolide)s(PLGA)and poly(amino acids)(PAA),both of which have been widely used as micelle-forming materials.However,either the low drug loading capacity or bad stability for these polymeric micelles limits their application to drug delivery.Based on these three blocks,hydrophilic mPEG,hydrophobic PLGA and functional PAA,a type of novel triblock copolymer,mPEG-PLGA-PAA,is promising to overcome these problems.Therefore,a type of novel triblock copolymers,mPEG-b-PLGA-b-Poly(L-glutamic)s(mPEG-PLGA-PGlu),have been designed and synthesized in the study,and the relevant studies including their self-assembling process,physiochemical properties and potential application as a useful drug-delivery vehicle have been also conducted carefully.In the study,firstly,series of diblock copolymers,mPEG5k-PLGA,were synthesized by the classical opening-ring polymerization using mPEG5k-OH(k denoted 1000Da)as initiator Then,a series of triblock copolymers,mPEG-PLGA-poly(?-benzyl glutamate)(mPEG-PLGA-PBZL),were synthesized via the ring-opening polymerization(ROP)of 5-Benzyl L-glutamate N-carboxyanhydride(NCA)initiated by amino-terminated mPEG5k-PLGA in anhydrous DCM.Finally,the mPEG5k-PLGA-PGlu was synthesized through removal of the y-benzyloxy end-group from mPEG5k-PLGA-b-poly(?-benzyl glutamate).The resulting polymer at every step of synthetic process was characterized by FT-IR,1H-NMR and GPC analysis.The critical aggregate concentrations(CAC)of two types of amphiphilic copolymers,mPEG5k-PLGA and mPEG5k-PLGA-PGlu,were determined using pyrene fluorescence probe.Results shown that its CAC was largely dependent on the hydrophobic length of PLGA block,the longer PLGA,lower its CAC,and triblock copolymer exhibited slightly higher CAC than that of corresponding diblock copolymer.The self-assembled morphologies of series of mPEG5k-PLGA-PGlu copolymers were studied using transmission electron microscope(TEM).TEM photographs suggested that mPEG5k-PLGA20.5k-PGlu7.9k could be self-assembled into negatively charged nanoparticles with a hybrid core of PLGA and PGlu,and a PEG shell,which was confirmed by 1H-NMR results of the solvated-nanoparticles in D2O.The pH sensitivity of mPEG5k-PLGA20.5k-PGlu with different PGlu length was investigated using transmittance method.The pH-transition range for mPEG5k-PLGA20.5k-PGlu7.9k is very narrow,pH6 to 5,which is comparable with that of cellular endolysosomes.Further,the pH sensitivity of mPEG5k-PLGA20.5k-PGlu7.9k was due to the pH-dependent helix-coil transition of PGlu segment,which was confirmed by circular dichroism technique.Besides,the degradation behavior of mPEG5k-PLGA20.5k-PGlu7.9k nanoparticles in presence of proteinase k was examined using GPC technique.Results shown that the polymeric backbone was rapidly degraded into multiple polymeric debris after incubation with proteinase k.Hydrochloride doxorubicin(DOX)as a model drug could be easily loaded into the mPEG5k-PLGA20.5k-PGlu7.9k nanoparticles prepared by dialysis method.The effect of variable factors including drug/copolymer ratio,types of copolymers and relative length of PGlu in mPEG5k-PLGA-PGlu copolymer,on drug-loaded nanoparticles were investigated in term of drug loading capacity(DL),entrapment efficiency(EE),particles size(Dh)and particle size distribution(PDI),and Zeta potential.Remarkably,when the ratio of drug/copolymer increased,the actual DL increased,to a maximum of 25%,and the EE was left largely unchanged at 80-97%for the triblock copolymer,mPEG5k-PLGA20.5k-PGlu7.9k.Inversely,as the ratio of drug/copolymer increased,the dynamic diameter(Dh)and Zeta potential of drug-loaded nanoparticles decreased,and finally remained minimum at the maximum drug loading level.Finally,an optimum formulation was established by using mPEG5k-PLGA20.5k-PGlu7.9k as optimum copolymer and fixing the ratio of drug/copolymer at 1:5.The high DOX-loaded nanoparticles(DOX-NPs)prepared by this optimum formulation have the average size of 107.2nm,Zeta potential of-17.54 mV,DL of 14.75%and EE of 90.12%.DOX-NPs exhibited a pH-dependent and enzyme-sensitive release profile in vitro.Within 72 h in pH 7.4 PBS,70%of the incorporated DOX was released from the NPs.However,a rapid release occurred in pH 5.0 acetate buffers,and more than 70%of entrapped DOX was released after incubation for 24h.In the presence of proteinase k,a rapid drug release was observed;about 80%and 60%of entrapped DOX was released in pH 5.0 and pH 7.4 medium within 6h,respectively.When HP-?-CD(1%,w/v)as a stabilizer was added to the aqueous DOX-NPs suspension for freeze-drying,the dried DOX-NPs power were easily reconstituted,having less than 10 nm increasing in average size after reconstruction with water.Then,the in vitro cytotoxicity of the DOX-NPs against human breast cancer MCF-7 and drug-resistant MCF-7/ADR was studied.Against MCF-7 cells,DOX-NPs exhibited the better cytotoxicity than DOX-S,having the IC50 value been 2-3 orders of magnitude lower than that for free DOX solution.Against MCF-7/ADR cells,more obvious cytotoxicity of DOX-NPs were observed,exhibiting IC50 value 3-4 orders of magnitude lower than that of DOX-S.Time-dependent cellular uptake of the DOX-NPs was observed and at least 4 hours was required for rapid internalization by MCF-7 cells.DOX-NPs were endocytosed into cytoplasm by caveolin-mediated endocytosis and macropinocytosis by MCF-7/ADR cells,far beyond the reach of trans-membrane localized drug efflux pumpsFinally,the in vivo evaluations of DOX-NPs including the pharmacokinetics,the anti-tumor efficacy,tissue distribution and toxicity were conducted in animals.The results of the pharmacokinetic study showed that the AUC0-t,AUC0-? and t1/2? for DOX-NPs were 2.65-,3.13-,and 8.68-times higher than those of free DOX solution,and CL and Vz was 3.19-and 1.91-times lower,indicating its longer circulation time and the slower clearance.DOX-NPs significantly reduced the drug distribution in the heart,liver,spleen,lung,and kidney of tumor-bearing nude mice at 2h following injection and produced a 2-fold increase in the drug distribution in the tumor at 8h compared with free DOX solution Serum toxicity-relevant enzyme analysis indicated that DOX-NPs exhibited no organ toxicity,but free DOX solution produced clear cardio-toxic effects reflected by increase in LDH and CPK at a DOX dose of 10mg/kg.