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Two-Dimensional Tantalum Carbide (MXenes) Composite Nanosheets for Multiple ImagingGuided Photothermal Tumor Ablation Chen Dai,† Yu Chen,*,§ Xiangxiang Jing,*,‡ Lihua Xiang,† Dayang Yang,‡ Han Lin,§ Zhuang Liu,∥ Xiaoxia Han,⊥ and Rong Wu*,† Downloaded via KAOHSIUNG MEDICAL UNIV on June 30, 2018 at 17:04:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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Department of Ultrasound in Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, PR China ‡ Department of Ultrasound, Hainan General Hospital, Haikou 570311, PR China § State Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China ∥ Department of Radiology, Fudan University Shanghai Cancer Center, Shanghai 200032, PR China ⊥ Second Affiliated Hospital, Institute of Ultrasound Imaging, Chongqing Medical University, Chongqing 400010, PR China S Supporting Information *
ABSTRACT: MXenes, an emerging family of grapheneanalogues two-dimensional (2D) materials, have attracted continuous and tremendous attention in many application fields because of their intrinsic physiochemical properties and high performance in versatile applications. In this work, we report on the construction of tantalum carbide (Ta4C3) MXene-based composite nanosheets for multiple imagingguided photothermal tumor ablation, which has been achieved by rational choice of the composition of MXenes and their surface functionalization. A redox reaction was activated on the surface of tantalum carbide (Ta4C3) MXene for in situ growth of manganese oxide nanoparticles (MnOx/Ta4C3) based on the reducing surface of the nanosheets. The tantalum components of MnOx/Ta4C3 acted as the high-performance contrast agents for contrast-enhanced computed tomography, and the integrated MnOx component functionalized as the tumor microenvironment-responsive contrast agents for T1-weighted magnetic resonance imaging. The photothermal-conversion performance of MnOx/Ta4C3 composite nanosheets not only has achieved contrast-enhanced photoacoustic imaging, but also realized the significant tumor-growth suppression by photothermal hyperthermia. This work broadens the biomedical applications of MXenes, not only by the fabrication of family members of biocompatible MXenes, but also by the development of functionalization strategies of MXenes for cancer-theranostic applications. KEYWORDS: MXene, tantalum carbide, theranostic, nanomedicine, cancer
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platforms with varied compositions and nanostructure have been developed as the photothermal-conversion nanoagents to enhance the therapeutic effects of PTT, including the wellknown Au nanoparticles,12−14 graphene and its derivatives,15−17 black phosphorus,18,19 transition-metal dichalcogenides,20−22 some organic nanosystems,23,24 etc. In addition, the accurate predetermination of tumor location is of high significance for improving the therapeutic accuracy and mitigating the damage of PTT to the surrounding normal cells/tissues, which can be
ngineering and tailoring materials at an atomic or molecular level have vastly propelled the development of scenarios for efficient cancer therapy.1−5 Due to sparse knowledge of the tumorigenesis metastasis and angiogenesis, and limitation of current diagnostic techniques and therapeutic modalities, cancer is still a major public health problem worldwide.6 Photothermal therapy (PTT), as an emerging protocol for tumor ablation by hyperthermia, is now developing fast.7−9 Typically, PTT generates heat by transforming near-infrared (NIR) light into thermal energy at a minimally invasive manner, which is regarded as the potential treatment substitution for conventional clinical cancertherapeutic modalities.10,11 On this ground, abundant nano© 2017 American Chemical Society
Received: October 12, 2017 Accepted: November 20, 2017 Published: November 20, 2017 12696
DOI: 10.1021/acsnano.7b07241 ACS Nano 2017, 11, 12696−12712
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Cite This: ACS Nano 2017, 11, 12696−12712
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Figure 1. Schematic illustration of the synthetic procedure and MR/CT/PA imaging-guided photothermal tumor therapy by MnOx/Ta4C3−SP composite nanosheets. (a) The scheme of exfoliation process for 2D Ta4C3 nanosheets based on ball-and-stick model, including HF etching and subsequent sonication exfoliation. (b) The scheme of the synthetic procedure for MnOx/Ta4C3−SP composite nanosheets, including HF etching, sonication exfoliation, in situ redox reaction between Ta4C3 nanosheets and postintroduced KMnO4, and subsequent surface SP modification. (c) Schematic illustration of theranostic functions of MnOx/Ta4C3−SP composite nanosheets, i.e., MR/CT/PA imaging-guided efficient PTT ablation of cancer.
accuracy.36−39 However, the rational integration of two or more different theranostic functional components into one nanoplatform generally suffers from complicated synthetic procedures, high cost, low production yield and instability, thus significantly hindering their further possible clinic translation. Recently, layer-structured two-dimensional (2D) transition metal carbides and nitrides (MXenes) have attracted considerable attention and interest due to their rich elemental compositions and 2D topologies as compared to graphene,40,41 endowing them with abundant/tunable physiochemical properties and high performance in versatile applications, ranging from mostly explored electrochemical energy storage materials36,42−44 to recently developed biomedical applications.45−47 Typically, MXenes are synthesized by exfoliation of their corresponding layer-structured 3D MAX-phase ceramics, which represent a large material family with over 70 members.48 The general formula of the MAX phase is Mn+1AXn (n = 1, 2, 3) where M is an early transition metal, A is a mainly A-group element and X is carbon or nitrogen.41,49,50 The A layer can be
achieved by the imaging guidance and monitoring before and during the PTT hyperthermia.25,26 It has been well-demonstrated that each diagnostic-imaging modality is featured with individual advantages. For instance, computed tomography is regarded as the powerful means for medical diagnosis because of its high spatial resolution and valuable tomography information on anatomical structure.21,27 In addition, magnetic resonance imaging (MRI) can offer higher anatomical structure information on soft tissue at a noninvasive and nonionizing manner as compared to other imaging modalities.28,29 As an emerging diagnostic-imaging modality, photoacoustic (PA) imaging can break the penetration limit of optical imaging by detecting the induced pressure waves of laser-irradiated tissues, which is based on its low tissue-attenuation coefficient, affording real-time detections of biological structure and functional information.30−35 It is highly expected to integrate different imaging modalities together to acquire comprehensive and synergetic information on living subjects for further enhancing the diagnosis 12697
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Figure 2. Characterization of bulk MAX-phase Ta4AlC3 ceramics, Ta4C3 nanosheets and MnOx/Ta4C3 composite nanosheets. (a) Photograph of bulk Ta4AlC3 ceramics. (b) SEM images and (c) TEM images of multilayer-structured Ta4C3 MXene after HF etching for 4 days. (d) Highresolution TEM (HRTEM) image of ultrathin Ta4C3 nanosheets. Inset shows SAED pattern of ultrathin Ta4C3 nanosheets. (e, f) TEM images of Ta4C3 nanosheets after 12 h sonication at different magnifications. (g) XRD patterns of Ta4AlC3 ceramics and Ta4C3 nanosheets. (h) TEM images of MnOx/Ta4C3 composite nanosheets. (i) Dark-field and (j) bright-field SEM images of MnOx/Ta4C3 composite nanosheets.
