Two-Dimensional Biodegradable Niobium Carbide (MXene) for

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Two-Dimensional Biodegradable Niobium Carbide (MXene) for Photothermal Tumor Eradication in NIR-I and NIR-II Bio-Windows Han Lin, Shanshan Gao, Chen Dai, Yu Chen, and Jianlin Shi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07818 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Journal of the American Chemical Society

Two-Dimensional Biodegradable Niobium Carbide (MXene) for Photothermal Tumor Eradication in NIR-I and NIR-II BioWindows Han Lin,1,2 Shanshan Gao,1,2 Chen Dai,1 Yu Chen,1* and Jianlin Shi1* 1

State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China. E-mail: [email protected]; [email protected] 2 University of Chinese Academy of Sciences, Beijing, 100049, P.R. China. KEYWORDS. Two-dimensional materials, Nb2C nanosheets, MXene, Phototherapy, Nanomedicine

ABSTRACT: Conventionally, ceramics-based materials, fabricated by high-temperature solid-phase reaction and sintering, are preferred as bone scaffolds in hard tissue engineering because of their tunable biocompatibility and mechanical property. However, their possible biomedical applications have rarely been considered, especially the cancer phototherapeutic applications in both first and second near infrared light (NIR-I and NIR-II) bio-windows. In this work, we explore, for the first time as far as we know, a novel kind of 2D niobium carbide (Nb2C) MXene with highly efficient in vivo photothermal ablation of mouse tumor xenografts in both NIR-I and NIR-II windows. The 2D Nb2C nanosheets (NSs) was fabricated by a facile and scalable two-step liquid exfoliation method combining stepwise delamination and intercalation procedures. The ultrathin, lateral-nanosized Nb2C NSs exhibited extraordinarily high photothermal conversion efficiency (36.4% at NIR-I and 45.65% at NIR-II), as well as high photothermal stability. The Nb2C NSs intrinsically features unique enzyme-responsive biodegradability to human myeloperoxidase, low phototoxicity and high biocompatibility. Especially, these surface-engineered Nb2C NSs present highly efficient in vivo photothermal ablation and eradication of tumor in both NIR-I and NIR-II bio-windows. This work significantly broadens the application prospects of 2D MXenes by rationally designing their compositions and exploring related physiochemical properties, especially on phototherapy of cancer.

1.

INTRODUCTION

Light is an external stimulus, providing considerable benefits in efficacy in cancer phototherapy. Photothermal therapy (PTT) for cancer ablation using near-infrared (NIR) laser radiation has attracted extensive interest, which exploits local heating effect to ablate tumors with poorly vascularized microenvironment. The employment of NIR light as an external and remote stimulus brings high temporal and spatial control of local heating while minimizing adverse side effects.1,2 Clinical medicine applications in which both soft tissues and blood are highly penetrable require NIR light with a considerably deeper penetration. To achieve effective tumor-tissue ablation, two essential criteria of NIR irradiation need to be met, i.e., minimized tissue scattering and absorption to allow potent light-penetration efficacy while keeping the minimum of tissue self-heating, and high photothermalconversion performance of the employed photoabsorbers. Great efforts have been devoted to exploring novel photoabsorbers with high optical cross sections and photothermal-conversion efficiency,3-12 while very few have developed the role of excitation light. Specifically, previous research mainly focuses on the first NIR (NIR-I) bio-window (750−1000 nm), but the second NIR (NIR-II) bio-window (1000−1350 nm) has been rarely investigated. Compared to the well-developed NIR-I bio-window, working in NIRII bio-window possesses two merits including larger maximum permissible exposure (MPE) and desirable penetration depth to laser.13 The expected maximal penetration depth is located at wavelength of 1000−1100 nm, and the corresponding MPE for skin exposure is 1 W cm-2 (American National Standard for Safe

Use of Lasers, ANSI Z136.1−2007).14,15 Though focusing on NIR-II bio-window has attracted some attentions in biological optical imaging,16,17 it has rarely been used for PTT up to now, which is mainly due to the lack of photosensitive agents with desirable strong absorption and high photothermal-conversion efficiency in NIR-II bio-window. Two-dimensional (2D) nanomaterials have been attracting great attention in the past decade due to their ultrathin structure and fascinating physiochemical property. Interest was further inspired since the discovery of graphene’s unique electronic properties.18 Especially, researches on graphene analogues such as layered inorganic materials have bloomed in the last few years.19,20 Very recently, MXenes, a new family of multifunctional 2D materials including a large group of carbides, nitrides, and carbonitrides with numbers of intriguing properties, have been developed by Gogotsi, Barsoum and colleagues.21,22 MXenes were produced by extracting A-element from the layered ternary carbides of MAX phases, where M is an early transition metal, A is an A group element, and X is C or N. MXenes exhibit both metallic conductivity and hydrophilicity, as well as good mechanical properties.23-25 Based on their physiochemical characteristics, MXenes have been explored in a number of applications including electrodes for Li-ion/Li-S batteries,26,27 aqueous supercapacitors,28 proton exchange membranes for fuel cells,29 water purification,30 and electromagnetic interference shielding.31 In the respect of biomedical applications, despite very recent few reports on the biomedical applications of MXenes regarding biosensors,32 antibacterial activity33 and PTT,34-37 the extensive and original inves-

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tigations on the cellular and animal levels, including cytotoxicity, cellular uptake, in vivo toxicity, and other medical applications such as cancer theranostics, are urgently expected to be further explored. Very recently, we and other groups reported on 2D ultrathin Ti3C2 nanosheets for photothermal conversion.36,37 However, their low photothermal-conversion efficiency and the only photo-response in NIR-I bio-window hinder their further broad biomedical applications.

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solution, J&K Scientific Co., Ltd., Beijing, China) under stirring for 3 d at room temperature. Then, the raw Nb2C were collected by centrifugation and washed for three times with ethanol and water to remove the residual TPAOH. 2.2 Surface Modification of Nb2C Nanosheets (Nb2C-PVP) The as-synthesized Nb2C nanosheets were hydrophilic and could not be stable in biological application. For good biocompatibility, 3 mmol Nb2C and 6 mmol PVP40 (polyvinylpyrrolidone, Average MW 40000) was dissolved in 100 mL anhydrous ethanol and refluxed at 50 °C for 6 h. After collecting by centrifugation and washing with water and ethanol, the precipitations, Nb2CPVP, were obtained. 2.3 Characterization

Scheme 1. Schematic illustration of 2D biodegradable Nb2C (modified with PVP) for in vivo photothermal tumor ablation in NIR-I and NIR-II biowindows.

In this work, we explore a newly ultrathin 2D niobium carbide (Nb2C) MXene, as novel phototherapeutic agent, for highly efficient in vivo photothermal ablation of mouse tumor xenografts at both NIR-I and NIR-II windows (Scheme 1). The synthesis and delamination of 2D ultrathin Nb2C nanosheets were achieved by a liquid exfoliation method combining HF etching (delamination) and tetrapropylammonium hydroxide (TPAOH) intercalation (disintegration). The ultrathin and lateral-nanosized Nb2C nanosheets exhibit an excellent NIR photothermal performance with an extraordinarily high photothermalconversion efficiency, as well as desirable photothermal stability. Nb2C nanosheets were further surface-modificated to produce Nb2C-PVP (PVP: polyvinylpyrrolidone) colloidal solutions, which substantially improves the biocompatibility and physiological stability of those nanosheets, exhibiting no noticeable toxicity as evaluated both in vitro and in vivo. Especially, these Nb2C nanosheets intrisically feature enzymeresponsive biodegradability. Furthermore, in vitro and in vivo evaluations of laser tissue penetration depths in both NIR-I and NIR-II bio-windows and their corresponding photothermalconversion performances were investigated, indicating the inconstancy of tissue penetration capability between NIR-I and NIR-II. Especially, highly effective in vivo photothermal ablation by Nb2C-PVP nanosheets against tumor xenografts has been successfully demonstrated in both NIR-I and NIR-II bio-windows.

