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  • br Preparation of BC and BC

    2020-08-18


    2.4. Preparation of BC and BC/LMNs membranes
    BC was biosynthesized from Gluconacetobacter xylinus (G. xylinus) (ATCC53582). The bacteria were grown in Hestrin and Schramm (HS) medium. The medium was sterilized for 20 min at 121 °C by the auto-clave (Yamato, SN310C, Japan), and then cooled to room temperature. We obtained multilayer and homogenized BC membranes by the static MPDL3280A method in the 12- and 24-well plates. The BC membranes were washed for 24 h with sterilized water, then dipped in 1 wt% NaOH for 60 min at 90 °C, and washed with sterilized water until the pH reached 7.0. The BC membranes were finally sterilized for 20 min at 121 °C and stored in physiological saline at either room temperature or 4 °C.
    The BC/LMN membranes were prepared based on the solution im-pregnation method under static magnetic field conditions. The com-posite membranes were prepared according to the following methods: the BC membranes were sterilized and then dried for three hours on an aseptic operating table at room temperature to allow more LMNs to impregnate the BC. The BC membranes were then dipped into 10 mL of PBS for 48 h at 25 °C. The LMNs was obtained through a 0.22 mm filter. Subsequently, 1-mL or 200-mL LMN solution was combined with the BC membrane in a 24- or 96-well plates, respectively, within 24 h. The preparation procedures are illustrated in Fig. 1.
    2.5. Encapsulation, loading efficiencies and release profiles for DOX and HMME in MHNP
    We dissolved 10 mg (Wa) MHNP nanoparticles and 3 mg (Wb) DOX in PBS [36], and we dissolved 1 mg (Wa) MHNP nanoparticles and 2 mg (Wb) HMME in PBS, respactively. After incubating for 72 h at 4 °C, the solution was dialyzed for eight hours. The DOX and HMME quantity (Wc) in the dialysate were determined using ultraviolet spectroscopy (UV, UV2450, SHI-MADZU, Japan) at 480 nm and 620 nm. The en-capsulation efficiency was calculated as following formula: encapsula-tion efficiency (%) = (Wb −Wc)/Wb, while the loading efficiency of NIPAm-AA was calculated as following formula: loading efficiency
    (%) = (Wb −Wc)/Wa. The release profiles for HMME High Perfor-mance Liquid Chromatography (HPLC, LC-20A, SHIMADZU, Japan) were performed to probe the characteristic peaks and curves.
    2.6. Characterizations of the samples.
    The distribution of Fe3O4-OA, MHNP, MHNP-FA, and LMNs in the PBS were evaluated by the dynamic light scattering (DLS) using the
    Fig. 1. Schematic strategies of the molecular structures and chemical processes for preparing LMNs and BC/LMNs.
    Zetasizer Nano-ZS90 (Malvern Instruments Ltd, England), respectively. Additionally, the size of LMNs in the cell culture medium were texted by using the Zetasizer Nano-ZS90. To detected the difference in the surface morphology and molecular structure for the samples. The FTIR spectra of samples were recorded on a Paragon 500 (Perkin Elmer, Waltham, USA). The TGA analysis was performed in a thermogravi-metric analyzer (STA449 F3, NETZSCH, Germany). Then, it is critical to check whether the photoactive LMNs are loading onto the surface of the BC or not. The surfacemorphology was investigated by atomic force microscopy (AFM, Cypher, Asylum Research, USA) and scanning elec-tron microscop (SEM, JSM-7600F, Japan). The details of these char-acterization analyses were referenced to our recent papers to detect the characteristics of nanoparticles [29,36].
    2.7. In vitro cell viability assay
    Cell Culture. Human breast carcinoma MCF-7 cells and Human skin fibroblasts HSF cells were grown in adhesion in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco 12800–017) supplemented with peni-cillin (50 IU·mL−1), streptomycin (50 μg·mL−1), and 10% fetal bovine serum (FBS). Both lineages were maintained at 37 °C in humidified atmosphere at 5% CO2.
    2.8. Apoptosis and migration analysis of MCF-7 by PDT in vitro
    Cells were seeded on 24-well plate and treated with PBS, the saline + M + L, LMNs, the LMNs + M + L and the BC/LMNs + M + L for 24 h. The samples were applied for the flow cytometry and wound healing assay. Data was collected and analyzed by the CellQuest soft-ware (FACS Calibur, Becton-Dickinson). And cell viability was also 
    assessed by confocal microscopy by live/dead staining (FDA, green; PI, red). The migrated cells were observed under an inverted microscope. Protein expression was detected using antibodies against p53, Bax, Bcl-2 (Boster, China), as shown in Fig. 4F1-4. The details of those can be referenced in our recent papers for detecting the effect of inhibiting cell growth in vitro [36].
    2.9. Breast tumor animal model construction and treatment