Preparation And Properties Of Ultra-pH-Sensitive Micelles Mediated Enhanced Uptake And Therapeutic Efficacy By DePEGylation

Drug delivery system(DDS)has been widely studied in antitumor,which could effectively reduce and improve the toxicity of small molecule drugs,such as nanoparticles,nano-micelles,nanogel,liposomes,etc.These nanocarriers have high drug capacity by loaded,the blood circulation stability,etc.,and could effectively enrich in the tumor tissues,and have killing effect on tumor cells.Based on the tumor tissue and intracellular environment,more and more stimuli-responsive nanocarriers have been designed and prepared.For example,redox-responsive nanocarriers and pH-responsive nanocarriers are the hot topics in drug delivery.The stimuli-responsive PEGylated nanoparticles can be stable in the blood circulation,avoid being cleared by the reticuloendothelial system(RES),and "hidden" in the blood,but the stimulation of responsive PEGylated nanoparticles has the disadvantage of reducing cellular uptake and the like.Therefore,it is necessary to design a PEGylated nanoparticle that can maintain stability in the blood circulation and enhance tumor cell uptake as well as anti-tumor effect.In this paper,the main work is to design and prepare a pH-supersensitive micelle that enhances cellular uptake by dePEGylation.Polyurethane(PUAL-g-NH2)was used as the hydrophobic backbone and grafting hydrophilic orthoesters(OEMPEG)on the side chains.The amphiphilic copolymer PUAL-g-OEMPEG with side-chain orthoester was prepared.The structure of the copolymer and the micelle properties were characterized.The main work of this article includes the following:.(1)The copolymer PUAL-g-NH2 were prepared by the polycondensation reaction under milder conditions,,and the hydrophilic orthoester OEMPEG was prepared by transesterification reaction.Finally,different proportions of OEMPEG were grafted onto the reactive groups of the copolymer PUAL-g-NH2 to obtain PUAL-g-OEMPEG copolymers.The structure of the product and the molecular weight of the polymer were determined by nuclear magnetic resonance(1HNMR)and gel permeation chromatography(GPC).The result shows that the structure of the polymer PUAL-g-OEMPEG was correct,and the molecular weight(Mn)of the polymer PUAL-g-OEMPEG was 3.91 x 104,3.25 x 104,and 2.74 x 104,respectively.(2)The amphiphilic PUAL-g-OEMPEG copolymer were utilized to self-assemble into micelles in water.Then the critical micelle concentration(CMC)of micelles was determined by fluorescence spectrophotometer using Nile Red and calculated to be 0.8-9 × 10-4mg/mL,respectively.And smaller CMC values indicating that PUAL-g-OEMPEG graft copolymer forms micelles with high propensity,and high stability in blood circulation.Characterization of micelles that the results showed that the size was 155-240 nm,and the morphology of micelles is spherical-like.In vitro degradation studies showed that the micelles were stable in the physiological condition(pH=7.4),and the degradation was complete at pH = 6.5 and in 24 hours and 12 hours,respectively.At pH 6.5,the size of micelles initially becoming small and then larger.It shows that the hydrolysis rate of ortho ester side chain was pH-dependent and time-dependent,and rapid dePEGylation could be achieved at pH=6.5.And the dynamic change of micellar sizes that because of de-pegylation.(3)Preparation of doxorubicin(DOX)loaded micelle with PUAL-g-OEMPEG copolymer.In vitro drug release studies showed that the release rates of the three drug-loaded micelles different because their hydrophilicity,but both had faster drug release rates at pH 5.0 and 6.5.Cytotoxicity of blank micelles and drug-loaded micelles at different pH was determined by cytotoxicity test(MTT).The results showed that the blank micelles were non-toxic and drug-loaded micelles showed concentration-dependent killing.In addition,compared with normal conditions,DOX-loaded micelles were more cytotoxic due to dePEGylation when mimicking the tumor extracellular acid environment(pH=6.5).Confocal laser scanning microscopy(CLSM)and flow cytometry was used to evaluate the qualitatively and quantitatively cellular uptake of DOX-loaded micelles towards human hepatic cancer cell line(HepG2)and human neuroblastoma cancer cell line(SH-SY5Y),respectively.The results showed that drug-loaded micelles at pH = 6.5 could achieve rapid dePEGylation and enhance the cellular uptake of both HepG2 and SH-SY5Y cells.And construction of 3-D multicellular spheroids to evaluate antitumor effects of PUAL-g-OEMPEG-DOX micelles.The results showed that the DOX-loaded micellar osmosis in a time-dependent manner,and the three DOX-loaded micelles showed different osmotic efficiency because different grafting rates,but both were superior to the DOX.And DOX-loaded micelle could well inhibit the growth of multicellular spheres.In conclusion,the ultra-pH-sensitive micelle,which are based on dePEGylation and dynamic change of particle size can effectively improve the uptake of cells and have great potential in drug delivery and anti-tumor.