2.

EXPERIMENTAL SECTION

2.1 Synthesis of Nb2C Nanosheets (MXenes) The Nb2AlC ceramics powder was purchased from Forsman Scientific (Beijing) Co., Ltd. The powder (roughly 10 g) was immersed and stirred in 60 mL of a 50% HF aqueous solution (Sinopharm Chemical Reagents Co., Ltd., Shanghai, China) for 2 d at room temperature. After collecting by centrifugation and washing with water and ethanol, the precipitations were dispersed in 60 mL TPAOH (Tetrapropylammonium hydroxide 25 wt.% aqueous

Transmission electron microscopy (TEM) images and Energy dispersive X-ray spectroscopy (EDS) spectrum were obtained on a JEM-2100F transmission electron microscope. Scanning electron microscopy (SEM) images, scanning transmission electron microscopy (STEM) images and element mapping scanning were acquired on field-emission Magellan 400 microscope (FEI Company). X-ray photoelectron spectroscopy (XPS) was operated on ESCAlab250 (Thermal Scientific, US). Dynamic light scattering (DLS) and Zeta potential measurements were recorded on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). Xray diffraction (XRD) was measured on a Rigaku D/MAX-2200 PC XRD system (parameters: Cu Kα, λ = 1.54 Å, 40 mA, and 40 kV). The confocal laser scanning microscopy (CLSM) images were acquired in FV1000 (Olympus Company, Japan). Atomic force microscope (AFM) measurement was collected by means of Veeco DI Nanoscope Multi Mode V system. UV-vis-NIR absorption spectra were collected by UV-3600 Shimadzu UV-vis-NIR spectrometer. The quantitative analysis of element was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent 725, Agilent Technologies, US). The temperature detection and thermal image record were conducted on an infrared thermal imaging instrument (FLIRTM A325SC camera, USA). NIR-I laser was produced using an 808 nm high power multimode pump laser (Shanghai Connect Fiber Optics Company). NIR-II laser was produced using a 1064 nm high power multimode pump laser (Shanghai Connect Fiber Optics Company). 2.4 Biodegradation of Nb2C Nanosheets Mixtures contained 50 µg MPO from human neutrophils (hMPO, Athens Research and Technology, USA) with an activity of 1.1 U µg-1 per 500 µg of Nb2C-PVP nanosheets suspended in 500 µl of 20 mM phosphate buffer containing 100 mM diethylene triamine pentaacetic acid (DTPA) and 140 mM NaCl. Hydrogen peroxide (H2O2) was added at a rate of 200 mM per hour for 5 h. As a result of loss of activity of hMPO in the phosphate buffer, the enzyme was replenished per 5 h and the incubation system was maintained at 37 oC for 24 h. 2.5 Intracellular Degradation Behavior of Nb2C Nanosheets The intracellular biodegradation behavior of Nb2C-PVP nanosheet and its structural evolution were directly observed by Bio-TEM. Typically, 4T1 cells were cultured with Nb2C-PVPdispersed DMEM medium (the concentration of Nb2C-PVP was 100 µg mL-1) for 1, 2, 3, and 5 d, respectively. After varied coincubation durations, the cells were harvested, fixed, and sectioned for Bio-TEM characterization. To quantify the biodegradation, 4T1 cells co-incubated with Nb2C-PVP (100 µg mL-1 in DMEM medium) for 1, 2, 3, and 5 d were washed by PBS and then collected and melted by chloroazotic acid. The residual Nb contents in 4T1 cells were determined by ICP-OES (n = 3).


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2.6 Photothermal Performance of Nb2C Nanosheets Photothermal performance of Nb2C nanosheets was obtained and analyzed by irradiating an EP tube (Eppendorf tube) containing Nb2C nanosheets dispersion. a. Calculation of the Extinction Coefficient To evaluate the NIR absorption capability of Nb2C nanosheets, the extinction coefficient ε(λ) of the Nb2C nanosheets is determined, according to the Lambert-Beer Law: A(λ) =εLC

(1)

tuting equation (3) for into equation (5) and rearranging to get =

38

Following Roper report, in this case, the energy balance for the whole system is ∑ ,

= + −

(2)

where Cp and m are the heat capacity and the mass of solvent (water), T is the solution temperature, is the energy input of Nb2C nanosheets, is the baseline energy input of the sample cell, and is conducting heat away from the system surface by air. The NIR laser induced source term, , expresses heat dissipated by electron-phonon relaxation of the plasmon on the Nb2C nanosheets surface under the irradiation of 808 or 1064 nm (λ) laser: = 1 − 10

(3)

where I is incident energy of NIR laser (mW), !" is the absorbance of the Nb2C nanosheets at NIR laser wavelength (λ) of 808 or 1064 nm, and η is the photothermal-conversion efficiency from incident NIR laser energy to thermal energy. Besides, represents heat dissipated from photo-absorption of the quartz cuvette sample cell itself, and it was measured independently to be = 5.4 × 10' (in unit of mW) using a sample cell containing pure water without Nb2C nanosheets. is temperature-dependent parameter, which is linear with the output of thermal energy: = ℎ)* − *

(4)

where h represents heat-transfer coefficient, S represents the surface area of the container, T represents the temperature of system surface and * represents the surrounding temperature. Once the NIR laser power is defined, the heat input + will be finite. Since the heat output is increased along with the rise in temperature according to the equation (4), the temperature of system will reach a maximum when the heat output is equal to heat input: + = +,- = ℎ)*+,- − *

(5)

where +,- represents conducting heat away from the system surface by air when the sample cell reaches the equilibrium temperature. *+,- is the equilibrium temperature, representing no heat conduction away from the system surface by air. The photothermal conversion efficiency (η) can be obtained by substi-

9:;; ?

(6)

where !" is the absorbance of Nb2C nanosheets at λ of 808 or 1064 nm. Thus, only the hS remains unknown for calculation of η. In order to obtain the hS, a dimensionless driving force temperature, θ is introduced, scaled using the maximum system temperature, *+,-

where A is the absorbance at a wavelength of λ, L is pathlength (1 cm), and C is the concentration of the Nb2C nanosheets (in g L-1). The extinction coefficient ε is calculated by plotting the slope (in L g-1 cm-1) of each linear fit against wavelength. The 808 and 1064 nm laser extinction coefficient (ε) of Nb2C nanosheets can be measured to be 37.6 and 35.4 L g-1 cm-1, respectively. b. Calculation of the Photothermal Conversion Efficiency

./01 2344 5678

@=

2344

(7)

/01 2344

and a time constant of sample system, A A =

∑7 B7 C,7

(8)

.

which is substituted into the equation (2) and rearranged to yield D

=

;

F

5GH I J5678

E8 ./01 2344

− @K

(9)

At the cooling period of Nb2C nanosheets dispersion, the laser radiation ceases, + = 0, reducing the equation (9) to LM = −A

D D

(10)

and integrating, giving the expression M = −A NO @

(11)

Therefore, time constant of heat transfer from the system was determined to be A = 250.66 s of 808 nm and 232.48 s of 1064 nm (Figure 3e,f). In addition, the m is 0.3 g and the C is 4.2 J g-1. Therefore, according to equation (8), the hS can be determined. Substituting hS into equation (6), the 808 nm laser photothermal conversion efficiency (η) of Nb2C NSs can be calculated to be 36.4%. Similarly, the 1064 nm laser photothermal conversion efficiency (η) of Nb2C NSs can be calculated to be 45.65%. 2.7 In Vitro Cytotoxicity Assay Murine breast cancer 4T1 cell line (noted as 4T1 cells, Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and U87 cell line (noted as U87 cells, Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were maintained at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, GIBCO, Invitrogen) and supplemented with 1% penicillin/streptomycin, and 10% fetal bovine serum (FBS) in a humidified incubator. 4T1 and U87 cells were preferred for the following in vitro and in vivo study. Cells were generally plated in cell culture flask (Corning, USA) and allowed to adhere for 24 h then harvested by treatment with 0.25% trypsin-EDTA solution (Gibco, USA). In vitro cytotoxicity of Nb2C-PVP was evaluated by a standard CCK-8 viability assay (Cell Counting Kit, Dojindo Laboratories, Kumamoto, Japan) of 4T1 cells. The cells were seeded in 96-well culture plates at a density of 1 × 105 cells/well in DMEM medium supplemented with 10% FBS and 1% penicillinstreptomycin at 37 °C and 5% CO2 for 24 h to allow the cells to attach. Then culture medium above was changed with fresh culture medium containing Nb2C-PVP with different concentration (0, 12, 25, 50, 100, and 200 µg mL-1). After 24 or 48 h of incubation, CCK-8 assay was used to evaluate the viability of cells (n=5). 2.8 In Vitro Photothermal Ablation of Cancer Cells



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4T1 or U87 cells were first seeded in 96-well plates at a density of 1 × 105 cells per well in DMEM at a 37 °C in the presence of 5% CO2 for 24 h before treatment. Then, the culture medium was removed, and then Nb2C-PVP dispersed in DMEM (100 µg mL-1) was added into the wells (100 µL per well). After 4 h of incubation, the culture medium with unbound nanosheets was removed and cells were rinsed three times with PBS (Runcheng Bio-tech Co., Ltd., Shanghai), and fresh DMEM medium (10% FBS and 1% penicillin-streptomycin) was added into the wells. These cells were then irradiated for 5 min under an 808 or 1064 nm laser at varied power densities (0, 0.5, 1.0, 1.5, and 2.0 W cm2 ). Finally, standard CCK-8 assay was used to evaluate the viability of cells (n=5). 4T1 cells were seeded in CLSM-exclusive culture dishes with a density of 1 × 104 cells/well and allowed to adhere overnight. For examining the photothermal effect of Nb2C-PVP to 4T1 cells in vitro, DMEM solution of Nb2C-PVP (100 µg mL-1, 100 µL) was added into the dishes and subsequently incubated for 4 h, when cells of each disk reached 80% confluence. The adherent cell solution was irradiated by an 808 or 1064 nm laser for 5 min under a power density of 1.0 W cm-2. After removal of the DMEM medium, the cells were rinsed with PBS for over three times. 4T1 cells were incubated with calcein-AM (100 µL) and PI solution (100 µL) for 15 min. Living cells and dead cells were stained with calcein-AM (green fluorescence) and PI (red fluorescence) solution, respectively. 2.9 Deep-Tissue Photothermal Therapy in NIR-I and NIR-II Windows The NIR-II window can achieve larger tissue penetration depth in comparison to the commonly studied NIR-I window, because of lower absorption and scattering by tissues in this specific spectrum. We evaluated the tissue penetration ability of the

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NIR-I and NIR-II windows by detecting the residual laser energy intensity after tissue penetrated under 808 and 1064 nm laser irradiation, where chicken breast muscles of varying thickness (0, 2, 4, 6, 8, and 10 mm) were prepared and used as model biological tissues. Then, the deep tissue photothermal ability in NIR-I and NIR-II windows was further assessed using the synthesized Nb2C NSs with above chicken breast muscles of varying thickness under 808 and 1064 nm laser irradiation. 100 µL of Nb2C NSsdispersed aqueous suspensions (80 µg mL-1) covered above the tissues of different thickness were subjected to 808 and 1064 nm laser irradiation (1 W cm-2) for 5 min, and the infrared thermographs were then captured by a thermal imaging camera. 2.10 In Vivo Toxicity Assay Animal experiment procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments and the care regulations approved by the administrative committee of laboratory animals of Fudan University. Healthy female Kunming mice (~ 18 g) were obtained and raised at Laboratory Animal Center, Shanghai Medical College of Fudan University. Sixty SBF-level mice (4 weeks old) were divided into four groups: (1) control group, (2) mice intravenously administered with Nb2C-PVP under NIR-I (808 nm) irradiation for 10 min, (3) mice intravenously administered with Nb2C-PVP under NIR-II (1064 nm) irradiation for 10 min and (4) mice intravenously administered with Nb2C-PVP under the artificial daylight for 24 h. The Nb2C-PVP dose for all groups is 20 mg kg-1. The histological, haematological and blood biochemical indexes were collected at varied time intervals of 1, 7 and 28 days after intravenous administration. 2.11 In Vitro and In Vivo PA Imaging For in vitro PA imaging, different wavelengths (from 680 to

Figure 1. Synthesis and characterization of Nb2C nanosheets (MXenes). (a) Schematic diagram for the fabrication of ultrathin Nb2C nanosheets, including HF etching (delamination) and TPAOH intercalation (disintegration). (b) Digital photograph, (c,d) SEM images, and (e) HRTEM image (Inset shows the corresponding SAED pattern) of Nb2AlC ceramic bulks (MAX phase). (f) Digital photograph, (g,h) SEM images, and (i) HRTEM image (Inset shows the corresponding SAED pattern) of multilayer Nb2C. (j) Digital photographs, (k) Bright-field TEM image, (l) Dark-field TEM image, and (m) Fourier transform patterns (Inset shows the original SAED pattern) of single-layer Nb2C.


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850 nm) of excitation light were used to collect the photoacoustic signals, which provided the following PA imaging test with an optimized excitation light of 704 nm. Different concentrations of Nb2C-PVP nanosheets (0.031, 0.062, 0.125, 0.25, 0.5, 1.0, and 2.0 mg mL-1) dissolved in purified water were used for PA signal detection, which also used to evaluate the linearity of the PA signal as a function of Nb2C-PVP concentration. For PA imaging in vivo, the 4T1 tumor-bearing mice intravenously administered with Nb2C-PVP nanosheets samples (20 mg kg-1). After the injection, the signal was recorded on the PA instrument at different time points (0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h). The PA signal before injection was used as the control. Imaging parameter: the region of interest is 20 mm. All the PA imaging were obtained by Vevo LAZR photoacoustic imaging system (VisualSonics Company, Canada) with the following parameters: frequency: 21 MHz; PA gain: 40 dB; 2D gain: 10 dB; wavelength: 704 nm.

3.

RESULTS AND DISCUSSIONS

3.1 Synthesis and Characterization of Nb2C Nanosheets (MXenes) 2D Nb2C nanosheets were synthesized by a modified chemical exfoliation process according to the method reported by our group previously for the synthesis of Ti3C2 MXenes.36 First, the MAX phase Nb2AlC was etched using 50% HF aqueous solution to remove the Al layer. In order to achieve the high dispersibility of ultrathin Nb2C nanosheets, the etched Nb2C powder was then intercalated in TPAOH aqueous solution to substantially reduce the planar dimensions (Figure 1a). The as-obtained Nb2C nanosheets (noted as Nb2C NSs) features lateral nanosize and atomic-scale thickness, making the biomedical application of MXene possible.

2.12 Pharmacokinetics, Biodistribution and Metabolism Studies To develop the tumor model, 4T1 cells (1 × 106 cell/site) suspended in 150 µL PBS solution were injected into the back of mice. In pharmacokinetic experiments, 4T1 tumor-bearing mice were intravenously administered with Nb2C-PVP (20 mg kg-1) in PBS (n = 3). The 15 µL of blood was collected at varied time intervals (2 min, 5 min, 10 min, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h) after injection. The quantitative analysis of Nb element was measured by ICP-OES. The in vivo blood terminal half-life of Nb2C-PVP was determined by a double-component pharmacokinetic model. Biodistribution of Nb2C-PVP in tumor and other organs was performed in 4T1 tumor-bearing mice (n = 3). 4T1 tumor-bearing mice were intravenously administered with Nb2CPVP (20 mg kg-1) in PBS. Mice were dissected at predesignated time intervals (2, 6, 12, 24, and 48 h). Dissected organs were weighed, homogenized, and treated with strong acid. The Nb2CPVP distributions in different tissues were calculated as the percentage of injected dose per gram of tissue. A long-term biodistribution (7 d) of Nb2C-PVP in tumor and other organs was also determined with the same above treatment. To investigate the metabolism process of Nb2C-PVP, Nb2CPVP NSs in PBS (20 mg kg-1 per mouse) were intravenously injected into the 4T1 tumor-bearing mice. The urine and faces were collected at different time intervals (2, 6, 12, 24, 36 and 48 h). The Nb contents in urine and faeces were quantitatively determined by ICP-OES. 2.13 In Vivo Photothermal Therapy in NIR-I and NIR-II BioWindows

Figure 2. Composition, surface modification and in vitro biodegradability of Nb2C nanosheets (MXenes). (a) XRD patterns of Nb2AlC bulks and Nb2C NSs. (b) Raman spectra of Nb2AlC bulks and Nb2C NSs. (c) XPS spectra of Nb2AlC bulks and Nb2C NSs. (d) Typical AFM image of Nb2C NSs. (e) Lateral size and thickness distribution analysis of Nb2C NSs. (f) AFM 3D view of area in panel d. (g) Photographs, (h) dynamic light scattering analysis, (i) visNIR spectra, and (j) TEM images of Nb2C-PVP nanosheets after receiving various biodegradation treatments for 24 h.

The 5-week old female Balb/c nude mice (~ 13 g) were obtained and raised at Laboratory Animal Center, Shanghai Medical College of Fudan University. To develop the tumor model, 4T1 cells (1 × 106 cell/site) suspended in 100 µL PBS solution were injected into the back of mice. In vivo photothermal therapy by intravenous administration was performed when the tumors volume reached ~200 mm3. All mice were anesthetized before NIR laser irradiation. The measurement of the tumors volume were conducted by a digital caliper every two days during half a month after the corresponding experiments. The tumor volume was measured according to the following formula: tumor volume = (tumor length) × (tumor width)2 /2. The tumors were dissected after the corresponding treatments and fixed in a 10% formalin. Thereafter, the tumor issues were sectioned into slices and stained with H&E, TUNEL and Ki-67 for histological analysis. Mice with tumors larger than 1000 mm3 should be euthanized according to the standard animal protocol.

The Nb2AlC MAX phase was fabricated by solid-phase sintering (Figure 1b). Scanning electron microscopic (SEM) images show that Nb2AlC MAX phase exhibits a unique morphology of layered ternary compound (Figure 1c,d). Highresolution transmission electron microscopy (HRTEM) image clearly shows that Nb2AlC belongs to hexagonal (P63/mmc) structure (Figure 1e and inset). SEM images show the microstructure of HF-etched Nb2C powder (Figure 1f) from Nb2AlC solid bulk, which is well stacked of uniform sheets (Figure 1g,h). HRTEM images clearly show the crystalline lattice of multilayer Nb2C NSs with hexagonal structure, and selectedarea electron diffraction (SAED) pattern also confirms that the basal plane hexagonal symmetry structure of the parent MAX phase has been preserved after HF etching (Figure 1i and inset). After further TPAOH intercalation, bright-field and dark-field TEM images reveals ultrathin, electron-transparent flakes of exfoliated Nb2C NSs (Figure 1k,l), which exhibit the typical



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sheet-like morphology with an average size of ~ 150 nm. HRTEM images of Fourier transform pattern show the crystalline lattice of few-layer or single-layer Nb2C NSs with hexagonal structure, and the corresponding original SAED pattern confirms the preserved hexagonal symmetry structure (Figure 1m and inset). The elemental mapping of MAX phase, HF-etched Nb2C powder and exfoliated Nb2C NSs demonstrates the successful etching-removal of Al element (Figure S1). The electron energy loss spectrum (EELS) also confirms the existence of Nb, C, O elements and the absence of Al element, indicating its complete removal from the structure (Figure S2). The as-prepared Nb2C NSs can be well dispersed in water for several weeks if tightly sealed in a bottle. Digital photographs of Nb2C NSs dispersed in water with typical Tyndall effect indicates their excellent hydrophilicity and dispersity (Figure 1j).

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X-ray diffraction (XRD) pattern shows the successful synthesis of Nb2AlC MAX phase (Figure 2a, blue curve). The Nb2C peak (red curve) intensities originating from the parent Nb2AlC bulk decreased substantially after HF treatment and TPAOH intercalation. Especially, the (002) peak of Nb2C NSs broadened and downshifted significantly toward a lower 2θ angle of 7.78°. The newly emerged low-angle (002) peak is typical for most reported MXenes, which implies that the sample has completely converted to MXenes39,40. In addition, XRD pattern of freezing-dried Nb2C NSs reveals the disappearance of the most intense peaks of Nb2AlC at 2θ ≈ 39° because of the exfoliation. Raman spectra of Nb2AlC and Nb2C NSs are shown in Figure 2b. Vibration modes ω1 and ω3 became suppressed or even disappeared after HF etching, indicating the elimination of Al layer or the exchange-out of Al atoms by lighter atoms.41 Modes ω4 have downshifted and shape-changed, while modes ω2 have

