Article Cite This: Chem. Mater. 2017, 29, 8637-8652
pubs.acs.org/cm
Biocompatible 2D Titanium Carbide (MXenes) Composite Nanosheets for pH-Responsive MRI-Guided Tumor Hyperthermia Chen Dai,†,§ Han Lin,‡ Guang Xu,† Zhuang Liu,∥ Rong Wu,*,† and Yu Chen*,‡ †
Department of Ultrasound in Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China ‡ State Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China § Department of Ultrasound, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P. R. China ∥ Department of Radiology, Fudan University Shanghai Cancer Center, Shanghai 200032, P. R. China S Supporting Information *
ABSTRACT: The emerging of two-dimensional (2D) MXenes significantly broadens the family members and versatile applications of 2D materials, but the rational design of MXene-based composites and their specific applications in theranostic biomedicine are still challenging. In this work, we report, for the first time, on the elaborate design of the Ti3C2based composite MXene (MnOx/Ti3C2) for highly efficient theranostic applications against cancer. These MnOx/Ti3C2 composite MXenes have been constructed by triggering a simple redox reaction to in situ grow small MnOx nanosheets on the surface of Ti3C2. These MnOx/Ti3C2 composite MXenes have been developed as multifunctional theranostic agents for efficient magnetic resonance (MR) and photoacoustic (PA) dual-modality imaging-guided photothermal therapy (PTT) against cancer. Especially, the decoration of the MnOx component onto MnOx/Ti3C2 realizes the unique tumor microenvironment-responsive T1-weighted MR imaging of tumors, and the high photothermal-conversion performance not only endows the MnOx/Ti3C2 with desirable contrast-enhanced PA-imaging property but also realizes the highly efficient tumor ablation and tumor-growth suppression. The in vivo biocompatibility of these MnOx/Ti3C2−SP composite nanosheets has also been systematically evaluated and revealed. This work not only paves a new way for the multifunctionalization of 2D MXenes simply by integrating other functional components but also broadens their versatile applications in theranostic nanomedicine.
■
phosphorus,22−25 and even their composites.26−28 The fast development of theranostic nanomedicine and 2D nanomaterials catalyzes the generation of new 2D nanosystems with improved photothermal-conversion efficiency, high biocompatibility, and specific responsibility to tumor microenvironments for enhanced theranostic nanomedicine.15,29−31 As a new 2D material, MXenes are featured with the general formula of Mn+1Xn, where M represents an early transition metal and X represents carbon and/or nitrogen, n = 1, 2, 3.32−34 These 2D MXenes are typically generated from their corresponding layer-structured MAX phases via the selective etching of the A layer.35−39 The A layer refers to the element from the group IIIA or IVA in the periodic table. Especially, we recently have demonstrated that 2D titanium carbide (Ti3C2) MXenes show the high photothermal-conversion efficiency for tumor ablation, demonstrating the promising application
INTRODUCTION Cancer has become one of the main causes of death nowadays.1 Although surgery, chemotherapy, and radiotherapy have been extensively adopted for clinical cancer therapy, these therapeutic modalities inevitably cause serious side effects and low therapeutic efficacy. Photothermal therapy (PTT), as a promising alternative therapeutic modality, has gained increasing attention due to its minimal damage to normal tissues and high ablation efficacy, which is typically triggered by nearinfrared (NIR) laser to in situ generate heat for the thermal ablation of cancers.2−5 In order to optimize the PTT efficiency, a variety of photothermal-transducing agents (PTAs) have been explored, such as Au-based nanomaterials,6−8 Pd nanosheets,9 carbon-based nanomaterials,10−13 etc. Especially, recent research has demonstrated that two-dimensional (2D) nanomaterials with planar topology and ultrathin thickness exhibit promising physiochemical performance and biological behavior for theranostic nanomedicine, especially for PTT against cancer. Diverse 2D nanosheets have been explored as the PTAs,14,15 including 2D MoS2,16−18 WS2,19,20 TiS2,21 black © 2017 American Chemical Society
Received: June 12, 2017 Revised: August 31, 2017 Published: August 31, 2017 8637
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 1. Schematic illustration of the synthetic process and MR/PA imaging-guided photothermal tumor therapy of MnOx/Ti3C2−SP composite nanosheets. (a) The scheme of the exfoliation process for 2D Ti3C2 nanosheets based on a ball-and-stick model, including HF etching and TPAOH intercalation. (b) The scheme of the whole synthetic procedure for MnOx/Ti3C2−SP composite nanosheets, including HF etching, TPAOH intercalation, in situ redox reaction between Ti3C2 nanosheets and postintroduced KMnO4, and subsequent surface SP modification. (c) Schematic illustration of theranostic functions of MnOx/Ti3C2−SP composite nanosheets, i.e., MR/PA imaging-guided efficient PTT ablation of cancer.
