Preparation Of Self-assembled Nanoparticles Of Cholesterol Conjugated Carboxymethyl Curdlan And The Study Of Their Potential As Novel Carriers Of Epirubicin

In this study, we synthesized novel polymeric amphiphiles by cholesterol conjugated carboxymethyl curdlan (CCMC) and prepared its self-assembled nanoparticles. Epirubicin (EPB) was chosen as a model drug to assess the potential of CCMC self-assembled nanoparticles as a carrier for anti-cancer drugs, the loading capability and release behavior were studied. The in vitro anti-tumor effects and cell toxicity of EPB-loaded CCMC self-assembled nanoparticles were studied by cell culture. The in vivo pharmacokinetics and biodistribution studies were investigated by animal experiments. The main contents and results are as follows:A series of CCMC conjugates with different degrees of substitution (DS) of the cholesterol moieties were synthesized and their phychemical properties were characterized by couductometric titration, fourier transform infrared (FT-IR), proton nuclear magnetic resonance (1H NMR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and high performance liquid chromatography (HPLC). The degree of substitution (DS) of cholesterol moiety was 2.3,3.5 or 6.4 cholesterol groups per hundred sugar residues of CM-curdlan determined by high performance liquid chromatography (HPLC) method. CCMC self-assembled nanoparticles were prepared by probe sonication and dialysis in distilled water and their characteristics were analyzed by transmission electron microscopy (TEM), dynamic laser light scattering (DLLS), Zeta-potential and fluorescence spectroscopy. TEM observation demonstrated that CCMC self-assembled nanoparticles were roughly spherical in shape, the mean diameter of CCMC self-assembled nanoparticles were in the range of 144-233 nm, which depending on the DS value. The mean diameter of self-assembled nanoparticles decreased as the DS increasing. The zeta potentials of CCMC self-assembled nanoparticles were very negative in distilled water, which indicated the negatively charged carboxyl groups of CMC covered nanoparticles. The critical aggregation concentration (CAC) values of the CCMC conjugates defined as the threshold concentration of self-assemble formation of polymeric amphiphiles by intra-and/or intermolecular association depends on the DS value of cholesterol, and the CAC values decrease with an increase in the DS of cholesterol.EPB was chosen as a model drug, and we used the remote loading method to physically entrap it into CCMC self-assembled nanoparticles in this study. The drug loading contents increased with the weight ratio of EPB to CCMC self-assembled nanoparticles increasing, while the drug entrapment efficiency decreased from 78.0% to 65.6% at the same time. Transmission electron microscopy (TEM) showed that EPB-loaded CCMC self-assembled nanoparticles were almost spherical in shape and had a coarser surface compared with the blank CCMC nanoparticles. We believed this is because some EPB was adsorbed to the surface of nanoparticles. Dynamic laser light scattering (DLLS) showed that the mean diameter of EPB-loaded CCMC self-assembled nanoparticles increased from 294.4 to 510.4 nm with the increase of EPB contents. In vitro release studies showed that EPB release from EPB-loaded CCMC self-assembled nanoparticles was sensitive to the pH of the release media as well as the drug loading contents, and the release rate decrease with the pH of the release media and the drug loading contents increase.The cellular cytotoxicity and cellular uptake were accessed by using Human Cervix Carcinoma (HeLa) cells. The blank CCMC self-assembled nanoparticles did not show any cytotoxic effect on HeLa cells even at the concentration of 100μg/mL. However, the EPB-loaded CCMC self-assembled nanoparticles and the free EPB showed anti-tumor activity at the drug concentration of 0.1 to 10μg/mL in vitro. EPB-loaded CCMC self-assembled nanoparticles exhibited slightly more cytotoxic to HeLa cells at low EPB concentrations (<1μg/mL), while the inhibitory effects of EPB-loaded CCMC self-assembled nanoparticles increased more rapidly with drug concentration increased from 1 to 10μg/mL compared with the free EPB. In addition, the cellular cytotoxicity increased significantly with the cell incubation time increases. Flow cytometry and confocal image anlysis revealed that the free EPB was mainly distributed within the nucleus, while the EPB-loaded CCMC self-assembled nanoparticles showed a broader distribution within the cells. The cellular uptake of EPB-loaded CCMC self-assembled nanoparticles was better than that of free EPB.The in vivo pharmacokinetics and biodistribution were investigated in Wister rats and revealed that the EPB-loaded CCMC self-assembled nanoparticles showed a much longer circulation time and sustained release effect in rats, which may implied reduced side effects and thus have clinical significance. The EPB-loaded CCMC self-assembled nanoparticles significantly changed the tissue biodistribution of the original drug which greatly reduced the systemic toxicity of EPB while helped to bypass glomerular filtration and was finally eliminated after the formulation yielded to metabolism in the liver. The results of in vivo anti-tumor efficacy of free EPB and EPB-loaded CCMC self-assembled nanoparticles indicated that both free EPB and EPB-loaded CCMC self-assembled nanoparticles could inhibit tumor growth obviously compared with the control group on day 14 after administration, in particular, the EPB-loaded CCMC self-assembled nanoparticles showed better anti-tumor activity as the time going compared with free EPB, with a significant difference on day 14 after administration. This maybe attributed that the EPB-loaded CCMC self-assembled nanoparticles could reach tumor site through EPR effect and maintain the effective therapeutic concentration for a long period of time.In short, cholesterol conjugated carboxymethyl curdlan derivatives could from nanoparticles by self-assembled manner, the method of prepaing nanoparticles was simple and feasible. CCMC self-assembled nanoparticles can be used as anti-tumor drug carrier of both amphipathic and hydrophobic drugs that sustain the release effect and reduce the systemic toxicity and is expected as a new carrier for anti-tumor drugs delivery.