Amplification of luminescence intensity by ytterbium(III) dopant in upconversion nanoparticles integrated with carbon dots for NIR-responsive targeted photodynamic therapy

Abstract

Photodynamic therapy (PDT) is one of the promising fields for cancer treatment, demonstrating precise and significant therapeutic outcomes. Despite their widespread use, conventional photosensitizers show significant limitations and suboptimal integration with other systems for PDT application. Furthermore, most of them require multistep synthesis procedures. To resolve these limitations, it is essential to develop simple methods to prepare more efficient materials for photodynamic therapy. In this work, we report the synthesis of a carbon dot-conjugated upconversion system (UCNP@CDs) via an in situ co-carbonization method, which gets activated under a dual-mode laser (980 nm and 660 nm) for enhanced photodynamic therapy. The whole system works based on the Förster resonance energy transfer (FRET) mechanism, where UCNPs are activated by a 980 nm laser and this energy is transferred to the carbon dots, which in turn behave as a photosensitizer to produce ¹O₂ with 72% cell mortality at a concentration of 100 μg mL⁻¹. Besides their photosensitizing property, the synthesized carbon dots are derived from folic acid and p- phenylenediamine to achieve active targetability towards cancer cells and exhibit high biocompatibility and water dispersibility. Notably, an in vitro study confirmed that the synthesized nanohybrid targeted the cytoplasm of cancer cells and exhibited considerably pronounced cytotoxicity in the presence of laser irradiation. Therefore, the results of this work demonstrate that the designed nanohybrid has great potential in cancer treatment.

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1. Introduction

Cancer has become a major public health issue, ranking as the second leading cause of mortality worldwide, after heart disease.^1,2 Global demographic characteristics predict an increasing trend in the number of cancer cases in the upcoming decades, and by 2025, more than 20 million fresh cancer cases are expected.^3–5 Conventional treatments against malignant tumors are limited by various causes such as cellular metastasis, heterogeneity, and their resilience towards radiotherapy and chemotherapy.⁶ It is very encouraging that there has been a major methodical change in the landscape of tumor treatment in recent times to resolve these limitations. Current methods such as stem cell therapy, targeted therapy with small molecule inhibitors and monoclonal antibodies, and photothermal/photodynamic therapy in oncology focus on the betterment of secure and efficient cancer treatment.^7,8 Among all the methods, photodynamic therapy has attracted attention as a promising approach due to its advantages such as non-invasive nature, tumor-specific selectivity, low systematic toxicity and good patient tolerance effects.⁹

PDT involves the presence of a light-activated photosensitizer (PS) and triplet oxygen (³O₂). Depending upon the type of reaction, PSs can convert triplet oxygen into two different types of reactive oxygen species (ROS), i.e. superoxide radical (O₂˙⁻) and hydroxyl radicals (˙OH) in type I and cytotoxic singlet oxygen (¹O₂) in type II reactions.^10,11 Conventional organic photosensitizers face several limitations, such as poor water solubility, skin photosensitivity, and toxic effects on healthy tissues, which hinder their broad clinical use.¹² To date, numerous PSs have been reported for clinical trials, which include some transition-metal coordination complexes and organic-fluorophores like BODIPY, xanthenes, and naphthalimides for their photodynamic applications.^11,13 Recently, as an emerging nanomaterial, carbon dots have been designed as photosensitizers for PDT applications, with the advantages of good water solubility, facile modifications, good biocompatibility, easy preparation, cost-effectiveness, and photo stability.^14–18 The nanoenzymatic activity of carbon dots for efficient PDT applications has been explored.^16,19,20 Zhang et al. synthesized three types of RCDs having tunable quantum yields by changing the reactive solvents for type I and type II ROS generation in the presence of a 635 nm laser to enhance the PDT effect.²¹ Most of the carbon dots (photosensitizers) are activated in the UV-visible light region, which has limited penetration depth and detrimental effects on tissue, leading to inefficient outcomes on internal or larger tumor sites. Hence, in terms of laser irradiation, near-infrared (NIR) light is more suitable compared to UV-visible light for PDT application to overcome these challenges. Notably, the NIR window (700–1000 nm) is considered the “optical tissue penetration window”. It offers the lowest photodamage to cells and higher penetration depth (e.g., <10 cm) compared to UV or Vis light (e.g., <1 cm).^22–25 Hence, in recent times, researchers have been focused on the development of NIR-activated carbon dots due to their high applicability in phototherapy.^26–29 However, there has been limited work in this particular area, which highlights the need for the synthesis of more biocompatible dots with enhanced energy transfer capability.