potentials of MXenes in biomedicine.40 In addition, the therapeutic complexity of tumors typically requires the predetermination of tumor sites and/or monitors the therapeutic process and efficiency. The fabrication of theranostic agents with concurrent diagnostic−imaging capability and therapeutic performance can achieve this goal to some extent.41−44 However, 2D titanium carbide (Ti3C2) MXenes only exhibit single photothermal capability for tumor ablation. It is still challenging to endow these Ti3C2 MXenes with more functionalities to satisfy the strict theranostic application requirements. This work reports, for the first time, on the exploring of 2D MXene-based composite theranostic agents for efficient cancer treatment. We developed a versatile but highly efficient “redox reaction-induced growth” (RR-IG) approach to in situ grow MnOx nanosheets onto the surface of 2D Ti3C2 MXene (MnOx/Ti3C2). The integration of MnOx with Ti3C2 is based on the consideration that magnetic resonance imaging (MRI) is
one of the most powerful imaging modalities in clinic due to its high spatial resolution and excellent tissue contrast,45 which has been extensively explored for tumor diagnosis. However, the US Food and Drug Administration (FDA) has warned that traditional clinic Gd-based contrast agents can increase the risk of potential nephrogenic systemic fibrosis (NSF), which severely limits their extensive practical applications.46 Comparatively, as one of the important essential trace elements in the human body, manganese (Mn)-based paramagnetic agents have been regarded as possible substitutes for those traditional Gd-based contrast agents.47−50 For instance, MnCl2 tetrahydrate (Lumenhance), an oral contrast agent, has been approved by the FDA for clinical use.51 Therefore, the rational integration of Mn-based agents with Ti3C2 MXenes is expected to realize the concurrent MR imaging and photothermal ablation of tumors. The elaborately designed MnOx/Ti3C2 composite nanosheets are featured with the following specific characteristics for 8638
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 2. Characterization of Ti3AlC2 bulk ceramic and Ti3C2 nanosheets. (a) Photograph of Ti3AlC2 bulk ceramic. (b) SEM images of multilayered Ti3C2 after HF etching for 3 days. (c) TEM images of ultrathin Ti3C2 nanosheets after exfoliation by TPAOH for 3 days. (d) SAED pattern and (e) HRTEM image of ultrathin Ti3C2 nanosheets. (f) AFM image of ultrathin Ti3C2 nanosheets after exfoliation. (g) The statistical diameter (top image) and thickness (down image) of Ti3C2 nanosheets obtained from the AFM image. (h) Three-dimensional AFM image of ultrathin Ti3C2 nanosheets.
efficient tumor theranostics. First, the in situ grown MnOx components on the surface of Ti3C2 nanosheets can act as the pH-responsive contrast agents for T1-weighted MR imaging. Second, these MnOx/Ti3C2 composite nanosheets exhibit promising photoacoustic (PA) imaging capability for efficient tumor detection. Third, the high photothermal-conversion performance of MnOx/Ti3C2 composite nanosheets efficiently induces the tumor-cell death by laser-induced hyperthermia. Last but not least, the high biocompatibility of MnOx/Ti3C2 composite MXenes can guarantee their further potential clinical translation, which has been systematically evaluated both in vitro and in vivo.
redox reaction on the surface of Ti3C2 nanosheets can in situ generate paramagnetic MnOx species, which are firmly anchored on the surface of Ti3C2 nanosheets (Figure 1b). Their surface has been further modified with soybean phospholipid (SP) to further improve the stability of MnOx/ Ti3C2 composite nanosheets (designated as MnOx/Ti3C2−SP). These elaborately designed MnOx/Ti3C2−SP composite nanosheets exhibit the unique structure−property relationship for cancer theranostics after transportation within the blood vessel and accumulation into tumor tissue via the enhanced permeability and retention (EPR) effect of tumors (Figure 1c). On one hand, the surface-anchored paramagnetic MnOx components act as the pH-responsive contrast agents for MR imaging of tumors. On the other hand, the ultrathin Ti3C2 nanosheets exert the high photothermal-conversion performance for efficient thermal ablation of tumors via PTT. The bulk Ti3AlC2 bulk ceramic was initially sintered by a solid-phase reaction at high temperature (Figure 2a), followed by HF etching to remove the Al layer. The SEM image (Figure 2b) shows that Ti3C2 after HF etching exhibits obvious layer structures with the expanded interlayer space. However, the HF-treated Ti3C2 is still in the form of sheet aggregates, which is not suitable for the biomedical applications, especially for intravenous administration and transportation within the blood vessels. Importantly, the further exfoliation assisted by TPAOH could directly transform the sheet aggregates into ultrathin and highly dispersed nanosheets with small planar sizes (Figure 2c). The selected area electron diffraction pattern (SAED, Figure 2d) and the high resolution TEM (HRTEM) image (Figure 2e) show that the as-fabricated Ti3C2 nanosheets are highly crystallized with hexagonal crystal structure, indicating that the crystallized feature of the basal planes of as-synthesized Ti3C2 nanosheets was well preserved after HF etching and subsequent TPAOH exfoliation. To characterize the sheet thickness, the typical atomic force microscopy (AFM) was
■
RESULTS AND DISCUSSION Design, Synthesis, and Characterization of 2D Ti3C2 (MXenes) Composite Nanosheets. 2D Ti3C2-based MXenes have been extensively explored for versatile applications,52,53 but their biomedical application only emerges very recently,40,54−56 not to mention the rational design of Ti3C2based composite nanosheets for theranostic biomedical purposes. Herein we focus on the multifunctionalization of ultrathin Ti3C2 nanosheets with paramagnetic MnOx components for tumor microenvironment-sensitive (acidity) T1weighted MRI-guided efficient PTT against tumors. The synthetic process of Ti3C2-based composite nanosheets (MnOx/Ti3C2−SP) is divided into three steps. First, ultrathin 2D Ti3C2 MXene nanosheets were synthesized by a two-step exfoliation procedure from the bulk Ti3AlC2 MAX-phase ceramic, including initial HF etching and subsequent tetrapropylammonium hydroxide (TPAOH) intercalation (Figure 1a, b). The surface of as-synthesized ultrathin Ti3C2 nanosheets has been demonstrated to be abundant with −OH groups with reducing property,52,57 which can in situ react with postintroduced KMnO4 with strong oxidative behavior. This 8639
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 3. Morphology and structural characterizations of MnOx/Ti3C2 nanosheets. (a, b) TEM images of MnOx/Ti3C2 nanosheets at different magnifications. (c) Dark-field and (d) secondary electron SEM images of MnOx/Ti3C2 composite nanosheets.
