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. 2025 Aug 28;10(35):39555–39566. doi: 10.1021/acsomega.5c02344

Harnessing Lignin-Derived Carbon Dots for Enhanced NIR Photothermal Ablation of Melanoma Cells

Vinita Satwani †, Badri Narayana Sahoo ‡, Kallayi Nabeela †, Abhijeet Joshi ‡, Shaikh M Mobin †,§,*
PMCID: PMC12423852  PMID: 40949255

Abstract

The integration of optical imaging modalities with photothermal therapy (PTT) enables simultaneous oncotherapy and bioimaging, offering improved therapeutic efficacy and enhanced treatment precision. As a result, this approach has emerged as a promising strategy for cancer treatment. Carbon dots (CDs) are highly stable, biocompatible materials with exceptional fluorescent properties, making them strong contenders for optical bioimaging and photothermal applications. However, challenges remain in synthesizing optimal CDs due to insufficient photothermal conversion efficiency, often requiring high laser power densities and higher CD concentrations for effective photothermal therapy (PTT). In this study, nitrogen-doped lignin-derived CDs (N-LCDs) are synthesized via a green, low-cost route using lignin as the carbon source and ethylenediamine as the nitrogen dopant. The resulting N-LCDs exhibit excellent absorption in the near-infrared (NIR) range, superior photothermal stability, and remarkable biocompatibility. Cellular uptake studies confirmed efficient internalization, while flow cytometry analysis revealed significant apoptosis upon NIR laser exposure, validating their photothermal therapeutic effect. The structural and optical characterization of N-LCDs demonstrates a high photothermal conversion efficiency (PCE ∼ 57.5%), highlighting their potential as effective and safe bioimaging and photothermal agents for in vitro cancer therapy.

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1. Introduction

Despite significant advancements in understanding, diagnosing, treating, and preventing cancer, the incidence and mortality rates of the disease continue to rise, necessitating the development of more effective and safer therapies to complement traditional treatments such as surgery, chemotherapy, and radiation therapy. , Photothermal therapy (PTT) presents a promising alternative by utilizing a therapeutic approach that increases the local temperature at the target site through near-infrared (NIR) laser irradiation. , This method penetrates tissues and induces a temperature rise by converting absorbed light into heat, mediated by photothermally active (PTA) agents-photosensitive molecules that cause localized damage to the target area. PTT offers significant advantages over conventional cancer treatments, providing a noninvasive, nonionizing option with high therapeutic specificity and efficiency. − Moreover, PTT is capable of directly eradicating cancer cells within primary tumors or localized lymph node metastases, making it particularly effective in addressing early stage cancer. Mostly, the light-sensitive moieties show absorbance in UV–visible regions in the range of 100–700 nm that usually have superficial tissue-penetration depth (0.5–2.5 mm) and hence are limited to the mucosa and peripheral applications in the skin. , Conversely, the photons in the NIR window in the range of 700–1100 nm show minimum photodamage with increased tissue penetration (∼1 cm). Hence, materials showing strong NIR absorption hold promising photoactivities in living cells and are considered an efficient PTA agent. Various types of PTA agents have been derived from organic NIR chromophores − and noble metal-based nanoparticles. − These agents generally show high NIR absorption, exhibiting relatively higher PCE. However, the complicated synthetic procedures of chromophores with high costs impede their practical applications. Also, metal-based nanoparticles possess a nonbiodegradable nature with delayed renal clearance and potential long-term toxicity. Additionally, the low hydrophilicity and poor photostability of traditional dyes showing good NIR absorbance restrict their use in therapeutic applications.

Interestingly, among the PTA agents developed so far, nanomaterials based on carbon, including graphene oxide (GO) and carbon nanotubes (CNTs), have been widely accepted as strong candidates as PTA agents. Still, they also show long-term toxicity and poor solubility. Additionally, the efficacy of PTT, however, is often limited by challenges in accurate diagnosis and difficulties in monitoring therapeutic effects during follow-up. To address this limitation, imaging modalities must be integrated into PTT. This integration allows for precise identification of a tumor’s size and location while providing real-time monitoring of the therapeutic effect within the targeted region. Such monitoring is critical for planning more effective interventions and improving therapeutic outcomes. Despite these advancements, the development of biocompatible PTT agents capable of simultaneously enabling imaging and therapy in a straightforward and efficient manner remains a significant challenge. −

Hence, carbon-based quantum dots are highly preferable classes of carbon-based nanomaterials due to their unique optical properties, extremely small size, high hydrophilicity, good thermal and photostability, biological safety, and low production cost. Also, it possesses excellent broadband absorption in the NIR region which is requisite in PTT. − Moreover, doped CDs show superior optical properties and a high photothermal conversion efficiency. The NIR responsiveness of CDs can be effectively tuned through heteroatom doping. , Introducing impurities such as metals, nonmetals, or organic species into the core or onto the surface of CDs alters their electronic structure, thereby modifying the energy band gap. Doping with different metal or nonmetal sources enhances the photothermal performance of CDs in various ways, improving their ability to absorb NIR light and convert it into heat efficiently.

