Recent Advances in Functional Carbon Quantum Dots for Antitumour

Abstract

Carbon quantum dots (CQDs) are an emerging class of quasi-zero-dimensional photoluminescent nanomaterials with particle sizes less than 10 nm. Owing to their favourable water dispersion, strong chemical inertia, stable optical performance, and good biocompatibility, CQDs have become prominent in biomedical fields. CQDs can be fabricated by “top-down” and “bottom-up” methods, both of which involve oxidation, carbonization, pyrolysis and polymerization. The functions of CQDs include biological imaging, biosensing, drug delivery, gene carrying, antimicrobial performance, photothermal ablation and so on, which enable them to be utilized in antitumour applications. The purpose of this review is to summarize the research progress of CQDs in antitumour applications from preparation and characterization to application prospects. Furthermore, the challenges and opportunities of CQDs are discussed along with future perspectives for precise individual therapy of tumours.

Introduction

At present, tumours are still one of the main culprits threatening human health. Due to the heterogeneity and metastatic nature of tumours, their diagnosis, treatment and control are not easy. Depending on the different sites and nature of the tumour, a comprehensive analysis of the clinical manifestations and signs of the patient, combined with laboratory examination and imaging and cytopathological examination, usually leads to a definitive diagnosis.¹ However, there is still a lack of ideal and specific methods for early diagnosis, especially for deep tumours. There are many kinds of malignant tumours, which have different properties, involve different tissues and organs, occur during different stages of diseases, and have different responses to various treatments, so most patients need comprehensive treatment.² Surgery is a good way to remove tumours, but it can only eradicate early or earlier solid tumours.³ Drug therapy is chemotherapy that can kill tumours. Since tumour cells differ most from normal cells in their rapid cell division and growth, antitumour drugs usually work by disrupting the mechanisms of cell division, such as DNA replication or chromosomal separation.^4,5 Nevertheless, most chemotherapeutic drugs are not specific, so they simultaneously kill normal tissue cells that divide, often harming healthy tissues that need to divide to function properly. Radiotherapy uses radiation to kill tumour cells, thereby shrinking the tumours.^6–8 Radiation can control the growth of tumour cells by damaging their genetic material, thus preventing them from growing or dividing.^9,10 The disadvantage of radiotherapy is that it is limited to areas of radiation exposure, and it kills tumour cells while affecting normal cells.¹¹ Therefore, the early detection and diagnosis of tumours and the specific elimination of tumour cells have become the direction of antitumour research.

Located in the second period of the IVA group on the periodic table, carbon is a simple, stable and common element that is widely present in nature.¹² Since the last century, carbon has played an important role in the development of nanomaterials.¹³ Although these nanomaterials are mainly composed of the same element, carbon, they have different functions and sizes due to their different structures, which ensures that they have different but extraordinary properties. Carbon nanomaterials have been widely reported and include carbon nanotubes (CNTs),¹⁴ carbon fibres (CFs)¹⁵ and carbon quantum dots (CQDs).¹⁶ CNTs, also known as bucky tubes, are one-dimensional quantum materials with a unique structure of coaxial circular tubes.¹⁷ Research on the nucleation thermodynamics of CNTs is ongoing and has made great progress.¹⁸ CF is a unique fibre with more than 90% carbon content, and it has great application potential in the development of advanced technology.¹⁹ CQDs are carbon-based zero-dimensional nanoparticles with particle sizes less than 10 nm that are composed of dispersed spherical carbon particles.²⁰ Their nuclei are generally formed by sp² hybridization, and they have two kinds of structures: lattices and nonlattices.²¹ In 2004, Professor Xiaoyou Xu of the University of South Carolina et al reported for the first time that during the electrophoretic purification of single-walled carbon nanotubes, CQDs that can emit bright fluorescence were found in the products,²² thus introducing CQDs and opening the door for scientific researchers to further explore them. In 2007, Cao et al reported that the two-photon absorption cross sections of prepared CQDs were comparable to the two-photon absorption cross sections of the best semiconductor quantum dots or core shell nanoparticles reported in previous literature and demonstrated that the CQDs could enter breast cancer cells.²³ In 2012, Lin et al mixed CQDs with oxidants such as KMnO₄ and Ce⁴⁺ to stimulate their chemiluminescence, which is the production of light through a chemical reaction.²⁴ In 2017, Yuan et al first fabricated CQDs that had the ability to emit fluorescence from red to blue light species by controlling the fusion and carbonization of citric acid and diaminonaphthalene, which provided a new clue for the development of light-emitting diodes.²⁵ In 2019, Guo et al embedded CQDs into alginate gel beads to form composite materials that could absorb rare earth elements, which could be applied as a rare earth element adsorbent for environmental protection.²⁶ In 2021, Tao et al developed CQDs that emitted blue light in water and redshifted in the solid state and reported that these CQDs could be utilized as fluorescent ink for anti-counterfeiting and for printing high-quality fluorescent images.²⁷ Figure 1 shows the development history of CQDs since their discovery.

With the in-depth exploration of CQDs, researchers have gradually obtained a thorough understanding of them. Generally, the following commonalities can be listed. Due to the influence of precursors, the surface of CQDs is covered with hydrophilic functional groups such as -COOH and -OH, and the particle sizes are small, which endows them with the potential for good biocompatibility.^33–35 Besides, the hydrophilic groups on the surface of CQDs promote the interaction with proteins in vivo, resulting in the formation of protein corona, which changes the original properties of CQDs.^36,37 Therefore, how to change the surface properties of CQDs to realize the controllable load of proteins and how to regulate the interaction between the two has become one of the hot topics that many scientists compete to study. CQDs also have excellent fluorescence properties, which gives them considerable application prospects in the field of bioluminescence imaging and sensors.³⁸ Due to their small structure, CQDs can be trapped in tumour sites by an enhanced permeability and retention effect (EPR).^39,40 In normal tissues, the microvascular endothelial space is dense and intact, and CQDs are difficult to penetrate the vascular wall, while in solid tumor tissues, blood vessels are abundant, and the wide vascular wall space, poor structural integrity and missing lymphatic return result in high selective permeability and retention of large CQDs.^41,42 Therefore, EPR effect promotes the selective distribution of CQDs in tumor tissues, which can increase antitumour efficacy and reduce systemic side effects. Moreover, the additional functions of CQDs can be added by modification and doping. We can utilize the abovementioned characteristics of CQDs to develop and expand their antitumour applications and provide new ideas for the discovery, diagnosis, treatment and monitoring of tumours in the future. This review outlines the features and characterizations of CQDs, introduces the preparation of CQDs, elaborates the application of CQDs in antitumour applications, including drug delivery, phototherapy, monitoring and auto-antitumour applications, and discusses the design of related topics.

Features and Characteristics of CQDs Preparation of CQDs

The synthesis methods of CQDs include the "top-down" method and the "bottom-up" method.^43–45 The "top-down" method refers to the separation of CQDs with small particle sizes from carbon-based materials (such as CNTs, CF, graphite) by chemical or physical methods, including arc discharge,²² electrochemical methods,⁴⁶ chemical oxidation,⁴⁷ laser ablation⁴⁸ and combustion.⁴⁹ Contrary to the "top-down" method, the "bottom-up" method mainly refers to the formation of CQDs by carbonization and polymerization of a series of small molecules through chemical reactions, including hydrothermal methods,⁵⁰ microwave methods⁵⁵ and template methods.⁶⁰ Table 1 summarizes the advantages and disadvantages of various methods for preparing CQDs.

"Top-Down" Method Arc discharge is a self-sustaining discharge method that uses gas plasma generated in a sealed reactor to drive anodic electrodes to decompose CQDs from bulk carbon precursors. To generate high-energy plasma, the heat of the reactor can be as high as tens of thousands of degrees Celsius under the action of the electric current, so arc discharge is the most intense method of gas discharge. The most obvious visual characteristics of arc discharge are bright arc columns and electrode spots, while energy balance is another important law that describes the arc discharge phenomenon. The generation of energy is the JHEAT of the arc, and the divergence of energy occurs through radiation, convection and conduction. Xu et al accidentally harvested three kinds of CQDs with different fluorescence characteristics when purifying single-walled carbon nanotubes by the arc discharge method. These three CQDs can emit blue-green, yellow and orange fluorescence at 365 nm.²² Although this method can synthesize CQDs with good water dispersion, the size of the CQDs formed in the discharge process is different, and they generally have a larger particle size distribution.

Electrochemical methods are simple and convenient preparation methods that can be carried out under normal temperature and pressure. With the function of inhibiting maltose activity, chiral CQDs were fabricated from L- or D-glutamic acid through electrochemical methods to be used as a candidate drug for controlling blood sugar.⁴⁶ Yen et al reported a three-electrode electrochemical method for the direct synthesis of high-quality CQDs in pure water electrolyte.⁶¹ This method provides a step for the preparation of aqueous CQD solutions without further postprocessing, such as filtration, dialysis, centrifugation, column chromatography and gel electrophoresis. The results show that the prepared CQDs have a catalytic effect on H₂O₂ and reduce the charge transfer resistance and ionic diffusion resistance. Although special equipment is needed, the particle size and fluorescence properties of CQDs synthesized by electrochemical methods can be easily adjusted, and the CQDs are formed in high yield.

Chemical oxidation has been widely used to decompose bulk carbon-based materials into CQDs and introduce hydrophilic groups such as -COOH or -OH, which can significantly improve the water dispersion and fluorescence of CQDs. Qiao et al prepared CQDs by a chemical oxidation method using activated carbon as a raw material in the treatment of nitric acid, which had excellent photostability and cell avirulence and could penetrate living cells, so they had the potential to be utilized as living cell imaging agents.⁴⁷ Hu et al utilized coal tar pitch as a raw material to prepare CQDs-based nanosensors by chemical oxidation for the detection of Cu²⁺, Fe³⁺ and L-ascorbic acid.⁶²

Laser ablation is used to irradiate the surface of carbon-based materials with large particle sizes with a high-energy laser pulse, causing the materials to reach a high temperature and high pressure, quickly heat up and evaporate into the plasma state, and finally crystallize through steam to form CQDs. Yu et al prepared triple valence CQDs in toluene by laser irradiation, which increased the fluorescence lifetime.⁴⁸ Moreover, graphene was observed during production through a real-time detection system. Kang et al transformed Earth's rich resources and low-cost coal into CQDs within 5 minutes through a simple, low-cost, environmentally friendly liquid pulse laser ablation method.⁶³ CQDs prepared by laser ablation have good water dispersibility and strong fluorescence properties, but their wide application is hindered by low yield, uneven particle size and complex operation.

Combustion is a method that involves collecting CQDs from the products of burning organic matter. Zinc oxide and zinc sulfide CQDs were synthesized by combustion using zinc nitrate and thioacetamide as raw materials and ethylene glycol as fuel, whose water solution exhibited three blue-green emission peaks at 420 nm, 486 nm and 520 nm.⁴⁹ Han et al prepared single- and double-doped CQDs by a combustion method, which could skillfully adjust the doping amount of heteroatoms by changing the precursor concentration.⁶⁴ Although combustion is easy to operate, the yield of CQDs is low, and the particle size is different.

"Bottom-Up" Method The hydrothermal method is one of the most commonly used methods to synthesize CQDs. The main process is to dissolve small organic molecules and/or polymers in water or organic solvents to form reaction precursors, followed by transferring them to a stainless steel autoclave lined with Teflon and finally forming CQDs at high temperature. Ye et al first prepared two-photon CQDs by a hydrothermal method for detecting intracellular pH, which depended on the disaggregation and aggregation of their physical states, thus contributing to future applications in living systems.⁵⁰ Atchudan et al utilized P. acidus and aqueous ammonia as raw materials to form CQDs by a hydrothermal method.⁵¹ The prepared CQDs can be used as fluorescence inks and Fe³⁺ detectors, which provides a new idea for the monitoring of Fe³⁺ in industrial wastewater. Jiang et al prepared room temperature phosphorescence (RTP)-based CQDs from trimellitic acid (TA) by a hydrothermal method; these CQDs were designed to be applied for anti-counterfeiting and information encryption.⁵² Zheng et al utilized amino acids and polymers as raw materials to fabricate CQDs for blood analysis and cell imaging by hydrothermal methods.⁵³ Our group reported a method of preparing iodine-doped CQDs as a high-efficiency CT contrast agent for the first time by hydrothermal carbonization.⁵⁴ Compared with traditional iodine contrast agents, the prepared CQDs not only have unique photoluminescence and X-ray attenuation characteristics but also have the characteristics of a long cycle and passive target CT imaging. The operation of the hydrothermal method is simple, the size of CQDs is almost the same, and the yield is high.

Among the bottom-up methods, the microwave method is the most rapid commercial method. The preparation process involves dissolving the precursor in the solution to form a transparent solution, followed by heating it in a microwave oven. CQDs with PEI modified on the surface were prepared by the microwave method.⁵⁵ Among them, PEI played two important roles: enhanced fluorescence and gene delivery. Singh et al prepared CQDs with quinolinate phosphoribosyl transferase (QPRTase) on the surface by the microwave method, which was used to detect the endogenous neurotoxin quinolinic acid (QA), thus providing a candidate for the treatment of neurological disorders.⁵⁶ In addition, to address the difference in pH between wounds and normal regions, CQDs with the function of monitoring pH were fabricated by the microwave method; these CQDs provided new clues for the identification, treatment and healing of wounds in the future.⁵⁷ In addition, Pierrat et al utilized citric acid and BPEI25K as raw materials to prepare CQDs by the microwave method to transfect nucleic acids in vivo and in vitro, thus providing a new method for drug delivery to the lungs.⁵⁸ Our group also reported a synthesis of nitrogen-doped CQDs that uses citric acid and ammonium acetate as precursors through a microwave method. These CQDs had strong photoluminescence, good water solubility and stable optical properties.⁵⁹ It is worth noting that an inhomogeneous multilayer structure was observed for the first time in nitrogen-doped CQDs. The microwave method is a simple, rapid and environmentally friendly method for the synthesis of CQDs.

The template method is a technique of preparing CQDs with special materials as templates and then removing the template by physical or chemical methods to collect the CQDs. Yang et al synthesized organic templated TiOS under solvent-free conditions, performed heat treatment at 300 °C in vacuum to prepare [email protected], and finally, they transformed the organic template into N-CDs and embedded them into a titanium sulfate matrix to collect the CQDs.⁶⁰ This kind of CQDs can be used as an efficient photosensitizer and has good application potential in visible light-driven photocatalysis, which provides an effective way to prepare CQDs by a template method for visible light catalysis. The CQDs prepared by the template method have good water dispersibility and uniform particle size, but the operation is cumbersome.

Photoluminescence of CQDs

Photoluminescence (PL), a basic characteristic of CQDs, has been widely researched in related fields. PL refers to the electrons of matter jump from the valence band to the conduction band and leave holes in the valence band under the excitation of light.⁶⁵ In the conduction band and valence band, electrons and holes reach the lowest unoccupied excited state by relaxation and enter a quasi-equilibrium state. In this quasi-equilibrium state, electrons and holes emit light through recombination, forming spectra of intensity or distributing energy across different wavelengths.⁶⁶ Although PL can only be used for qualitative analysis rather than quantitative analysis, it has high resolution.⁶⁷ In recent years, many studies have been performed on the PL mechanism of CQDs, but there has been no unified conclusion.^68–70 This lack of consensus may be because different raw materials, surface passivators or preparation methods have a great impact on the structure and PL properties of CQDs. It is widely believed that the mechanism of the PL properties of CQDs involves surface defects,⁷¹ lattice defects⁷² and quantum confinement effects.⁷³ The PL analysis of CQDs has the significant advantages of high sensitivity and strong selectivity, including an excitation spectrum and emission spectrum, making it possible to choose a variety of wavelengths for molecular fluorescence analysis.⁷⁴ It is noteworthy that the traditional CQDs are excited by single photon, which leads to photobleaching when they are used for tumour tissue imaging. Two-photon CQDs are excited by two-photon near-infrared light, which can improve the tissue penetration, and thus observe in deep tissues and prolong the observation time.^75,76 Therefore, their research in tumour tissue imaging is more popular. PL is an important characteristic of CQDs, and PL measurements are helpful for later research. The influence of external pressure on the fluorescence properties of CQDs can be judged by measuring the PL and UV-Vis spectra.⁷⁷ As shown in Figure 2A, Ding et al utilized urea and р-phenylenediamine to fabricate CQDs by a hydrothermal method; the CQDs were filtered through silica column chromatography into eight different components, each of which emitted a different fluorescence colour.⁷⁸ In addition, the cerium-doped CQDs (Ce-doped CQDs) prepared by our research group also showed excellent fluorescence properties.⁷⁹ As shown in Figure 2B, with increasing excitation wavelength, the fluorescence intensity of Ce-doped CQDs increased gradually, reaching a peak at 360 nm, and then decreased gradually.

UV visible (UV-Vis) spectrophotometry is a method for the determination of the absorbance of the substance in the range of 190–800 nm, which can be used to determine the linear optical absorption behaviour of CQDs.⁸⁰ When the light passes through the solution, the absorption of the substance changes with the wavelength of the light. In the ultraviolet absorption spectrum, electrons undergo four different types of transitions, σ→σ*n→σ*π→π* and n→π*which are arranged according to the decreasing order of energy required by various transition types.⁸¹ Among them, σ→σ* transitions indicate the presence of C-C bonds,⁸² n→σ* transitions demonstrate the presence of -OH and -NH₂ bonds,⁸³ π→π* transitions occur due to the double bond absorption of aromatic nuclei,⁸⁴ and n→π* transitions imply the presence of C=O, C=S, -N=O and -N=N-.⁸⁵ The same concentration of the solution to be measured has different absorbance for different wavelengths of light, while for the same solution to be measured, the higher the concentration, the greater the absorbance, but the wavelength corresponding to the maximum absorption peak (λmax) is the same. Therefore, by measuring the absorbance of the substance at different wavelengths and drawing the relationship between the absorbance and wavelength, the absorption spectrum of the substance can be obtained. As shown in Figure 3A, Qi et al prepared nitrogen-doped CQDs (N-CQDs), which fluoresced blue under ultraviolet light but were quenched when Fe³⁺ was encountered.⁸⁶ In addition, the prepared aqueous B-CD solution was colourless and transparent under normal environmental conditions.⁸⁷ With the extension of microwave irradiation time, the peak value of the aqueous B-CD solution at 360 nm gradually increased, and its colour gradually darkened, which indicated that microwave irradiation could shorten the reaction time (Figure 3B).

Characterization and Biocompatibility of CQDs

A requirement for the biological application of CQDs is that they must be harmless to the organism, so analysis of their biocompatibility is the primary task. The excellent biocompatibility of CQDs is due to their small size and surface groups,⁸⁸ as well as biocompatibility of precursors, distribution of the surface charges and even the structural properties of carbon elements.^89–91 The particle size of CQDs is generally less than 10 nm, which makes it impossible to observe through ordinary optical microscopy. Moreover, the wavelength of transmission electron microscopy (TEM) is much shorter than that of visible light and ultraviolet light, which enables the resolution of TEM to reach 0.2 nm.^92–94 The working principle of TEM is to project the accelerated and focused electron beam onto a very thin sample. The direction of the electron beam changes due to the collision between the electron and the atom in the sample, resulting in solid angle reflection.^95–97 The size of the scattering angle is related to the density and thickness of the sample, so different light and dark images are formed, which are displayed on the imaging device after the image is magnified and focused.⁹⁸

The three kinds of CQDs, including r-CDs (Figure 4A), c-CDs (Figure 4C), and l-CDs (Figure 4D), were distributed uniformly in water, and the particle size was approximately 2 nm.⁹⁹ Furthermore, as shown in Figure 4B, the r-CDs were magnified again, and their crystal structure was observed by high-resolution transmission electron microscopy (HRTEM). In addition, Li et al prepared two CQDs, including ACDs and OCDs.¹⁰⁰ The ACDs had a crystalline structure with a lattice spacing of 0.208 nm, while the OCDs did not, which implied that there were lattice and nonlattice conformations in the CQDs. In addition, TEM showed that the size of the N,P-CDs varied, and the maximum size was approximately 15 nm.¹⁰¹ In addition, the N,P-CDs were magnified again by HRTEM. Different N,P-CDs had different lattice spacings of 0.24 nm, 0.35 nm and 0.51 nm, which implied that the N,P-CDs did not have uniform lattice spacing, which may be related to the plane spacing of the N,P-CDs. Furthermore, energy dispersive X-ray (EDX) spectroscopy is usually used in combination with TEM because it can determine the content of elements in CQDs by comparing the intensities of spectral lines of different elements.

Fourier transform infrared spectroscopy (FTIR), which is mainly composed of a Michelson interferometer and computer, can be used to analyse the chemical structure and surface groups of CQDs.¹⁰² A Michelson interferometer is a precision measuring instrument that uses the interference principle of light to precisely measure the length and tiny changes in length.¹⁰³ The principle is that incident light is reflected back by the corresponding plane mirror after being divided into two beams by the spectroscope. Because the two beams have the same frequency, the same vibration direction and constant phase difference (ie, the interference conditions are satisfied), interference can occur.¹⁰⁴ The different optical paths of the two beams can be realized by adjusting the length of the interference arm and changing the refractive index of the medium so that different interference patterns can be formed.¹⁰⁵ The intensity distribution of the original light source can be calculated by Fourier transform of the interference function on a computer.¹⁰⁶ Compared with traditional spectrometers, FTIR has the following advantages: fast scanning speed, high resolution, high sensitivity and wide spectral range.¹⁰⁷ Zhang et al prepared layered double hydroxide-combined CQDs (LDH-carbon quantum dots) capable of adsorbing methyl blue.¹⁰⁸ As shown in Figure 5, compared with the fixed hydrogen bond peak of LDH, the hydrogen bond peak of LDH-carbon quantum dots blueshifted (3490 to 3479 cm⁻¹), suggesting that there was a hydrogen bond between LDH and CQDs. Moreover, the peaks at 1183 cm⁻¹ and 1038 cm⁻¹ were attributed to the -SO₃- of LDH and LDH-carbon quantum dots.

X-ray photoelectron spectroscopy (XPS), an advanced analytical technique for the microanalysis of electronic materials and components, can analyse the chemical elements in CQDs, and it is usually coordinated with auger electron spectroscopy (AES).¹⁰⁹ The principle of XPS is to irradiate the CQDs with X-rays so that the core electrons of the atoms or molecules (also known as photoelectrons) are excited to emit and then measure the energy of photoelectrons.¹¹⁰ With the kinetic energy/binding energy of photoelectrons as the abscissa and the relative intensity as the ordinate, the photoelectron spectrum can be created to obtain relevant information on the CQDs.¹¹¹ As a modern analytical method, XPS has the following characteristics: the ability to analyse all elements except H and He, strong identification, the ability to observe chemical shifts, quantitative analysis and high sensitivity.¹¹² Figure 6A–C correspond to the survey, C_1s and O_1s XPS spectra of the obtained CQDs, respectively. As shown in Figure 6D, two peaks at 459.4 eV and 465.2 eV corresponded to the Ti⁴⁺2p_3/2 core level, while the peaks at 457.8 eV and 460.9 eV belonged to Ti³⁺2p_1/2. XPS results demonstrated that CQDs were mainly composed of three elements, including carbon, oxygen and titanium.¹¹³

Interestingly, some researchers prepared green CQDs with food as raw material, which fully demonstrated the high biocompatibility of CQDs.^114,115 Nevertheless, although the biocompatibility of CQDs has been reported in many studies, some researchers have found that there is a "threshold" for the low toxicity of CQDs. Liu et al reported the CQDs prepared at high temperature by mixing folic acid and CuCl₂ with anhydrous ethanol.¹¹⁶ When the CQDs were mixed at different concentrations and added to cells, the cell viability was no less than 90% before 400 μg/mL but gradually decreased from 600 μg/mL. Therefore, it can be inferred that the toxicity and side effects of CQDs on cells are related to the type of cells co-incubated, the concentration of CQDs and the co-incubation time; these results are conducive to the subsequent exploration of the biocompatibility of CQDs and the design of their application in antitumour projects. It is expected that the toxicity assessment of therapeutic CQDs with potential biomedical applications will be carried out through animal models that conform to the norms of international organizations in the future.

Applications of CQDs in Antitumour Diagnosis

At present, clinical cancer is often found in the middle or even late stage, making later treatment much more difficult. When it is not discovered until the cancer develops to the late stage, the cancer is difficult to handle and shortens the life span.¹¹⁷ The treatment of advanced cancer can only rely on drugs,¹¹⁸ chemotherapy¹¹⁹ or radiotherapy¹²⁰ to kill other free tumour cells, but these methods cannot completely kill all tumour cells in vivo. In addition, advanced cancer easily metastasizes and spreads, leading to the invasion of tumour cells in other normal parts of the body, which makes it impossible to control the tumours simply by removing the original area.¹²¹ Therefore, the development of tumour cell probes for early cancer screening is vital,¹²² and CQDs have become candidates for probes of tumour cells due to their excellent fluorescence properties and high sensitivity.

Because they are fluorescent, CQDs become excited after receiving electromagnetic radiation, thus emitting the same or different emission spectrum as the excitation spectrum and showing fluorescence.¹²³ Due to this characteristic, CQDs have been widely used in fluorescence imaging and sensing.^124–126 When two fluorescent groups meet, if the emission spectrum of one fluorescent group is consistent with the excitation spectrum of the other, the fluorescence signal of the former weakens or even disappears, which is called fluorescence resonance energy transfer (FRET).¹²⁷ The conditions for effective FRET between the donor and the acceptor are specific.¹²⁸ First, the emission spectrum of the donor and the absorption spectrum of the acceptor must overlap.¹²⁹ Second, FRET requires that the donor and the energy acceptor be close enough, generally 7–10 nm apart, and the FRET decreases significantly with distance.¹³⁰ Finally, the fluorescent chromophores of the donor and the acceptor must be arranged correctly.¹³¹ Due to this principle, many researchers have combined FRET with CQDs for biological analysis.^132–134 Using CQDs as donors and AuNPs as acceptors, Somayeh et al assembled a FRET-based sensor that could detect CA125 on the surface of ovarian cancer cells. Due to the -NH₂ groups, anti-CA125 could be modified on the surface of AuNPs.¹³⁵ The sandwich structure of the AuNPs-CA125-CDs could be formed when the CA125-trapped AuNPs met the DNA-coated CQDs, which promoted the occurrence of FRET and led to the fluorescence quenching of the CQDs. As shown in Figure 7A, by -S- wrapping PAMAM-Dendrimer with -NH₂ on the surface of AuNPs, the -NH₂ on the surface of AuNPs could be amplified, which made the fluorescence quenching signal of CQDs more intense, thus greatly improving the sensitivity of the device to CA125. Moreover, the UV-Vis absorption spectrum showed that the absorbance of AuNPs-PAMAM/Ab was higher than that of AuNPs-Ab at any wavelength, which implied that the adjunction of PAMAM-Dendrimer could significantly enhance the fluorescence quenching signal (Figure 7B).

In recent years, as a combination of chemiluminescence and electrochemical methods, electrochemiluminescence (ECL) has attracted great interest due to its high sensitivity and selectivity.¹³⁶ ECL is an electricity generation phenomenon in which certain substances are generated by electrochemical methods, and then these substances or biomass and other substances react further.¹³⁷ It retains the advantages of chemiluminescence methods, such as high sensitivity, wide linear range and convenient observation, and has many incomparable advantages, including good reproducibility, easy control and partial repeatability.¹³⁸ ECL not only greatly promotes biochemistry and molecular biology research but also brings a technological revolution in clinical diagnosis, in which ECL probes play an important role.¹³⁹ Although CQDs have been developed as ECL probes,^140,141 they are not widely used because of their low ECL yield. Therefore, the modification of CQDs to amplify the ECL signal is conducive to the application of ECL technology in biological analysis. Qiu et al designed an ECL system by combining CQDs with magnetic beads.¹⁴² MCF-7 cells and MDA-MB-231 cells were captured on an ITO electrode by folic acid, and then HA-solid-state ZnCQDs could bind to CD44 on the surface of tumour cells through HA, resulting in an enhanced ECL effect. It can be utilized for monitoring tumour cells and evaluating the heterogeneity of CD44 expression, which is conducive to the early diagnosis and screening of breast cancer. Moreover, the greater amounts of H₂O₂ secreted by tumour cells can be used in active metabolism to induce stronger ECL emission from NHCDs combined with Au NPs, which provides another method of cancer monitoring.¹⁴³ Nevertheless, without electrode plates, DNA or RNAs as aptamers that can identify HER2 on the surface of MCF-7 cells are applied to amplify the ECL signal through CQDs or synergistic metal-organic frameworks.¹⁴⁴

In addition to FRET and ECL, CQDs can also be utilized to monitor the tumour environment by structural modification, spatial conformation or properties. Won et al combined gelatine and ureidopyriminone (Upy) into hydrogels (Gel-UPy) by performing isocyanate-amine group reactions, followed by mixing CQDs into the Gel-UPy to prepare a self-healing Gel-UPy/dsCD hydrogel.¹⁴⁵ The self-healing properties of the Gel-UPy/dsCD hydrogel depended on GSH and H₂O₂, which are produced by tumour cells rather than normal cells. As shown in Figure 8, after planting 5 pieces of Gel-UPy/dsCQD hydrogel into the cell inoculation site of mice, one hour later, the Gel-UPy/dsCQD hydrogel of the MDCK group was still a single slice, while the MDA-MB-231 group converged into a whole gel structure. This result indicates that the Gel-UPy/dsCQD hydrogel can self-heal at the tumour site but not in normal cells, so it can be used to monitor tumour cells through its self-healing function. In addition, Kalytchuk et al prepared CQDs with ICPA (a molecular fluorophore from the family of pyridines) on the surface, which caused fluorescence quenching when the CQDs were cooled to a solid and returned to the liquid state when heated, recovering their fluorescence.¹⁴⁶ However, when the CQDs were in a cool solid-state, the addition of alcohol prevented fluorescence quenching. Therefore, they can be used to measure alcohol, which is conducive to the early diagnosis of lung cancer. In addition, CQDs can monitor tumours by using the fluorescence change of CQDs as the detection signal. Li et al fabricated CQDs that could produce 430 nm and 642 nm emission under 360 nm laser irradiation.¹⁴⁷ When they encountered GSH, they only produced 430 nm excitation light, which made their fluorescence change from blue green to dark blue. The GSH content in tumour sites is much greater than that in normal tissues, so we can distinguish tumour cells from normal cells by the change in fluorescence colour of CQDs.

In contrast to normal cells, tumour cells have a unique acidic environment, tumour markers and related genes; these factors can be considered in the design of new treatments. Table 2 summarizes the application of CQDs for monitoring tumours or antitumour drugs in related research. At present, the optical properties of CQDs are mostly used in tumour monitoring research, in which FRET and ECL are the most widely used, while other properties are rarely involved. However, whether the other properties of CQDs, such as changes in surface functional groups, size and properties, could be utilized to monitor tumours remains to be explored in the future. In addition to the early diagnosis of tumours and the release of antitumour drugs, monitoring the heterogeneity, mutation, metastasis and recurrence of tumours is a possible application of CQD probes.

Drug Delivery

Traditional antitumour drugs have low bioavailability due to their nontargeting and metabolic dynamics, and they can damage normal cells.¹⁵⁶ Therefore, it is urgent to improve the bioavailability of antitumour drugs. The natural, ultrasmall structure endows the CQDs with EPR, which actively causes them to gather in the tumour site.¹⁵⁷ Using CQDs as carriers to deliver antitumour drugs to the body not only solves the drug targeting problem but also increases the time of drug aggregation in the tumour site.¹⁵⁸

Doxorubicin (DOX), as a broad-spectrum antitumour drug, can inhibit the synthesis of RNA and DNA, inducing the death of tumour cells in various growth cycles.^159–161 DOX can restrain DNA superhelix topoisomerase II by embedding and inhibiting the interaction between polymer biosynthesis and DNA, thus destroying the replication of the DNA strand.^162,163 It can also act on chromatin with transcriptional activity, which leads to histone changes. In the clinic, DOX has been widely used in the treatment of various diseases, including breast cancer,¹⁶⁴ lung cancer,¹⁶⁵ ovarian cancer,¹⁶⁶ leukaemia,¹⁶⁷ and gastric cancer.¹⁶⁸ Therefore, loading CQDs with DOX and incorporating them into the body for tumour treatment has become possible. Nitrogen- and phosphorus-doped CQDs (PNHCDs) loaded with DOX were prepared by spontaneously heating glucose to boiling without additional heating operation.¹⁶⁹ The tumour volume of tumour-bearing mice treated with PNHCDs-DOX was larger than that of tumour-bearing mice treated with free DOX. Moreover, the weight of tumour-bearing mice treated with free DOX gradually decreased, while that of the mice treated with PNHCDs-DOX showed an upward trend, which indicated that PNHCDs-DOX could not only inhibit tumour growth but also improve the tumour targeting of DOX, exhibiting the advantage of the EPR of CQDs. In addition, compared to free DOX, DOX-loaded and transferrin-modified C-Dot-Trans-Dox showed better therapeutic effects for brain cancer.¹⁷⁰ Since both tumour cells and the blood brain barrier (BBB) have transferrin receptors, it is possible for C-Dot-Trans-Dox to cross the BBB and gather in brain tumour sites, which is promising for the treatment of brain tumour-related diseases in the future. Moreover, due to the nitrogen functional groups on the surface of the CSCNP-R-CQDs, they could enter not only the tumour cell nucleus but also the cancer stem cell nucleus, which provides new possibilities for tumour eradication.¹⁷¹ As a pH-sensitive drug, DOX is more soluble under acidic conditions than under neutral conditions. Tumour tissues and cells can provide an appropriate pH environment for DOX release, which also leads to the tumour targeting of DOX. With the rapid development of CQDs technology, researchers are committed to combining it with other chemical compounds, biomaterials and even technology to play enhanced antitumour roles. Hou et al modified aminoethyl anisamide (AEAA) onto CQDs loaded with DOX and Fe ions to form [email protected]/DOX. Then, [email protected]/DOX were connected with each other by Asp-Ala-thl-gly-pro-Ala peptides (Pep) to form losartan (Los) wrapped in a mesoporous structure, thus finally forming [email protected]/DOX-LOS.¹⁷² Pep was sensitive to the fibroblast-activating protein-α (FAP-α), while FAP-α was overexpressed in cancer-associated fibroblasts (CAFs). When [email protected]/DOX-LOS flowed through the CAFs, Pep was degraded, while [email protected]/DOX and Los were released. Los, as a modifier of the tumour microenvironment, could deplete the tumour matrix. Along with Fe ions and DOX, Los had the ability to accelerate the infiltration of T cells and NK cells, promote the release of proinflammatory cytokines, and downregulate the recruitment of immunosuppressive cells (Figure 9). Kang et al coated silicon dioxide on the surface of a nanotube template and increased the temperature, forming CQDs on the surface of silicon dioxide.¹⁷³ The template simultaneously dissolved to form a hollow structure (C-hMOS), and then the C-hMOS was filled with DOX by soaking. Moreover, after heat treatment, the DOX-delivering C-hMOS showed more significant fluorescence intensity, which endowed them with the ability to observe the distribution of DOX in vivo. In addition, Türk et al fabricated pH-sensitive hydrogels by self-crosslinking hydroxyapatite, NCQDs and DOX through Schiff bases, hydrogen bonds and ion interactions for the first time.¹⁷⁴ After reaching the tumour site, the Schiff base broke away and DOX was released.

Similar to other platinum drugs, oxaliplatin (II) has an antitumour mechanism that targets DNA, and platinum atoms cross-bind with DNA to prevent its replication and transcription.^175–177 Nevertheless, due to drug resistance and side effects, it is possible to use Pt(IV) complexes instead of oxaliplatin (II).¹⁷⁸ Pt(IV) complexes are prodrugs of oxaliplatin(II) that can be transformed into oxaliplatin(II) by a reduction reaction, and tumour cells can provide an inherent reducing environment. Based on this principle, CQDs were prepared by modified thermal pyrolysis with citric acid as the carbon source and polyene polyamine as the passivator, following which a Pt(IV) complex (oxa(IV)-COOH) was combined with CQDs to form CD-Oxa by dehydration condensation between the amino carboxyl groups.¹⁷⁸ Under excitation light at 405 nm, 488 nm and 555 nm, CD-Oxa could produce bright polychromatic fluorescence, which was conducive to its effective monitoring of tumour cells (Figure 10A–E). Moreover, with increasing drug concentration, the viability of HepG2 human tumour cells decreased gradually, and the decrease in the CD-Oxa group was greater than that of the free oxaliplatin group, which indicated that the curative effect of CD-Oxa was better than that of free oxaliplatin (Figure 10F). In addition, Feng et al mounted PEG-(PAH/DMMA) on the surface of CQDs containing Pt(IV) (CDs-Pt(IV)). Among them, PEG-(PAH/DMMA) was equivalent to a "cap", which was not hydrolysed under normal conditions but hydrolyzed under the acidic conditions of tumour tissues, causing CDs-Pt(IV) to enter the cells. Finally, the Pt(IV) prodrug was reduced to oxaliplatin(II), which binds to the nucleic acid of tumour cells.¹⁷⁹ Similarly, PEG-RGD was modified on CDs-Pt(IV), in which PEG and RGD were linked by benzoimide bonds.¹⁸⁰ RGD could target the RGD receptor on the surface of tumour cells, but the RGD receptor was not specific to tumour cells. Therefore, PEG was designed to block RGD on the surface of CDs-Pt(IV). When CDs-RGD-Pt(IV)-PEG reached the tumour cells, the benzoimide bond was hydrolysed under acidic conditions to expose RGD to achieve the targeting of tumour cells.

Gene therapy refers to the transfer of exogenous "repair genes" into target cells to treat related diseases at the gene level.^181–183 The free gene is easily degraded by lysosomes after entering the cells, so it needs to be protected by the vector and then sent to the target cells to solve the problem.¹⁸⁴ Although single CQDs do not have the ability to carry genes, if gene carrying substances are modified on their surface, they can carry genes into target cells for gene therapy.^185,186 Three kinds of LNA-based oligonucleotides (anti-miR-K1, anti-miR-K4, and anti-miR-K11) were loaded onto CQDs by PEI modification.¹⁸⁷ As shown in Figure 11, after entering lymphoma cells, Cdots/LNA could knock down miR-K1, miR-K4 and miR-K11 of Kaposi's sarcoma virus (KSHV), thus leading to apoptosis and growth inhibition of KSHV-positive primary active lymphoma cells through the caspase-3 pathway. Moreover, a novel DNA self-assembly nanostructure based on nitrogen-doped CQDs known as NPNCD was reported.¹⁸⁸ The conjugation of NPNCD with KRAS siRNA showed a superior gene knockdown effect, which provided inspiration for the clinical treatment of non-small-cell lung cancer.

In addition to DOX, oxaliplatin and "therapeutic" genes, other forms of antitumour drugs have also been widely researched. Large neutral amino acid transporter 1 (LAT1) is highly expressed in various tumour cells but is rarely expressed in normal cells. LAT1 transports amino acids into tumour cells by binding with amino and carboxyl groups on the surface of amino acids. Li et al utilized 1,4,5,8-tetraminoanthraquinone (TAAQ) and citric acid as raw materials to prepare CQDs based on simulated large amino acids (LAAM TC-CQDs).¹⁸⁹ There were many paired amino and carboxyl groups on the surface of the LAAM TC-CQDs, which made the tumour cells mistakenly believe that they were amino acids; thus, the drug-loaded LAAM TC-CQDs could be transported into tumour cells through LAT1. It is exciting that LAT1 is expressed not only in tumour cells but also in the BBB, which suggests that LAAM TC-CQDs can be utilized to treat brain cancer across the BBB. In addition, microwave ablation (MWA) uses a special needle to puncture the lesion, transmits RF microwaves to the needle tip, generates a large quantity of heat at this position, and destroys the focus, thus treating the disease. Tumour cells killed by MWA can release tumour-associated antigens (TAAs). Some CQDs can capture TAAs, while some carbohydrate modifiers can target dendritic cells (DCs), and mannose-based CQDs (Man-CDs) combine the two.¹⁹⁰ Man-CDSs can deliver captured TAAs to DCs, activate the immune system and amplify MWA, thus skillfully combining drug delivery with immunotherapy. Silver nanoparticles are natural nanodrugs that kill tumour cells. Ghosal et al utilized fresh lemon slices as raw materials to prepare green CQDs by a hydrothermal method and then added solid silver nitrate to CQDs aqueous solution to synthesize CQDs loaded with silver nanoparticles ([email protected]) in situ.¹⁹¹ [email protected] kill tumour cells by producing ROS without any other external roles. Amygdalin, a natural chemotherapeutic drug, is nontoxic, but when it is metabolized by β-glucosidase, it produces toxic hydrocyanic acid, which can be catalysed into nontoxic thiocyanate by thiocyanase. Kalaiyarasan et al used a gold acid as a raw material to prepare CQDs with many carboxylic acids on the surface and then functionalized them with almond protein by dehydration or esterification to form [email protected]¹⁹² Malignant cells contain more β-glucosidase than normal cells, while normal cells contain more thiocyanase than tumour cells, so [email protected] can cleverly achieve selective killing of tumour cells after entering the body.

Table 3 summarizes the application of CQDs as drug carriers in antitumour research. To achieve good clinical antitumour effects, the selection of CQDs and drugs is very important. The choice of antitumour drugs can be made according to the different manifestations of drugs on normal tissues and tumour tissues. Tumour tissues have more acidic substances, H₂O₂, glutathione and tumour markers than normal tissues. If the antitumour drugs are nucleic acids, it is necessary to prepare CQDs with gene carrying materials such as PEI and chitosan or modify them on the surface of CQDs. Moreover, encapsulation of CQDs in macromolecules such as nanoparticles and hydrogels is also a possible strategy. In addition, CQDs can be reformed by doping, grafting and modification so that they can combine with antitumour drugs to target tumour sites to expand the therapeutic effect against cancer. In summary, the primary purpose of these methods is to improve the targeting of drug delivery and prolong the residence time of drugs in tumour sites, thus achieving better antitumour efficacy than that of free drugs.

Monitoring the Release of Antitumour Drugs

Antitumour drugs need to be released in time after reaching the tumour sites to exert antitumour effects, and CQDs can monitor their release in real time. P-CQDs were CQDs covered with PEI on the surface, which could emit blue fluorescence under the activation of the excitation signal under normal circumstances.²⁰¹ When hyaluronic acid-conjugated doxorubicin (HA-Dox) was coated on the surface of P-CQDs, the fluorescence of P-CQDs was quenched by FRET, in which P-CQDs and HA-Dox were the donor and the acceptor, respectively. When P-CQDs/HA-Dox reached and entered tumour cells, the HA on the surface of P-CQDs/HA-Dox was degraded into small fragments by intracellular hyaluronidase; thus, DOX was released, and the P-CQDs were exposed. Additionally, the fluorescence recovery of P-CQDs could be used to monitor drug release. Similarly, under the effect of FRET, CQDs-Pt(IV) emitted blue, green and red fluorescence, but the blue fluorescence was limited by the quenching agent dabsyl.²⁰² Under the condition of an intracellular reduction reaction, the three substances disintegrated and released Pt(IV), and the blue fluorescence of the CQDs was restored, which was used to monitor the activation of antitumour prodrugs in a complex biological microenvironment. Li et al encapsulated the CQDs and DOX into mesoporous silica nanoparticles to form DOX-CQDs/[email protected]²⁰³ PNVCL was folded on the surface of the DOX-CQD/[email protected] by hydrophobic phase transition, which blocked the orifices and hindered DOX release and concealed the fluorescence of the CQDs. Due to the acidic conditions of the tumour, the Schiff base linkage between MSNs and CQDs/PNVCL was broken, and then DOX and CQDs were released, so the release of antitumour drugs can be monitored by the fluorescence of CQDs.

