Synthesis of folate modified chitosan-based nanomicelles and its anti-tumor activity / 浙江大学学报·医学版

OBJECTIVE@#To design and synthesize folate-modified pH-responsive chitosan-based nanomicelles and investigate the anti-tumor activity of the drug-loaded micelles.@*METHODS@#CHI-DMA was obtained by reductive amination reaction of aldehyde-based chitosan and hydrophilic amine compounds, and CHI-DMA-LA was obtained by condensation reaction with lauric acid; FA-CHI-DMA-LA was obtained after modification with folic acid (FA). The drug-loaded nanomicelles FA-CHI-DMA-LA/DOX were assembled by solvent change method. The physicochemical properties of polymers were characterized by hydrogen nuclear magnetic resonance and transmission electron microscope. The particle size and surface potential were determined by dynamic light scattering method. Folic acid access rate, doxorubicin (DOX) loading rate and entrapped efficiency were measured by UV-vis spectrophotometer. The drug release properties of DOX-loaded micelles were monitored by fluorescence spectrophotometer at different pHs (7.4, 6.5, 5.0). The cytotoxicity against human oral cancer KB cells was detected by MTT assay. Fluorescence microscope and flow cytometry were applied to investigate the phagocytosis of DOX-loaded micelles on KB cells.@*RESULTS@#FA-CHI-DMA-LA was synthesized. The particle sizes of FA-CHI-DMA-LA-1 and FA-CHI-DMA-LA-2 micelles which used for the subsequent experiments were (73±14) nm and (106±15) nm, zeta potential were (15.59±1.98) mV and (21.20±2.35) mV, respectively. The drug loading rates of drug-loaded micelles FA-CHI-DMA-LA-1/DOX and FA-CHI-DMA-LA-2/DOX are (4.08±1.12)%and (4.12±0.44)%, respectively. drug release is pH-responsive, with cumulative release of DOX up to 37%and 36%at pH 5.0, which is about 1.5 times higher than that of pH 7.4. For FA-CHI-DMA-LA micelles with 1.25 to 125 μg/mL concentration, the survival rate of KB cells is more than 70%after incubation for 24 hours. The cell uptake of FA-CHI-DMA-LA/DOX micelles was enhanced compared to CHI-DMA-LA/DOX, and the cell uptake was higher in incubation without FA medium than that with FA. Compared with free DOX or CHI-DMA-LA/DOX, FA-CHI-DMA-LA/DOX nanomicelles showed higher cyctoxicity to KB cells, especially the FA-CHI-DMA-LA-2/DOX nanomicelles, the cell survival rate was about 17% after incubation for 24 hours.@*CONCLUSIONS@#FA-modified chitosan-based nanomicelle with good biocompatibility was successfully prepared, which exhibits tumor microenvironmental pH responsive drug release and tumor targeting.

[Bacterial outer membrane vesicles as nano carriers to study immunological activities].

Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 µg/mL, 100 µg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.