To show the uniformity of MnOx distribution, the X-ray energy dispersive spectroscopy (EDS) element mapping of MnOx/Ti3C2 composite nanosheets has been conducted where the corresponding element-mapping images show that the Mn element is present on the surface of MnOx/Ti3C2 with high uniformity (Figure S2, Figure 4a−c). The Mn content was determined to be around 6.5% by EDS results (Figure 4d). The absence of Al signals indicates the complete removal of the Al layer from Ti3AlC2 during the HF etching and TPAOH intercalation process. The electron energy loss spectrum (EELS) of MnOx/Ti3C2 shows the presence of obvious Mn, Ti, and O signals, demonstrating the desirable MnOx-decorated Ti3C2 composition (Figure 4e). Furthermore, the chemical status of MnOx/Ti3C2 composite nanosheets has been characterized by the typical X-ray photoelectron spectroscopy (XPS). The obvious Mn signal in XPS demonstrates the presence of MnOx on the surface of Ti3C2 nanosheets (Figure 4f), and the fitted peaks were at 641.2 and 658.2 eV, respectively (Figure S3). The relative contents of bivalent, trivalent, and quadrivalent Mn in MnOx/Ti3C2 composite MXenes were determined to be 21%, 40%, and 39%, respectively (Figure 4g). In Vitro Photothermal-Conversion Property of MnOx/ Ti3C2 Nanosheets. The Ti3C2 MXene component in MnOx/ Ti3C2 composite nanosheets is featured with high photothermal-conversion capability for potential PTT against cancer. UV−vis spectra of MnOx/Ti3C2 nanosheets exhibit the concentration-dependent light absorption in the NIR region (Figure 5a), especially at the typical wavelength of 808 nm for PTT application. The extinction coefficient MnOx/Ti3C2 composite at 808 nm at varied concentrations (80, 40, 20, 10, and 5 ppm) was measured to be 5.0 Lg−1 cm−1 (Figure 5a inset). In order to evaluate the photothermal-conversion performance of MnOx/Ti3C2 nanosheets, they were exposed to an 808 nm NIR laser (1.0 W cm−2) at elevated concentrations, and the temperature variation was recorded
adopted. It has been found that the as-prepared 2D Ti3C2 nanosheets exhibit a mean planar diameter of ∼227 nm and an average thickness of ∼2.8 nm (Figure 2f−h), clearly demonstrating the typical 2D ultrathin nanostructures suitable for biomedical applications. The as-synthesized Ti3C2 MXene has broad thickness distribution, which is considered to be attributed to the relatively complicated synthetic process involving two exfoliation steps (HF etching and TPAOH intercalation). The ultrathin Ti3C2 MXene was synthesized by the direct exfoliation from the solid and hard MAX ceramic, which makes it difficult to synthesize 2D MXene nanosheets with highly uniform thickness. Large amounts of reducing groups are present on the surface of as-synthesized Ti3C2 nanosheets such as −OH.57 Based on the specific advantages of large surface area and reducing surface of Ti3C2 nanosheets, we simply introduced oxidative KMnO4 into Ti3C2 aqueous solution, by which a simple redox reaction between KMnO4 and Ti3C2 could be in situ triggered to generate highly dispersed small MnOx nanosheets on the surface of initial Ti3C2 nanosheets. As shown in Figure 3a and b, highly uniform and dispersive small MnOx nanosheets have been grown onto the surface of Ti3C2 nanosheets. The darkfield TEM image (Figure 3c) of MnOx/Ti3C2 composite nanosheets further demonstrates the formation of a thin MnOx layer on the surface of Ti3C2 nanosheets. The secondary electron SEM image (Figure 3d) exhibits the clear planar morphology of MnOx/Ti3C2 composite nanosheets with small sheetlike MnOx components on the surface. The postformed MnOx nanoparticles exhibit the sheetlike morphology with an average planar size of ∼15.5 nm and an average thickness of ∼1.5 nm (n = 50, Figure S1). This RR-IG strategy for MnOx/ Ti3C2 composite nanosheets is highly facile and effective, which only requires the mild reaction condition at room temperature and short reaction duration, but the products are highly uniform and controllable. 8640
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 4. Composition and element analysis of MnOx/Ti3C2 composite nanosheets. (a-c) SEM images of elemental mappings of MnOx/Ti3C2 nanosheets (a: Ti, b: Mn, c: merged image). (d) X-ray energy dispersive spectrum (EDS) and (e) electron energy loss spectra (EELS) of MnOx/ Ti3C2 nanosheets. (f, g) XPS spectra of Ti3C2 and MnOx/Ti3C2.