Recently, bioinspired CDs have been explored as a green source for the synthesis of CDs as it aids in improved biocompatibility and optical properties. − Moreover, the use of biomass-derived carbon precursors reduces preparation costs and could aid in the green recycling of resources, for instance, in the utilization of waste. , Since most of the CDs synthesized using biomass show bare minimum or nil absorbance in the NIR region, they have been mainly exploited for sensing, bioimaging, and detection of toxin/heavy metals. Research using biomass-derived CDs for PTT application with a NIR laser has been conducted previously. As per the report by Li et al., watermelon juice was used as a carbon precursor for PTT with 808 nm laser irradiation at 1.4 W/cm2 power density at a very high dose (20,000 μg/mL). In another study, Meena et al. utilized plants of medicinal value as carbon precursors but used harsh chemicals like H2SO4 and HNO3 for CD synthesis, and in vitro photothermal studies were conducted under irradiation of a 750 nm laser at 0.5 W/cm2 power density with the dosage of 500 μg/mL. Similarly, other conventional CDs derived from polymers and synthetic small molecules have also been employed for PTT − but are still facing challenges because of the requirements of higher concentration and high power with large irradiation time that can prove harmful to surrounding cells causing cytotoxicity.

Lignin and its derivatives, being the second most occurring organic matter on earth, have also been explored for CD synthesis as a green precursor of carbon due to their unique natural aromatic structure and high carbon content. Additionally, lignin is considered an undervalued biomass byproduct, burned to produce heat energy in the paper pulping industry. , Hence, converting the biomass-derived precursors to CDs can be viewed as a “two-birds-one-stone” strategy for clean development and high-value utilization of CDs. It possesses a three-dimensional structure with cross-linked phenolic groups attached to sp2-hybridized benzene rings in high proportion. The abundance of sp2-hybridized benzene rings renders it an optimal precursor of CD synthesis and the loosely held π electrons promote photothermal conversion with its excitation and relaxation to their ground states making it an efficient candidate for photothermal applications.

Herein, we report the utilization of lignin-derived carbon dots synthesized via a one-step hydrothermal treatment for cellular uptake and efficient photothermal conversion using 808 nm NIR laser irradiation at 1 W/cm2 power density. The as-synthesized CDs were doped using ethylenediamine (EDA) as a nitrogen source that efficiently aids in reducing the energy band gap, and studies were conducted concerning NIR absorbance and photothermal behavior. The prepared N-LCDs showed excellent NIR absorbance and demonstrated high-efficiency heat generation proving to be a promising PTA agent. Moreover, N-LCDs also showed excellent fluorescent properties and hence were also utilized for bioimaging. Additionally, the cell viability studies confirmed the biocompatible nature of N-LCDs even at higher concentrations. Furthermore, the actual mechanism behind the cell death by apoptosis due to the photothermal effect of N-LCDs was explained.

2. Results and Discussion

2.1. Synthesis and Characterizations of N-LCDs

The photothermally active and fluorescent N-doped CDs were synthesized through the hydrothermal method using lignin and EDA as carbon and nitrogen sources, respectively (Scheme ). The hydrothermal treatment was carried out for 16 h at 200 °C to obtain N-LCDs as shown in Scheme . The as-prepared N-LCDs were characterized by using various techniques to unravel their morphological, optical, and chemical properties and further employed for bioimaging and photothermal therapeutic applications.

1. Synthesis of N-LCDs.

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2.1.1. Morphological and Structural Characterization of N-LCDs

The morphological and structural details of the prepared N-LCDs were determined by using transmission electron microscopy (TEM). As can be seen in Figure a, most of the particles were found to be well-dispersed and spherical in morphology. The diameters of these particles were found to range from 0.93 to 4.13 nm with an average particle size of 2.38 ± 0.73 nm (Figure b). The selected area electron diffraction (SAED) pattern also revealed poor crystallinity of the formed particles indicating its amorphous structure which is also supported by powder X-ray diffraction (PXRD). The PXRD pattern of N-LCDs revealed a broad peak centered at 21.34°, which is commonly witnessed for CDs and generally belongs to the amorphous structure and corresponds to the (002) plane of the graphite-filled carbon core (Figure c). To further assess the particle size distribution and colloidal behavior in aqueous dispersion, dynamic light scattering (DLS) and zeta potential measurements were performed (Figures S1 and S2, Supporting Information). The DLS analysis revealed a hydrodynamic diameter of 186.6 nm with a polydispersity index (PDI) of 0.117, indicative of a narrow size distribution. The larger hydrodynamic size compared with the TEM-derived core size is attributed to the hydration layer and surface functional groups. While DLS may also capture minor aggregation to some extent, the high zeta potential of −45.01 mV (measured at pH 7.4) confirms strong electrostatic stabilization, suggesting that N-LCDs are well-dispersed and colloidally stable in aqueous media. It also indicates the abundance of nitrogen-/carbonyl-containing groups on the surface imparting negative charge. The Raman spectra of N-LCDs exhibited G (sp2-hybrid) and D (sp3-hybrid) bands at approximately 1580 cm–1 and 1405 cm–1 which characterized the graphitic and disordered carbon, respectively, demonstrating the formation of graphitic structures. The I D/I G intensity ratio corresponding to the D and G bands is about 1.01, indicating the presence of certain defects in N-LCDs (Figure d).

