Page 53 - TD-1-1

Tumor Discovery Dual-targeting of cancer receptors by nanoparticles

(DynaMag TM-50 system) and dried using freeze-drying. 12.5 mg/mL) of Fe O -DPN-HA-FA NPs were used to treat
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The absorption of the final solution was examined by the cancer cells. Furthermore, four wells in every period of
ultraviolet (UV) spectrophotometer (CECIL, CE1021) at time were exposed to culture medium alone as a negative
278 nm to determine the percentage of TMX loading (%) control. At appropriate times, the media were removed and
where the line equation of the standard curve using five replaced with 200 µL of MTT solutions composed of 150 µL
concentrations of TMX was y = 5.7384x + 0.2061 (R = of fresh media plus 50 µL of MTT solutions (prepared as
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0.9991). 2 mg/mL in PBS), and were then incubated for 4 h. Finally,
MTT solutions were carefully aspirated off, cells were
2.7. Release assay of TMX washed (3×) by PBS, and formazan crystals formed were
The in vitro drug release study for TMX was conducted solubilized in 200 µL of DMSO. After 10 min incubation at
under physiological conditions at pH 7.4 and 37°C. For room temperature under light protection, the absorbance
this purpose, 10 mg of Fe O -DPN-HA-FA-TMX NPs were was recorded at 570 nm using a spectrophotometer (BioTek
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[24]
dispersed in 5 mL PBS and sealed inside a dialysis bag, then Instruments, Inc., Bad Friedrichshall, Germany) .
were put into a beaker with 40 mL of PBS with the same 2.10. RT-PCR analysis
pH condition for 120 h. At predetermined time intervals,
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proper amounts of buffer medium were withdrawn and MDA-MB-231 cells were seeded in T25 cm flasks with
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liberation of TMX from MNPs was measured using a UV a density of 1.0×10 before being treated overnight with
spectrophotometer. As a control, the release of free TMX 1 mg/mL and 0.11 mg/mL of Fe O -DPN-HA-FA-TMX
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was done in PBS with the same pH. NPs and TMX alone, respectively. One flask without any
treatment and one flask containing Fe O -DPN-HA-FA
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2.8. Characterization methods NPs were also prepared as a control. Thereupon, cells
All steps of the synthesis of Fe O -DPN-HA-FA-TMX NPs were washed with warm PBS, and 1 mL of 0.25% trypsin
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were validated by Fourier-transform infrared spectroscopy was added to dissociate adherent cells from the surface.
(FTIR; Shimadzu IR PRESTIGE 21 spectrophotometer, Next, the cells were centrifuged at 3000 rpm for 5 min
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Tokyo, Japan) in the spectral range of 4000–500 cm at to eliminate extra solutions and resuspended in 200 µL
room temperature . Furthermore, surface morphological PBS. High Pure RNA Isolation Kit (Roche) was used for
[22]
analysis was performed using field emission scanning purifying intact total RNA from cultured cells according
electron microscopy (FESEM) (Mira 3 TESCAN to the manufacturer’s protocol. Accordingly, cells were
instrument; TESCAN, Brno-Kohoutovice, Czech resuspended in 400 µL lysis/binding buffer and were then
Republic) with high magnification to achieve good image incubated for 15 min with 90 µL DNase incubation buffer
quality, depth of focus, and low noise in imaging. plus 10 µL DNase I at room temperature to remove genomic
DNA contamination. After washing with two different
2.9. In vitro cell viability and cytotoxicity assay using wash buffers (I and II), total isolated RNA was transferred
MTT assay into a clean, sterile 1.5 mL microcentrifuge tube by adding
100 µL elution buffer. The purity and concentration of
Human breast cancer MDA-MB-231 cell lines were routinely the isolated RNA were determined by the WPA Biowave
cultured for 6 days in 25 cm culture flasks using RPMI II spectrophotometer (Biochrom Ltd., Cambridge, UK)
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1640, 10% fetal bovine serum, and 100 µL of penicillin G/ which absorbs over the 230–260 nm range. The eluted
streptomycin mixture in a 5% CO incubator according RNA was stored at −80°C before the synthesis of cDNA .
[25]
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to existing standards . In appropriate equal intervals of
[23]
time, cell passaging is essential for cells to proliferate and In accordance to Pars Tous EasyTM cDNA synthesis kit,
reached confluence around 80%, and also, PBS washing is four separate mixtures composed of 10 µL 2× buffer mix,
needed to remove the media serum. Then, cells at a density 2 µL enzyme mix, 6 µL isolated RNA, and 2 µL DEPC-
of 1.5 × 10 cells/well were transferred into 96-well culture treated water were mixed to synthesize complementary
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plates and incubated in normal cell culture conditions for DNA (cDNA). Thereafter, a total of 20 µL of reaction
24 h. After that, under aseptic conditions, the cells were mixtures were incubated at 25°C for 10 min, 47°C for
treated with five different concentrations (i.e., ranging from 60 min, and 85°C for 5 min, respectively.
0.78 to 12.5 mg/mL) of Fe O -DPN-HA-FA-TMX NPs RT-PCR quantitative assays for genes (Table S1)
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and also five concentrations of TMX alone (i.e., ranging involved in apoptosis were performed using the Applied
from 0.088 to 1.4 mg/mL) for two periods of 48 and 72 h. Biosystems StepOne™ instrument (Thermo Fisher
The concentration of TMX alone was set according to the Scientific, Massachusetts, USA). The thermal cycling
amount of TMX loading on modified MNPs. As for placebo, conditions were as follows: A holding stage at 95°C for
five different concentrations (i.e., ranging from 0.78 to 12 min followed by 40 cycles of 95°C for 20 s, 55°C for

Volume 1 Issue 1 (2022) 4 https://doi.org/10.36922/td.v1i1.41