soybean phospholipid (SP), these biocompatible MnOx/ Ta4C3−SP composite nanosheets have been systematically evaluated on their biosafety and theranostic performance on combating cancer.
selectively removed to exfoliate the MAX-phase ceramics to produce 2D MXenes. The surface of MXene is typically terminated by −OH, −O, and/or −F, offering the possibility for engineering and functionalization of MXene’s 2D topological surface.49,51 Recently, we have demonstrated that 2D Ti3C2 MXenes have the high photothermal-conversion capability for cancer hyperthermia,45 but these MXenes cannot exert the specific functionality for diagnostic imaging because of the simple titanium and carbon compositions. In this work, we have successfully developed a MXene-based composite nanoplatform for multiple imaging-guided photothermal hyperthermia of cancer. 2D tantalum carbide (Ta4C3) MXene has been constructed based on its high photothermalconversion performance for photothermal cancer ablation and tantalum-based composition with high atomic number (Z = 73) for contrast-enhanced CT imaging.52,53 Furthermore, a surfaceengineering strategy has been proposed to in situ grow manganese oxide nanoparticles onto the surface of Ta4C3 MXene by triggering the redox reaction between reducing MXene’s surface and strongly oxidative MnO4− (MnOx/ Ta4C3). The integrated MnOx component endows the composite MXene nanosheets with tumor microenvironment (TME)-responsive T1-weighted MR imaging capability. Especially, the photothermal-conversion performance of Ta4C3 in MnOx/Ta4C3 composite nanosheets makes the as-synthesized MnOx/Ta4C3 become the desirable contrast agents for PA imaging. After the further surface organic modification by
RESULTS AND DISCUSSION Design, Synthesis, and Characterization of MnOx/ Ta4C3 Composite Nanosheets. As a MXene family member, 2D Ta4C3 nanosheets have been rarely explored because of the relatively high synthetic difficulty for the fabrication of Ta4C3 nanosheets with desirable 2D topology and nanostructures as compared to mostly explored 2D Ti3C2 MXene. Especially, the biomedical applications of nanoparticles typically require the strict controlling of their composition and structure at nanoscale. To satisfy the biomedical requirements, a two-step exfoliation strategy has been developed to fabricate nanosized 2D Ta4C3 nanosheets. The bulk MAX-phase Ta4AlC3 ceramics were initially sintered at high temperature, followed by chemical etching in 40% HF solution for 4 days to remove the middle Al layer. Subsequently, the delaminated Ta4C3 MXenes were treated by sonication for 12 h to fabricate nanosized 2D Ta4C3 ultrathin MXene nanosheets (Figure 1a and 1b). To integrate manganese oxide (MnOx) components onto the surface of 2D Ta4C3 MXene, we introduced highly oxidative KMnO4 into the Ta4C3 solution, which could react with the surface-exposed −OH groups (reducing sites) for in situ producing and growing MnOx nanoparticles (Figure 1b, 12698
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Figure 3. Detailed composition and element analysis of MnOx/Ta4C3 composite nanosheets. (a−c) Elemental mappings of MnOx/Ta4C3 composite nanosheets (a: O, b: Ta, c: Mn), and (d) corresponding X-ray energy dispersive spectroscopy (EDS) and electron energy loss spectrum (EELS) of MnOx/Ta4C3 composite nanosheets. (f) X-ray photoelectron spectroscopy (XPS) of Ta4AlC3, Ta4C3 and MnOx/Ta4C3, and (g) corresponding XPS spectra of Mn 2p region in MnOx/Ta4C3 spectrum.