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Characterization of the N-LCDs. (a) TEM image (inset shows the corresponding SAED pattern), (b) histogram for particle size distribution, (c) PXRD pattern, and (d) Raman spectra (inset shows deconvoluted spectra).

Moreover, the functional groups present over the surface of N-LCDs were further elucidated by FTIR spectroscopy (Figure a). The broad-ranging peak at 3400–3100 cm–1 is consistent with the stretching vibration of the O–H and N–H functionalities, and the peaks that appeared at 2980–2830 cm–1 can be allocated to the C–H vibrations. The peak centered at 1645 cm–1 is due to the stretching vibrations of CO/CC groups and at 1129 cm–1 due to the bending vibrations of the C–O group indicating oxygen-rich groups on the surface. The bending vibrations of N–H and stretching vibrations of C–N were observed at 1584 cm–1 and 1362 cm–1, confirming the presence of amine and nitrogen-rich functionality on the surface of N-LCDs. The chemical composition and functionalities were further confirmed through XPS analysis. According to the full-scan survey spectra, the synthesized N-LCDs contain three peaks that belong to carbon (C 1s), oxygen (O 1s), and nitrogen (N 1s) (Figure S3, Supporting Information). The atomic ratios of the observed carbon, oxygen, and nitrogen at 285.08, 533.08, and 400.08 eV were determined as contents of C (67.48%), O (21.86%), and N (3.3%) in the N-LCDs, respectively. The deconvoluted XPS spectra of C 1s as shown in Figure b revealed three peaks at 284.6, 285.8, and 288.3 eV that correspond to the C–C/CC, C–N/C–O, and CO/CN, respectively. In the high-resolution O 1s XPS spectra, the two peaks appearing at 531.5 and 532.8 eV belong to CO and C–O–C/C–OH, respectively (Figure c). In addition, the N 1s spectra were deconvoluted into three peaks that represent the pyridinic, pyrrolic, and amide N appeared at 399.5.6, 400.1, and 401.6 eV, respectively (Figure d). To further elucidate photophysical and fluorescent properties of N-LCDs, UV–vis absorption and fluorescence excitation and emission spectra were discussed.

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(a) FT-IR spectra of N-LCDs. XPS high-resolution spectrum for N-LCDs: (b) C 1s, (c) O 1s, and (d) N 1s.

2.1.2. Photophysical Properties of N-LCDs

To assess the photophysical properties of N-LCDs, their UV–vis absorption spectrum was recorded, as shown in Figure a. A strong absorption peak at 280 nm was observed, attributed to the π–π* transitions of the CN and CC bonds within the sp2 aromatic carbon core. Peaks at 340 and 380 nm correspond to the π–π*/n–π* transitions of CO bonds from surface functional groups. These surface functional groups play a key role in the application of N-LCDs in bioimaging following cellular uptake and in photothermal therapy. When excited at 380 nmone of the N-LCDs’ absorption peaksa broad emission peak at 500 nm was observed. This peak arises from the trapping of excited energy on the heteroatom-passivated surface of N-LCDs, producing a strong cyan fluorescence. This emission is predominantly determined by band-edge exciton-state decay rather than defect-state decay. Additionally, the emission maxima remain well-defined and do not shift when the excitation wavelength changes from 370 to 390 nm, indicating excitation-independent emission behavior. However, fluorescence intensity varies significantly, with the maximum intensity at 380 nm (Figure b). Digital photographs of N-LCD dispersions (Figure b, inset) display a yellow color under visible light (left) and a bright cyan fluorescence under UV light at 365 nm (right). These observations confirm the favorable photophysical properties of N-LCDs for fluorescence-based applications. The energy band gap of N-LCDs was measured to be 1.57 eV (Figure S4, Supporting Information) which is narrower than previous reports. Additionally, the intrinsic fluorescence characteristic is also exhibited in the decay curve as shown in Figure c which indicates the average fluorescence lifetime of N-LCDs, calculated to be 2.91 ns fitted biexponentially. The fluorescence quantum yield (eq S1, Supporting Information) of N-LCDs was estimated to be 3.33% at 380 nm using quinine sulfate as a reference. Given these favorable optical and structural properties, further investigations were carried out to evaluate the stability of N-LCDs under various physicochemical and biologically relevant conditions to ensure their robustness for therapeutic and imaging applications.