Recently, upconversion nanoparticles (UCNPs) have been reported to act as a modulator and can covert high tissue-penetrating NIR to visible light for activating PSs via an energy transfer mechanism and generating ROS to kill cancer cells.^30–33 Upconversion nanoparticles (UCNPs) having lanthanides are known as a promising new type of luminescent bioprobe material having anti-Stokes emission characteristics under the activation of low intensity light, showing extraordinary benefits in targeted bioimaging, photocatalysis and therapeutics.^34–37 UCNP-based nanocomposites frequently maintain the exceptional photoluminescence characteristics of UCNPs, while having other changeable properties of several functional materials. The diagnosis and treatment of malignant tumors can be achieved by combining different therapeutic agents (chemotherapy agents, photodynamic agents, photothermal agents, bioimaging contrast agents, etc.).³⁸ Tang et al. constructed a switchable DNA/UCNP nanoplatform with the conjugation of the photosensitizer chlorin e6 (Ce6) to generate ROS (¹O₂) and achieve successful photodynamic therapy under 980 nm NIR light excitation, showing new approaches for specific targeting and highly competent cancer therapy.³⁹ Shi et al. developed a new NIR light-active UCNP@Al(OH)₃/Au nanohybrid for UCL imaging and synergistic-targeted PTT effect.⁴⁰ Zhang et al. proposed a suitable mitochondria-targeted nanocomposite via the covalent attachment of UCNPs and GQD for effective PDT therapy. However, the main disadvantages of this system are its multistep synthesis procedure, depleted loading percentage of GQDs through EDC-NHS chemistry, and high power irradiation.⁴¹ Thus, to eliminate these disadvantages, we have synthesized a one-step UCNP@CD nanohybrid with efficient inhibition of the growth of tumor cells after low-intensity light irradiation. Considering the above-mentioned advantages and disadvantages, the combined nanohybrid of UCNPs and CDs could achieve PDT with higher efficiency and is likely to be used in many methods due to its strong adaptability and high performance.

Herein, we have designed a upconversion nanohybrid system containing carbon dots and UCNPs through an in situ co-carbonization method for the generation of type II PDT triggered by 980 nm NIR light (Scheme 1). Our synthesized nanohybrid has built-in targetability phenomena, thus avoiding the need for tedious reaction procedures for surface functionalization, and simultaneously avoiding surface passivation, thus reducing the effective radius of the nanohybrid for cellular uptake. In the presence of NIR light irradiation, UCNPs converted NIR light into visible light, which could activate the CDs to produce sufficient ROS for the PDT effect. In addition, the biological compatibility of the synthesized dual-mode-activated nanohybrid and cytotoxicity against tumor cells were studied systematically in in vitro experiments.

Scheme 1 Schematic of the preparation of dual-mode-activated UCNP@CD nanohybrid for PDT.

2. Materials and methods

2.1. Materials

Bismuth nitrate pentahydrate (Bi(NO₃)₃·5H₂O), >99%, potassium hydroxide and polyvinylpyrrolidone K30 (PVP K-30) were purchased from CDH Fine Chemical. Lanthanide nitrates (Yb(NO₃)₃·5H₂O and Er(NO₃)₃·5H₂O), ammonium fluoride (NH₄F) and p-phenylenediamine were obtained from Alfa Aesar. Folic acid (from TCI Chemicals) and ethylene glycol (from Finar Chemicals) were also used.

Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Thermo Fisher Scientific, USA). Reagents including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent were procured from Invitrogen (Thermo Fischer Scientific, USA). Calcein-AM/EthD-III was purchased from Biotium (Fremont CA, US). Additional reagents, including di-methyl sulphoxide (DMSO), and Triton-X, were obtained from HiMedia (Mumbai, India). 2′,7′-Dichlorofluorescence diacetate (DCFH-DA) was purchased from Thermo Fisher. The reagents listed above were employed in the cell culture-based experiments. Ultrapure water obtained from a Millipore system was utilized throughout the experiments. Unless otherwise specified, all commercially available materials and chemicals were utilized directly as received without any further purification.

2.2. Synthesis of UCNPs

In brief, PVP/KBF:Yb (20%), Er(3%) were first prepared according to a previous study.⁴² 0.96 mmol (0.46 g) Bi(NO₃)₃·5H₂O, 0.20 mmol Yb(NO₃)₃·5H₂O (0.135 g), and 0.03 mmol Er(NO₃)₃·5H₂O (0.013 g) were simultaneously dissolved in 10 mL of ethylene glycol in a 50 mL round-bottom flask. Then, a homogeneous solution was formed after the addition of polyvinylpyrrolidone K30 (0.5560 g) at 80 °C. Then, another mixture of 6 mmol NH₄F with 1 mmol KOH dissolved in 10 mL ethylene glycol at 80 °C was added dropwise to the main solution. Now, the solution was magnetically stirred for 10 minutes and the temperature was set at 160 °C for 2 h, followed by cooling. Afterward, the precipitate was extracted through centrifugation at 3000 rpm for 5 minutes and the precipitate was washed with ethanol. Finally, drying of UCNPs was carried out by keeping the precipitate overnight in an oven at 65 °C. Similarly, in the case of the preparation of UCNPs with 25% and 30% Yb³⁺ concentration, the same procedure was followed.

2.3. Synthesis of UCNP@CDs nanohybrid

The UCNP@CD nanohybrid was synthesized according to a previous procedure.⁴³ Typically, folic acid (0.353 g) and p-phenylenediamine (1.5 g) were well dispersed in water (20 mL). Then, 50 mg of pre-synthesized UCNPs (30% Yb³⁺) was dissolved in water (3 mL) and added to the above-mentioned solution. Afterward, the resulting solution was subjected to magnetic stirring for an extra 15 minutes. Subsequently, the solution was subjected to hydrothermal treatment at 200 °C for 10 h, followed by centrifugation at 8000 rpm for 10 min. Finally, the crude product was washed three times with water and ethanol to obtain UCNP@CDs and dried at 65 °C for 12 h.

2.4. Characterization of nanoparticles

To study the morphology of UCNPs and UCNP@CDs, a concentration of 100 μg mL⁻¹ sample was added to a copper mesh. Transmission electron microscopy (TEM) was employed to obtain the images. The crystallinity of particular systems was detected using an X-ray diffractometer (XRD). The chemical compositions of CDs, UCNPs, and UCNP@CDs were evaluated by Fourier transform infrared spectroscopy (FTIR).

