Abstract

   MXenes, a new class of two‐dimensional (2D) nanomaterials, have shown
   enormous potential for biological applications. Notably, the
   development of 2D MXenes in nanomedicine is still in its infancy.
   Herein, a distinct W[1.33]C i‐MXene with multiple theranostic
   functionalities, fast biodegradation, and satisfactory biocompatibility
   is explored. By designing a parent bulk laminate in‐plane ordered
   (W[2/3]Y[1/3])[2]AlC ceramic and optionally etching aluminum (Al) and
   yttrium (Y) elements, 2D W[1.33]C i‐MXene nanosheets with ordered
   divacancies are efficiently fabricated. Especially, theoretical
   simulations reveal that W[1.33]C i‐MXene possesses a strong
   predominance of near‐infrared (NIR) absorbance. The constructed
   ultrathin W[1.33]C nanosheets feature excellent photothermal‐conversion
   effectiveness (32.5% at NIR I and 49.3% at NIR II) with desirable
   biocompatibility and fast degradation in normal tissue rather than in
   tumor tissue. Importantly, the multimodal‐imaging properties and
   photothermal‐ablation performance of W[1.33]C‐BSA nanosheets are
   systematically revealed and demonstrated both in vitro and in vivo. The
   underlying mechanism and regulation factors for the W[1.33]C‐BSA
   nanosheets‐induced hyperthermia ablation are also revealed by
   transcriptome and proteome sequencing. This work offers a paradigm that
   i‐MXenes achieve the tailoring biomedical applications through
   composition and structure design on the atomic scale.

   Keywords: biodegradability, i‐MAX phase, i‐MXene, theranostics, vacancy
   ordering
     __________________________________________________________________

   A distinct two dimensional W[1.33]C i‐MXene with multiple theranostic
   functionalities, fast biodegradation, and satisfactory
   biocompatibilityhas been demonstrated. The underlying mechanism and
   regulation factors for the engineered W[1.33]C‐BSA nanosheetsinduced
   photothermal hyperthermia are revealed via genomics and proteomics.
   This work significantly expands the biomedical applications of
   i‐MXene‐based nanomedicines with the desirable biological effect and
   excellent theranostics performance.

   graphic file with name ADVS-8-2101043-g001.jpg

1. Introduction

   Two‐dimensional (2D) nanomaterials with unique structural features and
   physicochemical properties have shown substantial potential for cancer
   theranostics.^[ [44]^1 ^] For example, graphene, transition‐metal
   dichalcogenides, monoelemental 2D materials and metal–organic
   frameworks. Since the Ti[3]C[2] nanosheets first introduced in 2011,
   transition‐metal nitrides and carbonitrides (MXenes), as an emerging
   family of 2D materials with numbers of intriguing electronic and
   optical properties, have been explored for versatile applications in
   energy storage,^[ [45]^2 ^] electromagnetic interference shielding,^[
   [46]^3 ^] environmental science,^[ [47]^4 ^] and biology.^[ [48]^5 ^]
   MXenes are generally synthesized by optionally etching the A group
   element from the matching layer‐structured MAX, in which “M” refers to
   early transition metal and “X” corresponds to carbon (C) and/or
   nitrogen (N). Owing to the attractive physicochemical properties,
   MXenes stand out among 2D materials and are expected as distinct
   state‐of‐the‐art platforms to promote the biomedical researches.^[
   [49]^5 , [50]^6 , [51]^7 ^] For instance, the hydrophilic surface and
   low cytotoxicity highlight the biosafety and clinical translation
   potential of MXene‐based nanoplatforms. Furthermore, the broad and
   strong absorbance in near‐infrared (NIR) region as well as high
   light‐to‐heat conversion efficiency has rendered MXenes attractive for
   photoacoustic (PA) imaging and photothermal therapy (PTT). Based on
   abundant oxygen‐containing groups onto the surface, the nanoscale
   MXenes exhibit excellent surface‐engineering capability which can
   enhance the colloidal stability and prolong the in vivo blood
   circulation.^[ [52]^5 , [53]^6 , [54]^8 ^] However, up to now, even
   though more than 30 MXenes have been explored, only several types of
   MXenes including Ti[3]C[2], Nb[2]C, Mo[2]C, V[2]C, and Ta[3]C[4] have
   been used in biomedicine.^[ [55]^9 ^] Bio‐safety issues of these MXenes
   including biodegradation and excretion behaviors are considered as a
   crucial issue to be addressed. To the best of our knowledge, most types
   of MXenes are featured by high crystallinity and accompany with low
   defects, making them difficult to decompose in the physiological
   environment.^[ [56]^10 ^] It is highly necessary but challenging to
   explore new members of 2D MXenes with multiple theranostic
   functionalities, fast biodegradation, and satisfactory biocompatibility
   for guaranteeing their versatile biomedical applications and further
   clinical translations.

   Owing to the high atomic number (Z = 74), strong X‐ray attenuating
   potency and intense absorbance in the NIR region, tungsten (W)‐based
   nanosystems have been explored as a kind of potential candidates for
   cancer theranostics.^[ [57]^11 ^] It follows that a particular
   potential application of W‐based 2D nanomaterial is biomedical field.
   The driving force to explore W‐based MXenes is goal‐oriented; however,
   it has been predicted that none of the ternary W‐based MAX phases are
   stable, which makes it difficult to synthesize W‐based MXene.^[ [58]^12
   , [59]^13 ^] It is necessary to explore suitable fabrication strategies
   for W‐based MXenes. MAX phases containing different components and
   their corresponding MXenes could be obtained by structural designing at
   the atomic level. The reports show that out‐of‐plane and in‐plane
   chemical ordering, termed as o‐MAX and i‐MAX, have been explored in the
   MAX phase. The quaternary i‐MAX phase is characterized by in‐plane
   chemical order with two components M^1 and M^2 in a ratio of 2:1,
   following the general equation of (M^1 [2/3]M^2 [1/3])[2]AC.^[ [60]^13
   , [61]^14 , [62]^15 , [63]^16 ^] The selective removal of the A
   element, commonly Al, and M^2 elements during etching results in M^1
   [1.33]C MXene, called i‐MXene, with vacancies, in which the vacancies
   are ordered, endowing remarkable benefits.^[ [64]^14 ^] Owing to their
   unconventional structure and composition, together with advanced
   theoretical prediction and experimental measurements, it has promising
   applications in energy storage and conversion.^[ [65]^16 , [66]^17 ^]
   However, the application in other fields of i‐MXene is rarely reported,
   especially in biomedicine. Therefore, it is highly motivated to exploit
   appropriate strategies to synthesize W‐based i‐MXene and extend its
   biomedical application.

