Two-photon Photodynamic Therapy Research Articles

Trifluoromethyl-pyrrolidone phthalocyanine nanoparticles for targeted lipid droplet imaging and in vitro photodynamic therapy in breast cancer cells

Lipid droplets (LDs) serve as vital subcellular organelles, crucial for the maintenance of lipid and energy homeostasis within cells. Their visualization is of significant value for elucidating the intricate interactions between LDs and other cellular organelles. Despite the importance of LDs, the literature on the utilization of phthalocyanine-based photosensitizers for targeted LD imaging and two-photon imaging-guided photodynamic therapy (PDT) remains sparse. In this study, we have designed and synthesized trifluoromethyl-pyrrolidone silicon phthalocyanine (PyCF3SiPc). To enhance the water solubility of PyCF3SiPc and improve its tumor cells accumulation, we employed 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-mPEG2000) as a nanocarrier, thereby formulating DSPE@PyCF3SiPc nanoparticles. Our in vitro experiments in MCF-7 cells demonstrated that DSPE@PyCF3SiPc selectively targets and visualizes LDs, offering a reliable tool for tracking their dynamic movement. Moreover, DSPE@PyCF3SiPc demonstrates considerable phototoxicity against MCF-7 cells subjected to PDT underscoring its potential as an effective therapeutic agent. In conclusion, DSPE@PyCF3SiPc presents itself as a promising novel probe for the dual purpose of monitoring the dynamic movement of LDs and guiding imaging-assisted PDT. The development of this nanoparticle system not only advances our understanding of LD biology but also paves the way for innovative therapeutic strategies in oncology.

Advances in two-photon absorption photodynamic therapy of glioma based on porphyrin-based metal-organicframework composites

Gliomas of the brain are characterised by high aggressiveness, high postoperative recurrence rate, high morbidity and mortality, posing a great challenge to clinical treatment. Traditional treatments include surgery, radiotherapy and chemotherapy; they also have significant associated side effects, leading to difficulties in tumour resection and recurrence. Photodynamic therapy has been shown to be a promising new strategy to help treat malignant tumours of the brain. It irradiates the tumour site at a specific wavelength to activate a photosensitiser, which selectively accumulates at the tumour site, triggering a photochemical reaction that destroys the tumour cells. It has the advantages of being minimally invasive, highly targeted and with few adverse reactions, and is expected to be well used in anti-tumour therapy. However, the therapeutic effect of traditional PDT is limited by the weak tissue penetration ability of photosensitiser, hypoxia and immunosuppression in the tumour microenvironment. This paper reviews the current research status on the therapeutic principle of photodynamic therapy in glioma and the mechanism of tumour cell injury, and also analyses the advantages and disadvantages of the current application in glioma treatment, and clarifies the analysis of ideas to improve the tissue penetration ability of photosensitizers. It aims to provide a feasible direction for the improvement of photodynamic therapy for glioma and a reference for the clinical treatment of deep brain tumours.

A Single-Shot Technique for Measuring Broadband Two-Photon Absorption Spectra in Solution.

Applications involving two-photon activation, including two-photon fluorescence imaging, photodynamic therapy, and 3D data storage, require precise knowledge of the two-photon absorption (2PA) spectra of target chromophores. Broadband pump-probe spectroscopy using femtosecond laser pulses provides wavelength-dependent 2PA spectra with absolute cross sections, but the measurements are sometimes complicated by cross-phase modulation effects and dispersion of the broadband probe. Here, we introduce a single-shot approach that eliminates artifacts from cross-phase modulation and enables more rapid measurements by avoiding the need to scan the time delay between the pump and the probe pulses. The approach uses counterpropagating beams to automatically integrate over the full interaction between the two pulses as they cross. We demonstrate this single-shot approach for a common 2PA reference, coumarin 153 (C153), in three different solvents using the output from a Yb:KGW laser. This approach provides accurate 2PA cross sections that are more reliable and easier to obtain compared with scanning pump-probe methods using copropagating laser beams. The single-shot method for broadband two-photon absorption (BB-2PA) spectroscopy also has significant advantages compared with single-wavelength measurements, such as z-scan and two-photon fluorescence.