still sufficiently high to ablate the tumor. Therefore, the high in vitro photothermal-conversion capability and stability are expected to guarantee the potential efficient in vivo tumor ablation. The stability of as-fabricated MnOx/Ti3C2 nanosheets in physiological solution is rather low, which means that they tend to aggregate, making them unsuitable for intravenous administration. To further improve the stability of MnOx/ Ti3C2 in physiological solution, their surface was modified with the biocompatible soybean phospholipid (SP) by a facile rotary evaporation technique (designated as MnOx/Ti3C2−SP). After SP coating, the average particle size of MnOx/Ti3C2−SP nanosheets measured by dynamic light scattering (DLS) was determined to be around 230 nm (Figure 5e). Importantly, the surface-modified MnOx/Ti3C2−SP nanosheets have high colloidal stability in physiological solutions, such as Dulbecco’s Modified Eagle’s Medium (DMEM), saline, PBS, and simulated body fluid (SBF), which can guarantee their further intravenous administration (Figure 5f, Figure S6). Contrast-Enhanced pH-Responsive T1-Weighted MR Imaging of MnOx/Ti3C2−SP Nanosheets. The as-synthesized MnO x /Ti 3 C 2 −SP nanosheets exhibit the unique composition-property relationship for tumor theranostics. The integrated MnOx components exhibit the unique pH-
by an IR thermal camera. It has been found that the temperature increases significantly after exposing MnOx/ Ti3C2 nanosheets solution to laser irradiation (Figure 5b and c). The temperature was increased 15.3, 12.2, and 11.5 °C at the concentration of 50, 25, and 12.5 ppm, respectively. Comparatively, the pure water only showed a negligible increase after the laser irradiation. In addition, the MnOx/ Ti3C2 nanosheets exhibited power density-dependent photothermal-conversion efficiency where the high power density (1.5 W cm−2) caused much of the enhanced temperature increase (Figure S4). The photothermal stability was evaluated by testing five heating/cooling cycles where no obvious temperature decrease was observed, showing the high photothermal stability of MnOx/Ti3C2 composite nanosheets. The photothermal-conversion efficiency η of MnOx/Ti3C2 nanosheets was calculated to be 22.9% (Figure S5), which was comparable to that of traditional Au nanorods (21%)58 and Cu2−xSe NCs (22%).59 Compared to initial Ti3C2 nanosheets,40 the synthesized MnOx/Ti3C2 composite MXenes show the reduced photothermal effect (photothermal-conversion efficiency: from 30.6% to 22.9%) because the in situ triggered chemical reaction during MnOx integration could partially influence the nanostructure of ultrathin Ti3C2 nanosheets, but the photothermal effect of MnOx/Ti3C2 composite MXenes is 8641
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 5. (a) UV−vis spectra of MnOx/Ti3C2 nanosheets dispersed in aqueous solution at varied concentrations (80, 40, 20, 10, and 5 ppm). Inset: Normalized absorbance intensity at λ = 808 nm divided by the characteristic length of the cell (A/L) at varied concentrations (80, 40, 20, 10, and 5 ppm). (b) The temperature and thermal imagings and (c) the photothermal heating curves of MnOx/Ti3C2 nanosheets at elevated concentrations under 808 nm laser irradiation (1.0 W cm−2). (d) Recycling heating profiles of MnOx/Ti3C2 nanosheets aqueous solution after exposure to 808 nm laser at 1.0 W cm−2 for five laser on/off cycles. (e) Dynamic light scattering (DLS) of MnOx/Ti3C2 and MnOx/Ti3C2−SP nanosheets. (f) Digital images of MnOx/Ti3C2 and MnOx/Ti3C2−SP nanosheets dispersed in different physiological solutions.
