New features of edge-selectively hydroxylated graphene nanosheets as NIR-II photothermal agent and sonothermal agent for tumor therapy

Abstract

Second near-infrared (NIR-II) laser-mediated photothermal therapy and sonothermal therapy using low-intensity focused ultrasound exposure for tumors have attracted increasing attention owing to their ability to penetrate deep tissues and provide noninvasive ablation with high therapeutic efficacy. However, their applications were limited by the shortness of optimal NIR-II photothermal agents and sonothermal agents. In this study, we discovered that the edge-selectively hydroxylated graphene nanosheets (EHG NSs) with excellent water dispersibility and an “intact conjugated plane” were not only an outstanding NIR-II photothermal agent but also an effective sonothermal agent for tumor therapy. EHG NSs were incorporated into an injectable adhesive thermosensitive hydrogel with a characteristic sol–gel phase transition behavior. EHG NSs endowed the injectable hydrogel with an exceptional photothermal effect under the laser irradiation (1064 nm, 1.0 W cm⁻²) as well as an effective sonothermal effect under ultrasonic exposure (3.0 MHz, 2.1 W cm⁻²), effectively killing tumor cells in vitro and inhibiting tumor growth after intratumoral injection. Especially, the NIR-II photothermal therapy based on the hybrid hydrogel completely ablated the primary tumors and effectively activated systemic anti-tumor immune responses benefiting from the protein adsorption capacity of the injectable hydrogel, significantly inhibiting the growth of the distal tumors. Collectively, EHG nanosheets loaded in the injectable hydrogel will be a promising “all-rounder” for noninvasive deep penetrating thermotherapy and a potent platform that integrates various therapies.

---

Introduction

Cancer is a serious threat to human health. Surgery resection is a usually preferred treatment for solid tumors, but it cannot be carried out on medium and advanced cancer patients. Meanwhile, traditional chemotherapy and radiotherapy have huge toxic and side effects. Therefore, many new methods of treating cancer have been developed, such as noninvasive photothermal therapy (PTT), photodynamic therapy, sonodynamic therapy and chemodynamic therapy based on nanomaterials.¹ Among them, thermal therapy based on the physical thermal conversion of photothermal or sonothermal agents has the potential to completely ablating tumors, but the treatment efficiencies of the dynamic therapies are seriously limited by the short half-life and action distance of reactive oxygen species and insufficient concentration of oxygen and hydrogen peroxide in the tumor microenvironment.² Especially, second near-infrared (NIR-II) PTT and sonothermal therapy (STT) under low-intensity focused ultrasound (LIFU) exposure have advantages of deeper tissue penetration than NIR-I PTT^3–5 and minimal damage to the normal tissues in comparison with high-intensity focused ultrasound therapy.⁶ Therefore, NIR-II PTT and STT based on LIFU have received much attention in recent years.^6–10

Various materials have been developed for NIR-II photothermal therapy, including Au nanomaterials,⁸ inorganic 2D nanomaterials,^9,11 organic D–A-type small molecules and conjugated polymers,^12–14 and organic semiconducting dyes.¹⁵ These materials usually involve complicated preparation processes or have high cost. Graphene-based nanomaterials including graphene oxide (GO) and reduced GO nanosheets (NSs) are widely used as NIR-I photothermal agents due to their good biosafety, easy functionalization and strong photothermal stability.^16–18 However, they are seldom used alone in the NIR-II PTT instead by surface functionalization or combined with other therapies.^16,19–21 The reason could be attributed to a contradiction between “water dispersibility” and “intact conjugated plane” of graphene-based photothermal agents. The more flawless the planar conjugated structure of graphene-based material is the more its absorption wavelength shifts to longer wavelength, which will endow them with strong absorption in the NIR-II region. However, they are inevitably unstable and agglomerate in water.²¹ Although the introduction of hydrophilic groups can enhance the water dispersibility of graphene, it also destroys the basal plane structure and affects its photothermal effect in the NIR-II region.

Similar to NIR-II laser, acoustic energy is also a promising source for hyperthermia with deeper tissue penetration and lesser damage to normal tissue. Sonic energy can be converted into thermal energy via sonothermal effects.²² As reported, the temperature increase induced by ultrasound (US) exposure can be promoted by nanomaterials, such as gold nanoparticles (NPs),²³ porous silicon NPs,²⁴ magnetic NPs,²⁵ red phosphorus nanoparticles²⁶ and GO NSs.²⁷ The sonothermal mechanism of these nanomaterials mainly involves the enhancement of acoustical absorption, ultrasonic cavitation and thermal conductivity. In addition, GO NSs could absorb sound waves due to the microvibrations of the aligned layers²⁸ and convert the acoustic energy into heat by molecular friction of the solid domain in the vibration mode.^10,25,29,30 However, the increased temperature induced by these reported nanomaterials made it hard to reach an adequate temperature under LIFU irradiation for tumor hyperthermia. For example, the temperature of Fe₃O₄ NPs dispersion²⁵ and gold NPs³¹ increased no more than 10 °C under the LIFU irradiation. Though red phosphorous-coated titanium plates¹⁰ and solid powders of the phase change material loaded with Fe₃O₄/GO NPs³⁰ could reach a relatively high temperature under US irradiation, they were difficult to use in vivo. Therefore, it is critical to develop novel sonothermal nanomaterial with satisfactory sonothermal performance for applications in vivo.

For the edge-selectively oxidized graphene, such as edge-hydroxyl graphene nanosheets (EHG NSs) and edge-carboxylated graphene NSs, the hydrophilic groups are only introduced at the edges of graphene NSs. Therefore, they keep the relatively complete conjugated planar structure as well as hydrophilicity.^32–34 Our previous study indicated that EHG NSs possessed good aqueous dispersibility and high photothermal conversion efficiency in vitro.³⁵ It may be an excellent sonothermal agent for STT due to its unique structure. Therefore, in this work, EHG NSs were encapsulated in the adhesive injectable thermosensitive hydrogel based on poly(N-isopropylacrylamide-co-dopamine methacrylamide) (PND) nanogels (the mixture was named as EHG@PND), which allowed EHG NSs to stay in the tumor for a long time helping for multiple treatments. The anti-tumor effects based on EHG NSs-mediated NIR-II PTT and STT using LIFU were investigated (Scheme 1). In comparison with the reported injectable hydrogel used in NIR-II PTT,^36,37 the PND hydrogel was able to capture tumor antigens produced by PTT to enhance the photothermal immunotherapy due to the adhesive catechol groups.³⁸ Therefore, the injectable hybrid hydrogel-based NIR-II PTT and STT could effectively inhibit tumor growth. The former could also suppress the growth of the distal tumor via activated CD8⁺ T cells. Therefore, EHG nanosheets will be a novel and promising nanomaterial for noninvasive and deep-penetrating local thermotherapy.

Scheme 1 Schematic diagram showing the tumor treatment of NIR-II PTT and STT based on the injectable EHG@PND hydrogel.

Experimental

Materials, cells and animals

Graphite (8000 mesh, >99.5%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. GO aqueous dispersion (10 mg mL⁻¹) was obtained from Hangzhou Gaoxi Technology Co., Ltd. N-Isopropylacrylamide (NIPAM, >98.0%) were provided by Shanghai Macklin Biochemical Co., Ltd. N-[2-(3,4-Dihydroxyphenyl)ethyl]-2-methyl-2-propenamide (DMA, >95.0%) was purchased from Jiangsu Aicon Biopharmaceutical R&D Co., Ltd. Potassium persulfate (KPS) and other chemical agents were analytical reagents and obtained from Sinopharm Chemical Reagent Co., Ltd. Cell counting kit-8 (CCK-8) was obtained from Dongren Chemical Technology (Shanghai) Co., Ltd. Roswell Park Memorial Institute 1640 medium, fetal bovine serum, trypsin, penicillin–streptomycin were obtained from Gibco Invitrogen Corp. Bicinchoninic acid assay (BCA) protein assay kit was purchased from Beyotime Institute of Biotechnology Co., Ltd. Mice high mobility group protein 1 (HMGB1) enzyme-linked immunosorbent assay (ELISA) kit, cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) ELISA kits and other assay kits were purchased from Wuhan MSK Biotechnology Co., Ltd.

The mouse breast cancer 4T1 cells, mouse melanoma B16-F10 cells, mouse L929 fibroblasts and human umbilical vein vascular endothelial cells (HUVECs) were all obtained from the China Center for Type Culture Collection. The mice were female BALB/c mice (6–8 weeks) and purchased from the Laboratory Animal Center of China Three Gorges University. All animal experiments were carried out by qualified operators (Certificate Numbers TY2018601 and TY20220701) under the protocols approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (Wuhan, China).

Preparation of the injectable thermosensitive EHG@PND hydrogel

PND nanogels were prepared according to the method described previously using NIPAM and DMA as co-monomers with the feeding molar ratio of 9 : 1.³⁸ Similarly, pure PNIPAM without the catechol groups (denoted as PNI) was prepared as a control. ¹H NMR spectrum of the PND nanogels was obtained on a 400 MHz NMR spectrometer (Avance III, Bruker, Germany) using D₂O as a solvent. The thermosensitive property of the PND nanogels was characterized by measuring the absorbance change of the nanogel dispersion at 560 nm with increasing temperature. The average hydrodynamic diameter of the PND nanogels was measured by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments Ltd, UK) at 25 °C.

EHG NSs were prepared via a ball milling method using a planetary ball mill (YXQM-4L, Changsha MITR Instrument Equipment, China).³⁵ Briefly, 240 g of zirconia balls with various diameters, 0.40 g of graphite powder and 5.60 g of KPS as milling agents and oxidants were placed in a 250 mL milling jar. After milling at 350 rpm for 2 h, the mixture was washed with DI water and then re-dispersed under ultrasonication to obtain crude EHG dispersion. The dispersion was then centrifuged at 3000 rpm for 10 min to discard the big particles and the EHG NSs were obtained in the supernatant for further use. The obtained EHG NSs were named E1. In addition, 7.20 g of KPS instead of 5.60 g was adopted in the above procedure to obtain another EHG NSs named E2.

Subsequently, according to the absorption coefficient of and E2 at 660 nm of 3210 and 4150 L g⁻¹ m⁻¹, respectively, the concentration of the EHG NSs in the stock dispersion was determined using a UV-vis-NIR spectrometer (UV 3600, Shimadzu) and it was diluted to approximately 250 μg mL⁻¹ for stock.³⁵ The stock E1 and E2 dispersions were then diluted to a certain concentration and followed by the addition of the lyophilized powder of PND nanogels. The mixed dispersions were fully swollen overnight in a refrigerator at 4 °C to obtain the injectable E1@PND and E2@PND hydrogel for further use. The injectable E1@PND hydrogel was composed of 100 mg mL⁻¹ of PND nanogels and 50 μg mL⁻¹ of the E1. The injectable E2@PND hydrogel was composed of 80 mg mL⁻¹ of PND nanogels and 1.0 mg mL⁻¹ of the E2. In addition, the PNI lyophilized powder was mixed with the E1 dispersion to obtain the injectable hydrogel with the concentration of PNI of 100 mg mL⁻¹ and the E1 of 50 μg mL⁻¹ (named as E1@PNI), which was used as a control in the following NIR-II photothermal experiments.

Morphology and structure characterization of EHG nanosheets

The EHG NS aqueous dispersion (5 μg mL⁻¹) was dropped on the copper grid and followed by drying at room temperature. Its micromorphology was observed using a transmission electron microscope (TEM, Tecnai G2-F30, FEI) at an accelerated voltage of 200 kV. The Raman spectra of the freeze-dried GO and EHG powder were measured on a laser confocal Raman spectrometer (LabRAMHR800, Horiba) with a laser energy of 2.33 eV and a wavelength of 532 nm. The X-ray photoelectron spectroscopy (XPS) of EHG NSs was performed on an X-ray photoelectron spectrometer (AXIS Supra⁺, Shimadzu, Japan) using Al Kα rays (E = 1486.6 eV) as the excitation source. The absorbance of the EHG dispersion (including E1 and E2) and GO dispersion was recorded on a UV-vis-NIR spectrometer at 25 °C with a scanning wavelength range of 200–1200 nm.

Sol–gel phase transition behavior and protein-adsorption properties of the injectable EHG@PND hydrogel

The thermosensitive sol–gel phase transition behavior of the injectable E1@PND and E2@PND hydrogels was first observed using a bottle inversion method. Then, the storage modulus (G′) and loss modulus (G′′) of the injectable hydrogel varying with the temperature were measured on a rheometer (Kinexus, Malvern) using a parallel plate (diameter: 50 mm; gap: 0.5 mm). The temperature range was 25–50 °C with a heating rate of 3 °C min⁻¹. The frequency and shear strain were set as 1.0 Hz and 1%, respectively. The viscosity changes of the injectable hydrogel at 25 °C were measured when the shearing rate increased from 0.1 to 1000 s⁻¹. The micromorphology of the forming hydrogel at 37 °C was characterized by scanning electron microscopy (SEM, Sirion 200, FEI) with an accelerating voltage of 5.00 kV. The protein-adsorption properties of the forming E1@PND hydrogel were investigated using a BCA protein quantification kit with fetal bovine serum (FBS) as a model protein.

NIR-II photothermal properties of the injectable EHG@PND hydrogel in vitro

1.0 mL of the injectable hydrogel with 100 mg mL⁻¹ of PND and various amounts of E1 was placed in a 5 mL centrifuge tube and followed by irradiation under a 1064 nm laser (MDL-H-1064-2.5 W, Changchun Laser Optoelectronic Technology Co., Ltd) for 10 min at the laser power density of 1.0 W cm⁻² with the spot diameter of the laser of 1 cm and distance between laser head and sample of 10 cm. The temperature changes were recorded with a digital thermometer (DM6801A, Shenzhen Shengli Gao Electronic Technology Co., Ltd). Then, the injectable E1@PND hydrogel (E1: 50 μg mL⁻¹) was irradiated under different laser power densities (0.5, 0.8, 1.0 or 1.2 W cm⁻²) using a 1064 nm laser to investigate the effect of the laser power density. The photothermal stability of the injectable hydrogel was determined as follows. The injectable E1@PND hydrogel was irradiated by a 1064 nm NIR laser (1.0 W cm⁻²) for 5 min firstly. Then, the light source was turned off and the sample was cooled to room temperature naturally. The on–off cycle of laser irradiation was repeated 5 times. The entire temperature changes were recorded. In order to calculate the photothermal conversion efficiency (η) of the injectable E1@PND hydrogel, 1.0 mL of the system was irradiated under a NIR-II laser (1064 nm, 1.0 W cm⁻²) for 30 min, then cooled to room temperature naturally. The temperature changes were recorded and the η was calculated as previously reported.³⁸

In addition, to investigate the photothermal performance of the injectable E1@PND hydrogel under NIR-II irradiation in deep tissues, 200 μL of the injectable hydrogel was added in a 96-well plate, covered by the chicken breast tissue of various thicknesses (2 and 5 mm) and then irradiated by 1064 or 808 nm laser (1.0 W cm⁻²) for 15 min. The temperature was monitored using a digital thermometer.

Sonothermal properties of the injectable EHG@PND hydrogel in vitro

The temperature changes of the E1, E2 and E2@PND dispersions with different E2 concentrations under US exposure (3.0 MHz, 1.2 W cm⁻², 40% duty cycle) were monitored using a US probe (diameter of 2.0 cm, DM-300B, Shenzhen Dimip Technology Co., Ltd) for 10 min and recorded using a digital thermometer (VICTOR6801, Shenzhen Yisheng Victor Technology Co., Ltd). These dispersions (1.0 mL) were separately placed in a 4.5 mL polystyrene cuvette and its bottom was contacted with the US probe by the coupling gel. In addition, the effects of the US intensity on the temperature change of the E2@PND dispersion (concentration of E2: 1.0 mg mL⁻¹) were studied. The on–off cycle of US irradiation was repeated 5 times and the temperature changes were recorded.

Additionally, the deep-tissue sonothermal performance of the injectable E2@PND hydrogel under US exposure was explored by adding 200 μL of the injectable hydrogel in a 96-well plate and then covered with pork tissue of various thicknesses (0.5, 1.0 and 1.5 cm). The temperature change was recorded by a digital thermometer during ultrasonication using a US probe (3.0 MHz, 1.8 W cm⁻², and 60% duty cycle) for 5 min.

Cytotoxicity of the EHG@PND hydrogel-based PTT and STT in vitro

For the E1@PND hydrogel-based PTT, 4T1 cells and B16-F10 cells were placed in the lower chamber of a 24-well transwell plate (1 × 10⁵ cells per well) and cultured in 500 μL of RPMI 1640 complete medium in 5% CO₂ incubator (37 °C) for 24 h. Then, 200 μL of the formed E1@PND hydrogel at 37 °C was placed into the upper chamber of the transwell, followed by irradiation under NIR-II laser (1064 nm, 1.0 W cm⁻²) for 10 min. The cell viability was detected using CCK-8 assay after co-culture for 24 h. Similarly, the cytotoxicity induced by the injectable E2@PND hydrogel-based STT was detected under US exposure (3.0 MHz, 1.8 W cm⁻², and 60% duty cycle) for 5 min instead of NIR laser irradiation. The bottom of the 24-well plate was exposed to the US probe by the coupling gel.

Cytocompatibility of the EHG@PND hydrogel in vitro

As described above, mouse L929 fibroblasts and HUVECs (5 × 10⁴ cells well⁻¹) were firstly cultured in the lower chamber of a 24-well transwell plate for 24 h and then co-cultured with 200 μL of the forming E1@PND hydrogel for 24 h. The cell viability was detected by the CCK-8 method to evaluate the cytocompatibility of the E1@PND hydrogel. In addition, the viability of HUVECs co-cultured with the E2@PND hydrogel for 24, 48 and 72 h, respectively, were also evaluated using a similar method.

Immunogenic cell death induced by the EHG@PND hydrogel-based NIR-II PTT and STT in vitro

In order to investigate the immunogenic cell death (ICD) induced by the EHG@PND hydrogel-based NIR-II PTT and STT, 4T1 cells were cultured in 24-well plates (2 × 10⁵ cells well⁻¹) using a standard procedure. For the E1@PND hydrogel-based NIR-II PTT, the cells were irradiated immediately after the sample addition under NIR-II laser (1064 nm, 1.0 W cm⁻²) for 5 min. For the E2@PND hydrogel-based STT, the cells were treated by US exposure (2.1 W cm⁻², 70% duty cycle, and 5 min) immediately after adding the sample. Then, the medium was removed and replenished with a fresh serum-free medium. After incubation for another 4 h, 4T1 cells were digested with trypsin enzyme and collected to detect the content of the exposed calreticulin (CRT) using a flow cytometry (Cytoflex, Beckman Coulter, USA) and secreted HMGB1 using an HMGB1 ELISA kit.^38,39

NIR-II PTT based on the injectable E1@PND hydrogel for unilateral tumor-bearing mice

100 μL of PBS containing 2 × 10⁶ 4T1 cells was injected subcutaneously into the right flanks of female BALB/C mice (6–8 weeks). When the tumor grew up to about 100 mm³, the tumor-bearing mice were randomly divided into 4 groups (n = 5 per group) and intratumorally injected with 50 μL of various materials: PBS (pH 7.4) (named as PBS group); injectable E1@PND hydrogel dispersion (named as E1@PND group); injectable E1@PNI hydrogel dispersion and then exposing to 1064 laser (1.0 W cm⁻²) for 10 min (named as E1@PNI + NIR group); injectable E1@PND hydrogel dispersion and then exposing to 1064 laser (1.0 W cm⁻²) for 10 min (named as E1@PND + NIR group). The temperature at the tumor site was immediately recorded with a thermal imager (E50, FLIR, USA) after laser irradiation. The tumor volumes and body weights were recorded every other day after treatments.

On day 14 after treatments, the mice were sacrificed and the tumors were collected for photographing and weighing. The major organs including the heart, kidney, spleen, lung and kidney of mice in each group were harvested for hematoxylin and eosin (H&E) staining for pathological analysis.

Antitumor and immune activation effects of the injectable E1@PND hydrogel-based NIR-II PTT for bilateral tumor-bearing mice

Female BALB/c mice (6 to 8 weeks) bearing bilateral tumor model were established by subcutaneously injecting 1 × 10⁶ and 5 × 10⁵ 4T1 cells per mouse into the right flank and the left flank of mice, respectively. When the tumor volume on the right flank of the mice reached 150 mm³, the tumor-bearing mice were randomly divided into 4 groups with 8 mice per group. Then the primary tumors (right side) were treated with different materials. The PBS group and E1@PNI group were treated the same as those in the unilateral tumor experiments. As for the E1@PNI + NIR group and E1@PND + NIR group, laser irradiation (1064 nm, 1.0 W cm⁻²) for 10 min was performed on day 1 and day 4 after materials injection, respectively. The tumor volume on both sides and the body weight of the mice were measured every other day after treatments.

On day 8 after treatments, three mice in each group were randomly selected. The blood of mice was collected and the sera were obtained by centrifugal separation to determine the concentrations of IFN-γ and TNF-α in sera using ELISA kits according to the manufacturer's protocol. In addition, their distal tumors were collected to analyze the content of CD8⁺ T cells in the tumor tissue using the immunofluorescence method. On day 14 after the treatment, the distal tumors of the other mice were taken for TUNEL and Ki67 staining to evaluate the tumor cell apoptosis and proliferation, respectively. Major organs of mice (heart, kidney, spleen, lung, and kidney) were sliced for H&E staining to evaluate their pathological changes.

