QDs possess unique optical and electronic properties including tunable emission wavelength, broadband absorption spectrum, and photostability that make them useful materials in microelectronics and biomedical research [1]. QDs toxicity and surface coatings to render them biologically compatible have been intensively researched (Rizvi et al., 2012). QDs were appealing alternatives to conventional fluorophores due to their superior optical properties and have the potential to meet some of these outstanding challenges in biotechnology [2].

Cadmium QDs (Cd-QDs) including CdTe-QDs and CdSe-QDs have shown great potential for use as fluorescent tags in therapeutic targeting and in medical and molecular imaging but its toxicity has to be considered [1]. QDs toxicity limits their biomedical applicatio, although they can now be aqueously synthesized. QDs toxicity was multifactorial and was determined by their physiochemical properties, including composition of the core, size, surface charge, concentration, surface chemistry, bioactivity, oxidative, photolytic and mechanical stability, as well as their environmental interactions [3] [4]. Cd-QDs toxicity has been intensively researched. The toxicity of Cd-based QDs has been proposed to be associated with the oxidation reaction of the metal core. The reaction generates reactive oxygen species (ROS) and Cd²⁺, which were toxic to cells of animals, plants and microbes [5]. Another research found that the physicochemical characteristics of CdTe core QDs influenced subcellular localization and cytotoxicity; quantified as generation of ROS [6]. Cadmium (Cd), which was capable of inducing known toxicities in humans including hepatic, renal, neurologic, and/or genetic toxicities, was the most abundant component of QDs [7]. A research found that CdTe-QDs with a smaller size showed greater hematopoiesis toxicity and CdTe-QDs effects on immune system [5] [8]. CdTe-QDs can induce cytotoxicity, autophagy, oxidative stress, ER stress, chromatin condensation, reducing cell viability and apoptosis in cells [9]. Based on the above research, we intend to study the effect of cell inhibition with CdTe core QDs and CdTe QDs modified with functional group in vitro experiments. Our results provide new insights into Cd-QDs toxicology and provide evidence for the future application.

Preparation and Characteristics of QDs

The CdTe-QDs used in the study were synthesized by Janus New-Materials Co., Ltd and kept in a refrigerator at 4˚C until use. The physicochemical properties of these QDs were evaluated. Ultraviolet-visible (UV-vis) spectra were measuring with a PerkinElmer UV-VisNIR (Lambda 900) spectrophotometer (PerkinElmer, Waltham, MA, USA). Photoluminescence spectra were collected with a Cary Eclipse fluorescence spectrometer (Varian Medical Systems, Palo Alto, CA, USA). The concentration of Cysteine (Cys) and Thioglycolic acid (TGA)-modified CdTe/ZnS and CdTe QDs aqueous solution was calculated to be 8 μmol/L (3 mg/ml) by UV absorption and Lambert’s law. The Emission Voltage was 500 V. The emission wavelength of the resulting solution was 450 nm. Emission wavelength was 537 ± 5 nm. Maximum absorption peaks was 500 nm.

Cell Culture

Caco-2 cells were obtained from Fu Heng biology (Shanghai, China) and were maintained in DMEM (Gibco, NY, USA)supplemented with 10% fetl bovine serum (Gibco, NY, USA) and 1% penicillin-streptomycin-neomycin (Gibco, NY, USA). Cells were incubated in a humid atmosphere at 37˚C and 5% CO₂, and passaged with 0.25% trypsin-EDTA, confluence 70% - 80% began subculturing.

Cell Treatment

Confluence of about 80% of the logarithmic cells was collected. Cells were seeded in 96-well plates at 106 cells/well in 100 μl of medium and incubated at 5% CO₂ and 37˚C for 12 h. Cells were treated to concentration (0, 12.5, 25, 50, 100 μg/ml) of Cys-modified CdTe QDs (Cys-CdTe QDs), Cys-modified CdTe/ZnS QDs (Cys-CdTe/ZnS QDs), TGA-modified CdTe QDs (TGA-CdTe QDs) and TGA-modified CdTe/ZnS QDs (TGA-CdTe/ZnS QDs) at 5% CO₂ and 37˚C for 6 h, 24 h and 48 h in the following experiments.

