The CdX (X=Se, S, or Te) QDs are the most investigated QDs. However, the leaked cadmium ions are culprits for the observed cytotoxicity of cadmium-based QDs that hamper their further practical applications. However, with the emerging demand for more biocompatible QDs as the signal reporter, heavy metal-free QDs were developed such as group IV QDs including carbon-based QDs and silicon or germanium QDs (See Figure 1D for the core-type QDs).

Core-shell QDs are the second-generation products that have been widely used as a way to adjust the photophysical properties of simple QDs. Their shell is designed carefully to enhance simple QDs photostability and photoluminescence efficiency by several folds. The core and the shell are typically composed of type II-VI, IV-VI, and III–V semiconductors with configurations such as (CdS) ZnS, (CdSe) ZnS, (CdSe) CdS, and (InAs) CdSe (See Figure 1D for the core-shell type QDs).

Alloying QDs are one of the hot topics in QDs research. Alloyed QDs such as cadmium selenium sulfide (CdSeS) with both homogeneous and gradient internal structures are the newest generation of highly luminescent QDs. The homogeneous QDs have a uniform internal structure, thus the composition is same everywhere on a single QD, while in gradient QDs, alloy compositions are varied radially which means that the ratio of the first semiconductor and the second semiconductor changes gradually from the core to the surface in a gradient internal structure (7). Gradient and homogeneous alloyed QDs have varied optical and electrical properties due to their different structures, hence the internal structure of alloyed QDs is an important parameter in their applications (See Figure 1D for the alloying type QDs).

The ligand exchange process is simply defined as the substitution of a functional ligand for a nonfunctional ligand in order to provide QDs additional features such as solubility and stability ( Figure 2A ). Thiols (-SH), carboxyl (-COOH), and PEG are the most commonly used ligands. Thiol groups are frequently utilized as anchoring groups that bind to the surface of QDs, while carboxyl groups are frequently used as hydrophilic endings that provide extra stability, and PEG is frequently used to improve QDs solubility range from pH 5 to 12 for more than a year (8). However, there are a number of drawbacks of this method. For instance, in aqueous environments, the thiol molecules may form disulfides and detach from the surface, producing QDs aggregation and oxidation, and the surface change may result in an irreversible drop in QDs quantum yield.

Silanization, which involves the insertion of a silica shell over the QDs, is an effective covalent coating approach for modifying hydroxyl-rich material surfaces ( Figure 2B ). The main advantage of silanization is that the ligand molecules are highly cross-linked and form a chemically stable capping agent. Besides improving the biocompatibility, the end terminal groups of the silane shell can expose either their thiol, phosphonate, or methyl terminal ends for further coating of QDs. Moreover, silanization is also preferred because of less toxicity compared to other ligands (9). Finally yet importantly, QDs response to light can be finely tuned by controlling the thickness of the silica shell. CdSe/CdS/ZnS-QDs, for example, have been encapsulated in silica nanoparticles using a water-in-oil reverse microemulsion method (10). To test their applicability, the silica-coated QDs are next modified with amino, carboxyl, and epoxy groups and stabilized with polyethylene glycol (PEG) fragments. These modified QDs were found efficiently conjugated with antibodies and applied as a fluorescent label in the immunoassay detection (11).

Amphiphilic polymers are used to provide additional stability and flexibility to QDs under complex biological conditions. Poly (acrylic acid) copolymer, the highly charged linear polyelectrolytes containing carboxylic acid groups, are a class of amphiphilic polymers. Abdolahi and colleagues have reported the fabrication of a starch-g-poly (acrylic acid)/ZnSe-QDs hydrogel that may act as a dye adsorbent and photocatalyst (12). This approach has received much attention in designing efficient photocatalysts considering the high stability of QDs in the hydrogel system ( Figure 2B ). Phospholipids are another type of amphiphilic polymer with a polar and nonpolar portion in the structure that impart great biocompatibility and amphiphilicity in function. Besides, they also have a good emulsifying property that can stabilize the emulsions ( Figure 2B ). These unique features make phospholipids most appropriate for biological applications. For instance, in vitro and in vivo imaging by QDs encapsulated with phospholipid micelles was described by Dubertret et al. (13). This QD-based probes performed as in vitro fluorescent probes when coupled with DNA, hybridizing with particular complementary sequences, and showed to be stable, nontoxic, cell-autonomous, and photobleached slowly when injected into Xenopus embryos.

