**2. Semiconductor QDs (SQDs): synthesis methods, modification, cytotoxicity, and application in cancer cell imaging**

This class of QDs is usually core-shell (**Figure 1**), in the primary classification, the biocompatible fluorescent core consists of elements from groups II–IV, for example, CdSe, or groups III–V, for example, InP and is covered by a semiconductor shell which improves efficiency and photostability (ZnS, ZnSe). Finally, a variable outer layer such as silica can offer a large surface area for surface functionalization that enables their dispersion in water, and functionalized with other biomolecules (like peptides, antibodies, nucleic acids, and small-molecule ligands) to target specific proteins expressed on the surface of the cancer cells [12, 18, 19].

The methods of synthesizing SQDs are different depending on the used material, desired size, quantum yield and their applications [20]. Synthesis through high temperature, synthesis through γ-Irradiation, microwave-assisted method, sol–gel technique, and core-shell technique are among the common methods for synthesis of SQDs. In the high-temperature method, the NaHTe solution (mixture of potassium tellurite and sodium borohydride which were previously heated under N2 protection) was injected into the N2-saturated Cd2+ –MSA–alginate precursor. Then, the reaction mixture was heated to reflux (100°C) under atmospheric conditions with a condenser attached for different time intervals. Finally, the as-prepared CdTe alginate QDs were precipitated with ethanol, collected via centrifugation and dispersed in distilled water [21]. In the

#### **Figure 1.**

 *Schematic diagram of SQDs structure; QDs consist of an inorganic (fluorescent) core, an inorganic shell, and aqueous organic coating [ 17 ].* 

gamma irradiation method, the aqueous solution containing CdCl2 and SeO2 is aerated under N2. Then the solution is irradiated with gamma ray. After irradiation, the synthesized quantum dots are separated by centrifugation at 10,000 for 5 min [ 22 ]. In the microwave-assisted method, the 3-mercaptopropionic acid is added to an N2-saturated CdCl2 solution (solution 1). Then the desired pH is adjusted using NaOH (about 11). After that, the freshly prepared NaHTe solution is added to solution 1 and exposed to microwave irradiation at 120°C for 2–5 min to obtain monodispersed CdTe core QDs. Next, the GSH is added to an N2-saturated CdCl2 solution (solution 2) and adjusted pH to 11 by NaOH. Then freshly prepared NaHSe solution is added to solution 2 and exposed to microwave irradiation at 60°C for 3–20 min to obtain CdTe/CdSe core/shell QDs [ 23 ]. In the sol–gel technique, the materials exist in solution form and grow at low temperature to form a wet gel. This technique is utilized for the formation of Au, Cu, and Zn QDs [ 20 ]. In the core-shell technique, synthesis is done by the formation of metal droplet precursor and intermediate growth on the surface of quantum dots [ 20 ].

 In order to increase the luminous efficiency and stability of QDs, some modifications are made on their surface. Organic modification of SQDs which include organic ligand, organic polymer, proteins, and other organics modifiers. Inorganic modifications that include elements such as Co 2+, Ni 2+, Mn 2+ , and Cu 2+ doped into SQDs [ 24 ]. SQDs, especially Cadmium (Cd)-containing QDs, despite having effects such as high sensitivity and strong stability, but due to cytotoxicity, especially in normal cells, they are not very effective in cell imaging [ 25 ]. The released Cd ions are highly toxic and lead to cell damage. In addition, the small size of nanoparticles is also very important, where studies have shown that CdTe-QDs with a size of 2.2 nm are more toxic than large particles with a size of 5.2 nm [ 26 ]. Furthermore, different studies indicated that CdTe QDs cause damage to the DNA of HUVEC cells by inducing the production of reactive oxygen species (ROS) [ 27 ]. Thus, this type of QDs due to having heavy metals cannot be used for clinical treatment. Therefore, Copper indium sulfide (CuInS 2 , CIS) QDs proposed as a non-toxic and potential alternative. CIS QDS with a ZnS shell (CIS/ZnS) have a high photoluminescence quantum yield that can be easily transferred in aqueous medium and facilitate their application in biological imaging [ 25 ]. Liu et al. synthesized CuInS2/ZnS QDs as a near-infrared (NIR) fluorescence nanoprobe. They used Arg-Gly-Asp (RGD)-labeled bovine serum albumin-poly (ε-caprolactone)-coated CuInS2/ZnS QDs to further evaluate the cytotoxicity and in

#### **Figure 2.**

 *Application of CuInS 2 QDs in cancer cell imaging. (A) Confocal fluorescence image of U87 and HeLa cells treated with nontargeting nanoprobe (a, c), and treated with cRGD-functionalized nanoprobe (b, d). reprinted with permission from [ 26 ]. (B) NIR fluorescence images of RR1022 tumor-bearing mouse after intravenous injection with cRGDyk-GCM-QDs. reprinted with permission from [ 27 ]. (C) Confocal fluorescence image of MCF10CA1a breast tumor cells treated with GSH-capped CuInS2/ZnS QDs. reprinted with permission from [ 28 ].* 

vitro tumor targeting in U87 and HeLa cells. As shown in **Figure 2A** , the presence of cRGD significantly increases the internalization of QDs in two kinds of cancer cells. In addition, BSA as an outer shell QDs, significantly reduced non-specific cellular binding and improved biocompatibility [ 28 ]. In another study, Kim et al. synthesized Cd-free high-quality CuInS2/ZnS core/shell QDs and used for in vivo tumor targeting in RR1022 cancer cell xenograft mice ( **Figure 2B** ). Their results showed that after intravenous injection of cRGDyk-GCM-QDs, the NIR fluorescence emission increased with time in the supine and prone positions of the mice which indicates high tumor uptake [ 29 ]. In the study conducted by Zhao et al., the ability to use GSHcapped CuInS2/ZnS QDs in cancer cell imaging was investigated ( **Figure 2C** ). Their results indicated that the CuInS2/ZnS QDs can enter the cell and most of them were in the cytoplasm [ 30 ]. Previous studies have shown that CIS QDs without ZnS shells rapidly degrade and cause significant toxicity in blood chemistry, organ weight, and histology. Therefore, more caution should be taken in clinical practice [ 31 ].