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(a) UV–vis absorption spectra of N-LCDs. (b) FL spectra showing excitation and emission (inset shows digital photographs of N-LCD dispersion taken in visible light (left) and UV light (right)). (c) TCSPC measurement recorded for N-LCDs.

2.2. Stability Studies of N-LCDs

The long-term functionality and therapeutic reliability of photothermal nanomaterials depend strongly on their stability under diverse biological and therapeutic conditions. Hence, to assess the practical applicability of N-LCDs in such conditions, comprehensive stability studies were conducted on a wide range of parameters. To begin with, N-LCDs were incubated under continuous UV exposure (Figure S5a, Supporting Information), across a wide pH range (Figure S5b, Supporting Information), and at different temperatures (Figure S5c, Supporting Information), with no significant changes observed in their fluorescence intensity, suggesting excellent colloidal stability and resistance to photodegradation. In addition, the stability of N-LCDs was conducted in solutions with varying NaCl concentrations (Figure S5d, Supporting Information), where the optical properties remained unaffected, indicating strong tolerance to ionic strength typically encountered under physiological conditions. Since the primary application of the prepared carbon dots lies in cellular and biological systems, their stability was also assessed in different biological media, including deionized water, PBS, and DMEM, over a 24 h incubation period (Figure S5e, Supporting Information). The fluorescence intensity remained stable across all media, confirming that N-LCDs retain their structural and optical integrity under biologically relevant conditions. These findings not only confirm the excellent physicochemical and biological stability of N-LCDs but also highlight their potential for consistent performance in photothermal applications. Given their promising optical properties and robust physicochemical stability, we next evaluated the photothermal performance of N-LCDs under 808 nm laser irradiation.

2.3. Photothermal Performance Studies of N-LCDs

The absorption spectra of N-LCDs in the range of 650–1000 nm exhibit a broad absorption profile extending from the visible to the NIR region. As shown in Figure a, the entire absorption spectrum of N-LCDs from 650 to 1100 nm demonstrates concentration-dependent behavior, with absorbance increasing consistently as the concentration rises. The enhanced NIR absorption of N-LCDs is attributed to the heavily doped nitrogen species and associated surface functional groups. This broad absorption capability in the NIR region renders the synthesized N-LCDs highly NIR-responsive, making them excellent candidates for photothermal therapy (PTT) with efficient light-to-heat conversion. Given the strong NIR absorbance of the synthesized material, we further assessed its photothermal performance under 808 nm laser irradiation.

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Photothermal (PT) performance characterization of N-LCDs: (a) Vis–NIR absorption spectra of N-LCD dispersions at varied concentrations. (b) Temperature increment of N-LCD dispersions at varied concentrations (808 nm, 1.0 W/cm2). (c) Temperature increment of N-LCD dispersions (500 μg/mL) at varied laser power densities. (d) Temperature curves of N-LCDs under six laser turn-on/off cycles. (e) Heating–cooling curve obtained from the single laser turn on/off process. (f) Fitting curve of the cooling period v/s negative natural logarithm of the temperature driving force obtained from the cooling period data of (e).

The photothermal performance of N-LCDs was evaluated at different concentrations and varied power densities by plotting the temperature changes as a function of time. The N-LCDs showed excellent photothermal performance with a temperature rise of 22.9 and 29 °C for 5 and 10 min of laser irradiation achieving 47.9 and 54 °C. Figure b shows the concentration-dependent temperature change of N-LCDs with time, while the control exhibited a negligible change in temperature. With the increase in concentration, elevated temperatures were recorded under common laser power (1.0 W/cm2). Additionally, it was observed that the temperature of the solution increased with an increased power density of the laser (Figure c). The N-LCD solution was exposed to different power densities ranging from 0.4 to 1.4 W/cm2 with an 808 nm laser for 10 min. The temperature of the solution reached 32.6 and 55.1 °C within 5 min of laser exposure on varying power density from 0.4 to 1.4 W/cm2 and further 39.4 and 62.3 in 10 min, respectively, exhibiting power-dependent behavior. Accordingly, the optimum irradiation conditions were fixed at a power density of 1 W/cm2 at 500 μg/mL for 5 min to derive enough heat (47.9 °C) to destroy cancer cells.