and 7.26 mM−1 s−1 at the pHs of 5.0 and 6.0 after 0.5 h soaking, respectively (Figure S7a), which was nearly 5.8- and 5.6-fold as compared to that of MnOx/Ti3C2−SP nanosheets at neutral condition. The r1 relaxivity increased with the prolonged incubation time (Figure 6 and Figure S7), and the acidic microenvironment significantly enhanced the positive r1 relaxivity. The r1 relaxivity almost remains constant after 6 h soaking under acidic conditions, which might be attributed to the nearly complete disintegration of MnOx nanoparticles and release of manganese ions. Such an increased relaxivity is due to the enhanced accessibility of paramagnetic Mn centers to water molecules after the MnOx disintegration. This desirable pH response of MR imaging is highly favorable for the tumor imaging based on mild acidic conditions during the tumorigenesis.63−65 The structure evolution of MnOx on the surface of Ti3C2 was further characterized by TEM to directly show the disintegration of MnOx under acidic conditions. It has been found that the MnOx component is still present on the surface after soaking in neutral buffer solution (Figure 6d and e). Comparatively, the rough surface of initial MnOx/Ti3C2−SP
responsive T1-weighted MR imaging capability for tumor imaging because the Mn−O bonds are easily broken up under mild acidic conditions of tumor microenvironments.47,60,61 This behavior can release the Mn2+ ions that can maximize the interaction chances between paramagnetic Mn centers and water molecules, which further enhances the T1-weighted MRI performance (Figure 6a).49,62 The in vitro T1-weighted MRI performance was evaluated by testing the relaxivity (r1) of MnOx/Ti3C2−SP in the buffer solution at different pH values (pH = 7.4, 6.0, and 5.0) on a clinical 3.0 T human clinical MRI scanner. The neutral pH (7.4) represents the normal blood-circulation condition, and the acidic pHs (6.0 and 5.0) mimic the mild acidic microenvironment of tumors. It is interesting to find that an obvious concentration-dependent brightening effect was achieved in T1-weighted MR imaging, and acidic conditions induced significantly enhanced positive MRI signals (Figure 6b). The r1 relaxivity of MnOx/Ti3C2−SP was calculated to be 1.30 mM−1 s−1 after 0.5 h soaking at a neutral environment. Importantly, this r1 value increased to as high as 7.63 mM−1 s−1 8642
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 6. Contrast-enhanced pH-responsive MRI of MnOx/Ti3C2-SP nanosheets both in vitro and in vivo. (a) Schematic illustration of the disintegration of the MnOx component from MnOx/Ti3C2 nanosheets under mild acidic conditions for contrast-enhanced T1-weighted MR imaging. (b) In vitro T1-weighted MR imaging of MnOx/Ti3C2−SP nanosheets in buffer solution at different pH values after soaking for 3 h. (c) 1/T1 vs Mn concentration for MnOx/Ti3C2−SP nanosheets in buffer solution at different pH values after soaking for 3 h. TEM imaging (d, e, g, h, j, k) and the corresponding EDS (f, i, l) of MnOx/Ti3C2−SP nanosheets after soaking under different pH values (d, e, and f: pH = 7.4; g, h, and i: pH = 6.0; j, k, and l: pH = 5.0). (m) T1-weighted imaging and (n) corresponding MRI-signal intensity of 4T1 tumor-bearing mice after intravenous administration of MnOx/Ti3C2−SP nanosheets at different time intervals.
μL). After intravenous administration of MnOx/Ti3C2−SP composite nanosheets into mice, the T1-weighted MR images were acquired at the given time intervals. As shown in Figure 6m, a remarkable brightening effect of MRI signals was observed in tumors, which was further gradually enhanced with the prolonged imaging durations. This contrast-enhanced positive MRI signal was due to efficient accumulation of the MnOx/Ti3C2−SP nanosheet via the EPR effect and the mild acidic microenvironment of tumors, which further triggered the disintegration of the MnOx component in MnOx/Ti3C2−SP
became smooth after soaking in the acidic buffer solution (Figure 6g, h, j, and k) because of the disintegration of MnOx accompanying the release of Mn2+. The corresponding EDS results show that the Mn signals had a significant decrease in acidic conditions as compared to neutral microenvironments (Figure 6f, i, and l), further demonstrating this unique pH responsiveness. The in vivo T1-weighted MRI capability of MnOx/Ti3C2−SP composite nanosheets was further evaluated on nude mice bearing 4T1 breast tumor xenograft (dose: 2 mg mL−1, 100 8643
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 7. PA imaging of MnOx/Ti3C2-SP nanosheets both in vitro and in vivo. (a) In vitro PA imaging and (b) the corresponding quantitative concentration-dependent PA signals of MnOx/Ti3C2−SP buffer solution. (c) In vivo PA imaging and (d) PA-signal values of the tumor of 4T1 tumor-bearing mice after intravenous administration of MnOx/Ti3C2−SP nanosheets for prolonged time intervals.
time intervals after the injection. It has been found that the PA signal was gradually enhanced with the prolonging of the observation time (Figure 7c and d) because of the tumor accumulation via the typical EPR effect, and the signal intensity reached high value after the injection for 24 h. The excellent in vitro and in vivo PA-imaging evaluation demonstrates the possibility of MnOx/Ti3C2−SP composite nanosheets for the following PA imaging-guided PTT and monitoring the therapeutic process. In Vitro PTT against Tumor Cells by MnOx/Ti3C2−SP Composite Nanosheets. The photothermal-conversion capability of MnO x/Ti 3C2−SP nanosheets was initially evaluated at the cell level by choosing 4T1 breast cancer as the model cell line. The cell viability of 4T1 cells after coincubation with MnOx/Ti3C2−SP nanosheets at elevated concentrations (10, 20, 40, 80, 160 μg mL−1) was first assessed by adopting a typical cell-counting kit 8 (CCK-8) protocol. No obvious cytotoxicity was observed even at the high concentration of 160 μg mL−1 after coincubation for 24 and 48 h (Figure 8a), indicating the relatively high biocompatibility of MnOx/Ti3C2−SP composite nanosheets. The photothermal-ablation efficiency was further evaluated by the exposure of MnOx/Ti3C2−SP composite nanosheets to 808 nm laser at different power densities. After coincubation for 4 h followed by 808 nm laser irradiation, the cells were
