Quantum Dots for Cancer Therapy and Bioimaging

Abstract

Quantum dots (QDs) usually refer to very small nanoparticles of only few nanometers in size. The optical and electronic properties of QDs differ from those of larger particles. QDs will emit light of specific frequencies if electricity or light is applied to them, and these frequencies can be precisely tuned by changing the dots’ size, shape, and material, giving rise to many applications. In this chapter, apart from the most common QDs, e.g., the cadmium (Cd)-containing semiconductor QDs, other types of QDs, including silver chalcogenide quantum dots, carbon quantum dots, silicon quantum dots, black phosphorus quantum dots, germanium quantum dots, and polymer dots are also introduced with an emphasis on their cancer therapy and imaging applications.

1 Semiconductor Quantum Dots

Semiconductor quantum dots are one of the most important QDs, whose size and shape can be precisely controlled by the duration, temperature, and ligand molecules used in the synthesis [1]. In comparison with conventional molecular fluorophores, the emission wavelength of QDs can be tuned by varying their size and composition. Due to the narrow emission and broad excitation spectra, QDs perform multicolor imaging with minimal spectral overlap and have the possibility to excite all colors of QDs simultaneously with a single light source. Furthermore, QDs display excellent photostability over molecular fluorophores, so that long-term imaging can be achieved without artifacts from photobleaching. Because of these unique optical properties, QDs are of wide interest and have emerged as a strong competitor as fluorescent probes for biomedical imaging and diagnostics applications [2, 3]. In particular, as one type of multifunctional materials, QDs exhibit specific advantages in tumor imaging and tumor therapy due to the large surface area which enables them to be conjugated with different agents including imaging substances, targeting molecules, and therapeutic agents. Herein, we focus on QDs or QDs-based nanomaterials for tumor imaging and therapy in recent years.

1.1 QDs for Tumor Imaging

Cancer remains one of the leading causes of death in the world. Diagnostic tumor imaging has gained a major role in the management of tumor therapy with qualitative and quantitative analyses of the biological processes of tumors by monitoring changes of tumor cells at tissue, cellular, or subcellular levels. There are various kinds of imaging techniques such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and fluorescence imaging. These techniques and the combined use of them help to provide accurate information of tumors from a variety of aspects. For this section, we overview the recent progress of visible- and near-infrared (NIR)-emitting QDs-based fluorescence imaging and multi-model imaging of tumor.

1.1.1 QDs-Based Fluorescence Imaging

Fluorescence imaging is commonly used as one of the most potent tools for cancer diagnosis by tumor-targeted imaging from cells and tissues of living animals. On the other hand, functionalized QDs which are modified with biomolecules possess many advantages over conventional organic fluorophores, such as high photoluminescence efficiency, great photostability, size-dependent emission wavelength, and sharp emission profile. In addition to their good biocompatibility and low toxicity, QDs emitting in the visible and NIR ranges have been widely applied in fluorescence imaging in vitro and in vivo as novel fluorescent probes [4,5,6,7,8,9,10,11].

1.1.1.1 Visible Fluorescence Imaging

Fluorescence imaging using light in the visible wavelength regime (400–700 nm) is a useful technology for cancer diagnosis due to its fast feedback as well as relatively good spatial resolution. Visible-emitting QDs have gained considerable attention in the last decade for in vitro and in vivo tumor imaging [12,13,14,15,16,17,18,19,20]. Zhang et al. offered a strategy to synthesize DNA-functionalized Zn²⁺-doped CdTe QDs (DNA-QDs) through a facile one-pot hydrothermal route [13]. The as-prepared QDs exhibit high quantum yield (up to 80.5%), excellent photostability, and low toxicity. Moreover, DNA has been designed as an aptamer specific for mucin 1 overexpressed in many cancer cells including lung adenocarcinoma, and the aptamer-QDs are designed for the first time for application in active tumor-targeted imaging in vitro and in vivo (Fig. 1a). Furthermore, an intelligent “on-off” or “off-on” switch system for tumor-targeted fluorescence imaging without unnecessary background signal is urgently desired. Zhang et al. reported a novel fluorescence turn-on probe for targeted imaging of folate receptor (FR)-overexpressed cancer cells based on the self-assembly of folic acid (FA) and polyethyleneimine (PEI)-coated CdS/ZnS QDs (PEI-CdS/ZnS QDs) [14]. The primary fluorescence of PEI-CdS/ZnS QDs turns off first upon the electrostatic adsorption of FA onto PEI-CdS/ZnS QDs based on electron transfer to produce negligible fluorescence background. The presence of FR expressed on the surface of cancer cells then makes FA desorb from PEI-CdS/ZnS QDs due to the specific and high affinity of FA for FR. As a result, the primary fluorescence of PEI-CdS/ZnS QDs adhering to the cells turns on due to the inhibition of electron transfer (Fig. 1b). Besides tumor-targeted imaging, recognition of a specific cancer cell type among various cell types is also essential for cancer diagnosis and targeted cancer therapy. In a recent study, Wang et al. presented a pattern recognition of cells via multiplexed imaging with three types of monosaccharide imprinted QDs [18]. Aberrant expression of glycan structures on the cell surface is a universal hallmark of cancer cells. For instance, sialic acid (SA) and fucose (Fuc) are overexpressed on most cancer cells, while mannose (Man) is overexpressed on certain cancer cells, such as liver cancer cells. Therefore, the combination of multiple monosaccharides can be effective for specific cancer recognition. As shown in Fig. 1c, the three-dimensional (3D) plot intuitively shows the spatial distributions of these cell lines in the coordinate system. The results disclose the similarities and differences of different cell lines, allowing for not only the recognition of cancer cells from normal cells but also the recognition of specific cancer cells. Thus, the study paves a solid ground for the design and preparation of novel cancer cell-targeting reagents and nanoprobes.

Fig. 1

a Photograph of aqueous solution of DNA-QDs under UV condition, and schematic illustration of the synthesis and tumor-targeted fluorescence imaging of DNA-QDs. Reprinted with permission from Ref. [13]. Copyright 2013 American Chemical Society. b Schematic illustration of FA-assembled PEI-CdS/ZnS QDs as fluorescence turn-on probe for imaging FR overexpressed cancer cells. Reprinted with permission from Ref. [14]. Copyright 2013 American Chemical Society. c Spatial distribution of the 11 cell lines constructed by multiplexed imaging via simultaneously staining with Man-, SA-, and Fuc-imprinted QDs. Reprinted with permission from Ref. [18]. Copyright 2017 American Chemical Society. d Schematic in vitro microscopy and live-cell imaging of kinesin labeled with seven Gly-Ser repeats (SNAP-K560_14GS-GFP) and red sulfoBG QDs. Reprinted with permission from Ref. [19]. Copyright 2017 American Chemical Society. e Identification of single malignant tumor cells in a predominantly benign prostate gland by QD multiplexed staining of four protein biomarkers (E-cadherin, green; CK HMW, white; p63, red; and AMACR, blue). Reprinted with permission from Ref. [12]. Copyright 2010 American Chemical Society. f Fluorescence imaging of the tumors and normal brains using PEG-QDs or NGR-PEG-QDs after the tail vein injection. Reprinted with permission from Ref. [20]. Copyright 2017 Elsevier

On the other hand, QDs-based cancer molecular imaging has emerged as an important technique for cancer detection, personalized treatment, drug development, and imaging-guided surgery. Over the past decade, various types of cancer molecules, including nucleic acids [6, 8], proteins [12, 19], cell-surface receptors [17], and antigen [21] have been conjugated onto QDs to identify and target cancer cells. Notably, protein-targeting strategy plays a significant role and has attracted much attention. To improve protein labeling efficiency, Wichner et al. reported compact aqueous CdSe/CdS QDs with superior single-molecule optical property [19]. These QDs are able to label SNAP-tagged proteins ~10-fold more efficiently than existing SNAP ligands. Furthermore, the QDs show that 99% of time is spent in the fluorescence on-state, ~fourfold higher quantum efficiency than standard CdSe/ZnS QDs. Figure 1d shows that these bright QDs can track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables the tracking of single kinesins in live cells. Additionally, labeling protein biomarkers by multicolor QD probes is an effective method for investigating tumor heterogeneity and complexity. For example, Liu et al. utilized QD-based spectral imaging for high-throughput digital mapping of molecular, cellular, and glandular variations on surgical prostate cancer specimens [12]. They detected and identified a single malignant tumor cell from the complex microenvironments of radical prostatectomy and needle biopsy tissue specimens using a panel of just four protein biomarkers (E-cadherin, high-molecular-weight cytokeratin, p63, and α-methylacyl CoA racemase) (Fig. 1e). The multiplexed QD mapping provides correlated molecular and morphological information that is not available from traditional tissue staining and molecular profiling methods. For the in vivo tumor fluorescence imaging, delivery of imaging agents to brain glioma is challenging because the blood–brain barrier (BBB) functions as a physiological checkpoint guarding the central nervous system from circulating large molecules [22]. Thus, BBB limits drug delivery to brain parenchyma, attenuating the diagnosis and therapy effect of brain tumors. Fortunately, QDs offer great promises for crossing the BBB and reaching brain parenchyma based on their ultrasmall sizes, contributing to the development of theranostic nanoprobes for various neurological disorders. In a very recent study, Huang et al. synthesized a novel nanoprobe by conjugating biotinylated asparagines-glycine-arginine (NGR) peptides to avidin-PEG-coated QDs [20]. These QDs can cross the BBB and target CD13-overexpressing glioma and tumor vasculature in vitro and in vivo, contributing to the fluorescence imaging of the brain malignancy (Fig. 1f). This nanotechnology highlights a novel prospect for the molecular diagnosis and image-guided neurosurgery of glioma.

1.1.1.2 NIR Fluorescence Imaging

NIR-emitting QDs (NIR QDs), allowing lower tissue absorption and scattering, lower undesirable NIR autofluorescence, and deeper penetration depth, have recently been explored as highly promising imaging probes. These high-quality NIR-emitting QDs are especially useful in cellular labeling, deep-tissue imaging, and tumor targeting. In particular, two optimal wavelength ranges of 700–950 and 1000–1350 nm, known as the first and second biological windows (I-BW and II-BW), respectively, have been identified. In the I-BW (700–950 nm), the light is minimally absorbed by tissue components as compared to visible light, resulting in greater penetration and thus deeper imaging. At even longer wavelengths of the II-BW (1000–1350 nm), a greater reduction in the scattering cross section leads to a further improvement in the detection depth and resolution. In this section, we will summarize the recent advancements of the NIR QDs for in vitro and in vivo tumor imaging.