2.5 Cell culture conditions

The HeLa and HEK293 cell lines, obtained from the National Centre for Cell Science (NCCS), Pune, India, were selected for the study. Cells were cultured in DMEM supplemented with 4.5 g L⁻¹ glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg L⁻¹ sodium bicarbonate. The complete medium was prepared by adding 10% FBS, penicillin (100 units mL⁻¹), and streptomycin (100 μg mL⁻¹). Cultures were maintained in a CellXpert C170 incubator (Eppendorf, Germany) at 37 °C under a 5% CO₂ atmosphere.

2.6. Extracellular ROS detection

DCFH-DA (2′,7′-dichlorofluorescence diacetate) was used as the fluorescence probe to detect the generation of extracellular singlet oxygen in aqueous solution. The UCNP@CD nanohybrid was dispersed in an aqueous solution containing DCFH-DA (dissolved in ethanol, 25 μM) and subjected to dual irradiation of 980 nm and 660 nm from an NIR laser, respectively. The generation of singlet oxygen was monitored by the increase in the fluorescence emission spectrum at 540 nm at different intervals with excitation at 494 nm.

2.7. In vitro cytotoxicity assay of UCNP@CDs by MTT assay

Initially, the samples were sterilized by UV treatment for 6 h. Following four passages, both HeLa and HEK cells were seeded in a 96-well plate at a density of 1 × 10⁴ per well and incubated in a humidified atmosphere at 37 °C containing 5% CO₂ for 24 h. UCNP@CDs were dispersed in PBS and introduced into each well at varying concentrations of 0, 50, 75, and 100 μg mL⁻¹. Then, the cells were incubated with UCNP@CDs for 24 h to check the sample toxicity at the given concentrations under normal physiological conditions using both normal and cancerous cell lines. Following the designated incubation period, the medium in each well was replaced with fresh medium, and MTT reagent was added to achieve a final concentration of 0.5 mg mL⁻¹. The plates were then incubated for 4 h at 37 °C. After incubation, the medium was carefully aspirated and the formazan reaction product was dissolved in 150 μL of DMSO, followed by 15 min incubation at 37 °C with intermittent shaking. Subsequently, the absorbance was monitored at 570 nm using a Multiskan™ FC Microplate Reader (Thermo Scientific). All experiments were conducted in triplicate to determine the mean and standard deviation.

2.8. In vitro cellular uptake study

The progressive uptake study of UCNP@CDs was done on HeLa cells. Primarily, poly-D-lysine-coated glass coverslips were taken for the culture of HeLa cells at a count of 25 × 10³ cells per well and incubated for 2 days (48 h) to ensure adhesion. Afterward, the cells were exposed to 100 μg mL⁻¹ of the UCNP@CD nanohybrid, and then incubated for different durations of 2, 4, and 8 h at 37 °C. Subsequently, the cells were treated with 4% formaldehyde for 10 min at room temperature after each incubation period. Then, the cells were washed with PBS to affix the coverslips onto glass slides in the presence of Fluoromount-G mounting medium, which contained DAPI for nuclei labeling. Finally, the uptake study was analyzed with a Leica fluorescence microscope.

2.9. In vitro PDT effect

To evaluate the therapeutic effect of UCNP@CDs, HeLa cells were seeded into 96-well plates and exposed to varying concentrations of UCNP@CDs (0, 50, 75, and 100 μg mL⁻¹) for 12 h. The cells were then divided into two groups, where one group received laser treatment at 980 nm (0.4 W cm⁻²) for 10 min, while the other did not. Following 12 h incubation at 37 °C, the standard MTT assay was carried out to assess the cell viability. Relative cell viability (%) was calculated by comparing the absorbance of the treated wells with that of the untreated control wells, which were set at 100% viability. The absorbance values from the treated wells were normalized to the control to determine the cell viability under the experimental conditions. The results from three independent experiments were averaged, and the mean ± standard deviation was presented in a bar graph.

In the live/dead cell imaging assay, HeLa cells were seeded at a density of 1 × 10⁴ cells per well into 8-well chamber slides and incubated for 24 h. Following this initial incubation, the cells were treated with UCNPs and UCNP@CDs for an additional 12 h. The treated cells were divided into two groups, where one group was kept in the dark, while the other was irradiated with a 980 nm laser (0.4 W cm⁻²) for 10 min. The cells were then washed with PBS and incubated simultaneously with 2 μM calcein AM and 4 μM EthD-III staining solutions for 30–45 min at room temperature. The cells were incubated with the staining solution. Post incubation, the staining solution was aspirated and fresh PBS was introduced into the wells before imaging. Fluorescence imaging was conducted using a Leica DMi8 epi-fluorescence microscope equipped with the LAS-X software and a 20× objective lens. Calcein was visualized using an FITC filter set, and EthD-III was imaged using a TexasRed® filter.

2.10. ROS detection in living cells

ROS production was assessed using DCFH-DA. HeLa cells were first incubated overnight in 8-well chamber slides. After the initial incubation, the cells were subjected to treatment with 100 μg mL⁻¹ of UCNP@CDs and co-incubated at 37 °C with 5% CO₂ for an additional 12 h. Then, the cells were washed with PBS twice or thrice, followed by incubation with 5 μM DCFH-DA for 20 min. Post incubation, the cells were washed twice or thrice using PBS, followed by irradiation of the wells with a 980 nm laser (0.4 W cm⁻²) for 10 min. In addition, the above-mentioned procedure was also carried out in dark conditions without laser irradiation to evaluate the level of toxicity in both the radiated and irradiated treatment groups. The intense green fluorescence was captured by a Leica DMi8 epi-fluorescence microscope using the LAS-X software with a 20× objective lens.