Acceptor-planarized type I photosensitizer for lipid droplet-targeted two-photon photodynamic therapy by ferroptosis

Acceptor-planarized type I photosensitizer for lipid droplet-targeted two-photon photodynamic therapy by ferroptosis

Newly Designed Quasi-intrinsic Photosensitizers for Fluorescence Image-Guided Two-Photon Photodynamic Therapy with Type I/II Photoreactions.

In this work, a set of quasi-intrinsic photosensitizers are theoretically proposed based on the 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo[1,2-α]-1,3,5-triazin-4(8H)-one (P), which could pair with the 6-amino-5-nitro-3-(1'-β-d-2'-deoxyribofuranosyl)-2(1H)-pyridone (Z) and keep the essential structural characters of nucleic acid. It is revealed that the ring expansion and electron-donating/electron-withdrawing substitution bring enhanced two-photon absorption and bright photoluminescence of these monomers, thereby facilitating the selective excitation and tumor localization through fluorescence imaging. However, instead of undergoing radiative transition (S1 → S0), the base pairing induced fluorescence quenching and rapid intersystem crossing (S1 → Tn) are observed and characterized by the reduced singlet-triplet energy gaps and large spin-orbit coupling values. To ensure the phototherapeutic properties of the considered base pairs in long-lived T1 state, we examined the vertical electron affinity as well as vertical ionization potential for production of superoxide anions via Type I photoreaction, and their required T1 energy (0.98 eV) to generate singlet oxygen 1O2 via Type II mechanism.

Anthraquinone-Centered Type I Photosensitizer with Aggregation-Induced Emission Characteristics for Tumor-Targeted Two-Photon Photodynamic Therapy

Anthraquinone-Centered Type I Photosensitizer with Aggregation-Induced Emission Characteristics for Tumor-Targeted Two-Photon Photodynamic Therapy

Two-photon NIR-responsive carbon dots incorporated into NMOFs for targeted photodynamic therapy

In recent years, Photodynamic therapy (PDT) has shown great potential in clinics to treat cancer. Due to the limited depth range of UV or visible laser light penetration, the eradication of solid tumors using traditional photosensitizer is difficult. Here, we report the development of a new NIR active photosensitizer covalently conjugated to nanoscale metal-organic frameworks (NMOFs) for the eradication of deep-seated tumors. This photosensitizer can generate reactive oxygen species (ROS) in the presence of near-infrared light irradiation (980 nm) for PDT. Therefore, we use a strategy to develop two-photon absorbing carbon dots with a fluorescence quantum yield of 13%. The NMOFs are fabricated in the presence of folic acid, which enables them to target cancer cells that overexpress the folate receptor. NMOFs are used as a nanocarrier for CDs and are employed to achieve targeted PDT. This study is the first to observe that carbon dots embedded in NMOF composite could generate singlet oxygen (1O2) under the 980 nm laser light irradiation and also specifically target cancer cells. At a concentration of 100 μg/ml, the nanocomposite demonstrated a 73% rate of cell inhibition. The primary results reported here will show great potential in the future to develop multifunctional carbon dots for targeted two-photon PDT in the presence of higher wavelength light irradiation.

Quasi-intrinsic thiobase derivatives as potential targeted photosensitizers in two-photon photodynamic therapy

In this study, a set of potential quasi-intrinsic photosensitizers for two-photon photodynamic therapy (PDT) are proposed based on the unnatural 2-amino-8-(1′-β-ᴅ-2′-deoxyribofuranosyl)-imidazo[1,2-ɑ]-1,3,5-triazin-4(8H)-one (P), which is paired with the 6-amino-5-nitro-3-(1′-β-ᴅ-2′-deoxyribofuranosyl)-2(1H)-pyridone (Z) and can specifically recognize breast and liver cancer cells. Herein, the effects of sulfur substitution and electron-donating/electron-withdrawing groups on the photophysical properties in aqueous solution are systematically investigated. The one- and two-photon absorption spectra evidence that the modifications could result in red-shifted absorption wavelength and large two-photon absorption cross-section, which contributes to selective excitation and provides effective PDT for deep-seated tissues. To ensure the efficient triplet state population, the singlet–triplet energy gaps and spin–orbit coupling constants were examined, which is responsible for a rapid intersystem crossing rate. Furthermore, these thiobase derivatives are characterized by the long-lived T1 state and the large energy gap for radiationless transition to ensure the generation of cytotoxic singlet oxygen.