and enhanced the MRI signals afterward. This MRI-signal enhancement in tumor tissue was further clearly shown in quantitative positive signal values (Figure 6n). This pHresponsive T1-weighted MRI performance is highly favorable for the following PTT guidance and monitoring. PA Imaging of MnOx/Ti3C2−SP Nanosheets Both in Vitro and in Vivo. As a new but highly promising diagnosticimaging modality, PA imaging can break through the penetration limitation of traditional optical imaging based on its low tissue-attenuation coefficient, which can afford the realtime detection of the in vivo biological structure and functional information.66 The high photothermal-conversion capability of MnOx/Ti3C2−SP endows them with the potential contrastenhanced PA imaging performance. As expected, significantly enhanced PA signals could be observed in the solution containing MnOx/Ti3C2−SP composite nanosheets (Figure 7a and b). It is also confirmed that the PA signals produced by MnOx/Ti3C2−SP nanosheets are in linear relation with their concentration (Figure 7b, R = 0.988), indicating the desirable PA-imaging performance of MnOx/Ti3C2−SP composite nanosheets. The in vivo PA-imaging performance of MnOx/Ti3C2−SP composite nanosheets was evaluated on nude mice bearing 4T1 breast tumor xenograft after intravenous administration (dose: 5 mg mL−1, 100 μL). The PA images were acquired at different 8644
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 8. In vitro PTT against tumor cells. (a) Relative viabilities of 4T1 cells after incubation with various concentrations of MnOx/Ti3C2−SP nanosheets for 24 and 48 h. Error bars were based on the standard deviations (SD) of five parallel samples. (b) Relative viabilities of 4T1 cells after incubation with MnOx/Ti3C2−SP followed by exposure to 808 nm laser at different laser power densities. (c) Relative viabilities of 4T1 cells after incubation with elevated concentrations of MnOx/Ti3C2−SP composite nanosheets followed by laser irradiation at the power density of 1.0 W cm−2. (d) Schematic illustration of MnOx/Ti3C2−SP nanosheets as the photothermal agents for cancer cell ablation, including efficient uptake into cancer cells, the distribution within the cytoplasm of cells, and photothermal conversion by laser irradiation to elevate the intracellular temperature, causing the cell ablation and death. (e) CLSM images of 4T1 cancer cells after the different treatments, including the control group, NIR group, MnOx/ Ti3C2−SP group, and MnOx/Ti3C2−SP + NIR group.
In Vivo PTT Suppressing the Tumor Growth by MnOx/ Ti3C2−SP Composite Nanosheets. Encouraged by their high photothermal-conversion capability and efficient in vitro photothermal ablation of cancer cells, the in vivo performance of MnOx/Ti3C2−SP nanosheets was further systematically evaluated by the intravenous administration of the composite nanosheets into 4T1 breast tumor-bearing mice followed by NIR irradiation. Initially, 20 mice were subcutaneously injected with murine breast cancer 4T1 cells in 10 μL of PBS into the backs of each Balb/c female mouse. After the size of the tumors reached approximately 60 mm3, the mice were randomly divided into four groups with five mice in each group, including (a) the control group (no MnOx/Ti3C2−SP nanosheets injection and no laser irradiation), (b) the MnOx/Ti3C2−SP group (only intravenously injected with MnOx/Ti3C2−SP nanosheets); (c) the NIR group (only exposed to the laser irradiation); and (d) the MnOx/Ti3C2−SP + NIR group (intravenously injected with MnOx/Ti3C2−SP nanosheets followed by exposure to laser irradiation). The tumor-bearing mice in the laser and MnOx/Ti3C2−SP + NIR groups were treated under the 808 nm laser irradiation at the power density of 1.5 W cm−2 for 10 min after the intravenous injection for 4 h. It has been found that the tumor temperature in the MnOx/ Ti3C2−SP + NIR group increased remarkably from approx-
efficiently killed by photothermal ablation because of the presence of MnOx/Ti3C2−SP composite nanosheets as the photothermal agents, which is laser power density-dependent (Figure 8b) and MnOx/Ti3C2−SP concentration-dependent (Figure 8c). The small planar size of MnOx/Ti3C2−SP composite nanosheets could guarantee their efficient endocytosis into cancer cells, which is mainly distributed within the cytoplasm of cells. After 808 nm laser irradiation, the high photothermal-conversion capability of MnOx/Ti3C2−SP nanosheets triggers the intracellular temperature elevation and subsequent ablation of cancer cells (Figure 8d). Confocal laser scanning microscopic (CLSM) images of 4T1 cells were further acquired after treatment with laser, MnOx/Ti3C2−SP, and MnOx/Ti3C2−SP combined with laser irradiation followed by costaining with calcine AM (green) and propidium iodide (PI, red). It clearly shows that the cells kept the normal status after the treatment with laser and MnOx/Ti3C2−SP because of the presence of obvious green fluorescence that indicates the live cells with normal status (Figure 8e). Comparatively, the cells after the treatment with MnOx/Ti3C2−SP combined with laser irradiation exhibit strong red fluorescence that represents the dead cells, demonstrating the high therapeutic and killing effect of MnOx/Ti3C2−SP nanosheets after the photothermal conversion. 8645
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 9. (a) The elevated temperature at the tumor region of 4T1 tumor-bearing nude mice under laser irradiation for 600 s. (b) IR thermal images of 4T1 tumor-bearing nude mice with or without receiving intravenous injection of MnOx/Ti3C2−SP nanosheets followed by 808 nm laser irradiation (1.5 W cm−2) at different time intervals. (c) Time-dependent body-weight curves of nude mice of four groups after different treatments, including the control group, NIR group, MnOx/Ti3C2−SP group, and MnOx/Ti3C2−SP + NIR group. (d) Time-dependent tumor-growth curves of four groups after different treatments as indicated in the figure. (e) Digital images of tumors from each group after varied treatments. (f) H&E staining, TUNEL staining, and Antigen Ki-67 immunofluorescence staining in tumor tissues from each group after the treatment. All the scale bars are 100 μm.