2.11. Hemolysis assays

Blood samples were collected and immediately centrifuged at 1700 rpm for 5 min. The supernatant was aspirated, followed by the addition of 2 mL of PBS (pH 7). A similar step was repeated thrice or until the supernatant became clear. Following aspiration, the remaining pellet was suspended in PBS (pH 7) in the ratio of 1 : 100 to obtain a 1% erythrocyte suspension. Both PBS and 10% Triton-X-100 and PBS were treated as the negative and positive controls, respectively. Subsequent experiments were set up in the tubes by mixing 50 μL of the test compound, PBS, or Triton-X-100 with 50 μL of the 1% erythrocyte suspension. The samples were allowed to incubate for 1 h at 37 °C. Following incubation, the tubes were centrifuged at 1700 rpm for 5 min. After centrifugation, 60 μL of the supernatant was transferred to transparent, flat-bottom 96-well plates. Absorbance measurement was performed at 405 nm using a Multiskan™ FC Microplate Reader (Thermo Scientific). The following equation was used to calculate the hemolysis rate.⁴⁴
where A_s denotes the absorbance of the samples, while A_n and A_p represent the absorbance of the negative and positive controls, respectively.

3. Results and discussion

3.1. Preparation and characterization of UCNP@CDs

The preparation of UCNP@CDs is described in Scheme 1. Briefly, UCNPs were mixed with the CD precursor, followed by a feasible solvothermal treatment in which electrostatic interactions are responsible for the growth of CDs on the surface of UCNPs. The UCNPs were prepared according to a previous study.⁴² The morphologies and particle size of UCNPs and UCNP@CDs were characterized by SEM and TEM (Fig. 1). Here, the growth of carbon dots over UCNPs over a particular interval is observed. The TEM images depict the nucleation of carbon dots on the surface of the PVP-layered UCNPs at different intervals. After 4 h of reaction (Fig. 1b), there are no structural changes in the UCNPs, with only a few carbon dots attached to the surface. Upon completion of the reaction after 8 h, the morphology of the UCNPs changed due to the attachment of a higher amount of carbon dots (Fig. 1c). The possible underlying mechanism suggested the formation of CDs on the surface of UCNPs due to the co-carbonization of the PVP-layered UCNPs with the carbon dot precursors. According to Fig. 1d and Fig. S1, the synthesized UCNPs are irregular in shape with an average size of ∼70.819 nm and energy dispersive spectroscopy (EDS) mapping shows the distribution of different elements present in UCNPs (Fig. S2). Fig. 1e demonstrates that the morphology of UCNPs remained mainly unaffected after their modification with carbon dots, which is attributed to their small size. In addition, the HRTEM analysis (Fig. 1f) clearly indicates the purity and crystalline nature of the synthesized PVP/KBF nanoparticles by resolving the fringe lattices of the (111) crystallographic plane. The measured interplanar distance of 0.350 nm well matches the theoretical value for the cubic K_0.3Bi_0.7F_2.4 phase.⁴² The in vitro stability of the synthesized system was assessed, as shown in Fig. S3, based on the alteration in the particle size of UCNP@CDs in PBS buffer, FBS, and DMEM within different intervals. In addition, increasing the amount of Yb³⁺ from 20 to 30 mol% barely changed the shape and size of UCNPs, confirming that the morphology of the particles is independent of the Yb³⁺ concentration. The HR-TEM images also revealed that CDs were attached to the surface of the UCNPs, and after modification with CDs, the UCNP@CD nanohybrid showed no evident change in average diameter, and also acquired good dispersion in water.

Fig. 1 Transmission electron microscopy (TEM) images of (a) UCNPs, (b) UCNP@CDs after 4 h and (c) UCNP@CDs after 8 h. Scanning electron microscopy (SEM) images of (d) UCNPs, (e) UCNP@CDs after 8 h and (f) fringe pattern of UCNPs.

To confirm the formation of the desired nanostructure, its energy dispersive spectrum (EDS) was recorded, which depicts the characteristic peaks of the Bi, Yb, F, Er, and K elements of UCNPs as well as C, N, and O of CDs (Fig. S4). Thermogravimetric analysis was executed on the UCNP@CD nanohybrid to analyze the loading concentration of CDs on the surface of UCNPs and thermal stability of the system as well. According to the results, continuous weight loss was observed between 200 °C and 1000 °C, which indicated the attachment of CDs on the surface of UCNPs and the amount was determined to be 54.33% with respect to only UCNPs (Fig. 2a), whereas the carbon dots showed about 30% decay at 200 °C and a 60% decay at around 600 °C. This was attributed to fact that the weight loss of CDs at their synthesis temperature is possibly due to presence of free surface organic moieties, whereas the formation of the composite causes surface passivation of CDs, anchoring the organic moieties in the UCNP interface, and leading to high temperature durability.

Fig. 2 Thermogravimetric curves of UCNPs, CDs, and UCNP@CDs. (b ) FTIR spectra of UCNPs and UCNP@CDs. (c) XRD patterns of UCNP@CDs and UCNPs. (d) Zeta potential of UCNPs, CD solution and UCNP@CDs.