New Approaches to Photodynamic Therapy of Melanomas

SignificanceWe present new approaches to treat both primary cutaneous and ocular melanoma, with potential to reduce metastatic spread ApproachIn mouse models optical clearing combined with dual-photosensitizer PDT is used to treat primary cutaneous xenogafts. Two-photon PDT is used in ocular melanoma of extraocular (conjunctival) or intraocular (uveal) melanoma. Immune effects are investigated in immunocompetent mice using blood biomarkers and by tumor re-challenge. In vitro studies in pigmented and non-pigmented melanoma cells show melanin-mediated photosensitization as a novel mechanism of action of 2-γ PDT. ResultsFull-thickness primary tumor eradication has been achieved: ∼1 and 4 mm in immune deficient and competent mice, respectively. Marked reduction in metastatic tumor burden is observed in the latter. ConclusionsThese studies reopen the possibility of PDT as an effective modality in melanoma.

Thio-naphthalene-modified artificial bases as targeted photosensitizers for two-photon photodynamic therapy

In this work, a series of quasi-intrinsic photosensitizers (PSs) are designed based on 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (P), which could pair with the 6-amino-5-nitro-3-(1-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone (Z) and specifically recognize breast and liver cancer cells. The spectra calculations show that the modified bases possess efficient two-photon absorption in red and near-infrared spectra region that contribute to an efficient photodynamic therapy (PDT) for deep-seated tumors. After the vertical excitation, these modified bases in the excited singlet state exhibit a predominance of intersystem crossing to the triplet state channels, which can be predicted by the energy difference and the spin-orbit coupling values between the singlet and triplet states. Additionally, the triplet state lifetimes are examined by considering the radiative and non-radiative rates, thus reflecting the interaction time of the PS with molecular oxygen. To evaluate the direct usefulness in PDT, we further investigated the effect of base pairing and linking to deoxyribose on photosensitivity.

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy.

In this study, two water-soluble red fluorescent protein (RFP) dimers, FP2R' and FP2R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP2R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient (β) was 2.1 × 10-11 cm W-1. Further, the RFP dimer FP2R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1O2 and O2˙- simultaneously. Under 460 nm illumination, the photosensitizer FP2R'' showed high phototoxicity with an IC50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP2R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP2R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP2R'' effectively produced 1O2 and O2˙- in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP2R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP2R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.

Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging.

In this paper, three fluorescent protein chromophore analogs PFPAr (PFPP, PFPC, and PFPT) were synthesized and proved to be useful for photodynamic therapy and two-photon fluorescence imaging. By adding five- or six-membered aromatic heterocycles to the photosensitizer PFP, we obtained three fluorescent protein photosensitizers PFPAr with better performances. As a demonstration, compared with the reported photosensitizer PFP, photosensitizer PFPP exhibits larger emission wavelengths (701 nm) and achieves a slight enhancement in the efficiency of singlet oxygen (ΦΔ = 23%). Notably, PFPP can perform good two-photon fluorescence imaging with an 800 nm femtosecond laser in zebrafish. In in vitro cytotoxicity assays, PFPP shows good phototoxicity (IC50 = 4.12 μM) and acceptable dark toxicity (cell viability assay >90%). The reactive oxygen imaging experiments and AO/EB double staining assay indicate that PFPP can generate singlet oxygen to eliminate A-549 tumor cells effectively with photoexcitation of 460 nm blue light (20 mW cm-2). Furthermore, PFPP can label the lysosomes of tumor cells with high specificity for lysosomes (Pearson's correlation coefficient of 0.91). Thus, our study demonstrated that the rational introduction of aromatic heterocycles into fluorescent protein photosensitizers can effectively enhance the key parameters of photosensitivity and pave the way for further two-photon photodynamic therapy.