Figure 3. Photothermal-conversion performance and stability of Nb2C nanosheets (MXenes). (a) Vis-NIR absorbance spectra of Nb2C NSs-dispersed aqueous suspensions at varied concentrations (2.5, 5, 10 and 20 µg mL-1). Inset: the first near-infrared bio-window (NIR-I, 750–1000 nm) and the second near-infrared bio-window (NIR-II, 1,000–1,350 nm). (b) Mass extinction coefficient of Nb2C NSs at 808 nm (NIR-I) and 1064 nm (NIR-II). Normalized absorbance intensity at λ = 808 nm divided by the characteristic length of the cell (A/L) at varied concentrations (2.5, 5, 10 and 20 µg mL-1). (c, d) Photothermal heating curves of Nb2C NSs-dispersed aqueous suspensions under the irradiation with 808 nm (NIR-I) and 1064 nm (NIR-II) laser at varied power densities (0.5, 0.75, 1.0, 1.25 and 1.5 W cm-2). (e, f) Calculation of the photothermal-conversion efficiency at 808 nm (NIR-I) and 1064 nm (NIR-II). Black line: photothermal effect of an aqueous dispersion of Nb2C NSs under the irradiation with NIR-I or NIR-II laser for certain periods, and then the laser was switched off. Blue line: time constant (τs) for the heat transfer from the system determined by applying the linear time data from the cooling period. (g) Heating curves of a Nb2C NSs suspension in water for five laser on/off cycles (1.5 W cm-2) under the irradiation with NIR-I or NIR-II laser. Vis-NIR absorbance spectra of (h) Nb2C NSs, (i) Au NRs, and (j) ICG before and after laser radiation for 30 minutes. (k) TEM images of Nb2C NSs and Au NRs before and after laser radiation for 30 minutes.


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merged and weakened, which confirm the well-retained Nb2C layer and increased interlayer spacing of the MXene structure (Figure S3, Table S1, and Discussion S1). To determine the composition and surface terminations, the Nb2C NSs were ananlyzed by energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS). EDS results confirm the presence of Nb, C, and finite O elements (Figure S4). The high oxygen content measured by EDS is due to water intercalation between the MXene layers, which would be hard to be removed completely, or the partial surface oxidation of Nb2C layers. In addition to the inherent Nb-C bond, XPS results of the Nb2C NSs show the presence of Nb (V) and NbCxOy, indi-

cating the formation of Nb2O5 or Nb2C(OH) after the treatment (Figure 2c and S5). Atomic force microscopy (AFM) provided further evidence for the formation of freestanding Nb2C NSs (Figure 2d, f). The statistical thickness distribution of as-prepared Nb2C NSs measured by AFM shows a height of about 0.3~0.8 nm, which is consistent with the dimension of a single-layer or double-layer Nb2C NSs (Figure 2e). The lateral size is statistically ~150 nm, which matches well with the TEM results. It should be noted that small nanoparticles attached on the surface of single-layer Nb2C NSs occurred after Nb2C NSs dispersed in the water for several months, which was due to the surface oxidation of Nb2C NSs

Figure 4. Evaluation of in vitro and in vivo tissue penetration depth for NIR-I and NIR-II photothermal conversion. (a) Schematic diagram and equipment for detecting tissue-penetration capability of NIR laser at 808 nm and 1064 nm. (b) Different thicknesses (0, 2, 4, 6, 8, and 10 mm) of chicken breast tissues fixed in transparent pipes. (c) Schematic diagram and (d) equipment for the photothermal-conversion demonstration of Nb2C NSs-dispersed aqueous suspensions by tissue-penetrated NIR laser (1.0 W cm-2, 80 µg mL-1). (e) Energy intensities of NIR-I laser (808 nm) penetrated through tissues-of different thickness intervals. Inset: Normalized penetrated NIR-I energy through tissues of different depths. α808: the attenuation coefficient of 808 nm NIR-I laser. (f) Energy intensities of NIR-II laser (1064 nm) penetrated through tissues of different thicknesses. Inset: Normalized penetrated NIR-II energy through tissues of different depths. α1064: the attenuation coefficient of 1064 nm NIR-II laser. (g) Temperature elevations of Nb2C NSs-dispersed aqueous suspensions upon exposures to tissue-penetrated NIR-I and NIR-II laser via photothermal-conversion. (h) Schematic diagram of in vivo tumor tissue-penetration for photothermal conversion based on NIR-I and NIR-II. (i) Antigen Ki-67 immunofluorescence staining for cellular proliferation at different depths (0, 2, 4, 6, 8, and 10 mm) of dissected tumor tissues. All images share the same scale bar (50 µm).


Journal of the American Chemical Society caused by oxygen dissolved in water (Figure S6).

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3.2 Biodegradation Behavior of Nb2C Nanosheets (MXenes) Responsive to Human Myeloperoxidase (hMPO) Nanomaterials of 20 to 200 nm in dimensions would effectively avoid rapid renal filtration, achieving passive accumulation in tumors at enhanced concentrations and for a longer period than conventional organic particles by the enhanced permeability and retention (EPR) effect.42,43 However, inorganic nanoparticles, unlike other small biodegradable organic materials, generally possess poor biodegradability and long-term body retention, triggering the risk of adverse effects.44,45 Thus, clinical demand of nanoagents depends on the potential or controllable biodegradability as well as a long-term toxicity of the nanomaterials and their by-products. Therefore, it is of high desirability to exploit novel photoabsorbers which could not only have the appropriate dimensions ensuring effective tumor targeting, but also process satisfactory biocompatibility and biodegradability ensuring their harmless excretion from body in a specific period after therapeutic administration. Neutrophils are responsible for killing microorganisms invading living body by using their free radical species-generating enzymes, such as proteases, NADPH oxidase and myeloperoxidase.46 Human myeloperoxidase (hMPO) generates hypochlorous acid (HOCl) and reactive radical intermediates, which contribute to the degradation of polymeric substances or other carbon-based nanomaterials.47,48 Thus, we reason that the same hMPO oxidants could also biodegrade Nb2C nanosheets.

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regions, which was uncommon in the nanomaterials as a photothermal agent (Figure 3a). The normalized adsorption intensity over the length of the cell (A/L) at λ = 808 nm and 1064 nm with varied concentrations (C) was determined. Following the Lambert-Beer law (A/L = εC, where ε is the extinction coefficient), a linear dependence of A/L on the concentration was obtained, and the extinction coefficients at 808 nm and 1064 nm were measured to be 37.6 and 35.4 Lg-1cm-1 (Figure 3b), respectively, which was remarkably higher than that of traditional Au nanorods at 808 nm (13.9 Lg-1cm-1)49 or carbon nanodots (0.35 Lg-1cm-1),50 implying the strong NIR laser absorption capability of the Nb2C NSs. Furthermore, the photothermal-conversion efficiency (η) of Nb2C NSs was calculated based on the results of time constant for heat transfer and the maximum steady-state temperature (Figure 3e, f), which gave the value of as high as 36.5% of 808 nm and 46.65% of 1064 nm, significantly higher than those of representative Au nanorods (21%) 51, Cu9S5 nanocrystals (25.7%) 52, gold nanovesicles (37%) 53, and Prussian blue (41.4%) 54. In addition, the photothermal performance parameters for the classic inorganic photothermal agents and 2D inorganic photothermal agents have been summarized in Figure S13 and Table S2, which shows the noticeable advantages of Nb2C NSs using NIR-II laser than using NIR-I laser, even compared to most of the inorganic photothermal agents in the literatures.

