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  • br DLS studies were conducted to

    2020-08-18


    DLS studies were conducted to obtain the mean particle size and surface charge of the Coleus aromaticus extract-mediated CuO NPs. Fig. 4A shows the size distribution of the CuO NPs; a mean particle size of 33 nm was observed, and the size distribution was in agreement with the TEM results. The surface zeta potential of the CuO NPs is known to be −25.0 mV, signifying the colloidal stability of the biosynthesized CuO NPs using Coleus aromaticus extract polyphenols (Fig. 4B). This negative zeta potential may be due to the adsorption of Coleus ar-omaticus extract biomolecules onto CuO NPs. Additionally, this negative surface charge may generate strong electrostatic repulsive forces among the CuO NPs, resulting in stable CuO NPs through reduced aggregation [24].
    FT-IR studies were conducted to investigate possible functional groups existing on the surface of the CuO NPs. The FT-IR results showed the existence of major ABT-263 bands at 630, 1076, 1620 and 3428 cm−1 (Fig. 5); these absorption bands were due to the strong stretching vibrations of the OeH group (3428 cm−1), the tertiary al-cohol group (1620 cm−1) and the carboxylic –CeOeC stretching groups (1076 cm−1), indicating capping of polyphenolic constituents of Coleus aromaticus extract on the NP surface. Furthermore, the existence of an absorption band at 630 cm−1 corresponding to CueO stretching indicated the formation of CuO NPs. The FT-IR and zeta potential analysis results indicate the stabilization of bioconstituents of Coleus aromaticus extract on CuO NPs surfaces.
    Aptamer conjugation onto the CuO NPs surface was studied using fluorescence microscopy. From Fig. 6, the existence of a fluorescent green color in the micrograph indicates the successful conjugation of the aptamer onto the CuO NPs. In contrast, NPs that were not func-tionalized with the aptamer showed no fluorescent green color in the micrograph.
    3.1. In-vitro cytotoxicity of CuO NPs
    The cytotoxicity of the prepared CuO NPs was evaluated by con-ducting an MTT cell viability assay. A549 cell lines derived from lung carcinoma were incubated with CuO NPs of different concentrations: 0.05, 0.1 and 0.2 mg mL−1. After incubation for 24 h with CuO NPs, the cell viabilities of A549 cells were found to be more than 85% for the
    Fig. 10. Fluorescence images representing the interaction of FITC-MUC1 aptamer-conjugated CuO NPs with the cell membrane after 2-h incubation. (A) Aptamer-FITC (B) AlexaFluor-555-labeled wheat germ agglutinin (WGA-AF-555) (C) DAPI (D) Merged.
    three NP concentrations (Fig. 7), demonstrating the biocompatible nature of prepared CuO NPs, and their low toxicity. The biocompat-ibility of the prepared CuO NPs may be due to the capped polyphenolic constituents on the NP surfaces.
    3.2. In-vitro release study of miRNA
    3.3. Cellular uptake of aptamer-conjugated NPs
    The uptake of aptamer-conjugated NPs was studied using fluores-cence microscopy and flow cytometry. From Fig. 9, the internalization of MUC1 aptamer-conjugated siGLOFAM-loaded CuO NPs in lung cancer (A549) cells is possibly greater (P#0.001) in noninhibited cells compared to cells pretreated with MUC1 aptamer. The interaction of FITC-MUC1 aptamer-conjugated CuO NPs with the cell membrane of A549 cells was studied using fluorescence microscopy. Fig. 10 shows the existence of green colored FITC-MUC1 aptamer-conjugated CuO ABT-263 NPs on the cell membrane and the cytosol of lung cancer cells (A549) after incubation for 2 h. Cell membranes were stained with WGA-AF-555 (red color) to enable the identification of cell boundaries. The ex-istence of both red and green colors on the cell membrane of A549 cells indicates the interaction between the cell membrane and FITC-MUC1 aptamer-conjugated CuO NPs.
    4. Conclusions
    Here, we report a low-cost, facile and eco-friendly strategy for biofabrication of CuO NPs using Coleus aromaticus extract. The CuO NPs were conjugated with MUC1 aptamer and used for in-vitro delivery of microRNA29b to lung cancer cells. The results show that this delivery system can effectively deliver miRNAs to cancer cells, with superior performance compared to the traditionally used transfection agents, thus acting as an efficient platform for intracellular miRNAs delivery and improving therapeutic outcomes for lung cancer.
    Acknowledgments
    This work was supported by The National Natural Science Foundation of China (No. 81372567) Grant [2013]163 from the Key Laboratory of Malignant Tumor Molecular Mechanisms, and the Translational Medicine of Guangzhou Bureau of Science and Information Technology; Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes.