Aggregation-Induced-Emission Luminogens Functionalized MXene Nanosheets for Stimuli-Responsive Hydrogel in Pyroptosis-Mediated Choroidal Melanoma Therapy
Yingni Xu, Fei Wang, Wenfang Liu, Ruibin Lin, Cheng Liu, Qi Zhao, Guokang He, Guiping Yuan, Weidong Yin, Fei Yu, Jianwei Sun, Ryan T. K. Kwok, Jacky W. Y. Lam, Li Ren, Xuan Zhao, Jin Yuan, Ben Zhong Tang
TL;DR
A new nanoplatform combining imaging and therapy is developed for treating choroidal melanoma using a hydrogel that releases nanosheets over 72 hours.
Contribution
The integration of aggregation-induced emission luminogens with MXene nanosheets in a hydrogel for sustained, imaging-guided therapy of choroidal melanoma is novel.
Findings
The nanoplatform enables dual-modal imaging (FLI and PTI) and synergistic mPTT/PDT therapy.
The hydrogel allows controlled release of nanosheets over 72 hours, enabling single injection for multiple treatments.
In vitro and in vivo studies confirm the nanosystem's effectiveness in tumor ablation via pyroptosis.
Abstract
A pyroptosis mediated mild photothermal therapy (mPTT)/photodynamic therapy (PDT) enabled by aggregation induced emission married MXene nanosheets for choroidal melanoma is reported.The nanosheets realize near infrared Fluorescence Imaging (FLI)- Photothermal Imaging (PTI) dual imaging guided synergistic mPTT/PDT therapy , while Agar/PSBMA hybrid hydrogel allows controlled nanosheets release over 72 h , enabling single injection, multiple treatment for precise theranostics. The nanoplatform addresses the existed imaging diagnostics and therapeutic predicaments in choroidal melanoma. A pyroptosis mediated mild photothermal therapy (mPTT)/photodynamic therapy (PDT) enabled by aggregation induced emission married MXene nanosheets for choroidal melanoma is reported. The nanosheets realize near infrared Fluorescence Imaging (FLI)- Photothermal Imaging (PTI) dual imaging guided synergistic…
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Taxonomy
TopicsNanoplatforms for cancer theranostics · Ocular Oncology and Treatments · MXene and MAX Phase Materials
Introduction
Choroidal melanoma is one of the most prevalent intraocular malignancies in adults, accounting for approximately 5% of all melanoma cases [1]. Although multimodal therapeutic strategies, including chemotherapy and radiotherapy [2, 3], can manage localized tumors, nearly 50% of patients with choroidal melanoma develop liver metastases, leading to disease progression and treatment delays [4–7]. Current clinical interventions encompass photodynamic therapy (PDT), radiotherapy, enucleation and combinations of these modalities [8, 9]. However, these treatments face significant limitations. For instance, PDT is typically effective only in the early stages of the disease and has limited efficacy thereafter [10]. Radiotherapy often suffers from uneven dose distribution and may result in radiation-induced ocular complications [11, 12]. Enucleation remains a conventional approach for larger choroidal melanomas; however, it is a disfiguring procedure that can lead to severe physical and psychological consequences for patients and is associated with a high recurrence rate [13, 14]. Significant obstacles in the clinical management of choroidal melanoma include the lack of sensitive and noninvasive imaging modalities for tumor detection and the presence of the blood-ocular barrier [15, 16], which hampers the accumulation of therapeutic agents in the targeted area, particularly for traditional chemotherapy [17]. Collectively, these challenges underscore the urgent need for the development of multifunctional theranostic strategies that can achieve precise drug delivery, synergistic therapeutic effects, and real-time imaging capabilities for the effective management of choroidal melanoma [18].
Light-activated therapies utilizing photosensitizers have been established as safe and effective methods for tumor ablation across various cancer types [19–22]. Despite significant advancements in the development of phototherapeutic agents and devices over the past few decades [23], there remain several challenges that limit their wider clinical application beyond certain dermatological uses [24]. Photothermal therapy (PTT) and PDT represent two primary approaches that leverage light activation [25, 26]. The integration of these methods can effectively address their individual limitations, such as the heat-shock response and limited light penetration [27, 28]. Recent advancements have led to the development of multifunctional nanomaterials suitable for both PDT and PTT, which can selectively accumulate in tumors via the enhanced permeability and retention (EPR) effect [29–31]. The incorporation of photoactive nanoparticles not only enhances tissue heating and promotes reactive oxygen species (ROS) generation, but also introduces an additional layer of selectivity to these therapies [32]. Two-dimensional (2D) nanomaterials have gained significant attention over the past decade due to their ultrathin structures and superior physicochemical properties [33, 34]. MXenes (M_n+1_X_n_T_m_) [35], a new family of multifunctional 2D materials introduced in 2011, show great potential for tumor therapy due to their remarkable attributes, including superior drug loading capabilities, ease of surface modifications, and high photothermal conversion efficiency [36]. However, challenges still exist regarding precise tumor targeting and identification, highlighting the need for further research into modification techniques to enhance their therapeutic efficacy [34]. Aggregation-induced emission generators (AIEgens), as an emerging concept in luminescence, are gaining traction as molecular imaging and therapeutic agents in oncology [37]. Their large Stokes shift, high quantum yield, excellent biocompatibility, and impressive photostability make them attractive for cancer applications [38, 39]. AIEgens can be engineered for specific targeting of various cancer types using diverse strategies [40]. When integrated with other imaging modalities, AIEgen-based multimodal imaging offers a comprehensive view of cancer hallmarks from multiple perspectives [41, 42]. Furthermore, AIEgen-based phototherapy can be effectively applied in both PDT and PTT [43], enabling efficient cancer cell ablation while maintaining excellent biocompatibility and therapeutic efficacy in vivo [44, 45]. The synergy between AIEgens and functional nanomaterials enhances their versatility, leading to the development of novel multifunctional theranostic nanoplatforms tailored for treating choroidal melanoma [46, 47].
Herein, a multifunctional nanoplatform, namely MX@PEG-MeoTTPy, was prepared by coating a water-soluble positively charged AIEgen (PEG-MeoTTPy) onto the surface of MXene nanosheets via electrostatic interaction. These tumor-targeted nanosheets were subsequently encapsulated within an Agar/PSBMA hydrogel network to create a stimuli-responsive injectable hydrogel (GNSs) for the treatment of choroidal melanoma (Scheme 1), which facilitated the sustained release of MX@PEG-MeoTTPy nanosheets over 72 h. Once internalized by tumor cells, these nanosheets could induce pyroptosis through the synergistic effects of mild photothermal therapy (mPTT) and PDT. mPTT was achieved using near-infrared (NIR-I) laser irradiation, which minimized the risk of thermal damage to the ocular tissue while enhancing therapeutic efficacy. The incorporation of PEG-MeoTTPy, which generated type I reactive oxygen species (ROS), alongside MXene nanosheets enabled a collaborative approach to effectively target and eliminate cancer cells. This strategy not only imparted strong fluorescence emission in the NIR region and PDT capabilities to the theranostic nanoplatform, but also established a controlled delivery system for the nanosheets. Both in vitro and in vivo evaluations demonstrated that the multifunctional nanosheets can effectively achieve long-term retention at the tumor site to enable continuous monitoring and treatment, and precisely ablate tumors, thus presenting a NIR fluorescence imaging and photothermal imaging-guided synergistic mPTT-PDT theranostic solution for choroidal melanoma.Scheme 1. Schematic illustration of pyroptosis-mediated mPTT/PDT therapy enabled by stimuli-responsive nanosheets hybrid hydrogel platform for Choroidal Melanoma. a Nanosheets synthesis route. b Release behavior of the stimuli-responsive nanosheets-hybrid hydrogel. c Mechanism of choroidal melanoma therapy
Experimental Section
Preparation of MX@PEG-MeoTTPy Nanosheets
100 μg mL^− 1^ of MXene nanosheets dispersion were mixed with different concentrations of PEG-MeoTTPy (5 ~ 100 μM) in water. After tip sonication for 0.5 h on ice and stirring overnight, the excess unloading was filtered by centrifuging in Amicon tubes (MWCO 100 kDa, Millipore) at 4000 rpm for 20 min at 4 °C and then washed three times with water. The concentration of PEG-MeoTTPy in the MX@PEG-MeoTTPy nanosheet dispersion was determined by measuring the absorbance at 510 nm using a UV–vis-NIR spectrophotometer. The absorbance attributed to the MXene nanosheets was subtracted from the spectra of the MX@PEG-MeoTTPy nanosheets to obtain the corresponding normalized absorbance. Notably, the concentration mentioned in the text referred to the final concentration of PEG-MeoTTPy or MXene nanosheets containing in MX@PEG-MeoTTPy nanosheets dispersion after purification. Additionally, the loading efficiency of PEG-MeoTTPy was also determined via UV–vis-NIR spectra of MXene nanosheets (100 μg mL^− 1^) before and after PEG-MeoTTPy (100 μM) loading at 510 nm, and the loading efficiency of PEG-MeoTTPy was calculated to be 47.09%.
Selective Staining of the Cancer/Normal Cells
The MUM-2B (pre-stained with Calcein AM for 15 min) and C2C12 cells or HCECs (Human Corneal Epithelial Cells) were mixed and seeded onto the confocal dishes until the confluence reached ca. 75%. The corresponding adherent cells were washed with PBS to remove the remnant medium. Then, cells were incubated with MX@PEG-MeoTTPy nanosheets dispersion (containing 100 μg mL^− 1^ MXene nanosheets and 50 μM PEG-MeoTTPy) in DMEM medium for 4 h at 37 °C, followed by PBS washing for three times and used for bioimaging software. Calcein AM was excited at 405 nm, and the nanoparticles was excited at 561 nm. Data were expressed as means ± standard deviation (n = 3).
Intracellular Delivery Pathway Study
The intracellular delivery pathway of MX@PEG-MeoTTPy nanosheets on MUM-2B cells was studied by double-labeling assay with different specific organelle dyes including LysoTracker Green (lysosomes dye) and MitoTracker Green (mitochondria dye). Briefly, MUM-2B cells were seeded into confocal dishes and incubated for 24 h. Then, the cells were firstly treated with MX@PEG-MeoTTPy nanosheets (containing 100 μg mL^− 1^ MXene nanosheets and 50 μM PEG-MeoTTPy) for 1 h at 37 °C and washed with PBS three times then. At prearranged time intervals (2, 4, and 6 h), the cells were stained separately with LysoTracker Green and MitoTracker Green for 30 min at 37 °C. Finally, the cells were rinsed with PBS and observed by CLSM. The exciting wavelength and emission filter of LysoTracker Green and MitoTracker Green were 488 and 520–550 nm, respectively.
Cell Pyroptosis Monitoring
The MUM-2B cells were plated onto confocal dishes and incubated at 37 °C, 5% CO_2_ atmosphere until the confluence reached of 70%. The cells were incubated with MX@PEG-MeoTTPy nanosheets (containing 100 μg mL^− 1^ MXene nanosheets and 50 μM PEG-MeoTTPy) for 4 h. The cells were exposed to 808 nm NIR laser at a power density of 0.8 W cm^− 2^ for 3 min and white light at a power density of 20 mW cm^− 2^ for 5 min; then, the cellular behavior was observed under CLSM.
Preparation of Agar/PSBMA Hydrogel Containing MX@PEG-MeoTTPy Nanosheets (GNSs)
Agar/PSBMA hydrogel containing MX@PEG-MeoTTPy nanosheets (GNSs) was prepared by mixing nanosheets into the pre-hydrogel. First, a certain amount of Agar and deionized water was heated and stirred to completely dissolve the Agar. Once a certain amount of SBMA was added and completely dissolved in the mixed solution, it was quickly poured into a glass Petri dish to form a preformed hydrogel. Second, to prevent excessive cross-linking, the preformed hydrogel was immersed in an APS/TEMED mixed solution and placed at room temperature for 30 min for polymerization. Then, excess crosslinker was removed and washed with NaCl solution for three times to obtain Agar/PSBMA hydrogel. The GNSs was prepared by adding MX@PEG-MeoTTPy nanosheets into the Agar/PSBMA solution under 60 °C when the hydrogel was liquid. The final concentration of MX@PEG-MeoTTPy nanosheets contained in the hydrogel was 100 μg mL^− 1^ MXene nanosheets and 50 μM PEG-MeoTTPy, and the Agar/PSBMA hydrogel was 1% (w/v)/0.7% (w/v).
Antitumor Activity In Vivo
Orthotopic tumor models were built to evaluate the antitumor ability of these samples (PBS, NSs + NIR + WL, GNSs + Dark, GNSs + NIR, GNSs + WL, GNSs + NIR + WL) in vivo. Animal experiments were conducted in accordance with the animal policies of Sun Yat-sen University (SYT2024054). To establish the choroidal melanoma mouse model, 5 × 10^5^ Luc-A375 cells were injected into the middle between the retina and choroid in the right eye of each mouse. Seven days later, the tumor cells had invaded the vitreous cavity. Mice were randomly divided into six groups (five mice per group) according to the total intensity of luminescence [18]. Then, mice were treated with 2 μL of sterile PBS (control), NSs + NIR + WL, GNSs + Dark, GNSs + NIR, GNSs + WL or GNSs + NIR + WL. The concentration of NSs was 300 μg mL^− 1^ MXene nanosheets and 150 μM PEG-MeoTTPy, and the light irradiation was 808 nm NIR laser at a power density of 0.8 W cm^− 2^ for 5 min or/and white light at a power density of 20 mW cm^− 2^ for 10 min for two consecutive days. To evaluate the therapeutic results, mice were intraperitoneally injected with D-luciferin potassium salt (10 mg kg^− 1^) and imaged by the in vivo imaging instrument (IVIS) spectrum system with a 60-s exposure time. A caliper and electronic balance were used to measure the diameter and weight of the eyeballs.
Results and Discussion
Synthesis and Characterization of MX@PEG-MeoTTPy Nanosheets
The synthesis of PEG-MeoTTPy through a straightforward three-step reaction process is illustrated in Scheme S1. Initially, product 1 was obtained via a palladium-catalyzed Suzuki–Miyaura cross-coupling reaction between 4-bromo-N, N-bis(4-methoxyphenyl) aniline and 5-formyl-2-thiopheneboronic acid, utilizing a palladium catalyst. Following this, a condensation reaction between compound 1 and p-toluenesulfonic acid was conducted. This intermediate then reacted with Br-PEG-NH_2_ to produce the final product, PEG-MeoTTPy. All compounds were fully characterized by ^1^H NMR, high-resolution mass spectrometry (HRMS), and Fourier transform infrared (FTIR) spectroscopy with satisfactory results (Figs. S1-S6). The maximum absorption of PEG-MeoTTPy was observed at 510 nm (Fig. S7), displaying typical AIE characteristics (Fig. S8). Benefiting from the positively charged pyridinium moiety and poly-(ethylene glycol)-amine (PEG-NH_2_) fragment, PEG-MeoTTPy exhibited good water solubility. The MXene nanosheets were prepared based on an in situ HF etched method [48], where the MAX phase Nb_2_AlC was etched by HF solution to remove Al layer. The resulting material was then kept in deionized water and exfoliated via bath sonication to obtain few-layered Nb_2_C MXene nanosheets. Transmission electron microscopy (TEM) images demonstrated that Nb_2_AlC MAX phase exhibited the characteristic morphology of a layered compound (Fig. S10a). After HF etching, TEM images revealed ultrathin, electron-transparent flakes of exfoliated Nb_2_C nanosheets (Fig. S10b), which displayed a typical sheet-like morphology with an average size of approximately 200 nm. The SEM–EDS analysis also confirmed the existence of niobium (Nb) and carbon (C) elements, while the absence of aluminum (Al) indicated its complete removal from the structure (Fig. S10c). Raman spectra of Nb_2_AlC and Nb_2_C nanosheets are depicted in Fig. 1e. The vibration modes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega$$\end{document} 1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega$$\end{document} 3 were suppressed or even disappeared after HF etching, confirming the elimination of the Al layer or the exchange of Al atoms with lighter species. The mode \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega$$\end{document} 4 exhibited downshifting and shape changes, while the mode \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\omega$$\end{document} 2 merged and weakened, indicating the retention of the Nb_2_C layer and an increase in interlayer spacing [34]. However, MXene has poor tumor targeting capabilities [49]; it is essential to develop novel photoabsorbers that not only exhibit satisfactory biocompatibility and biodegradability, but also possess appropriate dimensions for effective tumor targeting, ensuring their safe excretion from the body within a specific timeframe following therapeutic administration.Fig. 1. Synthesis of MX@PEG-MeoTTPy nanosheets. a Zeta potential of samples. b TEM images of MX@PEG-MeoTTPy nanosheets and Tyndall effect of the suspension. c Particle size distribution of MX@PEG-MeoTTPy. d SEM–EDS analysis of MX@PEG-MeoTTPy. e Raman spectrum of samples. f–h Survey XPS spectrum of MX@PEG-MeoTTPy and high-resolution C 1s, and N 1s spectra. i UV–vis-NIR spectra of samples. Insets in (i): I–III) Photographs of: I) MXene (Concentration: 100 μg mL^− 1^), II) PEG-MeoTTPy (Concentration: 50 μM) and III) MX@PEG-MeoTTPy (MXene concentration: 100 μg mL^− 1^ and PEG-MeoTTPy concentration: 50 μM). j Fluorescence spectra of samples. Excitation wavelength: 510 nm
The desired nanomaterial, MX@PEG-MeoTTPy nanosheets, was successfully prepared by embedding AIE-active PEG-MeoTTPy on the surface of MXene, which was confirmed through zeta potential measurements, SEM–EDX, Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS) analyses. As shown in Fig. 1a, the zeta potential of PEG-MeoTTPy in water was + 36.9 mV, which facilitated the adsorption of negatively charged materials through electrostatic interactions. In contrast, the zeta potential of MX@PEG-MeoTTPy decreased from − 31.9 to + 10.3 mV compared to the bare MXene nanosheets. This positive charge enhanced the uptake of the nanosheets by cancer cells. TEM images revealed that the MX@PEG-MeoTTPy nanosheets had an irregular shape with sharp margins, with an average lateral size of approximately 200 nm, making them readily endocytosed by cells. Additionally, the presence of the Tyndall effect in digital photographs of MX@PEG-MeoTTPy dispersed in water indicated their excellent hydrophilicity and dispersibility (Fig. 1b). Furthermore, UV–vis measurements of the supernatant after 7 days of dialysis showed negligible AIEgen release (Fig. S13a), confirming the stable association between PEG-MeoTTPy and MXene, which also contributes to the long-term colloidal stability of the nanosheets under physiological conditions (Fig. S13b). Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of MX@PEG-MeoTTPy nanosheets was around 234 nm (Fig. 1c), exhibiting uniform morphology similar to that of the bare MXene nanosheets (Fig. S10b). This size is conducive to efficient accumulation at tumor sites, driven by the EPR effect. SEM–EDS images demonstrated the excellent colocalization of four different elements: niobium (Nb) from MXene, and oxygen (O), bromine (Br), and sulfur (S) from PEG-MeoTTPy (Fig. 1d). Raman spectra indicated similar peaks to those of bare MXene nanosheets, with some red shifts observed, confirming that the organic modification did not affect the structure of the corresponding bulk material (Fig. 1e). Furthermore, the chemical composition of MX@PEG-MeoTTPy was validated by XPS analysis (Fig. 1f-h). The peaks at 284.5, 286.1, and 288.4 eV correspond to C–O and C = O bonds, while the peaks at 399.4 and 398.3 eV are attributed to C–N bonds and pyridine nitrogen, respectively, indicating the presence of PEG-MeoTTPy on the surface of MXene nanosheets. The optical properties of MX@PEG-MeoTTPy were assessed through UV–Vis-NIR and fluorescence spectroscopy. As shown in Fig. 1i, MX@PEG-MeoTTPy in water exhibited a broad absorption band spanning the UV to NIR regions. Moreover, a broad emission band was observed, with maximum intensity at 754 nm, alongside partial fluorescence quenching of the loaded AIEgens, likely due to strong interactions between AIEgens and MXene nanosheets. These analyses collectively confirm the successful coating of AIEgens onto the MXene nanosheets. Notably, when MX@PEG-MeoTTPy was placed in an acidic solution, the fluorescence quenching was alleviated due to the electrostatic interactions between AIEgens and MXene nanosheets (Fig. 1j), enhancing its potential as an effective cell imaging photosensitizer.
Photothermal and ROS Generation Properties
The MX@PEG-MeoTTPy nanosheets exhibit broad and intense spectral absorption ranging from visible light to NIR region, indicating potential photothermal-conversion capability. To explore the photothermal property of MX@PEG-MeoTTPy nanosheets, the temperature changes were examined upon irradiation recorded by infrared thermal imaging camera. The photothermal heating curves of combined nanosheets revealed concentration and laser power density-dependent temperature variations under 808 nm NIR laser irradiation (Figs. 2a and S14). The temperature can be precisely regulated from 24.8 to 64.7 °C by adjusting the nanosheets concentration or laser power density. To assess the photostability of MX@PEG-MeoTTPy nanosheets, the temperature changes were recorded during five cycles of NIR laser irradiation (Fig. 2b). Notably, there was no obvious change in temperature throughout the cycling process, indicating satisfactory photothermal stability of MX@PEG-MeoTTPy nanosheets. The high-performance photothermal conversion and exceptional stability make MX@PEG-MeoTTPy nanosheets highly promising for PTT application.Fig. 2. Photothermal and ROS generation properties of MX@PEG-MeoTTPy nanosheets. a Temperature evaluation curves of MX@PEG-MeoTTPy nanosheets suspension at different concentrations. b Photothermal stability of MX@PEG-MeoTTPy nanosheets suspension under five cycles of 808 nm laser irradiation (0.8 W cm^− 2^) on/off processes. c–e Plots of I/I_0_ versus light irradiation times in the presence of different samples and c) DCFH-DA, d) DHR 123, and e) HPF. I_0_ and I in (c-e) represent the fluorescence intensities of DCFH-DA, DHR 123, and HPF before and after 808 nm NIR laser (NIR, 0.8 W cm^− 2^) and white light (WL, 20 mW cm^− 2^). f Plots of A/A_0_ versus light irradiation times in the presence of different samples. A_0_ and A represent the absorbance of ABDA before and after Light. g EPR spectra of MX@PEG-MeoTTPy with DMPO or TEMP with or without light irradiation. h Intracellular ROS detection by CLSM after MUM-2B cells was incubated under different conditions. Light irradiation condition in (g-h) was 808 nm NIR laser (NIR, 0.8 W cm^− 2^ for 5 min) and white light (WL, 20 mW cm^− 2^ for 10 min). Scale bar: 100 μm
ROS generation performance is critical for PDT application [50], so the related estimation of combined nanosheets was conducted by using commercially available ROS indicators. Given that PEG-MeoTTPy exhibited strong absorption in the visible light region, white LED light was employed as the light source for PDT. The photoinduced ROS generation capacity of the nanosheets was subsequently evaluated using 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA) as a broad-spectrum ROS indicator [51]. As illustrated in Fig. 2c, the ROS generation profiles of bare MXene nanosheets in the presence of DCF-DA almost unchanged throughout the 808 nm NIR laser (0.8 W cm^− 2^) and white light (20 mW cm^− 2^) irradiation process, indicating that the bare MXene nanosheets do not produce ROS under these conditions. In contrast, the presence of PEG-MeoTTPy and MX@PEG-MeoTTPy resulted in a rapid increase in DCF-DA fluorescence, with enhancements of 464-fold and 380-fold, respectively, after 5 min of irradiation (Fig. 2c), demonstrating highly efficient ROS generation. The relatively lower ROS generation efficiency of MX@PEG-MeoTTPy compared to PEG-MeoTTPy may be due to the ROS-scavenging capability of MXene nanosheets, primarily through surface-mediated adsorption and redox reactions, which reduced the local oxygen concentration around PEG-MeoTTPy. Additionally, chlorin e6 (Ce6), a well-known photosensitizer for PDT, was also investigated under similar conditions, yielding a 201-fold enhancement in DCF-DA emission intensity. This result was only half of the ROS production efficiency observed with MX@PEG-MeoTTPy, suggesting that the ROS generation performance of MX@PEG-MeoTTPy was far superior to Ce6. While MX@PEG-MeoTTPy did not generate ROS under NIR irradiation alone, it could generate ROS under white light with/without NIR irradiation, indicating white light was the key factor in ROS generation (Fig. S15). The superior ROS production of MX@PEG-MeoTTPy could be attributed to the more efficient intersystem crossing of PEG-MeoTTPy, resulting from both heavy atomic effects and high electron-donating (D)-withdrawing (A) strength. To further delineate the specific type of ROS, various fluorescent probes including dihydrorhodamine 123 (DHR123, for •O_2_^−^), hydroxyphenyl fluorescein (HPF, for •OH) and singlet oxygen indicator (ABDA, for ^1^O_2_) were introduced. As depicted in 2D-E, the fluorescence intensity of DHR123 at 530 nm and HPF at 520 nm increased sharply in the presence of PEG-MeoTTPy and MX@PEG-MeoTTPy under dual-light irradiation. As shown in Fig. 2f, when irradiating the mixture of ABDA and MX@PEG-MeoTTPy with 808 nm NIR laser (0.8 W cm^− 2^) and white light (20 mW cm^− 2^), the absorbance of ABDA barely decreased. A similar absorbance silencing phenomenon was observed for the other three groups. However, a significant decrease in absorbance was noted in the presence of Ce6, confirming the absence of ^1^O_2_ generation. As the most reliable technique in identifying short-lived ROS, electron paramagnetic resonance (EPR) spectroscopy was also employed to capture the ROS generated by the nanosheets. After irradiating the mixture of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, spin trap agent) and MX@PEG-MeoTTPy with 808 nm NIR laser (0.8 W cm^− 2^) and white light (20 mW cm^− 2^), a sharp four-line EPR signal in 1:2:2:1 intensity with hyperfine coupling constant of 14.8 G was detected (Fig. 2g), which matched with the characteristic resonance of the DMPO/•OH adduct. This finding indicated MX@PEG-MeoTTPy presented better photosensitized •OH generation capacity. For the mixture of 4-amino-2,2,6,6-tetramethylpiperidine (TEMP, a ^1^O_2_ trapping agent) and MX@PEG-MeoTTPy, the EPR signal was notably weak in the absence of light irradiation, and no EPR signal was observed following irradiation, supporting the conclusion that ^1^O_2_ was not produced. Taken together, it is clear that the MX@PEG-MeoTTPy could efficiently generate type-I ROS •O_2_^−^ and •OH radicals through an electron transfer pathway, which can overcome the anoxic environment of tumor. Notably, theoretical calculations also depicted that the energy transfer from triplet state (T_1_) of PEG-MeoTTPy to nearby ^3^O_2_ to produce ^1^O_2_ was forbidden, as the vertical emission energies from T_1_ to S_0_ (0.78 eV) were smaller than the oxygen sensitization threshold (0.98 eV) (Fig. S9) [52].
The intracellular ROS generation efficiency of MX@PEG-MeoTTPy nanosheets was further evaluated by using DCF-DA as a ROS probe, which produced green fluorescence upon oxidation by ROS. As shown in Fig. 2h, negligible green fluorescence signal was detected after 4-h incubation of PBS (as control) or bare MXene nanosheets with MUM-2B cells upon simultaneous irradiations by 808 nm NIR laser and white light. In contrast, cells incubated with MX@PEG-MeoTTPy nanosheets exhibited strong green fluorescence in the presence of white light with/without 808 nm laser (Fig. S16), demonstrating the efficient intracellular ROS production. Notably, no green fluorescent signal was observed when only the 808 nm NIR laser was used as the illumination source, indicating that white light was essential for triggering ROS generation from MX@PEG-MeoTTPy nanosheets. The ROS generation under hypoxia was comparable to that under normoxia, demonstrating the superiority of the nanosheets in producing Type I ROS (Fig. S17).
Cancer Cell Imaging and Killing
Benefiting from their excellent photothermal performance and effective ROS generation, MX@PEG-MeoTTPy nanosheets were utilized for synergistic PTT and PDT therapy by quantitative estimation on MUM-2B cells through MTT assay. Individually, both MXene nanosheets and PEG-MeoTTPy demonstrated good biocompatibility in the dark and were capable of killing cancer cells through PTT or PDT. However, they required longer exposure times and exhibited unsatisfactory effects when used alone (Figs. S18-S20). Moreover, the MX@PEG-MeoTTPy nanosheets also showed excellent cytocompatibility with cancer cells under dark conditions. To assess the in vitro phototherapeutic effects of MX@PEG-MeoTTPy, dose-dependent cytotoxicity experiments with or without 808 nm NIR laser and/or white light irradiation were conducted using MUM-2B cells. It was found that the IC50 values (inhibitory concentration of 50% cell death) of MXene nanosheets and PEG-MeoTTPy containing in the MX@PEG-MeoTTPy-associated light irradiations (white light and NIR laser irradiation) were determined to be 48.24 µg mL^− 1^ and 24.12 μM, respectively, which were significantly lower than that of treated with NIR laser (63.72 µg mL^− 1^ for PTT) or white light (48.26 μM, for PDT) alone, indicating that neither PTT nor PDT alone could achieve satisfactory therapeutic efficacy (Fig. 3a). Furthermore, the combination index (CI) value for MX@PEG-MeoTTPy in terms of PTT and PDT was calculated to be 0.87, suggesting that the combination of MXene nanosheets and PEG-MeoTTPy exhibited synergistic anticancer effects (Table S1). Additionally, the PDT therapeutic efficiency of the nanosheets in hypoxia was further evaluated. Encouraged by the efficient Type I ROS generation of MX@PEG-MeoTTPy, we measured the PDT efficiency in a simulated hypoxic environment. As depicted in Fig. 3a, the cell viability of MX@PEG-MeoTTPy with white light and/or NIR irradiation under hypoxia decreased, and it possess a similar PDT therapeutic efficiency under normoxia due to the exceptional anti-hypoxia activity resulting from superior Type I ROS productivity ability. Taken together, these results indicated that MX@PEG-MeoTTPy was significantly powerful to ablate cancer cells by means of synergistic PDT and PTT treatments.Fig. 3. Intracellular behavior of MX@PEG-MeoTTPy nanosheets. a Cell viability of MUM-2B cells after treated with MX@PEG-MeoTTPy nanosheets at different concentrations with or without 808 nm NIR laser (NIR, 0.8 W cm^− 2^ for 5 min) and/or white light (WL, 20 mW cm^− 2^ for 10 min), the groups without hypoxia were under normoxia, incubation time for 6 h. b Cell viability comparison between MUM-2B and C2C12 cells after treated with MX@PEG-MeoTTPy nanosheets at different concentrations (NIR, 0.8 W cm^− 2^ for 5 min. WL, 20 mW cm^− 2^ for 10 min), incubation time for 6 h. c Schematic illustration of cancer cell discrimination with normal cells by MX@PEG-MeoTTPy nanosheets. d CLSM images of mixed MUM-2B (pre-stained with calcein AM) and C2C12 cells incubated with MX@PEG-MeoTTPy nanosheets (MXene concentration: 100 μg mL^− 1^ and PEG-MeoTTPy concentration: 50 μM) for 4 h. Scale bar: 20 μm. e The corresponding fluorescence intensities in (d). f CLSM images of mixed MUM-2B (pre-stained with calcein AM) and C2C12 cells incubated with MX@PEG-MeoTTPy nanosheets (MXene concentration: 100 μg mL^− 1^ and PEG-MeoTTPy concentration: 50 μM) for 4 h. Scale bar: 20 μm
A key requirement for precise oncotherapy is the selective disruption of cancer cells while sparing normal cells [53]. Encouragingly, MX@PEG-MeoTTPy nanosheets demonstrated relatively low phototoxicity toward C2C12 cells compared to MUM-2B cells (Fig. 3b). Considering the unique energy metabolism characteristics of malignancies, we speculated that the cationic nature MX@PEG-MeoTTPy nanosheets would facilitate their preferential accumulation in cancer cells over normal cells, which was significantly helpful for the early diagnosis and accurate surgical resection of malignant tumors. To verify this hypothesis, MUM-2B cells were pre-stained with calcein AM to differentiate MUM-2B cells from other cell types (Figs. 3c and S22a). After coculturing the MUM-2B cells with C2C12/HCECs cells and then staining with MX@PEG-MeoTTPy nanosheets, distinct differences in imaging performance between cancer and normal cell lines were achieved. Besides, ROS generation in normal HCECs was relatively low and slightly fluorescent signal was observed, indicating the lower intracellular uptake levels (Fig. S22b). As shown in Fig. 3d, all cancer cell lines were successfully stained and exhibited intense fluorescence signals, whereas normal cell lines displayed significantly weaker signals. The extracted fluorescence intensities indicated that the cancer cells showed approximately three times higher fluorescence than the normal cells (Fig. 3e). This cancer-selective uptake was further confirmed by time-dependent imaging at 2 and 6 h (Fig. S23). These findings suggest that MX@PEG-MeoTTPy nanosheets hold promise for targeted cancer therapy by preferentially accumulating in malignant cells.
To further elucidate the mechanism underlying the efficient ablation of cancer cells by MX@PEG-MeoTTPy nanosheets upon dual light irradiation, we visualized the cytoplasmic distribution of these nanosheets in MUM-2B cells using double-labeling with different commercial organelle-targeting dyes, including LysoTracker Green and MitoTracker Green. As illustrated in Fig. 3f, after internalizing into cells for 2 h, MX@PEG-MeoTTPy nanosheets were perfectly colocalized with lysosomes. With prolonged incubation time to 4 and 6 h, the overwhelming majority of yellow fluorescence accumulation in mitochondrial could be clearly observed, confirming the effective lysosomal escape and mitochondria targeting. Accordingly, it was believed that after internalization into cells, MX@PEG-MeoTTPy nanosheets were sealed into lysosomes, and the amine groups in PEG-MeoTTPy were partially protonated in the acidic lysosomal environment, enabling PEG-MeoTTPy to be separated from MXene nanosheets. Subsequently, the discrete PEG-MeoTTPy escaped from the lysosome due to the proton sponge effect [54]. Because of the positively charged pyridinium moiety and protonated amine groups in the PEG-MeoTTPy structure, it then directly targeted the mitochondria through electrostatic interactions. It has been demonstrated that mitochondria played considerably important roles in physiological and pathological scenarios, making mitochondrial-targeting function an ideal approach for the destruction of cancer cell [55]. Moreover, the residual MXene nanosheets and PEG-MeoTTPy served as phototherapy agents for both hyperthermia and ROS-induced cell pyroptosis in the lysosomes. These findings indicated that the dual-subcellular killing ability of MX@PEG-MeoTTPy nanosheets can greatly boost the death of cancer cells through synergistic PTT and PDT pathways. Additionally, we examined the endocytosis behavior of MX@PEG-MeoTTPy nanosheets in normal C2C12 cells, as shown in Fig. S21. Most of the MX@PEG-MeoTTPy nanosheets showed perfect colocalization with lysosomes even the time was extended to 6 h. Weak fluorescence in the mitochondria indicated that the majority of the nanosheets were internalized into lysosomes, differing from the behavior observed in tumor cells. Tumor cells typically exhibited a more negative electrical potential, which facilitated the electrostatic binding of PEG-MeoTTPy and enhanced accumulation in the mitochondria. This characteristic allowed for the potential differentiation of tumor cells from normal cells based on the site of nanosheet accumulation.
Cellular Pyroptosis Investigation
To better evaluate the cytocidal effects of dual-organelles targeted phototherapy, real-time tracking of the cellular morphological changes was conducted. After staining with MX@PEG-MeoTTPy nanosheets, the MUM-2B cells were exposed to the dual-light irradiation and the cellular images were collected. Microscopy observations revealed that the MX@PEG-MeoTTPy-stained cells exhibited intact structures prior to light irradiation, and the distribution of nanosheets within the cells could be clearly visualized (Fig. S24a). However, after 5 min of irradiation, cells swelled (evidence of pyroptosis) rather than shrinking (evidence of apoptosis) [56], and the bubbles were generated from the plasma membrane and gradually expanded, demonstrating an obvious pyroptosis process (Figs. 4a and S24b). The presence of pyroptotic morphologies motivated us to conduct a detailed mechanism investigation of photosensitized MX@PEG-MeoTTPy nanosheets induced programmed cell death (PCD).Fig. 4. Cellular pyroptosis investigation. a TEM characterization of nanosheet internalization and pyroptotic morphology of MUM-2B cells, the red arrows indicate plasma membrane pores and cytoplasmic vacuoles. b Volcano plot of all differentially expressed genes of MUM-2B cells under light irradiation (NIR for 5 min and WL for 10 min), with the cells under dark as control. c Bubble pattern of KEGG pathway enrichment analysis. d Specific gene expression of NOD-like receptor signaling pathway components for NIR + WL and dark group. Red and blue blocks indicate upregulation and downregulation of gene expression compared to the dark group, and the genes in red squares are typical genes in the NOD-like receptor signaling pathway. e qPCR analysis of pyroptosis-related genes after different treatments. f The secretion of IL-18 and IL-1β in MUM-2B cells treated with MX@PEG-MeoTTPy nanosheets by ELISA under different conditions. g LDH release of MUM-2B cells treated with MX@PEG-MeoTTPy nanosheets. h Western blotting analysis of pyroptosis-related proteins after different treatments
The transcriptome of MUM-2B cells treated with MX@PEG-MeoTTPy nanosheets under light irradiation (NIR for 5 min and WL for 10 min) and under dark as control was compared then. From Fig. 4b, there are 154 highly expressed genes (red dots) and 106 downregulated genes (blue dots) compared to the dark group, indicating significant changes at the gene expression level. The bubble diagram in Fig. 4c highlights several signaling pathways associated with cell death, with the NOD-like receptor signaling pathway being most relevant to pyroptosis [57]. To determine this, the effects of various cell death inhibitors on the viability of MX@PEG-MeoTTPy-treated cells were investigated. Figure S26 showed that pretreatment pyroptosis inhibitors (Disulfiram) could effectively improve the viability of MUM-2B cells after MX@PEG-MeoTTPy treatment under white light and NIR irradiation, while pretreatment with autophagy (3-MA), necrotic apoptosis (Nec-1) and noncanonical inflammasome (NSA) inhibitors only exhibited negligible effect. Inhibitors of apoptosis (z-VAD-fmk) and ferroptosis (Ferrostatin-1 and Liproxstatin-1) showed a little increase in viability compared to the control group. These results showed pyroptosis is the predominant and decisive cell death mechanism for tumor therapy. From the identification of 145 genes in the NOD-like receptor signaling pathway (Fig. 4d), typical genes related to pyroptosis were studied, including *IL-1 * \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document} , IL-18, CASP 1, GSDMD, NLPR 3, CASP 4, etc. Based on above findings, we further identified several critical markers to investigate the mechanisms of pyroptosis induced by MX@PEG-MeoTTPy nanosheets. The mRNA expression level of GSDMD, CASP 1, NLPR 3, and IL-18 was significantly elevated in the light-treated groups as shown in Fig. 4e. Both the NIR and white light groups exhibited increased mRNA expression levels compared to the untreated group, with the NIR group showing a more pronounced upregulation. Moreover, the levels of inflammatory factors, including IL-18, IL-1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\beta}$$\end{document} , and LDH, were markedly higher in the groups treated with MX@PEG-MeoTTPy nanosheets under dual-light irradiation (Fig. 4f, g). To further demonstrate the above PCD mechanism, the key signaling proteins regulating pyroptosis were analyzed by a western blotting assay. As shown in Fig. 4h, NIR + WL group potently induced GSDMD cleavage, generating higher level of GSDMD-N fragment, while NIR/WL treatments also triggered moderate cleavage. This indicates synergistic activation of pyroptosis under combined irradiation. Above all, under white light irradiation, MX@PEG-MeoTTPy nanosheets generated reactive oxygen species (ROS), activating caspase-1 at the cell membrane, which cleaved GSDMD into its N-terminal domains (GSDMD-N). GSDMD-N then translocated to the cell membrane, forming pores that induced pyroptosis. Additionally, the photothermal effect of MX@PEG-MeoTTPy nanosheets accelerated the pyroptosis process, leading to the rapid release of inflammatory cytokines.