MnOx/Ta4C3). To improve the stability of as-synthesized MnOx/Ta4C3 composite nanosheets, soybean phospholipid (SP), as a biocompatible natural organic component, was further introduced to modify the surface of MnOx/Ta4C3 nanosheets (designated as MnOx/Ta4C3−SP), which could guarantee the possible in vivo evaluation and application. Each component in MnOx/Ta4C3 composite nanosheets has its own functionality for cancer theranostics, including MnOx component for tumor microenvironment-responsive MR imaging, Ta component for contrast-enhanced CT imaging and Ta4C3 component with high photothermal-conversion capability for simultaneous contrast-enhanced PA imaging and photothermal ablation of tumor (Figure 1c). The bulk MAX-phase Ta4AlC3 ceramics were initially sintered by solid-phase reaction at 1500 °C for 2 h (Figure 2a). After 40% HF etching for 96 h to remove the Al layer sandwiched between two Ta4C3 MXene layers, the exfoliated multilayer nanostructure of Ta4C3 MXene could be fabricated, which was clearly observed in SEM (Figure 2b) and TEM (Figure 2c) images, indicating the expanded interlayer space after the removal of Al layer. The high-resolution TEM (HRTEM) image and corresponding selected area electron diffraction (SAED) pattern clearly exhibited the crystalline lattice and hexagonal structure of as-prepared multilayered Ta4C3 MXenes, indicating that the crystallinity of the Ta4AlC3
MAX phase could be well preserved after long-term HF treatment (Figure 2d). To satisfy the strict biomedical application where the small nanoparticulate sizes is required, the as-prepared multilayer-structured Ta4C3 nanosheets were further treated by sonication for 12 h, which could reduce the planar size of Ta4C3 nanosheets. The SEM and TEM images of the obtained samples (Figure 2e and 2f, Figure S1) showed the successful fabrication of ultrathin Ta4C3 nanosheets with nanosizes in planar direction. X-ray diffraction (XRD) pattern of the synthesized Ta4C3 nanosheets is shown in Figure 2g. After 40% HF etching of MAX-phase Ta4AlC3 ceramics for 96 h, the (0002) peaks broadened and downshifted to lower angles due to its higher c value. In addition, more peaks in XRD pattern vanished or shrunk, especially over the range of 30−40 degree. These results suggested the successful removal of Al layer and desirable topological transformation from three 3D to 2D. The 2D topology of Ta4C3 MXenes endows it with large surface area, and the HF etching could bring with the special surface terminations such as −OH groups,40,54,55 which provides the possibility for further surface modification and functionalization. Herein, highly oxidative KMnO4 was introduced into Ta4C3 aqueous solution to generate in situ MnOx species onto the surface of the Ta4C3 nanosheets via a simple redox reaction between MnO4− ions and reducing −OH groups. The TEM images of MnOx/Ta4C3 composite nano12699
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Figure 4. In vitro photothermal-property characterization and dispersity characterization after surface SP modification. (a) UV−vis spectra of MnOx/Ta4C3 nanosheets dispersed in aqueous solution at elevated concentrations (5, 10, 20, 40, and 80 ppm). Inset: Normalized absorbance intensity at λ = 808 nm divided by the characteristic length of the cell (A/L) at elevated concentrations. (b) The photothermal-heating curves of MnOx/Ta4C3 composite nanosheets at elevated concentrations under 808 nm laser irradiation (2.0 W cm−2). (c) Recycling-heating profiles of MnOx/Ta4C3 composite nanosheets aqueous solution after 808 nm laser irradiation at 1.5 W cm−2 for five laser on/off cycles. (d) Photothermal effect of an aqueous dispersion of MnOx/Ta4C3 composite nanosheets under 808 nm laser irradiation (1.5 W cm−2). (e) Time constant for heat transfer from the system was determined to be τs = 225.8 s by using the linear time data from the cooling period versus negative natural logarithm of driving force temperature, which was obtained from the cooling stage. (f) Schematic of surface modification of MnOx/Ta4C3 composite nanosheets by SP. (g) Digital images of MnOx/Ta4C3 and MnOx/Ta4C3−SP composite nanosheets dispersed in different physiological solutions, including H2O, PBS, SBF, saline and DMEM. (h) Dynamic light scattering (DLS) of MnOx/Ta4C3−SP composite nanosheets in aqueous solution. (i) Zeta potential of Ta4C3, MnOx/Ta4C3 and MnOx/Ta4C3−SP.
and Mn elements, indicating the successful functionalization of the MnOx on the surface of Ta4C3 nanosheets. The atomic ratio of Mn and Ta was nearly 1:5. The electron energy loss spectrum (EELS) of MnOx/Ta4C3 (Figure 3e) shows the presence of typical Mn signal, further indicating the MnOxfunctionalized Ta4C3 composition. X-ray photoelectron spectroscopy (XPS) was adopted to investigate the chemical nature of Ta4AlC3, Ta4C3, and MnOx/Ta4C3 composite nanosheets. As shown in Figure 3f, after HF etching, Al-indexed peaks in the XPS spectrum of Ta4C3 nanosheets disappeared as compared to that of Ta4AlC3 MAX-phase ceramic. The obvious Mn-indexed peaks in XPS demonstrated the presence of MnOx components on the surface of Ta4C3 nanosheets. The fitted peaks were at 642.7 and 655.7 eV, respectively. The relative contents of bivalent, trivalent and quadrivalent Mn in MnOx/ Ta4C3 composite nanosheets were determined to be 18.5% 65.4%, and 16.1%, respectively (Figure 3g). In Vitro Photothermal-Conversion Property of MnOx/ Ta4C3 Composite Nanosheets. It has been found that the as-
sheets documented that the as-formed MnOx component in sheet-like morphology was firmly attached onto the surface of Ta4C3 nanosheets (Figure 2h and Figure S2a,b). The Brightfield SEM image (Figure S2c) of MnOx/Ta4C3 composite nanosheets evidenced the formation of a thin MnOx layer on the surface of Ta4C3 MXene. The Dark-field SEM image (Figure 2i), corresponding the high-angle annular detector dark-field (HAADF) image (Figure S2d) and the TEM image at high magnification (Figure S2e) of MnOx/Ta4C3 composite nanosheets showed the sheet-like morphology of MnOx species anchored on the surface of Ta4C3, which was further demonstrated by the corresponding secondary electron SEM image (Figure 2j). The elemental distribution mappings of Ta, Mn and O in MnOx/Ta4C3 composite nanosheets (Figure 3a−c) confirmed the coexistence of the Ta and Mn elements and high uniformity of MnOx distribution on the surface of Ta4C3 nanosheets. X-ray energy dispersive spectroscopy (EDS) of MnO x /Ta4 C 3 composite nanosheets (Figure 3d) exhibit the presence of Ta 12700
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Figure 5. In vitro PTT against tumor cells. (a) 3D schematic illustration of MnOx/Ta4C3−SP composite nanosheets as the photothermal agents for 4T1 cell ablation after the exposure to 808 nm laser irradiation. (b) CLSM images of 4T1 cells after coincubation with FITClabeled MnOx/Ta4C3−SP composite nanosheets for 0, 1, and 4 h. All the scale bars are 20 μm. (c) CLSM images of 4T1 cancer cells stained by calcein AM and PI after the varied treatments, including control, NIR laser only, MnOx/Ta4C3−SP only and MnOx/Ta4C3−SP + NIR laser. (d) Relative viabilities of 4T1 cells after incubation with MnOx/Ta4C3−SP composite nanosheets at elevated concentrations (25, 50, 100, 200, and 400 μg mL−1) for 24 and 48 h. Error bars were based on the standard deviations (s.d.) of five parallel samples. (e) Relative viabilities of 4T1 cells after incubation MnOx/Ta4C3−SP composite nanosheets at various concentrations (12.5 25, 50, 100, and 200 μg mL−1) followed by laser irradiation at the power density of 2.0 W cm−2. (f) Relative viabilities of 4T1 cells after incubation with MnOx/Ta4C3−SP composite nanosheets (200 μg mL−1) followed by the exposure to 808 nm laser at different laser power densities (0, 0.5, 1, 1.5, and 2.0 W cm−2).