To date, a variety of QDs emitting within I-BW (NIR-I QDs) with well-controlled structure and multifunctional properties as novel biolabeling agents have been developed [4, 23,24,25,26]. For instance, Liu et al. synthesized N-acetyl-l-cysteine (NAC)-capped CdHgTe/CdS core/shell QDs. The QDs have NIR-I fluorescence (Fig. 2A, a) and are successfully applied for in vivo tumor imaging of nude mice by passive targeting (Fig. 2A, b), indicating that these highly fluorescent probes can be very effective in long-term diagnostics and therapy in in vivo observation [26]. Miyashita et al. developed a new immunohistochemical (IHC) technique with NIR-I QD-conjugated trastuzumab using single-particle imaging to quantitatively measure the HER2 expression level (Fig. 2B) [25]. Moreover, they precisely calculated the number of QD-conjugated trastuzumab particles binding specifically to a cancer cell as the IHC-QD score. The use of IHC-QD score is believed as a predictive factor for trastuzumab therapy.

Fig. 2

A UV-PL spectra (a) and in vivo tumor targeting (b) of the NIR CdHgTe/CdS/ZnS QDs. Reprinted with permission from Ref. [26]. Copyright 2017 Royal Society of Chemistry. B Images of IHC stained with DAB-labeled HER2 antibody (top row) or QD-conjugated trastuzumab (middle and bottom rows). The middle row shows the images observed with 695–740 nm band-pass filter. The bottom row shows the images subtracting autofluorescence in tissues from the images of middle row to visualize the fluorescence of QDs (bright spots) only. Reprinted with permission from Ref. [25]. Copyright 2016 Wiley. C (a) PL spectra of initial PbS QDs, PbS/CdS QDs, and final PbS/CdS/ZnS QDs emitting at 1220 nm. (b) Optical (left) and luminescence (right) images of 930 nm emitting PbS/CdS/ZnS core/shell/shell QDs. (c) In vivo optical + fluorescence images of the mouse after injection with the two differently sized QDs solutions emitting in the first and second biological windows, respectively. Reprinted with permission from Ref. [28]. Copyright 2017 Wiley. D A schematic overview of the synthesis of core–shell and core–shell–shell short-wave infrared (SWIR) QDs and the subsequent functionalization for next-generation imaging applications. Reprinted with permission from Ref. [29]. Copyright 2017 Nature Publishing Group. E (a) Three different emission spectra from different PbS/CdS/ZnS QDs (blue, QDs emitting at 930 nm; green, QDs emitting at 1270 nm; red, QDs emitting at 1450 nm). The black line shows the extinction coefficient (comprising both absorption and scattering contributions) of biological tissues for the spectral range under consideration. (b) Fluorescence image of four HeLa cells incubated with a solution of 1270 nm emitting QDs with the excitation of 808 nm. (c) Sub-tissue fluorescence images taken at two different temperatures from the chicken breast previously injected with QDs emitting at 1270 nm. Reprinted with permission from Ref. [27]. Copyright 2015 Wiley

Compared to the widely used NIR-I QDs, the QDs emitting in the II-BW (NIR-II QDs) can realize in vivo imaging with much higher signal-to-noise (S/N) ratio and spatial resolution. Thus, the development of an effective aqueous synthetic route to high-quality and biocompatible NIR-II emitting QDs is highly appealing. Many efforts have been devoted to developing NIR-II QDs for in vivo fluorescence imaging [9, 11, 27,28,29]. Ren et al. reported water-dispersible PbS/CdS/ZnS core/shell/shell QDs emitting NIR-II fluorescence (Fig. 2C, a and b) with excellent colloidal stability and photostability [28]. These QDs are injected into mice for tumor imaging in small animals and show high brightness even at quite low concentrations, under significantly reduced NIR laser density excitations, with very short signal integration time and deep injection (Fig. 2C, c). Bruns et al. introduced a class of short-wavelength infrared region (SWIR: 1000–2000 nm)-emissive indium-arsenide-based QDs that are readily modifiable for various imaging applications [29]. These QDs exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. Then, they quantify, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generate detailed 3D quantitative flow maps of the mouse brain vasculature (Fig. 2D). Benayas et al. used water-dispersible core/shell/shell PbS/CdS/ZnS QDs as NIR imaging probes fabricated through a rapid, cost-effective microwave-assisted cation exchange procedure [27]. The emission wavelength of the QDs probe is within the second biological window (1000–1350 nm) (Fig. 2E, a). The in vitro (Fig. 2E, b) and ex vivo (Fig. 2E, c) experiments prove that the QDs are capable of high-resolution thermal sensing in the physiological temperature range. Together with their intense fluorescence, these PbS/CdS/ZnS QDs represent multifunctional probes both for in vitro and in vivo applications in biomedicine.

1.1.2 QDs-Based Multimodal Imaging

With the outstanding optical properties of QDs, the QDs-based fluorescence imaging techniques show great potential in extracting detailed biomedical information with high imaging sensitivity and low-cost imaging facilities in comparison to clinically used MRI, CT, and PET methods. However, the poor tissue penetration restricts the better biomedical research and related clinical applications of light-emitting QDs as fluorescence imaging probes. Therefore, the combination of QDs-based fluorescence imaging and other imaging techniques within a single nanoplatform has emerged as an effective approach to collect reliable biomedical information, thus improving the efficiency and sensitivity of clinical imaging diagnostics. In recent years, multimodal imaging with the combination of fluorescence imaging and other imaging techniques such as dark-field imaging, PET, CT, photoacoustic (PA), and MRI offers revolutionary imaging tools for biomedical applications and has ignited intense research interest worldwide [30,31,32,33,34].

Recently, the integration of MRI agents with QDs has been designed as a useful imaging modality pair for more accurate biomedical detections [30, 35, 36]. MRI is one of the most powerful medical diagnosis tools, which can visualize the anatomical structure of the body and extract physiological information with high spatial resolution and soft tissue contrast [37]. The MRI/fluorescence imaging technology enables significant improvement in diagnostic accuracy and therapeutic strategy, in comparison with standalone imaging. For example, Lai et al. presented In₂S₃/ZnS core/shell QDs co-doped with Ag⁺ and Mn²⁺ (referred to as AgMn:In₂S₃/ZnS) [36]. Ag⁺ can alter the optical properties of the host QDs, whereas the spin magnetic moment (S = 5/2) of Mn²⁺ efficiently induces the longitudinal relaxation of water protons. This is the first report of the aqueous synthesis of color-tunable AgMn:In₂S₃/ZnS core/shell QDs with magnetic properties. The obtained QDs have a satisfactory quantum yield (45%), high longitudinal relaxivity (6.84 mM⁻¹ s⁻¹), and robust photostability. As seen by confocal microscopy and MRI, AgMn:In₂S₃/ZnS conjugated to hyaluronic acid (referred to as AgMn:In₂S₃/ZnS@HA) can efficiently and specifically target to cancer cells. Moreover, AgMn:In₂S₃/ZnS@HA shows negligible cytotoxicity in vitro and in vivo, rendering it a promising diagnostic probe for dual-modal imaging in clinical applications (Fig. 3a).

Fig. 3

a Synthesis of AgMn:In₂S₃/ZnS core/shell QDs and their application in optical imaging and MRI. Reprinted with permission from Ref. [36]. Copyright 2017 Elsevier. b Schematic illustration, in vivo NIR fluorescence images, and T₁-weighted MRI of QDs@DTDTPA-Gd NPs. Reprinted with permission from Ref. [34]. Copyright 2017 American Chemical Society. c In vivo PA and CT images of 4T1 tumors on mice after intravenously injected with MoS₂ QD@polyaniline nanohybrids. Reprinted with permission from Ref. [33]. Copyright 2016 American Chemical Society. d Illustration of the intrinsically radioactive [⁶⁴Cu]CIS/ZnS QDs for PET/Cerenkov resonance energy transfer (CRET) luminescence imaging. Reprinted with permission from Ref. [32]. Copyright 2015 American Chemical Society. e Illustration of ZCIS QDs used as theranostic nanomedicines with intrinsic fluorescence/MSOT imaging and PTT/PDT therapy abilities. Reprinted with permission from Ref. [40]. Copyright American Chemical Society

Apart from the visible light-emitting QDs, the NIR-emitting QDs have gained more attention when integrating with MRI agents since they can minimize light absorption and diffusion and therefore maximize imaging depth [34, 38]. Yang et al. developed a bimodal contrast nanoagent by chelating gadolinium ions to 2-[bis[2-[carboxymethyl-[2-oxo-2-(2-sulfanylethyl-amino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA)-modified CuInS₂/ZnS QDs [34]. The longitudinal relaxivity of the resulted QDs@DTDTPA-Gd NPs is calculated to be 9.91 mM⁻¹ s⁻¹, which is 2.5 times as high as that of the clinically approved Gd-DTPA (3.9 mM⁻¹ s⁻¹). In addition, the in vivo imaging experiments show that QDs@DTDTPA-Gd NPs can enhance both NIR fluorescence and T₁-weighted MRI of tumor tissue through passive targeting accumulation. Moreover, the high colloidal and fluorescence stabilities and good biocompatibility ensure the potential use of QDs@DTDTPA-Gd NPs as an efficient nanoagent to integrate the extremely high sensitivity of fluorescence imaging to the high resolution of MRI (Fig. 3b).

CT and PA imaging, with higher density resolution and deeper tissue penetration, are widely used as diagnostic tools in biomedical applications. Wang et al. introduced a versatile nanomaterial based on MoS₂ QD@polyaniline nanohybrids, which exhibit not only fluorescence imaging of cancer but also enhanced PA imaging and CT signal in vivo (Fig. 3c) [33]. This versatile nanohybrid shows good potential to facilitate the multimodal imaging for a better imaging-guided tumor therapy.