MTT

Cells were incubated with four QDs at 5% CO₂ and 37˚C for 6, 24, and 48 h. 20 μl of MTT (5 mg/ml) solution was added to each well. After 4 hours of incubation, the remaining MTT solution was removed and 150 µl of DMSO was added to each well, under standard assay conditions, shaking shaker on the low-speed shock for 5 minutes to completely dissolve the precipitation. After the formazan crystals had dissolved, the optical density (OD) was measured by enzyme-linked immunosorbent assay at 490 nm. The cell inhibitory rate was calculated according to the following equation: the cell inhibitory rate = [1 − OD experiment/OD control] × 100%. All the experiments were performed three times.

Statistical Analysis

The experiments were performed in triplicate. Statistical analyses were carried out using the statistical software SPSS 20.0. Pearson’s correlation coefficient (r) was calculated. The difference between the experimental groups and the control group was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. The statistically significant difference was considered to * P ≤ 0.05 compared to control cells and ** P ≤ 0.01 compared to control cells.

Figure 1 shows that the excitation wavelength of CdTe aqueous solution with concentration of 8 umol/L (3 mg/ml) (calculated by UV absorption and Lambert Beer’s law) is 450 nm.

The emission wavelength is 537 nm (due to different detection conditions, the wavelength will fluctuate up and down 5 nm).

Figure 2(a) and Figure 2(b) had the similar pattern. The inhibition rate of Cys-CdTe/ZnS QDs and Cys-CdTe QDs increased with the increase of concentration and reached maximum for 48 h (66.72% for Figure 2(a), 57.01% for

Figure 2(b)). The inhibition rate (0.96% - 27.86% for Figure 2(a), 6.54% - 30.92% for Figure 2(b)) was linear positive correlation at 6 h (r = 0.95, P ≤ 0.05 for Figure 2(a), r = 0.98, P ≤ 0.05 for Figure 2(b)). The inhibition rate increased with the increase of time and was linear positive correlation at the concentration of 12.5 μg/ml (r = 0.96 P ≤ 0.05 for Figure 2(a), r = 1.00 P ≤ 0.05 for Figure 2(b)). The inhibition rate was almost similar with concentrations from 25 to 100 μg/ml at 24 h and 48 h. Figure 2(c) and Figure 2(d) had the similar pattern. The inhibition rate of TGA modified QDs reached maximum 65.88% at the lowest concentration with a slight fluctuate until 100 g/ml at three exposure times. The inhibition rates both reached maximum for 48 h (79.21% for Figure 2(c), 72.24% for Figure 2(d)).

Figure 3(a) showed the relative ratio of Cys-CdTe/ZnS QDs to Cys-CdTe QDs (Cys-ZnS/Cys). The ratios were closed to 1 (0.901 - 1.07) with concentrations from 25 to 100 μg/ml for 6 h and 24 h. The average ratios of Cys-ZnS/Cys reached 1.11 (1.07 - 1.17) for 48 h. The lowest ratio reached 0.15 at concentration of 12.5 μg/mll for 6 h. The largest ratio reached 1.4 at the concentration of 12.5 μg/ml for 24 h. Figure 3(b) showed the relative ratio of TGA-CdTe/ZnS QDs to TGA-CdTe QDs (TGA-ZnS/TGA). The ratio in all concentrations were closed to 1 (0.89 - 1.14) at three exposure times.