Stimuli-Responsive Injectable Hydrogel Delivery System
During the process of eyeball injection treatment, a therapeutic hydrogel should be squeezed out from a specialized extremely fine needle and maintain a stable shape at the aimed position. Hydrogels with stimuli-responsive and shear-thinning properties are advantageous for loading nanosheets and facilitating injection. As illustrated in Fig. 5a (Scheme 1), we developed an injectable and NIR/TME (Tumor Microenvironment) smart controlled release Agar/PSBMA hydrogel containing MX@PEG-MeoTTPy nanosheets (referred to as GNSs) through a straightforward two-step procedure. SBMA (sulfobetaine methacrylate) is a typical zwitterionic polymer containing anionic and cationic groups, which can form a chemically cross-linked network via free radical polymerization. By mixing SBMA powders with the Agar solution at heating status, we can create a preformed hydrogel where hydrogen bonding in Agar acts as the physical cross-linking point at lower temperatures. This physical cross-linking allows for the uniform dispersion and support of other reactive components, ensuring consistent formation of the secondary network. The free radical polymerization reaction of SBMA was initiated by small molecule additives that penetrate the hydrogel network, creating a second chemical cross-linking network and resulting in Agar/PSBMA hydrogels. Due to the upper critical solution temperature (UCST) behavior arising from strong intramolecular and intermolecular electrostatic interactions between zwitterionic groups in the PSBMA polymers, the Agar/PSBMA hydrogel exhibited temperature self-sensing capabilities. The sol–gel transition temperature of GNSs was approximately 42.5 °C (Fig. S27), indicating its temperature sensitive properties. The Agar/PSBMA hydrogels can transit into liquid when the temperature increased to 50 °C, facilitating easy injection into the eyeball (Fig. S28). The resulting GNSs were an adorable milky pink, inheriting a similar gel–sol transformation through a heating–cooling procedure, but with a distinct color compared to the Agar/PSBMA hydrogel (Fig. 5b). After immersing the GNSs in saturated NaCl solution for 48 h, the hydrogel network degraded due to the infiltration of Na^+^ and Cl^−^ (Fig. 5c), allowing the PSBMA hydrogel to dissociate under physiological conditions. The porous structure of GNSs facilitated the loading of nanosheets. As shown in Fig. 5d, the addition of PSBMA enhanced cross-linking, resulting in a denser porous structure (Fig. S30), which can prolong the retention time of GNSs in the tumor environment.Fig. 5. Controlled release of stimuli-responsible Agar/PSBMA hydrogel (GNSs). a Schematic diagram of the stimuli-responsive release behavior of nanosheets in Agar/PSBMA hydrogel. b Digital images of GNSs under different temperatures. c The degradation images of GNSs in water and saturated NaCl solution for 48 h. d SEM images of GNSs. Scale bar: 100 μm. e Photothermal stability GNSs under five cycles of 808 nm laser irradiation (0.8 W cm^−2^) on/off processes. f Corresponding percent of MX@PEG-MeoTTPy nanosheets release from the Agar/PSBMA hydrogel. g Cell viability of MUM-2B cells treated with the released MX@PEG-MeoTTPy nanosheets from hydrogel under different conditions. h E. coli and S. aureus viability of GNSs with or without 808 nm NIR laser (NIR, 0.8 W cm^− 2^ for 5 min) and/or white light (WL, 20 W cm^− 2^ for 20 min). i Infrared thermal images in vivo when eyeballs were injected by GNSs under 0.8 W cm^− 2^ laser irradiation for 4 min. j Corresponding temperature change curves of eyeballs of (i)
It is widely acknowledged that the eyes are delicate structures, and exposure to high temperatures (above 50 °C) during photothermal therapy (PTT), as well as the introduction of large quantities of nanoparticles, can potentially harm ocular tissues. To evaluate the photothermal effects of the GNSs, we exposed them to 808 nm laser irradiation for 5 min, during which a clear increase in temperature over time was observed (Fig. S31). Thereafter, the photostability of GNSs was characterized, it showed little difference after five cycles turning the 808 nm laser on/off, and the average temperature could reach 60 °C, which was adjustable to change the nanosheets concentration and NIR laser power.
The remarkable stimuli-responsive and photothermal effects of the GNSs motivated us to explore the release behavior of MX@PEG-MeoTTPy nanosheets under NIR light irradiation, and the release behaviors of GNSs before and after NIR irradiation (0.8 W cm^− 2^) were investigated. In the absence of irradiation, less than 15% of MX@PEG-MeoTTPy nanosheets were released after 40 min of incubation. In contrast, under NIR irradiation, the cumulative release reached 42%, with a notable NIR-triggered “on–off” phenomenon observed after four rounds of irradiation (Fig. 5f). The NIR-responsive MX@PEG-MeoTTPy nanosheets release of GNSs was related to the NIR- induced gel–sol transformation and ionic immersion. MX@PEG-MeoTTPy nanosheets were tightly entrapped within the hydrogel and difficult to diffuse out due to the porous structure. However, the gel–sol transition under irradiation disrupted the physical cross-linking, thereby accelerating the diffusion of nanosheets from the loosened network. Additionally, the hydrogel network can respond to Na^+^ and Cl^−^ in PBS solution, further accelerating the nanosheets release (Fig. S29). By adjusting the laser irradiation time and nanosheet concentrations, we can achieve controlled accumulation and release of the nanosheets. The released MX@PEG-MeoTTPy nanosheets from the GNSs exhibited a satisfactory cancer cell killing effect, outperforming other groups as shown in Fig. 5g, attributed to the enhanced release efficiency of the nanosheets. While ocular injection was a common administration route, it carried a risk of infection. In our study, GNSs not only effectively killed cancer cells but also eliminated bacteria, including E. coli and S. aureus. From Fig. 5h (Fig. S32), the synergistic PTT and PDT under NIR laser and white light irradiation demonstrated superior bacterial killing efficiency. Notably, GNSs were more effective against S. aureus than E. coli, likely due to differences in cell wall composition between the two bacteria. The modest antibacterial effect in the dark group may be attributed to the physical disruption of bacterial membranes by MXene’s sharp edges and positive surface charge. Single PTT or PDT was also effective against E. coli, with bacterial viability recorded at 29.2% and 28.4%, respectively. These results suggest that the synergistic effects of PTT and PDT surpass those of single modalities in the antibacterial context, highlighting the advantages of the proposed GNSs in preventing ocular infections. After 2 µL GNSs were injected into the eyeballs of mice, the temperature of eyeball alone (control) showed a slight upward trend and the GNSs groups showed an increase from 33 to 43 °C (0.8 W cm^− 2^, 4 min, Fig. 5i, j). Collectively, these results demonstrated that the GNSs could maintain outstanding photothermal conversion effects and was suitable for PTT treatment of the eyeball.
