Preparation Of Self-aggregated Nanoparticles Of Cholesterol-bearing Pullulan Derivatives And The Primary Study On Using Them As The Novel Carriers Of Drugs

Polysaccharides are highly stable,safe,non-toxic,hydrophilic,biodegradable, abundant resources in nature and low cost in their processing.Moreover,there are various derivable groups on molecular chains,so that polysaccharides can be easily modified and result in many kinds of derivatives by chemically and biochemically.Due to above outstanding merits,polysaccharides and their derivatives have received more and more attention in the area of drug delivery systems.Pullulan has many advantages as a macromolecular drug carrier,e.g.highly water-soluble,non-toxic,multiple hydroxyl groups that can readily be modified chemically,lacks immunogenicity,and usefulness as a plasma expander.Cholesterol has the hydrophobic property due to the presence of hydrophobic moieties such as cyclopentenophenanthrene nucleus in its molecule. Succinic acid is a natural compound existing in the human body which can be used as a linker between hydrophobic groups and hydrophilic polysaccharides skeleton.Therefore, using succinic acid,small endogenous molecule of the body's metabolic process,replace the exogenous 1,6-hexyl diisocyanate.This provides a new strategy for the effective and safe synthesis cholesterol-modified pullulan.It also is of great significance to further expand its application.Based on above information,we developed a new method to synthesize CHSP or CHS-CMP by directly grafting proper cholesterol residues onto pullulan or O-carboxymethyl pullulan.The main content of this research are shown as follows.(1) Cholesterol-modified pullulan(CHSP) conjugate with succinyl linkage was synthesized and characterized by fourier transform infrared(FT-IR),proton nuclear magnetic resonance(~1H NMR),and X-ray diffraction(XRD).The degree of substitution (DS)of cholesterol moiety determined by ~1H NMR ranged from 3.87 to 5.70 cholesterol groups per hundred glucose units.The CHS-CMP was also synthesized by using cholesterol succinate reacted with O-carboxylmethyl pullulan,and the intermediates and pullulan derivatives were characterized by FT-IR and ~1H NMR.The synthesis of FITC-labeled CHSP was reacted CHSP and FITC catalyzed by DBTDL.The DS of FITC grafted onto CHSP was characterized by NMR and fluorescence spectrophotometry.(2) CHSP self-aggregated nanoparticles were prepared by probe sonication in aqueous media and analyzed by dynamic laser light-scattering(DLS),zeta potential, transmission electron microscopy(TEM) and the fluorescence probe technologies.These novel nanoparticles were almost spherical in shape,and their size,ranging from 51.8 to 73.0 nm,could be controlled by DS of cholesterol moiety.The zeta potentials of CHSP self-aggregated nanoparticles were near zero in aqueous media.The value of critical aggregation concentration(cac) was dependent on the DS of cholesterol moiety. FITC-labeled CHSP self-aggregated nanoparticles with D_h about 50 nm can be obtained by a dialysis method.(3) Mitoxantrone(MTO) was loaded into the CHSP nanoparticles by dialysis method.MTO-loaded CHSP self-aggregated nanoparticles were almost spherical in shape and their size increased from 153.1 to 174.2 nm with the MTO-loading capacity increasing from 4.35%to 14.29%.The encapsulation efficiency(EE) of the process and loading capacity(LC) of the nanoparticles increased with increasing cholesterol DS. XRD powder patterns showed that crystal peaks of MTO disappeared when MTO was entrapped into CHSP nanoparticles.DSC experimental results also proved the same conclusions.The release behavior of MTO from CHSP self-aggregated nanoparticles was studied in vitro.The results showed that the release behavior of MTO from CHSP nanoparticles exhibited a sustained release,and MTO release rate decreased with the pH increase of the release media.Epirubicin(EPI) was also physically entrapped inside the CHCS self-aggregated nanoparticles by remote loading method,and the EE and LC of EPI-loaded CHSP nanoparticles appeared rather lower than those of MTO-loaded CHSP self-aggregated nanoparticles.(4) The study of cell cytotoxicity in vitro showed CHSP self-aggregated nanoparticles had no antitumor activity even the CHSP concentration at 200ug/mL. However,whether MTO loaded CHSP or EPI loaded CHSP nanoparticles showed antirumor activity when adjusted the drug concentration at 1～10μg/mL.Moreover,the cytotoxicity of the MTO or EPI loaded nanoparticles was higher than free drug with the same concentration.The result of flow cytometry and confocal image ananlysis revealed that EPI loaded CHSP self-aggregated nanoparticles appeared much easier cellular endocytic than free EPI.The analysis of intracellular distribution of FITC-labeled CHSP self-aggregated nanoparticles in HeLa cell was carried out,and the result showed that the FITC labeled nanoparticles accumulated in cell membrane firstly,and then the nanoparticles passed through cell membrane and arrived at cellular nuclear with incubation time prolonged.EPI loaded FITC-CHSP nanoparticles cell entry also confirmed by the same way.(5) The EPI loaded CHSP nanoparticles and free drugs were injected intravenously to wistar mice respectively,and the preliminary study about Pharmacokinetics was carried out.The concentration of EPI at different times in blood was detected by high performance liquid.The result showed that the EPI-loaded CHSP nanoparticles achieved long-circulating and sustained-release effect.