Besides, PET is also a powerful biomedical imaging technique widely used for diagnostic applications in clinical oncology with the advantages of high sensitivity and quantitative accuracy. Attaching positron-emitting radioisotopes onto QDs has also attracted intense interest in both preclinical research and clinical applications. As a representative example, Guo et al. described a straightforward synthesis of intrinsically radioactive [⁶⁴Cu]CuInS/ZnS QDs by directly incorporating ⁶⁴Cu into CuInS/ZnS nanostructure with ⁶⁴CuCl₂ as a synthetic precursor (Fig. 3d) [32]. The [⁶⁴Cu]CuInS/ZnS QDs are demonstrated to have an excellent radiochemical stability with less than 3% free ⁶⁴Cu detected even after exposure to serum containing EDTA (5 mM) for 24 h. The PEGylated radioactive QDs show high tumor uptake (10.8% ID/g) in a U87MG mouse xenograft model. Overall, these [⁶⁴Cu]CuInS/ZnS QDs are successfully applied as an efficient PET/self-illuminating luminescence in vivo imaging agent.

On the other hand, multispectral optoacoustic tomography (MSOT) is also an indispensable imaging technique that overcomes the optical diffusion limitation by integrating the spectral selectivity of molecular excitation with the high resolution of ultrasound detection based on the PA effect [39]. Therefore, it is highly desirable to achieve theranostic nanomedicines with intrinsic fluorescence/MSOT dual-modal imaging ability. Lv et al. exploited CuInS/ZnS quantum dots (ZCIS QDs) as “all-in-one” versatile nanomedicines that possess intrinsic fluorescence/PA imaging for synergistic photothermal therapy (PTT)/photodynamic therapy (PDT) (Fig. 3e) [40]. The ZCIS QDs allow noninvasively monitoring tumor site localization profiles and thus hold great potential as precision theranostic nanomedicines.

Other multimodal technologies including fluorescence/CT and fluorescence/plasmic imaging based on QDs have also acquired wide attention [31, 41]. These nanomaterials have highly integrated multifunctions. For example, combining CT contrast agents and QDs into one nanoplatform provides more complementary and accurate information about the anatomical structure, as well as high-resolution and sensitive imaging capability at both tissue and cell levels. Besides, the integration of the fluorescence and plasmic imaging is potentially powerful in precise identification of subcellular targets by co-localization of fluorescence and light scattering (dark field) imaging.

1.2 QDs in Cancer Therapy

QDs are luminescent nanocrystals with rich surface chemistry and unique optical properties, which make them useful as probes or carriers for drug delivery in cancer therapy. Besides, due to their unique properties, QDs can also produce reactive oxygen species (ROS) or generate heat under irradiation to kill cancer cells. In this part, we summarize the recent advancements of QDs in cancer therapy.

1.2.1 QDs as Photosensitizers

PDT involves the administration of photosensitizers (PSs) followed by local illumination of the lesion using light of a specific wavelength to activate the PS. A series of photochemical reactions triggered by the PS can lead to the death of cancerous or bacterial cells [42]. Due to their unique optical properties, QDs can act as PSs and kill cancer cells under light irradiation. Dong et al. synthesized photoluminescent MoS₂ QDs with superior singlet oxygen (¹O₂) production ability exceeding the commercial photosensitizer PpIX, which has great potential for PDT [43]. He et al. demonstrated that CdSe/ZnS QDs with illumination can cause ultrastructural changes in pancreatic cancer cells, such as organelle degeneration and chromatin condensation and aggregation at the periphery of the nucleus due to the ROS generation [44].

Although QDs can be PS candidates, their photosensitizing efficacy is still not satisfactory for clinical applications. Thus, a new type of photosensitizer consisting of CdTe QDs with good photosensitizing efficacy, excellent water dispersibility, and stability was reported by Sun et al. [45]. Different from most of the previous reports, the as-prepared QDs do not inhibit the growth of normal cells in the experimental concentration range, but can act as a photosensitizer to specifically and remarkably inhibit the proliferation of human hepatoma cells. Mechanistic studies reveal that the QDs can be specifically internalized by hepatoma cells, considerably induce the generation of intracellular ROS under light illumination, and significantly induce the necrosis of hepatoma cells. This work provides an inspiration for the direct application of QDs as a new type of photosensitizer to treat human hepatoma through PDT.

1.2.2 QDs as Photothermal Agents

Besides being as PSs, QDs can also act as photothermal agents in photothermal therapy (PTT). The QDs which have strong absorbance in NIR region can produce heat for selectively killing/disrupting cancer cells. Wang et al. prepared MoS₂ QD@polyaniline nanohybrids, and realized successful PTT of cancer [33]. Yong et al. demonstrated that WS₂ QDs with small size (3 nm) possess not only significant X-ray CT/PA imaging signal enhancement but also remarkable PTT/radiotherapy (RT) synergistic effect for tumor treatment [46]. Chu et al. reported that CdTe and CdSe QDs can rapidly convert light energy into heat upon 671-nm laser irradiation [47]. The growth of mouse melanoma tumors injected with silica-coated CdTe QDs is significantly inhibited after laser irradiation.

1.2.3 QDs as PSs and Photothermal Agents

As stated above, QDs can be PSs and photothermal agents simultaneously. The combination therapy can be more effective than a single treatment. Lv et al. synthesized CuInS/ZnS quantum dots (ZCIS QDs) for synergistic PTT/PDT therapy [40]. Under a single 660 nm laser irradiation, the ZCIS QDs have simultaneous photothermal and photodynamic effects, resulting in high therapeutic efficacy against tumors. Ding et al. realized PA imaging-guided PTT/PDT in a single material MoO_3−x QDs [48]. Due to their strong NIR harvesting ability, MoO_3−x QDs can convert incident light into hyperthermia and sensitize the formation of singlet oxygen synchronously as evidenced by the in vitro assay.

1.2.4 QDs in Drug Delivery Systems

QDs represent a versatile platform for designing and engineering drug delivery systems. QDs facilitate the in-depth studies on the interactions between nanocarriers and biological systems through real-time monitoring of biodistribution, intracellular uptake, drug release, and long-term nanocarrier fate. At the same time, the compact size and compatibility with a variety of surface modification strategies of QDs enable the substitution of any NP core with a QD in single-NP drug delivery systems, or the incorporation of QD tags within larger multicomponent vehicles.

1.2.4.1 QDs as Carriers in PDT

In PDT, the excellent tissue-penetrating ability of an external excitation light and the good match between the wavelength of laser emission and the absorption wavelength of each PS are the two main factors that affect the generation of ROS and cell killing behavior. In an attempt made by Hsu et al., Renilla luciferase-immobilized QDs-655 (QD-RLuc8) was used for bioluminescence resonance energy transfer (BRET)-mediated PDT to solve the abovementioned problems [49]. The bioluminescent QD-RLuc8 conjugate exhibits self-illumination at 655 nm after coelenterazine addition, which can activate the photosensitizer Foscan-loaded micelles for PDT. This nanotechnology-based PDT possesses several clinical benefits, such as overcoming light penetration issues and treating deeper lesions that are intractable by PDT alone. Tsay et al. developed peptide-coated QD-photosensitizer conjugates using covalent conjugation strategies [50]. Rose bengal and chlorin e6, which generate singlet oxygen in high yield, are covalently attached to phytochelatin-related peptides. The photosensitizer–peptide conjugates are subsequently used to coat green- and red-emitting CdSe/CdS/ZnS nanocrystals. Generation of singlet oxygen can be achieved via indirect excitation through Förster resonance energy transfer (FRET) from the QDs to PSs, or by direct excitation of the PSs. In the latter case, by using two color excitations, the conjugate can be simultaneously used for fluorescence imaging and singlet oxygen generation. Singlet oxygen quantum yields as high as 0.31 were achieved using the 532-nm excitation wavelength. Similarly, Martynenko et al. prepared ZnSe/ZnS QDs and chlorin e6 complexes [51]. These complexes have shown ~50% intracomplex FRET from QDs to chlorin e6. The PDT test shows that the complexes had a twofold enhancement of the cancer cell photodynamic destruction as compared to free chlorin e6 molecules. They believe the enhanced PDT effect is attributed to two factors: the efficient QD–chlorin e6 photoexcitation energy transfer and the enhanced cellular uptake of the photosensitizer in the presence of ZnSe/ZnS QDs.

Owing to the two-photon excitation (TPE) property of QDs, enhanced photodynamic therapeutic efficacy through combining QDs with PSs under TPE was achieved by several research groups [52,53,54]. The singlet oxygen generation under TPE in the QD–PS systems is much higher than that in the free PSs. These studies underline the potential of QD-combined PSs for TPE PDT.

1.2.4.2 QDs as Carriers in Chemotherapy

Although chemotherapy is widely used in cancer treatment, it is usually ineffective due to the low cellular uptake and low tumor-targeting efficiency of the chemotherapeutics used. To solve these problems, Zhou et al. prepared 3-mercapitalpropionic acid (MPA)-capped CdTe QDs (MPA-CdTe QDs) to facilitate the interaction of the anticancer agent daunorubicin (DNR) with leukemia cells and kill drug-resistant leukemia K562/A02 cell lines [55]. In another study, Ye et al. synthesized ZnO QDs with polymer shells, onto which Gd³⁺ ions and the anticancer drug doxorubicin (DOX) were adsorbed form a new kind of multifunctional ZnO-Gd-DOX nanoplatform [56]. The as-prepared nanoplatforms are pH-sensitive and can release DOX to cancer cells in vitro and to mouse tumors in vivo, and have better specificity and lower toxicity than free DOX, and even better therapeutic efficacy than an FDA-approved commercial DOX-loading drug DOX-Liposome Injection (DOXIL, NDA#050718). The ZnO-Gd-DOX nanoplatforms exhibit strong red fluorescence, which is beneficial to the fluorescence imaging on live mice. Further, the nanoplatforms possess a high longitudinal relaxivity of 52.5 mM⁻¹ s⁻¹ at 0.55 T, which is superior to many other Gd³⁺-based nanoparticles. Thus, both fluorescence imaging and MRI can be applied simultaneously on the tumor-bearing mice along with drug delivery.

1.2.4.3 QDs as Labels in PTT

In PTT, a photothermal agent can heat and kill abnormal cells or tissues under light irradiation. However, this strategy usually has low selectivity. Thus, an imaging agent is urgently needed to “see” the tumor site in PTT. Due to the excellent photoluminescence property, QDs are commonly used as an imaging agent in PTT. Xia et al. developed multifunctional NPs by incorporating gold nanorods (GNRs) and CdSe/ZnS QDs into silica [41]. Cell imaging experiments reveal that the NPs exhibit strong X-ray attenuation for X-ray CT imaging and strong fluorescence for fluorescence imaging. Nair et al. developed a hybrid nanosystem based on QDs and single-wall carbon nanotubes (SWCNTs) which is found to be useful not only in imaging applications but also in selective cancer cell destruction [57]. Besides, a novel, multifunctional, and low-toxic QD-reduced graphene oxide (rGO) nanocomposite was designed to serve as an imaging agent in the visible light region and a photothermal agent in the NIR region by Hu et al. [58]. Since the photothermal effect of the irradiated rGO can cause not only cell killing but also the degradation of the QDs, the QDs also serve as an optical indicator for the heat dosage and therapeutic progress.

