The Study About Drug Delivery System Based On Pluronic Modified Polymer

PurposeCancer is a serious disease that threatens human life and health.According to statistics,the incidence of cancer in China is close to the world level,but the mortality rate is higher than the world level.Among them,breast cancer is the most common cancer in women worldwide.Currently,chemotherapy is the most common cancer treatment in our society.However,chemotherapy drugs still facing their own limitations in the treatment,such as:poor water solubility,serious side effects,low bioavailability,which limits the clinical application.Therefore,how to improve the therapeutic effect of anti-cancer drugs is particularly important.The development of nanotechnology provides new hope for new anti-tumor drug formulations.The polymer micelle has a hydrophobic core and hydrophilic shell,which formed by self-assembly of amphiphilic polymers in aqueous solutions.Micelles can deliver the poorly soluble drug to tumor sites and increase therapeutic effect of the drug.Pluronic,also known as poloxamer,is a triblock amphiphilic copolymer composed of polyoxyethylene(PEO)-polyoxypropylene(PPO)-polyoxyethylene(PEO),which is accepted as an excipient by the U.S.and British Pharmacopoeia and widely used in clinical applications.In this study,Pluronic F68(PEO80-PPO27-PEO80)and Pluronic F127(PEO101-PPO56-PEO101)were modified into Pluronic F68-Vitamin E succinate polymer(F68-VES)and redox sensitive polymer(F127-SS-TOC),respectively.These polymeric micelles could reduce Pluronic CMC values,improve their anti-dilution capacity and reduce the risk of drug leakage.Besides,the micelles also facilitate high tumor accumulation based on enhanced permeability and retention(EPR)effect.MethodThis research mainly revolves two drug delivery systems,including the synthesis of polymers,the preparation and evaluation of drug-loaded micelles and pharmacodynamic evaluation.1.F68-VES/MIT drug delivery system.(1)We synthesized polymer F68-VES via esterification reaction and verified the structure by 1H NMR and FTIR.(2)F68-VES/MIT micelles were prepared by solvent evaporation method.Then the micellar particle size,Zeta potential and stability were observed by DLS.Besides,the micellar morphology was observed by TEM.The entrapment efficiency and drug loading efficiency were determined by HPLC and the micellar CMC was measured by fluorescence probe method.(3)MTT method was used to compare the cytotoxicity of MIT and F68-VES/MIT micelles in breast cancer cells MCF-7 and MDA-MB-231.(4)The cell apoptosis of MIT and F68-VES/MIT micelles were measured by flow cytometry and the morphology of nucleus was observed by IN Cell Analyzer 2000.(5)The cellar uptake of MIT and F68-VES/MIT micelles in MDA-MB-231 cells to MIT and F68-VES/MIT micelles was assessed by using flow cytometry and autofluorescence of MIT was observed by IN Cell Analyzer 2000.2.F127-SS-TOC/RES redox-sensitive drug delivery system.(1)F127-SS-TOC polymer was synthesized by two steps and characterized by 1H NMR and FTIR.(2)The entrapment efficiency and drug loading efficiency,particle size,Zeta potential,micelle morphology,stability,redox sensitivity,CMC value,in vitro drug release properties,biocompatibility of F127-SS-TOC/RES micelles were evaluated.(3)MTT assay was used to investigate the anti-tumor effect of free RES,F127-SS-TOC/RES micelles and non-redox sensitive micelles F127-TOC/RES on breast cancer cells MCF-7 and MDA-MB-231.(4)In vitro anti-tumor activity of F127-SS-TOC/RES micelles in breast cancer cell MDA-MB-231 was evaluated by the expression of intracellular ROS.Results1.The study about F68-VES/MIT drug delivery system.(1)1H NMR,FTIR and 13C NMR verified that F68-VES polymer was successfully synthesized.(2)F68-VES/MIT micelles have been prepared by solvent evaporation method.The mean particle size(184.33 ± 6.53 nm,PDI=0.06 ±0.031)and Zeta potential(-32.67±0.87 mV)were measured by dynamic laser light scattering(DLS),which conformed to the TEM image.The encapsulation efficiency and drug loading rate of F68-VES/MIT micelles were 91.88±2.20%and 5.85±0.89%,respectively.The critical micelle concentration of micelles was about 3.311 mg/L measured by fluorescence probe method.MIT-loaded F68-VES micelles had sustained release ability,which was beneficial for keeping an effective concentration during the treatment.(3)MTT assay showed that F68-VES/MIT micelles exerted significantly enhanced cytotoxicity on MCF-7 cells and MDA-MB-231 cells.(4)Cell apoptosis showed that free MIT and F68-VES/MIT micelles showed a significant effect with apoptotic rates of 38.9%and 60.8%,respectively.In other words,F68-VES/MIT micelles had a greater effect on inducing cell apoptosis in MDA-MB-231 cells than free MIT.Besides,we found a higher apoptotic rate in the F68-VES/MIT micelles group than in free MIT group through nuclear morphology observation.(5)The cellular accumulation of MIT occurred in a time-dependent manner in both free MIT and F68-VES/MIT micelles treatment groups.Besides,compared with free MIT,there was an increased trend of cellular uptake of F68-VES/MIT micelles in MDA-MB-231 cancer cells.2.The study about F127-SS-TOC/RES drug delivery system.(1)In this study,F127-SS-TOC polymer was successfully synthesized and verified by 'H NMR and FTIR.(2)The encapsulation efficiency and drug loading rate of F127-SS-TOC/RES micelle were 99.09 ± 1.11%and 13.25 ± 2.29%,respectively.The average particle size of the micelles was 39.50 ± 1.39 nm(PDI:0.16 ± 0.01)and the Zeta potential was-4.55 ± 0.39 mV.The CMC value of F127-SS-TOC micelles was 7.58 mg/L,which was lower than the CMC value of F127.The micelles were stable during 7 days,and the particle sizes of F127-SS-TOC/RES micelles containing different FBS concentrations were stable without sharp changes as time increased up to 24 hours.In addition,the obvious reduction sensitivity of the F127-SS-TOC micelles was verified by measuring the size change in the presence of 10 mM DTT.The in vitro drug release profile of F127-SS-TOC/RES micelles indicated that the micelles might be stable under extracellular conditions over a prolonged period with little drug release while exhibited a rapid drug release property in the presence of 10 mM DTT.The hemolytic test showed that the biocompatibility of this polymer was safe.(3)MTT assay showed that F127-SS-TOC/RES micelles could significantly enhanced cytotoxicity on MCF-7 cells and MDA-MB-231 cells compared with free RES and F127-TOC/RES micelles.(4)Furthermore,F127-SS-TOC/RES micelles had a greater effect on inducing cell apoptosis in MDA-MB-231 cells than free MIT and and F127-TOC/RES micelles.(5)The intracellular ROS level of F127-SS-TOC/RES micelles was obviously up-regulated than free free RES and F127-TOC/RES micelles in breast cancer cell MDA-MB-231.ConclusionIn this study,the effects of polymeric micelles on delivery of anti-tumor drug have been explored based on the synthesised amphiphilic block polymers F68-VES and F127-SS-TOC.The properties,in vitro anti-tumor activity and mechanisms of drug-loaded micelles were evaluated by a series of tests,which suggested Pluronic-based polymer carriers have the potential to deliver antitumor drugs,improve the drug concentration in the tumor site,enhance the efficacy and reduce the side effects.