Preparation And Study Of Glutathione-response Nano-drug Delivery System Based On Polymeric Prodrug

6-mercaptopurine（6-MP）is the main drug for the maintenance treatment of children with acute lymphoblastic leukemia.However,its poor water solubility（about180μg/mL）,short plasma half-life（0.5-1.5 h）,variable bioavailability（about 16%）,and accompanied by severe bone marrow suppression and liver toxicity and other shortcomings greatly limited its application in clinic.In order to improve its shortcomings in clinical application,in this paper,we prepared a kind of glutathione（GSH）-sensitive long-circulation micelles which based on 6-MP prodrugs.Different from the commonly used disulfide-based GSH-sensitive design,herein,the GSH-sensitive strategy is based on a Michael addition-elimination reaction.That is,the amphiphilic polymeric prodrug which containsα,β-unsaturated carbonyl group acts as a Michael acceptor to receive the attack of nucleophile-GSH,and undergoes elimination reaction to release the original drug.Furthermore,the polyethylene glycol shell helps the polymeric prodrug micelles to obtain stealth properties in the body.The main works and results are summarized as follows:Ⅰ.Preparation and characterization of amphiphilic polymeric prodrugs of 6-MP:Firstly,the small prodrug PTA was prepared by 6-MP and propiolic acid through a reaction of Michael addition.Then,PTA was conjugated onto the terminal hydroxyl of mPEG by esterification reaction to obtain amphiphilic prodrug PTA-mPEG.The chemical structures of PTA and PTA-mPEG were characterized by IR and¹H NMR.Ⅱ.Polymeric prodrug micelles were prepared from PTA-mPEG,and the morphological characteristics,critical micelle concentration（CMC）,storage stability and GSH sensitivity of the polymeric prodrug micelles were also studied.TEM images showed that the polymeric prodrug micelles had spherical core-shell morphology and a relatively uniform size distribution.The critical micelle concentration（CMC）experiment results indicated that the polymeric prodrug micelles had low critical micelle concentration values(4.50×10^-33 mg/mL for PM-1;2.72×10^-2 mg/mL for PM-2)which mean the micelles had the ability to withstand the dilution of body fluids as well as maintaining the structural integrity.That is,the polymeric prodrug micelles possessed good dilution stability.Nano ZS90 was adopted to measure the mean diameter（MD）and polydispersity index（PDI）of polymeric prodrug micelles before and after storage for 15 days.By comparing the data,the values of MD and PDI changes a little which suggest the high storage stability.GSH sensitivity experiment results showed that the polymeric prodrug micelles were more sensitive in millimolar level GSH than that in micromolar level GSH.Ⅲ.The drug release kinetic study of GSH response polymeric prodrug micelles:Investigate the GSH response drug release kinetics of polymeric prodrug micelles.The result showed that the 6-MP release was GSH concentration dependent.Compared with the stimulation of micromolar GSH,the drug release rate of polymeric prodrug micelles was much higer than that in milimolar GSH.Ⅳ.The long-circulation experiment of polymeric prodrug micelles:HL-60 cells were chosen as the leukemia cells to study the in vitro cytotoxicity of polymeric prodrug micelles.And the study results suggested that the cell inhibition was 6-MP dose-dependent.The higher the dose led to the greater inhibition rate to cells.The cellular uptake result indicated that the PMs were internalized by HL-60cells and the 6-MP could be released from the PMs in thecellular.Murine monocyte macrophage cell J774 A.1 was adopted as the simulation circumstance of organism immune clearance system.Study results suggested that the polymeric prodrug micelles had a more pronounced long-circulation ability compared with 6-MP-CMCS（6-mercaptopurine-carboxymethyl chitosan which previously studied by our group）nanoparticles in vitro.What’s more,the PM-2 with a longer chain of PEG shows a smaller phagocytosis rate relative to PM-1,indicating a more excellent long-circulation performance.