| In order to transport the drug to the molecular target in the cells, the antivirus drug delivery system using polymeric micelle with glycolipid-like structure for the treatment of hepatitis B virus infection was prepared. Moreover, due to the intracellular ability, the nanostructured lipid carrier (NLC) was prepared to increase the degree of the uptake of antivirus drug and markedly enhance the curative effects. This study tried to explore totally inhibiting the virus replication, reduce the side effects of antivirus drugs and evade the resistant occurrence, which provides the academic foundation for highly effective and safety antivirus treatment.To increase lipophilicity of antiviral model drug (Lamivudine, LA), the prodrug of LA, Lamivudine stearate (LAS) was synthesized via ester linkage between LA and stearic acid in the presence of DCC and DMAP. The crude compound was purified by column chromatography method over silica gel, and the structure of LAS was confirmed by melt point (mp), IR, NMR and ESI-MS. The yield was 34.2%. The mp of LAS was 115-117℃, and the molecular weight of LAS was 495. The concentration of LA and LAS was determined by high-performance liquid chromatography (HPLC) method with gradient elution. The octanol-water partition coefficients (log P) of LA and LAS were determined to be-0.95 and 1.82 by shake-flask method. The hydrolysis of LAS in PBS medium with different pH followed apparent first-order kinetics, and the hydrolyzing rate enhanced with the decreasing pH. Furthermore, in the simulative in vivo condition, the hydrolysis of LAS also followed apparent first-order kinetics and the half-life was 0.32 h.The chemical conjugate of stearate-grafted chitosan oligosaccharides (CSO-SA) was synthesized by coupling carboxyl group of stearic acid (SA) and amine group of chitosan oligosaccharides (CSO) with 18.0 kDa molecular weight in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and identified by’H-NMR. The substitution degree (SD) of CSO-SA was 3.79%, which was determined by the 2,4, 6-trinitrobenzene sulfonic acid (TNBS) method. CSO-SA copolymers could self-assemble into micelles in the aquous medium, and the critical micelle concentration of CSO-SA micelles was 0.32 mg/mL, which was determined by fluorescence measurement using pyrene as a probe. The average size and zeta potencial of CSO-SA micelles determined by Zetasizer were 460.8 nm and +29.7 mV respectively, and the diameters of the CSO-SA micelles increased with decreasing pH of the aquouse medium. The surface morphology of CSO-SA micelles in deionized water (DI water) was observed by an atomic force microscopy. It was found that surface morphology of CSO-SA micelles represented sphericity, and the diameter was 94.01 nm. HBV transfected human hepatoblastoma cells (HepG2.2.15) were chosen as model cells. By MTT assay, the 50% cellular growth inhibition (IC50) of CSO-SA micelles with treatment for 48 h and 72 h against HepG2.2.15 cells was 750μg/mL and 680 p.g/mL, respectively. The CSO-SA was labled with fluorescein isothiocyanate (FITC), and the intracellular uptake of fluorescent micelles (FITC-CSO-SA) was observed under a fluorescence microscope. CSO-SA micelles possessed the ability of rapid internalization into cells. With the incubation time prolonged, the uptake amount of CSO-SA micelles increased.LAS loaded CSO-SA micelles (CSO-SA/LAS) were prepared by dialysis method. The entrapment efficiency (EE%) and drug loading (DL%) of CSO-SA/LAS was determined by ultrafiltrate centrifugation and extraction with organic solvent. It was found that the EE% of CSO-SA/LAS was all over 97%, and the DL% could be as much as 39.04%. The average size and zeta potencial of CSO-SA/LAS micelles determined by Zetasizer were 270-300 nm and above +30 mV, respectively. However, the diameters of the CSO-SA/LAS micelles didn’t change a lot in the aquouse medium with different pH. The surface morphology of CSO-SA/LAS micelles in DI water represented sphericity, which was observed by an atomic force microscopy. The size distribution was homogeneous, and the diameter was about 168nm. In