Anti-tumor effects of the injectable E2@PND hydrogel-based STT in vivo

A unilateral 4T1 tumor-bearing mice model was established as described above. Firstly, the tumor-bearing mice were subcutaneously injected with 50 μL of the injectable E2@PND hydrogel and sacrificed on days 1, 4 and 7 to monitor the stability of the hydrogel in vivo. Then, the tumor-bearing mice were randomly divided into 4 groups (n = 4 per group): PBS, E2@PND, PBS + US and E2@PND + US with intratumorally injected with 50 μL of PBS or E2@PND dispersion. For PBS + US and E2@PND + US groups, the tumors were treated by US exposure (3.0 MHz, 2.1 W cm⁻², 70% duty cycle, 5 min) after 5 min of the sample injection. The temperature of the tumor site was immediately recorded using a thermal imager after US exposure. Then, the US irradiation was conducted on days 2, 4 and 6 again. The tumor volumes and body weight were recorded every other day after treatments. On day 14 after treatments, the mice were sacrificed and the tumors and major organs were harvested for H&E staining. In addition, the mice's blood was collected for the biochemical analysis including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urine nitrogen (BUN), and creatinine (CREA) using clinical analysis method.

Statistical analysis

The data were shown as mean ± standard deviation (n ≥ 3). Statistical analyses were performed based on 2-tailed Student's t-tests using the GraphPad Prism software. P < 0.05 was considered as with statistically significant difference (*P < 0.05, **P < 0.01, and ***P < 0.001), while P > 0.05 was regarded as no significant differences (ns).

Results and discussion

Preparation and characterization of the injectable EHG@PND hydrogel

EHG NSs including E1 and E2 were prepared by the mechanochemical method using graphite flakes as the raw material and potassium persulfate as the grinding agent and oxidant in mass ratios of 1 : 14 and 1 : 18, respectively.³⁵ TEM images showed that E1 and E2 had an irregular thin lamellar structure (Fig. 1A and Fig. S1A, ESI†). FTIR spectra (Fig. 1B) showed obvious stretching vibration peaks of O–H (3500 cm⁻¹), C=O (1720 cm⁻¹), C=C (1580 cm⁻¹ for the conjugated double bond and 1623 cm⁻¹ for the non-conjugated one), and C–O (1045 cm⁻¹ for alkoxy and 1209 cm⁻¹ for the phenolic hydroxyl group). According to the relative intensity of peaks, E1 and E2 had better conjugation effects than commercially available GO. The XPS spectrum of E1 suggested the existence of oxygen and carbon atoms (Fig. S1B, ESI†). Raman spectra indicated that EHG and GO had a graphite characteristic peak (G band) generated by stretch vibrations of sp² carbon at 1567 cm⁻¹ and 1590 cm⁻¹, respectively (Fig. 1C).⁴⁰ The defect peak (D band) generated by the disordered vibration of the suspended carbon atoms in the inner or edge defects of graphene appeared at 1338 cm⁻¹ and 1352 cm⁻¹, respectively. It was noted that the peak width at half heights of E1 and E2 was narrower than that of GO. The 2D band of EHG NSs appeared at 2676 cm⁻¹. The disappearance of the D′ band in GO indicated the transition of GO from nanographite to sp³ amorphous carbon. In addition, the I_D/I_D′ values of E1 and E2 were calculated to be about 2.2 and 2.1, respectively, indicating that their defects were mainly located at the edges of graphene.⁴¹

Fig. 1 Basic characteristics of the EHG and injectable EHG@PND hydrogel. (A) TEM image of E1. (B) FTIR spectra of the E1, E2 and GO. (C) Raman spectra of the E1, E2 and GO. (D) Normalized UV-vis-NIR spectra of the E1, E2 and GO aqueous dispersion. (E) Transmittance of the PNI and PND nanogel dispersion at 560 nm versus temperature. (F) and (G) Modulus of the injectable E1@PND and E2@PND hydrogel versus temperature. (H) Shearing-thinning properties of the injectable E1@PND and E2@PND hydrogel at 25 °C. (I) SEM image of the formed E1@PND hydrogel.

Moreover, the defect density (n_D) and the average defect distance (L_D) of the graphene can be calculated based on the ratio of I_D/I_G according to the following equations reported elsewhere.⁴²Eqn (1) and (2) were used to calculate the n_D and L_D of EHG NSs, while eqn (3) and (4) were used for GO.

In this work, the laser energy (E_L) utilized was 2.33 eV, while the I_D/I_G ratio of E1, E2 and GO was determined as 1.03, 1.21 and 0.92, respectively, based on Fig. 1C. Consequently, the n_D and L_D values for E1 and E2 were calculated as 2.22 × 10⁻³ nm⁻², 11.90 nm; and 2.60 × 10⁻³ nm⁻², 10.98 nm, respectively. While GO exhibited a higher defect density of 0.22 nm⁻² and shorter adjacent defect space with 1.21 nm than those of the EHG NSs. These findings suggested that the planar conjugated structure of the EHG NSs was more complete, enabling them to efficiently absorb long-wave light for photoelectron generation and induce thermal radiation through electron transition. Notably, UV-vis-NIR spectra demonstrated a red shift in the maximum absorption peaks of the EHG NSs to 266 nm from GO's 230 nm (Fig. 1D). Importantly, the absorption capacity of E1 in the near-infrared region, including NIR-II, was stronger than those of GO and E2, indicating its superior potential for NIR-II photothermal effects. According to the ratio of the absorbance at 1064 nm to 660 nm, the absorption coefficient of the E1 and E2 at 1064 nm was calculated to be 2553 and 3184 g L⁻¹ m⁻¹, respectively, and that of the GO was nearly zero due to its negligible absorption at 1064 nm. However, the molar absorption coefficient of the E1 and E2 could not be determined because their molecular weight was unknown. Due to the lower defect density, longer adjacent defect space and higher conductivity induced by less KPS used during its preparation as we reported obviously,³⁵ E1 was used as NIR-II photothermal agent in the further study.

PND nanogels were prepared according to our previous reports.^38,431H NMR spectra of the PND and PNI hydrogel are shown in Fig. S2A (ESI†). Compared to PNI, the peaks observed at 6.62–6.78 ppm and 7.69–7.87 ppm could be assigned to the characteristic peaks of dopamine in PND.⁴⁴ The average hydrodynamic diameter of the PND nanogels at 25 °C was approximately 234 nm with a polydispersity index of 0.083 measured by DLS. The changes in transmittance of the PND and PNI dispersions with increasing temperature, as well as their corresponding differential curves, indicated a similar lower critical solution temperature of ∼33 °C (Fig. 1E). The injectable E1@PND, E1@PNI and E2@PND hydrogels were prepared by dispersing lyophilized nanogel powders in an aqueous dispersion of EHG NSs at a nanogel concentration of 100 and 80 mg mL⁻¹, respectively. The concentrations of E1 and E2 in their dispersions were 50 μg mL⁻¹ and 1.0 mg mL⁻¹, respectively. Visual observations depicted in Fig. 1F and G illustrated that the injectable EHG@PND hydrogels, including both E1@PND and E2@PND, exhibited flowability at room temperature (25 °C) but formed bulk hydrogels at ∼32 °C due to the thermosensitive nature of the PND nanogels. The loss modulus (G′′) of the hydrogel was higher than its storage modulus (G′) at a temperature below ∼31.5 °C, but reversed as the temperature increased, indicating a sol–gel phase transition occurring at this temperature. Similar behavior was observed in the E1@PNI dispersion at 32 °C (Fig. S2B, ESI†). In addition, the injectable EHG@PND hydrogel exhibited significant shear-thinning properties (Fig. 1H) and could be injected at room temperature and formed a hydrogel in situ after intratumoral injection. SEM images showed that both the PND (Fig. S2C, ESI†) and E1@PND hydrogels (Fig. 1I) had typical porous three-dimensional network structure, while UV-vis-NIR absorbance spectra indicated that neither PND nor PNI interfered with the strong NIR-II absorbance of the E1 (Fig. S2D, ESI†).