   Herein, we report a W‐based 2D i‐MXene for multimodal‐imaging‐guided
   cancer theranostics (Scheme [67] 1 ). Quaternary i‐MAX phase,
   (W[2/3]Y[1/3])[2]AlC with in‐plane chemical order, was fabricated
   through structural design on the atomic level. W[1.33]C‐based i‐MXenes
   could be produced by specifically remove both Y and Al elements from
   the corresponding i‐MAX phase bulks, which feature ordered basal‐plane
   vacancies. Given the inefficiency of current experimental
   trial‐and‐error methods, theoretical simulations are strategically used
   as an efficient strategy to predict the photonic properties of W[1.33]C
   i‐MXene. The results of theoretical simulations suggest that W[1.33]C
   i‐MXene possesses strong absorbance in the NIR region. Experimentally,
   we systematically assess the biocompatibility, degradability and
   light‐to‐heat conversion performance of the W[1.33]C i‐MXenes as a
   photothermal agent (PTA) by laser activation in NIR range at both the
   first (NIR I) and second (NIR II) biowindow. Bovine serum albumin
   (BSA)‐modified W[1.33]C i‐MXene (W[1.33]C‐BSA) exhibits an excellent
   photothermal‐conversion efficacy both in vitro and in vivo. Considering
   the acidic and hypoxic tumor microenvironment (TME), the as‐prepared
   W[1.33]C‐BSA nanosheets with pH‐dependent biodegradation properties are
   comparatively stable in acidic environments and can rapidly degrade
   under physiological conditions. Therefore, W[1.33]C‐BSA nanosheets
   could efficiently accumulate into tumors for inducing longer tumor
   retention and would be rapidly degraded and excreted without obvious
   systemic toxicity. Taking advantage of superior multimodal‐imaging
   properties for computed tomography (CT)/PA/photothermal imaging, we
   further demonstrate that W[1.33]C‐BSA nanosheets can be used as a
   robust, versatile, and noninvasive imaging contrast agent (CA) to
   monitor cancer therapy in vivo. In addition, we further reveal the
   underlying biomedical mechanism of PTT as enabled by W[1.33]C i‐MXene.
   Joint analysis of proteome and transcriptome uncovers that W[1.33]C‐BSA
   nanosheets‐induced PTT activate the “complement and coagulation
   cascades” signaling pathway, resulting in cell membrane rupture and
   cell lysis. Therefore, this work presents a distinct kind of i‐MXenes
   with ordered vacancies that possess fast biodegradation and rapid
   excretion, accompanying with efficient tumor retention as well as
   theranostic functionalities, which significantly broadens the
   biomedical applications of 2D MXenes.

Scheme 1.

   Scheme 1
   [68]Open in a new tab

   Schematic illustration on the construction of 2D W[1.33]C‐BSA
   nanosheets as a 2D phototherapeutic agent for multimodal‐imaging‐guided
   cancer treatment.

2. Results and Discussion

2.1. Synthesis and Characterization of 2D W[1.33]C‐BSA Nanosheets

   The quaternary i‐MAX phase (W[2/3]Y[1/3])[2]AlC bulk was fabricated by
   sintering W, Y, Al, and C powders under argon (Ar) atmosphere up to
   1450 °C and then immersed into the etchant solution mixing fluoride
   (LiF, 4 g) with hydrochloric acid solution (HCl, 60 mL, 12 m) to remove
   both Y and Al layers (Figure [69] 1a ). The as‐obtained
   (W[2/3]Y[1/3])[2]AlC powders were initially characterized by scanning
   electron microscopy (SEM). The corresponding SEM images of
   (W[2/3]Y[1/3])[2]AlC and element mapping reveal the presence and
   distribution of W, Y, Al, and C (Figure [70]S1a, Supporting
   Information). After etching and exfoliation treatment, the compact
   layers expanded, leading to the accordion‐like multilayered topology,
   further demonstrating the exfoliation of (W[2/3]Y[1/3])[2]AlC and
   selective removal of Y/Al for the production of 2D W[1.33]C i‐MXene
   (Figure [71]S1b, Supporting Information). To explore more detailed
   microarchitecture information, the high‐angle annular dark field
   (HAADF) imaging in scanning transmission electron microscopy (STEM) was
   acquired in the [110] and [100] direction of i‐MAX phase
   (Figure [72]1b,c). The W/Y/C atoms are on the same layer and
   alternately arranged with Al atoms. In addition, according to the HAADF
   imaging and corresponding energy dispersive X‐ray spectrum (EDX)
   linear‐scanning analysis (Figure [73]1d), the mean distance between two
   adjacent Al atom is 0.68 nm. Transmission electron microscopy (TEM)
   images exhibit the ultrathin structure of exfoliated W[1.33]C
   nanosheets with a typical lateral size of ≈150 nm (Figure [74]1e). From
   EDX elemental mapping results of W[1.33]C i‐MXene (Figure [75]S2,
   Supporting Information), it is concluded that both the Y and Al atoms
   were selectively etched.

Figure 1.

   Figure 1
   [76]Open in a new tab

   a) Scheme of exfoliation process for 2D W[1.33]C nanosheets. b)
   High‐resolution STEM (HSTEM) images and corresponding schematic in
   [110] direction. c) HSTEM images and corresponding schematic in [100]
   direction. d) EDX linear‐scanning along [100] direction (the line
   depicted in inset), mapping the W, Y, Al, and C element signals of
   (W[2/3]Y[1/3])[2]AlC. e) TEM image and corresponding SAED (inset image)
   of W[1.33]C. f) XRD patterns of (W[2/3]Y[1/3])[2]AlC i‐MAX and W[1.33]C
   i‐MXene.