vitro LA release from CSO-SA/LAS micelles was carried out by dialysis method. It was clear that the release rate of LA from the micelles was retarded compared with that of free LAS and was pH dependent. The release rate was enhanced with the decreasing pH of the dissolution medium, and decreased with the increasing DL%. The stability of LAS encapsulated in CSO-SA micelles was enhanced compared with that of LAS in PBS medium, and the degradation of LAS encapsulated in CSO-SA micelles was markedly retarded from 0.32 h to 9.36 h in the simulative in vivo condition. HepG2.2.15 cells were chosen as model cells. By MTT assay, the cytotoxicity of CSO-SA/LAS micelles was quite low. When the concentration of CSO-SA micelles was 0.1 mg/mL, the cell survival rate was 70%-86%. The intracellular uptake of fluorescent micelles (FITC-CSO-SA/LAS) was observed under a fluorescence microscope. CSO-SA/LAS micelles possessed the ability of rapid internalization into cell. Through the fragmentation of cells, the intracellular concentration of LAS was determined at predefined time. After encapsulated in CSO-SA micelles, the highest uptake percentage of the drug was elevated from 4.02% to 65.64%. The in vitro anti-HBV activities of CSO-SA/LAS micelles on HBV-induced HBsAg, HBeAg and DNA productions were investigated against HepG2.2.15 cells. It was revealed that the inhibitory activities of CSO-SA/LAS micelles were enhanced dose-dependently and time-dependently, and the inhibitory effects of LAS were obviousely elevated after incorporated by CSO-SA micelles.Monostearin and oleic acid (OA) were chosen as solid lipid material and liquid lipid material for the nanosrtructured lipid carrier (NLC), and LAS was used as model drug. The LAS loaded NLC (NLC/LAS) was prepared by the solvent diffusion method in an aqueous system. The EE% and DL% of NLC/LAS was determined by filtration method and extraction with organic solvent. It was found that the EE% of NLC/LAS was all over 98%, and the DL% could be as much as 20.29%. When the DL% was 4.85-20.29%, the average size and zeta potencial of NLC/LAS nanoparticles determined by Zetasizer were 358-432 nm and below-30 mV, respectively. The surface morphology of blank NLC nanoparticles and NLC/LAS nanoparticles represented sphericity, which was observed by transmission electron microscopy (TEM). The size distribution was homogeneous, and the diameter was about 25-75 nm. In vitro LA release from NLC/LAS nanoparticles was carried out by dialysis method. It could be seen that the release rate of LA from the nanoparticles was retarded compared with that of free LAS. Moreover, the release rate was enhanced with the increasing content of OA, and decreased with the increasing DL%. HepG2.2.15 cells were chosen as model cells. By MTT assay, the blank NLC nanoparticles and NLC/LAS nanoparticles possessed low cytotoxicities. The conjugate of octadecylamine-fluorescein isothiocyanate (ODA-FITC) was synthesized by the reaction between amino group of ODA and isothiocyanate group of FITC. To prepare FITC labled nanoprticles, ODA-FITC was used instead of the same amount of monostearin. The intracellular uptake of fluorescent nanoparticles (blank FITC-NLC and FITC-NLC/LAS) was observed under a fluorescence microscope. NLC/LAS nanoparticles had the ability of internalization into cells and cytoplasm targeting. With the increasing concentration of nanparticles and incubation time, the uptake amount of nanoparticles augmented. Through the fragmentation of cells, the intracellular concentration of LAS was determined at predefined time. After encapsulated in NLC nanoparticles, the highest uptake percentage of the drug was elevated from 4.02% to 54.27%. The in vitro anti-HBV activities of NLC/LAS nanoparticles on HBV-induced HBsAg, HBeAg and DNA productions were investigated against HepG2.2.15 cells. It was found that the inhibitory activities of NLC/LAS nanoparticles were enhanced dose-dependently and time-dependently, and the inhibitory effects of NLC/LAS nanoparticles were much stronger than free drugs. |
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