**2. Cytotoxic effects of quantum dots on diverse cell lines**

The cell membrane is the first barrier that divides intracellular from extracellular mechanisms. The process by which QDs enter the cell is not well defined, although it includes anchoring of QDs to the cell membrane, transmembrane transport, distribution and localization within subcellular compartments, and intracellular accumulation. All these processes are linked to their future application, their potential toxic effects, and the adverse effects induced in a dose-time-dependent manner [21]. Tests such as i*n vitro* cytotoxicity are important because of the significant morphological changes caused by QDs at the cellular and subcellular levels. In recent years, a huge variety of *in vitro* studies suggest that QDs have toxic effects on cells at different levels [22, 23]. In addition, the passage of QDs across the cell membrane has been demonstrated, the effects are oxidative stress, direct damage to membrane, morphological alterations, and various types of cell death.

*In vitro* models are necessary for safety assessment in preclinical testing of nanomaterials for diagnostic purposes. Although some models for cytotoxicity are not sufficient due to lack of human cells available for culture or even lack of reproducibility in assays. Therefore, the predictability about the safety of a nanodrug is a difficult task for nanotoxicology researchers [24]. However, there are *in vitro* models considered as standard patterns for toxicological studies of nanomedicines such as the use of human renal Hek293 cells [25]. Over a decade, our research group has focused its interest on the study of dextrin-coated 3.5 nm sized cadmium sulfide QDs (CdSdex) [26] and their potential biomedical application as is the case of doxorubicinconjugated CdS-dex QDs (CdS-dex/dox) [27]. Therefore, we have established several *in vitro* tests using Hek293, HeLa (cervix adenocarcinoma), and HepG2 (hepatic cells) cells for preclinical studies on CdS-dextrin quantum dots and with maltodextrin. Therefore, our results demonstrate that CdS-dex QDs and CdS-dex/dox QDs induce exposure to dose-dependent cytotoxic effects. In addition to this, we consider that one of the main evaluations to be performed on QDs is the monitoring of their cellular uptake and distribution. We observed that Hek293, HeLa and HepG2 cells when being treated with concentrations of 0.01 and 1 μg/mL, CdS-dex QDs cross the cell membrane, induce morphological changes, and distribute uniformly at different cellular level. Due to their nanometer size, QDs caused cytotoxicity in the three different cell types by crossing the cell membrane. However, morphological changes varied significantly between Hek293, HepG2, and HeLa cells and the concentration of CdSdex QDs (**Figure 2**). When QDs have contact with the extracellular membrane, they interact with components of the plasma membrane which allows them to somehow enter the cell by some mechanism such as endocytosis. Endocytosis engulfs the QDs by invagination of the membrane to form endocytic vesicles, which transport the QDs to subcellular compartments. Depending on the cell type, as well as some biomolecules involved in the process, endocytosis can occur in different types [28, 29]. Some authors refer to the uncertainty about the toxic effect that quantum dots may cause as they are transported through the bloodstream and leach into the kidneys.

#### **Figure 2.**

*Fluorescent microscopic visualization of CdS-dex QD in human cell lines. Cells were treated for 24 h with CdSdex QD (0,01–1 μg/mL). Cells were seeded on slides by smearing and allowed to dry, then analyzed using confocal epifluorescence microscope. Green fluorescence shows the presence of QD surrounding the cytoplasm of Hek293, HeLa, and HepG2 cells. Scale bar 20 μm.*

However, there is no information on the nephrotoxic effects of quantum dots both *in vitro* and *in vivo*. Nevertheless, some studies aim to understand the cytotoxic effect on renal cells caused by quantum dots. Therefore, quantum dots, such as titanium oxide (TiO2), zinc oxide (ZnO), and cadmium sulfide (CdS), have been evaluated in tubular cells (HK-2) in which the cellular and molecular mechanism through oxidative stress induced by quantum dots was demonstrated. In which it was observed that the cytotoxicity of quantum dots was size and solubility dependent. Furthermore, quantum dots that were soluble such as CdS and ZnO were found to cause dose-dependent cell death and degradation/discharge of their ions, respectively [30].