   The X‐ray diffraction (XRD) pattern reveals that the
   (W[2/3]Y[1/3])[2]AlC i‐MAX phase was successfully synthesized
   (Figure [77]1f, black curve). After etching, the peak intensities from
   (W[2/3]Y[1/3])[2]AlC bulk decrease significantly, indirectly
   demonstrating that the bulk crystal has been converted to W[1.33]C
   i‐MXenes. The thickness of the fabricated W[1.33]C nanosheets is
   determined using atomic force microscopy (AFM) to be around 0.25 nm
   (Figure [78]S3, Supporting Information). To improve the stability in
   the physiological environment for further biomedical application, BSA
   as a bioactive macromolecule was used to modify the surface of W[1.33]C
   nanosheets through hydrogen bonding and/or van der Waals attractive
   interactions. The successful synthesis of W[1.33]C‐BSA nanosheets is
   confirmed by Fourier transform infrared (FTIR) spectroscopy (Figure
   [79]S4, Supporting Information), revealing that two distinct and strong
   absorption peaks at ≈1542 and 1665 cm^−1 , assigning to the stretching
   vibrations of —NO[2] and —C═O— in BSA.^[ [80]^18 ^] As expected, the
   obtained W[1.33]C‐BSA nanosheets exhibit high dispersity in different
   physiological solvents such as phosphate buffer saline (PBS) and cell
   culture media. Furthermore, the average hydrodynamic diameters of
   W[1.33]C‐BSA nanosheets is around 190 nm, which is a little bigger than
   bare W[1.33]C nanosheets (≈160 nm), indicating the successful BSA
   modification (Figure [81]S5, Supporting Information). Meanwhile, the
   change of zeta potential is induced by the enhanced superficial
   hydrophilicity and bio‐macromolecular charge‐shielding effect attached
   to the surface (Figure [82]S6, Supporting Information). Owing to the
   successful surface modification of the W[1.33]C nanosheets with BSA,
   the W[1.33]C‐BSA nanosheets exhibit favorable stability in various
   physiological conditions such as saline, PBS, Dulbecco's modified Eagle
   medium (DMEM), and whole blood diluent (Figure [83]S7, Supporting
   Information).

2.2. Optical Properties of W[1.33]C Nanosheets (i‐MXenes)

   The strong light absorption of PTA is essential to guarantee efficient
   light‐to‐heat conversion. The absorption spectra could be simulated by
   the typical density functional theory (DFT) calculations. Based on the
   first‐principle calculation, the density of states (DOS) reveals that
   W[1.33]C i‐MXene exhibits metallic properties (Figure [84] 2a ).
   Notably, the simulated optical absorption spectrum acquired on W[1.33]C
   i‐MXene exhibits a broadband absorption in NIR region, which offers
   high potential for photonic nanomedicine. The further experimental
   results show that W[1.33]C i‐MXene nanosheets have broadband absorption
   across both NIR I and II biowindows, consistent with the theoretically
   calculated spectrum, implying their potential as a PTA candidate
   (Figure [85]2b,c). The results reveal that the extinction coefficients
   (ε) of W[1.33]C nanosheets are 8.54 and 4.94 L g^−1 cm^−1 at 808 and
   1064 nm, respectively(Figure 2d). Furthermore, the
   photothermal‐conversion efficiency (η), another essential parameter for
   the photothermal performance of W[1.33]C nanosheets is 32.5% (NIR I,
   808 nm) and 49.3% (NIR II, 1064 nm), indicating that W[1.33]C
   nanosheets could serve as a powerful PTA for their applications in PTT
   in the bio‐windows, especially in NIR II biological window
   (Figure [86]2e,f). Especially, the W[1.33]C nanosheet dispersion
   exhibits quick temperature increase in a concentration‐dependent manner
   from 26.0 to 60.5 °C under NIR I laser irradiation and from 26.3 to
   60.9 °C under NIR II laser irradiation (Figure [87]2g,h), respectively.
   In addition, the photothermal temperature of W[1.33]C nanosheet
   dispersion can also be elevated with the increase of laser radiation
   power density (Figure [88]2i,j), further demonstrating the controllable
   photothermal performance of 2D W[1.33]C nanosheets. For the
   photothermal stability, it is demonstrated that no significant change
   in the thermal performance was recorded during four laser on/off cycles
   (Figure [89]2k,l).

Figure 2.

   Figure 2
   [90]Open in a new tab

   a) The total and local DOS of W[1.33]C i‐MXene. b) Calculated
   photo‐absorption spectrum of W[1.33]C i‐MXene. c) Absorption spectra of
   W[1.33]C nanosheets dispersion at various concentrations (4.7, 9.4,
   18.8, 37.5, and 75 µg mL^−1). d) Mass extinction coefficient of
   W[1.33]C nanosheets at λ = 808/1064 nm. e) Photothermal conversion
   efficiency (PCE) at 808 nm irradiation. f) PCE at 1064 nm laser
   irradiation. Concentration‐dependent temperature‐changing curves under
   g) 808 nm and h) 1064 nm laser illumination. Photothermal heating
   curves of W[1.33]C nanosheets solutions irradiated by a series of power
   densities of i) 808 nm and j) 1064 nm laser. Heating curve of W[1.33]C
   nanosheets dispersion for four laser on/off cycles irradiated by k)
   808 nm and l) 1064 nm laser at 1.25 W cm^−2.

   Afterward, to confirm the superiority of W[1.33]C‐BSA nanosheets in NIR
   II photothermal performance, the deep‐tissue photothermal‐penetration
   experiments were further carried out using 4T1 tumor‐bearing mice where
   4 mm‐thickness chicken breast tissue is on the top of the tumor area.
   As expected, the highest increase in temperature (△T) after
   illumination under NIR II laser for 10 min is 13.7 °C, whereas the △T
   is only 6.5 °C under NIR I laser irradiation for 10 min (Figure [91]S8,
   Supporting Information). Therefore, these results verify that
   W[1.33]C‐BSA nanosheets could serve as a high‐performance photothermal
   converter for PTT treatment, featuring high photothermal‐conversion
   efficiency and favorable tissue penetration, especially in NIR II
   biowindow.

2.3. Degradation Behavior of W[1.33]C i‐ MXene

   Rapid biodegradation of PTA could reduce long‐term toxicity and
   guarantee high in vivo biosafety. To evaluate the degradation behavior
   of W[1.33]C‐BSA nanosheets, a series of experiments were executed by
   observing the ultraviolet–visible–NIR (UV–vis–NIR) absorption spectrum
   changes of W[1.33]C‐BSA nanosheets in PBS and fetal bovine serum at
   different pHs (Figures [92]S9 and [93]S10, Supporting Information). It
   could be visually found that W[1.33]C‐BSA nanosheets exhibit a
   significant pH‐dependent degradation effect. W[1.33]C‐BSA nanosheets
   rapidly degrade in basic circumstances, while they are comparatively
   stable under the acidic environment, implying that W[1.33]C‐BSA
   nanosheets could deliver more durable therapeutic effects in the tumor
   microenvironment with weak acidic and hypoxia conditions. Notably, the
   estimated pH within the 4T1 breast cancer tumor is about 6.0–6.5.^[
   [94]^19 ^] We thus infer that W[1.33]C‐BSA nanosheets degrade more
   slowly in tumour compared to normal tissue.