1.2.4.4 QDs as Labels in Other Treatments

Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, a further understanding of their biological behaviors and potential applications has been hampered by the limitations of current labeling techniques. Chen et al. proposed a universal donor-cell-assisted membrane biotinylation strategy for labeling MPs by utilizing the natural membrane phospholipid exchange of their donor cells [59]. This innovative strategy conveniently leads to the specific, efficient, reproducible, and biocompatible QD labeling of MPs, thereby reliably conferring the valuable traceability of MPs. By further loading with small interfering RNA (siRNA), QD-labeled MPs that have inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. Kim et al. designed and synthesized immunomodulatory hybrid nanoconjugates (HNCs) based on polymer nanocomposites containing QDs (as imaging tracers) conjugated with CpG oligodeoxynucleotides (ODNs) (as a TLR9 ligand) and STAT3 siRNAs (as an immunosuppressive gene silencer) [60]. These HNCs can efficiently target immune cells, induce TLR activation, and silence immunosuppressive genes. Simultaneous delivery of STAT3 siRNAs and CpG ODNs to the tumor microenvironment causes the inhibition of STAT3 along with the activation of dendritic cells (DCs) by CpG ODNs, and their antitumor effects are found to be synergistic. By using NIR-emitting QDs, the migration of DCs to lymph nodes was tracked by real-time NIR fluorescence imaging.

To be able to label a gene and monitor its migration are key important approaches for the clinical application of cancer suicide gene therapy. One of the most promising suicide genes—herpes simplex virus thymidine kinase (HSV-TK) gene was successfully linked with CdTe/CdS core/shell QDs [61]. From confocal microscopy, it is demonstrated that plasmid TK intracellular trafficking can be traced via monitoring the luminescence of the QDs up to 96 h after transfection of QDs-TK conjugates into HeLa cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results show that the QDs-TK conjugates have a high cytotoxicity after adding ganciclovir (GCV) into HeLa cells, whereas the QDs exert no detectable deleterious effects on the cellular processes. These results indicate that the QDs-based labeling technique is suitable for monitoring TK gene delivery and anticancer activity.

1.3 Limitations and Future Perspectives

QDs as one type of multifunctional materials, have shown promising advantages in tumor imaging and therapy due to their specific physicochemical properties. Using the rich surface functionalization chemistry of QDs, targeting biomolecules and drug formulations can be integrated with QDs for traceable drug delivery and therapy in vitro and in vivo. Many studies have demonstrated that the incorporation of drug formulations with QDs do not compromise the drug efficacy. More importantly, the QD-involving nanostructures are able to serve as an excellent platform for the development of a new generation of traceable drug delivery strategies for real-time monitoring of the drug biodistribution in vitro and in vivo.

Because of toxicity concerns, heavy-metal-based QDs might not be the best candidate for in vivo drug delivery and therapy. Thus, many researchers are currently synthesizing cadmium-free QDs for in vivo applications. However, for in vitro-based drug studies, heavy-metal-based QDs will still be used, since toxicity is not a key concern. Another potential concern for the use of QDs in delivery and therapy is the overall QD size. In general, it is preferable to minimize the overall size of QDs for in vivo applications to reduce their accumulation in the reticuloendothelial system. Further, passivation of the QD surface with a long-lasting and robust polymer coating is essential to prevent the breakdown of QDs in the biological environment that gives rise to their toxicity. Some reports have suggested that capping the QD core with a higher bandgap semiconductor or biomolecule can minimize the toxicity of the QDs. However, it is worth noting that each additional step toward functionalizing the QDs will contribute to their final hydrodynamic size and could directly or indirectly affect their biodistribution. If these issues could be solved, we envision that QDs will become one type of promising material for real-time tumor-targeted imaging and therapy in the future.

2 Silver Chalcogenide Quantum Dots

Silver chalcogenide QDs (SCQDs) composed of silver and a chalcogen (S, Se, or Te) are another type of newly emerged semiconductor QDs. Their narrow and controllable bandgap and extremely low solubility in aqueous solutions endow SCQDs with outstanding photoluminescence properties like NIR emission, adjustable emission peaks, high photostability, and good biocompatibility. These properties make SCQDs competitive candidates in bioimaging, detection, photocatalysis, thermal-electric applications, and QDs-sensitized solar cells. Currently, the synthetic methods and applications of SCQDs have been systemically reviewed by Gui et al. [62] In this part of the chapter, we will focus on utilizing SCQDs in bioimaging and cancer therapy. Subsequently, we are going to introduce SCQDs in three parts: Ag₂S QDs, Ag₂Se QDs, and Ag₂Te QDs. Considering that Ag₂Te QDs are seldom reported in the literature, our discussion will be mainly concentrated on Ag₂S QDs and Ag₂Se QDs.

The applications of traditional QDs (most commonly CdSe@ZnS QDs) in in vivo bioimaging are limited because these QDs usually have fluorescence emissions in the regime of visible spectrum (400–700 nm) in which the autofluorescence of living tissues makes the results ambiguous. Subsequently, NIR-emissive QDs emerge as a new group of luminescent probes benefiting from their deeper tissue penetration and less skin scattering. Compared with other heavy-metal-containing NIR QDs including PbS, PbSe, and CdHgTe QDs, SCQDs may be more biocompatible and more suitable for biomedical applications.

2.1 Silver Sulfide (Ag₂S) QDs

Although the synthesis of Ag₂S nanocrystals has been intensively explored in the past 20 years, there were no reports about the photoluminescence of this type of nanomaterials prior to 2010. The discovery of the NIR emission property of SCQDs has attracted great interest from the researchers in the world because it makes the noninvasive imaging in deep tissues possible. In addition, their extremely low aqueous solubility ensures that a minimum amount of silver ions is released to the environment, ensuring their safety in biomedical applications. Besides Ag₂S QDs, there are a number of NIR-II emitting fluorophores, such as single-walled carbon nanotubes (SWCNTs), organic dyes, rare-earth-doped nanoparticles, and conjugated copolymers [63]. However, SWCNTs have relatively low photoluminescence quantum yields (PLQYs) and their safety is under discussion. Organic dyes are prone to be photobleached easily. In contrast, Ag₂S QDs are bright, biocompatible, and resistant to photobleaching. Concerning that the utilization of Ag₂S QDs in bioimaging and cancer therapy has been mainly reported in the recent 5 years, we will review the work from 2012 to 2017.

In 2012, the application of Ag₂S QDs for bioimaging was demonstrated by several research groups [64,65,66,67]. For example, Jiang et al. synthesized water-dispersible Ag₂S QDs using 3-mercaptopropionic acid (3-MPA) as capping agent in ethylene glycol medium [64]. The photoluminescence of the as-prepared Ag₂S QDs can be tuned from 510 to 1221 nm and the 910 nm emitting sample with QY = 2.1% was employed for in vivo imaging (Fig. 4a–d). Under either subcutaneous or celiac injection occasion, the Ag₂S QDs display bright fluorescence, which is significantly distinct from the autofluorescence of body. Zhang et al. synthesized NIR-II emitting Ag₂S QDs modified by dihydrolipoic acid (denoted as DHLA-Ag₂S QDs) [65]. They realized in vitro cell imaging with high selectivity by conjugating DHLA-Ag₂S QDs with specific molecules (Erbitux protein for targeting to epidermal growth factor receptor (EGRF), and cyclic arginine-glycine-aspartic acid (RGD) peptide for targeting to α_vβ₃ integrin overexpressed on the surface of several types of cancer cells). In another study, Hong et al. conjugated the DHLA-Ag₂S QDs with six-armed polyethylene glycol (6PEG) to yield 6PEG-Ag₂S QDs (QY = 15.5%) [67]. Due to its high brightness and proper size, 6PEG-Ag₂S QDs were used for fast tumor detection guided by enhanced permeability and retention (EPR) effect (Fig. 5, a–f). The biodistribution and pharmacokinetics results (Fig. 5, g–i) show that  >10% ID/g 6PEG-Ag₂S QDs accumulate in the tumor site and 6PEG-Ag₂S QDs can be metabolized by biliary excretion.

Fig. 4

Reprinted with permission from Ref. [64]. Copyright 2012 Elsevier

In vivo NIR fluorescence imaging (pseudocoloured image) of nude mice. Control experiment (a), with subcutaneous injection (b) and with celiac injection (c) of Ag₂S QDs emitting at 910 nm; Unmixed image of Ag₂S QDs fluorescence signal (c, inset); The corresponding emission spectra of the autofluorescence and QDs fluorescence of mice with celiac injection (d). (In images a–d, the blue corresponds to the mice autofluorescence and the red corresponds to QDs fluorescence.)

Fig. 5

Reprinted with permission from Ref. [67]. Copyright 2012 Wiley

NIR-II fluorescence imaging of a xenograft 4T1 tumor with high uptake of 6PEG-Ag₂S QDs. a–e Time course of NIR-II fluorescence images of the same mouse injected with 6PEG-Ag₂S QDs. f A white-light optical image of the same mouse at 24 h postinjection (p.i.). The tumor mass was visibly darkened owing to high uptake of 6PEG-Ag₂S QDs. g The tumor-to-background ratio (TBR) plotted as a function of time p.i. for NIR-II images, indicating a 100% certainty of the tumor being identified from the NIR-II image after 4 h p.i., according to the Rose criterion. h A representative plot of the %ID/gram of the 6PEG-Ag₂S QDs in the blood versus time after tail vein injection, as determined by the NIR-II fluorescence of the blood samples. A first-order exponential fits the data points with a half-life of circulation for the 6PEG-Ag₂S QDs of 4.37 ± 0.75 h. i Quantitative biodistribution of 6PEG-Ag₂S QDs in various organs and the tumor 72 h after injection based on inductively coupled plasma mass spectrometry (ICP-MS).