Figure 4(a) showed the relative ratio of Cys-CdTe/ZnS QDs to TGA-CdTe QDs (Cys-ZnS/TGA-ZnS). Figure 4(b) showed the relative ratio of Cys-CdTe QDs to TGA-CdTe QDs (Cys/TGA). Figure 4(a) and Figure 4(b) had the similar pattern. The relative ratio is less than 1 in all concentrations at three exposure times. With the concentration increases, the ratio significant increased and exhibited linear positive correlation for 6 h (r = 0.97, P ≤ 0.01 for Figure 4(a), r = 0.99, P ≤

0.01 for Figure 4(b)). With the concentration increases, the ratio increased slowly for 24 h. The ratios reached lowest (0.025 for Figure 4(a), 0.18 for Figure 4(b)) at concentration of 12.5 μg/ml for 6 h. The ratios were closed to 0.85 at maximum concentration (100 μg/ml) at three exposure times. Figure 4(b) exhibited time-dependent at concentration of 12.5 μg/ml (r = 0.951, P ≤ 0.05).

Table 1 showed the t-test result on the inhibition rate of Cys-ZnS/Cys, TGA-ZnS/TGA, Cys-ZnS/TGA-ZnS and Cys/TGA. The ratios of Cys-ZnS/Cys and TGA-ZnS/TGA had significant differences at high concentrations for 48 h (P ≤ 0.05). The ratios of Cys-ZnS/TGA-ZnS and Cys/TGA had significant differences at most concentrations (P ≤ 0.05) at three exposure times.

Our result showed that four QDs (CdTe-QDs, TGA and Cys-modified CdTe/ZnS and CdTe QDs) have inhibited cell proliferation. A study reported CdTe QDs have inhibited to HepG2 and HELF cells proliferation and have dose dependent

*P ≤ 0.05; **P ≤ 0.01.

and time dependent [10], which supports our observations.

It has reported that cytotoxicity of QDs depend on their surface modification [11]. Our result showed that the inhibition rate of TGA-modified QDs was higher than Cys-modified QDs. For TGA-modified QDs, the inhibition on cell proliferation exhibited obviously time-dependent and no effected by design-concentration range. The inhibition was almost consistent in all concentrations and had already reached this level at the lowest concentration. At a low concentration, TGA-CdTe QDs also showed the cytotoxic effects to HepG2 cells [12]. Contrary to TGA, Cys-modified QDs of the inhibition exhibited obviously dose-dependent and no obviously time-dependent. The inhibition was similar at 24 h and 48 h. The inhibition was almost no effect at the designed-lowest concentration and shortest time. At a low concentration and short time, Cys-CdTe QDs also had no apparent effect in cell metabolic activity and apoptosis [13].

The chemical composition of the QDs core is important factors affecting QDs toxicity [14]. For Cys-modified QDs, the inhibition of CdTe/ZnS QDs was higher than CdTe QDs at the higher concentration in the maximum time despite they both inhibited to cell proliferation. For TGA-modified QDs, the inhibition of CdTe/ZnS QDs and CdTe QDs were almost consistent at all concentrations and three exposure times.

We found that four CdTe-QDs, modified with TGA or Cys, with ZnS core or not, all have inhibited cell proliferation. The inhibition of TGA-modified QDs was higher than Cys-modified QDs. Inhibition exhibited obviously time-dependent for TGA-modified QDs while exhibited obviously dose-dependent for Cys-modified QDs. The inhibition of CdTe/ZnS QDs was higher than CdTe QDs for Cys-modified QDs. The inhibition of CdTe/ZnS QDs was almost consistent at all concentrations and three exposure times. The study facilitates the further understanding of the inhibition effect on cell proliferation of CdTe QDs with core or functional group and provides useful information for the use of CdTe QDs.

This research was financially supported by the National Natural Science Foundation of China (Grant No. 31460249) and the science plan project of Inner Mongolia Autonomous Region (Grant No. 201505016).

The authors declare no conflicts of interest regarding the publication of this paper.

Su, X., Gao, X.Y., Jiang, L., Zhang, J., Wang, Z.F., Chi, B.F., Zhang, H.R., Bao, S.B., Zhang, R., Yun, B.H., Sun, D.J. and Sun, J. (2021) MTT Detection of the Toxicity on CdTe Quantum Dots. Open Journal of Preventive Medicine, 11, 1-8. https://doi.org/10.4236/ojpm.2021.111001