   For further investigation of W[1.33]C‐BSA nanosheets in blood
   degradation, the PBS containing 10% mouse blood UV–vis–NIR absorption
   spectra over varied incubation durations (2, 4, 8, 12, and 24 h) were
   performed. The degradation of W[1.33]C‐BSA nanosheets in blood was
   revealed to be a time‐dependent procedure that exhibits an increase
   with the extension of time (Figure [95]S11, Supporting Information).
   Additionally, TEM observation was used to monitor the structure changes
   during the degradation process (Figure [96]S12, Supporting
   Information). It is found that the significant surface oxidation on the
   layer structure of partial nanosheets takes place at the initial stage,
   and then the severe surface oxidation induces a slight breakup of
   planar dimensions on the layer structure of W[1.33]C‐BSA nanosheets.
   Finally, the complete decomposition for planar dimensions of
   W[1.33]C‐BSA nanosheets occurred at the final stage. The intracellular
   degradation behavior and structural evolution of W[1.33]C‐BSA
   nanosheets were further evaluated on the cellular level (Figure
   [97]S13, Supporting Information). Initially, W[1.33]C‐BSA nanosheets
   could be effectively endocytosed into cancer cells and surface
   oxidation occurred within 1 d. Subsequently, prolonged incubation lead
   to severe surface oxidation and almost complete breakdown of
   W[1.33]C‐BSA nanosheets in planar dimensions after 3 d, indicating that
   the nanosheets underwent significant biodegradation.

   PA imaging combines the advantages of optical and ultrasound imaging.
   The intensity of PA signal is proportional to the absorbed optical
   energy and the thermoelastic energy of PTA. When W[1.33]C‐BSA
   nanosheets degraded, the light absorption and the thermoelastic
   performances of W[1.33]C‐BSA would also decrease, which leads to a
   decrease in the PA signal. Therefore, PA imaging has been applied to
   monitor the in vivo degradation of W[1.33]C‐BSA nanosheets in muscle
   and tumors. Intratumorally or intramuscularly injections of
   W[1.33]C‐BSA nanosheets were administered to 4T1 tumor‐bearing mice and
   PA imaging was acquired at varied time points of post‐injection (Figure
   [98]S14, Supporting Information). As expected, compared to original PA
   signals in muscles, the intensity of PA signal is only about 30% of
   their original signals 1 h later. On the contrary, 60% of original PA
   signals were observed in the tumor within 1 h, which demonstrates the
   obviously slower degradation rate of W[1.33]C‐BSA nanosheets in tumor
   than that in muscle. Therefore, these biodegradable W[1.33]C‐BSA
   nanosheets would enable their longer tumor retention and harmless
   degradation during exerting their therapeutic functions. The clearance
   of W[1.33]C‐BSA nanosheets was monitored by measuring W contents in
   urine and feces of mice using inductively coupled plasma
   atomic‐emission spectroscopy (ICP‐AES) (Figure [99]S15, Supporting
   Information). It is found that the W contents were high in urine and
   feces during the 48 h of post‐injection, demonstrating that
   W[1.33]C‐BSA nanosheets could be quickly cleared from the mouse via
   renal and fecal routes. The biodistribution of W[1.33]C‐BSA nanosheets
   in different tissues was investigated at different time points. The
   results show that W[1.33]C‐BSA nanosheets mainly accumulate in the
   liver. The intratumor passive accumulation efficiency based on W has
   been quantified as 4.87%, 3.42%, and 3.20% ID g^−1 in 4, 24, and 48 h,
   respectively, which is competitive compared to current 2D nanomaterials
   used in cancer(Figure [100]S16, Supporting Information). Taken
   together, it could be deduced that W[1.33]C‐BSA nanosheets would be a
   biodegradable PTA, which could not only be relatively stable in tumors
   but also enable rapid excretion for avoiding long‐term in vivo
   toxicity.

2.4. In Vitro Photothermal Cancer Cell Ablation and Endocytosis

   For in vitro cytotoxicity assessment, W[1.33]C‐BSA nanosheets
   dispersion was incubated with both cancer cell (4T1 mouse breast cancer
   cell line) and normal cell lines (L929 mouse fibroblast cell line) at
   pre‐supposed concentrations. As shown Figure [101] 3a , negligible
   cytotoxicity was detected from both cell lines even at the
   concentration up to 200 µg mL^−1 after 24 h co‐incubation, which
   signifies that these W[1.33]C‐BSA nanosheets possess low cytotoxicity.
   Furthermore, the in vitro photothermal ablation capability of
   W[1.33]C‐BSA nanosheets was also assessed via Cell Counting Kit‐8
   (CCK‐8) assay. It is revealed that as power density and concentration
   W[1.33]C‐BSA nanosheets increased, more cancer cells are killed upon
   NIR laser illumination (Figure [102]3b,c). The survival rate is down to
   29% or 28% when 4T1 cells are irradiated with NIR I or NIR II for
   10 min, respectively

Figure 3.

   Figure 3
   [103]Open in a new tab

   a) Relative survival rate of 4T1 and L929 cells after incubated with
   W[1.33]C‐BSA nanosheets dispersion at elevated concentrations for 24 h
   . Relative survival rate of 4T1 cells treated with W[1.33]C‐BSA
   nanosheets dispersion at b) different power densities and c) various
   concentrations under 808/1064 nm laser irradiation for 10 min (n = 5,
   mean ± SD). d) CLSM images of 4T1 cells after various treatments,
   followed by staining with calcein‐AM (green) and PI (red). e) CLSM
   images of cellular organelles, including LysoTracker DND‐26 stained
   Lyso (green), MitoTracker FM stained Mito (green), Gi‐Tracker stained
   Gi (red), and Alexa Fluor 488 conjugated phalloidin stained Cyto
   (green). f) FCM apoptosis assay of 4T1cells after different treatments
   with the staining of annexin V‐FITC and PI. g) Corresponding FITC
   fluorescence signals of CLSM images of 4T1 cells incubated with
   FITC‐labeled W[1.33]C‐BSA nanosheets for 2, 4, and 8 h. h) Cellular
   uptake amounts of W[1.33]C‐BSA nanosheets as analyzed by FCM, and the
   corresponding FITC intensity of 4T1 cells incubated with FITC‐labeled
   W[1.33]C‐BSA nanosheets for 0, 2, 4, and 8 h.