Moreover, the thermal stability and PCE of the material are crucial factors to intervene in the overall photothermal performance of the PTA agent. Hence, the thermal stability of N-LCDs at 500 μg/mL concentration was explored by repetitive irradiation with an 808 nm laser at a power density of 1 W/cm2 for several consecutive on/off cycles. As shown in Figure d, the N-LCD did not show obvious temperature decay during 6 heating–cooling cycles indicating excellent photothermal stability that resists photobleaching. Furthermore, the PCE was determined to be 57.5% using equations S2 and S3, Supporting Information, and was evaluated based on the data obtained through the heating–cooling curve (Figure e,f) as per the reported method. , Remarkably, the PCE of N-LCDs is relatively higher than that of previously reported PTA agents such as polydopamine CDs (35%), urea and citric acid CDs (24.3%), Cu2–x Se nanocrystals (22%), and other biomass-derived and chemical-based CDs (Table S1, Supporting Information) and comparable to polypyrroles, cyanine-anchored silica nanochannels, and nanodiamonds rimmed polydopamine–indocyanine green conjugates.

The high PCE of the CDs is primarily attributed to nitrogen doping, which effectively reduces the band gap (∼1.57 eV) while enhancing their light-harvesting capability. Additionally, the presence of graphitic structures further promotes nonradiative transitions, leading to efficient heat generation and contributing to the high PCE. The plausible heat generation mechanism for cancer PTT with carbon-based nanoparticles is primarily explained by the excitation of loosely bound π-electrons and their subsequent relaxation through nonradiative processes, which efficiently convert absorbed light energy into heat. − The nonradiative transitions are the primary factor contributing to their low quantum yield in graphene quantum dots, as a significant portion of the absorbed energy is dissipated as heat rather than re-emitted as light. , This phenomenon is also observed in N-LCDs, which exhibit a low quantum yield of 3.33%. A low quantum yield indicates that the majority of the energy absorbed by the nanomaterial at a given wavelength is dissipated as heat rather than emitted as light. Given that N-LCDs demonstrate minimal light emission upon excitation, it is likely that nearly all the energy absorbed is converted into heat through nonradiative transitions. The heat generated during this process leads to an increase in the local temperature surrounding the N-LCD particles, which can be effectively utilized for cancer PTT. To further validate the biosafety and cytocompatibility of N-LCDs, their cytotoxic effects were evaluated through in vitro studies.

2.4. In Vitro Studies of N-LCDs

The cytotoxic effect of the synthesized N-LCDs was investigated on HEK 293 cells by performing an MTT assay. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is a tetrazolium salt that gets reduced with mitochondrial oxidoreductase enzyme of living cells and forms a yellowish formazan crystal and then solubilizes with DMSO and turns into a purple color solution which indicates the population of live cells. The in vitro cytotoxic effects were analyzed for N-LCDs with HEK 293 (normal cells). Different concentrations of N-LCDs (100 μg/mL, 200 μg/mL, 300 μg/mL, 500 μg/mL, 700 μg/mL, 1000 μg/mL, and 2000 μg/mL) were added to the HEK 293 cell line and incubated for 24 h.

Experimental data (Figure ) confirm that the synthesized N-LCDs exhibit no toxic effects, even at very high concentrations. Their nontoxic and biocompatible nature can be attributed to naturally occurring hydrophilic functional groups, such as hydroxyl (−OH), carboxyl (−COOH), and amine (−NH2), which enhance water solubility and minimize cytotoxicity. Additionally, their small size (∼2.5 nm) and biodegradable nature facilitate efficient cellular uptake. These findings establish the safety and suitability of N-LCDs for bioimaging and photothermal therapy applications.

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In vitro cell viability analysis using MTT assay of N-LCDs on HEK 293 cells after incubation of 24 h.

Furthermore, as bioderived nanomaterials with intrinsic autofluorescence properties, N-LCDs are particularly well-suited for bioimaging. Their cellular uptake was analyzed using the A375 cell line, further supporting their potential in biomedical applications.

The fluorescence signal of the synthesized N-LCDs can be monitored in real-time internalization into the cell through the cell membrane and imaged. 200 μg/mL synthesized N-LCDs were incubated to analyze the uptake into the cells. The green fluorescence signal of the N-LCDs (525 nm) was imaged, which ensured the internalization of the N-LCDs through the plasma membrane as the clear fluorescence images of N-LCDs internalized cells with confocal laser microscopy were observed. These studies (Figure a,b) reveal that after 6 h of incubation, no signal was detected in the control cells, while N-LCD-treated cells exhibited a green emission, likely originating from the N-LCDs internalized by the cells. This confirms the potential of N-LCDs as a bioimaging agent.

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Bioimaging: Confocal images of cellular uptake of the N-LCDs. (a) Control and (b) N-LCD treatment after 24 h of incubation in A375 cells.