Cyclometalated iridium(III) tetrazine complexes for mitochondria-targeted two-photon photodynamic therapy.

The fast-moving field of photodynamic therapy (PDT) has provided fresh opportunities to expand the potential of metallodrugs to combat cancers in a light-controlled manner. As such, in the present study, a series of cyclometalated Ir(III) complexes modified with a tetrazine functional group (namely, Ir-ppy-Tz, Ir-pbt-Tz, and Ir-dfppy-Tz) are developed as potential two-photon photodynamic anticancer agents. These complexes target mitochondria but exhibit low toxicity towards HLF primary lung fibroblast normal cells in the dark. When receiving a low-dose one- or two-photon PDT, they become highly potent towards A549 lung cancer cells (with IC50 values ranging from 24.0 nM to 96.0 nM) through the generation of reactive oxygen species (ROS) to induce mitochondrial damage and subsequent apoptosis. Our results indicated that the incorporation of tetrazine with cyclometalated Ir(III) matrices would increase the singlet oxygen (1O2) quantum yield (ΦΔ) and, meanwhile, enable a type I PDT mechanism. Ir-pbt-Tz, with the largest two-photon absorption (TPA) cross-section (σ2 = 102 GM), shows great promise in serving as a two-photon PDT agent for phototherapy.

Near-infrared BODIPY photosensitizers for two-photon excited singlet oxygen generation and tumor cell photodynamic therapy.

In this paper, two near-infrared BODIPY photosensitizers, Id-BDPI and Cz-BDPI, were obtained by modifying the indole and carbazole aromatic heterocycles in the core of BODIPY. The maximum absorption wavelengths of Id-BDPI and Cz-BDPI were 694 nm and 722 nm, and their singlet oxygen yields were 48% and 48.4%, respectively. In the simulated tumor cell photodynamic therapy, Id-BDPI and Cz-BDPI could effectively inhibit the growth of A549 tumor cells under near-infrared light. Meanwhile, the lysosomal co-localization coefficients of Id-BDPI and Cz-BDPI with A549 tumor cells were 0.94 and 0.89, respectively, showing high lysosomal targeting ability and biocompatibility. The two-photon absorption cross sections measured at 1050 nm by the Z-scanning method were 661.8 GM and 715.6 GM, respectively, and Cz-BDPI was further successfully applied to two-photon fluorescence imaging and two-photon excited singlet oxygen generation in zebrafish. The above results indicate that the introduction of aromatic heterocycles can effectively enhance the photodynamic efficacy of BODIPY photosensitizers, and the larger two-photon absorption cross section also brings potential for two-photon photodynamic therapy applications.

Heavy-atom-free BODIPY dendrimer: utilizing the spin-vibronic coupling mechanism for two-photon photodynamic therapy in zebrafish.

In this study, the heavy-atom-free BODIPY dendrimer TM4-BDP was synthesized for near-infrared photodynamic therapy, and was composed of a triphenylamine-BODIPY dimer and four 1-(2-morpholinoethyl)-1H-indole-3-ethenyl groups. The TM4-BDP could achieve near-infrared photodynamic therapy through two different photosensitive pathways, which include one-photon excitation at 660 nm and two-photon excitation at 1000 nm. In the one-photon excitation pathway, the TM4-BDP could generate singlet oxygen and superoxide radicals under 660 nm illumination. In addition, the one-photon PDT experiment in human nasopharyngeal carcinoma (CNE-2) cells also indicated that the TM4-BDP could specifically accumulate in lysosomes and show great cell phototoxicity with an IC50 of 22.1 μM. In the two-photon excitation pathway, the two-photon absorption cross-section at 1030 nm of TM4-BDP was determined to be 383 GM, which means that it could generate reactive oxygen species (ROS) under 1000 nm femtosecond laser excitation. Moreover, the two-photon PDT experiment in zebrafish also indicated the TM4-BDP could be used for two-photon fluorescence imaging and two-photon induced ROS generation in biological environments. Furthermore, in terms of the ROS generation mechanism, the TM4-BDP employed a novel spin-vibronic coupling intersystem crossing (SV-ISC) process for the mechanism of ROS generation and the femtosecond transient absorption spectra indicated that this novel SV-ISC mechanism was closely related to its charge transfer state lifetime. These above experiments of TM4-BDP demonstrate that the dendrimer design is an effective strategy for constructing heavy-atom-free BODIPY photosensitizers in the near-infrared region and lay the foundation for two-photon photodynamic therapy in future clinical trials.