power-dependent photothermal features. Such a high photothermal-conversion capability of MnOx/Ta4C3 composite nanosheets could guarantee the further in vivo tumor ablation. In contrast, the temperature increase of only 1 °C in pure water was recorded after 300 s irradiation at the same conditions, indirectly indicating the functionality of MnO x /Ta 4 C 3 composite nanosheets for elevating the aqueous temperature. The photothermal stability of MnOx/Ta4C3 composite nanosheets was further tested. Insignificant changes were observed in elevating temperature after 5 cycles of NIR laser irradiation (1.5 W cm−2) for nearly 1 h (Figure 4c). Especially, the photothermal-conversion efficiency η of MnOx/Ta4C3 composite nanosheets was measured to be nearly 34.9% (Figure 4d, and 4e), much higher than that of Au nanorods (21%)56 and Cu2‑xSe NCs (22%).57 Although the Ta4C3 nanosheets show high hydrophilicity and dispersity in water solution, MnOx/ Ta4C3 is easily aggregated in physiological solutions. In order to address this issue, soybean phospholipid (noted as SP) was further used to modify the surface of MnOx/Ta4C3 composite nanosheets (Figure 4f) to endow the MnOx/Ta4C3 composite nanosheets with excellent stability and high dispersity in various
synthesized Ta4C3 MXene was featured with high photothermal-conversion capability. Therefore, it was expected that the MnOx/Ta4C3 composite nanosheet was also featured with similar photothermal-conversion performance for potential photothermal hyperthermia due to the presence of Ta4C3 component in the composite nanosheets. UV−vis spectra of MnOx/Ta4C3 composite nanosheet displayed the specific concentration-dependent light absorption in the NIR region (Figure 4a). The extinction coefficient MnOx/Ta4C3 composite nanosheet at 808 nm was tested and calculated to be 8.67 L g1− cm−1 (Figure 5a inset), which was much higher than that of mostly explored graphene oxide (3.6 L g1− cm−1).16 Then, we evaluated the in vitro photothermal performance of MnOx/ Ta4C3 composite nanosheets after exposing to an NIR laser (808 nm) at various concentrations under the laser power density of 1.5 and 2.0 W cm−1 (Figure 4b and Figure S3). The maximum temperature of the aqueous solution containing MnOx/Ta4C3 composite nanosheets could reach up to 49 °C at the power density of 1.5 W cm−2 after 300 s irradiation, and to nearly 65 °C at a power density of 2.0 W cm−2 under the same irradiation duration, which also exhibited a concentration-/laser 12701
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Figure 6. Contrast-enhanced CT imaging capability of MnOx/Ta4C3-SP composite nanosheets both in vitro and in vivo. (a) In vitro CT images and (b) CT contrasts of MnOx/Ta4C3−SP composite nanosheet solutions and iopromide solutions at varied concentrations (0.1, 0.3, 0.6, 1.3, 2.5, 5.0, and 10 mg mL−1 with respect to Ta and I, respectively). (c) Schematic of in vivo CT imaging by using MnOx/Ta4C3−SP composite nanosheets as the contrast agents. (d) In vivo CT contrasts of tumor tissue before and after i.v. administration of MnOx/Ta4C3−SP composite nanosheets. (e) In vivo 3D reconstruction CT (left) and contrast (right) images of mice before and after i.v. administration MnOx/Ta4C3−SP composite nanosheets (20 mg kg−1, 100 μL) for 2 h.
the cancer cells as evidenced by the presence of obvious intracellular green fluorescence originated from fluorescein isothiocyanate (FITC)-labeled MnOx/Ta4C3−SP composite nanosheets after 1 h coincubation. The fluorescence intensity was substantially enhanced with the prolonged coincubation duration for 4 h (Figure 5b). Then, confocal fluorescence images of calcine AM (green) and PI (red) costained cancer cells confirmed the effective photothermal ablation of 4T1 cells in the presence of MnOx/Ta4C3−SP composite nanosheets after exposing to NIR irradiation. The majority of 4T1 cells after the incubation with MnOx/Ta4C3−SP composite nanosheets was destructed under the NIR laser irradiation as demonstrated by the presence of strong red fluorescence (PI staining). In contrast, the viability of 4T1 cells treated without MnOx/Ta4C3−SP composite nanosheets or the only NIR laser irradiation was not obviously affected as evidenced by the strong green fluorescence (calcine AM staining, Figure 5c).
solvents such as H2O, saline, phosphate buffer saline (PBS), simulated body fluid (SBF), and Dulbecco’s Modified Eagle’s Medium (DMEM) (Figure 4g). The hydrodynamic size of MnOx/Ta4C3 by means of SP surface modification was measured to be around 190 nm by dynamic light scattering (DLS, Figure 4h). The series changes on Zeta potential of each modification step further indicated the successful grafting of SP on the surface of MnOx/Ta4C3 composite nanosheets (Figure 4i). In Vitro PTT against Tumor Cells by MnOx/Ta4C3−SP Composite Nanosheets. The in vitro cytotoxicity of MnOx/ Ta4C3−SP nanosheets as photothermal agents was initially evaluated. Nanosized MnOx/Ta4C3−SP could efficiently enter the cancer cells, trigger the intracellular hyperpyrexia and subsequently induce the death of cancer cells after 808 nm laser irradiation (Figure 5a). The confocal laser scanning microscopic (CLSM) images exhibited that MnOx/Ta4C3−SP composite nanosheets could be efficiently endocytosed into 12702
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Figure 7. Contrast-enhanced tumor microenvironment-responsive MR imaging by MnOx/Ta4C3-SP composite nanosheets both in vitro and in vivo. 1/T1 vs Mn concentration for MnOx/Ta4C3−SP composite nanosheets in buffer solution (a) at different GSH concentrations and (b) at different pH values after soaking for 3 h. (c) In vitro T1-weighted MR imaging of MnOx/Ta4C3−SP composite nanosheets in buffer solution at different GSH concentrations and (d) at different pH values after soaking for 3 h. (e) Schematic illustration of the disintegration of MnOx components from MnOx/Ta4C3 composite nanosheets under the specific tumor microenvironment (mild acidity and elevated GSH level) for contrast-enhanced T1-weighted MR imaging. (f) MRI-signal intensity and (g) corresponding T1-weighted imaging of 4T1 tumor-bearing mice after i.v. administration of MnOx/Ta4C3−SP composite nanosheets for prolonged time intervals.