In 2013, the Ag₂S QDs were further functionalized and their long-term potential toxicity was systematically evaluated [68, 69]. Wang et al. reported a method of preparing ultrasmall bovine serum albumin (BSA)-stabilized Ag₂S QDs with tunable NIR fluorescence and vascular endothelial growth factor (VEGF) antibody (antiVEGF) was additionally conjugated to enhance the in vivo U-87 MG human glioblastoma tumor-targeting efficiency on mice [68]. However, the QY of the as-prepared BSA-Ag₂S QDs was determined to be only 1.8%. In another example, Zhang et al. provided a detailed study of long-term in vivo biodistribution and examined the potential toxicity of PEGylated Ag₂S QDs [69]. The results showed that the PEGylated Ag₂S QDs are mainly accumulated in liver and spleen after intravenous injection and mostly eliminated by fecal excretion. Negligible toxicity is observed with an intravenous injection dose of as high as 30 mg/kg during the 2-month-long experiments, which is evidenced by the blood biochemical, hematological, and histological analyses. Besides tumor-targeting imaging, Tan et al. also used Ag₂S QDs as NIR fluorescent probes in nitric oxide delivery system [70, 71].

In view of the excellent imaging properties of Ag₂S QDs with ultrahigh spatial (30 μm) and temporal resolution (<50 ms), they were applied in more circumstances in 2014 [72,73,74]. Li et al. used PEGylated Ag₂S QDs to develop a noninvasive approach of visualizing lymphatic drainage and vascular networks and tracking angiogenesis mediated by a minuscule tumor in vivo [72]. Chen et al. further modified the PEGylated Ag₂S QDs with TAT peptide to form TAT-Ag₂S QDs, which can efficiently label human mesenchymal stem cells (hMSCs) [73]. Applying the TAT-Ag₂S QDs, the dynamic monitoring of the transplanted hMSCs in vivo can be realized with a temporal resolution of 100 ms. Further, Chen et al. conjugated 3-MPA-capped Ag₂S QDs with doxorubicin (DOX, a commercial drug for cancer chemotherapy) and cyclic RGD peptide as a multifunctional nanoplatform to achieve NIR imaging-guided tumor-targeting therapy [74]. This work implies that Ag₂S QDs can not only be used as NIR fluorescent probes but also have potential applications in cancer therapy.

Because of their remarkable NIR fluorescence property and proper size for tumor targeting by EPR effect, Ag₂S QDs are increasingly regarded as one of the ideal nanoplatforms for detecting tumors, tracking biological process, and monitoring drug biodistribution in 2015 [75,76,77,78,79,80,81]. Other modalities such as CT and MRI were employed together with NIR fluorescence imaging by combining Ag₂S QDs with iodinated oil and Gd³⁺, respectively [77, 78]. Since 2016, multimodal imaging has become popular for the reason that each individual imaging method has its own disadvantages. Wu et al. reported the synthesis of ICG@PEG-Ag₂S QDs using an FDA-approved NIR dye indocyanine green (ICG) [82]. The as-prepared ICG@PEG-Ag₂S QDs exhibit dual-modal imaging ability (fluorescence and PA imaging) of atherosclerosis. Furthermore, after precisely targeting atherosclerosis, the photothermal effect of ICG can be employed to treat the disease. Zhang et al. prepared Ag₂S@BSA-DTPA^Gd via a biomineralization method to realize fluorescence imaging and MRI of tiny tumors [83]. Inspired by the strong absorption of Ag₂S QDs in NIR regime, Gao et al. directly utilized these QDs to achieve cancer PTT [84]. They used aptamer to further functionalize the Ag₂S QDs to increase the tumor-targeting efficiency. The results show that Ag₂S QDs are not only NIR fluorescent probes but also excellent photothermal agents. Subsequently, in 2017, Yang et al. reported the synthesis of size-dependent Ag₂S QDs in hollow human serum albumin (HSA) nanocages for dual-modal imaging (fluorescence and PA imaging) and PTT [85]. The as-prepared Ag₂S QDs show various advantages including photobleaching resistance, preferable endocytosis, effective tumor accumulation, and in vivo body elimination. Also in 2017, Li et al. developed an Ald/DOX@Ag₂S nanoplatform for bone tumor chemotherapy and osteolysis inhibition [86]. In their design, alendronate (Ald) which is conjugated to the surface of Ag₂S QDs plays a role of osteolysis inhibition and pain alleviation. Meanwhile, DOX as the antitumor drug is encapsulated around Ag₂S QDs by hydrophobic interaction. The key component Ag₂S QDs is responsible for prolonging drug circulation time and visualizing the drug delivery process.

2.2 Silver Selenide (Ag₂Se) QDs

Selenium is in the same group with sulfur, so Ag₂Se QDs are akin to Ag₂S QDs in many properties. Ag₂Se QDs were first used in bioimaging in 2012 [87]. As shown in Fig. 6, the Ag₂Se QDs were injected into the abdominal cavity of a nude mouse and the emitting light successfully penetrated the tissues even on the back side. In 2013, C₁₈-PMH-PEG-Ag₂Se QDs with bright photoluminescence centered at 1300 nm, excellent water-dispersity, great colloidal stability and photostability, and good biocompatibility were employed for deep imaging of organs and vascular structures in vivo [88]. Compared with intravenously injected ICG, the C₁₈-PMH-PEG-Ag₂Se QDs show a much clearer image of liver which is buried deep in the body and can visualize the branched blood vessels even as cramped as 123 μm. Although Ag₂Se QDs are thought to be safe, there has been no detailed investigation about their in vivo behavior and toxicity until 2016 [89]. As reported by Tang et al., PEG-Ag₂Se QDs are quickly cleared from the circulation system in mice with a half-life of 0.4 h. Additionally, the PEG-Ag₂Se QDs are mainly amassed in liver and spleen and are converted to Ag and Se in a week. Similar to Ag₂S QDs, Ag₂Se QDs can also be used in multimodal imaging-guided theranostics. Zhao et al. functionalized Ag₂Se QDs with Mn²⁺ to form Ag₂Se@Mn QDs, which integrated the remarkable NIR fluorescence of Ag₂Se QDs and MRI ability of Mn²⁺ [90]. Further, the Ag₂Se@Mn QDs were loaded into circulating MPs (CMPs) freshly purified from the peripheral blood of oral squamous cell carcinoma (OSCC) patients via electroporation, and the thus-obtained Ag₂Se@Mn QD-labeled CMPs were developed as dual-modally traceable and actively tumor-targeted nanoplatform for cancer theranostics [91]. A similar design was reported by Zhu et al., in which cetuximab (a clinical drug for tumor therapy) was conjugated to Ag₂Se QDs to achieve simultaneous imaging and therapy of orthotopic tongue cancer [92].

Fig. 6

Reprinted with permission from Ref. [87]. Copyright 2012 American Chemical Society

MTT assay and NIR images of a living nude mouse after injection of Ag₂Se QDs: a MTT assay on A549, Vero, and MDCK cells exposed to Ag₂Se QDs at different concentrations from 0 to 47.4 μg/mL for 24 h; b fluorescence image of the nude mouse; c fluorescence imaging from the abdominal cavity of the nude mouse with Ag₂Se QDs injected into the abdominal cavity; d fluorescence imaging on the back side of the nude mouse with Ag₂Se QDs injected into the abdominal cavity; e, f merged images of the bright-field and the threshold false color of (c) and (d), respectively.

2.3 Silver Telluride (Ag₂Te) QDs

In comparison with Ag₂S QDs and Ag₂Se QDs, Ag₂Te QDs seem to be enigmatic. Because of the high electron mobility and low thermal conductivity of Ag₂Te, it is usually used for thermoelectric applications rather than biomedicine. The photoluminescence property of Ag₂Te QDs was first reported by Yarema et al. [93]. They synthesized 3.2 nm Ag₂Te QDs with the emission peak centered at 1300 nm. Chen et al. prepared luminescent Ag₂Te QDs via a cation exchange method [94]. The as-synthesized Ag₂Te QDs possess photoluminescence ranging from 900 to 1300 nm and the PLQY was determined to be 2.1%. By further growth of ZnS, the PLQY of Ag₂Te/ZnS core/shell QDs can be increased to 5.6%. Neither the Ag₂Te QDs nor the Ag₂Te/ZnS core/shell QDs at concentrations ranging from 50 to 200 nM show cytotoxicity after incubation with HeLa cells for 24 h. Nevertheless, their relatively low PLQYs limit their further imaging applications. In 2015, Yang et al. employed a multivalent polymer poly(maleic anhydride)-graft-cysteamine (PMAC) as both stabilizer and capping agent to fabricate Ag₂Te QDs in aqueous solution [95]. The as-prepared Ag₂Te QDs with adjustable NIR fluorescence peaks ranging from 995 to 1068 nm have relatively high PLQYs of 13.1–15.9%. They also show excellent photostability and aqueous stability as illustrated in Fig. 7b–f. Until now, the bioimaging and cancer therapy applications of Ag₂Te QDs are still lacking. Considering that Ag₂Te has even lower solubility than Ag₂S and Ag₂Se but similar NIR-II photoluminescence properties as compared with Ag₂S and Ag₂Se, we assume that Ag₂Te QDs are probably an alternative in bioimaging. Furthermore, the intrinsic heavy atom Te may endow the Ag₂Te QDs with satisfying performance as a CT contrast agent.

Fig. 7

Reprinted with permission from Ref. [95]. Copyright 2015 Elsevier

a Bright-field images of L929 cells, b NIR-II images acquired immediately upon irradiation (808 nm), and c acquired after irradiation for 2 h of L929 cells. a–c The cells were incubated with Ag₂Te QDs (0.1 mg/mL) for 1 h. d Bright-field and NIR-II images of Ag₂Te QDs suspended in PBS (1 mM, pH = 7.4) or 100-fold diluted mouse whole blood. e Mean PL intensities of the two samples determined based on the NIR-II images. f PL stability of Ag₂Te QDs in mouse whole blood within 48 h.