Additionally, to further confirm the cellular uptake of N-LCDs, we incubated cells with N-LCDs and analyzed their internalization using flow cytometry. The results show that the fluorescence intensity of cells treated with N-LCDs significantly increased in comparison to the untreated control, indicating efficient internalization of the N-LCDs (Figure S6, Supporting Information). This suggests that N-LCDs were successfully taken up by cells in a dose-dependent manner. The uptake was observed to increase over time. The observed uptake pattern suggests that the N-LCDs may enter the cells via an endocytosis/phagocytosis mechanism, as indicated by previous studies. These findings are consistent with the ability of N-LCDs to penetrate cell membranes, which is essential to their potential use in targeted delivery systems for cancer therapy.

The synthesized N-LCDs exhibit strong absorption in the NIR region and a significant temperature rise under 808 nm laser irradiation, demonstrating their potential to kill cancer cells, as carcinomas are highly susceptible to elevated temperatures. The photothermal cell-killing ability of the N-LCDs was evaluated by using A375 cells.

Various concentrations of N-LCDs (100 μg/mL, 200 μg/mL, 300 μg/mL, 500 μg/mL, 700 μg/mL, 900 μg/mL, and 1000 μg/mL) were incubated with A375 cell lines. After 6 h of incubation, one set of cells was exposed to an 808 nm laser (1 W/cm2) for 5 min, while another set of cells with the same N-LCD concentrations received no laser exposure. As shown in Figure , N-LCDs exhibited dose-dependent cytotoxicity. In the absence of laser, cell viability gradually decreased with increasing concentration but remained above 50% even at 1000 μg/mL. However, laser irradiation significantly enhanced cytotoxic effects at higher concentrations wherein MTT assays revealed that N-LCDs exhibited a half-maximal inhibitory concentration (IC50) of 403.88 μg/mL. Statistical analysis revealed that laser-treated groups showed significantly lower cell viability compared to nonirradiated groups starting at 300 μg/mL (p < 0.01), with highly significant reductions at 500 μg/mL and above (p < 0.001). These results confirm the synergistic photothermal cytotoxicity of N-LCDs under NIR exposure confirming the high efficiency of N-LCDs in phototherapeutic applications. Additionally, a live/dead analysis was performed to visually evaluate the therapeutic effect by distinguishing between co-stained live and dead cells.

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In vitro cell viability analysis using MTT assay of N-LCDs A375 cells after incubation for 6 h at varying concentrations with laser and no laser (1 W/cm2, 808 nm) exposure for 5 min. Data represent mean ± SD from triplicate experiments. Statistical significance was determined using Student’s t-test comparing laser and no laser groups at each concentration. p < 0.01 (**), p < 0.001 (***).

The live–dead assay was conducted to confirm cell death induced by the photothermal effect. Live cells, stained with glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) dye, emitted a blue signal, while dead cells, stained with propidium iodide (PI) dye, showed a red signal. As shown in Figure a, control samples exhibited a prominent population of live cells (blue signal) after laser exposure. In contrast, N-LCD-treated samples (Figure b) displayed a significant reduction in the blue signal and a prominent red signal, confirming that cell death is caused by the photothermal effect. Additionally, strong green fluorescence was observed in N-LCD-treated cells, attributed to their efficient cellular uptake. This intense uptake led to a higher heat generation during laser exposure, causing substantial cellular damage. The apparent decrease in live cancer cells and the emergence of dead cells after treatment highlight the anticancer effect of the N-LCDs. To further elucidate the mechanism of cell death, apoptosis studies were performed.

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Confocal image of live and dead cell analysis after treatment with N-LCDs followed by NIR laser treatment on A375 cells for 6 h. Cells were stained with GF-AFC and PI dye. (a) Control cells and (b) N-LCD-treated cells.

The mechanism of cell death induced by the photothermal effect of N-LCDs was analyzed using Annexin V-fluorescein isothiocyanate (FITC) and PI staining. Annexin V-FITC binds to apoptotic cells, while PI stains dead cells. Following laser exposure, cells treated with N-LCDs exhibited apoptotic cell death due to the temperature rise. The photothermal effect caused by NIR laser exposure and N-LCDs led to damage to intracellular membrane proteins, potentially disrupting cell regulatory pathways and triggering programmed cell death (Figure a,b). Additionally, Annexin V-FITC-stained apoptotic cells were prominently observed in the N-LCD-treated samples. Red fluorescence from PI was detected within the green signal of Annexin V-stained phosphatidylserine (PS) moieties, confirming that all of the dead cells underwent apoptosis as a result of NIR laser exposure.

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Apoptosis analysis; confocal images showing apoptotic cell death after treatment with N-LCDs followed by laser exposure on A375 cells for 6 h. Cells were stained with Annexin V dye. (a) Control cells and (b) N-LCD treated cells.