It is noted that the as-fabricated MnO x /Ta 4 C 3 −SP composite nanosheets exhibited negligible cytotoxicity to 4T1 cells as evaluated by a standard Cell Counting Kit-8 (CCK-8) protocol (Figure 5d). After coincubation of MnOx/Ta4C3−SP nanosheets with cells at elevated concentrations (25, 50, 100, 200, 400 μg mL−1) for 24 and 28 h, MnOx/Ta4C3−SP composite nanosheets exhibited no obvious effect on the survival of 4T1 cells, even at a high evaluated concentration of 400 μg mL−1, indicating the excellent cytocompatibility of
MnOx/Ta4C3−SP composite nanosheets. The in vitro photothermal-ablation efficiency of MnOx/Ta4C3−SP composite nanosheets as photothermal agents for 4T1 cell ablation at different concentrations from 12.5 to 200 μg mL−1 was assessed after coincubation with cancer cells for 4 h, followed by 808 nm laser irradiation (2.0 W cm−2) for 5 min (Figure 5e). It has been shown that with the increase of the concentration of MnOx/Ta4C3−SP nanosheets, the viability of 4T1 cells sharply decreased after 808 nm laser irradiation. In addition, the 12703
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Figure 8. Contrast-enhanced PA imaging by MnOx/Ta4C3-SP composite nanosheets both in vitro and in vivo. (a) PA imaging of MnOx/ Ta4C3−SP buffer solution. (b) In vitro concentration-dependent PA signals of MnOx/Ta4C3−SP. (c) Schematics of in vivo PA imaging. (d) In vivo PA imaging and (c) PA signal values of the 4T1 tumor-bearing mice after the administration of MnOx/Ta4C3−SP composite nanosheets for prolonged time intervals.
viability of 4T1 cells incubated with the MnOx/Ta4C3−SP composite nanosheets at the concentration of 200 μg mL−1 were investigated after 808 nm laser irradiation at different power densities of 0, 0.5, 1.0, 1.5, and 2 W cm−2. The results showed that the enhanced 4T1 cell ablation was observed with the increased NIR power density, indicating the concentrationdependent and power density-dependent photothermal features of MnOx/Ta4C3−SP composite nanosheets for PTT (Figure 5f). The desirable in vitro PTT efficiency of MnOx/Ta4C3−SP composite nanosheets guarantees their further in vivo photothermal hyperthermia of tumor. Multiple Imaging Performances (CT, MRI and PA) of MnOx/Ta4C3 Composite Nanosheets. X-ray computed tomography (CT) imaging has been widely used in clinic due to its high spatial resolution, deep tissue penetration and excellent high-resolution 3D tomography imaging of anatomical structure and functional information.58 Tantalum, an element with high atomic number (Z = 73) and high X-ray attenuation coefficient (Ta: 4.3 cm2 kg−1, Au: 5.16 cm2 kg−1 at 100 eV),53 has been demonstrated as the desirable CT contrast agents.58−60 On this ground, we initially evaluated the X-ray attenuation ability of as-prepared Ta-containing MnOx/Ta4C3 composite nanosheets as CT contrast agents in vitro. Figure 6a and 6b showed that a significantly improved brightness of CT
images and the corresponding enhanced Hounsfield units (HU) values of MnOx/Ta4C3−SP composite nanosheets were observed with the elevation of Ta concentrations, revealing a perfect linear relationship with the Ta concentration. The HU value induced by MnOx/Ta4C3−SP nanosheets was nearly three folds higher than that of clinically used iodine-based Iopromide 300 under the same element concentration (10 mg mL−1), indicating the high contrast-enhanced CT imaging capability of MnOx/Ta4C3−SP nanosheets. Then, we further investigated whether these MnOx/Ta4C3− SP composite nanosheets could be used as in vivo CT contrast agents. Balb/c mice bearing 4T1 tumors were intravenously injected with MnOx/Ta4C3−SP composite nanosheets (dose: 20 mg kg−1, 100 μL) and then scanned by the clinical CTscanning instrument. Strong contrast in tumor of mice was observed from CT imaging, and the corresponding HU value increased to 132 HU after i.v. injection as compared to 78 HU before i.v. injection (Figure 6c−e), suggesting that MnOx/ Ta4C3−SP composite nanosheets could act as the promising contrast agents for in vivo contrast-enhanced CT imaging of tumors. Transition metal manganese (Mn), as one of the necessary elements in human bodies, shows relatively high biosafety and low toxicity as compared to clinically used Gd3+-based agents as 12704
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Figure 9. In vivo photothermal hyperthermia of tumor by MnOx/Ta4C3-SP composite nanosheets. (a) IR thermal images of 4T1 tumorbearing nude mice with or without intravenous injection of MnOx/Ta4C3−SP nanosheets under 808 nm laser irradiation (2 W cm−2) taken at different time intervals. (b) The corresponding elevated temperature curves at the tumor sites of 4T1 tumor-bearing nude mice under 808 nm laser irradiation for 600 s. (c) Time-dependent body-weight curves of 4T1 tumor-bearing nude mice of four groups after receiving different treatments, including control, NIR laser only, MnOx/Ta4C3−SP only and MnOx/Ta4C3−SP + NIR laser. (d) Time-dependent tumor-growth curves of four groups (control group, NIR laser group, MnOx/Ta4C3−SP group and MnOx/Ta4C3−SP + NIR laser group) after receiving different disposes. (e) Survival rates of 4T1 tumor-bearing mice within the feeding duration after different treatments (n = 5). (f) H&E staining, TUNEL staining and Antigen Ki-67 immunofluorescence staining in tumor tissues from each group after varied treatments. All the scale bars are 50 μm. (g) Digital images of tumors from each group at the end of various treatments.
