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Effective Tumor-targeted Delivery Of Etoposide Using Chitosan Nanoparticles Conjugated With Folic Acid And Sulfobetaine Methacrylate

Posted on:2017-02-22Degree:MasterType:Thesis
Country:ChinaCandidate:S HuaFull Text:PDF
GTID:2284330488956177Subject:Radiation Medicine
Abstract/Summary:PDF Full Text Request
Objective: We demonstrated the chitosan-based biocompatible nanoparticles coated with folic acid(FA) and poly(sulfobetaine methacrylate)(PSBMA) as an effective tumor-specific drug delivery system. Etoposide could be incorporated into the hydrophobic inner core of the nanoparticles and exerted promising tumor-targeting effects and therapeutic efficacy both in vitro and in vivo..Methods: 1.Poly(sulfobetaine methacrylate) is grafted onto chitosan(CS) by reversible addition-fragmentation chain transfer(RAFT) polymerization under high dose γ-ray irradiation. 2. FA was grafted onto CS-g-PSBMA by the reaction between amino grounp and carboxyl grounp on the condition of catalysis of EDC and NHS. 3.FA-CS-g-PSBMA was characterized by Fourier transform infrared(FT-IR) spectra, 1H nuclear magnetic resonance(NMR) spectra, dynamic light scattering(DLS) and field-emitting scanning electron microscopy(SEM). 4. Human dermal fibroblast(HDF)cells were used to investigate the cytotoxicity of FA-CS-g-PSBMA by MTT assays. 5.FA-CS(VP-16)-g-PSBMA nanoparticles were prepared and then the drug loading efficiency and encapsulation efficiency were measured by using UV/visible spectroscopy. 6. Hela cells were used to investigate the cytotoxicity of VP-16,CS(VP-16)-g-PSBMA and FA-CS(VP-16)-g-PSBMA by MTT assays. The confocal laser scanning microscope and flow cytometer were used to monitor the cellular uptake of FITC-labeled CS(VP-16)-g-PSBMA and FA-CS(VP-16)-g-PSBMA. The apoptosis of Hela cells treated with VP-16 in different formulations was detected by flow cytometer.7.Cy5.5-labeled nanoparticles were injected via the tail vein into SD rat, blood samples were collected from SD rat at different time points. The Cy5.5 fluorescence intensities in serum were measured by using a microplate reader. 8. The targeting efficiency of Cy5.5-labeled nanoparticles in subcutaneous tumor-bearing nude mice were evaluated by the In-Vivo Fx Pro imaging system and the tumor size of mice was used to evaluatedthe treatment efficacy of VP-16 and FA-CS(VP-16)-g-PSBMA nanoparticles.Results: 1.The result of 1H NMR spectrum and FT-IR spectra suggested that PSBMA and FA were successfully grafted onto chitosan. The diameter of FA-CS-g-PSBMA was 163.9 ± 32 nm with narrow size distribution. After loading of VP-16, the diameter of nanoparticles was swelled to 200.5 ± 17 nm while maintaining their spherical shapes. In addition, the Zeta potential values of FA-CS-g-PSBMA and FA-CS(VP-16)-g-PSBMA were-6.7 ± 2.31 m V and-5.36 ± 1.8 m V in PBS(p H = 7.4).2. The payload of VP-16 in nanoparticles is high up to 27% determined by gravimetry.The in vitro release study indicated that VP-16 release rate was much faster in p H 6.4than in p H 7.4. 3. Confocal laser scanning microscope and flow cytometer indicated the uptake amount of FA-CS(VP-16)-g-PSBMA was higher compared to CS(VP-16)-g-PSBMA. MTT assay showed that VP-16 treatment decreased the cell viability in a dose-dependent manner. The cell viability of CS(VP-16)-g-PSBMA-treated group was equivalent to that of VP-16. In accordance with the amount of the internalized nanoparticles, FA-CS(VP-16)-g-PSBMA exerted the highest cytotoxic effects when He La cells were treated the nanoparticles containing the corresponding amount of VP-16. The results of cell apoptosis were consistent with MTT.4. Both nanoparticles showed similar pattern of retention behavior in blood with a half-life of nearly 10 h calculated by a one-compartment open model. 5. In vivo distribution of Cy5.5-labeled nanoparticles suggested that FA-CS(VP-16)-g-PSBMA had a better tumor targeting ability than CS(VP-16)-g-PSBMA. The antitumor assay in vivo revealed that FA-CS(VP-16)-g-PSBMA performed enhanced anti-tumor efficacy than VP-16.Conclusions: In this study, a novel amphiphilic polymer FA-CS-g-PSBMA was synthesized for the targeting delivery of the hydrophobic anti-cancer drug VP-16. FA modification nanoparticles FA-CS(VP-16)-g-PSBMA showed a good anti-tumor efficacy both in vitro and in vivo. FA-CS-g-PSBMA nanoparticles hold the promising prospects in the area of targeted tumor therapy.
Keywords/Search Tags:Chitosan, Folic acid, Nanoparticles, Active targeting, Hela cells
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