In another investigation, carboxylated CdTe QDs were used and the induced cytotoxicity was evaluated in HeLa cells treated at concentrations from 0.1 to 1000 ng/mL during different exposure times. The effect of CdTe QDs on cell death type, genotoxic effect, and cellular uptake was also evaluated. In this study, they demonstrated that carboxylated QDs did not prove to be less cytotoxic compared to CdTe alone in a concentration-dependent manner. Furthermore, they concluded that CdTe-COOH QDs have genotoxic properties and antiproliferative effects in HeLa cells [31].

#### *Toxicity of Quantum Dots DOI: http://dx.doi.org/10.5772/intechopen.112073*

Although CdS-dex quantum dots produced different cytotoxic effects on human tumor cells, these effects are not necessarily benign. In fact, our study showed that these nanoparticles had the ability to enter even subcellular compartments. Thus, their biological behavior could trigger pathophysiological effects in a concentrationdependent intrinsic manner. Our CdS quantum dots are coated with a polymeric layer of dextrin. However, many nanomaterials are known to have an inorganic or polymer layer protecting the core to prevent degradation. Even so, heavy metal ions such as cadmium can be released through low stability [32–34]. Studies are needed to know if the cadmium core degrades and releases metal ions and what effects are related to this degradation.

Despite the remarkable effects caused by CdS-dex quantum dots, we clearly need to reinforce the studies and strategies that allow us to learn more about their toxicity. We are getting closer and closer to obtaining biocompatible semiconductor nanoparticles with useful capabilities in diagnosis, treatment, and monitoring of pathologies such as cancer.

Evidently, QDs have physicochemical properties and capabilities and characteristics similar to biological molecules that allow them to be used in biodiagnostics, bioimaging, and targeted drug delivery. For a drug to be effectively delivered using nanocarriers such as QDs, the core component of the QD, the drug or molecule with which it will bioconjugate, and the core shell must be considered. That is, this set of components must be carefully selected to have therapeutic efficiency and optimal safety for use in a biological system [35, 36]. Currently, QDs are considered a tool with promising uses and applications in nanomedicine. However, their cytotoxic effects remain among the main challenges regarding their biocompatibility. The QDs with the highest capacity to emit luminescence and with the highest efficiency in carrying molecules with active principle are those containing cadmium (Cd). However, one of the limitations for the use of Cd QDs in nanomedicine and clinical research is that it is suggested that the core disintegrates and is potentially toxic. That is, it has been considered that it is the core of the QD that largely determines the cytotoxic response and pathophysiological effects [37–39].

Some authors refer that the safety assessment of QDs alone or conjugated is of vital importance since it will allow predicting the effects when interacting with a biological system. They suggest that a nanomaterial is small enough to enter a cell and its cellular compartments, regardless of the route of administration [40–42]. For systemic drug delivery, the intravenous (IV) route is used, which is a major challenge in the development of nanotherapies [43]. The US Food and Drugs Administration (FDA) has approved NMs that have been studied in rigorous preclinical studies combining therapeutic and biological targets as drug delivery agents [44–46].

Our working group has been given the task of synthesizing colloidal CdS-dex/dox QD and evaluating on HeLa cell. We treated HeLa cells with CdS-dex and CdS-dex/ dox to compare the selectividad of uptake alone as well as bioconjugated (1 μg/mL) in both cases and with doxorubicin at the same concentration. After 24 h of incubation and in order to investigate the cellular absortion of QD, cells were fixed on slides for visualization by confocal fluorescence microscopy. Through visualization of fluorescence and cellular uptake, we can observe that in cells treated with CdS-dex QDs without bioconjugation, there was a higher distribution in cytoplasm, nucleus, and nucleoli of the cell. However, this cellular uptake and distribution were not the same in the case of HeLa cells treated with doxorubicin and CdS-dex/dox. Nevertheless, in cells treated with doxorubicin and CdS-dex/dox, a significant increase in cell size was observed compared to cells treated with QDs alone. Although, QDs did not appear

homogeneous throughout the cytoplasm and with lower fluorescence intensity in the nucleus (**Figure 3**). They can also induce not only cytotoxic but also genotoxic effects in both normal and cancer cells [47–50].