UCNPs and UCNP@CDs were further characterized by the Fourier transform infrared spectroscopy (FT-IR) technique. Fig. 2b exhibits that the presence of functional groups such as PVP and KOH could influence the chemical and physical characteristics of UCNPs. The absorption bands at 3371 and 1630 cm⁻¹ are attributed to the vibrations of the O–H and C=O groups; the absorption bands at 1434 and ∼1100 cm⁻¹ are assigned to the characteristic vibration peaks of the C–H and C–N bonds, respectively. After the formation of UCNP@CDs, the intensity of the peaks at 1630 and 1434 cm⁻¹ was reduced and the peak for the C–N bond diminished significantly, which was possibly due to the attachment of CDs on the surface of the UCNPs. This study showed that UCNP@CDs had been synthesized effectively. Moreover, the Raman spectrum of CDs was recorded to describe other structural information. Fig. S5 demonstrates two characteristic Raman bands at around 1365 cm⁻¹ and 1516 cm⁻¹ with the I_D/I_G value of 0.91, corresponding to the disordered (D band) and graphitized (G band) structure in CDs, respectively. According to the X-ray diffraction (XRD) pattern (Fig. 2c), it can be observed that the significant peaks of UCNPs well matched with the standard cubic K_0.3Bi_0.7F_2.4 (JCPDS no. 01-84-0534) and no peaks of subsequent phases are observed. When the UCNP@CDs were formed, the XRD pattern showed some significant new peaks, along with the UCNP peaks, confirming the synthesis of the nanohybrid. Moreover, zeta potential values were recorded to evaluate the potential difference of every nanoparticle formed. According to Fig. 2d, the zeta potential value decreased from +12.77 to +1.48 mV due to the presence of negatively charged carbon dots and this offered the further confirmation of the synthesis of UCNP@CDs.

Furthermore, we investigated the elemental composition on the surface of UCNP@CDs and CDs by X-ray photoelectron spectroscopy (XPS). The peaks of the characteristic elements Yb, Er, and Bi of UCNP@CDs and C, N, and O of CDs are observed in the XPS full spectrum (Fig. 3a and c). The co-carbonization mechanism between UCNPs and carbon dot precursors was confirmed by the high-resolution spectra of CDs and UCNP@CDs, separately. The deconvolution of the O 1s high-resolution spectra of CDs (Fig. 3d) exhibited two peaks at 532.2 eV and 531.2 eV, which suggested the presence of C=O and C–O bonds, respectively.⁴⁵ When CDs are attached on the surface of the UCNPs, two other peaks were observed at 530.4 eV and 529.6 eV in the O 1s spectra, along with the two peaks of C=O and C–O bonds. These two newly formed peaks can be credited to the presence of Bi–O and Yb–O (Fig. 3b), respectively,^46,47 which also confirmed the formation of UCNP@CDs.

Fig. 3 (a) XPS survey spectra and (b) deconvolution peak of O 1s of UCNP@CDs. (c) XPS survey spectra and (d) deconvolution peak of O 1s of CDs.

3.2. Optical properties

The optical properties of the nanohybrid were further studied (Fig. 4a). The absorption spectrum of UCNP@CDs showed an absorption band in the range of 450–660 nm compared to the UCNPs, which suggests the modification of CDs to form UCNPs. In addition, only CDs exhibit broad absorption bands within the same range as the UCNP@CD nanohybrid, as represented by their UV visible spectra (Fig. S6) and photoluminescence spectra (Fig. 4b) upon excitation at different wavelengths.

Fig. 4 (a ) Absorption spectra of UCNPs and UCNP@CDs. Inset: zoom of the absorption in the visible region. (b) Emission spectra of UCNP@CDs at different excitation wavelengths. (c) Upconversion emission spectra of UCNP at different dopant concentrations of Yb³⁺. (d) Upconversion emission spectra of UCNPs at different concentration loading of CDs.

The PL spectra clearly depict the fact that modification of CDs on the surface of UCNPs resulted in excitation-independent phenomena. Upon variation of the wavelength from 330 to 400 nm, the equivalent emission maximum was observed at around 452 nm, whereas in the case of only CDs, slightly excitation-dependent PL behavior was observed (Fig. S7).

3.3. Upconversion photoluminescence and energy transfer mechanism of UCNPs and UCNP@CDs

The upconversion luminescence characteristics of UCNPs were studied. In this work, we examine the upconverted light emission of UCNPs in detail, where KBF phosphors are co-doped with Er³⁺ and Yb³⁺ in different dopant concentration ratios. When the UCNPs are stimulated with a 980 nm laser, the upconversion luminescence spectra of all the samples mainly showed a slightly green emission band at 550 nm (²H_11/2/⁴S_3/2 → ⁴I_15/2) and a highly intense red emission centered at 654 nm (⁴F_9/2 → ⁴I_15/2) (Fig. S9).⁴²