   To better visualize the treatment performance of W[1.33]C‐BSA
   nanosheets on 4T1 cells in vitro, co‐stained imaging of calcein‐AM
   (green) and propidine iodide (PI, red) was acquired using confocal
   laser scanning microscopy (CLSM) (Figure [104]3d). It is found that the
   majority of 4T1 cells are killed after treatment with W[1.33]C‐BSA
   nanosheets under NIR I or II laser illumination, while the 4T1 cells in
   other groups ( control, W[1.33]C‐BSA nanosheets only, NIR I only, and
   NIR II only) are not significantly influenced. Generally, the cell
   death is concomitant with the alternation of the cellular morphology,
   structure and organelle function. Cellular organelles, which mainly
   include lysosomes, mitochondria, Golgi apparatus, endoplasmic
   reticulum, and cytoskeleton, play vital roles in regulating cellular
   activities and survival.^[ [105]^20 ^] Therefore, after W[1.33]C‐BSA
   nanosheets‐mediated PTT treatment, detailed subcellular structure
   evolution was further tracked by corresponding cellular structure
   fluorescent probe staining (Figure [106]3e and Figure [107]S17,
   Supporting Information). For lysosome, punctate green signals in the
   cytoplasm are observed from the imaging of 4T1 cells in control groups.
   Adversely, disseminated green fluorescence staining appears throughout
   the cytoplasm in treatment groups implying that W[1.33]C‐BSA nanosheets
   are able to initiate the damage of lysosomes under both NIR I and NIR
   II laser irradiation. Similarly, hierarchical mitochondrial
   fragmentation, Golgi apparatus destruction, cytoskeleton damages, and
   endoplasmic reticulum shrunken induced by hyperthermia are discovered.
   By contrast, there is negligible morphology changes in mitochondria,
   Golgi apparatus, cytoskeleton, and ER of 4T1 cells incubated with
   W[1.33]C‐BSA nanosheets or under laser irradiation alone.

   Subsequently, the quantification ratio of viable and apoptotic cells
   during the process of PTT was revealed by flow cytometry (FCM)
   apoptosis assay (Figure [108]3f). It is found that the apoptotic cell
   populations are 76.5% (34.9% for the early apoptotic ratio, 41.6% for
   late apoptotic ratio) and 70.2% (consisting of the early and late
   apoptotic ratio of 26.2% and 44%, respectively) for the group of
   W[1.33]C‐BSA + NIR I and W[1.33]C‐BSA + NIR II, respectively.
   Comparatively, 4T1 cells incubated with W[1.33]C‐BSA nanosheets groups
   or only laser irradiation remain at very high survival rates,
   indicating the high biocompatibility of W[1.33]C‐BSA nanosheets. Cell
   cycle analysis was further performed using FCM to obtain more insights
   into the anti‐cancer effect of PTT. After co‐incubated with
   W[1.33]C‐BSA nanosheets plus NIR I or II laser illumination, an obvious
   increment in the percentage of 4T1 cells at the sub‐G1 phase (Figure
   [109]S18, Supporting Information) is observed. Conversely, the
   percentage of 4T1 cells in the sub‐G1 phase of the other groups shows
   no significant increase. These results indicate that W[1.33]C‐BSA
   nanosheets‐triggered hyperthermia resultes in significant cell‐cycle
   inhibition and 4T1 cells apoptosis. Efficient cellular endocytosis of
   the nano‐agents is a prerequisite for anticancer therapy. To understand
   the mechanism of in vitro cancer cell inhibition by W[1.33]C‐BSA
   nanosheets, the intracellular uptake of W[1.33]C‐BSA nanosheets was
   further assessed. Because W[1.33]C‐BSA nanosheets show no
   autofluorescence and fluorescein isothiocyanate (FITC)‐labeled
   W[1.33]C‐BSA nanosheets exhibite a strong fluorescence signal at 525 nm
   (Figure [110]S19, Supporting Information), the FITC‐labeled
   W[1.33]C‐BSA nanosheets were used to observe the intracellular uptake
   of W[1.33]C‐BSA nanosheets. CLSM images clearly show the efficient
   intracellular uptake of W[1.33]C‐BSA nanosheets (FITC conjugated BSA,
   green) after the co‐incubation for 8 h (Figure [111]3g and Figure
   [112]S20, Supporting Information). As shown in Figure [113]3h, the
   internalization amount of W[1.33]C‐BSA nanosheets was determined by FCM
   over 8 h. The mean fluorescence intensities exhibit time‐dependent
   increment. Especially, the green fluorescence of W[1.33]C‐BSA‐FITC
   rarely overlapped with the red fluorescence of LysoTracker red and
   MitoTracker red when 4T1 cells were co‐incubated with W[1.33]C‐BSA
   nanosheets, indicating that these materials scattered within the
   cytoplasm (Figures [114]S21 and [115]S22, Supporting Information).

2.5. In Vivo Anticancer Activity

   A systematic investigation of in vivo toxicology for W[1.33]C‐BSA
   nanosheets was further conducted before subsequent treatment. Healthy
   Kunming mice were raised and classified into four groups based on the
   different dosages of W[1.33]C‐BSA nanosheets injected intravenously.
   The mice after different treatments were euthanized on the 28th day. It
   has been found that there is no abnormality in the blood routine and
   biochemical indexes in four groups (Figure [116]S23, Supporting
   Information). In addition, no significant histological abnormality is
   recorded in major organs after 28 d feeding via the hematoxylin and
   eosin (H&E) staining (Figure [117]S24, Supporting Information).

   In respect of the W[1.33]C‐BSA nanosheets have strong NIR‐absorbance in
   the NIR region, W[1.33]C‐BSA nanosheets are expected to be an ideal PA
   probe(Figure [118] 4a ). To confirm this, PA imaging was further
   performed using W[1.33]C‐BSA nanosheets as a probe. As shown in
   Figure [119]4b, the signal intensity of the PA images increases
   linearly with the increasing concentration of W[1.33]C‐BSA nanosheets.
   Subsequently, 4T1‐tumor‐bearing mice were intravenously injected with
   W[1.33]C‐BSA nanosheets and PA images were recorded at pre‐assigned
   time points. The PA intensity value of W[1.33]C‐BSA nanosheets after 4
   h accumulation in tumor site increases from 0.1 to 1.04 a.u.
   (Figure [120]4c,d), which is ten fold stronger than that before
   administration. After 4 h, the PA intensity starts to decrease,
   suggesting the degradation or metabolism of W[1.33]C‐BSA nanosheets in
   the tumor section.

Figure 4.