Incorporating QDs in microspheres or microbeads is one of the great interests in biological applications. For example, Liu et al. designed the QD-microsphere-based immunochromatographic quantitative ciprofloxacin (CIP) test strips (14). The QD-monoclonal antibody probes adhered to CIP and were unable to be caught by the CIP-bovine serum albumin conjugation dispersed along the T lines, resulting in reduced fluorescence intensities. These test strips provide a low detection limit and a wide linear detection range with high sensitivity and accuracy along with good selectivity, reproducibility, and stability which might be used in rapid on-site testing.

In mouse models, QDs coupled with several cancer indicators have been examined in vivo. For instance, Gao et al. were the first to apply QDs-antibody conjugates in vivo (6) ( Figure 4A ). They administered QDs-PSMA antibody systemically to a mouse bearing subcutaneous human prostate cancer. The QDs-antibody conjugates distributed efficiently and uniformly in the prostate tumor due to the specific binding between PSMA antigen in prostate cancer cells and QDs-PSMA antibody conjugates. Similarly, Liu et al. introduced a nanosystem that allows selective background quenching to gain exceptional tumor-specific fluorescent signals (46). This system uses near-infrared QDs and a membrane-impermeable etchant as a cation donor. Briefly, QDs were delivered intravenously into orthotopic breast and pancreas tumors in mice using the tumor-penetrating iRGD peptide. Subsequently, etching quenches excess QDs, leaving intact QDs in the extravascular tumor cells to deliver a highly tumor-specific signal and facilitating the renal clearance of metal ions released from QDs.

Apart from that, QDs can also detect multiple cancer markers such as circulating tumor cells (CTCs) and multiple biomarkers with more sensitivity than standard immunohistochemistry (47, 48). CTCs are a kind of tumor cells playing vital roles in cancer metastases, as their capture, isolation, and genetic profiling are meaningful for the early diagnosis and control of metastatic. Nondestructive and sensitive detection is essential for learning more about CTCs. For example, Kuo et al. used antibody (anti-CD24 or anti-CD133) coupled QDs to image serum CTCs in real time in living mice (49). They developed a noninvasive cancer model by injecting pancreatic cancer cells containing fluorescent proteins into the earlobes of mice. CTCs with fluorescent proteins in the bloodstream may be seen consistently after breaking off from the solid tumor. In another study, QDs were employed for CTCs “omics” (50, 51). In 90% of cases, CTCs could be trapped individually, and the expression level of protein biomarkers on a single CTC could be measured (52). As showed in Figure 4B , a typical model has been used for detecting CTCs.

Because various emission wavelengths activated by a single light source may be created by altering chemical composition and size, QDs offer considerable benefits in multiplexed diagnostic detection ( Figure 5A ). Multiple tumor markers in clinical samples can be diagnosed simultaneously with great accuracy and consistency by combining QDs-encoded microbeads with flow cytometry (53). Guo et al. constructed a multiplex electrochemiluminescence (ECL) immunoassay for simultaneous assessment of two unique tumour markers, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), using multicolor QDs as labels and graphene as a conducting bridge (CEA) (54). A standard sandwich immune complex was established on the glass carbon electrode to obtain recognizable ECL signals, with QDs525 and QDs625 tagged on secondary anti-AFP and anti-CEA antibodies, respectively. With a working range of 0.001-0.1 pg/ml and no noticeable cross-reactivity, the multiplex ECL-immunoassay allowed simultaneous monitoring of AFP and CEA in a single run. This immunoassay provides a simple, sensitive, specific, and repeatable approach for simultaneously detecting tumor markers in clinical situations. Qu et al. used three QDs-encoded microbeads (Qbeads) to simultaneously identify three miRNA biomarkers, miRNA-21, miRNA-221, and miRNA-16, in ~500 human hepatoma cancer cells (55). These tests can be completed in a single step, resulting in low cost and simple operation. However, the majority of the biosensors described thus far can only perform double or triple analysis.

Collectively, these approaches not only identify multiple biomarkers or CTCs but also assess circulating microRNAs (56). As shown in Figure 5B , QDs have been applied in a model of circulating miRNAs detection and exosomes to acquire integrated information (57).