A significant increase in the emission spectra was witnessed when the concentration of Yb³⁺ increased from 20% to 30% (Fig. 4c). An alteration in the emission was also observed when the concentration of carbon dots and the interval were varied. As shown in Fig. 4d, a change in the concentration of carbon dots led to less energy transfer from UCNPs to CDs. Hence, the emission did not decrease as much compared to higher concentrations of carbon dots. In terms of the growth of carbon dots with time, the emission was also altered (Fig. S8). Under 980 nm laser excitation, the electronic transition of the Yb³⁺ ions from the ground ²F_7/2 energy state to the higher excited ²F_5/2 energy state was observed. A portion of the population present in the respective excited state of Yb³⁺ ions transmits their energy to the nearest Er³⁺ ions and settles into the ground-state energy level. Now, activator ions (Er⁺³) are energized by absorbing a photon, and firstly, they move from the stable ground state ⁴I_15/2 to the higher excited state ⁴I_11/2. Then, the second photon may be absorbed by the excited state mechanism of absorption of the higher energy level ⁴I_11/2 of Er³⁺ ions, causing excitation to the next higher energy level ⁴F_7/2. An Er³⁺ ion is transferred to ⁴F_9/2 from ⁴I_13/2 by the second energy transfer step at the same time. Now, the whole energy transfer mechanism is processed between Yb³⁺ and Er³⁺, facilitating the excitation of Er³⁺ from its ground state of ⁴I_15/2 to the higher energy levels of ⁴I_11/2 and ⁴F_7/2. Consequently, non-radiative relaxation of Er³⁺ ions occurs, leading to energy transitions between ⁴S_3/2 to ⁴I_15/2, and resulting in a green emission centred at 544 nm, along with an intense red emission at 654 nm caused by the electronic transitions of Er³⁺ ions between ⁴F_9/2 to ⁴I_15/2.⁴⁸ An enhancement in the concentration of Yb³⁺ ions reduces the usual distance created between the Er³⁺ and Yb³⁺ ions, and as a result a higher cross-section area for photon absorption is acquired by Yb³⁺ ions under 980 nm irradiation. Moreover, the difference in the energy gap of the energy levels ⁴I_15/2 and ⁴I_11/2 followed by ⁴I_11/2 and ⁴F_7/2 of Er³⁺ perfectly resonates with the energy difference between the ²F_7/2 and ²F_5/2 energy levels of Yb³⁺, respectively. Therefore, a high Yb³⁺ doping concentration leads to a strong ET process towards the excited-state population. This phenomenon shows the regulation of the green and red emission of Er³⁺. Interestingly, a significant amplification in the red/green emission ratio is witnessed with an increase in the Yb³⁺ ion concentration. The population density of ⁴F_9/2 levels is responsible for generating red color, which is pushed through two probable mechanisms (Fig. S10). Firstly, two-photon excitation is observed from ⁴I_15/2 to ⁴I_11/2 (GSA), followed by non-radiative decay of ⁴I_11/2 to ⁴I_13/2, and afterward the electronic transition from ⁴I_13/2 to ⁴F_9/2via ESA2. In the other mechanism, cross-relaxation (CR), i.e. exchange of energy is observed in neighboring Er³⁺ ions through the transition of ⁴F_7/2 (Er³⁺) + ⁴I_11/2 (Er³⁺) → 2 ⁴F_9/2 (Er³⁺), considering the almost similar energy difference in the energy level of ⁴F_9/2–⁴I_11/2 (∼5080 cm⁻¹) and ⁴F_7/2–⁴F_9/2 (∼5140 cm⁻¹). However, the population density of the ⁴F_7/2 and ⁴I_11/2 energy levels is enriched constantly via different transition processes such as ET, GSA, and ESA1 and the population distribution of excited states (⁴F_9/2) is also stimulated.

As represented in Fig. 4d, it is evident that the photoluminescence intensity of UCNPs modified with CDs significantly decreased with different loading concentrations of CDs compared to only UCNPs. This observation demonstrates effective energy transfer in the heterogeneous structure of UCNP@CDs. Low-energy photons present in the near infrared spectrum can be generated by UCNPs to produce shorter wavelength emissions, which are absorbed by CDs through the FRET mechanism. The carbon dots on the surface of UCNPs are excited by red light, leading to an electronic transition from the ground state S₀ to a higher energy state S₁. Since the S₁ state is unstable, a non-radiative process known as intersystem crossing (ISC) occurs between the S₁ and triplet state T₁. The triplet state then releases its energy back to the ground level through a chemical de-excitation process. Meanwhile, this emitted energy is absorbed by molecular oxygen, resulting in the production of singlet oxygen (¹O₂) (Fig. 5). To further validate the FRET mechanism, the upconversion fluorescence lifetime of UCNPs and UCNP@CDs was studied. As depicted in Fig. S11, a higher decay of UCNPs (τ = 186.7 μs) is witnessed as compared to UCNP@CDs (τ = 91.3 μs), signifying a steady decrease in the fluorescence lifetime under 980 nm irradiation. This phenomenon highlights the effective energy transfer from UCNPs to CDs to alleviate ROS generation.

Fig. 5 FRET mechanism between UCNPs and CDs.

3.4. In vitro ROS detection

Here, the presence of CDs on the surface of UCNPs makes this nanosystem an ROS-generating agent. CDs can generate ¹O₂ under 660 nm light irradiation (Fig. S12). Therefore, in this UCNP@CD heterogeneous system, CDs can be activated effectively by absorbing light at 654 nm from the UCNPs and generating toxic ¹O₂ to improve photodynamic type II treatment.

We further investigated the capability of the UCNP@CD nanosystem to generate ¹O₂ in the presence of NIR light irradiation. 2′,7′-Dichlorodihydrofluorescein (DCFH-DA) was used as a probe to detect ¹O₂. DCFH-DA is non-fluorescent in nature and can be hydrolyzed in weak alkaline medium and converted to DCFH. Under laser irradiation in the dark, UCNP@CDs produced ¹O₂, which oxidized DCFH to green fluorescence DCF. The detection of ¹O₂ was confirmed by monitoring the intense characteristic fluorescence band of DCF centered at 540 nm under 490 nm excitation. Fig. S13 and S14 show the variation in the DCF fluorescence intensity observed in the solution of nanosystem with different illumination times under 980 nm and 660 nm laser irradiation. It is obvious that a significant increase in the DCF fluorescence intensity occurred with an extended irradiation time, which confirms the production of ¹O₂, but in the case of only UCNPs, no such change was observed (Fig. S15).