To further validate the apoptotic effect of N-LCDs upon NIR laser exposure, we conducted flow cytometry using Annexin V-FITC/PI dual staining. While CLSM had already indicated that N-LCDs, in combination with an NIR laser (1 W, 808 nm), can induce apoptosis, flow cytometry was performed to quantitatively assess the extent of apoptotic cell death. Annexin V-FITC selectively binds to phosphatidylserine residues that translocate to the outer leaflet of the plasma membrane during early apoptosis, which is detected in the green fluorescence channel. In the absence of a NIR laser, cells treated with N-LCDs alone exhibited a minimal apoptotic population of 16.4%, indicating relatively low cytotoxicity under dark conditions. However, upon NIR laser irradiation, the apoptotic cell population increased drastically to 58.2%, confirming that photothermal activation significantly enhances cell death. This represents a 3.82-fold increase in apoptosis compared to N-LCDs treatment without laser exposure (Figure S7, Supporting Information).

These findings demonstrate that N-LCDs can act as effective photothermal agents, triggering apoptosis through hyperthermia upon NIR irradiation. The substantial increase in the number of apoptotic cells upon laser activation reinforces the potential of N-LCDs for targeted photothermal therapy applications.

3. Conclusions

In summary, N-LCDs were successfully synthesized using lignin as the carbon precursor and EDA as the nitrogen dopant. These N-LCDs exhibited strong NIR absorption and excellent photothermal performance under 808 nm laser irradiation (1 W/cm2), which is attributed to their high nitrogen content and favorable electronic structure. The N-LCDs also demonstrated remarkable stability under different conditions and across repeated laser on/off cycles, supporting their suitability for biomedical applications.

In vitro studies confirmed that N-LCDs possess a strong green fluorescence and are readily internalized by cancer cells, enabling effective imaging. Flow cytometry revealed that NIR laser-irradiated N-LCDs induced substantial apoptotic cell death, showing a 3.82-fold increase in apoptosis compared to nonirradiated conditions. Cytocompatibility assays further confirmed their low toxicity and high biocompatibility, even at elevated concentrations in the absence of laser exposure.

Taken together, these results demonstrate that the synthesized N-LCDs are promising candidates for safe, fluorescent, and efficient photothermal therapy. Furthermore, the use of lignina low-cost, renewable biomassas a carbon source supports a sustainable and scalable synthetic approach. Given their biocompatibility, optical functionality, and green synthesis route, this work provides a strong platform for future in vivo studies and the clinical translation of lignin-derived carbon-based nanotherapeutics in cancer treatment.

4. Experimental Section

4.1. Photothermal Performance Studies of N-LCDs

To evaluate the photothermal performance of N-LCDs, samples were diluted to varying concentrations (0–1000 μg/mL), transferred into quartz cuvettes, and irradiated with an 808 nm laser at a power density of 1 W/cm2 for 10 min. Temperature changes were monitored using an infrared thermal camera (FLIR TG267) at 30 s intervals. To assess the impact of different laser power densities (0.4–1.4 W/cm2), a fixed N-LCD concentration of 500 μg/mL was used, with water serving as the control (0 μg/mL). Additionally, photothermal stability was tested by repeatedly heating and cooling the 500 μg/mL dispersion with an 808 nm NIR laser at 1.0 W/cm2 for six cycles, confirming the material’s stability and reliability.

4.2. Cellular Uptake and Bioimaging

The cellular uptake of N-LCDs was investigated using human melanoma cell line A375. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in T25 culture flasks until ∼70% confluency was achieved. For imaging studies, 7,000 cells per well were seeded in confocal wells and incubated for 24 h, followed by treatment with N-LCDs (200 μg/mL) for an additional 6 h. After incubation, the cells were washed thrice with 1× Dulbecco’s Phosphate-Buffered Saline (DPBS) to remove any noninternalized N-LCDs and then imaged using a confocal laser scanning microscope (40× magnification) to confirm intracellular localization.

For the quantitative assessment of cellular uptake, flow cytometry analysis was also performed. A375 cells were incubated with N-LCDs (200 μg/mL) for 6 and 12 h. Postincubation, cells were washed thoroughly with PBS to eliminate unbound N-LCDs, resuspended in PBS, and analyzed using the green fluorescence channel of the flow cytometer. The fluorescence intensity directly correlated with the level of the internalized N-LCDs.

4.3. In Vitro Thermal Ablation with NIR Laser

The photothermal therapeutic efficacy of the synthesized N-LCDs was assessed by using the A375 human melanoma cell line via the MTT assay. Cells (1 × 104 per well) were seeded in 96-well plates and incubated for 24 h under standard culture conditions. Subsequently, cells were treated with varying concentrations of N-LCDs and incubated for 6 h. Post-treatment, one group of cells was exposed to an 808 nm NIR laser (1 W/cm2) for 5 min, while another set was kept as a control, receiving only N-LCD treatment without laser exposure. Following irradiation, a 0.25% (w/v) MTT solution was added to each well and incubated for 3 h to allow the formation of formazan crystals. The MTT solution was then discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. Absorbance was measured at 590 nm using a microplate reader. The percentage of cell death was calculated by comparing the absorbance values of laser-exposed and nonexposed samples, highlighting the enhanced cytotoxicity of N-LCDs under NIR laser exposure.

