br Mel Rm cells were cultured in
Mel-Rm 94421-68-8 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.
result indicates that the L-Dopa molecules were intercalated into the interlayers of LDHs.
In order to investigate the morphology of [email protected]@L-Dopa, the surface structure has been studied using SEM technique (Fig. 3). Fig. 3a indicates the nearly spherical and uniform shape of the Fe3O4 nanoparticles. Also, the spherical structure and layered surface of [email protected] and [email protected] are clearly verified (Fig. 3b, d). The mapping analysis of [email protected]@L-Dopa was shown in Fig. 3d–h. The high intensity of the elemental dispersion of O, C, Ca and Al shows the presence of loaded L-Dopa on the surface of LDH structures. In addition, low intensity of Fe dispersion, confirms the presence of Fe3O4 nanoparticles in the interior of the [email protected]@L-Dopa.
Elementary analysis of the surface was further studied by EDX analyses. The EDX pattern of [email protected] shows the major peaks of Al, Ca, Fe, O, C (Fig. 4a). Also, after intercalation of L-Dopa in the [email protected] structure, the strong band of N was appeared which confirms the presence of surface loaded L-Dopa in the structure of our synthesized [email protected]@L-Dopa (Fig. 4b).
In order to study more on the morphology of [email protected]@L-Dopa, TEM images were investigated. As expected, the results clearly indicate the core-shell structure of synthesized [email protected]@L-Dopa and obviously confirmed the structural information obtained from SEM technique (Fig. 5).
Fig. 5a clearly shows the spherical and uniform shape of the Fe3O4 nanoparticles. The low magnification image of [email protected]@L-Dopa in addition to the spherical [email protected] nanostructure shows the presence of thin layer of L-Dopa on the surface of carrier, which creates the structure with approximately 120 nm of dimensions (Fig. 5b). The high magnification image indicates the layered structures of CaAl-LDH and L-Dopa around Fe3O4 nanoparticles (Fig. 5c).