Owing to the steric hindrance of organic chains, the surface modification by biocompatible PVP chains endows Nb2C NSs with excellent colloidal stability in physiological environments (Figure S8a, b, c, and Figure S15). To investigate the biodegradability of Nb2C-PVP nanosheets (noted as Nb2C-PVP), hMPO and H2O2 in PBS was used as incubation medium in an experimental group. Compared to the three control groups (Nb2C-PVP in water, Nb2C-PVP in PBS, Nb2C-PVP with H2O2 in PBS), the Nb2C-PVP in the experimental group does degrade over time, and the suspension turned translucent in 24 h (Figure 2g). Dynamic light scattering (DLS) measurements of Nb2C NSs treated with hMPO and H2O2 show several peaks that corresponds to the size reduction of the treated nanosheets compared to untreated controls (Figure 2h). Furthermore, visible-near infrared (vis-NIR) absorbance spectra of treated nanosheets show that absorbance decreases significantly during biodegradation (Figure 2i). Drastic changes in nanosheet morphology were further confirmed in TEM images. The characteristic 2D planar structure of the original nanosheets was completely lost in the presence of hydrogen peroxide in 24 h of incubation, and in the meantime, the nanosheets were even no longer visible when both hMPO and H2O2 are present in PBS (Figure 2j) in the same period of incubation. Therefore, a novel route of enzyme-triggered biodegradation of Nb2C NSs has been achieved, which enables its harmless degradation under simulated condition in a reasonable in-body period after fulfilling their therapeutic functions.

Nb2C NSs were exposed to an 808 nm or 1064 nm laser at varied power densities (0.5, 0.75, 1.0, 1.25 and 1.5 W cm-2) to investigate their photothermal performance. At a relatively low Nb2C concentration (40 µg mL-1), exposure to either 808 nm or 1064 nm laser at 1.5 W cm-2 resulted in the solution temperature increases to 60 °C in 5 min. In contrast, the temperature of pure water shows no significant temperature change, indicating that the presence of Nb2C NSs can efficiently and rapidly convert NIR light into thermal energy (Figure 3c, d). More detailed experiments were conducted to investigate the photothermal performance of Nb2C NSs at varied concentrations (80, 40, 20, 10 ppm, and water) under 808 nm or 1064 nm laser at the power density of 1.0 W cm-2 (Figure S10). To further evaluate the photothermal stability of the Nb2C NSs, the recycling temperature variations of Nb2C NSs dispersion were recorded under an NIR laser radiation for 5 min (laser on) followed by natural cooling to room temperature (laser off) for five laser on/off cycles. The photothermal performance of the Nb2C NSs does not show any significant deterioration during the recycling, highlighting the potential of Nb2C NSs as a durable photothermal agent for PTT cancer treatment (Figure 3g). Compared with the mostly reported photothermal agents, Au nanorods (Au NRs) and indocyanine green (ICG), the absorbance maxima for Au NRs and ICG molecules diminished significantly upon laser irradiation under the same condition, clearly demonstrating that the photostability of Nb2C NSs is superior to these traditional PTT agents (Figure 3hk and Figure S11). UV-vis-NIR spectra of Nb2C NSs and Nb2CPVP dispersed in water show almost no decline in optical absorption after the PVP surface modification, indicating the wellmaintained photothermal performance of Nb2C-PVP (Figure S8d).

3.3 Optical Absorption and Photothermal Property of Nb2C Nanosheets (MXenes)

3.4 Deep-Tissue Photothermal Therapy in NIR-I and NIR-II Bio-windows

The photothermal performance of an agent used for photothermal conversion is determined by two main parameters: the extinction coefficient (ε) and photothermal-conversion efficiency (η). The extinction coefficient reveals the light absorption ability while the photothermal-conversion efficiency indicates the performance of the agent in converting the light into heat. The optical absorption spectra acquired on Nb2C NSs show a board and strong absorption band covering the NIR-I and NIR-II

The NIR-II bio-window can offer higher tissue-penetration depth than commonly explored NIR-I bio-window, due to its lower absorption and scattering by tissues in this spectral range. We initially evaluated the NIR tissue-penetration performances in the NIR-I and NIR-II bio-windows by detecting the residual laserenergy intensity after tissue penetration under 808 and 1064 nm laser irradiations, where chicken breast muscles of varying thicknesses (0, 2, 4, 6, 8, and 10 mm) were prepared and used as model


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biological tissues (Figure 4a,b). Then, the deep tissue photothermal capability in NIR-I and NIR-II windows was further assessed using the synthesized Nb2C NSs with above chicken breast muscles under the laser irradiations (Figure 4c). 100 µL of Nb2C NSs-dispersed aqueous suspensions covered with the tissues of different thicknesses were subjected to 808 and 1064 nm laser irradiation (1 W cm-2) for 5 min, and the infrared thermographs were then captured by a thermal imaging camera (Figure 4d). The energy residues of NIR-I and NIR-II laser after penetration through the tissues of increasing thicknesses were measured, showing a stronger attenuation trend of 808 nm NIR-I laser than 1064 nm NIR-II laser (Figure 4e, f). Furthermore, in order to quantitatively describe the attenuation difference between NIR-I and NIR-II laser in the tissues, the residue NIR laser energy penetrated though the tissues were normalized to that without the tissue presence, plotted as a function of tissue thickness, and numerically fitted by an exponential function. From the fitting curve of NIR-I and NIR-II, attenuation coefficients for 808 nm and 1064 nm laser of 1.02 and 0.73 were obtained (Figure 4e, f inset), suggesting the much stronger NIR tissue penetration in NIR-II window than that in NIR-I window. Subsequently, efficient heating of the Nb2C NSs solution was achieved without introducing significant heating inside the tissue. The measured temperature changes at varied depths of the tissues under NIR-I and NIR-II laser radiations show that, penetrated through the same thickness of the tissues, less attenuation of photothermal heating could be achieved with the NIR-II laser than with the NIR-I laser (Figure 4g). Especially, in order to evaluate the in vivo effective tissuepenetration depth for photothermal ablation based on NIR-I and NIR-II, 4T1 tumor-bearing mice with intravenous administrations of Nb2C-PVP were treated under 808 nm or 1064 nm laser radiations (Figure 4h). After the treatments, the tumor tissues were dissected to observe the cellular proliferation at different depths inside the tumor tissues, which exhibit that the effective photothermal ablation of subcutaneous xenograft tumor in nude mice could be achieved at the depth of ~4 mm (Figure 4i). This is in accordance to the value calculated by Bashkatov et al.13, showing the great promise of using Nb2C-PVP NSs for deep tissue photothermal therapy in both NIR-I and NIR-II bio-windows. 3.5 In Vitro Photothermal Cell Ablation and Endocytosis The in vitro toxicities of Nb2C-PVP to cells were tested using a standard CCK-8 assay. Two cancer cell lines of breast 4T1 cancer cells and glioma U87 cancer cells were incubated with Nb2C-PVP at varied concentrations (0, 12, 25, 50, 100, and 200 µg mL-1) for 24 h and 48 h. Nb2C-PVP shows negligible effect on the survival of both 4T1 cells and U87 cells, even at the concentration up to 200 µg mL-1 (Figure 5a and S14). Then, Nb2C-PVP as photothermal agents for in vitro cancer-cell ablation under laser irradiation was evaluated. 4T1 cells and U87 cells were incubated with Nb2C-PVP nanosheets at 40 µg mL-1 for 4 h, respectively, and then exposed to 808 nm or 1064 nm laser of varied power densities (0, 0.5, 1.0, 1.5, and 2.0 W cm-2). It was revealed that with the increase of laser energy, more cells incubated with Nb2C-PVP were killed upon the NIR-I and NIR-II laser irradiation (Figure 5b,c). Additionally, the significant cell apoptosis after photothermal ablation was further demonstrated by confocal microscopic imaging. After NIR laser irradiation, the live and dead cells were differentiated by calcein-AM and PI costaining, respectively. The control groups including the 4T1 cells without any treatment, NIR-I laser irradiation only, NIR-II laser irradiation only, and Nb2C-PVP only exhibits negligible of insignificant effects on the cell viability. In contrast, the majority of 4T1 cells have been killed by the photothermal ablation after treated with Nb2C-PVP under NIR-I or NIR-II laser irradiation

