Preparation And In Vitro Evaluation Of PH/Enzyme Dual-response Polymer Prodrug Micelles Based On Melphalan

To improve hydrolytic stability and biological half-life and anticancer efficacy of the anti-cancer drug melphalan(Mel),a novel esterification strategy was developed to fabricate a p H/enzyme dual-responsive polymer prodrug(m PEG-Mel)via the formation of an ion bond between the carboxyl group in Mel and diazotized p-aminobenzoate-terminated poly(ethylene glycol)monomethyl ether(m PEG-N2~+)followed by light-induced conversion of the ionic bond to an ester bond.The prodrug could self-assemble into nanomicelles.The main work and conclusions are listed below:1.Preparation and characterization of the polymer prodrugs(m PEG-Mel):The synthetic route of the amphiphilic Mel polymer prodrug(m PEG-Mel)could be divided into three parts.Firstly,m PEG reacted with Ts-Cl to synthesize m PEG-Ts;Next,m PEG-Ts reacted with PNBC to prepare m PEG-NH2.Finally,the m PEG-NH2was diazotized and subsequently reacted with Mel under ultraviolet light to obtain m PEG-Mel prodrug.The structure was characterized by infrared,nuclear magnetic,ultraviolet and gel permeation chromatography.The percentage of the drug was determined by high performance liquid chromatography.The results showed that the percentage of Mel decreased as the molecular weight of m PEG increased.2.Preparation of polymer prodrug nanomicelles,and study their morphology,stability,long cycle and p H/enzyme sensitivity:The critical micelle concentration(CMC)result indicates that with higher percentage of Mel,it is easier for nano-micelles to form.At p H 7.4,the micelles exhibited a regular core-shell structure with a particle size of about 100 nm.And the nanomicelles exhibited good storage stability and shear stability,and the hydrolysis of the Mel alkylated part of the micelle was significantly suppressed.In addition,mimicking the physiological environment,the interaction between micelles and plasma was weak,and micelles may have the ability of long circulation.When the p H dropped to 5.0,a significant increase in the size of the nanomicelles was observed and increased with the increase of cathepsin B(CTB)concentration.In the presence of2.0μg/m L CTB,the size of the nanomicelles reached 675 nm or more.3.In vitro release of enzyme/p H-responsive polymer prodrug micelles:Nanomicelles showed the least drug release(less than 10%in 24 hours)at p H7.4;however,drug release can be significantly accelerated in CTB-containing media at p H 5.0.In the presence of 2.0μg/m L CTB,the cumulative release of nano-micelles was up to 71.50%in 128 hours.Both p H/enzymes can synergistically promote the drug release.4.Cytotoxicity experiments,cell uptake and intracellular release of enzyme/p H-responsive polymer prodrug micelles:Cell experiments show that the semi-inhibitory concentration(IC50)of nanomicelles is 1.00μg/m L,which is about 5 times lower than free Mel(5.00μg/m L),indicating that nanomicelles are more cytotoxic to cancer cells(4T1 cells).However,a significantly reduced cytostatic effect was observed for normal cells(3T3cells).In addition,cell uptake experiments with Nile Red(NR)-loaded nanomicelles further supported the above experimental results,and stronger red fluorescence was observed in 4T1 cells,which means that nanomicelles can respond to p H/enzyme dual stimulate and release the drug.Thus,m PEG-Mel based p H/enzyme double-response polymer prodrug nanoparticles could effectively enhance drug stability and anticancer efficacy.