4.4. Live–Dead Analysis

The viability of A375 melanoma cells following treatment with synthesized N-LCDs and NIR laser irradiation was analyzed by using confocal laser microscopy. Cells (1.5 × 104 per well) were seeded in confocal wells and cultured under optimal conditions for 24 h. The cells were then treated with N-LCDs (500 μg/mL) and incubated for 6 h. Following incubation, the treated wells were exposed to an 808 nm NIR laser (1.0 W/cm2) for 5 min. To differentiate live and dead cells, GF-AFC (10 μg/mL) and PI (5 μg/mL) dyes were added. After a 20 min incubation, the stained cells were visualized using confocal laser microscopy at 40× magnification, confirming the photothermal therapeutic effect of N-LCDs.

4.5. Annexin V-FITC/PI Staining

The mechanism of cell death induced by N-LCDs and NIR laser treatment was investigated by using an Annexin V-fluorescein isothiocyanate (FITC) assay. Annexin V-FITC specifically binds to phosphatidylserine (PS) moieties, which translocate to the outer membrane in apoptotic cells, emitting green fluorescence (488 and 525 nm) upon FITC conjugation. To conduct the experiment, A375 melanoma cells (1.5 × 104 per well) were seeded in confocal wells and cultured under optimal conditions until confluency. The cells were then treated with N-LCDs and incubated for 6 h before being exposed to an 808 nm NIR laser (1 W/cm2) for 5 min. Following treatment, the cells were stained with Annexin V-FITC and PI to distinguish apoptotic and necrotic populations, and fluorescence imaging was performed by using confocal laser microscopy. This study confirmed the apoptotic nature of cell death induced by N-LCD-based photothermal therapy.

The apoptotic cell deaths that occurred due to treatment with N-LCDs followed by NIR laser exposure were also analyzed by flow cytometry for which 50,000 cells/well were seeded in a 24-well plate. After attaining confluency, the cells were treated with N-LCDs (IC50 concentration) and later incubated under similar conditions as above. Cells were then harvested and incubated with Annexin V-FITC (5 μg/mL) and propidium iodide dye (5 μg/mL) for 20 min. The samples were analyzed by flow cytometry to quantify the percentage of apoptotic cells by using the FITC and PE channel. The results were compared to those of control groups (untreated cells) to evaluate the apoptotic potential of the N-LCDs.

4.6. Statistical Analysis

All cell-based data were collected from three independent experiments (n = 3), and results are presented as the mean ± standard deviation. For comparison between two groups (e.g., laser vs no laser), a two-tailed unpaired Student’s t-test was used.

Supplementary Material

ao5c02344_si_001.pdf (968.7KB, pdf)

Acknowledgments

S.M.M. gratefully acknowledges A.J. for his help in conducting biostudies. V.S. would like to thank UGC, New Delhi, for the research fellowship. B.S. thanks the Ministry of Education for the fellowship. K.N. thanks the DBT-RA program in Biotechnology and Life Sciences (DBTHRDPMU/DBT-RA/2022-23/EXT/59). S.M.M. thanks NTTM, Ministry of Textile, New Delhi, India, and IIT Indore for financial support. The authors gratefully acknowledge the Sophisticated Instrumentation Centre (SIC), IIT Indore, for all the characterization facilities, MNIT-MRC for the Raman facility, AMRC-IIT Mandi for the XPS facility, and IISER Bhopal for flow cytometry (FACS) facility. V.S. is also thankful to Ravindra Vishwakarma and Dr. Tridib K. Sarma, Department of Chemistry, IIT Indore, for providing a zetasizer facility.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02344.

  • Instruments; materials; synthesis of N-LCDs; calculation of quantum yield; photothermal conversion efficiency; in vitro cytotoxicity; DLS of N-LCDs showing a hydrodynamic diameter of 186.6 nm and polydispersity index (PDI) of 0.117; zeta potential measurement of N-LCDs; full-survey XPS scan of N-LCDs; plot (Tauc plot) of (αhν)2 versus photoenergy (hν) for N-LCDs; stability profile of N-LCDs under different physicochemical conditions: relative FL intensity (%); flow cytometry histograms showing cellular uptake of N-LCDs; flow cytometric analysis of apoptosis by Annexin V/PI staining; and comparison table of photothermal conditions and efficiency between N-LCDs and other CDs (PDF)

The manuscript was written with the contributions of all the authors. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

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