QDs have a lot of potential in cancer bio-imaging because of their brilliant fluorescent signals and multiplex capabilities. They have a lot of selectivity and sensitivity in detecting early-stage tumors and their metastases. Akerman et al. originally investigated the application of QDs for in vivo imaging (58). They discovered that CdSe/ZnS QDs coated with peptides were preferentially distributed among endothelial cells in the lung blood vessels after injecting them into mice tail veins. However, QDs’ efficiency is severely limited by tissue autofluorescence, absorption, and photon scattering. Thus, to separate tissue autofluorescence from QD signal in transplanted malignancies, a spectral demixing technique must be created. Kim et al. were the first to address this issue by injecting NIR-emitting QDs intradermally into living mice and pigs to improve the signal-to-noise ratio by reducing photon scattering and penetrating deeper into tissues (59). Since then, QD-based immunohistochemistry combined with NIR-I (650-950nm) and NIR-II (1000-1350nm) fluorescence imaging has been widely used in personalized oncology to assess tumor origin and progression.

In recent years, many heavy element-free QDs with emission in the NIR-I and NIR-II windows have been developed. Ag2X (X=S, Se, and Te) QDs are great candidates since they are brilliant and photostable (60, 61). After biofunctionalization, these QDs could be used for in vivo fluorescent tumor detection and imaging (62–65). NIR QDs with various components have also been synthesized for bio-imaging, including dazzling CuInS2/ZnS QDs with tunable emission from 750 to 1100 nm (66), and 1200nm emitting PbS/CdS/ZnS QDs (67). However, developing NIR-II QDs with a high photoluminescence quantum yield (PLQY) and good biocompatibility is difficult. In a mouse model using multiplexed lymph node imaging, Saeboe et al. reported the reddest emitting indium phosphide quantum dots (InPQDs) to date. In the first optical tissue window, they exhibited tunable NIR photoluminescence (PL) as well as PL multiplexing while avoiding hazardous components (68). By widening the range of controllable direct-bandgap emission from InP-based nanostructures, these nanoparticles efficiently overcome a synthetic barrier that has stopped InPQDs from reaching their full potential. Many technologies have been employed to give real-time cancer imaging in vivo, as illustrated in Table 2 (58, 69–73). Overall, such high-quality NIR QDs could provide unrivalled sensitivity, speed, and real-time dynamic in vivo imaging.

High spatial and temporal resolutions, 3D tomography, a high signal-to-noise ratio, and noninvasiveness are all sought for in modern imaging technologies, whether for basic research or biomedical applications. However, owing of differences in biodistribution and other pharmacologic properties, a single imaging method cannot address all of these needs, and separate deployment of numerous imaging probes with diverse modalities is not an appropriate solution either. As a result, in order to achieve multimodal imaging technology, it has been attempted to unite the features of many imaging modalities in the same chemical entity. Because they can integrate a variety of probe properties, QDs are one of the most exciting multimodal probes. When fluorescence imaging with QDs is combined with molecules/materials that exhibit paramagnetism and radioactivity, for example, MRI and radiography imaging can be combined (74). MRI-fluorescence imaging is one type of bimodal imaging using QD probes, which has the advantage of being durable in multimodal imaging. Mulder et al. looked into multifunctional CdSe/ZnS QD probes as an example of QD-based MR-fluorescence bimodal imaging. They used their multifunctional probe to successfully target endothelial cells, which was detected using both fluorescence and MRI imaging (75). Mn-doped QDs particularly Mn-doped ZnSe QDs are another type of QDs-based MR-fluorescence bimodal imaging with a quite low concentration of dopant (Mn) (76). To achieve fluorescent/MRI dual-modal bio-imaging in vivo, Mn-doped ZnSe QDs were loaded into pores of mesoporous silica nanoparticles. Similarly, magnetically tailored Cd-free CulnS2 @ ZnS: Mn QDs were developed as potential dual-modality probes for fluorescence and MRI imaging of malignancies in vivo and found to be effective against both subcutaneous and intraperitoneal tumors (77). Individual imaging technology has advanced to the point that biomedical cancer imaging could gain a new dimension and momentum with the design and synthesis of appropriate multimodal probes based on QDs that meet in vivo cancer imaging standards. Magneto-fluorescent GdNS@CQDs with remarkable water dispersibility, high stability, high quantum yields, and outstanding magnetic characteristics were successfully synthesized in an independent investigation, and these qualities made them suitable for dual model imaging (78). Multimodal probes appears to be realistic, given the rapid expansion of QDs and the abundance of information on the molecular basis of cancer and other illnesses.