ment-triggered T1-weighted MRI contrast agent, in vitro T1 relaxation time of MnOx/Ta4C3−SP composite nanosheets in buffer solutions at different GSH concentrations and different pH values was measured by using a clinical 3.0 T human clinical scanner. We used buffer solutions at different GSH concentrations (5.0 mM L−1 and 10.0 mM L−1) to imitate tumor reducing microenvironments,67,73 and buffer solutions of lowed pH values (pH = 5.0 and pH = 6.0) to mimic the tumor acidic microenvironments and buffer solutions at pH of 7.4 to mimic the normal blood circulation. The increase of relaxivity r1 and corresponding concentration-dependent brightening effect of T1-weighted MR imaging in the buffer solution at different GSH concentrations and at lowed pH values could be directly observed and calculated (Figure 7a−d). The initial relaxivity r1 of MnOx/Ta4C3−SP composite nanosheets in neutral SBF was calculated to be 3.77 mM−1 s−1, which further increased to 4.71 mM−1 s−1 and 5.38 mM−1 s−1 at GSH concentrations of 5.0 and
MRI contrast agents, which is related to the occurrence of nephrogenic systemic fibrosis (NSF).61,62 The introduced MnOx-functional motifs on the surface of Ta4C3 nanosheets could achieve both pH-responsive and glutathione (GSH)sensitive T1-weighted MR imaging,29,63−67 which are the two most representative characteristics (mild acidity and overexpressed GSH level) in the tumor microenvironments. Especially, a great many bioresponsive materials have been recently constructed in view of the lowed pH values of tumor microenvironments as compared to the normal physiological milieus.68−70 The pH-responsive T1-weighted MR imaging capability for tumor imaging was bestowed on the MnOx components-based nanocomposites, attributed to the instability of Mn−O bonds under the mildly acidic and reductive conditions of tumor microenvironments.29,64,71,72 To access the possibility of using the as-synthesized MnOx/ Ta4C3−SP composite nanosheets as a tumor microenviron12705
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MnOx/Ta4C3−SP composite nanosheets only, (iv) intravenously injected with MnOx/Ta4C3−SP composite nanosheets + NIR laser irradiation. After 4 h of injection, NIR laser (808 nm) irradiation toward 4T1 tumor ablation was performed at the power density of 2 W cm−2 for 10 min. The whole-body thermal images of the 4T1 tumor-bearing mice were taken at given time intervals (Figure 9a). The surface temperature of tumors monitored by infrared thermal (IR) imaging instrument increased to ∼55 °C for MnOx/Ta4C3−SPtreated mice within 10 min, which was sufficiently high for tumor ablation (Figure 9b). In contrast, the tumor temperature of the mice injected without MnOx/Ta4C3−SP under 808 nm laser irradiation at the same power density only increased by 5 °C. After the different treatments, the mice weights were recorded every 2 days, which showed on obvious weight changes among these four groups (Figure 9c). The length and width of the tumors were measured by a digital caliper every 2 days and the corresponding digital photos of tumor regions were taken every 2 days during the next 2 weeks (Figure 2g and Figure S4). It is important to find that MnOx/Ta4C3−SPtreated mice with 808 nm laser irradiation experienced a significantly suppressed tumor growth, while the other three groups showed no obvious inhibition effect on the tumor growth (Figure 9d). These MnOx/Ta4C3−SP-treated mice were still healthy after 60 days post treatment (Figure 9e), and no tumor recurrence was observed. Comparatively, the mice death occurred continuously in the other three groups, confirming the high in vivo therapeutic efficiency of photothermal hyperthermia assisted by MnOx/Ta4C3−SP composite nanosheets. Hematoxylin-eosin (H&E) staining, TdT-mediated dUTP Nick-End Labeling (TUNEL) and Ki-67 antibody staining of tumor slices were collected from each group of mice to further reveal the mechanism of photothermal effect at 24 h after NIR laser irradiation. Severe destruction including changed cell shapes and even condensed nuclei of tumor cells was discovered from micrographs of H&E-stained tumor slices of MnOx/Ta4C3−SP + NIR group (Figure 9f). Comparatively, no noticeable abnormal sign was observed in the other three groups. TUNEL results also showed much higher necrosis of tumor cells in MnOx/Ta4C3−SP + NIR laser group as compared to other three groups, which was in accordance with the result of H&E staining. Ki-67 antibody staining was introduced to evaluate the proliferative activities of cancer cells. The results of MnOx/Ta4C3−SP + NIR laser group exhibited significantly suppressed proliferative activities of cancer cells while no significant change of suppression effect on the cell proliferation was detected in the other three groups, confirming the high photothermal ablation effect of MnOx/Ta4C3−SP as the photothermal-conversion nanoagents for ablating the tumors (Figure 9f). On the basis of the systematic in vitro and in vivo evaluations, the as-designed MnOx/Ta4C3−SP nanosheets could achieve simultaneous PA/CT/MR multimodality imaging where the T1-weighted MR imaging is TME responsive. Comparatively, traditional photothermal nanoagents (e.g., Au28, graphene and its derivatives,15,16 black phosphorus,19 WS221 and MoS274) are difficult to be designed with multiple-imaging capability where the tedious multiple functionalization steps are typically involved. Especially, the Ta component in MnOx/Ta4C3−SP nanosheets is capable of enhancing the contrast of CT imaging, which takes the composition feature of the MnOx/Ta4C3−SP photothermal nanoagent. Especially, the photothermal-con-
