Self-assembled Nanoparticles Based On Oleoyl-chitosan As Antitumor Drug Carriers

In cancer therapy, effective treatment of chemotherapy is often limited by its unfavorable specificity, great side effects and multidrug resistance (MDR). The development of nanotechnology enables nano-drug carriers, which can improve drug solubility, enhance anti-tumor effect and reduce side effects, to be used in chemotherapy. Chitosan (CS) is a positive polysaccharide with good biocompatibility. Hydrophobically modified chitosan-based drug delivery systems show great potential as antitumor drug carriers. In this paper, oleoyl-chitosan (OCS) is synthesized by reacting chitosan with oleoyl chloride. OCS nanoparticles are prepared by an O/W emulsification method and investigated as antitumor drug carriers.CS samples with different molecular weight are prepared by acetic acid hydrolysis. OCS is synthesized by grafting oleoyl onto the-NH2 at C-2 in CS molecule. The FTIR spectrum and 1HNMR spectrum indicate that oleoyl groups are introduced into CS molecule. The degrees of substitution (DS) are 5%,11% and 27%, respectively. OCS nanoparticles are prepared using an O/W emulsification method. The nanoparticles are spherical shape with narrow size distributions. The mean diameters of OCS nanoparticles increased with decreasing of the molecular weight and increasing of the DS.The hemolysis rate of OCS nanaoparticles is less than 5% and is regarded as non-toxic. The amount of bovine serum albumin (BSA) and bovine calf serum (BCS) proteins adsorbed on OCS nanoparticles decrease with increasing of the DS of OCS. The adsorption of proteins on OCS nanoparticles increase with increasing of the incubation time and the concentration of OCS. OCS nanoparticles exhibit no cytotoxicity to mouse embryo fibroblasts (MEF) and show good biocompatibility. The drug loading and release properties of OCS nanoparticles in vitro are investigated by using doxorubicin (DOX) as a model drug. The FTIR spectrum and the characterization of DOX-OCS nanoparticles indicate DOX loaded to OCS nanoparticles. The loading efficiency and loading capacity of OCS nanoparticles with different DS and molecular weight are all higher than 40%, and increase with increasing of the DS and decreasing of the molecular weight. DOX is rapidly and completely released from DOX-OCS nanoparticles at pH 3.8, whereas at pH 6.8 DOX is burst released within 6 hours and followed by a sustained release till 72 hours. At pH 6.8, OCS nanoparticles with different DS and molecular weight show sustained release and the in vitro release rates of DOX decrease with increasing of the DS and molecular weight.The in vitro cytotoxicity of blank OCS nanoparticles, DOX-OCS nanoparticles and DOX to human A549 cells, Bel-7402 cells, HeLa cells, and SGC-7901 cells are analyzed by MTT assay. Blank OCS nanoparticles show no toxicity to cancer cells. With the increase of DOX concentration and extension of incubation time, the inhibitory rates of DOX and DOX-OCS nanoparticles to A549 cells, Bel-7402 cells, HeLa cells, and SGC-7901 cells increased, and DOX-OCS nanoparticles showed better inhibition than DOX. There was a significant or very significant difference between the inhibitory rates of DOX-OCS nanoparticles and DOX, except that there was no significant difference between the inhibitory rates of DOX-OCS nanoparticles and DOX for Bel-7402 cells at a concentration above 5μg/mL and SGC-7901 cells at 10μg/mL. The inhibitory rates of DOX-OCS nanoparticles decrease with increasing of the molecular weight of CS.The in vivo antitumor effect of blank OCS nanoparticles, DOX-OCS nanoparticles and DOX are studied in S180 bearing mice after i.v. injection. The DOX-OCS nanoparticles can obviously inhibited the growth of tumor, and have better inhibition than DOX. Otherwise, the loading of DOX in OCS nanoparticles can decrease the toxicity of DOX to normal tissues and deduce the side effects of DOX.FITC-labeled OCS (FITC-OCS) nanoparticles are prepared for the in vitro cellular uptake and the in vivo tissue distribution tests. The cellular uptake of FITC-OCS nanoparticles by A549 cells and RAW264.7 cells is obviously time-dependent and concentration-dependent. The uptake efficiency of FITC-OCS nanoparticles increases with extension of the incubation time and increasing of the particle concentration, and no saturation is found. It should be emphasized that the cellular uptake of FITC-OCS nanoparticles by A549 cells is higher than that of RAW264.7 cells beyond 2 h of incubation, indicating that OCS nanoparticles is suitable to be a long circulating carrier for sustained release of doxorubicin. The uptake efficiency increases with increasing of the DS.The biodistributions of FITC-OCS nanoparticles are investigated in normal mice after i.v. injection. Distributions of FITC-OCS nanoparticles in liver, spleen and lung are at low levels and in kidney and blood are at high levels. The amount of FITC-OCS nanoparticles distribute in liver, spleen and lung increase with extension of the incubation time while in kidney and blood decrease with extension of incubation time.Based on the above considerations, OCS nanoparticles have good biocompatibility and sustained drug release property, and drug-loaded OCS nanoparticles show better inhibition to cancer cells than DOX in vitro and in vivo, thus are suitable to be antitumor drug carriers.