Choroidal Melanoma Treatment In Vivo
To further investigate the synergistic antitumor effect of GNSs in vivo, we constructed an orthotopic model of choroidal melanoma to mimic the intraocular microenvironment in which choroidal melanoma developed (Fig. 6a) [58, 59]. Briefly, a murine choroidal melanoma tumor model was established by subretinal injection of Luc-A375 cells into the right eye of each mouse using a Hamilton microsyringe. Seven days post-injection, the mice were imaged by an in vivo fluorescence imaging system (IVIS). Strong luminescence signals from the cancer cells confirmed the successful establishment of the choroidal melanoma model. The mice were then randomly divided into six groups: PBS (control), NSs (nanosheets) + NIR + WL, GNSs + Dark, GNSs + NIR, GNSs + WL and GNSs + NIR + WL. Prior to the therapeutic experiment, we first studied controlled nanosheets release properties of the GNSs in vivo by detecting MX@PEG-MeoTTPy fluorescence using IVIS. After being injected for 24 h, GNSs were predominantly localized within the eyeball, with no detectable signals in other organs, indicating successful accumulation at the tumor site (Fig. 6b). To further assess the controlled release of GNSs, MX@PEG-MeoTTPy nanosheets alone (NSs) were used as a control. As shown in Fig. 6c, d, there was no difference in fluorescence intensity between GNSs and NSs immediately after sample injection. Both groups were then subjected to irradiation for two consecutive days. For mice injected with the simple NSs, the fluorescence signal was reduced to 47.2% at 24 h, and the fluorescent signal almost invisible at 48 h. In contrast, the fluorescence signals of GNSs remained at 59.1% of the initial signals at 72 h, and there was no signal on day 5, indicating GNSs were degraded then (Fig. S33). These findings indicated that GNSs not only prolonged the retention of MX@PEG-MeoTTPy nanosheets within the eye but also maintained a higher local concentration of the nanosheets over an extended duration. Furthermore, the programmed release of the loaded MX@PEG-MeoTTPy nanosheets was successfully achieved.Fig. 6. Stimuli-responsive nanosheets-hybrid hydrogel for choroidal melanoma treatment. a Schematic illustration of establishing choroidal melanoma and the design of animal experiments. b Ex vivo NIR fluorescence images of major organs and tumors after injection with GNSs and PBS for 24 h. c Fluorescence imaging depicting the retention of NSs and GNSs in the eye after being injected at different time points. d Corresponding statistical fluorescence intensity of NSs and GNSs retained in the eye. e Tumor growth kinetics corresponding to bioluminescence signals in different groups. f In vivo bioluminescence images of mice bearing choroidal melanoma after different treatments. Five mice per group are shown. Body weight g, eyeball weight h and eyeball diameter i in different treatment groups; n = 5
The therapeutic results after different treatments were evaluated based on the bioluminescence signals from Luc-A375 cells imaged by the IVIS. As shown in Fig. 6e, f, the eyes of mice treated with GNSs + NIR + WL exhibited relatively low bioluminescence signals at day 8, indicating a significant therapeutic effect on choroidal melanoma in this group. In contrast, other treated groups, including NSs + NIR + WL, GNSs + NIR, and GNSs + WL, showed a bioluminescence signals decrease at day 2, but tumors reoccurred by day 8. This phenomenon could be attributed to the limited utilization of NSs and the restricted therapeutic efficacy of single PTT or PDT modalities [60]. The weights of mice in different groups during the treatment were also monitored. Nearly no weight reduction was observed in mice with different treatments, indicating that intraocular injection of GNSs induced minimal systemic side effects. After eight days of treatment, the eyeballs of mice were collected, and we measured the weight (Fig. 6h) and diameter (Fig. 6i). The results revealed that the eyeballs from the GNSs + NIR + WL group maintained normal weight and diameter, verifying the excellent antitumor efficacy of synergistic PTT and PDT, consistent with the bioluminescence signals. The external appearances of the eyeballs eight days post treatment are shown in Fig. S34. Although the biocompatibility of the GNSs had been assessed in vitro previously, further toxicity tests in vivo were still needed. Hematoxylin and eosin (H&E) staining assays of major organs, including the heart, liver, spleen, lung, kidney, and brain, indicated no signs of toxicity across all six groups (Fig. S36). And the biochemical analysis of the blood of the mice revealed no damage to the liver and kidney functions (Fig. S38), demonstrating that GNSs possessed excellent biosafety and biocompatibility.
Figure 7a showed the external appearance of the eyeballs eight days post-treatment. Eyeballs treated with PBS or GNSs under dark were filled with tumor tissue, exhibiting significant vascular proliferation and disturbances in eyeball mobility. In comparison, the degree of tumor proliferation was relatively lower but the eyeball still appeared muddy and swollen in the NSs + NIR + WL, GNSs + NIR, and GNSs + WL groups, indicating certain therapeutic benefits of PTT or PDT alone, the NSs group showed unsatisfactory therapeutic effect because of the rapid release of nanosheets and lower nanosheets concentrations. Additionally, corneal epithelium loss was observed as a result of tumor overgrowth and distortion of the eyeball, with significant immersion of tumor cells into the eyeball evident from OCT sections. Remarkably, the eyeball of the GNSs + NIR + WL group treated with synergistic PTT and PDT was clear and without tumor proliferation, resembling that of the normal control group. Besides, the GNSs + NIR + WL group maintained intact ocular structures (sclera, choroid, retina) and stable corneal thickness compared to the normal group (Fig. S35), demonstrating good biosafety. To better visualize the proliferation of choroidal melanoma cells, eyeballs were subjected to H&E and Ki67 staining. Figure 7b demonstrated that tumor cells invaded the vitreous and massively proliferated extensively within the eyeball in the PBS or GNSs under dark groups, and tumors proliferated outside the vitreous merely in the NSs + NIR + WL, GNSs + NIR and GNSs + WL groups. In contrast, no tumor cells were observed inside the eyeball in the GNSs + NIR + WL group, which was similar to the normal control. After being treated with GNSs + NIR + WL plus dual-light exposure, the suppressed cell proliferation of the tumor region was further confirmed according to the Ki67 staining (Figs. 7b and S37a). Meanwhile, the denouement of severe pyroptosis of the treated tumor cells (underwent PTT plus PDT) was proved by TUNEL staining (Fig. S37b) and GSDMD-N staining (Fig. S37c). Overall, these results confirmed the excellent synergistic antitumor effects of PTT plus PDT with benefit of the GNSs under dual-light irradiation, presenting a novel method for choroidal melanoma treatment without any associated toxicity.Fig. 7. Antitumor effects of different treatments in vivo. a Postoperative observation using slit-lamp microscopy and anterior segment optical coherence tomography (AS-OCT). Representative slit-lamp and AS-OCT images of balb/c corneas taken at day 8. The white arrows showed the unepithelialized area. Scale bar: 500 μm. b Representative H&E, Ki67, TUNEL and GSDMD-N staining analysis of tumor tissues after various treatments. TUNEL/ GSDMD-N and nucleus were stained red and blue, respectively (scale bar of 1 mm and 100 μm)
Conclusion
In this study, we have successfully developed a pyroptosis-mediated synergistic photodynamic and photothermal 2D nanosheets, encapsulated into a stimuli-responsive Agar/PSBMA hydrogel, for the therapy of choroidal melanoma. The AIEgen-conjugated 2D MXene nanosheets exhibited a remarkable ability to generate type-I ROS, which effectively induce pyroptosis by targeting dual organelles within tumor cells, and its photothermal performance further accelerated the pyroptosis process of cancer cells. Meanwhile, the NIR light-triggered photothermal transition, gel–sol transformation and TME responsive characteristics of the Agar/PSBMA hydrogel enabled mild PTT effect and smart nanosheets release. These exceptional features endowed it with significant performance for in vitro and in vivo choroidal melanoma theranostics involving NIR FLI-PTI dual imaging-guided synergistic PDT-PTT. Importantly, the controlled release of GNSs and mild hyperthermia exhibited minimal impact on normal cells, ensuring the safety of intraocular tissue. The constructed GNSs thus emerged as a promising strategy for choroidal melanoma therapy by a “single injection, multiple treatment” method, and the mPTT/PDT/antibacterial platform with enhanced NIR imaging and easy metabolism may provide a novel strategy for sensitive and noninvasive imaging and phototherapy in ocular tumors.
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file 1 (DOCX 11223 KB)