Aggregation-induced emission-active iridium (III)-based mitochondria-targeting nanoparticle for two-photon imaging-guided photodynamic therapy

The efficacy of imaging-guided photodynamic therapy (PDT) is compromised by the attenuation of fluorescence and decline in reactive oxygen species (ROS) generation efficiency in the physiological environment of conventional photosensitizers, limited near-infrared (NIR) absorption, and high systemic cytotoxicity. This paper presents the synthesis of two cyclometalated Ir (III) complexes (Ir-thpy and Ir-ppy) by using a triphenylamine derivative (DPTPA) as the primary ligand and their encapsulation into an amphiphilic phospholipid to form nanoparticles (NPs). These complexes exhibit aggregation-induced emission features and remarkably enhanced ROS generation compared to Chlorin e6 (Ce6). Moreover, Ir-thpy NPs possess the unique ability to selectively target mitochondria, leading to depolarization of the mitochondrial membrane potential and ultimately triggering apoptosis. Notably, Ir-thpy NPs exhibit exceptional photocytotoxicity even towards cisplatin-resistant A549/DDP tumor cells. In vivo two-photon imaging verified the robust tumor-targeting efficacy of Ir-thpy NPs. The in vivo results unequivocally demonstrate that Ir-thpy NPs exhibit excellent tumor ablation along with remarkable biocompatibility. This study presents a promising approach for the development of multifunctional Ir-NPs for two-photon imaging-guided PDT and provides novel insights for potential clinical applications in oncology.

Theoretical explore of bridge and donor engineering for novel naphthalimide based photosensitizers for two-photo photodynamic therapy

Naphthalimide derivatives are widely used in the development of clinical drugs and fluorescent probes because of their high light stability and low dark toxicity. Among these applications, as the photosensitizer (PS) used in the two-photon photodynamic therapy (TP-PDT) process is a new application condition of naphthalimide derivatives. Nevertheless, the development of such PSs is still hampered by their short triplet state lifetime and insufficient two-photon absorption (TPA) cross section. In order to explore the influence of different π-linkers on the efficient naphthalimide based PSs and solve the above problems, this paper characterizes the photophysical properties related to TP-PDT process of a series of naphthalimide based molecules using density functional theory (DFT) and time-dependent DFT (TD-DFT). The results reveal that compared to molecules without linkers and with phenyl as the linker, the compounds using thiophene as the π-linker have better planarity and optical properties, such as larger TPA cross section, lower energy gap of the singlet−triplet state, larger spin-orbit coupling value, and longer triplet state lifetime. Specifically, molecules 3s-ph, 2s-thio∼4s-thio have large TPA cross section value (1028–1384 GM, 8.3–11.2 times of molecule 1s) and triplet state lifetime (1337–11609 μs, 1.6–14.2 times of molecule 1s), making them effective for TP-PDT process. Furthermore, molecule 1s-thio acts as a promising PS candidate that can effectively undergo type I process and circumvent type II process. It is hoped that our theoretical research can provide some worthy ideas for the design and synthesis of effective naphthalimide based PSs in experiments.