2.4 Limitations and Future Perspectives

SCQDs possess tunable, bright NIR photoluminescence and have been extensively reported as fluorescent probes in vitro and in vivo. Their proper sizes and functional surfaces suggest their promising cancer theranostic applications. Compared with Cd/Pb/Hg-based NIR QDs, there are no concerns on SCQDs about toxicity caused by release of heavy metal ions. In recent years, the synthetic methods of SCQDs have been greatly explored and the various SCQDs have been developed into multimodal imaging probes and drug delivery platforms. However, there are still some problems which hamper the further applications of SCQDs in bioimaging. For example, the small size of SCQDs endows them with large surface area to be functionalized but also means more defects on the surface which decrease the PLQY of SCQDs. For Cd-based QDs, this issue can be solved by wrapping the QDs with ZnS to form a core–shell structure. Nevertheless, the materials that match the lattice of SCQD cores and form a shell on SCQD cores have been seldom reported. That is why the PLQY of Cd-based QDs can achieve even 60%, while it is difficult to find a kind of SCQDs with PLQY above 20%. If this problem can be solved, the PLQY and photostability of SCQDs could be further improved. We believe that SCQDs are a competitive class of promising theranostic agents in the future.

3 Carbon Quantum Dots

Carbon quantum dots (also known as carbon dots, C-dots, CDs, or CQDs), first discovered by Xu et al. during the electrophoretic purification of single-walled carbon nanotubes [96], are a novel class of carbon-based nanomaterials which are typically discrete, quasispherical nanoparticles, with sizes below 10 nm [97, 98]. Due to their fantastic features, such as simple low-cost synthesis and scalability, superior optical properties (e.g., tunable and wide emissions, high photostability, and two-photon excited fluorescence), facile functionalization, excellent biocompatibility, and good chemical inertness and solubility, CDs have attracted increasing attention in a wide range of applications including bioimaging, sensing, catalysis, photoelectric devices, and theranostics [99,100,101,102,103,104]. Synthetic approaches for CDs are generally classified into two categories—top-down and bottom-up [105]. Herein, we focus on the recent progress of CDs in cancer cell imaging and cancer therapy.

3.1 CDs for Cancer Bioimaging

CDs usually show the excitation wavelength-dependent emission property, which is mainly attributed to the surface state of CDs [106, 107]. A surface defect-based luminescence mechanism has been suggested for CDs [108, 109]. In addition, the use of surface passivating agents to provide uniform photoluminescence trapping sites on the CD surface and the introduction of electron-donating heteroatoms as dopants help to tune the photoluminescence properties of CDs [98]. The emission spectra of CDs are usually broad, ranging from deep ultraviolet to visible, or even extended to NIR. Jiang et al. prepared red, green, and blue emissive CDs using three kinds of phenylenediamine through a solvothermal method [110]. Ding et al. reported the first one-pot syntheses of full-color light-emitting CDs, and then collected the CDs exhibiting excitation-independent luminescence from blue to red via column chromatography following the hydrothermal treatment [111].

Bioimaging is one of the most important applications of CDs because of their unique optical properties and low cytotoxicity. Compared to the conventional semiconductor QDs, which usually contain cadmium or other heavy metals, CDs comprise nontoxic elements, ensuring their good biocompatibility [108, 112]. The first preliminary assessment of the bioimaging potential of CDs was presented by Sun et al., who observed cellular uptake of CDs by Caco-2 cells by confocal microscopy [113], realizing the optical cell tracking with CDs. Diverse CDs have witnessed the effects of size and surface nature (charge and chemistry) on their cellular uptake. Recent studies demonstrated that CDs are mostly internalized in the cytoplasm, especially in endosomes/lysosomes, but also in mitochondria or endoplasmic reticulum [114,115,116]. Very few CDs were reported to stain the cell membrane or nucleus [117, 118]. The two-photon fluorescence imaging capability of CDs internalized in cancer cells has also been demonstrated (Fig. 8a) [119]. Chizhik et al. realized super-resolution optical fluctuation bioimaging (SOFI) with their dual-color CDs (Fig. 8b) [120]. On the other hand, one kind of positively charged CDs fabricated by using polyethyleneimine and folic acid (FA) was reported to be able to selectively image the folate receptor-positive cancer cells (Fig. 8c) [121]. Furthermore, since deep-red and NIR light exhibits deeper tissue penetration, the development of CDs with long wavelength emissions is highly desired for in vivo imaging [122]. Ge et al. successfully utilized red fluorescent CDs with the emission peak at ~640 nm for in vitro and in vivo imaging (Fig. 8d, e) [123]. CDs intravenously injected into tumor-bearing nude mice accumulate in the tumor area through the EPR effect and a significant FL signal is observed in the tumor area in comparison with other tissues.

Fig. 8

a Representative two-photon luminescence images (800 nm excitation) of MCF-7 cells with internalized CDs. Reprinted with permission from Ref. [119]. Copyright 2007 American Chemical Society. b Fluorescence wide-field image and second-order SOFI image of a Saos-2 cell, respectively. Reprinted with permission from Ref. [120]. Copyright 2016 American Chemical Society. c Confocal images of 293T cells (top) and HeLa cells (bottom) incubated with CDs for 4 h. (i) bright-field, (ii) confocal fluorescence, and (iii) overlay images. Reprinted with permission from Ref. [121]. Copyright 2016 Elsevier. d Confocal images of the CDs in HeLa cells with bright field and excitation at 543 and 638 nm, respectively. Reprinted with permission from Ref. [123]. Copyright 2015 Wiley. e In vivo and ex vivo FL imaging. Top: Real-time in vivo FL images after intravenous injection of CDs in nude mice at different time points. Bottom: Ex vivo FL images (left) and average FL intensities (middle) of main organs and tumor tissue at 24 h postinjection, and average FL intensities of the tumor area at different time points postinjection (right). Reprinted with permission from Ref. [123]. Copyright 2015 Wiley

3.2 CDs for Cancer Therapy

Besides the strong fluorescence of CDs, their unique chemical structure allows the integration of active therapeutic molecules into the sp² carbon frame, and their surface functional groups enable further conjugation with other molecules such as biological affinity ligands [124]. Choi et al. used FA-functionalized CDs as carriers for the photosensitizer (PS) zinc phthalocyanine via π–π stacking interactions, leading to simultaneous imaging and targeted PDT after irradiation in vitro and in vivo (Fig. 9A) [124]. After loading doxorubicin (DOX) through π–π stacking, the resultant CD-DOX nanoagent show controlled drug release and efficient tumor therapy. Besides, Gong et al. fabricated innovative phosphorus and nitrogen dual-doped hollow carbon dots (PNHCDs) with negative surface charge and proved that PNHCDs (as the carriers) have strong electrostatic and hydrogen bonding interactions with the DOX drug molecules [125]. Apart from the physical interactions between CDs and drugs, the covalent bonding is also an important choice for loading drugs on CDs. In this regard, Yang et al. prepared CDs–DOX complexes using DOX-SH and amine-functionalized CDs via a coupling linker, N-hydroxysuccinimide ester-poly(ethylene glycol)₁₀-maleimide, ensuring a slower and prolonged DOX accumulation in the nucleus, which results in enhanced anti-tumor efficacy and less side effects compared to free DOX (Fig. 9B) [126]. Hua et al. utilized the CDs with intrinsic mitochondrial targeting ability to deliver PSs to mitochondria after covalent conjugation, and achieved significantly enhanced PDT efficacy (Fig. 9C) [127]. Zheng et al. developed a novel theranostic nanomedicine via the condensation reaction between the amine groups on the CD’s surface and the carboxyl group of Pt(IV) complex (Oxa(IV)–COOH, a chemotherapeutic prodrug), which may hold great potential for cancer diagnostics and therapy (Fig. 9D) [128]. Huang et al. designed novel multifunctional chlorin e6-conjugated CDs (CDs-Ce6, via amide condensation) as a light-triggerable theranostics agent for simultaneous enhanced-photosensitizer fluorescence detection (PFD) and PDT by FRET mechanism (Fig. 9E) [129]. The CDs as the carriers to load PSs or dyes can improve the stability and solubility of PSs or dyes in aqueous/biological media, extend blood circulation time, and enhance biocompatibility, thus making CDs-Ce6 a good candidate with excellent imaging and tumor-homing ability for FL imaging-guided PDT treatment. Furthermore, many pH-, redox-, enzyme-, or light-responsive CDs-involving complexes have been developed for imaging-guided drug delivery with enhanced therapeutic efficiency [130,131,132,133]. For instance, Wang et al. designed the biocompatible PEG-chitosan@CDs hybrid nanogels to realize pH and NIR light dual-responsive drug release and combined chemo-photothermal treatment (Fig. 9F) [130].

Fig. 9

A Schematic illustration of the preparation of carbon nanodots from α-cyclodextrin and targeted PDT with folic acid-functionalized carbon nanodots loaded with zinc phthalocyanine (CD-PEG-FA/ZnPc). Reprinted with permission from Ref. [124]. Copyright 2014 Wiley. B Schematic illustration of the synthesis of CDs and their applications in cell imaging and anticancer drug delivery (a). The interaction of CDs–DOX with cancer cells (b). Reprinted with permission from Ref. [126]. Copyright 2017 Royal Society of Chemistry. C Carbon quantum dots with intrinsic mitochondrial targeting ability for enhanced mitochondria-based PDT. Reprinted with permission from Ref. [127]. Copyright 2017 Royal Society of Chemistry. D Synthetic scheme for CD-Oxa and its applications in bioimaging and theranostics. Reprinted with permission from Ref. [128]. Copyright 2014 Wiley. E FRET process between CDs and Ce6. Reprinted with permission from Ref. [129]. Copyright 2012 Wiley. F PEG-chitosan@CDs hybrid nanogels for pH/NIR dual-responsive drug release and two-photon fluorescence cellular imaging. Reprinted with permission from Ref. [130]. Copyright 2015 Wiley

Besides being used as drug carriers, CDs can also be used as nanomedicines. In 2011, Christensen et al. found that CDs can be used as PSs to generate ROS under the irradiation of blue light in vitro [134]. However, the shallower tissue penetration of blue light limits the applications of the above CDs in vivo. In 2013, Hsu et al. validated that CDs without laser have a remarkable inhibitory effect on the growth of MCF-7 and MDA-MB-231 cancer cells, with lower toxicity to the MCF-10A normal cells, which may be explained by the significantly increased cellular ROS levels in the two types of cancer cells upon CD treatment [135]. Besides, some red-emitting or red light-excitable CDs have been considered as promising PSs or PT agents. For example, Ge et al. demonstrated the application of CDs for imaging-guided PDT and PTT both in vitro and in vivo [136]. The obtained CDs exhibit dual photodynamic and photothermal effects under 635 nm laser irradiation with a singlet oxygen (¹O₂) generation efficiency of 27% and high photothermal conversion efficiency of 36.2% (Fig. 10a). Since NIR has a deeper tissue penetration as compared with visible light, NIR-absorbing CDs are more suitable for in vivo bioimaging and PTT. Lan et al. prepared S, Se-codoped CDs with photothermal conversion efficiency of ~58.2% as new multifunctional phototheranostic agents for the TPE fluorescence imaging (with the excitation wavelength at 880 nm) and PTT (with the excitation wavelength at 635 nm) of cancer cells [137]. Another kind of CDs (named CyCD) showing strong absorption and NIR emission within the range from 600 to 900 nm was proved to be an ideal theragnostic agent for NIR fluorescence imaging and photothermal therapy in vitro and in vivo (Fig. 10b) [138]. Jia et al. prepared CD nanospheres via the self-assembly of individual CDs and sodium dodecyl benzene sulfonate (SDBS) to achieve NIR-light-responsive FL imaging and PDT of cancer (Fig. 10c) [139].