NIR-II photothermal and sonothermal effects of the injectable EHG@PND hydrogel in vitro

Based on the stronger absorption of NIR-II light of the E1 compared to the E2 and GO, the E1 was chosen as a NIR-II photothermal agent for subsequent experiments. Fig. 2A–E showed the photothermal effects of the injectable E1@PND hydrogel under irradiation of a 1064 nm laser. Among them, the effect of the E1 concentration in the injectable hydrogels on the photothermal effect was shown in Fig. 2A. The results demonstrated that the temperature increment (ΔT) of the injectable hydrogel increased with the increase of E1 concentration, and the tendency slowed down with the further concentration increasing. Furthermore, ΔT exhibited an increase with higher laser power density (Fig. 2B). Considering minimal differences in ΔT when both concentration and laser power density were further increased, subsequent photothermal experiments were conducted under the irradiation of 1.0 W cm⁻² and the E1 concentration of 50 μg mL⁻¹. To validate the enhanced immunity effect of the E1@PND hydrogel-based NIR-II PTT due to its potent antigen capture capability, a non-adhesive control consisting of E1@PNI hydrogel with an E1 concentration of 50 μg mL⁻¹ and PNI concentration of 100 mg mL⁻¹ was employed.³⁸ It was found that both injectable hydrogels exhibited similar temperature rising under the same power density of 1064 nm laser (Fig. 2B). In addition, the injectable E1@PND hydrogel showed good photothermal stability during five cycles of on–off laser irradiation (Fig. 2C), indicating its suitability for multiple photothermal treatments. Then, the photothermal conversion efficiency (η) of the injectable E1@PND hydrogel was determined according to previously reported methods.³⁸ The heating and cooling curves of the E1@PND dispersion (1.0 g) under NIR irradiation (1064 nm, 1.0 W cm⁻²), as shown in Fig. S3A (ESI†), were used for the calculation of the η. The absorbance of the E1@PND dispersion at 1064 nm was 1.506. The resulting cooling time versus ln θ curve is presented in Fig. S3B (ESI†), where θ represents the driving force temperature. The slope of this linear fitting curve was 704.46, which corresponds to the system's time constant (τ_s). In addition, considering that water had a specific heat capacity of 4.186 J (g °C)⁻¹, and the maximum ΔT values observed for water and the dispersion under NIR irradiation were 17.4 °C and 40.2 °C, respectively, the η of the injectable E1@PND hydrogel was found to be 20.5%.

Fig. 2 Photothermal and sonothermal effects of the injectable EHG@PND hydrogel in vitro. (A)–(E) Photothermal effects of the injectable E1@PND hydrogel under an irradiation of 1064 nm laser. (A) Effect of the concentration of E1 on the temperature increment under 1064 nm laser irradiation (1.0 W cm⁻²) (n = 3); (B) effects of the laser power density on the temperature increment (n = 3); (C) periodic changes in the temperature during the on–off cycles of the laser irradiation; (D) viabilities of 4T1 cells; and (E) viabilities of B16–F10 cells after various treatments (n = 5). (F)–(I) Sonothermal effects. (F) Heating curves of the E2 dispersion with various concentrations under US exposure (1.2 W cm⁻²) (n = 3); (G) heating curves of the E2@PND dispersion under US exposure with different US power density (n = 3); (H) temperature change of the E2@PND dispersion under on–off cycles of the US exposure (1.8 W cm⁻²); and (I) viabilities of 4T1 cells after different treatments (n = 4).

Two kinds of tumor cells (mouse breast cancer 4T1 cells and mouse melanoma B16-F10 cells) were used to test the phototoxicity of the injectable E1@PNI and E1@PND hydrogels under the irradiation of 1064 nm laser (1.0 W cm⁻²) for 10 minutes. The results (Fig. 2D and E) showed that the survival ratio of the cells in the non-NIR group was similar to that in the control group without obvious toxicity. However, the cell viabilities decreased significantly below 10% in both E1@PNI + NIR and E1@PND + NIR groups, indicating that the injectable E1@PNI and E1@PND hydrogels exhibited strong NIR-II photothermal effects to effectively kill tumor cells under laser irradiation.

Furthermore, the sonothermal effects of E1 and E2 were investigated in comparison with those of GO under US exposure (3.0 MHz, 1.2 W cm⁻², 40% duty cycle) for 10 min. As shown in Fig. 2F, the ΔT increased with the sequence of water, GO, E1 and E2 when the latter three had the same concentration (1.0 mg mL⁻¹). Therefore, E2 was chosen as the preferred sonothermal agent. The ΔT of the injectable E2@PND hydrogel depended on both the E2 concentration (Fig. 2F) and the US power density (Fig. 2G). Notably, higher concentrations and power density resulted in a faster and higher temperature rise. When exposed to US (1.2 W cm⁻²) for 10 min, the ΔT of the injectable E2@PND hydrogel with an E2 concentration of 1.0 mg mL⁻¹ was found to be 26.2 °C, meeting the requirements for thermal therapy in tumors. Moreover, the injectable E2@PND hydrogel showed excellent sonothermal stability even after five on–off cycles of US exposure (Fig. 2H). Fig. 2I shows significantly decreased cell viabilities in the E2@PND group under US irradiation due to its strong sonothermal effect.

In addition, considering the advantages of the deep tissue penetration capacity of NIR-II laser and ultrasonic waves, the photothermal effects of the injectable E1@PND hydrogel and sonothermal effects of the injectable E2@PND hydrogel in deep tissue were investigated. As shown in Fig. S4A (ESI†), the ΔT of the injectable E1@PND hydrogel was 7.2 °C under 1064 nm laser irradiation when it was covered by chicken breast tissue of 2 mm thickness, while there was no significant temperature change in other cases. A higher ΔT might be achieved by increasing the concentration of E1. As for the deep-tissue sonothermal performance of the injectable E2@PND hydrogel, ΔT was 19.8, 15.4 and 9.8 °C in case of pork tissue of 0.5, 1.0 and 1.5 cm thickness, respectively (Fig. S4B, ESI†). These results suggested the promising applications of the E1@PND and E2@PND for deep-tissue thermal therapy.

To investigate whether NIR-II PTT and STT based on the injectable EHG@PND hydrogel induced immunogenic cell death (ICD), representative markers including HMGB1 release amount and CRT exposure were measured accordingly. The results indicated that HMGB1 release amount and CRT exposure in the group of E1@PND + NIR significantly increased compared to those in the non-irradiation groups (Fig. S5A and B, ESI†) while both increased slightly in the group of E2@PND + US (Fig. S5C and D, ESI†). Therefore, NIR-II PTT based on the injectable E1@PND hydrogel not only effectively killed tumor cells, but also induced ICD. However, the induction of ICD by STT based on the injectable E2@PND hydrogel was limited.