(Figure 5d). These results clearly demonstrate the remarkable in vitro photothermal effect of the Nb2C-PVP nanosheets in promoting cancer cell ablation. Confocal laser scanning microscopy (CLSM) images clearly show the efficient intracellular uptake of FITC (fluorescein isothiocyanate)-labeled Nb2C-PVP after the co-incubation for 4 h, which was further quantitatively supported by the inductively coupled plasma atomic emission spectroscopy (ICP-OES) test in determining the intracellular uptake amount of Nb2C-PVP nanosheets (Figure 5e,f and S16).

Figure 5. In vitro photothermal ablation and endocytosis of cancer cells. (a) Relative viabilities of 4T1 and U87 cells after being incubated with Nb2C-PVP nanosheets of varied concentrations (0, 12, 25, 50, 100, and 200 µg mL-1). Relative viabilities of (b) 4T1 and (c) U87 cells after Nb2C-PVP (40 µg mL-1)induced photothermal ablation at different laser power densities (0, 0.5, 1.0, 1.5, and 2 W cm-2). Error bars were based on the standard deviations (s. d.) of five parallel samples. (d) Confocal fluorescence imaging of Nb2C-PVP induced photothermal ablation (1.0 W cm-2, 40 µg mL-1) after various treatments (Control, Nb2C-PVP only, NIR-I laser only, NIR-II laser only, Nb2C-PVP + NIR-I laser and Nb2C-PVP + NIR-II laser group). Images share the same scale bar (50 µm) (e) Confocal laser scanning microscopic (CLSM) images of 4T1 cells incubated with FITC-labeled Nb2C-PVP at the concentration of 100 µg mL-1 for 0, 1, 2, 4, and 8 h. 4T1 cells nuclei stained by DAPI (blue: DAPI = 4’, 6-diamidino-2-phenylindole), FITC fluorescence from Nb2C-PVP in cells (green). Images share the same scale bar (30 µm). (f) Nb2C-PVP intake concentrations, the corresponding FITC and DAPI fluorescence signals of 4T1 cells incubated with FITC-labeled Nb2C-PVP for 0, 1, 2, 4, and 8 h. Inset: threedimensional (3D) confocal fluorescence reconstructions of Nb2C-PVP endocytosized by 4T1 cells.

Owing to the strong NIR-absorbance and marked photothermal conversion efficiency of Nb2C-PVP in the NIR region, PA imaging was further carried out by using the Nb2CPVP nanosheets as contrast agents (CAs) (Figure S17). 3.6 In vivo toxicity of Nb2C-PVP. A detailed investigation of in vivo toxicology of Nb2C-PVP was further conducted to explore its in vivo translation potential. Sixty SBF-level mice were divided into four groups based on various experimental conditions: (1) control group, (2) mice intravenously administered with Nb2C-PVP under NIR-I (808 nm) irradiation for 10 min, (3) mice intravenously administered with Nb2C-PVP under NIR-II (1064 nm) irradiation for 10 min and (4) mice intravenously administered with Nb2C-PVP followed by



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exposure to artificial daylight for 24 h.

cant renal and hepatic toxicity in mice.

The normal haematology parameters including white blood cells (WBC), red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets (PLT) and mean corpuscular volume (MCV) were measured (Figure 6a). It could be found that no meaningful changes can be observed from the Nb2C-PVP-treated groups at different time points in comparison to the control group. These results demonstrate that the Nb2CPVP have caused no significant inflammation and infection in the treated mice.

Besides, the excretion pathways including urine and faeces show that near 20% of Nb content was excreted out of mice in 48 hours post-intravenous injection, which exhibits the possibility of Nb2C-PVP NSs excreted out of the body via the urine and faeces (Figure S19b), showing relatively high iv vivo safety for potential clinical translation.

The standard blood biochemical indexes were performed and various makers including alanine transaminase (ALT), aspartate transaminase (AST), total protein (TP), globulin (GLB), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA) and albumin (ALB) were examined (Figure 6b). All the indexes in the Nb2C-PVP-treated groups at different time points show no abnormity in comparison to the control group and the changes are of no statistical significance. Hence, the Nb2C-PVP treatment does no negative impact on the blood chemistry. Moreover, since ALT, AST and CREA are related functional indexes for the kidney and liver of mice, indicating the Nb2C-PVP induce no signifi-

Furthermore, the corresponding histological changes of major organs, including the heart, liver, spleen, lung and kidney, were collected and sliced for hematoxylin and eosin (H&E) staining (Figure 6c). No significant acute, chronic pathological toxicity and adverse effects can be found during the treatment period for all groups, suggesting no significant histological abnormalities in the treatment groups. Based on the above practices, no significant cytotoxicity has been observed from the Nb2C-PVP treatment without NIR laser irradiation. Even though the Nb2C-PVP-treated mice are exposed to artificial daylight for 24 h, no significant adverse effects could be observed, which indicates that the Nb2C-PVP could induce no apparent phototoxicity. These results introduce that Nb2C-PVP is of high biocompatibility for further safe in vivo photothermal

Figure 6. In vivo toxicity evaluation. (a) Haematological index of the mice with intravenous administration of Nb2C-PVP in 1, 7 and 28 days post-injection under various conditions (exposure to NIR-I, NIR-II and daylight). The results show the mean and s.d. of WBC, RBC, HGB, HCT, MCH, MCHC, PLT and MCV. (b) Blood-biochemical analysis of the Nb2C-PVP-treated mice in 1, 7 and 28 days post-injection under various conditions (exposure to NIR-I, NIR-II and daylight). The terms include ALT, AST, TP, GLB, TBIL, BUN, CREA and ALB. (c) Histological data (H&E stained images) obtained from the major organs (heart, liver, spleen, lung, and kidney) of the Nb2C-PVP-treated mice at 1, 7 and 28 days post-injection under various conditions (exposure to NIR-I, NIR-II and daylight) (scale bars: 100 µm).


