br Cell cytotoxicity was quantified
Cell cytotoxicity was quantified by measuring the release of lactate dehydrogenase (LDH) from damaged or destroyed DMAPP into the media. Cytotoxicity was measured with the LDH Cytotoxicity Detection Kit (Roche, Germany). This kit detects LDH release from dead cells. Therefore, the increase of LDH activity in each treatment shows that the treatment solution has further dead cells or cytotoxicity eﬀects on cells. Cells were plated in 24 well culture plates with 104 cells/mL densities for 12 h. Afterward, cells have been cultured with diﬀerentiation the medium for 24 h. The percentage of cytotoxicity was measured by the protocol of the company; colorimetry of LDH activity measured by calculating the absorbance of samples at 490 or 492 nm using an ELISA Reader (EL800; USA). The references wavelength should be > 600 nm. Whole the tests have been replicated partly at least 3 times. Within each experiment, we replicated each condition 4 times. The viability of cells for every concentration has been computed applying the following formula:
Mel-Rm cells were cultured in diﬀerent treatment media condition. The caspase-3 activity of lysates from the cells treated was measured with the caspase activity colorimetric assay kit (Bio-techne) according to the manufacturer's protocol using a plate reader. Data were obtained from two independent experiments.
2.13. Detection of mitochondrial membrane potential
For quantitative analysis, MMP was measured using the cell permeable cationic fluorescence probe rhodamine 123. Briefly; Mel-Rm cells 3 × 104 cells/well were cultured and treated in diﬀerent treatment media, then they were washed with PBS and incubated by 1 μM rho-damine 123 in the dark for 30 min at 37 °C. Then, the absorbance of cells was measured by calculated the absorbance of samples at 488 excitation and 525 nm emission using an ELISA Reader. The reference wavelengths should be > 630 nm. All the tests have been repeated in-dependently at least 3 times. Within each experiment, we replicated each condition 4 times.
2.14. Quantification of apoptosis incidence
Fixation for all cells in this study was done by 4% w/v paraf-ormaldehyde in PBS, pH=7.4 for 10 min at room temperature. An in situ cell death detection kit (Roche) was used to identify the apoptotic cells by TUNEL (Terminal Uridine deoxynucleotidyl transferase dUTP Nick End Labeling) staining, following the manufacturer's protocol.
Scheme 1. Synthetic route of [email protected]@L-Dopa.
Briefly, all cells were fixed, permeabilized, blocked and incubated with a mixture of fluorescently labeled nucleotides on tdt (terminal deox-ynucleotidy transferase) catalyzed the polymerization of labeled nu-cleotides to 3/0H terminals of DNA fragments. The cells were then counterstained with 10 μg/mL of propidium iodide (red) at room tem-perature for 15 min and washed with PBS. A positive apoptosis control, cells induced into apoptosis by 5% ethanol treatment, were included in each assay. TUNEL positive cells were counted in eight randomly se-lected fields from each culture under a fluorescent microscope (Olympus AX-70), and apoptotic index was calculated by dividing the number of apoptotic cells by the total cells.
3. Results and discussion
3.1. Structural characteristic
In this study, we report the synthesis of [email protected] as a magnetic pH-responsive nanocarrier of L-Dopa for the first time. Scheme 1 shows the synthetic route for the preparation of the designed [email protected]@L-Dopa as a therapeutic system for cancer therapy.
The [email protected]@L-Dopa was prepared using a method that involves the separated synthesis of Fe3O4 nanoparticles, immobilization of CaAl-LDH on the surface and intercalation of L-Dopa into the [email protected] structure.