br Preparation of CuO NPs br About mL
2.2. Preparation of CuO NPs
About 50 mL of fresh Coleus aromaticus leaf extract was added to
10 mL of 0.01 M CuSO4 aqueous solution and mixed well by manual shaking. The reaction mixture was then heated for 30 min at 90 °C in a temperature controlled water bath. A visual color change in the reac-tion solution, from dirty yellow to brown, was observed after 10 min indicating the formation of CuO NPs.
2.3. In-vitro cytotoxicity and MTT assay
The in-vitro cytotoxicity of the synthesized CuO NPs was studied by investigating the cell viability of A549 cell lines. Cell viability was determined after incubation of A549 Ko143 with CuO NPs at concentra-tions of 0.05 mg mL−1, 0.1 mg mL−1 and 0.2 mg mL−1 in Dulbecco's modified Eagle's medium (DMEM). A control experiment was also performed without the addition of CuO NPs in a complete growth culture medium. Cell viability was determined using the MTT reduction test. The MTT test was carried out in a 96-well plate following a stan-dard experimental procedure with slight modifications. Initially, the cells were seeded in a 96-well plate at a concentration of 1 × 104 cells/ well, followed by incubation for about 24 h at 37 °C. The medium was then discarded and a new medium containing NPs was added to each well and incubated at 37 °C for 24 h. The medium in all wells was then discarded again, followed by the addition of 10 μL of MTT (5 mg mL−1 in phosphate-buﬀered saline [PBS]) solution and 90 μL medium into each well. The medium in the wells was removed after incubation at 37C for 4 h, followed by the addition of 100 μL DMSO containing so-luble formazan crystals. The solutions were then allowed to rest at room temperature for 15 min. The optical absorbance of the solutions was measured at 560 nm using an ELISA plate reader. Cell viability was expressed as a percentage relative to the optical absorbance of the control.
2.4. Conjugation of aptamer to biofabricated CuO NPs
About 50 μL of CuO NPs (10 μg/mL in DNase/RNase-free water) was added to EDC (40 mM, 100 μL) and NHS (10 mM, 100 μL) followed by stirring for 15 min, resulting in the activation of the carboxyl groups of CuO NPs for conjugation of the aptamer. Then, about 50 μL of aptamer in DNase, RNAse-free water (1 μg/mL) was mixed with the NP solution and stirred gently at room temperature for 2 h. The subsequent solution was centrifuged at 4000 rpm for 5 min to remove unreacted N-hydro-xysuccinamide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide hydrochloride (EDC). The obtained aptamer-conjugated NPs Materials Science & Engineering C 97 (2019) 827–832
Fig. 1. X-ray diﬀraction (XRD) patterns of biosynthesized copper oxide nano-particles (CuO NPs).
2.5. In-vitro miRNA release study
The study of in-vitro miRNA release from aptamer-conjugated CuO NPs was conducted at various pH values (5, 6.6 and 7.4). About 3 mg of aptamer-conjugated CuO NPs were dispersed in 0.5 mL of buﬀer and dialyzed using a tightly secured tubular cellulose dialysis membrane, followed by incubation in a 5 mL buﬀer solution at 37 °C during agi-tation. The quantity of miRNAs released (percentage cumulative re-lease) at diﬀerent time intervals was measured using ion-pair high performance liquid chromatography (HPLC).
A Waters 2695 separation module combined with an HPLC system (Waters, Milford, MA, USA) containing a Waters 2998 photodiode array detector was used for quantification of released miRNA. A 3 μm Oligo-RP Phenomenex column (50 × 2.0 mm; Phenomenex, Torrance, CA, USA) was used for sample separation. The mobile phase was prepared using an acetonitrile (5%–12%) and 20 mM triethylamine–acetic acid (pH 7) gradient elution. Samples for HPLC analysis were prepared by mixing 1 μL of miRNA with the mobile phase. The measurements were performed at a flow rate of 0.2 mL/min and the optical absorbance was measured at 269 nm.
2.7. Fluorescence microscopy
Lung cancer (A549) cells were initially seeded at a concentration of 2 × 104 per well and incubated for about 48 h. After incubation, the cells were washed with PBS followed by incubation with fluorescein isothiocyanate-mucin 1 (FITC-MUC1) aptamer-conjugated miRNA-29b-loaded CuO NPs in medium, at a density of 100 μg/mL for 2 to 4 h. Then, the obtained cells were washed twice with PBS, and 2% paraf-ormaldehyde was added followed by incubation for 20 min at room temperature. The cells were then blocked with 5% bovine serum al-bumin (BSA) for 30 min and stained with either WGA-AF-555 or LysoTracker-Red, before staining with DAPI for nucleus visualization.
A concentration of about 1 million A549 cells/well was placed in a six-well plate followed by incubation for about 48 h. The cells were then exposed to siGLO-FAM-loaded equivalent NPs (100 μg/mL) in Opti-MEM medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) followed by incubation for about 4 h. The resulting cells were then pretreated with free MUC1 aptamer, followed by treatment with