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ablation of cancer.55

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3.7 In Vivo Photothermal Tumor Eradication in NIR-I and NIR-II Bio-windows The blood circulation and biodistribution of Nb2C-PVP were investigated before the in vivo PTT experiments. The circulation

compartment model, and the blood circulation half-time of Nb2CPVP was calculated to be 1.31 h (Figure 7a). The biodistribution of Nb2C-PVP in main organs and tumor was investigated at varied time points post-intravenous injection on 4T1 tumor-bearing mice (Figure 7b and S19a). In order to determine precise tumor uptake of Nb2C-PVP NSs, the coating amount of PVP on the surface of

Figure 7. In vivo photothermal cancer therapy, pharmacokinetic and biodistribution analysis. (a) Blood circulation lifetime of Nb2C-PVP after intravenous injection into mice (n = 3). The half-time (T1/2) was calculated to be approximately 1.31 h. (b) Biodistribution of Nb (% ID of Nb per gram of tissues) in main tissues and tumor after intravenous administration of Nb2C-PVP dispersed in PBS for varied time intervals (2, 6, 12, 24, and 48 h) (n = 3). (c) Temperature elevations at the tumor sites of 4T1 tumor-bearing mice in groups of NIR-I, NIR-II, Nb2C-PVP + NIR-I, and Nb2C-PVP + NIR-II during laser irradiation. (d) The corresponding IR thermal images at the tumor site of 4T1 tumor-bearing mice in groups of NIR-I, NIR-II, Nb2C-PVP + NIR-I, and Nb2C-PVP + NIR-II during laser irradiation in different time intervals. (e) Time-dependent tumor growth curves (n = 5, mean ± s.d.) after different treatments (control, Nb2C-PVP only, NIRI, NIR-II, Nb2C-PVP + NIR-I, and Nb2C-PVP + NIR-II). The treatments were performed only once. (f) Time-dependent body-weight curves of nude mice after different treatments as indicated in (e). Inset: tumor weights of mice in 16 days after the treatments. (g) Survival curves of mice after various treatments. (h) Photographs of 4T1 tumor-bearing mice and its tumor regions in 16 days after different treatments. (i) H&E staining for pathological changes in tumor tissues from each group to reveal the effectiveness of in vivo photothermal therapy by intravenous/intratumoral administrations of Nb2C-PVP. (j) TUNEL staining for pathological changes in tumor tissues. (k) Antigen Ki-67 immunofluorescence staining for cellular proliferation in tumor sections. All images share the same scale bar (50 µm).

of Nb2C-PVP in bloodstream was analyzed to follow a two-

Nb2C NSs has been determined by ICP-OES and TGA. The re-



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sults show that the coating amount of PVP on the Nb2C-PVP NSs is 20.36 % (Figure S9), and the intratumor passive accumulation efficiency based on formula Nb2C-PVP NSs has thus been quantified as 2.24% ID/g. The prolonging blood circulation time and effective local tumor accumulation of Nb2C-PVP make the high efficient in vivo photothermal therapy possible.

nanosheets with human myeloperoxidase have been demonstrated, which provides the possibility of MXene-based nanotherapeutic agent for safe in vivo applications. Presently such an enzymeresponsive biodegradability of Nb2C-PVP has been obtained only under in vitro stimulated conditions, the biodegradation mechanism and its in vivo verification need to be explored.

Encouraged by the in vitro high NIR-I and NIR-II laser absorbances, high photothermal-conversion efficiency and in vivo circulation and accumulation effect of Nb2C-PVP nanosheets, we further carried out the in vivo PTT experiments on tumor-bearing mice. After intravenous (i.v.) administration of Nb2C-PVP in PBS (20 mg kg-1), 4T1 tumor-bearing mice were anesthetized and exposed to 808 or 1064 nm NIR laser at a power density of 1.0 W cm-2, and the tumor-site temperatures rapidly increased from ~ 30 °C to ~ 61 °C and from ~ 30 °C to ~ 65 °C, respectively, in 10 min of laser irradiation. In comparison, the tumor temperature under only 808 or 1064 nm NIR laser irradiation showed very slight change (Figure 7c,d).

Furthermore, in vitro and in vivo evaluations of laser tissue penetration depth in both NIR-I and NIR-II windows and their corresponding photothermal-conversion ability have been investigated, indicating the unconsistency in the tissue penetration capability between NIR-I and NIR-II. Especially, highly effective in vivo photothermal ablation by Nb2C-PVP nanosheets against tumor xenografts has been successfully demonstrated in both NIR-I and NIR-II bio-windows. To the best of our knowledge, this is the first report to show in vitro and in vivo photothermal cancer therapy in both first and second NIR bio-windows. Therefore, such a Nb2C-based PTT agent with the unique combination of biodegradability and biocompatibility may find promising clinical-translation potentials.

Further in vivo photothermal therapeutic experiments were performed upon the tumor sizes having reached around 200 mm3. The 4T1 tumor-bearing mice were divided into six groups: control, Nb2C-PVP (i.v.) only, NIR-I only, NIR-II only, Nb2C-PVP (i.v.) + NIR-I and Nb2C-PVP (i.v.) + NIR-II. NIR laser irradiations were carried out in 24 h post-injection (i.v.) of Nb2C-PVP. In two days after photothermal ablation, tumors in two treated groups (Nb2C-PVP (i.v.) + NIR-I and Nb2C-PVP (i.v.) + NIR-II) disappeared, leaving black scars at the initial tumor sites. The tumor volumes of six groups were measured every two days using a digital caliper (Figure 7e), and the digital photos of tumor regions were taken every two days during half a month after the treatments (Figure S20). Remarkably, mice in the groups of control, Nb2C-PVP (i.v.) only, NIR-I only, NIR-II only showed average life spans of 16-20 days with the volumes reaching 1000 mm3. In comparison, tumors in two treated groups (Nb2C-PVP (i.v.) + NIR-I and Nb2C-PVP (i.v.) + NIR-II) were completely eradicated without further reoccurance after the treatment and all treated mice survived over 50 days (Figure 7f inset, h). All the mice demonstrate negligible weight fluctuations, thus confirming negligible adverse effects of these treatments on the health of mice (Figure 7g). H&E and TUNEL staining results show the highly significant necrosis of tumor cells of Nb2C-PVP (i.v.) + NIR-I and Nb2C-PVP (i.v.) + NIR-II groups compared to the mice groups of control, Nb2C-PVP (i.v.) only, NIR-I only, NIR-II only. (Figure 7i,j) The in vivo proliferative activities were measured by Ki-67 antibody staining, and the groups of Nb2C-PVP (i.v.) + NIR-I and Nb2C-PVP (i.v.) + NIR-II presented strong suppression effect on the cell proliferation, while the other four groups showed almost no adverse effects on the proliferative activity of cancer cells (Figure 7k).

4.

CONCLUSIONS

A novel kind of ultrathin 2D niobium carbide (Nb2C) MXene has been successfully fabricated via a two-step liquid exfoliation method combining HF etching (delamination) and TPAOH intercalation (disintegration), and its highly efficient in vivo photothermal ablation of mouse tumor xenografts in both NIR-I and NIR-II bio-windows has been revealed and demonstrated for the first time. The Nb2C nanosheets are ultrathin and lateralnanosized, and exhibit an extraordinarily high photothermal conversion efficiency, as well as desirable photothermal stability. After surface modification with PVP, the produced Nb2C-PVP colloids exhibits excellent biocompatibility and physiological stability, and no noticeable toxicity both in vitro and in vivo. In addition, a novel route of enzymatic biodegradation of Nb2C-PVP

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected]; *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We greatly acknowledge the financial support from the National Key R&D Program of China (Grant No. 2016YFA0203700), National Nature Science Foundation of China (Grant No. 51672303, 51722211), Young Elite Scientist Sponsorship Program by CAST (Grant No. 2015QNRC001), Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant No. 2013169) and Development Fund for Shanghai Talents (2015).

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Two-Dimensional Biodegradable Niobium Carbide (MXene) for

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