Although, it has been shown that the effect after cellular uptake of various QDs depends on their size, shape, concentration, and cell type. The cytotoxic effect and mechanisms of nanotoxicity by the interaction of QDs with cells remain complex to assess and far from fully understood. However, this nanotoxicity has been shown to occur intracellularly or extracellularly [51]. QDs can even interact directly with biomolecules once inside the cell, due to their minute size. As a result of this interaction, an alteration in cellular equilibrium coexists, as well as irreversible morphological

#### **Figure 3.**

*Fluorescent microscopic visualization of doxorubicin, CdS-dex, and CdS-dex/dox QD in HeLa cell. Cells were treated for 24 h at 0,01–1 μg/mL concentration of doxorubicin, CdS-dex, and CdS-dex/dox QD. Cells were seeded on slides by smearing and allowed to dry, then analyzed using a confocal epifluorescence microscope. Green fluorescence shows the presence of CdS-dex QD. Red emission shows fluorescence in the presence of doxorubicin and CdS-dex/dox QD. The yellow arrow represents the increase in size and the white arrow indicates the absence of QD.*

#### *Toxicity of Quantum Dots DOI: http://dx.doi.org/10.5772/intechopen.112073*

and functional damage [51]. Even if indirectly the outside of the interacts with QDs through membrane receptors that cause activation and inhibition of different signaling pathways, causing toxic reactions or cell death [52].

Therefore, the cytotoxicity of QDs is more complex than we can imagine, it can cause not only the interaction with heavy metals contained in QDs but the disintegration of the core and the release of Cd ions, which increases their toxic potential. Under this condition, researchers have expressed concern about the use of NM and the parameters to be evaluated for future medical applications. This question arises from the association of adverse effects derived from the ability of QDs to enter cells and lodge in various subcellular compartments. This implies that they could evade the defense mechanisms of the human body, cross biological barriers and even interact with components of blood circulation [53]. Moreover, the blood circulation is the primary passage of NMs to the distribution of target organs. Thus, vascular endothelial cells serve as the first barrier and are tasked with maintaining vascular integrity [54]. In a study with ZnO nanoparticles, it has been shown that they are capable of causing cytotoxicity in HUVEC cells due to the increase of intracellular reactive oxygen species (ROS) in a dose-dependent manner [55]. Our studies have shown that at concentrations of 0.01 μg/mL, CdS-dex QDs already cause cytotoxic effects in HUVEC cells. The QDs are distributed around the cytoplasm, producing an increase in cell size and completely changing the characteristic morphology of the endothelial cell (**Figure 4**). Although it does not penetrate into the nucleus and nucleoli, cellular uptake occurs in a dose-dependent manner. In addition, endosome formation is observed, suggesting that cell deformation and toxicity are caused by cellular stress following the passage of the QD into the cell. The cytotoxicity produced by QDs is the

#### **Figure 4.**

*Fluorescent microscopic visualization of HUVEC cells treated with CdS-dex QD at 0,01–1 μg/mL concentration and 24 h time exposure. Cells were seeded on slides by smearing and allowed to dry, then analyzed using a confocal epifluorescence microscope. Green fluorescence shows the presence of CdS-dex QD.*

main parameter limiting their use in bioimaging research. The idea of applying QDs that produce morphological changes and ultimately cell death is a determining factor. Currently, joint efforts are being made for the development of innovative QDs capable of meeting the needs in healthcare areas. This progress in QD design and synthesis has resulted in improved safety *in vitro* studies. However, a myriad of factors that lead to cytotoxicity of QDs in normal, cancer, and endothelial cells remain in question. It has also been demonstrated that when QDs come into contact with organisms, they produce toxicity that is size-dependent, concentration threshold-dependent, and varies according to cytosensitivity [56]. However, factors such as concentration range are responsible for the intracellular distribution, which necessitates storage and bioaccumulation and thus increases cytotoxicity [57]. There is still a long way to go to achieve an accurate understanding and standardized parameters on safety for the use of quantum dots in the field of biomedicine.

In a whole decade, we have been dedicated to the design, synthesis, and nanotoxicological evaluation of quantum dots so we are very clear that, quantum dots can be improved in their design and composition. In addition, the nanoparticle size must be strictly controlled as it is one of the main factors influencing the toxicological effects of quantum dots [53]. The idea of having a complete profile of a type of nanomaterial is not unrealistic. However, it is necessary to demonstrate with studies on its preclinical evaluation. These evaluations include physicochemical characterization, *in vitro* evaluations with different types of human tumor and healthy cells, biodistribution, bioaccumulation, and pharmacokinetic studies. In addition, to perform exhaustive evaluations on its hemocompatibility as a starting point to rule out the toxic effect of a nanomaterial.