Optical modulation of Iridium(III) complexes for enhanced ROS generation: Multi-photon absorption and near-infrared photodynamic therapy insights

In this study, we harness optical modulation techniques to tailor an auxiliary ligand, thereby optimizing the photophysical behavior of cyclometalated iridium (III) complexes, Ir–B and Ir-Q, for enhanced reactive oxygen species (ROS) generation. Intriguingly, the optical properties of Ir-Q's auxiliary ligands instigate a non-radiative intersystem crossover process, yielding a superior singlet oxygen (1O2) quantum efficiency of 0.92. Further, Ir-Q's robust multi-photon absorption spectra, particularly in the Two-photon (750–800 nm) and Three-photon (1350–1600 nm) domains, open avenues for potential applications in two-photon imaging-guided photodynamic therapy targeting deep-seated tumors. This investigation underscores the pivotal role of optical structure modulation in amplifying the therapeutic potential of the iridium(III) complex, particularly in the realm of near-infrared photodynamic therapy.

Iridium(III)-Based Infrared Two-Photon Photosensitizers: Systematic Regulation of Their Photodynamic Therapy Efficacy.

Cyclometalated iridium(III) complexes are of significant importance in the field of antitumor photodynamic therapy (PDT), whether they exist as single molecules or are incorporated into nanomaterials. Nevertheless, a comprehensive examination of the relationship between their molecular structure and PDT effectiveness remains awaited. The influencing factors of two-photon excited PDT can be anticipated to be further multiplied, particularly in relation to intricate nonlinear optical properties. At present, a comprehensive body of research on this topic is lacking, and few discernible patterns have been identified. In this study, through systematic structure regulation, the nitro-substituted styryl group and 1-phenylisoquinoline ligand containing YQ2 was found to be the most potent infrared two-photon excitable photosensitizer in a 4 × 3 combination library of cyclometalated Ir(III) complexes. YQ2 could enter cells via an energy-dependent and caveolae-mediated pathway, bind specifically to mitochondria, produce 1O2 in response to 808 nm LPL irradiation, activate caspases, and induce apoptosis. In vitro, YQ2 displayed a remarkable phototherapy index for both malignant melanoma (>885) and non-small-cell lung cancer (>1234) based on these functions and was minimally deleterious to human normal liver and kidney cells. In in vivo antitumor phototherapy, YQ2 inhibited tumor growth by an impressive 85% and could be eliminated from the bodies of mice with a half-life as short as 43 h. This study has the potential to contribute significantly to the development of phototherapeutic drugs that are extremely effective in treating large, profoundly located solid tumors as well as the understanding of the structure-activity relationship of Ir(III)-based PSs in PDT.

Single-sized N-functionality graphene quantum dot in tunable dual-modality near infrared–I/II illumination detection and photodynamic therapy under multiphoton nonlinear excitation

Doping sorted graphene quantum dots (GQDs) with heteroatoms and functionalizing them with amino acid could improve their radiative recombination and two-photon properties—including their excitation-wavelength-independent photoluminescence from the ultraviolet to the near-infrared-I (NIR-I) region, absorption, quantum yield, absolute cross section, lifetime, and radiative-to-nonradiative decay ratio—under two-photon excitation (TPE) at a low excitation energy and short photoexcitation duration, as determined using a self-made optical microscopy system with a femtosecond Ti–sapphire laser. Four types of sorted GQDs were investigated: undoped GQDs, nitrogen-doped GQDs (N-GQDs), amino-functionalized GQDs (amino-GQDs), and N-doped and amino-functionalized GQDs (amino-N-GQDs). Among them, the sorted amino-N-GQDs are effective as a two-photon photosensitizer and generate the highest quantity of reactive oxygen species for the elimination of multidrug-resistant cancer cells through two-photon photodynamic therapy (PDT). Larger amino-N-GQDs result in a greater number of C–N and N–functionalities, leading to a superior photochemical effect and more favorable intrinsic luminescence properties, making the dots effective contrast agents for tracking and localizing cancer cells during in-depth bioimaging in a three-dimensional biological environment under TPE in the NIR-II region. Overall, this study highlights the potential of large amino-N-GQDs as a material for future application to dual-modality two-photon PDT and biomedical imaging.