The cytotoxicity of QDs is one of the major concerns that has slowed their development. Much of the research shows that this toxicity is dose-dependent (79, 80). For example, Lu et al. studied the effects of CdSe/ZnS core/shell QDs in hepatic L02 cells and observed that cytotoxicity rises with QD concentration (from 5 to 80 nM) (81). Similarly, Yang et al. found that a high dose (5 nM per rat) of QDs causes severe toxicity in lactating rats, including splenomegaly, multiple organ injuries and inflammation, endocrine disruption, and rat death, while a low dose (1 nM per rat) causes mild toxicity, including weight loss, mild hematology, serum biochemistry, and histopathological changes (82).

The cytotoxicity of QDs is mostly governed by their physiochemical characteristics, including hydrodynamic diameter (HD) size, surface properties like charge or ligand, and shape. The HD size, determined by both the inorganic core size and the surface coating, has a tremendous impact on the uptake and clearance of QDs (83, 84). Choi et al. used a rodent model to test renal filtration and urine excretion of QDs with five distinct HD diameters ranging from 4.36 to 8.65 nm (85). When the size was smaller than 5.5 nm, a rapid and efficient urinary excretion from mice was clearly observed. However, QDs with larger HD size were not captured for renal excretion. Apart from excretion, the blood half-life increased 25-fold, from 48 min to 20 h, with the HD size increase from 4.36 to 8.65 nm. The presence or lack of a shell, the surface charge, and other surface characteristics of QDs can have a variety of effects on their toxicity. The better protected the shell is, the slower the discharge of heavy metal ions, which is one source of QD toxicity. According to Kirchner et al., ZnS coating protects the QD core from oxidation, reducing Cd²⁺ leakage and cytotoxicity (86). On the other hand, drug transport studies in the kidney show that in contrast to negatively-charged QDs, positive-charged QDs readily pass through the anionic glomerular basement membrane and are rapidly excreted into urine which means less cytotoxicity exists in the body (87). Because most QDs are spherical or spheroid-like, their form isn’t an apparent influencing element. In endocytosis and phagocytosis, the shape of QDs is useful in the membrane packing process. Endocytosis of spherical QDs is quicker than that of other non-spherical nanomaterials, such as carbon nanotubes, according to certain research, which indicates less harmful consequences (88, 89).

Pharmacokinetic factors such as half-life duration, biodistribution, degration, rate and route of excretion, and others can be used to quantify the impact of the physicochemical qualities listed above on toxicity. A lot of research into the pharmacokinetics of QDs in cells and small animals have been done so far. Ballou et al. investigated the impact of surface coating on blood circulation and organ biodistribution in the first place (90). Polyacrylic acid-coated QD (PAA-QD) conjugated to low molecular weight PEG (750 Da) and intravenously injected into nude mice exhibited short blood circulation half-life (t1/2 < 12 min) with predominant uptake by the liver, spleen, lymph nodes, and bone marrow. When the same QD was decorated with PEG5000, the blood t1/2 was raised to 3 hours, with reduced uptake in the liver, spleen, and lymph nodes (90). Similar studies by other groups showed that 15-20 nm QD coated with PEG5000 exhibited long t1/2 of 5-8 h (3, 6, 91, 92). Although rapid clearance of QDs from the body may reduce the potential toxic damage influence on the organs, tissues or cells, sufficiently long blood half-life time is desired for enhancing their accumulation in the targeted site. Furthermore, in all these reports, the QD biodistribution was qualitatively determined based either on QD fluorescence in tissue sections using fluorescence and confocal microscopy or the whole body fluorescence imaging of living animals. Fischer et al. described the first quantitative biodistribution study of QD by detecting the Cd atoms in the blood and organs of rats injected intravenously with QD (93). Once QDs have been taken up by the target cells, they are mainly directed to the endosomal/lysosomal pathway where they are degraded. Some QDs disintegrate quickly, but others can linger in the body for weeks or months, causing serious harm. Previously, Ballou et al. showed that QD could be found in the liver, lymph nodes, and bone marrow of mice for up to many months (90). Frangioni and colleagues found a link between the size of the QD and the degree of elimination (85, 94). Four (4) hours after injection, QD with an average diameter of 5-6 nm, which is below the renal filtration threshold, were eliminated via urine. Larger QDs stayed undesirably in the liver, potentially increasing the toxicity of these nanoparticles in the long run. The fate of QD after various administration routes has also been investigated. After subcutaneous, intradermal, or intraparenchymal injection in live animals, polymer-coated QD with an average diameter of 15-20 nm were observed to move quickly to the sentinel lymph nodes (SLN) (59, 95–98). This observation of detecting SLN resident nodules, can aid in the detection of cancer metastasis. Overall, it can be observed that depending on the properties of the QD (size, surface charge, and coating) and the method of administration, QD can remain in various organs in living animals. Furthermore, these investigations have revealed that QD may accumulate in the body for long periods of time, indicating that more research is needed to determine QD’s long-term toxicity before it is used in clinical applications.

