Drug Encapsulation Efficiency WDrug on the nanoparticles WTo
Drug Encapsulation Efficiency (%) = (WDrug on the nanoparticles/WTotal used drug)
Knowing the amount of drug left in the filtrate and total con-centration of L-Dopa (3 mM), the concentration of L-Dopa loaded on/in the surface/interlayer of [email protected] and the amount of Drug loading was determined as follow :
Drug Loading (%) = (WDrug on the nanoparticles /(WNanoparticles + WDrug on the nanoparticles)) × 100
2.6. In vitro drug release investigation
In order to evaluate the release of the L-Dopa from the [email protected] carrier, 1.738 mg of [email protected]@L-Dopa was suspended in 2 mL of phosphate buﬀer saline with diﬀerent pHs, and resulted sus-pension were transferred into 3500 MWCO dialysis bag. In the next step, the dialysis bag was placed into the 50 mL of the same buﬀer and agitated in the shaker at 37 °C (90 rpm). To determine the release of L-Dopa, 2 mL of buﬀer was removed and replaced with 2 mL of fresh buﬀer at time points of every 5 or 10 min until 120 min. The amount of released L-Dopa was measured by study on the absorbance of the re-sulting samples at 280 nm and using Beer-Lambert law to assess the concentration of L-Dopa present to calculate the amount of release.
In this study, we applied the Mel-Rm cells Melanoma (NCIt: C3224) which was derived from the metastatic site: Lymph node. The Mel-Rm cells were grown as a monolayer in DMEM culture medium supple-mented with 5% FCS in 25 cm2 flasks (Orange Scientific, Belgium). The cell line was seeded in 75 cm2 tissue culture flasks and maintained in Dulbecco's MEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL−1 penicillin and 100 μg/mL streptomycin. The medium was renovated every two days and the cell cultures were in-cubated at 37 °C in a humid KPT-185 (95% air and 5% CO2). While cell cultures reached 70 to 80% confluency, they were trypsinated using trypsin-EDTA 0.25% (Sigma) and were subcultured in 24-well culture plates at a density of 1 × 104 cells/well.
24 h after plating the cells, they have been washed with PBS (pH 7.4). There were three Groups: Group I: incubated with L-Dopa, Group II: incubated with [email protected]@L-Dopa, and Group III: incubated with [email protected] There were control and six treat-ments in every group, including; control: 0.0 μM, treatment 1: 1 μM, treatment 2: 2 μM, treatment 3: 4 μM, treatment 4: 8 μM, treatment 5: 16 μM, and treatment 6: 32 μM of any materials in each group respec-tively. Thereupon, the cells have been placed in the incubator at 37 °C with 5% CO2. The cells were cultured in DMEM, culture medium con-taining 0.2% BSA.
Trypan blue viability measurement has been done by standard methods. Trypan blue is the necessary stain. The usual procedure of performing trypan blue (0.4 g/100 mL in PBS) cell viability analysis contains manual staining and applying hemocytometer for counting.
Cell proliferation inhibition (PI) was quantified by measuring the Materials Science & Engineering C 101 (2019) 472–486
MTT assay. In order to perform the test, 15 × 103 cells were loaded into a 96-well plate and 200 μL of DMEM media containing 0.2% BSA was added. After 24 h of incubating, 200 μL of treatments media as de-monstrated was added to the wells. The cells were separately incubated with various treatments medium for 24 h. After incubation, the MTT test was accomplished in which the supernatant from each well was removed and 50 μL of MTT solution (5 mg/mL) was added to each well and incubated for 3 h. The supernatant from each well was then re-moved and 100 μL of dimethyl sulfoxide was added in order to dissolve the formazan crystals at room temperature for 30 min. The optical density of each well was measured using an enzyme-linked im-munosorbent assay (ELISA) reader at 570 and 630 nm. The cell pro-liferation inhibition of the cells for each concentration was calculated using the following formula (45):
2.11. Cell cytotoxicity measurement
Cell cytotoxicity was quantified by measuring the release of lactate dehydrogenase (LDH) from damaged or destroyed cells 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: