Light-responsive nanotherapeutic carbon dots: a next generation tool for cancer phototherapy

Abstract

Carbon dots (CDs) have emerged as promising nanomaterials in cancer phototherapy due to their unique optical properties, biocompatibility, and tunability. This review comprehensively examines the role of CDs in photodynamic therapy (PDT) and photothermal therapy (PTT), highlighting their mechanisms of action, including the induction of apoptosis, necrosis, and necroptosis in cancer cells. Key factors influencing the efficacy of cancer phototherapy—such as the tumor microenvironment (TME), nanoparticle stability, and light parameters—are thoroughly analyzed. Advancements in surface modifications, targeted, drug delivery and multifunctional applications, including their integration with chemotherapy and immunotherapy, further expand the clinical potential of CDs. While preclinical studies have demonstrated the efficacy of CDs in selectively targeting cancer cells and inducing therapeutic effects, challenges related to biosafety and regulatory approval hinder clinical translation. By addressing existing challenges and leveraging interdisciplinary innovations, CDs hold great promise for personalized and less invasive cancer treatments. Their future success in clinical application will depend on overcoming biological barriers and optimizing the safety profile to facilitate their seamless integration into oncology.

1. Introduction

Cancer remains one of the most challenging diseases worldwide, driving the continuous search for innovative diagnostic and therapeutic approaches to enhance patient survival and quality of life. Conventional cancer treatments such as chemotherapy, radiotherapy, and surgery often face major drawbacks, including systemic toxicity, limited selectivity, and the development of multidrug resistance. These challenges have accelerated the integration of nanotechnology into oncology, offering new strategies for targeted, minimally invasive, and highly efficient therapies. Among various nanomaterials, carbon dots (CDs) have emerged as a new class of light-responsive nanotherapeutics with remarkable potential in cancer phototherapy.1, 2, 3, 4 Although fluorescent nanomaterials have long played a central role in biomedical research, carbon-based nanomaterials have transformed the field due to their eco-friendly nature, stability, and versatile optical properties. The carbon nanomaterial family includes carbon nanodots (CNDs), graphene quantum dots (GQDs), carbon nanoparticles (CNPs), carbon nitride quantum dots (CNQDs), fullerenes, carbon quantum dots (CQDs), and carbonized polymer dots (CPDs).¹ Unlike conventional semiconductor quantum dots containing toxic heavy metals, CDs provide a biocompatible and eco-friendly alternative with comparable or superior optical properties, making them ideal for bioimaging, drug delivery, biosensing, and cancer therapy.2, 3, 4 Since their first discovery in 2004 by Xu et al.⁵ during the purification of single-walled carbon nanotubes, CDs have undergone extensive evolution in synthesis, structural optimization, and biomedical applications.^3,6,7 Typically ranging from 1 to 10 nm in diameter, CDs exhibit excellent fluorescence tunability, photostability, and high surface functionality. Their abundant oxygen- and nitrogen-containing groups enhance water solubility and allow conjugation with biomolecules, targeting ligands, and therapeutic agents.^2,6 These versatile characteristics have positioned CDs as multifunctional platforms capable of integrating diagnostic and therapeutic roles for cancer theranostics.^4,8

Among their various biomedical applications, the light-responsive properties of CDs have drawn significant attention in oncology. Light-mediated therapeutic modalities such as photodynamic therapy (PDT) and photothermal therapy (PTT) utilize photosensitive agents to generate reactive oxygen species (ROS) or localized heat upon light irradiation, leading to selective cancer cell destruction.^2,6,9, 10, 11 CDs are particularly suitable for these applications due to their broad light absorption in the visible and near-infrared (NIR) regions, which allows deep tissue penetration and efficient energy conversion. Their tunable photoluminescence (PL) and excellent photostability further enable simultaneous imaging and therapeutic functionalities, making them valuable for image-guided cancer phototherapy.^9,10 Functional modification of CDs can significantly improve therapeutic efficiency. For example, surface conjugation with tumor-targeting ligands enhances selective accumulation at tumor sites, while heteroatom doping and photosensitizer integration increase ROS generation and photothermal conversion.^12,13 In PDT, CDs function as photosensitizers that produce singlet oxygen and other ROS to induce apoptosis or necrosis in cancer cells.^9,10,14, 15, 16, 17 In PTT, they efficiently convert NIR light into heat, resulting in localized hyperthermia and tumor ablation. Combining both PDT and PTT within a single CD-based platform generates synergistic effects, improving treatment efficacy and reducing side effects. The tumor microenvironment (TME) also influences the performance of CD-based nanotherapeutics. Tumors typically exhibit abnormal pH, hypoxia, and disordered vasculature, which can affect therapeutic outcomes.¹⁸ CDs can exploit these conditions for controlled and site-specific drug release, enhancing selectivity and reducing systemic toxicity.19, 20, 21, 22, 23 Moreover, ROS generation during CD-mediated phototherapy can induce immunogenic cell death, releasing tumor-associated antigens that stimulate antitumor immune responses.²⁴ This dual ability to destroy tumors and activate immune mechanisms highlights CDs as multifunctional agents for integrated phototherapy and immunotherapy. Despite these advantages, clinical translation faces challenges such as limited tissue penetration of light, potential long-term toxicity, and variations in biodistribution.¹⁸ Addressing these issues requires optimization of CD synthesis, surface modification, and photophysical properties to achieve higher precision, safety, and therapeutic selectivity.^12,13

In addition to therapeutic efficacy, CDs exhibit strong diagnostic capabilities. Their bright, tunable fluorescence enables high-resolution bioimaging for early tumor detection and real-time monitoring of therapeutic progress. Furthermore, light-triggered drug release from CDs allows spatially controlled delivery of anticancer agents, minimizing off-target effects and maximizing therapeutic response.25, 26, 27 The integration of diagnostic and therapeutic functionalities into a single CD-based system marks a major step toward precision oncology and personalized nanomedicine. In summary, carbon dots represent a new generation of light-responsive nanotherapeutics with exceptional potential for cancer treatment. Their superior optical properties, excellent biocompatibility, tunable surface chemistry, and ability to unify imaging and therapy make them promising candidates for next-generation phototherapy. This review focuses on the classification, synthesis, and characterization of CDs, emphasizing their mechanisms in cancer phototherapy and the factors influencing therapeutic outcomes. By addressing existing challenges and advancing functionalization strategies, CDs could revolutionize oncological treatment by providing a targeted, minimally invasive, and highly effective approach for combating cancer.

2. Carbon dots

2.1. Classification, synthesis, and characterization for advanced applications

CDs represent a new class of carbon-based nanomaterials with outstanding optical, structural, and biocompatible characteristics, making them highly attractive for biomedical and technological use (Fig. 1). Generally sized between 1 and 10 nm, CDs are composed mainly of sp² and sp³ hybridized carbon atoms functionalized with oxygen-, nitrogen-, or sulfur-containing groups that determine their fluorescence, solubility, and biological interactions. Based on structure and composition, CDs can be divided into several types, including GQDs, CQDs, CNDs, CNQDs, and CPDs.¹ GQDs are nanosized graphene fragments with graphitic domains that influence their optical and electronic performance.^3,28, 29, 30, 31, 32 Their fluorescence depends on edge states and heteroatom doping, which regulate the bandgap. CQDs are quasi-spherical with crystalline or nanocrystalline cores surrounded by polymeric shells, where fluorescence arises from surface defects and quantum confinement.^3,29,33 CNDs have amorphous carbon cores with structural disorder yet strong luminescence and high dispersibility, making them cost-effective for sensing and imaging.^3,34,35 CNQDs, derived from graphitic carbon nitride (g-C₃N₄), possess a layered tri-s-triazine structure whose optical behavior is controlled by delocalized π-electrons and chemical modification.^3,36 CPDs contain polymer–carbon hybrid frameworks formed by partial carbonization of cross-linked polymers, providing high photostability for imaging, drug delivery, and PTT.^3,28,37,38

Fig. 1.

Overview of CDs, including their classification, characterization techniques, key properties and potential applications. AFM, atomic force microscopy; CDs, carbon dots; FTIR, fourier transform infrared spectroscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy; UV, ultraviolet; UV–Vis, ultraviolet–visible spectroscopy; XRD, X-ray diffraction.

CDs can be synthesized using either top-down or bottom-up approaches.^4,39 Top-down techniques break down bulk carbon materials such as graphite, soot, or carbon nanotubes using laser ablation, electrochemical oxidation, ultrasonic treatment, or chemical oxidation.^39,40 Laser ablation creates CDs via intense laser pulses but often yields low quantum efficiency.40, 41, 42 Electrochemical oxidation offers controlled particle size,43, 44, 45, 46 while ultrasonic treatment fragments carbon through cavitation.47, 48, 49 Chemical oxidation produces oxygen-rich CDs but raises environmental concerns.^50,51 Bottom-up strategies, on the other hand, assemble CDs from small organic or natural precursors using hydrothermal, solvothermal, microwave-assisted, or pyrolysis methods.^47,52,53 These allow control over size, crystallinity, and optical properties. Green synthesis using plant extracts, fruit peels, or biomass precursors has gained interest due to its sustainability and biocompatibility.54, 55, 56, 57, 58, 59, 60, 61, 62 Heteroatom doping with nitrogen, sulphur, or phosphorus enhances fluorescence, stability, and electron-transfer capability.¹³ Purification steps such as dialysis or centrifugation are essential to remove impurities.

Characterization is vital to link structure with function. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) reveal spherical particles (1–10 nm) with lattice fringes of about 0.21 nm for the (100) plane.63, 64, 65, 66 Scanning electron microscopy (SEM) and atomic force microscopy (AFM) provide surface and thickness information.^52,67 X-ray diffraction (XRD) identifies partial graphitization at 25–35° (2θ),^2,34 while Raman spectroscopy distinguishes disordered (D) and graphitic (G) carbon bands.^26,52,65 Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) identify functional groups and elemental composition.67, 68, 69 Zeta potential indicates colloidal stability when values exceed ±30 mV.⁷⁰ Optical studies using ultraviolet–visible (UV–Vis) and fluorescence spectroscopy show π–π* and n–π* transitions and excitation-dependent PL.^52,69,71,72 Quantum yield (QY) and fluorescence lifetime measurements assess efficiency, which improves significantly with doping and surface passivation (from 5.31% to 28.46%).^73,74 Collectively, these techniques provide comprehensive insight into CDs’ structure–property relationships, guiding their rational design for advanced biomedical and oncological applications.

2.2. Advancements, applications, and future perspectives of CDs

CDs have gained significant interest as a new class of nanomaterials with unique optical, electronic, and chemical properties suitable for biomedical, environmental, and industrial applications. CDs exhibit strong ultraviolet (UV) absorption between 200–400 nm due to π–π* and n–π* transitions within their carbon core.²⁷ Their excitation-dependent PL enables emission that can be adjusted from the visible to the NIR region,75, 76, 77, 78 attributed to quantum confinement and surface state variations. CDs possess excellent photostability, biocompatibility, and chemical versatility,^8,79 while their electron-donating and -accepting properties and high thermal stability make them suitable for catalysis and optoelectronic applications.^80,81 In biomedicine, CDs have demonstrated strong potential for bioimaging due to their intense fluorescence, water solubility, and low cytotoxicity.^2,79 Ding et al.⁸² synthesized DNA-carbon quantum dots (DNA-CQDs) that were efficiently internalized by HEK 293 cells for confocal imaging, and Zheng’s group found that CQDs could cross the blood-brain barrier (BBB) and target C6 glioma cells without additional targeting ligands.⁸³ Red- and NIR-emitting CDs further enhance in vivo imaging depth and tumor visualization.⁸⁴ Doped CDs have enabled multimodal imaging; for example, iodine-doped CDs improve X-ray CT contrast, while gadolinium-doped CDs provide strong T1-weighted magnetic resonance imaging (MRI) contrast along with fluorescence.85, 86, 87, 88

CDs find extensive use in biosensing because of their emission flexibility and rich surface chemistry. They act as fluorescent probes for detecting metal ions such as Zn²⁺,⁸⁹ Pt²⁺, Au³⁺, Pd²⁺,⁹⁰ Fe³⁺,⁹¹ Hg²⁺,⁹² ONOO⁻,⁹³ and ClO⁻,⁹⁴ through coordination or electrostatic interactions.²⁷ Functionalization with biomolecules such as antibodies or nucleic acids enhances selectivity toward disease biomarkers and environmental toxins. In nanomedicine, CDs serve as efficient carriers for targeted drug delivery and controlled release.^6,95 Ligand modification with folic acid (FA) or peptides enhances cancer cell specificity.^6,96 CDs also play key roles in PDT and PTT: in PDT, they generate ROS under light irradiation to destroy tumor cells,¹¹ while in PTT, they convert light energy into heat for localized hyperthermia.¹⁰ Outside biomedical applications, CDs hold great potential in catalysis, light-emitting diodes (LEDs), solar cells, and photodetectors because of their high quantum yield and emission versatility.97, 98, 99 However, issues such as inconsistent synthesis, low yields, and limited understanding of long-term toxicity remain challenges.^6,100 Standardized synthesis, enhanced biocompatibility, and mechanistic insights are essential for advancing CDs toward clinical and technological applications.

3. Biosafety, cytotoxicity, and pharmacokinetics of CDs

CDs have emerged as promising nanomaterials in biomedical science due to their remarkable optical properties, high stability, and biocompatibility, supporting their use in bioimaging, biosensing, drug delivery, and cancer therapy. With increasing application in PDT and PTT, understanding their biosafety including cytotoxicity, biodistribution, and metabolism is essential for clinical translation. Cytotoxicity refers to the potential of a material to induce cell death via apoptosis or necrosis.⁷⁹ Numerous studies indicate that CDs display low cytotoxicity and high compatibility with normal cells.¹⁰¹ For example, Sharma et al.¹⁰² showed that urea-derived CDs promoted dermal fibroblast proliferation and exhibited hemocompatibility in red blood cells. Lu et al.¹⁰³ reported that nitrogen-doped CDs maintained over 80% cell viability in 293T cells at 5 mg/mL. Yan et al.¹⁰⁴ observed that weakly charged CQDs were biocompatible with human umbilical cord-derived mesenchymal stem cells, while Vale et al.¹⁰⁵ confirmed negligible toxicity of hydrothermal CDs in MCF-10A cells. Interestingly, CDs often show selective cytotoxicity toward cancer cells through ROS-mediated apoptosis while sparing normal cells. Ginger-derived CDs inhibited HepG2 liver cancer cells,¹⁰⁶ and date pit-derived CDs suppressed MCF-7, A549, and PC3 cancer cells with minimal effects on HEK293 cells.¹⁰⁷ Walnut oil-derived CDs induced apoptosis via caspase-3 activation,¹⁰⁸ highlighting their potential as safe and selective anticancer agents.

A comprehensive understanding of CD pharmacokinetics, including absorption, distribution, metabolism, and excretion (ADME), is vital for assessing biosafety. CDs are efficiently internalized by cells due to their nanoscale size and are often administered via intravenous, subcutaneous, or intertumoral routes depending on application.²⁹ Huang et al.¹⁰⁹ found that polyethylene glycol (PEG)-1500N- and ZW800-functionalized CDs showed greater tumor retention after subcutaneous injection, while intravenous delivery resulted in systemic distribution and renal clearance within 24 hours. Smaller CDs (<5 nm) diffuse through continuous endothelium, while larger particles pass through fenestrated or discontinuous endothelium.110, 111, 112, 113 Internalization occurs via clathrin- or caveolae-mediated endocytosis, macropinocytosis, or passive diffusion, influenced by surface charge and modifications such as PEGylation or ligand conjugation.^29,114,115 Biodistribution studies show primary accumulation in the liver, spleen, lungs, and kidneys, though small CDs are mostly excreted through renal pathways.^109,116 While CDs resist enzymatic degradation, oxidative enzymes can partially decompose them.^117,118 Renal excretion dominates for small or neutral CDs, while larger or hydrophobic ones may undergo hepatic clearance.119, 120, 121, 122 Understanding these mechanisms is crucial for improving biosafety, minimizing toxicity, and enhancing the clinical applicability of CDs in nanomedicine.²⁹

4. Application of CDs in cancer phototherapy

Cancer is a major global health concern, causing millions of deaths annually. It encompasses a diverse group of diseases characterized by the uncontrolled proliferation of abnormal cells, often driven by genetic damage, with the potential to metastasize to other organs.¹²³ Cancer progression is associated with dysfunctions in critical biological processes, including cell survival, proliferation, immune responses, apoptosis, and DNA repair.¹²³ Due to the complexity of cancer, multiple treatment strategies have been developed to improve patient outcomes. Each year, more than 10 million individuals are diagnosed with cancer, necessitating a variety of therapeutic approaches, such as surgical resection, chemotherapy, radiation therapy, and immunotherapy.¹²⁴ Traditional treatment methods, however, have significant limitations. Surgery is restricted by tumor location and may not completely remove malignant tissues. Chemotherapy relies on highly toxic drugs that can induce systemic side effects, reducing patients’ overall quality of life. Radiation therapy, while effective, often damages surrounding healthy tissues due to its lack of specificity. Immunotherapy can provoke immune-related adverse reactions, including inflammation and autoimmune effects.¹ These challenges have driven the search for targeted, minimally invasive alternatives, with nanomedicine emerging as a promising field. Nanotechnology has transformed cancer diagnosis and therapy by enabling precise and efficient delivery of therapeutic agents.

Among these emerging strategies, cancer phototherapy has gained attention as a light-triggered treatment modality, encompassing PDT and PTT. Compared to conventional approaches, phototherapy offers advantages such as minimal invasiveness, high spatial and temporal precision, reduced systemic toxicity, and the possibility for repeated treatments without cumulative adverse effects.125, 126, 127 PDT employs photosensitizers (PSs) that, when activated by light, generate ROS to induce oxidative stress and apoptosis in cancer cells.128, 129, 130 PTT uses photothermal agents (PTAs) that convert light energy into heat, causing hyperthermia-induced tumor cell death.^127,131 Combining PDT and PTT can enhance therapeutic efficacy by integrating oxidative and thermal damage, reducing tumor recurrence and resistance mechanisms.

The development of nanomaterials has further advanced phototherapy by improving PS and PTA stability, targeting capacity, and photoconversion efficiency. Carbon-based nanomaterials, including graphene, carbon nanotubes, quantum dots, and gold nanoparticles (AuNPs), have demonstrated notable optical properties suitable for phototherapy applications.¹²⁵ CDs have emerged as promising next-generation nanotheranostic agents, attributed to their remarkable biocompatibility, flexible PL behavior, high ROS generation, and excellent photothermal conversion efficiency (PCE).¹²⁷ CDs absorb strongly in the visible and NIR regions, enabling deep tissue penetration and efficient light-to-heat or light-to-ROS conversion for both PDT and PTT.^127,131

CDs outperform conventional phototherapy agents in terms of photostability, emission versatility, and the duration of therapeutic effects. Unlike indocyanine green (ICG), which is prone to photobleaching and limited accumulation,¹³² CDs maintain stable optical properties, boosting treatment efficiency. Their PCE can match or exceed that of titanium dioxide (TiO₂) nanoparticles while overcoming tissue penetration and toxicity limitations.^133,134 CDs also provide remarkable biocompatibility, biodegradability, water solubility, and minimal accumulation concerns.¹¹ They maintain high ROS production under physiological conditions, unlike traditional PSs that aggregate and experience fluorescence quenching.^127,131

The broad absorption spectrum of CDs supports ROS generation under a wide range of wavelengths,^135,136 enhancing PDT efficacy in deep tumors compared to UV-activated TiO₂ nanoparticles.¹³⁷ Their high PCE allows rapid, localized hyperthermia upon NIR irradiation, minimizing damage to healthy tissues. Recent studies have explored multimodal therapy integrating PDT and PTT for synergistic effects.¹³⁸ Gas-assisted strategies have addressed tumor hypoxia to improve PDT,¹³⁹ while advanced light-responsive nanomaterials and photodynamic-chemodynamic cascade reactions have further improved therapeutic outcomes.^140,141 Combining remarkable photostability, adaptable optical features, high biocompatibility, and efficient photothermal and photodynamic performance, CDs offer a next-generation platform for cancer phototherapy. Future research should optimize CD synthesis, engineer surface modifications for targeted delivery, and conduct translational in vivo studies to fully realize their clinical potential.

4.1. Application of CDs in PDT

4.1.1. Overview of PDT and the role of CDs in cancer treatment

PDT is a non-invasive treatment with origins tracing back over 3000 years to ancient light-based therapies used in Egypt, India, and China for conditions such as psoriasis, rickets, vitiligo, and early forms of skin cancer.¹²⁵ Modern PDT began with Niels Finsen in the 19th century, who discovered the synergistic effects of light and chemicals, laying the foundation for current PDT frameworks.¹⁴² PDT relies on three main components: (1) a light-activated compound called a PS, (2) an appropriate light source, and (3) molecular oxygen within the target tissue or cells.¹⁴³ Upon light irradiation, the PS generates ROS, particularly singlet oxygen (¹O₂), inducing oxidative stress that selectively destroys cancer cells. This therapy offers advantages such as repeatability, minimal invasiveness, and compatibility with conventional treatments like chemotherapy, radiotherapy, and surgery. The effectiveness of PDT depends on the PS, which must selectively accumulate in cancer cells. Ideal PSs absorb strongly in the NIR range of 600 to 800 nm, known as the “optical window,” ensuring deep tissue penetration and efficient ¹O₂ generation.^21,125 Traditional PSs, including phthalocyanine,¹⁴⁴ porphyrin,¹⁴⁵ and hypocrellin,^146,147 demonstrate antitumor activity but face limitations such as poor water solubility, aggregation, photobleaching, and low tumor specificity, requiring high-intensity light that can damage healthy tissues.^11,148 As advanced PSs, CDs exhibit outstanding water solubility, flexible fluorescence behavior, superior photostability, and enhanced ROS production under reduced light exposure.^2,149,150 Their ultrasmall size (< 10 nm) allows efficient tumor accumulation via the EPR effect, while surface functionalization supports targeted delivery, improved cellular uptake, and reduced off-target effects.^6,149,150 CDs exhibit high fluorescence quantum yield, superior biocompatibility, and lower toxicity, making them highly promising for selective PDT applications and clinical translation.

4.1.2. Mechanism of PDT-mediated cell death induced by CDs

PDT employs PSs that, upon light activation, generate ROS to induce cancer cell death. CDs, due to their unique physicochemical properties, offer advantages over conventional PSs such as porphyrins and phthalocyanines.¹⁴³ The flexible optical behavior of CDs allows broad-spectrum excitation, promoting deeper tissue penetration with minimal impact on healthy tissues.^2,149,150 Recent advances have produced NIR-responsive CDs through elemental doping, surface passivation, and structural modifications. For example, nitrogen doping enhances electronic transitions for NIR absorption, while rare-earth doping with samarium or europium improves ROS generation at longer wavelengths, enhancing PDT efficacy in deep-seated tumors.^151,152 The PDT mechanism, illustrated in Fig. 2, involves CDs transitioning from the ground state (S₀) to the excited single state (S₁) upon light absorption, followed by intersystem crossing to the triplet state (T₁).153, 154, 155 ROS are generated via Type I reactions, producing free radicals like superoxide anion (O^2-), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH), or Type II reactions, producing ¹O₂.^{11,21,125,128,156} ROS induce oxidative stress, damaging proteins, membranes, lipids, and DNA, leading to cell death through apoptosis, autophagy, or necrosis.^128,157 Apoptosis is the most common PDT outcome, triggered by mitochondrial ROS accumulation, cytochrome c release, and caspase activation.^6,21,158,159 Higher ROS levels can cause necrosis, while prolonged autophagy may result in autophagic cell death.^21,160,161 CDs-mediated apoptosis is light-dependent and activates intrinsic pathways via Bid, Bax, Bak, Apaf-1, and caspases, and extrinsic pathways through death receptors like FADD.162, 163, 164, 165 CDs can also induce lysosome-dependent cell death by releasing cathepsins upon lysosomal membrane permeabilization.166, 167, 168 These mechanisms highlight CDs as versatile and highly efficient PDT agents.

Fig. 2.

Jablonski energy level diagram that describes the mechanism of action of CDs as phototherapy agents in PDT and PTT. CD, carbon dots; ISC, intersystem crossing; PDT, photodynamic therapy; PTT, photothermal therapy; ROS, reactive oxygen species.

4.1.3. Representative studies on CDs in PDT

The application of CDs in PDT has been extensively explored through both in vitro and in vivo studies, demonstrating their potential as effective PSs in cancer treatment (Table 1). CDs outperform conventional PSs with their exceptional water solubility, minimized aggregation, flexible photophysical behavior, and multifunctional therapeutic potential, establishing them as promising agents for targeted phototherapy (Fig. 3). Pang et al.¹⁶⁹ reported nucleolus-targeted PDT using renal-clearable CDs synthesized from citric acid (CA) and ethylenediamine (EDA). These CDs exhibited strong absorption in the 400–700 nm range and potent photodynamic activity against HeLa cells. Under LED irradiation for 30 minutes at 500 µg/mL, treated cells showed significant membrane disruption.

Table 1.

Reported studies on the application of CDs in cancer PDT.

CDs type	Targeted cell lines / organism	Light source and conditions	Absorption peaks	Max emission	ROS quantum yield	Mechanism of action	Key findings	References
Red emissive CDs	4TI cancer cells, mice	Laser, 635 nm, 5 mins	NA	680 nm	18% (e-RCDs) 42% (p-RCDs)	Tunable ROS generation, tunable Type I and Type II ROS generation, apoptosis	Tunable ROS generation for precise control in mitochondrion-targeted PDT, significant reduction in tumor size.	154
Renal-clearable CDs	HeLa cells, mice	LED, 400–700 nm, 30 mins	350 nm	NA	4.8%	ROS generation, membrane nucleus targeting capability, apoptosis	Intrinsic nucleus-targeting, efficient ROS generation, significant reduction in tumor size in irradiated mice.	169
S-CDs	U87-MG, A375 cells	LED, 420 nm, 5 mins	NA	650 nm	95%	Mitochondria-mediated apoptosis via P13K/Akt pathway inhibition	Modulated intracellular signaling, efficient ROS generation, induce apoptosis through mitochondria dysfunction.	170
F, N-CDs	HepG2 cells	LED, 400–500 nm	NA	NA	NA	Type I ROS generation, apoptosis	Efficient ROS generation, strong efficacy in Type I PDT under light irradiation.	171
Red emissive TP-CDs	HeLa cells	Laser, 638 nm, 5 mins	274 nm	605 nm	NA	ROS (¹O₂) generation, RNA cleavage	Efficient ROS generation under light irradiation, real-time monitoring of nucleus dynamic during PDT, significant affinity for RNA.	172
Phosphorescent CDs	CT26 cancer cells, mice	Laser, 685 nm, 5–10 mins	400 nm	672 nm	NA	ROS generation	Large tissue penetration (10 nm), efficient ROS generation under light irradiation, significant tumor growth inhibition in vivo.	173
CDcf	H413 cancer cells	Laser, 780 nm, 0–10 mins	277 nm	NA	NA	ROS generation, folate-receptor-mediated pathway, nucleus targeting	Enhanced ROS production and internalization in nucleus, targeting, damaged in DNA.	174
T-CDs	4T1 cancer cells, mouse	LED, 400–500 nm, 12 mins	210 nm	436 nm, 673 nm	NA	ROS (•OH) generation	Efficient ROS generation under light irradiation, effective tumor inhibition in solid tumors.	175
Riboflavin-based CDs	4T1 cancer cells, mice	Laser, 365 nm, 1–20 mins	365 nm	502 nm	71.6%	ROS (¹O₂) generation	Demonstrated efficient ROS generation under light irradiation.	153
Red-emissive MMCDs	HepG2 cancer cells, mice	LED, 400–500 nm, 12 mins	226 nm	642 nm	78.36%	ROS and NO generation, lysosome membrane destruction	Enhanced PDT efficiency, significant reduction in tumor volume in vivo.	176
N-CDs	CAL-27 cancer cells, mice	Laser, 660 nm, 10 mins	349 nm	583 nm	NA	ROS generation, mitochondrial pathway apoptosis.	Efficient in inducing apoptosis, significant reduction in tumor volume in vivo, inhibiting angiogenesis and cancer cell proliferation, damaged in DNA.	151

Abbreviations: CDcf, curcumin and folic acid-derived carbon dots; CDs, carbon dots; DNA, deoxyribonucleic acid; e-RCD, electron-rich red-emissive carbon dots; F, N-CDs, fluorine and nitrogen co-doped carbon dots; LED, light-emitting diode; mins, minutes; MMCDs, metformin and methylene blue-derived carbon dots; NA, not applicable; N-CDs, nitrogen-doped carbon dots; nm, nanometer; NO, nitric oxide; PI3K/Akt, phosphoinositide 3-kinase/protein kinase B; PDT, photodynamic therapy; p-RCD, protonated or passivated red-emissive carbon dots; RNA, ribonucleic acid; ROS, reactive oxygen species; S-CDs, sulfur-doped carbon dots; T-CDs, tea polyphenol-derived carbon dots; TP-CDs, two-photon carbon dots.

Fig. 3.

Schematic illustration of the PDT mechanism using CDs. Upon internalization into cancer cells, CDs are activated by light irradiation, leading to the generation of ROS. The elevated ROS levels induce oxidative stress, triggering cellular damage and apoptosis, ultimately resulting in cancer cell death. CDs, carbon dots; PDT, photodynamic therapy; ROS, reactive oxygen species.

The small size and renal-clearable nature of these CDs enabled efficient nucleolar accumulation, enhancing ROS generation at the DNA level and inducing apoptosis. In vivo studies further confirmed their efficacy, as treated tumor-bearing mice displayed substantial tumor reduction.¹⁶⁹ Li et al.¹⁷⁰ developed sulfur-doped CDs (S-CDs) to improve ROS production and selectively modulate intracellular signaling pathways. In U87-MG and A375 cells, S-CDs inhibited the PI3K/Akt survival pathway while activating the pro-apoptotic p38/JNK pathway, causing mitochondrial dysfunction, Bcl-2/Bax imbalance, and caspase activation, thereby enhancing apoptosis.¹⁷⁰ Wu et al.¹⁷¹ synthesized fluorine and nitrogen co-doped CDs (F, N-CDs) optimized for Type I PDT. These CDs generated superoxide anions and hydroxyl radicals under 400–500 nm LED irradiation, enhancing oxidative stress in HepG2 cells. Fluorine improved electron transfer efficiency, while nitrogen doping optimized surface charge for enhanced ROS generation.¹⁷¹

Red-emissive CDs (RCDs) from Hypericum perforatum were developed for mitochondrion-targeted PDT, absorbing in the 600–650 nm range for deeper tissue penetration. RCDs induced mitochondrial membrane collapse, triggering apoptosis, and inhibited tumor growth in BALB/c nude mice bearing 4T1 tumors.¹⁵⁴ Similarly, tea polyphenol-derived CDs generated ROS under white LED light, selectively inducing apoptosis and necrosis in 4T1 breast cancer cells.¹⁷⁵ In vivo studies further validated CDs for PDT. Diketopyrrolopyrrole-based CDs (DPP-CDs) under 540 nm laser irradiation significantly inhibited tumor growth,¹⁷⁷ and riboflavin-derived CDs under 365 nm light selectively accumulated in cancer cells, efficiently generating ¹O₂.¹⁵³ These studies collectively highlight the exceptional phototherapeutic potential of CDs. Through rational design, including elemental doping, structural engineering, and surface functionalization, CDs achieve enhanced ROS generation, deeper tissue penetration, and superior photostability, establishing them as next-generation PSs for effective cancer phototherapy.

4.2. Application of CDs in PTT

4.2.1. Overview of PTT and the role of CDs in cancer treatment

The therapeutic use of heat dates back to ancient civilizations, but it was not until the 18th century that hyperthermia was recognized as a possible cancer treatment.¹²⁵ During this period, researchers found that elevating the temperature of cancerous tissues above normal physiological levels, typically between 40–45 °C, could inhibit tumor growth and lead to cancer cell destruction.¹²⁵ Hyperthermia exerted cytotoxic effects through protein denaturation and physiological alterations, and when used alongside chemotherapy or radiotherapy, it enhanced their therapeutic efficiency.¹²⁵ However, conventional hyperthermia lacked precision and posed risks to healthy tissues, which spurred efforts to develop more targeted heat-based cancer treatments, ultimately leading to the advent of PTT.

The introduction of laser technology allowed more controlled heat generation in tumors. Laser-induced hyperthermia demonstrated potential, especially in the treatment of retinal and choroidal tumors.¹²⁵ Yet, this approach relied on high-powered lasers, raising safety and practicality concerns. PTT evolved as an advancement over laser hyperthermia, offering better selectivity and control. It employs PTAs that absorb light, particularly in the NIR range, and convert it into localized heat to elevate tumor temperature and induce cell damage.¹⁷⁸ The success of PTT depends on the optical properties of PTAs, light source, and tissue characteristics. Biocompatible PTAs with high NIR absorption enable efficient tumor ablation and suppression, making PTT a promising cancer treatment.

Compared with conventional treatments such as surgery, chemotherapy, and radiotherapy, PTT offers distinct advantages. Its targeted nature allows selective destruction of cancer cells while minimizing harm to surrounding tissues,¹⁷⁹ reducing side effects and improving patient outcomes. However, its application is limited by the restricted penetration of light into deep tissues.^180,181 Tumors located deep within the body or behind bones are difficult to treat because biological tissues absorb and scatter light.^180,181 Current research therefore focuses on optimizing PTA design, improving light delivery, and combining PTT with multimodal therapies to overcome these constraints.

With flexible optical behavior, high water solubility, biocompatibility, and strong photostability, CDs have recently emerged as promising PTAs.⁷⁶ Unlike traditional PTAs such as gold nanoparticles or organic dyes, CDs show lower toxicity and superior biological compatibility.^11,182 Although their native UV–visible absorption limits deep-tissue use, heteroatom doping with nitrogen (N), sulfur (S), phosphorus (P), or fluorine (F) can shift absorption into the NIR range, enhancing PCE.^183,184 For instance, nitrogen-oxygen co-doped CDs exhibit improved NIR absorption and superior PTT performance.¹⁸⁵ Surface modification with ligands like arginine or PEG further enhances NIR response, prolongs circulation, and improves tumor targeting.¹⁸⁶ D-arginine-functionalized CDs demonstrate strong NIR fluorescence and high photothermal efficiency.¹⁸⁷

Despite progress, limited light penetration remains a major obstacle. Strategies such as two-photon excitation, plasmonic nanomaterial integration, and photoacoustic imaging-guided PTT show potential to increase treatment precision. Overall, CDs represent an emerging generation of PTAs with unique advantages, paving the way for safer and more effective cancer phototherapy.

4.2.2. Mechanism of PTT mediated cell death induced by CDs

PTT employs PTAs to convert NIR light into heat, generating localized hyperthermia that destroys cancer cells. Traditional PTAs, such as organic dyes and metallic nanoparticles, have shown effectiveness in PTT but are often hindered by poor water solubility, high synthesis costs, limited biodegradability, and potential toxicity.^178,188 CDs have emerged as next-generation PTAs, combining high biocompatibility, flexible optical behavior, photostability, and straightforward synthesis.¹⁸⁹ Through heteroatom doping and surface functionalization, researchers have successfully extended CDs’ optical absorption into the NIR region, thereby enhancing their photothermal conversion efficiency. These engineered CDs convert absorbed NIR light into heat via non-radiative relaxation processes, which are determined by their electronic structure.¹⁹⁰

When CDs are illuminated with NIR light, they absorb photons and transition from the ground state (S₀) to an excited state (S₂) (see Fig. 2). This excitation produces electron-hole pairs that facilitate energy transfer within the system.^188,191,192 The absorbed energy is then released as thermal energy through non-radiative relaxation, creating localized hyperthermia in the tumor region due to the formation of hot nanoparticle lattices.^178,191 The subsequent temperature increase induces various biological effects, such as protein denaturation, membrane destabilization, and mitochondrial dysfunction, leading to cancer cell death.¹⁹³ Additionally, this controlled heating can modulate the TME by improving oxygenation, enhancing blood flow, reducing interstitial fluid pressure, and normalizing pH levels.¹⁹⁴

The effective temperature range for PTT-induced cancer cell death is generally between 42 °C and 60 °C. At around 42 °C, protein denaturation begins, leading to temporary inhibition of cancer cell activity. Between 43 °C and 45 °C, oxidative stress and irreversible cellular injury occur, while temperatures above 45 °C cause rapid necrosis. When temperatures exceed 48 °C, microvascular thrombosis and ischemia may arise, resulting in immediate cell death.^182,195 CD-mediated PTT induces two major pathways of cell death: apoptosis and necrosis (Fig. 4).¹⁸⁸ Apoptosis typically occurs at 42–50 °C through caspase activation, leading to controlled cell elimination.^161,196 CDs can trigger apoptosis by generating ROS, inducing DNA damage, and promoting endoplasmic reticulum stress,^159,162 which releases cytochrome c and Apaf-1 to activate caspases.^197,198 In contrast, necrosis occurs at higher temperatures (above 50 °C) and involves cellular swelling, mitochondrial collapse, and membrane rupture.¹⁶¹ The mechanism is light-dependent: under NIR irradiation, CDs induce apoptosis or necrosis depending on power density and exposure duration.¹⁹⁹

Fig. 4.

CDs induced apoptosis (programmed cell death) is light-dependent, which can be categorized into two different pathways: intrinsic pathway and extrinsic pathway. Bak, Bcl-2 antagonist/killer; Bax, Bcl-2–associated X protein; Bcl-2, B-cell lymphoma 2; BH3 protein, Bcl-2 homology 3–only protein; Bid, BH3-interacting domain death agonist; DNA, deoxyribonucleic acid; DISC, death-inducing signaling complex; ER stress, endoplasmic reticulum stress; FADD, Fas-associated death domain; ROS, reactive oxygen species; tBid, truncated Bid.

Beyond apoptosis and necrosis, PTT-induced hyperthermia can activate necroptosis, a regulated necrotic pathway that occurs when apoptosis is inhibited (Fig. 5).^161,200 Necroptosis shares morphological similarities with necrosis, such as cell swelling and membrane rupture, but involves distinct receptor-interacting protein kinase (RIPK)-mediated signalling.¹⁶¹ CDs offer a highly promising PTA platform, combining superior biocompatibility, flexible NIR absorption, and multifunctionality. Continued optimization of CD design, tumor-targeting capability, and integration with synergistic therapies such as PDT and immunotherapy will be critical for advancing their clinical translation in cancer treatment.

Fig. 5.

Schematic illustration of the PTT mechanism using CDs. After being internalized by cancer cells, CDs are activated upon light irradiation, converting light energy into heat, lead to localized hyperthermia. The elevated temperature induces protein denaturation, membrane disruption, and mitochondrial dysfunction, ultimately leading to cancer cell death through: apoptosis, necrosis, or necroptosis. CD, carbon dots; PTT, photothermal therapy.

4.2.3. Representative studies on PTT using CDs and their therapeutic outcomes

CDs have emerged as highly promising PTAs for PTT owing to their excellent PCE, outstanding biocompatibility, and versatile surface functionalization capabilities. Unlike standard PTAs such as gold nanostructures, graphene derivatives, and transition-metal sulfides, CDs demonstrate several benefits, including high aqueous dispersibility, strong photostability, flexible optical behavior, and minimal cytotoxicity. Their ultrasmall size enables deep tumor tissue penetration, while their surface chemistry allows facile modification for improved targeting specificity.¹⁸⁹ Moreover, CDs present a lower risk of long-term toxicity and metal ion-related complications commonly associated with metallic nanostructures. However, traditional CDs predominantly absorb light in the UV-visible spectrum, which limits their efficiency in deep-tissue applications. To overcome this limitation, recent progress in heteroatom doping, surface passivation, and π-electron conjugation has extended their optical absorption into the NIR region, significantly enhancing their PCE and allowing more effective tumor ablation through improved tissue penetration.

Numerous studies have reported the selective photothermal killing of cancer cells using CDs under NIR irradiation (Fig. 6), highlighting their therapeutic potential in PTT for cancer therapy (Table 2). Zhao et al.²⁰¹ developed NIR-responsive lysosome-targetable CDs via hydrothermal synthesis using coronene derivatives. Nitrogen doping reduced the electronic bandgap, promoting strong NIR absorption and efficient photothermal conversion at 808 nm (10 min irradiation). These CDs induced significant cytotoxicity in 4T1 cancer cells and demonstrated effective tumor volume reduction in vivo in a 4T1 tumor-bearing mouse model after NIR exposure. Importantly, histological analyses of major organs (heart, liver, spleen, lung, and kidney) revealed no pathological damage, confirming the biocompatibility and safety of the CDs.²⁰¹ This work demonstrated the dual functionality of CDs in imaging and photothermal ablation, showing selective cytotoxicity toward tumor cells while sparing normal tissues.

Fig. 6.

Schematic illustration of key findings from studies on CDs for PTT. CDs demonstrated strong potential as PTAs, effectively inducing cancer cell death in vitro and inhibiting tumor growth in vivo. CDs, carbon dots; NIR, near-infrared; PTAs, photothermal agents; PTT, photothermal therapy.

Table 2.

Reported studies on the application of CDs in cancer PTT.

CDs type	Targeted cell lines / organism	Light source and conditions	Absorption peaks	Max emission	PCE	Key findings	References
Cu-doped CDs derived from Alcea leaves	4T1 cancer cells	Laser, 800 nm, 10 mins	398 nm, 444 nm, 800 nm	460 nm	39.3%	Eco-friendly synthesis approach, demonstrated good photothermal conversion and thermal ablation of cancer cells, contributing to sustainable PTT approaches.	196
CDs derived from coronene derivatives	4T1 cancer cells, mice	Laser, 808 nm, 10 mins	315 nm	930 nm	54.7%	Efficient photothermal properties, significant cytotoxicity in 4T1 cells after light exposure, demonstrated potential for imaging and PTT, significantly inhibit tumor growth after treatment.	201
CDs derived from mixture of citric acid, formamide, and PEG	4T1 cancer cells, mice	Laser, 808 nm, 1064 nm, 10 mins	1033 nm	670 nm	41.19%	Dual function in NIR emission and absorption, significant reduction in cancer cell survival after light irradiation, significant tumor growth inhibition, showcasing multifunctional imaging and PTT capabilities.	202
ICGCDs	MDA-MB-435 cells, mice	Laser, 808 nm, 5–8 mins	215 nm, 780 nm, 895 nm	406 nm, 600 nm, 697 nm	23.9%	Significant enhanced thermal stability, efficient photothermal effect in inhibiting cancer cells, significant tumor growth inhibition.	203
Asphaltenes-derived CDs	4T1 cancer cells, mice	Laser, 808 nm, 5 mins	808 nm	472 nm	41.86%	Efficient photothermal effect in inhibiting cancer cells, significant tumor growth inhibition.	204
PCD derived from citric acid	MCF-7 cancer cells, mice	Laser, 808 nm or 1064 nm, 5 mins	NA	NA	77.25% at 1064 nm, 83.72% at 808 nm	Excellent deep tissue penetration, efficient photothermal effect in killing cancer cells, significant tumor growth inhibition.	205
Red-emission and SCDs derived from citric acid and dicyandiamide	HepG2 cancer cells	Laser, 808 nm, 3–10 mins	337 nm, 600 nm	Absence	41.7%	High specificity and selectivity in damaging cancer cells while sparing normal cells, mitochondria-targeted.	206
MP-CDs	4T1 cancer cells	Laser, 808 nm, 5 mins	375 nm	476 nm	18.4%	Efficient photothermal effect in killing cancer cells after light irradiation, enhanced immune response (immune activation via dendritic cells).	207
CDs@PDA derived from egg yolk	HepG2 cancer cells	Laser, 808 nm, 3 mins	280 nm	443 nm	NA	Excellent photothermal conversion capability, efficient photothermal effect in killing cancer cells after light irradiation.	208
Osmanthus-derived CDs	HeLa cancer cells	Laser, 808 nm, 10 mins	NA	500 nm	46.7%	High PCE, excellent photothermal stability, efficient photothermal effect in killing cancer cells after light irradiation.	209
S, N-CDs	HeLa and MCF-7 cancer cells, mice	Laser, 660 nm, 5 mins	420 nm	520 nm	34.4%	Two-photon emission, efficient photothermal/photodynamic effect in killing cancer cells after light irradiation, significant tumor growth inhibition.	210

Abbreviations: CDs, carbon dots; CDs@PDA, polydopamine-coated carbon dots; Cu-doped, copper-doped; ICGCDs, indocyanine green–derived carbon dots; MP-CDs, manganese-coordinated polyphenol carbon dots; mins, minutes; NA, not applicable; NIR, near-infrared; nm, nanometer; PCD, permeable carbon dots; PCE, photothermal conversion efficiency; PEG, polyethylene glycol; PTT, photothermal therapy; SCDs, supra-carbon dots; S, N-CDs, sulfur- and nitrogen-co-doped carbon dots.

In another study, Kim et al.²¹¹ synthesized FA-functionalized CD/polypyrrole nanoparticles (FA-CD/PPy-NPs) for enhanced bioimaging and PTT. The FA functionalization increased specificity toward folate receptor-overexpressing cancer cells, reducing off-target interactions. Incorporation of polypyrrole (PPy), a strong NIR-absorbing photothermal material, further improved PCE. Upon NIR irradiation, FA-CD/PPy-NPs significantly decreased HeLa cell viability, confirming high photothermal therapeutic efficacy.²¹¹ This work underscores the potential of hybrid nanoplatforms that combine CDs with other nanomaterials to optimize optical performance and cancer selectivity.

Similarly, Najaflu et al.¹⁹⁶ synthesized copper-doped CDs using an eco-friendly hydrothermal approach with Alcea leaves as the carbon precursor, representing a sustainable route for biomedical applications. Copper incorporation enhanced free electron density, boosting NIR absorption and PCE. Under 800 nm NIR irradiation, the Cu-doped CDs effectively ablated 4T1 cancer cells through localized heating and cell death induction.¹⁹⁶ This study emphasizes how heteroatom doping broadens the optical absorption range of CDs and enhances their suitability for deep-tissue photothermal treatment while also promoting green synthesis methodologies.

To further augment the efficacy of CDs, Zhao et al.²⁰² designed CDs with localized excited and charge-transfer states, enabling dual NIR-I/II emission and absorption. This structural engineering allowed multiphoton imaging and deep-tissue PTT within the NIR-II window (1000–1700 nm), where light scattering and tissue absorption are minimized.²⁰² These CDs exhibited broad absorption and achieved efficient tumor cell eradication upon 808 nm irradiation, as confirmed by MTT assays. In vivo experiments showed significant tumor suppression and tissue necrosis in treated mice without systemic toxicity. Histological evaluation revealed no damage or inflammation in major organs, and body temperature remained stable throughout treatment.²⁰² Collectively, these studies confirm the versatility and safety of CDs as PTAs in PTT. Through advancements in heteroatom doping, hybrid nanostructuring, and NIR-II engineering, CDs have demonstrated superior photothermal efficiency, excellent biocompatibility, and precise tumor-targeting potential. These characteristics position CDs as next-generation nanoplatforms for effective, non-invasive, and sustainable cancer phototherapy.

5. Factors influencing the efficacy of CDs in cancer therapy

In the rapidly evolving field of cancer treatment, photosensitive nanoparticles, particularly CDs, offer significant potential in phototherapy. Their therapeutic efficacy is influenced by multiple factors, including light source parameters, the TME, and nanoparticle stability. With unique optical behavior and flexible surface chemistry, CDs can be functionalized to enhance targeting, imaging, and therapeutic efficacy. Understanding the complex interactions between these factors is essential to fully exploit CDs’ potential in cancer therapy. This requires careful consideration of nanoparticle design, microenvironmental conditions, and treatment parameters to optimize delivery and therapeutic outcomes. By exploring these interrelated aspects, researchers can advance precision phototherapy, improving efficacy and minimizing off-target effects in cancer treatment.

5.1. Tumor microenvironment

The TME is a highly dynamic and heterogeneous system, presenting both challenges and opportunities for the application of CDs in phototherapy. Tumor-associated factors, including pH, oxygen levels, and vascular structure, strongly influence the behavior of nanoparticles, such as CDs, within this biological context.^18,212 These unique characteristics of the TME allow the rational design of stimuli-responsive CDs capable of enhancing drug delivery, improving phototherapeutic efficacy, and minimizing off-target toxicity. A defining feature of the TME is its acidic nature, which arises from accelerated glycolysis, proton pump activity, and inadequate blood perfusion.¹⁸ Whereas normal tissues typically maintain a near-neutral pH of 7.0–7.2, tumor tissues generally exhibit lower pH values between 6.2 and 6.9.²¹³ This differential can be harnessed to achieve targeted drug release and selective phototherapy activation, allowing CDs to act specifically within tumor regions while sparing healthy tissues.¹⁹

CDs can be functionalized with pH-sensitive groups that undergo conformational or electronic changes in acidic environments, triggering drug release or modulating fluorescence for imaging purposes. For example, a carbon dot–Proximicin-A peptide conjugate demonstrated pH-responsive behavior, in which acidic conditions induced structural and electronic transformations that enhanced selective interactions with cancer cells.²¹⁴ Density functional theory simulations confirmed that this conjugate remained stable at physiological pH but experienced protonation of key functional groups in acidic environments, resulting in localized therapeutic activation at tumor sites.²¹⁴ Such targeted mechanisms improve efficacy while reducing systemic toxicity. Additionally, certain CDs are engineered to exhibit enhanced fluorescence or self-disassembly in acidic conditions, thereby improving imaging precision and drug accumulation in tumors. In one study, researchers developed pH-responsive self-disintegrating nanoassemblies that were stable at neutral pH but rapidly dissociated under acidic TME conditions, efficiently releasing therapeutic agents.²¹⁵ This approach improved tumor penetration, enhanced bioavailability, and reduced premature clearance by the mononuclear phagocyte system (MPS).

Hypoxia, another hallmark of solid tumors caused by rapid proliferation and abnormal vasculature, poses a critical challenge for PDT, which relies on molecular oxygen to generate ROS.^18,21 To overcome hypoxia, CDs have been engineered to enhance oxygen availability. One strategy involves integrating CDs with oxygen-generating catalytic nanomaterials, such as MXene heterojunctions, which decompose endogenous H₂O₂ into oxygen upon ultrasound activation, alleviating hypoxia and enhancing ROS-mediated cytotoxicity in sonodynamic therapy.²¹⁶ Hybrid CD assemblies functioning as ROS nanogenerators maintain ROS production even under hypoxic conditions, improving both PDT and PTT performance.²¹⁷ Mesoporous silica nanospheres containing CDs can catalytically decompose H₂O₂ to generate oxygen, further overcoming hypoxia-related resistance and improving PDT efficiency.²¹⁸ Oxygen-loaded CDs utilizing hemoglobin, perfluorocarbon, or nanobubbles provide controlled oxygen release within tumor tissues, sustaining ROS production and increasing oxidative stress in cancer cells.²¹⁸ Additionally, MnO₂-doped CDs can catalyze H₂O₂ decomposition in oxygen-deficient environments, generating oxygen to enhance PDT activation.^219,220 Such self-adaptive oxygenation ensures continuous ROS generation, improving treatment precision while minimizing damage to healthy tissues.

Tumor vasculature abnormalities significantly hinder nanoparticle delivery. Irregular, dilated, and disorganized tumor blood vessels cause uneven nanoparticle distribution.^22,23,221 Although the enhanced permeability and retention (EPR) effect promotes selective accumulation, poor vascular perfusion limits uniform delivery and therapeutic efficacy.²²² The extracellular matrix (ECM), mainly composed of collagen and hyaluronic acid (HA), also restricts nanoparticle penetration.²²³ Surface modification of CDs can enhance vascular permeability and tumor infiltration. PEG functionalization prolongs circulation, minimizes protein adsorption, and improves dispersion, enhancing tumor accumulation via the EPR effect.²²⁴ Enzyme-functionalized CDs degrade ECM components, promoting deeper penetration. Molybdenum-doped CD nanozymes exhibit strong PL and catalytic activity, breaking ECM barriers and improving diffusion.²²⁵ CDs responsive to TME-specific cues such as pH or redox potential enable controlled drug release.²²⁶ chlorine e6 (Ce6)-loaded CDs with Cu²⁺ ions form TME-responsive nanoassemblies for dual imaging and synergistic cancer therapy.²²⁷

The structural adaptability and multifunctionality of CDs make them ideal candidates for intelligent nanocarriers capable of responding dynamically to TME variations, including acidity, hypoxia, and abnormal vasculature. By exploiting these tumor-specific traits, CDs can enhance drug delivery, optimize phototherapeutic effects, and minimize systemic toxicity. Continued advances in enzyme-functionalized, oxygen-generating, and pH-responsive CDs will be crucial for overcoming the biological and physiological barriers presented by the TME. These innovations are expected to enable the development of more effective, targeted, and patient-specific phototherapeutic strategies, improving clinical outcomes and reducing side effects in cancer treatment.

5.2. Stability of nanoparticles

The stability of CDs is a key factor determining their effectiveness in phototherapy, as fluctuations in the TME, including temperature, pH, and oxidative stress, can compromise their structure and therapeutic efficiency. In general, nanoparticle stability refers to the ability to maintain essential structural features such as aggregation state, crystallinity, composition, particle size, shape, and surface chemistry.²²⁸ Preserving the structural integrity of CDs during repeated light exposures is vital to ensure consistent efficacy in PDT and PTT.²¹

CDs possess high photostability due to their π-conjugated graphitic structure, which provides resistance to structural degradation during continuous irradiation. The presence of stable sp²-hybridized carbon atoms minimizes photobleaching and maintains fluorescence intensity even after prolonged light exposure.²²⁹ Efficient surface passivation, such as PEGylation or amino-rich functionalization, further enhances stability by reducing oxidative damage and ensuring sustained optical and therapeutic performance. PEGylation, for example, improves the PL and overall stability of CDs, making them highly suitable for bioimaging and therapeutic applications.¹⁸⁶ Moreover, the QY of CDs is closely linked to their photostability, as efficient non-radiative decay pathways allow excess energy to dissipate safely, minimizing thermal degradation and maintaining fluorescence.²³⁰

To enhance stability and therapeutic outcomes, strategies such as surface functionalization and encapsulation have been widely explored. Functionalization with biocompatible coatings improves photostability, prevents aggregation, and mitigates oxidative stress (Fig. 7). PEGylation increases hydrophilicity, decreases protein adsorption, and extends circulation time in vivo, resulting in better tumor accumulation and treatment efficacy.¹⁸⁶ A study reported that PEG-functionalized CDs displayed higher photoluminescence quantum yield (PLQY), improved stability, and prolonged retention, confirming their suitability for imaging and therapy.²³¹ Functionalization with targeting ligands or peptides can further promote tumor-specific accumulation. For example, CDs conjugated with nuclear localization signal (NLS) peptides achieve efficient nucleus-targeted imaging, facilitating precise therapeutic delivery.²³²

Fig. 7.

Schematic diagram of illustrating three different surface modification strategies of CDs to enhance the stability, safety, and therapeutic precision of CDs. CDs, carbon dots; PEG, polyethylene glycol.

Encapsulation using protective matrices such as biodegradable liposomes offers another route to improve photostability and controlled drug release. Liposomal encapsulation shields CDs from enzymatic degradation, enhances bioavailability, and allows targeted drug transport to tumor sites.²³³ CDs encapsulated in liposomes exhibit enhanced stability and controlled release, improving PDT performance.²³⁴ Additionally, encapsulation designs can be made responsive to tumor microenvironmental conditions. For instance, CDs embedded in mesoporous silica nanospheres decompose endogenous H₂O₂ into oxygen, increasing PDT efficiency under hypoxia.²¹⁸

Despite these advances, environmental factors such as extreme pH and high-intensity UV exposure still threaten CD stability.²³⁵ Improving synthesis methods and incorporating protective modifications are necessary to maintain long-term performance. The structural and functional stability of CDs can be characterized using XRD, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), X-ray spectroscopy, and electron microscopy to assess size, aggregation, and integrity under physiological conditions.²²⁸

Beyond stability, issues such as biodegradability, lymphatic clearance, and immunogenicity remain critical for clinical translation. Combining CDs with chemotherapy or immunotherapy enhances efficacy but requires careful formulation to prevent rapid degradation or immune activation. Tumor heterogeneity, patient variability, and nanoparticle diversity further complicate therapeutic design.²³⁶ Long-term toxicity of non-biodegradable nanoparticles also requires investigation.²³⁶

To achieve clinical success, the stability and safety of CDs must be verified in vivo. Functionalization with polymers such as PEG reduces immune reactions and prolongs circulation, while encapsulation within biodegradable liposomes protects against enzymatic degradation and promotes targeted delivery. Engineering CDs to respond to tumor-specific cues, such as pH or oxidative stress, can improve therapeutic precision. Overall, the stability and structural properties of CDs determine their success in cancer phototherapy. Through strategic surface engineering, encapsulation, and advanced characterization, researchers can improve stability, safety, and efficacy, paving the way for clinical translation into next-generation cancer treatments.

5.3. Light parameters

In cancer phototherapy, optimizing light parameters is essential to improve the therapeutic performance of photosensitive nanoparticles such as CDs. Their remarkable optical properties make them suitable for both PDT and PTT. Precise adjustment of parameters, including light source type, wavelength, power density, energy density, pulse mode, irradiation duration, and distance from the target tissue, is necessary to activate CDs effectively while minimizing damage to healthy tissue. Common light sources include lasers, LEDs, and lamps due to their controllability and wavelength specificity. While lasers emit coherent, monochromatic light for precise, localized treatments,²³⁷ LEDs feature flexible wavelength output, reduced cost, and enhanced adaptability.²³⁷ Lamps such as halogen or fluorescent types offer broad-spectrum illumination for large-area exposure.²³⁷ Recently, X-rays and natural sunlight have been investigated; X-rays enable deeper tumor targeting,²³⁸ whereas sunlight provides a cost-effective option for surface cancers. The optimal light source depends on tumor depth, activation wavelength, and economic considerations.^239,240

The structural and optical characteristics of CDs strongly influence their light absorption and therapeutic performance. Synthesis conditions and precursor types determine their absorption and emission spectra, impacting how efficiently they convert light energy. CDs with extended π-conjugation structures exhibit superior NIR absorption and deep-tissue penetration for PTT.⁶ Functionalization with carbonyl or sulfoxide groups can shift absorption toward the NIR region, enhancing PCE.²⁴¹ Sulfur-doped CDs synthesized from Camellia japonica flowers achieved a PCE of 55.4% under an 808 nm laser, confirming their effectiveness for cancer phototherapy.²⁴²

Wavelength selection is critical, as it must align with the CDs’ absorption peak for optimal activation and energy transfer.^{2, 154} Jamali et al.²⁴³ reported that blue light caused higher cytotoxicity in human glioma cells than red light, emphasizing wavelength precision. Power density, or irradiance, defined as the energy delivered per area and time (mW/cm²), must be optimized to balance efficacy and safety.²⁴⁴ High intensities can induce necrosis, while lower intensities promote apoptosis. Melamed et al.¹⁹⁹ observed that 30 W/cm² caused necrosis, whereas 5 W/cm² favored apoptosis, indicating that adjusting power density influences the therapeutic mechanism.

Energy density, or fluence (J/cm²), represents the total light energy applied during treatment and is determined by both power density and exposure duration.²⁴⁴ Proper fluence calibration ensures adequate tumor ablation while avoiding overheating. Pulse mode modulation enhances precision by allowing intermittent cooling and reducing thermal injury.²³⁹ Likewise, irradiation duration must be controlled to maintain an optimal “Goldilocks Zone” between sufficient activation and tissue preservation.²⁴⁵ Prolonged exposure increases ROS generation but risks thermal damage, while shorter exposure may yield incomplete tumor ablation. Modern systems incorporate real-time temperature and light-dose monitoring for dynamic control.²⁴⁵ The distance between the light source and tissue affects light distribution and penetration. Maintaining optimal spacing ensures uniform irradiation and prevents localized overheating or energy loss due to scattering. Adjustment based on tissue type and penetration depth is essential for consistent outcomes.

Table 3 summarizes recent studies employing CDs in PDT and PTT, showing that lasers and LEDs remain preferred sources for their reproducibility and precision. However, hybrid systems combining multiple wavelengths are being developed to overcome limited penetration and tissue heterogeneity. In conclusion, the therapeutic efficiency of CDs in phototherapy depends on careful regulation of illumination parameters. Future research should integrate adaptive light delivery systems, advanced dosimetry, and multimodal imaging to personalize therapy. Combining CD-based phototherapy with immunotherapy or targeted drug delivery may produce synergistic antitumor effects. With continued progress in NIR-responsive CD design and optimized irradiation control, CD-assisted phototherapy is poised to become a powerful, minimally invasive approach for next-generation cancer treatment.

Table 3.

Light parameters used in the application of CDs in phototherapy.

CDs carbon source	Type of light	Wavelength	Energy density (Fluence)	Irradiation time	Reference
Chitosan and diketopyrrolopyrrole	Laser	540 nm	15 mWcm-2	10 mins	177
Hypocrella bambusae	Laser	635 nm	0.8 Wcm-2	10 mins	246
CA and PEI	Laser	671 nm	500 mWcm-2	20 mins (in vitro) 10 mins (in vivo)	247
1,3,6-trinitropyrene	Laser	635 nm (in vitro) 800 nm (in vivo)	1–2 Wcm-2 (in vitro) 500 mWcm-2 (in vivo)	10 mins	117
O-phenylenediamine	Laser	532 nm	100 mWcm-2	4 mins	248
Poly (acrylic acid)	LED	400–700 nm	40 mWcm-2	12 mins	249
PT2	LED	420 nm	NA	5 mins	170
Coronene	Laser	808 nm	2 Wcm-2	10 mins	201
FePc	Laser	660 nm	0.5 Wcm-2	10 mins	250
Riboflavin	Laser	365 nm	60 mWcm-2	1–20 mins	153
CA and urea	Laser	650 nm	1 Wcm-2	10 mins	251
Polydopamine and FA	Laser	808 nm	1.5 Wm-2	10 mins	252
CA and Ru-Aphen	White light	NA	6.5 mWcm-2	30 mins	253
CA and urea	Laser	808 nm and 1064 nm	1 Wcm-2	5 mins	205
Lysine and o-phenylenediamine	Laser	660 nm	1 Wcm-2	5 mins	210
Triethylenetetramine hexaacetic acid	LED	365 nm	NA	30 mins	254
CA and tea polyphenol	LED	400–500 nm	15 mWcm-2	12 mins	175
Hypericum perforatum extract	Laser	635 nm	100 mWcm-2	10 mins	154,196
Alcea leaves	Laser	808 nm	NA	10 mins	196
L-Glycine and CA	Laser	660 nm and 808 nm	0.6 Wcm-2 and 2 Wcm-2	5 mins	255

Abbreviations: CA, citric acid; FA, folic acid; FePc, iron (II) phthalocyanine; LED, light-emitting diode; mW cm⁻², milliwatts per square centimeter; min, minutes; NA, not applicable; nm, nanometer; PEI, polyethyleneimine; PT2, polythiophene; Ru-Aphen, 5-amino-1,10-phenanthroline ruthenium(II) complex.

6. The future of CDs for clinical use in cancer treatment

The future of CDs in human-centered cancer therapy holds transformative potential, offering solutions to major challenges in current treatment approaches. Traditional methods such as surgery, radiotherapy, and chemotherapy, though effective, often cause severe side effects and damage to healthy tissues. With ongoing research, CDs are emerging as multifunctional nanoplatforms in phototherapy, drug delivery, and theranostics, providing targeted, efficient, and minimally invasive alternatives. Their distinctive properties, such as robust PL, high biocompatibility, flexible surface chemistry, and straightforward functionalization, make them highly promising for PDT and PTT.^{256, 257} However, challenges in biocompatibility, targeted delivery, and regulatory approval remain. Innovative strategies, such as photo-responsive CD aggregation and their integration as sealing agents, may further enhance therapeutic efficacy in precision oncology.