In addition, studies have also shown that QDs can cause immunotoxicity (99–105) and genotoxicity (106, 107). QDs cause apoptosis and necrosis in immune cells, and poor clearance of apoptotic cells by scavenger phagocytes may contribute to autoimmunity (108), while QD interactions with the immune response might alter immune-specific signaling pathways, resulting in alterations in immune cell function (109). There are direct and indirect pathways of QD-induced genotoxicity, according to published research (107, 110, 111). When cells absorb QDs, they may come into direct contact with genetic material, inflicting physical or chemical harm. However, the most likely mechanism of QD-induced genotoxicity is indirect, and oxidative stress is thought to be the most important indirect mechanism (112).

In conclusion, to pave the way for clinical utilization of QDs, more long-term toxicological and pharmacokinetic studies addressing QDs degradation, excretion, persistence, and immune response and precise control over the construction of QDs-based probes from the core to surface coating are needed before QDs-based probes may be declared as verified safe nanoparticles. Furthermore, more studies are needed in synthesis process to develop heavy metal free or robust covering QDs to reduce toxicity Last but not least, monitoring of the quantitative application dosage of QDs to set up safe ranges under different conditions is also urgently required.

According to all in vivo animal imaging investigations described thus far, a number of naked and non-targeted QDs may accumulate in the reticuloendothelial system (RES), which comprises of phagocytic cells located in the liver, spleen, lymph nodes, and bone marrow, after systemic delivery. This non-specific absorption prevents QDs from being targeted and raises concerns regarding RES toxicity.

One way to improve tumor targeting specificity is through modifying the surface of QDs to make them persist longer in the bloodstream by introducing high molecular weight PEG molecules (113). Of course, the surface coating and QDs must be stable enough to circulate for an extended period of time. For instance, mice were injected with peptide-conjugation iron oxide nanoparticles after being pre-treated with decoy liposome particles to eliminate plasma opsonin that bind to nanoparticles and prevent RES absorption. By increasing the half-life of iron oxide nanoparticles, intravenously given decoy liposomes greatly improved active targeting of xenograft breast cancers (114). Similarly, another research conjugated streptavidin-coated QDs (SA-QDs) with hydrazinonicotinamide (HYNIC) to boost QDs negative charge to suppress surface opsonization (115). When delivered intravenously to mice, HYNIC attachment exactly reduced SA-QD engulfment by macrophages, drastically enhanced SA-QD circulation, and lowered their RES absorption, according to confocal microscopy and fluorescence-activated cell sorting. Overall, surface functionalization could minimize toxicity by reducing non-specific RES phagocytosis.

Selective tumor targeting of QDs in vivo is more difficult than in vitro for a variety of reasons. To begin with, complicated anatomical structure and physiology, such as vascular endothelium, provide challenges for QDs in tissues and organ systems. Secondly, protein-based ligands are sensitive to degradation, resulting in a loss of targeting capability, and most conjugation chemistries do not allow controlling over complex macromolecules tethered to QDs, such as antibodies, resulting in partial or entire loss of cell-binding activity. Finally, only a few cell targeting ligands are truly tumor specific, meaning that QDs bind solely to cancer cells and not to normal cells.

QDs targeting detection and imaging should be adjusted or aided by other reagents to pass through various biological barriers and reach the target areas to improve selectivity. For example, Yong et al. used permeation enhancers based on organic solvents in modest percentages to improve intracellular targeted distribution of QDs transverse intracellular obstacles, particularly vesicle entrapment (116). In another study, Li et al. demonstrated that carbon QDs functionalized with multiple paired -carboxyl and amino groups that bind to the large neutral amino acid transport 1 (LAT1), which is expressed in most tumors, selectively accumulate in human tumor xenografts in mice and in an orthotopic mouse model of human glioma with high specificity, by mimicking large amino acids (117). Furthermore, other tumor specific antigens (TSA), such as Mucin 1 (MUC1), which is absent in normal tissues but overexpressed in almost all human epithelial cell malignancies, should be investigated in QDs targeting applications.