imately 25 to 60 °C under the laser irradiation for 10 min (Figure 9a and b), which is sufficiently high to destroy the cancer cells. Comparatively, no obvious temperature increase was observed in the NIR group, indicating the high photothermal effect of MnOx/Ti3C2−SP nanosheets. After each treatment, the tumor volumes and mice weights in each group were measured every 2 days over 2 weeks, which were then plotted as a function of time (Figure 9c and d). The mice weight shows no obvious change in each group (Figure 9c). Importantly, the tumor growth in the MnOx/Ti3C2−SP + NIR group was significantly suppressed after the photothermal ablation (Figure 9e, Figure S8). Comparatively, the tumor volumes in the other three groups had gradual and substantial increases. This high tumor-suppressing effect was attributed to the high photothermal-conversion efficiency of MnOx/Ti3C2− SP that can efficiently ablate the tumor and induce the tumorcell death. Hematoxylin-eosin (H&E), TdT-mediated dUTP Nick-End Labeling (TUNEL), and Ki-67 antibody staining of tumor sections were collected from each group of mice to evaluate the therapeutic effects at 24 h after 808 nm laser irradiation. The
H&E and TUNEL results show much higher necrosis of tumor cells in the MnOx/Ti3C2−SP + NIR group as compared to the other three groups, indicating the high antitumor efficiency of PTT induced by MnOx/Ti3C2−SP. Ki-67 antibody staining was carried out to assess the proliferative activities of cancer cells. The results of the MnOx/Ti3C2−SP + NIR group exhibit the strong suppressed proliferative activities of cancer cells, while the other three groups show no significant change in proliferative activities of cancer cells, confirming the high therapeutic effects of MnOx/Ti3C2−SP after exposure to 808 nm laser irradiation (Figure 9f). Systematic in Vivo Biocompatibility Assay. The biocompatibility and biosafety of MnOx/Ti3C2−SP composite nanosheets are of high significance for further potential clinical translation. Therefore, their in vivo biocompatibility was further systematically evaluated by intravenous administration of MnOx/Ti3C2−SP into healthy mice at different doses (5 mg kg−1, 10 mg kg−1, and 20 mg kg−1). The healthy Kunming mice were randomly divided into four groups (n = 6). After intravenous injection of MnOx/Ti3C2−SP nanosheets, the body weight of mice was monitored every 2 days. These mice were 8646
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Figure 10. (a−i) Hematological assay of mice from the control group and three treated groups at different MnOx/Ti3C2−SP dosages of 5 mg kg−1, 10 mg kg−1, and 20 mg kg−1 after intravenous administration and further feeding for 30 days. (m) H&E-stained tissue sections of major organs, including the heart, liver, spleen, lung, and kidney from mice after intravenous injection with saline (control) or MnOx/Ti3C2−SP nanosheets at elevated doses. All the scale bars are 100 μm.
observed (Figure S9). The blood indexes were measured, including the white blood cells (WBC), red blood cells (RBC), platelets (PLA), lymphocytes (LYM), hemoglobin (HGB), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), hematocrit (HCT), and liver and kidney function
sacrificed after further feeding for 30 days. Their blood was collected for biochemical analysis, and their major organs (heart, lung, liver, kidney and spleen) were harvested and fixed in 10% formalin for histological characterization. During the whole feeding period, no mouse died, and no obvious abnormal behavior or body-weight loss of mice was 8647
DOI: 10.1021/acs.chemmater.7b02441 Chem. Mater. 2017, 29, 8637−8652
Article
Chemistry of Materials
Ti3C2 aqueous solution (1 mM, 5 mL) under magnetic stirring for 3 h at room temperature. The resulting MnOx/Ti3C2 was collected by centrifugation and further washed with deionized water several times. Surface Modification of MnOx/Ti3C2 Nanosheets (MnOx/ Ti3C2−SP). The as-prepared MnOx/Ti3C2 composite nanosheets were easily aggregated in biological application. To facilitate the following biomedical applications, the surface of initial MnOx/Ti3C2 nanosheets was modified with soybean phospholipid (SP, Sigma-Aldrich, Shanghai, China; L-α-phosphatidycholine from soybean, Type II-S, 14−23% choline basis, contents of approximately 13% C16:0 (palmitic), 4% C18:0 (stearic), 10% C18:1 (oleic), 64% C18:2 (linoleic), and 6% 18:3 (linolenic) with other fatty acids being minor contributors; an average molecular weight of 776) following a “thinfilm” approach. Typically, MnOx/Ti3C2 nanosheets in ethanol solution (2 mg mL−1, 1 mL) were added into an SP chloroform solution (1 mg mL−1, 15 mL). Then, the mixture was heated up to 60 °C under the vacuum condition in a rotary evaporator for 20 min to evaporate the solvent. Finally, the obtained product was dissolved in 5 mL of phosphate buffer saline solution and sonicated for 5 min for further use. Characterization. Transmission electron microscopy (TEM) images and corresponding EDS spectrum were acquired on a JEM2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) images and element mapping were obtained on a field-emission