Photothermal Therapy

In recent years, photothermal therapy (PTT) has attracted extensive research, which aims to use a photothermal conversion agent to absorb near-infrared light (650~900 nm) and convert the absorbed light energy into heat energy to further achieve the deep penetration of tissues and the minimum heating of nontarget tissues.^204–206 Therefore, the choice of photothermal agent is the key factor in determining the success of photothermal therapy. First, to obtain effective treatment results, near-infrared photothermal agent-mediated tumour treatment usually requires a near-infrared laser irradiation power density significantly less than the maximum allowable skin exposure.²⁰⁷ Second, photothermal agents are applied for photothermal treatment of the tumour, and they should have good biocompatibility with surrounding tissues.²⁰⁸ Third, only when the size of the photothermal agent is small enough can it enter the tissue cells for photothermal therapy, so it must have superior tissue targeting.²⁰⁹ At present, widely used photothermal materials include noble metal nanoparticles such as Au, Ag and Pt,^210,211 carbon materials such as graphene and carbon nanorods,²¹² metal and nonmetal compounds such as CuS and ZnS,²¹³ and organic dyes such as indocyanine green and Prussian blue,²¹⁴ while few studies have mentioned CQDs.

CuS nanoparticles carrying CQDs and bortezomib were synthetized, coated with macrophage membranes and modified with the T7 peptide to form [email protected]²¹⁵ After [email protected] enters the body, the immune system does not attack them because they are protected by the macrophage membrane, but instead, it transports them to tumour cells through the T7 peptide. CQDs are used for photothermal ablation under 808 nm NIR, while bortezomib is a proteasome inhibitor that can protect tumour suppressor proteins from degradation, thus uniting in opposition to tumours (Figure 12A). As shown in Figures 12B and C, the photothermal conversion efficiency of the composites increased with increasing irradiation time. Compared to the PBS, CuSCD and [email protected] groups, the [email protected] group could reach 50 °C in 3 minutes, indicating its superior photothermal conversion rate, thus demonstrating the feasibility of thermal ablation therapy for tumour cells. Wang et al prepared a self-crosslinked chitosan hydrogel (CCHN) coated with DOX and CQDs with an average particle size of 65 nm to endow injectability.²¹⁶ When CCHNs reached the tumour site, chitosan hydrolysed and released CQDs and DOX. CQDs kill tumour cells through PTT; however, DOX is an antitumour drug, so this system achieves a dual antitumour effect of PTT synergistic drug delivery. In addition, Qian et al developed a CQDs-based mesoporous silica scaffold ([email protected]) for photothermal synergistic immunotherapy.²¹⁷ [email protected] targeted tumour sites and participated in photothermal ablation, while biodegradable [email protected] fragments could obtain TAAs from photothermally lethal tumour cells and then carry them away from necrotic tissue, initiating the immune response by stimulating the proliferation and activation of NK cells and macrophages.

Photodynamic Therapy

Photodynamic therapy (PDT) is a new method for treating tumour diseases with photosensitive drugs and laser activation, whose mechanism of action is to produce ROS by the interaction between a photosensitizer and oxygen to kill tumour cells.^218–220 The photosensitizer absorbed by the tissue is stimulated by laser irradiation of a specific wavelength, and the excited photosensitizer transfers energy to the surrounding oxygen to generate highly active singlet oxygen.²²¹ Singlet oxygen reacts with adjacent biological macromolecules to induce cytotoxicity, which leads to cell damage and even death.²²² In the treatment of oesophageal cancer,²²³ lung cancer,²²⁴ skin cancer,²²⁵ breast cancer²²⁶ and other diseases, PDT is a new tumour treatment technology that has risen and developed in recent years. As a cold photochemical reaction, the basic elements of PDT are oxygen, photosensitizer and visible light (commonly applied with a laser). Only by choosing an appropriate photosensitizer can PDT achieve the purpose of killing tumours. First, photosensitizers have a certain selectivity and affinity for tumour tissues and remain in tumour sites for a long time so that the concentration difference between tumour sites and normal tissues is maximized.²²⁷ Second, a photosensitizer can produce singlet oxygen in tumour tissue by illumination.²²⁸ Last, the photosensitizer is excited by light of an appropriate wavelength.²²⁹

Jia et al utilized manganese phthalocyanine (Mn-PC) as a raw material to prepare hydrophobic magnetic fluorescent CQDs (Mn-CDs) by a high-temperature method, followed by inserting PEG on the surface to improve their hydrophilicity.²³⁰ Mn²⁺ in Mn-CDs could be used as a catalyst to decompose a large amount of H₂O₂ at the tumour sites into oxygen, and ¹O₂ could be converted into ROS under irradiation with NIR light (635 nm), killing tumour cells (Figure 13). The lack of O₂ in tumour tissues limits the efficacy of PDT in antitumour therapy. Therefore, increasing O₂ content in tumour sites becomes the primary premise of PDT phototherapy. In addition, although C₃N₄, a water splitting material, can decompose water into O₂ by PDT, the decomposition efficiency is not high. To solve this problem, Zheng et al embedded CQDs and protoporphyrin IV (PpIV) on C₃N₄ to form PCCN.²³¹ Among them, CQDs were used to enhance the water decomposition of C₃N₄, thereby increasing the production of O₂, followed by transforming O₂ into ROS by PpIV under light and killing the tumour cells. Nevertheless, traditional PDT applies ultraviolet and visible light as the excitation light source, which cannot penetrate the tissues sufficiently, so PDT can only be used for shallow tumours. It is not effective for deep tumours, which greatly limits the scope of PDT in clinical applications. At present, there are studies that combine upconversion nanophosphors (UCNPs) with PDT; such methods use low-energy near-infrared light as the excitation light source and upconvert it into high-energy light to achieve the required spectrum of PDT photosensitizers.^232,233 Near-infrared light has a deeper light transmission depth, which allows PDT to be utilized for deeper tissue treatment. Chan et al combined UCNPs with CQDs for the treatment of tumours through PDT.²³⁴ Among them, UCNPs were used as light converters to transform 808 nm near-infrared light into ultraviolet light for CQDs to produce ROS and then kill tumour cells.