In addition to the DCFH-DA test, the confirmation of singlet oxygen (¹O₂) generation was done by electron spin resonance (ESR) spectroscopy, where 2,2,6,6-tetramethylpiperidine (TEMP) was used as a trapping agent. Fig. 6a exhibits the characteristic triple peak of ¹O₂ with a 1 : 1 : 1 ratio under 980 nm irradiation. This observation further demonstrated that UCNP@CDs produced a significant amount of ¹O₂via the energy transfer process.

Fig. 6 (a) EPR spectrum of UCNPs and UCNP@CDs under an NIR laser (λ = 980 nm) irradiation for 5 minutes. (b) In vitro cytotoxicity of HEK 293 and HeLa cells determined in the presence of UCNP@CDs. (c) HeLa cells treated with different concentrations of UCNP@CDs in the presence (980 nm laser) and absence of light. (d) Percentage hemolysis of different concentrations of UCNP@CDs. In vitro experiments were performed in triplicates (n = 3). (p*** < 0.001 compared to the respective treatments using two way Anova test by OriginPro 8.5).

The mechanism involved in the production of type I/II ROS is facilitated by the UCNP@CD composite in solution, and its performance regarding the production ROS in tumor cells was evaluated. Firstly, DCFH-DA was employed as the fluorescence probe with green emission for O₂˙⁻ and ¹O₂ recognition. The fluorescence microscopy results showed that all the HeLa cells treated with different concentrations of the sample emitted green fluorescence (Fig. 7). There was no significant fluorescence emission in the control group or UCNP@CDs under dark conditions. The results indicated the efficient intracellular generation of ROS by the synthesized nanohybrid under light irradiation.

Fig. 7 ¹O₂ generation evaluated in the presence of DCFH-DA: (a) DIC, (b) GFP, and (c) combined images for (i) control (L−), (ii) control (L+) and (iii) UCNP@CDs (L+) in HeLa cells (scale bar = 50 μm).

3.5. Cytotoxicity evaluation

The biocompatibility analysis of UCNP@CDs was conducted using both the HeLa and HEK293T cell lines. The MTT assay was initially performed for the cytotoxicity evaluation of UCNP@CDs under dark conditions. As presented in Fig. 6b, the HeLa and HEK293 cells were exposed to UCNP@CDs at 50–100 μg mL⁻¹ to detect the cell viability. The cells exhibited no significant signs of biological damage even at a UCNP@CD concentration as high as 100 μg mL⁻¹, indicating the extensive biocompatibility of the prepared product.

3.6. Time-dependent cellular uptake study

The in vitro cellular uptake study of UCNP@CDs was monitored qualitatively using fluorescence microscopy. The study was performed in the HeLa cell line, and uptake images were taken under 535 nm excitation wavelength, as shown in Fig. 8. The cells were plated in six-well tissue culture dishes at a density of 2 × 10⁵ cells per well and incubated for different durations (2 h, 4 h, and 8 h) after adding the sample. Here, nuclei labeling was done by DAPI (blue fluorescence), and the orange emission observed in the images was due to the localization of the UCNP@CDs in the cytoplasm. Combined images from both channels were given as required. As shown in the images, the intensity of the orange fluorescence signal from UCNP@CDs was observed to be initially weak, and the samples were attached to the surface of the HeLa cells after incubation for 2 h. This suggested that internalization started after 2 h. However, an enhancement in the fluorescence intensity over time was observed in the microscopy images, showing the steady uptake of the nanohybrid by the cancer cells. Complete internalization was observed after 8 h co-incubation. A comparative study of cellular uptake was also performed between the HEK293T cell line and the HeLa cell line, as given in Fig. S16. There was no significant cellular internalization of the synthesized nanohybrid UCNP@CDs in the normal HEK293T cell line due to the absence of folate receptors on the cell surface.

Fig. 8 Cellular uptake study of UCNP@CDs in HeLa Cells: (a) DIC, (b) DAPI, (c) UCNP@CDs and (d) combined frames at different intervals of (i) 0 h, (ii) 2 h, (iii) 4 h, and (iv) 8 h (scale bar: 50 μm).

3.7. In vitro PDT effect

The phototoxic effects of UCNP@CDs on cancer cells were investigated using the MTT assay at various concentrations under 980 nm laser irradiation. As illustrated in Fig. 6c, the survival rates of HeLa cells decreased with increasing concentrations of UCNP@CDs under light exposure, in contrast to the minimal effects observed under dark conditions. These findings highlight the cytotoxic effects of UCNP@CDs within the concentration range of 50 to 100 μg mL⁻¹, achieving approximately 72% cell mortality at a concentration of 100 μg mL⁻¹ compared to HEK 293 cells.

The PDT efficacy of the synthesized material was further assessed using calcein-AM and EthD-III double-staining of the cells (Fig. 9). Calcein-AM is a widely used fluorescent dye for labeling live cells. It easily crosses the cell membrane and is cleaved by intracellular esterases into calcein, a membrane-impermeable polar molecule that remains inside the cell, emitting green fluorescence.