6.1. Challenges in the clinical translation of CDs

Despite encouraging preclinical outcomes, the clinical translation of CDs for cancer therapy faces significant challenges. These include issues of biocompatibility, biodistribution, clearance, and tumor-targeting efficiency. Although CDs are generally regarded as biocompatible, variations in synthesis routes, precursor materials, and surface functionalization can markedly alter their toxicity. Therefore, comprehensive assessments of long-term biosafety are essential before CDs can be approved for clinical use. One major concern is biodistribution and clearance, which depend on physicochemical properties such as particle size, surface charge, and hydrophobicity.^119,120 Studies have shown that CDs may accumulate in key organs, including the heart, liver, spleen, and kidneys, potentially leading to long-term toxicity.^109,116 Smaller CDs (<6 nm) with neutral or positive charges are efficiently eliminated through renal excretion within 24 hours.^119,120 In contrast, larger or highly negatively charged CDs tend to accumulate in the liver and are cleared more slowly via hepatic excretion through bile and feces.¹¹⁹ Moreover, unfavorable surface modifications may impede clearance, resulting in prolonged exposure, oxidative stress, and inflammatory responses that heighten toxicity risks.²⁵⁸

Beyond safety, achieving precise tumor targeting remains a critical obstacle. Although the EPR effect is often employed for passive targeting, its effectiveness varies among tumor types and individuals. To enhance specificity, active targeting strategies have been developed through ligand, peptide, antibody, or biomolecule conjugation.²⁵⁹ For example, FA-functionalized CDs, which bind to folate receptors overexpressed in many cancer cells, significantly improve tumor selectivity.²⁶⁰ Likewise, coating CDs with biocompatible materials such as PEG and FA enhances stability, prolongs circulation, and promotes tumor accumulation.²⁶¹ The versatile surface chemistry of CDs thus supports their application in precision medicine with reduced off-target effects. Nevertheless, a substantial translational gap persists. The “Valley of Death” between preclinical and clinical stages stems from limitations in animal models, high clinical trial failure rates, and development costs.²⁶² Rigorous evaluations of toxicity, long-term biodistribution, and pharmacokinetics remain prerequisites for FDA approval. Future directions include pre-illumination of CDs to enhance photodynamic efficiency, development of nanocarriers for targeted delivery,²⁶³ and exploration of topical applications, particularly for superficial cancers such as melanoma, where the optical properties and light responsiveness of CDs can be fully utilized.

6.2. Future application of CDs as photo-responsive aggregation agents for cancer therapy

A promising strategy in cancer phototherapy involves the photo-induced aggregation of CDs, which enhances their tumor retention, PCE, and therapeutic efficacy. Under light irradiation, CDs can aggregate selectively within tumor tissues, increasing local concentration, improving PTT outcomes, and minimizing systemic toxicity. Recent advancements demonstrate that CDs can be engineered to aggregate in response to external stimuli, enabling controlled therapeutic actions at tumor sites. For instance, Song et al.²⁶⁴ designed depolymerizable enzymatic cascade nanoreactors that self-target tumor vasculature and disassemble upon exposure to tumor-specific enzymes, inducing localized CD aggregation and controlled drug release.²⁶⁴ This mechanism enhances treatment precision and prolongs CD retention within the TME, reducing off-target effects. Integrating enzyme- or photo-responsive mechanisms into CD-based systems can thus improve aggregation control, retention, and overall phototherapy performance.

Beyond PTT, photo-induced CD aggregation can augment chemotherapy. CDs conjugated with chemotherapeutic drugs such as DOX form stable nanocomplexes that remain inert in circulation but aggregate at tumor sites under light exposure.²⁶⁵ This aggregation triggers localized drug release, minimizes systemic toxicity, and enhances synergistic photodynamic–chemotherapeutic effects.²⁶⁵

CDs also possess remarkable PL properties, making them ideal for real-time imaging in cancer diagnosis and treatment monitoring.^95,226 Photo-induced aggregation increases fluorescence intensity, improving tumor visualization and enabling image-guided therapy. Dual-modal theranostic platforms could allow clinicians to track CD aggregation, assess treatment efficacy, and tailor therapies in real time.

Furthermore, integrating photo-aggregating CDs with immunotherapy offers significant promise. CDs can deliver immunomodulators and recruit immune cells to tumor sites, amplifying antitumor immunity.²⁶⁶ pH-sensitive CDs loaded with Ce6 have demonstrated selective aggregation, ROS generation, and immune activation.²⁰ Such dual photo-activated and immune-enhancing mechanisms could synergize with immune checkpoint inhibitors for superior tumor eradication. Overall, stimuli-responsive CD aggregation represents a transformative approach, merging PTT, chemotherapy, imaging, and immunotherapy to enable personalized, minimally invasive cancer treatments and improved patient outcomes.

6.3. CDs as sealing agents in tumor therapy

Sealing agents play a critical role in modern cancer management, particularly in surgical oncology and postoperative wound care. These biomaterials are engineered to rapidly close biological tissues, prevent bleeding, and support tissue repair and regeneration.²⁶⁷ In cancer therapy, sealing agents are applied after tumor removal to minimize hemorrhage, suppress inflammation, and accelerate recovery. Conventional sealants such as fibrin glues, gelatin sponges, and chitosan-based adhesives have long been used clinically,²⁶⁷ but they often face limitations, including immunogenicity, infection risk, and slow degradation. The emergence of nanotechnology, especially CDs, has led to the development of next-generation multifunctional sealants. CDs are ultrasmall, fluorescent carbon-based nanoparticles characterized by high biocompatibility, versatile surface functionality, and outstanding stability.² These attributes make them promising candidates for bioactive sealants that integrate imaging, drug delivery, and PTT to improve surgical outcomes in cancer treatment.

CDs also possess intrinsic hemostatic properties that make them particularly attractive as sealing materials in surgical oncology. Their interactions with platelets and plasma proteins can enhance coagulation and tissue adhesion, promoting rapid wound closure following tumor excision. Several studies have validated the hemostatic efficacy of CDs derived from natural precursors. Luo et al.²⁶⁸ synthesized CDs from Cirsium setosum Carbonisata and demonstrated that they accelerated clot formation through activation of the fibrinogen system and extrinsic coagulation pathways. Likewise, Zhang et al.²⁶⁹ produced CDs from Rubia cordifolia L. Carbonisata, which significantly reduced bleeding and promoted fibrin network formation in a mouse liver injury model. These results confirm the potential of CDs as effective bioactive sealants capable of rapid and safe hemostasis.

Beyond hemostasis, CDs can be engineered into multifunctional platforms that surpass the limitations of traditional sealants. Their modifiable surface chemistry enables functionalization with therapeutic agents to promote wound healing, inhibit microbial growth, and enable localized cancer therapy. One innovative approach involves embedding CDs into biodegradable hydrogels or self-healing nanomaterials to provide both mechanical strength and therapeutic activity. Zhao et al.²⁷⁰ designed a nanoplatform incorporating CDs within paclitaxel-loaded hollow mesoporous carbon (HMC). Upon photothermal activation, the “meteorolite” structures disassembled, releasing CDs to enhance local PTT efficacy and induce synergistic therapeutic effects.²⁷⁰ Such designs suggest future CD-infused bioadhesives could both seal wounds and deliver localized tumor ablation to prevent recurrence.

In another study, Zhao et al.²⁷¹ combined CDs with HA-gated hollow mesoporous silica nanocarriers for redox- and enzyme-triggered drug release. CDs provided fluorescence imaging while ensuring controlled release of anticancer drugs in tumor regions characterized by high glutathione and enzymatic activity.²⁷¹ This system could act as a smart sealing agent for localized chemotherapy following surgery. Similarly, Feng et al.²⁷² developed a triple stimuli-responsive nanoplatform integrating zinc oxide (ZnO) QDs with HMC. ZnO QDs functioned as sealing agents for HMC mesopores, preventing premature doxorubicin (DOX) leakage. Under NIR irradiation, the platform achieved dual photothermal and pH-responsive drug release, enhancing anticancer efficacy.²⁷²

Future advancements in CD-based sealants will focus on their multifunctionality. During tumor resection, these sealants could provide instant hemostasis while delivering chemotherapeutic or photosensitizing agents to residual cancer cells. Their photoluminescent properties also enable real-time imaging, allowing precise visualization of tumor margins and reducing collateral damage. Postoperatively, CD-based sealants could serve as biosensors for infection or recurrence detection. By integrating hemostatic, therapeutic, and imaging capabilities, CD-based sealing agents offer a transformative strategy for postoperative cancer care, promoting faster recovery, minimizing recurrence, and advancing personalized, minimally invasive oncological treatment.

7. Conclusions

CDs have demonstrated remarkable potential as multifunctional nanomaterials in cancer phototherapy, leveraging their photodynamic and photothermal properties for precise and efficient tumor ablation. Their ability to generate ROS in PDT and induce localized hyperthermia in PTT positions them as powerful tools for precisely targeting cancer cells. Additionally, their unique optical characteristics enable real-time bioimaging, making them valuable theranostic agents. This exploration highlights the factors influencing the efficacy of CDs in cancer phototherapy, such as TME, nanoparticles stability, and light parameters, providing a comprehensive guide to navigating the intricate interplay of variables. However, the road to clinical translation presents challenges, including biocompatibility, targeted delivery, and long-term safety concerns. Addressing these issues through surface functionalization, the development of nanocarriers, and the integration of CDs with other therapies will be key to harnessing their full therapeutic potential. Future research should focus on the CDs’ long-term safety profile, refining the pharmacokinetic properties of CDs, optimizing their interactions with biological systems, and ensuring their regulatory approval for clinical applications. Interdisciplinary collaboration among material scientists, biomedical researchers, and clinicians will be essential in overcoming existing barriers and advancing CDs toward clinical implementation. With continued innovation, CDs have the potential to revolutionize cancer therapy, offering targeted, minimally invasive, and highly effective treatment modalities tailored to individual patient needs.

Author contributions

Hong Hui Jing, Mohd Adnan, and Sreenivasan Sasidharan: Conceptualization, design, data collection and analysis, project administration, and original manuscript draft. Mitesh Patel, Mohd Adnan and Sreenivasan Sasidharan: methodology, data curation, investigation, visualization, analysis, review and editing. Hong Hui Jing, Mohd Adnan and Sreenivasan Sasidharan: critical revision of the manuscript, validation, formal analysis. Sreenivasan Sasidharan: study supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.