Preparation And In Vitro Evaluation Of Enzyme/pH Dual-Response Polymer Prodrug Micelles Based On 10-HCPT

10-Hydroxycamptothecin(10-HCPT)is a small molecule drug with high anticancer effect.However,it has some shortcomings,such as poor water solubility,instability of lactone E-ring,short plasma half-life,low bioavailability,gastrointestinal reaction and bone marrow suppression,which greatly limit the further use of 10-HCPT in clinical application.In order to enhance the stability and anti-cancer effect of the drug,the 20-hydroxyl group of 10-HCPT was directly bonded with pH/enzyme sensitive succinic anhydride(SA).Then,grafted onto natural macromolecule carboxymethyl chitosan(CMCS),and the pH/enzyme-responsive polymeric prodrug micelles based on 10-HCPT structure were obtained by ultrasonic dispersion.The preparation process is simple,which can bind to the target site without adding hydroxyl protection reagent.In addition,unlike common enzyme-sensitive strategies,the pH/enzyme-sensitive polymeric prodrug micelles belong to ester trigger-response,and cathepsin shows ester bond-responsive,which is benefit to the design of the experiment.PH/enzyme dual-response polymeric prodrug nanoparticles based on HCPT-g-CMCS are promising nanodrug delivery systems for cancer treatment.The main work and conclusions are as follows:1.Preparation and characterization of 10-hydroxycamptothecin polymeric prodrug:Firstly,10-HCPT-SA was prepared from 10-HCPT and succinic anhydride.Then,10-HCPT-SA was grafted onto the backbones of CMCS by amidation reaction to obtain 10-hydroxycamptothecin polymeric prodrug(HCPT-g-CMCS).The structures of the intermediate and the target compound were determined by ~1H-NMR,FT-IR,mass and UV.2.The self-assembly behavior,morphology,storage stability,plasma stability,Serum stability and pH/enzyme sensitivity of 10-hydroxycamptothecin polymeric prodrug micelles:The results of critical aggregation concentration(CAC)of pcrodrug mielles showed that the higher the grafting ratio was,the easier the micelles aggregate.TEM showed that the prodrug micelles were spherical with obvious core-shell structure and uniform size.After 30 days of storage and 48 hours of plasma incubation,the particle sizes of the polymeric micelles before and after treatments were slightly larger,which can be negligible,indicating that the micelles had good storage stability and plasma stability.The results of pH and papain sensitivity experiments showed that the polymeric prodrug micelles were more sensitive to tumor cells than normal ones.3.In vitro release of pH/enzyme daul-responsive polymeric prodrug micelles:In vitro release of the polymeric prodrug micelles,the results showed that the prodrug micelles were stable under pH 7.4 conditions,and the acumulative release of drugs increased slightly even papain was added.However,under the condition of pH5.0,HCPT-g-CMCS tends to form poly-core nanogel,which showed higher release rate in 2.0?M papain compared with 0.2?M papain.Under the same conditions(pH5.0 PBSwith 2?M papain),the higher the grafting rate is,the more cumulative drug release from the micelles.4.Cytotoxicity assay,cell uptake and internal release of pH/enzyme dual-responsive polymeric prodrug micelles:Cell inhibition of polymer prodrug micelles was determined by using 4T1 cells and normal cell s(L-O2)as models.The experimental results showed that the blank carrier was non-toxic to both cells.But in cancer cells,nanoparticles containing10-HCPT at the same dose showed higher toxicity than that of free 10-HCPT,while in normal cells,it showed lower toxicity.Cell uptake experiments showed that micelles could release drugs rapidly in cancer cells and continuously release the drug,but slowly release in healthy cells.