| objective: The purpose of this study was to develop and demonstrate a novel drug delivery system for a sustained and targeted delivery of honokiol(HK) to the nasopharyngeal carcinoma HNE-1 cell lines, and investigate the anti-tumor mechanism.Methods: Folate has been employed to modify amphiphilic copolymerpoly(ε-caprolactone)- poly(ethylene glycol) - poly(ε-caprolactone) (PCL-PEG-PCL,PCEC) through introducing amidogen of Polythylenimine (PEI) as a targeting vehicle (PCEC-PEI-FA) of various anti-cancer agents to increase their cellular uptake within nasopharyngeal carcinoma HNE-1 cell lines. Then, self-assembly emulsion-solvent evaporation method was used to develop the active targeting nanoparticles loaded HK (ATNH) with the PCEC-PEI-FA. Transmission electron microscopy and Fourier transform infra-red (FTIR) spectrum were employed to study the shape and the construction, respectively. In vitro cell culture studies were carried out in HNE-1 cell lines, a human nasopharyngeal carcinoma cell line, with the folate receptor was over-expressed on its surface,were treated with the ATNH. And nanoparticles size distribution, morphology,drug loading, drug release profile and anticancer activity in vitro were studied in detail. The cytotoxicity of nanoparticles loaded HK was determined by a 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay.The apoptosis was evaluated by flow cytometry. The anti-tumor activity of drug-loading NPs was evaluated in nude mice bearing HNE-1 tumors. The in vivo synergistic effect of the ATNH was observed in a HNE-1 xenograft model.The antitumor efficacy was detected by analysis of tumor growth delay and median survival time. Micro fluorine-18-deoxyglucose PET/computed tomography (18F-FDG PET/CT) was performed to assess early tumor response to ATNH. Apoptosis and analysis of cell cycle redistribution by flow cytometry,Ki 67 and CD 31 expression by immunohistochemistry was performed to evaluate ATNH mechanism of action.Results: The ATNH were obtained by self-assembly emulsion-solvent evaporation in water, and achieved a hydrodynamic diameter about of 188.34±0.54nm. HK release from the nanoparticles exhibited no initial burst but showed a sustained release profile. The results of MTT and fluorescence microscopy demonstrated that the ATNH had superior cytotoxicity against HNE-1 cells compared to free HK, and that they exerted this effect by folate receptor (FR)-mediated endocytosis. The percentage of apoptotic cells in the ATNH group was 86.07±4.95%. The in vivo studies demonstrated that compared with the control group (the tumor growth day and the median survival time of the HK group were 8 days and 34 days respectively ) and NATNH treatment groups (the tumor growth day and the median survival time were 18 days and 42.5 days respectively), the ATNH group has a better treatment efficacy with a tumor growth day of 24 days and median survival time of 57.7 days (p < 0.01). In the 18F-FDG PET/CT imaging, the T/M values are significant differences in the ATNH group 1.03±0.42 (P<0.01).Immunohistochemistry showed the Ki 67 positive cells in the ATNH group 37.24±3.89% (P<0.01). In addition, immunohistochemistry showed the CD 31expression level that in the ATNH group 2.16±0.76 (P < 0.01). A remarkable increase of cells in Gi phase in ATNH group 51.24±3.95% (P <0.01).Conclusion: The FA modified active targeting nanoparticles loaded HK (ATNH)showed gently release and target uptake by HNE-1 cell. Apoptosis and cytotoxicity studies confirmed that the ATNH generated higher cytotoxicity than free HK on HNE-1. Moreover, drug-loading ATNH treatment effectively inhibited tumor metabolism and growth in BALB/c nude mice bearing HNE-1 cell xenografts, and prolonged the survival time of mice. The HK-loaded nanoparticles inhibit the tumor through suppression of the metabolism and causing cell apoptosis, arresting cell cycle at G1 phase and decreasing the Ki 67 and CD 31 expression. |
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