Studies On Antitumor Activities Of Drug Delivery Systems Using Stearic Acid Grafted Chitosan Oligosaccharide Copolymer Micelles

During the past decade there has been a growing interest in polymeric micelles as a potential carrier for anticancer drug delivery.Polymeric micelles have core-shell structure composing of hydrophobic segments as the internal core and hydrophilic segments as a surrounding corona in aqueous medium.The internal hydrophobic core provides a storeroom for loading hydrophobic drug.The hydrophilic shell allows the stability of polymeric micelle in aqueous environment,and the active targeting to tumor cells can be reached by further ligand or antibody modification.The nanoscale dimensions of polymeric micelles permit the efficient accumulation in tumor tissue via the enhanced permeability and retention(EPR) effect,which termed as passive targeting.Comparing with traditional micelles of low-molecular-weight surfactant,polymer micelles are generally more stable with a relatively lower CMC,and show slower dissociation and enhanced stabilization in aqueous environment.However,polymeric micelles are a dynamic system,the shell of the core-shell architecture serves as a membrane-like "gate" to control the escape or import of drug.In fact,interactions between drug and core domain of polymeric micelles rely on the delicate balance of relatively weak interactions,such as hydrophobic,hydrogen-bonding,and electrostatic.As a result,polymeric micelles have a limitation as a drug delivery system due to the initial burst drug release or dissociation after dilution by the body fluids.Stearic acid grafted chitosan oligosaccharide copolymer(CSO-SA) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)-mediated coupling reaction of stearic acid(SA) and low-molecular-weight chitosan(CSO,Mw=15.0 KDa).Substitute degrees of amino groups(SD%) means the graft number of SA groups per 100 amino groups of CSO,which was measured by TNBS method.The critical micelle concentration(CMC) of CSO-SA in water was determined by fluorescence measurement using pyrene as a probe. Aggregation number of stearate groups per CSO-SA molecules was estimated by steady-state fluorescence quenching method.Size and Zeta potential of CSO-SA micelles were measured by dynamic light scattering.To control drug release,the shells of CSO-SA micelles were cross-linked by glutaraldehyde,and the effects of glutaraldehyde amount and reaction time on the micelle size were determined by dynamic light scattering.To terminate the crosslink of CSO-SA micelles by glutaraldehyde,glycine and sodium bisulfate were selected as termination agent.Cellular uptake percentage of CSO-SA micelles and shell crosslinked CSO-SA micelles in A549,LLC,and SKOV3 cells were carried out.Doxorubicin alkali (DOX) was chosen as typical anticancer drug.Size and Zeta potential of CSO-SA micelles before and after loading DOX were determined.The drug encapsulation efficiency(EE%), drug loading(DD%),release profiles,and cellular uptake percentage of CSO-SA/DOX and shell crosslinked CSO-SA/DOX were further investigated.Utilizing the carboxyl group of folic acid(FA) and remained amino groups of CSO-SA,the FA modified CSO-SA (FA-CSO-SA) was obtained.The properties of FA-CSO-SA micelles were determined,and the shell crosslinked FA-CSO-SA micelles were prepared.A549(FR=(-)),LLC(FR=37%) and SKOV3(FR=93%) were chosen as typical tumor cells,the cellular uptakes of FA-CSO-SA against the three cell lines were estimated.The properties of DOX loaded micelles were evaluated,and the cytotoxicities of FA-CSO-SA/DOX in the three cells were also determined.Male C57BL/6 mice were implanted with B16 cells(melanoma cells) and LLC cells(Lewis lung carcinoma cells) in the right armpit,respectively.The antitumor activities of CSO-SA/DOX and FA-CSO-SA/DOX with different drug loading on tumor-bearing mice were explored.The tumor volume was measured for calculating the rate of inhibiting tumor.In this study,low molecular weight chitosan with 15.0 KDa wieitht average molecular weight was obtained by enzymatic hydrolysis which well soluble in water.CSO-SA was then synthesized by the coupling reaction of SA and CSO.The substitute degrees of amino groups (SD%) was determined as 3.48%,and the CMC value of CSO-SA was determined to be 0.035 mg·mL^-1 in DI water.Aggregation number of hydrophobic microdomain per CSO-SA molecule was 2.8 under 1.0 mg·mL^-1 CSO-SA concentration.The mean number diameter of CSO-SA micelles with 1.0 mg·mL^-1 was 35.0 nm and the Zeta potential was 57.1±1.5 mV.The shell of CSO-SA micelles was cross-linked by glutaraldehyde.It was clear that the higher glutaraldehyde amount(n_CSO-SA:n_glu=1:20) induced the size increase during reaction due to the Schiff's reaction between micelles.In the lower glutaraldehyde amount(n_CSO-SA: n_glu=1:2.5),no obvious size change was found.In the middle glutaraldehyde amount(n_CSO-SA: n_glu=1:5,1:10,1:15),the particle size and Zeta potential were reduced.Using A549,LLC, and SKOV3 cells as model tumor cells,the cellular uptake of CSO-SA micelles and shell crosslinked CSO-SA micelles were carried out.It was found the cellular uptake of CSO-SA micelles and shell crosslinked CSO-SA micelles were increased with incubation time,and the cellular uptake percentage of micelles in LLC and SKOV3 cells were higher than that of micelles in A549 cells.There were no significantly differences in cellular uptake percentage between CSO-SA micelle and shell crosslinked micelle with 1:5 of n_CSO-SA:n_glu.The drug encapsulation efficiencies of DOX loaded micelles were higher than 90%.The cellular growth inhibition experiment was carried out.When DOX molecules were encapsulated into the micelles of CSO-SA,the cytotoxicity of CSO-SA/DOX was much greater than free DOX, and the effect against SKOV3 was higher than that on lung cancer cells,which might be due to the poor cellular uptake of free DOX in SKOV3 cells.Moreover,the cytotoxicity of crosslinked CSO-SA/DOX was much greater than that of CSO-SA/DOX.It might be attributed to the significant reduction of drug leakage before DOX-loaded micelles being internalized into cells.Modification of FA promoted self-assembly of FA-CSO-SA in water,the aggregation number of hydrophobic microdomain per FA-CSO-SA was more than CSO-SA.It was found that cellular uptakes of FA-CSO-SA in LLC and SKOV3 cells which with higher FA receptor expression were faster than that in A549 in the short incubation time,but the differences decreased as the incubation time prolonged.There were no significantly differences on the properties of drug loaded between CSO-SA micelles and FA-CSO-SA micelles.Drug release in vitro from FA-CSO-SA/DOX was slower than CSO-SA/DOX.The cytotoxicity of FA-CSO-SA/DOX was much greater than free DOX,but there were no obviously differences between CSO-SA/DOX and FA-CSO-SA/DOX.It was due to the nearly same cellular uptake percentage of FA-CSO-SA and CSO-SA at longer incubation time.There were no significantly differences in cellular uptake percentage between FA-CSO-SA micelle and shell crosslinked one.The shell crosslinked FA-CSO-SA/DOX could further enhance the cytotoxicity of DOX due to the reduced drug loss in diluting process.The antitumor actitivies results of DOX preparation in mice bearing B16 and LLC showed that the inhibitory rate of preparations were higher than positive control.When dosage was reduced to 1/5 of positive control,preparations also indicated higher inhibitory rate.The antitumor activities of all preparations against B16 and LLC were similar.FA modified preparations showed higher inhibitory rate than that of unmodified preparations.The antitumor activity of ED50 was nearly same with positive control of ED100,1/5 dosage was higher than that of unmodified preparations.