Magellan 400 microscope (FEI Company). Atomic force microscope (AFM) measurement was performed by means of a Veeco DI Nanoscope Multi Mode V system. The X-ray photoelectron spectroscopy (XPS) spectrum was recorded by ESCAlab250 (Thermal Scientific). UV−vis-NIR absorption spectra were recorded by a UV3600 Shimadzu UV−vis-NIR spectrometer with QS-grade quartz cuvettes at room temperature. The MnOx/Ti3C2 concentration was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies, US). Size and zeta potential measurements were conducted on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). The confocal laser scanning microscopy (CLSM) images were recorded by the FV1000 (Olympus Company, Japan). In Vitro MR Imaging and PA Imaging. For in vitro T1-weighted MR imaging evaluations, MnOx/Ti3C2−SP nanosheets were dispersed into buffer solutions with different pH values (5.0, 6.0, and 7.4). Then, the solutions were shaken at a speed of 150 rpm at 37 °C for 3 h. The MnOx/Ti3C2−SP buffer solutions at different Mn concentrations were diluted with corresponding xanthan gum buffer solution (2 mg mL−1) and transferred into 2 mL Eppendorf tubes for in vitro T1-MRI scanning. The in vitro MR imaging experiment was performed on Signa HDXT 3.0 T equipment (GE Medical System, Fudan University Cancer Hospital). For the T1-weighted fast-recovery fast spin−echo (FR-FSE) sequence, the following parameters were adopted: TR = 1000, 2000, 3000, and 4000, Slice = 3 mm, Space = 0.5 mm, Fov = 20, Phase fov = 0.8, Freq × Phase = 384 × 256, Nex = 2, ETL = 2. Meanwhile, to evaluate the linearity of the PA signal as a function of MnOx/Ti3C2−SP concentration, MnOx/Ti3C2−SP nanosheets with different Ti concentrations (0.3125, 0.625, 1.25, 2.5, 5 mg mL−1) dissolved in purified water were used for in vitro PA imaging. The scanning was performed using the Vevo LAZR PA Imaging System. The Mn and Ti content of the MnOx/Ti3C2 nanocomposites in PBS was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). In Vivo MR Imaging and PA Imaging. For in vivo T1-weighted MR dynamic-imaging evaluations, the nude mice with a 4T1 mammary xenograft tumor were established via the injection of 1 × 106 4T1 murine breast cancer cells suspended in PBS into the tested mouse. When the tumor size reached about ∼200 mm3, T1-weighted MR imaging and PA imaging of tumor-bearing mice were taken before and after the intravenous injection of MnOx/Ti3C2−SP at a dose of 2.5 mg kg−1. The in vivo MR imaging and PA imaging experiment and the corresponding parameters were the same as the in vitro experiment. The animal procedures were in agreement with the guidelines for the Animal Care Ethics Commission of Shanghai Tenth People’s Hospital, Tongji University School of Medicine.
indexes. The results show no obvious changes during the whole feeding period where the main indexes of the mice of MnOx/ Ti3C2−SP nanosheets-treated groups were close to that of the control group, indicating no notable toxicology profiles to mice at the treated doses (Figure 10a−i). The H&E staining of the major organs was employed to assess the pathological change after the injection of MnOx/Ti3C2−SP nanosheets at elevated doses. No obvious tissue damage in the main organs was observed after the treatment with different dosages of MnOx/ Ti3C2−SP (Figure 10m). These preliminary in vivo evaluations strongly demonstrate the relatively low biotoxicity and high biocompatibility of MnOx/Ti3C2−SP nanosheets, which can guarantee their further potential clinical translations.
■
CONCLUSIONS In summary, the Ti3C2-based composite MXene (MnOx/ Ti3C2) has been, for the first time, constructed by triggering a simple redox reaction on the surface of ultrathin Ti3C2 nanosheets, which has been further successfully developed as a multifunctional theranostic agent for efficient MR/PA imaging-guided PTT against cancer. These MnOx/Ti3C2 composite MXene nanosheets were further surface-engineered with SP (MnOx/Ti3C2−SP) for systematic in vitro and in vivo biomedical antitumor applications. The MnOx component in MnOx/Ti3C2−SP exhibits the unique tumor microenvironment (mild acidic)-responsive T1-weighted MRI capability. Importantly, the high photothermal-conversion performance not only endowed the MnOx/Ti3C2−SP composite nanosheets with excellent contrast-enhanced PA-imaging property but also realized the highly efficient tumor ablation and tumor-growth suppression. The in vivo biocompatibility of these MnOx/ Ti3C2−SP composite nanosheets has also been systematically evaluated, which guarantees their further potential safe clinical translation. This work not only provides a multifunctional theranostic agent for benefiting the precise personalized biomedicine but also paves a new way for the multifunctionalization and application of 2D MXenes, especially in the promising theranostic biomedical fields.
■
EXPERIMENTAL SECTION
Synthesis of Ti3AlC2. The Ti3AlC2 bulk ceramic was sintered by a solid-phase reaction. Typically, Ti powder (−325 mesh, 99.5%, Alfa Aesar, Ward Hill, USA), aluminum, Al powder (−325 mesh, 99.5%, Alfa Aesar, Ward Hill, USA), and graphite C (−300 mesh, 99%, particle size