10.0 mM, respectively (Figure 7a). Such a contrast-enhanced responsiveness was also observed in lowed pHs condition (pH = 5.0 and 6.0), where r1 value reached up to 4.89 mM−1 s−1 and 8.27 mM−1 s−1 at lowed pH values of 5.0 and 6.0, respectively (Figure 7b). This pH- and GSH-responsive MRI performance was attributed to the fact that the Mn−O bonds were easily cleaved under mildly acidic and elevated GSH condition of tumor microenvironments. The released Mn2+ could effectively improve the interaction probability of paramagnetic Mn centers to water molecules, further resulting in enhanced T1-weighted MRI performance (Figure 7e). Once recognized that the presence of the released Mn2+ originated from Ta4C3 nanosheets could efficiently enhance the relaxivity of hydrogen protons, MnOx/Ta4C3−SP composite nanosheets were further evaluated for their performance as the in vivo MRI contrast agents. The MR imaging of 4T1 tumorbearing mice was recorded before and after i.v. injection of MnOx/Ta4C3−SP composite nanosheets (20 mg kg−1, 100 μL). During the tested 60 min period after injection of MnOx/ Ta4C3−SP composite nanosheets, the MRI-signal enhancement in tumor tissues and their corresponding increased T1-weighted MR images were clearly observed (Figure 7f and 7g), suggesting that MnOx/Ta4C3−SP composite nanosheets is highly desirable for the subsequent PTT guidance and monitoring. Taking the high photothermal-conversion capability of MnOx/Ta4C3−SP composite nanosheets into account, the MnOx/Ta4C3−SP composite nanosheets were expected to act as the contrast-enhanced contrast agents for PA imaging. As shown in Figure 8a, the enhanced PA signals of MnOx/Ta4C3− SP composite nanosheets were observed as the Ta in MnOx/ Ta4C3−SP concentration increased, and the PA signals of MnOx/Ta4C3−SP composite nanosheets followed a linear relationship with the Ta concentration (Figure 8b), indicating the desirable PA-imaging capability of MnOx/Ta4C3−SP composite nanosheets. Furthermore, the in vivo PA imaging capability of MnOx/ Ta4C3−SP composite nanosheets was evaluated on 4T1 tumor bearing mice after the subcutaneous administration into mice (20 mg kg−1, 100 μL). As expected, the PA effect of MnOx/ Ta4C3−SP composite nanosheets as induced by a laser pulse illumination to generate rapid heating and subsequent acoustic emission signals could be clearly observed in tumor tissues with the prolonged observation time window, which could be further used to image the distribution of nanosheets as the contrast agents in tumors (Figure 8c). The PA signals were quickly enhanced, and the signal intensity reached the high value after the short time of subcutaneous administration (Figure 8d). The excellent in vitro and in vivo PA-imaging performance demonstrates the possibility of MnOx/Ta4C3−SP composite nanosheets as the contrast agents for PA imaging and detection of tumors. In Vivo Photothermal Ablation of Tumor by MnOx/ Ta4C3−SP Composite Nanosheets. To shed more light on the photothermal effects of MnOx/Ta4C3−SP composite nanosheets on the tumor therapy, 4T1 breast tumor-bearing mouse xenograft was established to further investigate the therapeutic efficiency of MnOx/Ta4C3−SP composite nanosheets as the photothermal-conversion nanoagents for tumor hyperthermia. After the tumor volumes reached about ∼60 mm3, the 4T1 tumor-bearing mice were randomly separated into four groups to receive different disposes (n = 5 per group): (i) control, (ii) NIR laser only, (iii) intravenously injected with 12706
DOI: 10.1021/acsnano.7b07241 ACS Nano 2017, 11, 12696−12712
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Figure 10. In vivo biocompatibility assay of MnOx/Ta4C3-SP composite nanosheets. (Top) Hematological assay of mice from the control group and three other treated groups at different MnOx/Ta4C3−SP doses of 5 mg kg−1, 10 mg kg−1 and 20 mg kg−1 after intravenous administration and further feeding for 30 days. (Bottom) H&E-stained tissue sections of major organs (heart, liver, spleen, kidney and lung) from mice after intravenous injection with saline (control) and MnOx/Ta4C3−SP composite nanosheets at different MnOx/Ta4C3−SP doses of 5 mg kg−1, 10 mg kg−1 and 20 mg kg−1 for another 30 days feeding. All the scale bars are 100 μm.
version efficiency of MnOx/Ta4C3−SP nanosheets (34.9%) is much higher than that of traditional Au nanorods (21%),56 Cu2‑xSe (22%),57 and Ti3C2 nanosheets (30.6%),45 indicating their high photothermal-hyperthermia performance for tumor ablation. Therefore, these MnOx/Ta4C3−SP nanosheets are intrinsically featured with structural, compositional and physiochemical property for efficient cancer theransotics. In Vivo Biocompatibility Assay of MnOx/Ta4C3−SP Composite Nanosheets. The in vivo biocompatibility and
biosafety of MnOx/Ta4C3−SP composite nanosheets were systematically investigated to ensure their safe bioapplications and guarantee further clinical translations. Twenty healthy Kunming mice were randomly divided into four groups, including control group and three other treated groups at three different MnOx/Ta4C3−SP doses of 5 mg kg−1, 10 mg kg−1 and 20 mg kg−1. After another 30-days feeding, these mice were sacrificed and their major organs including heart, lung, liver, kidney and spleen were collected and fixed in 10% 12707
DOI: 10.1021/acsnano.7b07241 ACS Nano 2017, 11, 12696−12712
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Synthesis of Ta4C3 Nanosheets. The resulting Ta4AlC3 products were immersed into a 40% HF aqueous solution (50 mL, Sinopharm Chemical Reagents Co., Ltd., Shanghai, China), and stirred for 96 h at room temperature. Then, the products were collected by centrifugation and washed for several times with ethanol and water. The HFetched Ta4C3 nanosheets were then immersed into water for another 12 h sonication to obtain ultrathin Ta4C3 nanosheets. Synthesis of MnOx/Ta4C3 Composite Nanosheets. The MnOx/Ta4C3 composite nanosheets were obtained via a facile redox reaction between KMnO4 (Sinopharm Chemical Regaent Co.) and terminated −OH groups on the Ta4C3 nanosheets’ surface to in situ generate MnOx components onto the surface of Ta4C3 nanosheets. Typically, KMnO4 aqueous solution (10 mg, 5 mL) was mixed with Ta4C3 aqueous solution (300 mg, 10 mL) under magnetic stirring for 24 h at room temperature. The resulting MnOx/Ta4C3 nanocomposites were purified with water and ethanol for several times by centrifugation at a speed of 20 000 rpm. Surface Modification of MnOx/Ta4C3 Composite Nanosheets (MnOx/Ta4C3−SP). The as-prepared MnOx/Ta4C3 composite nanosheets