   Figure 4
   [121]Open in a new tab

   a) Schematic illustration of W[1.33]C‐BSA nanosheets enabling in vivo
   multimodal imaging and b) in vitro PA images of the W[1.33]C‐BSA at
   elevated concentrations (0.06–1 mg mL^−1). c) In vivo PA images and d)
   quantitative changes in PA signals of tumor region. e) In vitro CT
   contrasts and CT images (inset) of W[1.33]C‐BSA nanosheet solutions at
   varied concentrations and corresponding CT intensities. f) In vivo CT
   contrasts before and after i.v. injection. g) Coronal plane images
   (inset) and corresponding signal intensities of CT imaging before and
   after intravenous injection. h) In vivo fluorescence images and i)
   corresponding quantified fluorescence intensity changes of tumor
   tissues at different time points (0, 1, 2, 4, 8, and 12 h) after i.v.
   injection with FITC‐labeled W[1.33]C‐BSA.

   Another intriguing property of W[1.33]C‐BSA nanosheets results from
   high atomic number element W (Z = 74), which enables them as a contrast
   agent (CA) for CT imaging.^[ [122]^21 ^] Therefore, contrast‐enhanced
   CT imaging was further explored using W[1.33]C‐BSA nanosheets as CAs.
   As shown in Figure [123]4e, the Hounsfield unit (HU) values of CT image
   enhance linearly with elevation concentration of W[1.33]C‐BSA
   nanosheets. 4T1 subcutaneous tumor‐bearing mice were administrated
   intravenously with W[1.33]C‐BSA nanosheets at concentration 20 mg kg^−1
   for in vivo CT imaging. After 4 h post‐injection of W[1.33]C‐BSA
   nanosheets, the CT signal at the tumor site significantly increase from
   37.5 ± 2.6 to 98.2 ± 14.6 HU (Figure [124]4f,g). Therefore, this result
   provides a strong evidence that W[1.33]C‐BSA nanosheets could be used
   as an efficient CA for contrast‐enhanced CT imaging.

   The blood circulation and biodistribution of W[1.33]C‐BSA nanosheets
   were assessed before the in vivo photonic hyperthermia experiments. The
   biodistribution of W[1.33]C‐BSA nanosheets was evaluated through in
   vivo fluorescence imaging system at the prespecified time, in which the
   fluorescence signal was generated by the conjugated FITC. As shown in
   Figure [125]4h,i, the in vivo fluorescence signal changes can be
   detected obviously with the prolonged time, showing that the strongest
   signal in the tumor sites is at 4 h after intravenous (i.v.) injection
   and gradually decreases thereafter. It suggests that W[1.33]C‐BSA
   nanosheets can be accumulated within tumor and excreted from the body
   to avoid long‐term toxicity. The morphology of nanomaterials could not
   only be a factor influencing the biodistribution in living bio‐system
   but also impact the behavior of cellular uptake, which might be
   ascribed to the surface area of nanomaterials in contact with cell
   films and the distortion energy of the cellular films during uptake.
   Although the contribution of shape to biology in every type of
   nanostructure might be different, the regulation of this property can
   be investigated to enhance the performance of nanomaterials‐mediated
   PTT. Additionally, the circulation of W[1.33]C‐BSA nanosheets in blood
   was initially analyzed and the half‐time of W[1.33]C‐BSA nanosheets is
   calculated to be about 1.5 h (Figure [126] 5a ).

Figure 5.

   Figure 5
   [127]Open in a new tab

   a) Blood circulation curve of W[1.33]C‐BSA after intravenous injection
   and T [1/2] was calculated to be about 1.5 h. b) CEUS images before and
   after PTT. c) Tumor‐growth curves after different treatments (n = 5,
   mean ± SD) (control, NIR I only, NIR II only, W[1.33]C‐BSA only,
   W[1.33]C‐BSA + NIR I, and W[1.33]C‐BSA + NIR II). (d) The curve of mice
   weight changes during different treatments (n = 5, mean ± SD). e)
   Analysis of the survival of mice during different treatments. f) The
   corresponding TUNEL, Ki‐67, and CD31 staining for various changes in
   tumor section (scale bar = 25 µm).

   The in vivo therapeutic performance was further evaluated on
   4T1subcutaneous tumor‐bearing mice, which were randomized into six
   groups, including control (G I), NIR I only (G II), NIR II only (G
   III), W[1.33]C‐BSA only (G IV), W[1.33]C‐BSA + NIR I (G V), and
   W[1.33]C‐BSA + NIR II (G VI). 4T1 tumor‐bearing mice were anesthetized
   and performed with different treatments when the volume of the tumor
   reached ≈150 mm^3. Meanwhile, the temperature of tumor site was
   measured by infrared thermal camera. It is worth noting that the tumor
   site temperatures of G V increased by 23 °C. Meanwhile, in G VI, the
   tumor‐site temperature increased by 25 °C. Comparatively, the tumor
   temperature of the mice in G II and G III only increased by 5 °C within
   5 min (Figure [128]S25, Supporting Information). In particular, the
   contrast‐enhanced ultrasound (CEUS) was adopted to precisely evaluate
   the degree and margination of ablation by real‐time monitoring the
   microcirculation perfusion of the photothermal ablation foci. As shown
   in Figure [129]5b, there is a clear edge between ablated tissues and
   normal tissues, indicating that W[1.33]C‐BSA nanosheets mediated PTT
   allows for precise image‐guided treatment. Remarkably, the tumors in
   two treated groups (G V and VI ) disappeared, leaving black scars in
   2 d after PTT treatment. Comparatively, the tumor size of mice in the
   groups of G I–IV grew up rapidly, indicating no tumor inhibition during
   these treatments (Figure [130]5c). Additionally, no significant
   bodyweight fluctuation is observed during the period of treatment,
   indicating that W[1.33]C‐BSA nanosheets could be applied as an
   effective PTA without adverse effects on mice health (Figure [131]5d).
   It is gratifying that tumors in G V and VI were entirely eliminated
   without further reoccurrence and the 60‐d‐survival rate of these groups
   was 100%, much higher than that in G I–IV (Figure [132]5e). The results
   of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
   assay show that apoptotic cells in the G V and VI are significantly
   more than that in other groups. As further demonstrated by Ki‐67
   antibody staining, G V and VI showed a strong inhibitory effect on cell
   proliferation, unlike the other four groups which exhibited almost no
   effects on the proliferation of cancer cells. Additionally, we found a
   significant reduction of CD31^+ vasculatures in two treated groups (G V
   and VI), while the other four groups showed almost negligible changes
   on cancer cells (Figure [133]5f).

