| Objective and ContentsTumor MDR is the main reason for cancer chemotherapy failure and patients death. Nanotechnology that uses nanomaterials to load chemotherapeutic drugs is an exploratory research for reversing tumor MDR. In the first part, we explored the effects and related mechanisms of ADR loaded pH-sensitive pullulan nanoparticles in reversing the MDR of resistant breast cancer cell MCF-7/ADR.In recent years, graphene oxide (GO) attracts more attention to be used as a novel carrier for the antitumor drug. In the second part, we explored the effects and related mechanisms of ADR loaded GO in reversing the MDR of resistant breast cancer cell MCF-7/ADR. To further evaluate the potential of GO as the drug carrier, we detected the genotoxicity of GO and explored the related molecular mechanisms through genechip technology in the third part.Methods1) We synthesized the urocanic acid modified pullulan derivative (URPA) and prepared pH-sensitive pullulan nanoparticles through self-assembly technology. Antitumor drug (ADR) was physically loaded into URPA nanoparticles and its pH sensitive release behaviors were determined by the dialysis method.2) MTT assay was used to detect the cytotoxicity of ADR-URPA nanoparticles in MCF-7and MCF-7/ADR cells. Flow cytometry was used to determine the drug intakes of MCF-7and MCF-7/ADR cells. Confocal assay was used to assess the introcellular distribution of ADR loading by URPA nanoparticles.3) GO nanosheets were prepared by the sonication method. ADR was loaded onto the surface of GO through the π-π stacking and hydrophobic interactions. ADR release behavior was determined in vitro by the dialysis method.4) MTT assay was used to detect the cytotoxicity of ADR-GO in MCF-7and MCF-7/ADR cells. Flow cytometry was used to determine the drug intakes of MCF-7and MCF-7/ADR cells. Confocal assay was used to assess the introcellular distribution of ADR loading by GO. Immunofluorescence assay was used to explore the action mechanisms of ADR-GO to reverse tumor MDR.5) Micronucleus test was used to detect the genotoxicity of GO. PCR and agarose gel electrophoresis was used to assess the interaction of GO with genomic DNA and the influence of GO on DNA replication. Genechip technology was used to explore the molecular mechanisms of genotoxicity induced by GO. MTT assay was used to assess the cytotoxicity of GO in human breast cancer cell MDA-MB-231. Flow cytometry was used to evaluate the influence of GO on the cell cycle.Results1) Urocanic acid modified pullulan was successfully synthesized by ester linkage; ADR-URPA nanoparticles were prepared and the in vitro release of ADR showed significant pH sensitivity.2) Compared to free ADR, ADR-URPA nanoparticles enhanced the cytotoxicity in MCF-7and MCF-7/ADR cells, especially in MCF-7/ADR cells, which had a significant lower IC50value.3) Compared to free ADR, ADR-URPA nanoparticles significantly increased ADR intakes in MCF-7/ADR cells.4) ADR-URPA nanoparticles significantly enhanced ADR accumulation in MCF-7/ADR cells, which mainly distributed in cell nucleus.5) ADR was successfully loaded onto GO surface by simple mixture method, and the in vitro release of ADR showed remarkable pH sensitivity.6) Compared to free ADR, ADR-GO significantly enhanced the cytotoxicity in MCF-7/ADR cells.7) Compared to free ADR, ADR-GO significantly increased ADR intakes of MCF-7/ADR cells.8) ADR-GO significantly enhanced ADR accumulation in MCF-7/ADR cells, which mainly distributed in cell nucleus.9) GO had mutagenic effects in mouse bone marrow polychromatic erythrocytes and induced the damage and crack of chromosomes.10) GO had strong interaction with genomic DNA, and significantly influenced the DNA replifi cation.11) GO altered gene expression profile of MDA-MB-231cells, and the differential gene expression involved a number of signaling pathways such as cell cycle, translational control, DNA damage, cell apoptosis, cellular metabolism et al.12) GO with a lower concentration had no obvious cytotoxicity for MDA-MB-231cell, however, GO with a higher concentration significantly inhibited the proliferation of MDA-MB-231cells.13) GO with a lower concentration decreased the DNA content in S phase and inhibited the DNA synthesis speed, however, GO with a higher concentration induced the arrest of G2/M phase and inhibited cell proliferation.Conclusions1) ADR-URPA nanoparticles reversed the drug resistance of MCF-7/ADR cells. ADR-URPA nanoparticles entered into cells mainly through endocytosis, thus avoided the identification and efflux effects of P-gp.2) ADR-GO reversed the drug resistance of MCF-7/ADR cells. ADR-GO entered into cells mainly through endocytosis and passive diffusion, thus avoided the identification and efflux effects of P-gp.3) GO showed a concentration-dependent genotoxicity, which production mechanisms involved a number of signaling pathways. |
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