Study Of Hydrophobized Polysaccharides And Their Folate Conjugates As Nano-drug Delivery Carriers

In present study,natural polysaccharides were hydrophobized and conjugated with folic acid to provide tumor targeted drug delivery carriers.The paper includes two parts as following:1.Study of pullulan acetate and its folate conjugate as nano-drug delivery carriersPullulan acetate(PA),as hydrophobized pullulan,was synthesized by the reaction of pullulan with acetic anhydride.Then folate was coupled to PA(FPA) by N,N'-Dicyclohexylcarbodiimide(DCC) and 4-Dimethylamino-pyridine(DMAP) mediated ester formation.The products were characterized by FT-IR,~1H NMR spectroscopy and X-ray diffraction(XRD).PA and FPA were insoluble in water, whereas completely soluble in common organic solvents.The solvent diffusion method was used to prepare PA and FPA nanoparticles and investigated various factors to determine the optimization method.The diameters of nanoparticles were affected by degree substitution of acetate,concentration of materials solution,PVA concentration in aqueous phase,organic solvent phase/ aqueous phase ratio and kind of organic solvent. The optimization method was explored.Epirubicin(EPI) was loaded into the nanoparticles as a model drug and investigated various affect factors.The results revealed that drug loading efficiencies of PA nanoparticles increased as the degree of acetate substitution,drug/materials ratio and ethylamine/epirubicin molar ratio.The TEM indicated that PA and FPA nanoparticles could form spherical nanoparticles.The size of nanoparticles was increased with the EPI-loading content increasing.The release behavior of EPI from PA or FPA nanoparticles was studied in vitro by dialysis method and the results showed that total EPI release rate was controlled by the degree substitution of acetate(FPA＞PA1＞PA2＞PA3) and pH value of release medium (pH6.4＞pH7.0＞pH7.4).The analysis of intracellular distribution of EPI was carried out in KB cells. Examination was done with inverted confocal laser scanning microscope.The results revealed that free EPI,PA/EPI and FPA/EPI nanoparticles gained access to the cell,but the route and the kinetics of uptake were difference apparently.Experiments tracking EPI fluorescence indicated that free EPI was rapidly internalized and localized in the nucleus,followed by the FPA/EPI nanoparticles and the last for PA/EPI nanoparticles. Cellular uptake extents of PA/EPI and FPA/EPI nanoparticles were evaluated using flow cytometry.Delivery of EPI by various EPI formulations to KB cells increased continuously with time of exposure.After lh incubation,fluorescence intensity in cells was:free EPI＞PA/EPI NPs＞FPA/EPI NPs＞FPA/EPI NPs + FA.By 4 h,the order of fluorescence intensity was:FPA/EPI NPs≥EPI＞PA/EPI NPs＞FPA/EPI NPs+FA.In addition,for FPA/EPI nanoparticles,more fluorescently labeled cells can be clearly visualized in the absence of folate in the medium than presence of 1mM folate, suggesting FPA/EPI nanoparticles were endocytosed in a folate receptor-mediated manner.The blank PA and FPA nanoparticles showed no significant cytotoxicity in KB and L929 cells by MTT assay.Cytotoxicity of free EPI,EPI-loaded PA and FPA nanoparticles increased with the time of incubation,especially FPA/EPI nanoparticles. The cytotoxicity of FPA/EPI NPs against KB cells was inhibited by excess free folate, which suggested that free folate molecules prevented the cellular uptake of FPA nanoparticles by competitive binding to the folate receptors on the cell surface.The free EPI exhibited a higher cytotoxicity than the EPI loaded PA and FPA nanoparticles against L929 cells for the same concentration of EPI.2.Study of deoxycholic acid-bearing chitosan and its folate conjugate as nano-drug delivery carriersFolate(FA) modified deoxycholic acid-beating chitosan(FA-CS-DA) was synthesized by the reaction of conjugate carboxyl groups of FA and DA to the amino groups of chitosan.The structures were characterized by FTIR,~1H NMR and XRD.The degree of deoxycholic acid(DA) group substitution(DS) was determined by elemental analysis was 9.6,7.7 and 2.8,respectively.An extent of folate conjugation with deoxycholic acid-beating chitosan(FA-CS-DA1,2) measured by spectrophotometric was 170μmol/g polymer and 186μmol/g polymer,respectively.CS-DA and FA-CS-DA self-aggregated nanoparticles were prepared by dialysis method in physiological saline.The critical micelle concentration(CMC) of CS-DA conjugates depended on the DS of deoxycholic acid over a range of 0.015mg/ml～0.046 mg/ml,and that FA-CS-DA1 and FA-CS-DA2 conjugates were 0.028mg/ml and 0.049mg/ml,respectively.The mean diameters of CS-DA self-assembled nanoparticles were 115.7nm～196.5nm,and the mean diameters of FA-CS-DA were about 200 nm～300nm.All trans-retinoic acid(ATRA) efficiently loaded into CS-DA and FA-CS-DA nanoparticles up to 12 wt%using ultrasonication method.The size of diameter increased with the increasing of EPMoaded content.Cell uptake studies were carried out in KB cells using fluorescein isothiocyanate labeled nanoparticles.The nanoparticles were taken up to the cells were increased with the time of incubation for 0.5～2h.The levels of FA-CS-DA nanoparticles were higher than those of CS-DA nanoparticles.The association of FA-CS-DA nanoparticles to KB cells was inhibited by an excess amount of folic acid,suggesting that the binding and/or uptake were mediated by the folate receptors.In conclusion,these hydrophobized polysaccharides and their folate conjugates could form nanoparticles by self-aggregate manner.The methods of preparing nanoparticles were simple and feasible.The self-aggregate nanoparticles could be used as drug delivery carriers of both amphipathic and hydrophobic drugs,which could control release of drugs and protect them.The folate conjugate nanoparticles were taken up by KB cells mainly mediated by folic acid receptor,which indicated that folate conjugated nanoparticles offer considerable potential for employment as cancer-targeted carriers for the efficient delivery of anticancer drugs.