Fig. 9 Evaluation of in vitro PDT effect by Calcein-AM/EthD-III double staining of HeLa cells: (a) live, (b) dead, and (c) merged after completion of different treatments of (i) control, (ii) control (L+), (iii) UCNPs, (iv) UCNPs (L+), (v) UCNP@CDs and (vi) UCNP@CDs (L+) (scale bar = 50 μm).

In contrast, EthD-III cannot penetrate the intact membranes of living cells but can enter damaged regions of the membranes in dead cells, emitting red fluorescence upon binding to the nucleus. Calcein-AM and EthD-III were combined to evaluate the phototoxicity of UCNP@CDs. The HeLa cells cultured with PBS (10 mM, pH 7.4) were employed as the control group and the HeLa cells co-cultured with the UCNP@CDs kept in dark were employed as another group. The images show green fluorescence in all the groups without laser irradiation, suggesting that the cytotoxicity of the sample is minimal in the absence of laser exposure. Further, in the case of UCNPs, bright green fluorescence was observed in presence and absence of laser, signifying no cell death.

The strong red fluorescence indicated that almost all the cells in the group treated with UCNP@CDs were destroyed following light exposure. The figure clearly shows that cells treated with the UCNP@CD nanohybrid (with over 50% cell death) after 980 nm laser irradiation exhibited greater cytotoxicity compared to the non-irradiated UCNP@CD-treated cells, highlighting the enhanced effectiveness. These findings suggest that UCNP@CDs hold considerable promise as an anticancer agent for photodynamic therapy (PDT).

3.8. Hemolysis test

Hemolysis is a critical factor in evaluating the biocompatibility of biomaterials. Hence, we performed a hemolytic activity assay to evaluate the biocompatibility of the UCNP@CDs and the results showed that UCNP@CDs at concentrations between 0.5 and 2.5 mg mL⁻¹ induced minimal hemolysis (less than 10%) (Fig. 6d and Fig. S17), whereas the positive control, Triton X, exhibited significant hemolysis.

Here, As denotes the absorbance of the samples, while An and Ap represent the absorbance of the negative and positive controls, respectively.

4. Conclusion

To intensify the effectiveness of PDT, herein, we report a dual-channel (980 nm and 660 nm) activated novel nanohybrid based on upconversion nanoparticles modified with carbon dots (PVP/KBF:Yb (30%), Er (3%)@CDs) through in situ modification. Here, carbon dots acting as a photosensitizer could be activated by the light emitted from UCNPs for the generation of ¹O₂ under low power 980 nm laser irradiation (0.4 W cm⁻²). Here, we amplified the emission intensity (660 nm) of UCNPs by varying the dopant Yb³⁺ concentration to get a better PDT effect via the FRET mechanism. Additionally, NIR-I light has higher penetration ability, making this system more capable of PDT in vitro. The designed nanohybrid retains good dispersibility and high physiological adaptability and shows better fluorescence properties under 980 nm laser. The in vitro investigation showed that the synthesized composite achieved an efficient anticancer outcome with 72% cell mortality toward HeLa cells in the presence of laser and the cytotoxicity result also revealed almost the same cell viability toward HEK-293 and HeLa cells, indicating the non-toxic nature of the composite. Therefore, this study suggests an idea for developing and designing integrated systems for enhancing ROS production to overcome the existing limitations in PDT.

Author contributions

Bijay Saha: writing – original draft, methodology, writing, formal analysis, data curation, conceptualization. Antara Ghosh: methodology, formal analysis and writing, Archana Singh: data curation and analysis. Sumanta Kumar Sahu: review, supervision, project administration, funding acquisition, conceptualization.

Conflicts of 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.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article its SI. Size distribution curve of UCNPs, Elemental mapping of UCNPs, Stability of UCNP@CDs in PBS, FBS and DMEM at different time durations, EDX spectrum of UCNP@CDs, Raman spectra of CD, Absorption spectra of CD, PL spectra of carbon dots, PL spectra of UCNP and UCNP@CDs (UC) at different time intervals, Schematic energy states diagram of UCNP indicating the different energy transfer mechanism in the Upconversion photoluminescence process, Schematic energy states diagram of UCNP, showing energy transfer mechanism during the variation of Yb³⁺ dopant concentration, Upconversion PL decay curve of UCNP and UCNP@CDs, ROS generation by CD under 660nm laser, ROS production by UCNP@CDs under 980 nm laser, ROS generation by only UCNP@CDs under 660 nm laser, ROS generation of only UCNP under 980 nm laser, Comparative cellular uptake study of folate receptor active HeLa cells and HEK293T cells, Hemolysis test of UCNP@CDs. See DOI: https://doi.org/10.1039/d5dt01208b.

Acknowledgements

S. K. Sahu and B. Saha is grateful to MoE-STARS/STARS-2/2023–0340 for awarding a scholarship and research grant. The authors are thankful to the Central Research Facility, Indian Institute of Technology (Indian School of Mines), Dhanbad, for HRTEM, FESEM, XPS, XRD, PL, Raman Study, and cell culture studies. B. Saha would like to acknowledge the Department Research Facility of the Department of Chemistry and Chemical Biology, Indian Institute of Technology (Indian School of Mines), Dhanbad, for UV and FTIR analysis. B. Saha acknowledges with gratitude the support of CIMFR (Central Institute of Mining and Fuel Research), Dhanbad, for the TGA analysis, and Shiv Nadar University, Delhi NCR, for the EPR study. The authors would like to thank Dr Kaushal Kumar, Department of Physics IIT (ISM) Dhanbad, India, for lifetime measurement studies.

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