To confirm the ability of the EHG@PND hydrogel to capture tumor antigens produced during PTT, we investigated the adsorption capacity of the PND and EHG@PND hydrogel for FBS. The results (Fig. S5E and F, ESI†) showed that after co-incubation, the amount of protein adsorbed by the forming PND, E1@PND, and E2@PND hydrogel was approximately equal and three times higher than that of the PNI, E1@PNI, and E2@PND hydrogel, respectively. Therefore, the catechol groups in the PND significantly enhanced the protein adsorption capacity of the hydrogel.

Anti-tumor effects of the NIR-II PTT based on the injectable E1@PND hydrogel in vivo

The 4T1 tumor-bearing mice model was constructed to validate the photothermal therapeutic efficacy of the injectable E1@PND hydrogel under 1064 nm laser irradiation. After intratumoral injection of different samples, the local temperatures of the tumors after 10 min under 1064 nm laser irradiation (1.0 W cm⁻²) were recorded using an infrared thermal imager. It was indicated that the temperature in both PTT groups (E1@PNI + NIR and E1@PND + NIR) reached 55 °C, while those in the PBS group and the E1@PND group remained below 32 °C (Fig. 3A). Correspondingly, the tumor volume increased rapidly in PBS and E1@PND groups, exceeding an average volume of 1000 mm³ on day 14 (Fig. 3B). In contrast, tumors in both PTT groups almost completely disappeared on day 8 post treatments and were indeed completely ablated without recurrence verified by the removed tumor tissues on day 14 after treatments (Fig. 3C and D). These data indicated that NIR-II PTT utilizing injectable thermosensitive hydrogels containing E1 nanosheets exhibited potent anti-tumor effect.

Fig. 3 Anti-tumor effects of the injectable E1@PND hydrogel-based NIR-II PTT on unilateral-tumor bearing mice. (A) Infrared thermal imaging of the tumor-bearing mice under 1064 nm laser irradiation (1.0 W cm⁻²) for 10 min. (B) Tumor-growth curves of different groups (n = 5). (C) Photographs of the tumors harvested from mice on day 14 after treatments. (D) Weight of the tumors harvested from mice on day 14 after treatments (n = 5). ns P > 0.05 and ***P < 0.001.

Anti-tumor effects of the STT based on the injectable E2@PND hydrogel in vivo

Because the sonothermal effect of the EHG was weaker than its photothermal effect, multiple sonothermal treatments were performed to improve the anti-tumor effect. Therefore, the stability of the E2@PND hydrogel in vivo was firstly evaluated by subcutaneous injection. As shown in Fig. S6 (ESI†), the hydrogel was still present at the injection site after 7 days, which demonstrated the good stability of the E2@PND hydrogel in vivo. To investigate the antitumor efficiency of the STT based on the injectable E2@PND hydrogel in vivo, the mice bearing subcutaneous 4T1 tumors were intratumorally injected with the injectable E2@PND hydrogel and subsequently exposed to ultrasound (2.1 W cm⁻², 5 min) on day 0, 2, 4 and 6, respectively (Fig. 4A). The tumor site injected with PBS showed a slight temperature increase (38.6 °C) under the first ultrasound irradiation for 5 min (Fig. 4B). In contrast, the tumor site in mice receiving injectable E2@PND hydrogel showed a rapid temperature rise from 30.1 °C to 47.1 °C under the same ultrasound exposure. Consequently, significant inhibition of tumor growth was observed in the E2@PND + US group in comparison with the other three groups (Fig. 4C and D). The therapeutic efficacy of the E2@PND-based STT was also investigated by H&E staining of tumor tissues, which revealed severe damage appeared in the group of the E2@PND + US, while no notable damage was found in the other three groups (Fig. 4E). These results demonstrated that sonothermal effects of the E2 contributed to effective suppression of tumor growth.

Fig. 4 Anti-tumor effects of sonothermal therapy based on the injectable E2@PND hydrogel in vivo. (A) Schematic illustration of the experimental approach. (B) Infrared thermal images of the tumor-bearing mice under US exposure (2.1 W cm⁻²) for 5 min. (C) Tumor-growth curves of different groups (n = 4). (D) Photographs of the tumors harvested from mice on day 14 after the first treatments. (E) Representative H&E-stained images of the tumor tissues in different groups. ns P > 0.05, **P < 0.01 and ***P < 0.001.

Photothermal immunity effects of the injectable E1@PND hydrogel using bilateral tumor model

In the above experiments on the treatment of unilateral tumor in mice, both the injectable E1@PNI and E1@PND hydrogels demonstrated ablation of tumor tissue under 1064 nm laser irradiation, with no discernible difference in treatment efficacy, which could be mainly ascribed to the favorable NIR-II photothermal effect of the E1. As we reported previously, in comparison with the injectable PNI hydrogel, the injectable PND hydrogel could promote photothermal anti-tumor immune response by capturing tumor antigens generated by NIR-I PTT due to the strong adhesive properties of catechol groups in the PND.³⁸ Therefore, based on robust ICD induced by NIR-II PTT of the injectable E1@PND hydrogel and the tumor antigens-capturing capacity of the PND, a bilateral tumor model was established to validate the effectiveness of NIR-II photothermal immune response using the injectable E1@PND hydrogel in comparison with the E1@PNI hydrogel.

As shown in Fig. 5A, the primary tumors in both PTT groups were irradiated with a 1064 nm laser for 10 min on day 0 and day 4. Similar to the results obtained from unilateral tumor experiments, primary tumors in both PTT groups were completely eradicated on day 6 after the NIR-II laser irradiation without any recurrence while rapid tumor growth was observed in PBS and E1@PND groups (Fig. 5B and C). In addition, growth curves for distal tumors in each group are shown in Fig. 5D. Although the average volume of the distal tumors was smaller in the E1@PNI + NIR group compared to that in the PBS and E1@PND groups, no significant differences were found. However, the growth rate of distal tumors was obviously slower in the E1@PND + NIR group compared to the other three groups. Distal tumor photographs on day 14 after the first NIR-II laser irradiation also verified that the tumors in the E1@PND + NIR group were the smallest in these groups (Fig. 5E). These results indicated that NIR-II PTT based on the injectable E1@PND hydrogel also activated systemic anti-tumor immune response and inhibited the growth of distal tumors.

Fig. 5 Anti-tumor effects of the injectable E1@PND hydrogel-based PTT on bilateral tumor-bearing mice. (A) Schematic illustration of the experimental approach. (B) Tumor growth curves of the treated tumors in different groups (n = 5). (C) Photos of the treated tumors on day 14 after material injection. (D) Tumor-growth curves of distal tumors in different groups (n = 5). (E) Photos of distal tumors on day 14 after materials injection. (F) Representative TUNEL immunofluorescence staining images of the distal tumor tissues in each group. (G) Representative Ki67 immunofluorescence staining images of the distal tumor tissues in each group. (H) Representative H&E-stained images of the distal tumor tissues. ns P > 0.05, **P < 0.01 and ***P < 0.001.

After 14 days of the first NIR-II laser irradiation, distal tumor tissues were collected and subjected to TUNEL, Ki67 immunofluorescence staining, and H&E staining for cell apoptosis, proliferation and tumor necrosis, respectively. Among these groups, the E1@PND + NIR group exhibited the highest level of TUNEL positive (green fluorescence in Fig. 5F) and the lowest expression of Ki67 (red fluorescence in Fig. 5G), indicating a high degree of apoptosis and lower proliferation in this group. The pathological H&E-stained images revealed significant tissue necrosis in the distal tumor tissue of the E1@PND + NIR group, compared to dense and dark blue cell nuclei, representing normal tumor cells in the other three groups (Fig. 5H).

Furthermore, on day 8 after the first NIR-II laser irradiation, distal tumor tissues were analyzed for CD8⁺ T cell levels using an immunofluorescence method. No significant difference was observed in the content of CD8⁺ T cells in the distal tumor tissues in the PBS, E1@PND and E1@PNI + NIR groups, while the content of CD8⁺ T cells was more than 3 times higher in the E1@PND + NIR group compared to that in the other groups (Fig. S7A and B, ESI†). These results indicated that NIR-II PTT based on the injectable E1@PND hydrogel significantly increased the infiltration of CD8⁺ T cells into the distal tumor. In addition, the levels of IFN-γ and TNF-α in mice sera on day 8 were analyzed by ELISA kits. The results showed that concentrations of IFN-γ (Fig. S7C, ESI†) and TNF-α (Fig. S7D, ESI†) were significantly elevated in the E1@PND + NIR group compared to those in the other groups, suggesting that the injectable E1@PND hydrogel-based NIR-II PTT effectively improved anti-tumor immune responses mediated by CD8⁺ T cells in tumor-bearing mice, which should be attributed to the tumor antigens-capturing capacity of catechol groups in the PND.

Biosafety of the injectable EHG@PND hydrogel

PND dispersions (100 mg mL⁻¹) containing varied concentrations of the E1 were incubated with mouse L929 fibroblasts and HUVECs at 37 °C for 24 h. The viabilities of the two kinds of cells were determined to be more than 90% using the CCK-8 method (Fig. 6A and B), indicating excellent cytocompatibility of the injectable E1@PND hydrogel. Furthermore, the average body weight of mice in each group in the E1@PND-based PTT experiment fluctuated within the normal range throughout the experimental period for either unilateral tumor (Fig. 6C) or bilateral tumors (Fig. 6D). On day 14, after the tumor treatment, H&E staining images revealed no obvious damage or lesions in main organs (heart, liver, spleen, lung and kidney) among all groups (Fig. 6E and F). These results indicated that the injectable E1@PND hydrogel exhibited remarkable biocompatibility with a potential application in NIR-II photothermal immunotherapy.

Fig. 6 Biosafety of the E1@PND injectable hydrogel. (A) and (B) Cell viability of L929 cells and HUVECs, respectively, after co-incubation with the injectable PND hydrogel containing varied amounts of the E1 for 24 h (n = 5). (C) and (D) Changes in the body weight of mice during the experiments (n = 5). (C) Unilateral tumor model and (D) bilateral tumors model. (E) and (F) H&E staining images of the main organs of mice on day 14. (E) Unilateral tumor model and (F) bilateral tumors model.

Furthermore, the injectable E2@PND hydrogel also exhibited excellent cytocompatibility even after co-incubation with HUVECs for 72 h (Fig. S8A, ESI†). Besides, there was no significant variation in the average body weights among different treatment groups in the E2@PND-based STT experiments (Fig. S8B, ESI†). The biochemical index levels including AST, ALT, ALP, BUN, and CREA in tumor-bearing mice (Fig. S8C and D, ESI†) suggested that these mice had normal liver and kidney functions. In addition, histological examination of major organs from mice treated with different treatments revealed no obvious organ damage, inflammatory lesions, or other abnormalities (Fig. S8E, ESI†). These results collectively indicated that the injectable E2@PND hydrogel held a promising potential as a safe and effective sonothermal therapeutic agent.

Conclusions

Injectable thermosensitive EHG@PND hydrogels including E1@PND and E2@PND hydrogels were prepared and utilized as a novel NIR-II photothermal agent and sonothermal agent for tumor treatment, respectively. The injectable EHG@PND hydrogel exhibited a typical thermosensitive sol–gel phase transition behavior, which allowed EHG NSs to stay for a long time in the tumor helping with multiple treatments. In vitro and in vivo studies indicated that the injectable E1@PND hydrogel showed a remarkable photothermal effect under 1064 nm laser irradiation and the injectable E2@PND hydrogel possessed excellent sonothermal effect due to the superior water dispersibility and relatively complete conjugated plane of the encapsulated EHG NSs. After intratumoral injection of the E1@PND hydrogel followed by irradiated with 1064 nm laser (1.0 W cm⁻²) for 10 min, the local temperature in mice reached 55 °C, resulting in complete tumor eradication on day 8 after treatments. Meanwhile, the injectable E2@PND hydrogel-based STT also effectively suppressed tumor growth. Moreover, the injectable thermosensitive EHG@PND hydrogel possessed excellent biocompatibility and held great promise for noninvasive and deep penetrating therapy in NIR-II PTT and STT against tumors. These encouraging findings also suggest their potent applications as a versatile platform integrating various therapies for antibacterial infection, wound healing and tissue engineering.

Author contributions

W. Zhang and M. Fan contributed equally to this work: methodology, investigation, data curation, and writing-original draft. R. Yang, Z. Li: methodology, investigation. Y. Qiu, M. Dong, P. Song: methodology. N. Wang, Y. Yang: methodology, writing-review and editing. Q. Wang: conceptualization, supervision, writing-review and editing.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21976063). We thank the Testing Centre in the School of Chemistry and Chemical Engineering, the Analytical and Testing Centre and Core Facilities of Life Science and the Analytical and Testing Centre of Huazhong University of Science and Technology for the related analysis.