Photothermal Therapy and Synergetic Photodynamic Therapy

PTT or PDT alone cannot completely eliminate tumours, so the combination of these two methods improves the curative effects.^235–237 Based on the upconversion luminescence (UCL) principle described above, the combination of PDT and PTT for tumour phototherapy provides a new opportunity to apply CQDs in tumour treatment. Using ZnPc as a photosensitizer and CQDs as thermal sensitizers, Lv et al prepared a nanomaterial with CQDs and UCNPs, in which the CQDs could gradually heat and ablate tumour cells while the UCNPs could transfer energy to ZnPc to generate ROS to kill the tumour cells under 980 nm near-infrared light irradiation; this method was expected to yield twice the result with half the effort.²³⁸ The CQDs grafted with the photosensitizer Ce6 were mixed with Cu²⁺ to form nanoparticles (Cu/CC NPs).²³⁹ A shown in Figure 14, Under the low pH, glutathione (GSH) overexpression and H₂O₂ surplus of the tumour environment, the Cu/CC NPs disintegrated and released CDs-Ce6 and Cu²⁺. GSH can eliminate ROS, which seriously hinders the PDT pathway of killing tumour cells. Therefore, the addition of Cu²⁺ can initiate Fenton-like reactions to eliminate H₂O₂ and produce O₂ for PDT while also removing GSH and strengthening PDT. Moreover, due to FRET, the assembled Cu/CC NPs hide the fluorescence of the CQDs, but they disintegrate and release CQDs when entering tumour cells, thus exposing the fluorescence of the CQDs and providing the possibility for monitoring their entrance into tumour cells. Hua et al grafted HPPH into CQDs doped with Cu²⁺ and Gd³⁺ to form BCCGH.²⁴⁰ CQDs, as thermal sensitizers, enabled PTT under 808 nm laser irradiation, while HPPH was a photosensitizer that produced ROS to kill tumour cells under 671 nm laser irradiation. In addition, Zhang et al combined PTT, PDT, starvation-like therapy and immunotherapy to fight tumours, in which Mn,Cu-CDs were utilized as both photosensitizers and thermal sensitizers, while the glucose-metabolic reaction agent glucose oxidase (GOx) snatched glucose from the tumour cells, causing them to starve.²⁴¹ Moreover, the tumour-associated antigens released by the above three ways of killing tumour cells stimulated cytotoxic T lymphocytes to penetrate distant tumours through the PD-1/PD-L1 pathway, which not only killed the primary tumours but also blocked the growth of distant tumours.

Compared with that of other nanoparticles, research on the application of CQDs in phototherapy is very limited. Table 4 summarizes the application of CQDs as thermal sensitizers/photosensitizers in antitumour research. Moreover, most CQD-mediated PTT systems are designed to encapsulate CQDs into nanoparticles. PTT requires that the thermal sensitizer has a superior photothermal conversion rate, and CQDs are candidates for thermal sensitizers owing to their naturally dark colour. The efficiency of photothermal conversion can be enhanced by screening raw materials and reforming synthetic CQDs. To design materials for CQD-mediated PDT, we need to bind a photosensitizer to the CQDs, and because tumour sites lack enough oxygen to produce ROS, so we also need to add specific materials to produce O₂. Among them, some transition metals, such as Co²⁺, Cd²⁺, Cu²⁺, Ag⁺, Mn²⁺ and Ni²⁺, can accelerate or replace Fe²⁺ to promote a Fenton-like reaction, thus becoming oxygen suppliers. Moreover, due to the low tumour eradication rates of PTT and PDT alone, the combination of PTT and PDT or with other therapies, including immunotherapy, radiotherapy, chemotherapy, hormone therapy and precision medicine, to achieve a higher tumour eradication rate needs further research.

Tumour-Targeting Lethal CQDs

Tumour-targeting lethal CQDs have antitumour effects without any other synergism and depend on the production of ROS after they are absorbed by tumour cells. Such CQDs only kill tumours, while normal cells can escape them. Using polyethylene glycol (PEG) as a carbon source, imidazole as a nitrogen source and phosphoric acid as a phosphorus source, Bajpai et al prepared N- and P-doped CQDs (NPCDs) by a heating method that could induce cell cycle arrest, autophagy and apoptosis in B16F10 melanoma cells.²⁵³ Yao et al utilized ginsenoside as a raw material, adding appropriate amounts of citric acid and EDA to improve the fluorescence performance to prepare antitumour Re-CQDs,²⁵⁴ while Emam and Ahmed used NaOH to dissolve carrageenan and pullulan and then fabricated two kinds of CQDs by a hydrothermal method, which were used for antitumour and antiviral activities.²⁵⁵

Summary and Outlook

In this review, the preparation methods, features and characterizations of CQDs were briefly introduced, and the application prospects of CQDs in the antitumour field were also summarized. The preparation methods of CQDs include "top-down" methods and "bottom-up" methods. The difference is that the former separates the CQDs from matter with a large molecular weight, while the latter produces CQDs by carbonization of small molecules. CQDs have excellent biocompatibility due to their small structure and hydrophilic groups on the surface. Moreover, although there is no consensus on the luminescence mechanism of CQDs, it is known to be related to the vacancies of surface groups and epitopes.

The applications of CQDs in antitumour therapy mainly include the detection and diagnosis of early tumours, delivery of antitumour drugs as carriers, monitoring the release of antitumour drugs, phototherapy as photosensitizers or thermosensitizers, and targeted killing of tumour cells by autonomic production of ROS. Early-stage tumours have no obvious clinical manifestations, and the changes in tumour markers are very small, which makes them difficult to detect, diagnose and treat early. CQDs have excellent specificity and sensitivity, and they can be used to effectively monitor early-stage tumours by means of FRET, ECL, and structural modifications to enable the detection of tumour markers or the tumour microenvironment and to release antitumour drugs. CQDs are most widely utilized in antitumour therapy for drug delivery, including traditional drugs and gene therapy drugs. The small size of CQDs increases their specific surface area, which is conducive to the improvement of the encapsulation and loading efficiency of a series of antitumour drug molecules. In addition, the high biocompatibility, low relative cytotoxicity and good cell uptake ability of CQDs also make them excellent materials for drug delivery. CQDs can combine with drugs by covalent bonds, noncovalent bonds, or both, so they can create different types of connections to exhibit high performance while maintaining their original properties. Moreover, because tumour cells are more sensitive to the thermal environment than normal cells, CQDs can be utilized as thermal sensitizers to achieve the targeted killing of tumour cells. Moreover, a photosensitizer can be doped into CQDs or encapsulated in nanoparticles with CQDs to achieve the antitumour effect of synergistic PTT and PDT. The mechanism of PDT is to produce ROS to damage tumour cells, which is consistent with the mechanism of killing tumour cells by tumour-targeting lethal CQDs.

Although CQDs have made significant contributions to the discovery, diagnosis and treatment of tumours, some challenges remain to be further explored. First, the combination of CQDs with other therapies, such as immunotherapy, gene therapy, endocrine therapy, cryotherapy and radiotherapy, needs to be further studied and promoted. Second, the biocompatibility of CQDs is concentration-dependent. Once the concentration threshold is exceeded, CQDs become toxic. The ameliorative concentration threshold for improving the biocompatibility without affecting the properties of CQDs remains to be further studied. Finally, PTT therapy with CQDs is only suitable for photothermal ablation of superficial tumours, so its treatment of deep tumours is a direction of future research.

Acknowledgments

We greatly acknowledge the financial support from the National Nature Science Foundation of China (82074473), the Natural Science Foundation of Jiangsu province (BK20191201, BE2020666), the Suzhou Health Personnel Training Project (GSWS2019074 and GSWS2020103), the Zhangjiagang Health Personnel Training Project (ZJGWSRC2020002), the Zhangjiagang Health System Youth Science and Technology Project (ZYYQ2005).

Disclosure

The authors report no conflicts of interest in this work.