   To further explore the underlying bio‐mechanism of W[1.33]C‐BSA
   nanosheets‐induced PTT, transcriptomic and proteomic analysis were
   performed to study the gene and protein changes of tumor on nude mice
   with different treatments. Transcriptome sequencing results indicate
   that there are 979 differential genes (fold change ≥ 1 and p < 0.05)
   between control and PTT treatment groups, including 740 upregulated
   genes and 239 downregulated genes (Figure [134] 6a ). Protein, the
   executor of life functions, plays an important role in regulating
   biological process. Thus, we performed label‐free quantitative
   proteomics technology (LFQPT) to explore the protein changes in tumor
   site on nude mice with different treatments. The results of heatmap
   analysis (Figure [135]6b) of the LFQPT‐based proteomics show 4830
   differential proteins, where the 200 downregulated proteins (|fold
   change| ≥ 2) and 163 upregulated proteins (|fold change| ≥ 2) represent
   a wide range of biological changes due to PTT treatment.

Figure 6.

   Figure 6
   [136]Open in a new tab

   Joint analysis of transcriptome and proteome analysis. a) Volcano plot
   of genes altering after the treatment of PTT (P < 0.05, |fold change| ≥
   2). b) Heat map of differential proteins (P < 0.05), including 200
   downregulated proteins and 163 upregulated proteins following PTT
   treatment. c) Analysis of the correlation between protein and gene
   expression (red for both in transcriptome and proteome; blue for only
   in proteome; green for only in transcriptome; black for either
   transcriptome or proteome). d) Venn diagram of the “mRNA” and
   “protein.” e) KEGG enrichment analysis for studying the underlying
   pathways PTT treatment. Heat map for showing the differentially
   expressed genes of differential expression of f) related transcripts
   and g) proteins in the “complement and coagulation cascades” pathway.
   h) PPI network of these proteins in the “complement and coagulation
   cascades” pathway.

   It is well known that the process from mRNA to protein involves a
   complete set of precise expression control mechanisms, such as
   transcription regulation, post‐transcriptional regulation, translation
   regulation, post‐translational regulation, etc. Therefore,
   simultaneously detecting the expression of mRNA and protein and
   conducting joint analysis are highly important to comprehensively
   explore the biological disease mechanism. Through joint analysis of
   proteome and transcriptome, we could implement the complementation and
   integration of mRNA and protein, and conduct a comprehensive analysis
   of the mechanism in a specific state of the organism. To
   comprehensively explore the underlying biological mechanism of the
   effects induced by 2D W[1.33]C‐BSA nanosheets, we performed a joint
   analysis of proteome and transcriptome. The relationship between gene
   and protein expression is exhibited by the volcano plot
   (Figure [137]6c). As shown in Figure [138]6d, there are 33 categories
   of different proteins and transcripts with corresponding relationships,
   including 30 co‐upregulated and 3 co‐downregulated genes and proteins.
   Next, we performed functional and pathway enrichment analysis on the
   final differential genes corresponding to above‐mentioned differential
   proteins and transcriptome. It is clear that more of identified
   proteins are concentrated in cellular components (especially
   extracellular components) in Gene Ontology (GO) enrichment analysis,
   suggesting that the essential changes taken place in the extracellular
   region (Figure [139]S26, Supporting Information). Various components of
   the extracellular matrix (ECM) are intimately involved in cell
   recognition, adhesion, proliferation, and migration, especially in
   tumor‐tissue stress states.^[ [140]^22 ^] To explore potential
   signaling pathways after PTT treatment, the identified proteins were
   annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG). As
   shown in Figure [141]6e, the “complement and coagulation cascades”
   signaling pathway was the most salient pathway among the top 20 terms
   of enriched KEGG pathway.

   Complement and coagulation are essential for an adequate congenital
   response to injury. The system could be activated by exposure to
   alleged danger signals or damage related molecular patterns (e.g.,
   pathogens and damaged host cells). With exposure to pathogens or
   damaged cells, C3 is cleaved to produce a series of complements,
   including C3b, C3a, C5a, C5b, etc., of which C5b‐9 is the primary
   component needed for the spontaneous formation of the membrane attack
   complex (MAC), which aggregates and invokes cleavage of the target
   cell. Thus, C3 plays an important role in this pathway, which is
   consistent with the enrichment results in this study. In this work, PTT
   induced hyperthermia induced can lead to tumor vascular damage, which
   could be seen as a danger signal that activates the complement and
   coagulation cascade. On the one hand, hyperthermia can lead to the
   tumor vascular damage, activating the exogenous thrombin pathway,
   converting fibrinogen (Fgb) into fibrin and then blocking tumor blood
   vessels, which ultimately starve tumor to death. In addition, after PTT
   treatment, a set of membrane attack complexes on the surface of cancer
   cells, formed by a cascade reaction in the classical pathway, lead to
   the entry of water into the cells due to the great difference in
   osmotic pressure, which eventually leads to the rupture of the cell
   membrane and cell lysis (Figure [142]6f–h).

3. Conclusions

   In summary, the distinct 2D W[1.33]C nanosheets (i‐MXene) with ordered
   divacancies and desirable biocompatibility/biodegradability were
   successfully constructed for cancer nanotheranostics. These intriguing
   MXene nanosheets have been demonstrated to feature distinct PTA
   functions for multimodal imaging (PA/CT/photothermal imaging) and
   photonic tumor hyperthermia. Especially, theoretical simulation has
   been employed to predict W[1.33]C nanosheets with a strong NIR
   absorption band. After surface modification with BSA, these ultrathin
   2D W[1.33]C‐BSA nanosheets exhibit high biocompatibility, rapid
   degradation, and high photothermal‐conversion efficiency. Importantly,
   these W[1.33]C‐BSA nanosheets could be rapidly cleared from normal
   organs, but their unique pH‐responsive characteristics allowed them to
   enrich and remain longer at tumor sites. Benefiting from the superior
   X‐ray attenuation ability and high NIR absorbance of W[1.33]C‐BSA
   nanosheets, remarkably enhanced in vivo CT and PA multimodal imaging of
   tumors has been executed. Especially, highly effective in vivo PTT by
   W[1.33]C‐BSA nanosheets of tumor has been systematically assessed on
   tumor‐bearing mice. The underlying mechanism and regulation factors for
   the engineered W[1.33]C‐BSA nanosheets induced PTT are also revealed
   via genomics and proteomics. Therefore, this work significantly expands
   the biomedical applications of i‐MXene‐based nanomedicines

4. Experimental Section

Synthesis of (W[2/3]Y[1/3])[2]AlC Powders (i‐MAX)

   The powders of W, Y Al and C were mixed at a molar ratio of
   1.33:1.33:1:1 to prepare (W[2/3]Y[1/3])[2]AlC MAX‐phase ceramics. After
   that, the hybrid powders were cool‐pressed into the disc, and then
   heating to 1450 °C with a rate of 8 °C min^−1 for 2 h with flowing
   argon (Ar) atmosphere. After cooling to room temperature, the sample
   was gently pulverized to yield the final product ((W[2/3]Y[1/3])[2]AlC
   i‐MAX phase powders).