Fig. 10

a In vitro PDT/PTT. Relative viability of B16-F0 skin cancer cells incubated with various concentrations of CDs under dark or irradiation by 635 nm laser at power densities of 0.1 W cm⁻² (left, PDT) or 2 W cm⁻² (right, PDT/PTT) for 10 min. Reprinted with permission from Ref. [136]. Copyright 2015 Wiley. b In vivo PTT. Top: Photographs of a CT26 tumor-bearing mouse after PTT (the tumor is circled with a red dotted line) and the excised tumors on the 11th day. Bottom: The tumor volume and body weight of the CT26 cancer bearing mice after different treatments (saline, CyCD, and CyCD + irradiation). Reprinted with permission from Ref. [138]. Copyright 2016 American Chemical Society. c Top: Schematic representations of the fabrication of CD nanosphere (CDNS) from individual CDs and amphipathic SDBS and the application as a nanophotosensitizer for simultaneous NIR-light-responsive FL imaging and PDT of cancer. Bottom: In vivo FL imaging and PDT efficacy of CDNS. Reprinted with permission from Ref. [139]. Copyright 2017 Wiley

4 Silicon Quantum Dots

Silicon or silicon-containing nanomaterials, a series of important nanomaterials with attractive properties including huge surface-to-volume ratios, favorable biocompatibility, improved multifunctionality, and excellent electronic/mechanical properties [140, 141], have been developed for various applications ranging from electronics to biology. Various silicon nanomaterials have been developed, such as silicon nanorods [142], silicon noanowires [143], and silicon nanodots [144]. Among these silicon nanomaterials, silicon nanodots or silicon quantum dots (SiQDs) are especially suitable for bioimaging and cancer therapy due to their ultrasmall size, bright fluorescence, and good biocompatibility.

4.1 Silicon Quantum Dots for Bioimaging Applications

The room-temperature synthesis of water-soluble silicon quantum dots that exhibit strong blue photoluminescence (PL) has been reported by Warner et al. [145]. The ease of synthesis and optical properties make the SiQDs excellent candidates for the imaging of cancer cells, as demonstrated in HeLa cells. For the applications in biological imaging and diagnosis, SiQDs must remain luminescent and be stably dispersed in biological fluids with a wide pH range of pH and a high salt concentration. Erogbogbo et al. reported the preparation of highly stable aqueous suspensions of SiQDs using phospholipid micelles, where the optical properties of SiQDs are well retained [146]. The micelle-encapsulated SiQDs were used as luminescent labels for pancreatic cancer cells in vitro (Fig. 11), thereby highlighting their potential as a nontoxic optical probe for biomedical diagnostics.

Fig. 11

Reprinted with permission from Ref. [146]. Copyright 2008 American Chemical Society

Confocal microscopic visualization of live pancreatic cancer cells treated with a amine-terminated micelle-encapsulated SiQDs, b transferrin-conjugated micelle-encapsulated SiQDs.

To overcome the shortcomings of severe photobleaching and cytotoxicity associated with the traditional dyes and the fluorescent II/VI QDs, He et al. reported a type of silicon-based nanospheres with the merits of excellent water-dispersibility, strong photoluminescence, and robust photostability, which can be used for cellular imaging [147]. To establish their utility as cellular probes, HEK293T human kidney cells were chosen to be labeled by the as-prepared SiQDs. As shown in Fig. 12, the confocal images indicate that the PL of the SiQDs-labeled HEK293T cells is intense and can be directly observed under excitation at different wavelengths. Besides, robust anti-photobleaching of the obtained SiQDs was verified by comparing with the fluorescein isothiocyanate (FITC) dye and fluorescent II/VI QDs.

Fig. 12

Reprinted with permission from Ref. [147]. Copyright 2009 Wiley

a Fluorescence microscopy images of HEK2-293T cells labeled with the as-prepared SiQDs. b Comparison of fluorescence signals of HEK293T cells imaging with the nanospheres (top) and FITC (bottom). c Temporal evolution of fluorescence of the HEK293T cells labeled with the as-prepared nanospheres (top) and FITC (bottom). d Photostability comparison of fluorescent II/VI QDs and the obtained SiQDs.

A novel kind of oxidized silicon nanospheres (O-SiNSs) prepared via thermal oxidation of the precursor SiNSs was reported by He et al. [148]. The O-SiNSs possess the properties of excellent aqueous dispersibility, high PLQY of 25%, wide pH stability, superior photostability, and favorable biocompatibility. Moreover, the O-SiNSs are conjugated with antibody (abbreviated as O-SiNSs/antibody bioconjugates), which are successfully applied in immunofluorescence cell imaging. Moreover, simultaneous detection of two biological targets and removal of background autofluorescence can also be realized by antibody-conjugated silicon quantum dot nanoparticles and organic dyes, which was reported by Tu et al. [149]. To realize selective cancer imaging using SiQDs, Erogbogbo et al. reported the development of SiQDs modified with folate and antimesothelin [150]. The successful targeted cancer cell imaging will be highly beneficial for targeted cancer diagnosis and therapy.

4.2 Silicon Quantum Dots for Drug Delivery and Cancer Therapy

Owing to the wide and growing applications of fluorescence in biomedicine and bioengineering fields, fluorescence imaging-guided cancer therapy has attracted great interest. Moreover, due to the low cytotoxicity of silicon materials, SiQDs are good carriers for delivering drugs into cells. SiQDs for delivering siRNA into tumor cells have been realized by Klein et al [151]. The internalization of SiQDs was found to occur via endocytosis, which was observed by transmission electron microscopy (TEM) and confocal microscopy. Moreover, the SiQD-siRNA complexes can significantly reduce the transporter efficiencies for the P-glycoprotein substrate Rhodamine [123].

Although there are many ways for the modification of SiQDs, attention should be paid to the organic groups on the surface of SiQDs which can increase toxicity. Ruizendaal et al. reported that amine-functionalized SiQDs exhibit cytotoxicity, whereas carboxylic acid-terminated analogues do not [152]. Multifunctional nanocarriers with a core–shell structure for drug delivery have been developed by Xu et al., in which SiQDs serve as the core and a water-soluble block copolymer serves as the shell [153]. Besides, as reported by Wang et al., poly(ethylene glycol)-block-polylactide (PEG-PLA) NPs were also used to encapsulate fluorescent SiQDs to deliver the anticancer drug quercetin, which suppress human hepatoma HepG2 cell proliferation more effectively than the free-standing form [154]. Further, Ji et al. used the ultrasmall silicon nanoparticles (SiNPs) featuring strong fluorescence, high photostability, and adjustable drug-loading capacity to load the anticancer drug DOX for long-term live cell tracking and realize in vivo cancer treatment (Fig. 13) [155]. Very recently, the applications of SiQDs for fluorescence imaging-guided high photodynamic cancer therapy have been realized by Liu et al., who selected phthalocyanine (Pc) as a representative drug and prepared SiQD-based composite nanoparticles. The as-prepared Si/Pc nanocomposite particles emit dual channel fluorescence signals and show high photodynamic cancer therapeutic efficiencies both in vitro and in vivo [156].

Fig. 13

Reprinted with permission from Ref. [155]. Copyright 2015 Wiley

a In vivo fluorescence images of mice at different time points after intratumoral injection of physiological saline (control), free DOX, SiNP-DOX, and RGD@SiNP-DOX. b Inhibition of tumor growth at different times points after intratumoral injection of physiological saline (control), pure SiNP, free DOX, SiNP-DOX, and RGD@SiNP-DOX.

5 Black Phosphorus Quantum Dots (BP QDs)

As a new member of two-dimensional (2D) layered materials, black phosphorus (BP) has attracted considerable research interest and exhibits many potential applications in various areas such as nanoelectronics, optoelectronics, bioimaging, and phototherapy [157,158,159,160]. In the BP family, zero-dimensional BP quantum dots (BP QDs, the ultrasmall BP nanosheets) have been successfully synthesized through chemical methods recently. In 2015, BP QDs were synthesized for the first time by Zhang’s group through a facile top-down method [161]. Besides, the approaches of solvothermal synthesis and ultrasonication were reported by Xu et al. and Gao et al. to prepare BP QDs, respectively [162, 163]. Compared with traditional semiconductor QDs, BP QDs show superior biocompatibility due to their in vivo stability and their final nontoxic degradation products (including phosphate and phosphonate) [164,165,166]. Consequently, BP QDs may be suitable for various biomedical applications, such as bioimaging, drug delivery, and cancer therapy (especially photothermal therapy). Sun et al. reported a new type of BP QDs (synthesized by a simple liquid exfoliation technique), which possesses excellent NIR photothermal properties (with a large extinction coefficient of 14.8 Lg⁻¹ cm⁻¹ at 808 nm and a photothermal conversion efficiency of 28.4%), good biocompatibility, as well as high PTT efficiency, suggesting great potential of the BP QDs in PTT applications [160]. Besides, Shao et al. synthesized biodegradable BP-based nanospheres using BP QDs and poly(lactic-co-glycolic acid) (PLGA), which have improved stability, good biocompatibility, excellent tumor targeting ability, and high PTT efficiency [167]. For cancer imaging application, BP QDs with excellent fluorescence properties were also obtained by ultrasonication-assisted solution method, and were successfully utilized in HeLa cell imaging [164].