Notes and references

1. J. Shin, N. Y. Kang, B. Kim, H. Y. S. Hong, L. Yu, J. Kim, H. M. Kang and J. S. Kim, Chem. Soc. Rev., 2023, 52, 4488–4514.
2. M. Overchuk, R. A. Weersink, B. C. Wilson and G. Zheng, ACS Nano, 2023, 17, 7979–8003.
3. Y. Liu, P. Bhattarai, Z. Dai and X. Chen, Chem. Soc. Rev., 2019, 48, 2053–2108.
4. J. Hu, Y. Cheng and X. Zhang, Nanoscale, 2018, 10, 22657–22672.
5. A. Pattyn, K. Kratkiewicz, N. Alijabbari, P. L. Carson, P. Littrup, J. B. Fowlkes, N. Duric and M. Mehrmohammadi, Med. Phys., 2022, 49, 6120–6136.
6. Y. Qi, S. S. Ren, J. W. Ye, S. N. Bi, L. Shi, Y. G. Fang, G. Y. Wang, Y. Z. Finfrock, J. Li, Y. Che and G. L. Ning, Adv. Healthcare Mater., 2023, 12, e2300291.
7. C. Xu and K. Pu, Chem. Soc. Rev., 2021, 50, 1111–1137.
8. Y. G. Zhang, S. Y. Zhang, Z. H. Zhang, L. L. Ji, J. M. Zhang, Q. H. Wang, T. Guo, S. M. Ni, R. Cai, X. Y. Mu, W. Long and H. Wang, Front. Chem., 2021, 9, 728066.
9. D. An, J. Fu, B. Zhang, N. Xie, G. Nie, H. Ågren, M. Qiu and H. Zhang, Adv. Funct. Mater., 2021, 31, 2101625.
10. W. Guan, L. Tan, X. M. Liu, Z. D. Cui, Y. F. Zheng, K. W. K. Yeung, D. Zheng, Y. Q. Liang, Z. Y. Li, S. L. Zhu, X. B. Wang and S. L. Wu, Adv. Mater., 2021, 33, 2006047.
11. Q. Zhang, Q. Guo, Q. Chen, X. Zhao, S. J. Pennycook and H. Chen, Adv. Sci., 2020, 7, 1902576.
12. Y. C. He, Y. Pan, X. Zhao, W. J. Fan, Y. Cai and X. Z. Mou, Acta Biomater., 2022, 152, 546–561.
13. Y. Chen, S. Chen, H. Yu, Y. Wang, M. Cui, P. Wang, P. Sun and M. Ji, Adv. Healthcare Mater., 2022, 11, e2201158.
14. Y. Chen, B. Sun, X. Y. Jiang, Z. Y. Yuan, S. Y. Chen, P. F. Sun, Q. L. Fan and W. Huang, J. Mater. Chem. B, 2021, 9, 1002–1008.
15. C. Yin, X. Z. Li, Y. Wang, Y. Y. Liang, S. Zhou, P. C. Zhao, C. S. Lee, Q. L. Fan and W. Huang, Adv. Funct. Mater., 2021, 31, 2104650.
16. V. Revuri, J. Mondal and Y.-K. Lee, Adv. Exp. Med. Biol., 2022, 1351, 177–200.
17. Z. Li, H. Lei, A. Kan, H. Xie and W. Yu, Energy, 2021, 216, 119262.
18. Y. Wang, J. Li, X. B. Li, J. P. Shi, Z. T. Jiang and C. Y. Zhang, Bioact. Mater., 2022, 14, 335–349.
19. Y.-W. Chen, Y.-L. Su, S.-H. Hu and S.-Y. Chen, Adv. Drug Delivery Rev., 2016, 105, 190–204.
20. Z. J. Gu, S. Zhu, L. Yan, F. Zhao and Y. L. Zhao, Adv. Mater., 2019, 31, 1800662.
21. G. Huang, Z. Chen, M. Li, B. Yang, M. Xin, S. Li and Z. Yin, Acta Chim. Sin., 2016, 74, 789–799.
22. X. R. Song, Q. Zhang, M. Q. Chang, L. Ding, H. Huang, W. Feng, T. M. Xu and Y. Chen, Adv. Mater., 2023, 35, e2212259.
23. J. Beik, M. B. Shiran, Z. Abed, I. Shiri, A. Ghadimi-Daresajini, F. Farkhondeh, H. Ghaznavi and A. Shakeri-Zadeh, Med. Phys., 2018, 45, 4306–4314.
24. A. P. Sviridov, V. G. Andreev, E. M. Ivanova, L. A. Osminkina, K. P. Tamarov and V. Y. Timoshenko, Appl. Phys. Lett., 2013, 103, 193110.
25. A. Józefczak, K. Kaczmarek, T. Hornowski, M. Kubovcíková, Z. Rozynek, M. Timko and A. Skumiel, Appl. Phys. Lett., 2016, 108, 263701.
26. Z. Ma, M. Yuan, Z. Cheng, Z. Yang, L. Yang, B. Liu, Y. Bian, A. A. Al Kheraif, P. Ma and J. Lin, Chem. Eng. J., 2024, 482, 148711.
27. J. Beik, Z. Abed, A. Ghadimi-Daresajini, M. Nourbakhsh, A. Shakeri-Zadeh, M. S. Ghasemi and M. B. Shiran, J. Therm. Biol., 2016, 62, 84–89.
28. J. H. Oh, J. Kim, H. Lee, Y. Kang and I. K. Oh, ACS Appl. Mater. Interfaces, 2018, 10, 22650–22660.
29. A. Akay, J. Acoust. Soc. Am., 2002, 111, 1525–1548.
30. L. Liu, J. Hu, X. Q. Fan, Y. Zhang, S. F. Zhang and B. T. Tang, Chem. Eng. J., 2021, 426, 130789.
31. J. Beik, Z. Abed, A. Shakeri-Zadeh, M. Nourbakhsh and M. B. Shiran, Phys. E, 2016, 81, 308–314.
32. I.-Y. Jeon, S.-Y. Bae, J.-M. Seo and J.-B. Baek, Adv. Funct. Mater., 2015, 25, 6961–6975.
33. J. Park, Y. S. Kim, S. J. Sung, T. Kim and C. R. Park, Nanoscale, 2017, 9, 1699–1708.
34. I. Y. Jeon, Y. R. Shin, G. J. Sohn, H. J. Choi, S. Y. Bae, J. Mahmood, S. M. Jung, J. M. Seo, M. J. Kim, D. W. Chang, L. M. Dai and J. B. Baek, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 5588–5593.
35. C. Huang, J. Lin, H. Tang, Q. Wang, T. Majima, N. Wang, Z. Luo and L. Zhu, ChemSusChem, 2022, 16, e202201496.
36. G. Wang, N. Zhang, Z. Y. Cao, Z. H. Zhang, Z. M. Zhu, G. Y. Sun, L. J. Jin and X. Z. Yang, Biomater. Sci., 2021, 9, 3516–3525.
37. Y. J. Kong, Y. N. Dai, D. S. Qi, W. Y. Du, H. Y. Ni, F. Zhang, H. H. Zhao, Q. M. Shen, M. X. Li and Q. L. Fan, ACS Appl. Bio. Mater., 2021, 4, 7595–7604.
38. M. Fan, L. Jia, M. H. Pang, X. L. Yang, Y. J. Yang, S. K. Elyzayati, Y. G. Liao, H. Wang, Y. H. Zhu and Q. Wang, Adv. Funct. Mater., 2021, 31, 2010587.
39. Y. Qiu, M. Fan, Y. Wang, X. Hu, J. Chen, S. Kamel, Y. Yang, X. Yang, H. Liu, Y. Zhu and Q. Wang, J. Controlled Release, 2023, 360, 647–659.
40. C. Y. Du, M. Cao, G. Li, Y. Hu, Y. C. Zhang, L. R. Liang, Z. X. Liu and G. M. Chen, Adv. Funct. Mater., 2022, 32, 2206083.
41. A. Eckmann, A. Felten, A. Mishchenko, L. Britnell, R. Krupke, K. S. Novoselov and C. Casiraghi, Nano Lett., 2012, 12, 3925–3930.
42. A. C. Ferrari and D. M. Basko, Nat. Nanotechnol., 2013, 8, 235–246.
43. M. Fan, Y. Liu, Y. Ren, L. Gan, Y. Yang, H. Wang, Y. Liao, X. Yang, C. Zheng and Q. Wang, Adv. Healthcare Mater., 2022, 11, e2200544.
44. X. Xiong, Y. M. Liu, F. Shi, G. W. Zhang, J. Weng and S. X. Qu, J. Bionic Eng., 2018, 15, 461–470.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb00866a

‡ These authors contributed equally to this work.

---