Synthesis of W[1.33]C Nanosheets (i‐MXenes)

   The as‐obtained (W[2/3]Y[1/3])[2]AlC precursor (2 g) was immersed into
   HCl aqueous solution (60 mL, 12 m) with LiF (4 g), and stirred for 48 h
   at 35 °C in an oil bath. After etching, the products were collected by
   centrifugation, adding HCl (50 mL) and shaking manually for 1 min,
   followed by centrifugation for 1 min (5000 rpm), and this process was
   repeated three times. The etched W[1.33]C nanosheets were then washed
   with deionized water (ddH[2]O) for several times until delamination
   occurred to obtain ultrathin W[1.33]C nanosheets. In order to inhibit
   the possible oxidation, the HCl and ddH[2]O were degassed for 30 min.

Surface Modification of W[1.33]C Nanosheets (W[1.33]C‐BSA)

   The as‐prepared nanosheets were water‐soluble, while it was not stable
   in the biological environment without further modification. To improve
   stability in physiological solution, W[1.33]C nanosheets were blended
   with BSA aqueous solution at room temperature with stirring and
   maintained in vacuum for 2 h. Then, the as‐prepared W[1.33]C‐BSA
   nanosheets were gathered by centrifugation and washed several times
   with degassed ddH[2]O to remove excess and free BSA. Finally, the
   acquired W[1.33]C ‐BSA sediment was redispersed in degassed DI water
   and stored at 4 °C.

In Vivo Deep‐Tissue Photothermal‐Penetration Exam in Both NIR I and NIR II
Biowindows

   To simulate the clinical situation, the in vivo deep‐tissue
   photothermal capability of the W[1.33]C‐BSA nanosheets was evaluated
   4T1 breast tumor‐bearing mice model with the tumor areas covered by a
   4 mm thick chicken breast tissue. The experimental conditions were as
   follows: laser wavelength, 808/1064 nm; laser power density, 1.5 W
   cm^−2; duration time, 5 min. Meanwhile, IR thermal camera was used to
   record the temperature change.

In Vitro Cell Toxicity of W[1.33]C‐BSA Nanosheets

   4T1 cells and L929 cells were used to assess the cytotoxicity of
   W[1.33]C‐BSA nanosheets. The 4T1 cells and L929 cells were seeded in
   96‐well plates (1 × 10^4 cells) for 12 h to attach. Then coincubation
   with W[1.33]C ‐BSA nanosheets at various concentrations (12–200 µg
   mL^−1) for 24 h, the cell survival rate of 4T1 and L929 cells was
   tested using the typical CCK‐8 assay.

In Vitro Photothermal Ablation Performance of W[1.33]C‐BSA Nanosheets

   4T1 cells (10^4 cells per well) were pre‐seeded in 96‐well plates and
   cultured overnight. Then, the culture medium was replaced by a fresh
   medium (100 µL) containing W[1.33]C‐BSA nanosheets at different amounts
   ( 12 to 200 µg mL^−1) . After 4 h incubation, the cells were exposed to
   the 808 or 1064 nm laser at a power density of 1.25 W cm^−2 for 10 min
   irradiation. After 2 h of treatment, the standard CCK‐8 assay was
   performed to access the cell viabilities.

Investigation on Photothermal Ablation Performance of W[1.33]C‐BSA Nanosheets
by Cell Apoptosis

   4T1 cells were pre‐seeded in six‐well plates for 24 h. Then culture
   mediums containing different ingredients were added to the cell plates
   and incubated for another 4 h. The cells were disposed of as mentioned
   above and were collected by centrifugation. After washing with PBS, the
   cells were resuspended in 100 µL binding buffer and stained by Annexin
   V FITC and PI for 20 min. At last, flow cytometry was performed to
   observe cell apoptosis and necrosis.

Intracellular Endocytosis as Observed by CLSM Observation

   4T1 cells were incubated for 24 h in glass bottom culture dish (1 ×
   10^5). Subsequently, the culture medium was replaced by an Roswell Park
   Memorial Institute （RPMI）‐1640 medium containing FITC‐labeled
   W[1.33]C‐BSA nanosheets (1 mL, 200 µg mL^−1). Then the cells were
   incubated for 2, 4, and 8 h. After incubation , 100 µL
   4',6‐diamidino‐2‐phenylindlole（DAPI） was supplemented for cell nuclei
   staining. After 15 min, the cells were then lightly washed with PBS and
   visualized by CLSM.

In Vivo Photothermal Therapy

   A 4T1 tumor‐bearing model was established by subcutaneous inoculation
   of 4T1 cells (1 × 10^6) at the right back. Then the mice were
   randomized into six groups (n = 5), including control (G I), NIR I only
   (G II), NIR II only (G III), W[1.33]C‐BSA only (G IV), W[1.33]C‐BSA +
   NIR I (G V), and W[1.33]C‐BSA + NIR II (G VI). The dose of W[1.33]C‐BSA
   nanosheets was 20 mg kg^−1. The mice were anesthetized 4 h after
   material injection and then subjected to laser irradiation. The
   experimental conditions were as follows: laser wavelength, 808/1064 nm;
   laser power density, 1.25 W cm^−2 ; duration time, 5 min. The volume of
   tumors was measured every 2 d. In addition, temperature changes at the
   tumor site were recorded by IR thermal imaging camera (FLIRTM A325SC
   camera, USA). Meanwhile, CEUS was carried out on the GE Logiq S7
   equipped with a linear array transducer of 3–5 MHz to assess to tumor
   elimination. Further, TUNEL staining, Ki‐67 antibody and CD31^+
   antibody were applied to observe tumor cell apoptosis, cell
   proliferation and vascular density, respectively.

Statistical Analysis

   All of the above quantitative data were shown as mean ± standard
   deviation (SD). Statistical analysis was carried out through Origin
   2017 software. The results were considered to be statistically
   significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [143]Click here for additional data file.^ (2.8MB, pdf)

Acknowledgements