6 Germanium Quantum Dots (Ge QDs)

Group IV materials (C, Si, and Ge), especially the Ge quantum dots (Ge QDs), which were synthesized for the first time in 1982 [168, 169], have attracted much attention and hold great potential for the applications in the biomedical field [169]. Compared with II–VI (e.g., CdX, X=S, Se, Te), III–V (e.g., GaAs, InP, InAs), and IV–VI (e.g., PbX, X=S, Se) QDs, Ge QDs possess various superior properties, such as valuable semiconducting and special optical properties, ultralow cytotoxicity, and electrochemical stability [169,170,171].

Based on their excellent fluorescence properties and good biocompatibility, Ge QDs may have excellent performance in the applications of cancer imaging. Li et al. developed blue-emitting pH-sensitive Ge QDs via a facile and green aqueous solution-based route and employed the Ge QDs to monitor the lysosome pH via cancer fluorescence imaging [172]. Meanwhile, Ge QDs with core–shell structure and size of ~3 nm were synthesized by Karatutlu et al. using a bench-top colloidal method [173]. This type of Ge QDs exhibits excitation-dependent fluorescence property with excellent photostability and superior biocompatibility and these superior properties render them a promising fluorescent probe for cancer imaging. In summary, considering the undesirable toxicity of traditional heavy-metal-based QDs and poor photostability of various organic dyes, the Ge QDs may hold great promise for cancer imaging and therapy.

7 Semiconducting Polymer Dots

In recent years, semiconducting polymer nanoparticles have emerged as attractive fluorescent probes in biomedical applications due to their outstanding optical characteristics [174]. Generally, these nanoparticles refer to nanomaterials consisting of π-conjugated polymers which in their pristine state are wide-band-gap semiconductors. These semiconducting polymers possess a direct band gap, resulting in an efficient absorption or emission at the band edge. According to the semiconductor band theory, an electron is excited from the highest occupied energy band (the π band) to the lowest unoccupied energy band (the π* band), which leaves a hole in the π band. The recombination of the excited electron with the hole generates a fluorescent photon. By tuning the π–π* band gaps, different semiconducting polymers can emit fluorescence of various wavelengths [174]. Therefore, semiconducting polymer-based fluorescent nanomaterials possess tunable electrical and optical properties.

Polymer dots (Pdots), a small subset of semiconducting polymer nanoparticles, possess a particle size (typically smaller than 20–30 nm) comparable to that of conventional QDs. To distinguish Pdots from conventional dye-loaded latex spheres or nanoparticles which possess only a small fraction of semiconducting polymers, Pdots are required to contain semiconducting polymers with a volume or weight fraction higher than 50%, preferably 80–90% [174]. Besides, Pdots should also contain a hydrophobic polymer interior which is essential to their colloidal stability, packing density of fluorophores, and fluorescence brightness. To date, a number of researches have demonstrated that Pdots exhibit extraordinary fluorescence brightness, fast emission rate, excellent photostability, and nonblinking feature [175,176,177,178]. These advantageous optical properties make them well-suited for applications in light-emitting devices [179]. In addition, the past decade has also witnessed the rapid development of Pdots in a wide range of biological applications, including cellular imaging [177, 178, 180,181,182,183], biological detection (e.g., pH [184], temperature [185], oxygen [186], and blood glucose level [187]), and high-resolution single-particle tracking [188]. Particularly, Pdots have attracted considerable interest in cancer diagnosis and therapeutics, due to their proper size, extraordinary brightness, and minimal cytotoxicity.

7.1 Cancer Cell-Specific Imaging and in Vivo Tumor Imaging

One of the most important applications of Pdots is the fluorescence imaging for cancer therapy. Benefiting from their flexible polymer matrix, Pdots can be functionalized via surface modification to realize cancer cell-specific fluorescence imaging [177, 189, 190]. For example, Zhang et al. prepared streptavidin (SA)-conjugated Pdots and successfully labeled the cancer cell-surface marker HER2 in human breast cancer cells through the specific recognition between biotin and streptavidin (Fig. 14) [189]. Besides, Geng et al. embedded Pdots into silica nanoparticles for targeted cellular imaging of HER2-overexpressed SK-BR-3 breast cancer cells [190]. Apart from cellular imaging, Pdots were also utilized for in vivo tumor targeting [191, 192]. Wu et al. developed a type of highly emissive Pdots (approximately 15 times brighter than the commercial QDs) modified with a tumor-specific peptide ligand and demonstrated their specific targeting to malignant brain tumors [192]. In view of the advantage that NIR light has a large penetration depth in tissue and causes minimal cellular autofluorescence, Pdots with NIR emission are highly desirable for bioimaging. To solve this challenge, Wu et al. synthesized squaraine-based Pdots with large Stokes shifts and narrow-band emissions in the NIR region and employed the Pdots to label EpCAM receptors on the surface of human breast cancer MCF-7 cells [193].

Fig. 14

Reprinted with permission from Ref. [189]. Copyright 2012 American Chemical Society

Fluorescence images of SK-BR-3 breast cancer cells treated with Pdot–SA probes. a Positive labeling of cancer cells using PFBT-C2-SA probe. b Negative labeling of cancer cells in the absence of EDC in the bioconjugation step. c Positive labeling of cancer cells using red-emitting PFTBT/PFBT-C2-SA probe.

7.2 Pdots-Based Cancer Therapy

Apart from bioimaging applications, Pdots are also widely used in designing nanomedicines for cancer treatment, especially PDT. Recently, nanoparticle-based photosensitizing agents have established their great potential in PDT as a result of the enhanced selectivity toward cancerous tissues and improved therapeutic efficacy [194, 195]. However, many PDT nanoagents are limited by low absorptivity and less efficient energy transfer. Owing to the light-harvesting and energy transfer properties of semiconducting polymers, Pdots provide new opportunities to overcome these limitations. One strategy is to entrap photosensitizers into Pdots where the polymer can efficiently absorb and transfer energy to the photosensitizer, thereby enabling enhanced ¹O₂ generation [196,197,198,199]. For example, Li et al. developed an energy transfer-mediated Pdot platform doped with a molecular photosensitizer tetraphenylporphyrin (TPP) for in vitro and in vivo PDT studies. In this system, the highly fluorescent Pdots are completely quenched, realizing an energy transfer efficiency of nearly 100% and ¹O₂ generation quantum yield of ~50% [197]. Similarly, Zhang and co-workers reported folic acid and horseradish peroxidase (HRP)-bifunctionalized Pdots incorporated with meta-tetra(hydroxyphenyl)-chlorin (m-THPC) for targeted PDT and cancer cell imaging [198]. On the other hand, considering the relatively large triplet energy values of most semiconducting polymers (typically exceed 0.98 eV), the energy from the excited state of Pdots can be directly delivered to the ground state of molecular oxygen, resulting in the generation of ¹O₂. Therefore, Pdots can serve as not only imaging agents but also photosensitizers for enhanced PDT. Shi et al. have successfully synthesized a series of ultrasmall phosphorescent Pdots composed of phosphorescent Ir(III) complexes and fluorescent fluorene units [200]. As shown in Fig. 15, the Pdots can transfer the energy from excited state upon irradiation to molecular oxygen for ratiometric oxygen sensing as well as photodynamic cancer therapy.

Fig. 15

Reprinted with permission from Ref. [200]. Copyright 2014 Wiley

a Chemical structures of phosphorescent conjugated Pdots with the Ir(III) complexes. b High-resolution transmission electron microscopy (HR-TEM) image of WPF-Ir4 in aqueous solution. c Schematic illustration of the oxygen sensing and PDT.

7.3 Non-conjugated Pdots and Their Biomedical Applications

Recently, non-conjugated Pdots which are different from traditional conjugated Pdots have emerged as a new type of fluorescent materials. Non-conjugated Pdots contain no typical fluorophore groups but only sub-fluorophores (such as C=O, C=N, and N=O), and thereby are not supposed to possess strong photoluminescence. Nonetheless, the photoluminescence of these sub-fluorophores can be significantly enhanced via polymerization and crosslinking, hydrothermal treatment, self-assembly, or physical immobilization, which is called crosslink-enhanced emission (CEE) effect [201]. Such CEE effect endows non-conjugated Pdots with outstanding photoluminescence property. Besides, these Pdots also possess good stability and low toxicity, which promote their applications in the biomedical field, including bioimaging [202] and drug delivery [203]. For example, Sun et al. utilized a general route to construct multifunctional non-conjugated Pdots by conjugating polyethyleneimine with hydrophobic polylactide [203]. The as-formed Pdots exhibit ultrabright and multicolorful fluorescence with excellent drug-loading capacity. The drug (paclitaxel)-loaded Pdots show not only improved therapeutic effect but also substantial accumulation around tumor, demonstrating their great advantage for imaging-guided drug delivery. Unfortunately, the potential application of non-conjugated Pdots in cancer therapy is still largely unexplored despite of a few reports.

In summary, Pdots hold great promise in bioimaging, especially in cancer cell-specific imaging, benefiting from their extraordinary fluorescence brightness, fast emission rate, excellent photostability, and low toxicity. Furthermore, Pdots also serve as a unique platform for developing multifunctional nanomedicines which integrate excellent imaging performance as well as improved anticancer effect.

8 Final Remarks and Future Perspective

In this chapter, we have presented a comprehensive overview of recent advancements in the area of photoluminescent QDs for cancer imaging and therapy. These quantum dots can be mainly classified into metal-containing or metal-free groups. The most common QDs, the semiconducting, metal-containing QDs, have been extensively investigated due to their superior optical properties. To date, several new types of QDs, especially the metal-free QDs (such as carbon, silicon, phosphorus, germanium, and organic molecule-based QDs) have attracted increasing interest from researchers in the field of cancer theranostics.

There are several issues that must be considered if these QDs are to be used for cancer imaging and therapy. First, the QDs should have suitable fluorescence emission property and good aqueous dispersibility and stability. Second, these QDs should have good biocompatibility, if they are used as fluorescent probes or drug carriers. Third, they can interact adequately with cells or can reach tumor tissues via passive targeting or active targeting. All these three issues are related with the structures and properties of the QDs. To design and synthesize QDs with suitable core materials, sizes, and surface chemistries are still the main challenges in the field of cancer theranostics. The development of highly effective, low-cost, and eco-friendly synthetic approaches and the elaborate control of the chemical, optical, and biological properties of these QDs are highly desired for future applications of QDs in the biomedical field. On the other hand, since a deep understanding of the interactions between QDs and cancer cells or tumor microenvironments will largely affect the imaging and therapeutic outcomes, developing new imaging techniques or analytical methods to monitor and decipher the details of the interaction processes will promote the clinical translation of these QDs.