were not stable and easily aggregated in biological environment. A “thin-film” approach by coating MnOx/Ta4C3 nanosheets’ surfaces with soybean phospholipid (SP, Sigma-Aldrich, Shanghai, China) was carried out to facilitate the subsequent biomedical applications. Briefly, MnOx/Ta4C3 nanosheets in ethanol solution (1 mg mL−1, 1 mL) were immersed into SP chloroform solution (1 mg mL−1, 30 mL). Then, the mixture solution in the flask fixed at a rotary evaporator was heated to 60 °C under vacuum for 20 min to evaporate the solvent to yield MnOx/Ta4C3−SP composite nanosheets. Finally, 10 mL phosphate buffer saline solution was added into the flask and sonicated for 20 min for later use. Characterization. X-ray diffraction (XRD) was carried out on a Rigaku D/MAX-2200 PC XRD system with Cu Kα radiation (λ = 1.54 Å) at 40 kV and 40 mA. Size and Zeta potential measurements were measured on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). X-ray photoelectron spectroscopy (XPS) spectrum was recorded by ESCAlab250 (Thermal Scientific). UV−vis−NIR absorption spectra were recorded by UV-3600 Shimadzu UV−vis−NIR spectrometer with QS-grade quartz cuvettes at room temperature. The optical absorbance per cell length (A/L) was measured from the optical absorbance intensity at 808 nm. The Ta4C3 extinction coefficient was extracted from the slope of a plot of A/L versus concentration from Beer’s law (A/L = αC). Transmission electron microscopy (TEM) images and corresponding EDS spectrum were obtained on a JEM-2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) images/scanning transmission electron microscopy (STEM) images and corresponding element mapping were obtained on a field-emission Magellan 400 microscope (FEI Company). The Ta4C3 concentration was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies, US). The confocal laser scanning microscopy (CLSM) images were obtained in FV1000 (Olympus Company, Japan). In Vitro Photothermal Performance of MnOx/Ta4C3 Composite Nanosheets. To evaluate photothermal performance of MnOx/ Ta4C3, the aqueous solution (0.1 mL) of MnOx/Ta4C3 with different Ta concentrations was measured and analyzed by continuously irradiating with an 808 nm NIR laser (Shanghai Connect Fiber Optics Company) at a power density of 1.5 and 2.0 W cm−2 for 5 min, respectively. The temperature at different time duration was recorded using the FLIR A325SC camera. Pure water was set as control. The detailed calculation is provided in the Supporting Information. In Vitro PTT against Tumor Cells by MnOx/Ta4C3−SP Composite Nanosheets. Murine breast cancer line 4T1 cells were used to assess the photothermal performance of MnOx/Ta4C3−SP composite nanosheets. The cell viability of 4T1 cells after coincubation with MnOx/Ta4C3−SP nanosheets at elevated concentrations (25, 50, 100, 200, and 400 μg mL−1) was first tested by using a typical cellcounting kit 8 (CCK-8) protocol. The 4T1 cells were maintained at 37 °C under 5% CO2 at in grown 96-well plates at 1 × 104/well were Cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, high
formalin for histological characterization. During 30-days feeding period, no obvious weight variation or abnormal behavior in the three treated groups were observed as compared to the control group (Figure S5). A series of blood indexes including the white blood cells, red blood cells, platelets, lymphocytes, hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular volume, hematocrit, and liver and kidney function in all four groups were measured at the 30th day post injection. All the indexes in the three MnOx/ Ta4C3−SP-treated groups were found to be normal as compared to that of control group, indicating no adverse effects induced by MnOx/Ta4C3−SP composite nanosheets (Figure 10 a−i). H&E staining of the key organs from different groups of mice treated at the elevated doses was carried out to evaluate the pathological change. Each group showed no shrunk malignant cells, loss of contact, or nucleus damage on the examined tissue sections, indicating that no obviously histological abnormalities or lesions were induced in the main organs after the injection of MnOx/Ta4C3−SP composite nanosheets (Figure 10m). Encouraged by these preliminary in vivo biosafety results at tested doses for 30 days, the as-prepared MnOx/Ta4C3−SP-treated MnOx/Ta4C3−SP composite nanonosheets are expected be nontoxic, which favors their further bioapplications and possibly clinical translation.
CONCLUSIONS In summary, we herein have successfully construct the 2D tantalum carbide (Ta4C3) MXene-based composite nanosheets for efficient multiple imaging-guided photothermal tumor ablation. Especially, a redox reaction was activated on the surface of tantalum carbide (Ta4C3) MXene for in situ growth of manganese oxide nanoparticles (MnOx/Ta4C3) based on the reducing surface of the nanosheets. By rational selection on the composition of MXene and further functionalization, the asconstructive MnOx/Ta4C3−SP composite nanosheets acted as the high-performance contrast agents for simultaneous computed tomography (tantalum-based component), tumor microenvironment-responsive T1-weighted magnetic resonance imaging (MnOx component) and photoacoustic imaging (photothermal-conversion property). Importantly, the high photothermal-conversion performance of MnOx/Ta4C3−SP composite nanosheets has realized the significant tumor-growth suppression by photothermal hyperthermia. Systematic in vivo evaluation has demonstrated the high biocompatibility and biosafety of the as-designed MnOx/Ta4C3−SP composite nanosheets. This work broadens the biomedical applications of 2D MXenes by rational choice of desirable chemical composition (Ta4C3) and surface engineering for functionalization (MnOx integration), which also provides the desirable high-performance theranostic nanoagents for efficiently combating cancer. METHODS Synthesis of Ta4AlC3. Ta4AlC3 MAX-phase ceramics were prepared by ball-milling element Ta (−325 mesh, 99.97%, Alfa Aesar, Ward Hill, USA), Al (−325 mesh, 99.5%, Alfa Aesar, Ward Hill, USA), and C (−300 mesh 99%, particle size
