Surface Modified Ti3C2 MXene Nanosheets for Tumor Targeting

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Surface Modified Ti3C2 MXene Nanosheets for Tumor Targeting Photothermal/Photodynamic/Chemo Synergistic Therapy Gongyuan Liu, Jianhua Zou, Qianyun Tang, Xiaoyan Yang, Ye-Wei Zhang, Qi Zhang, Wei Huang, Peng Chen, Jinjun Shao, and Xiaochen Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13421 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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ACS Applied Materials & Interfaces

Surface Modified Ti3C2 MXene Nanosheets for Tumor Targeting Photothermal/Photodynamic/Chemo Synergistic Therapy Gongyuan Liu,a Jianhua Zou,a Qianyun Tang,a Xiaoyan Yang,a Yewei Zhang,c Qi zhang,b Wei Huang,a Peng Chen,d* Jinjun shao,a* Xiaochen Donga,e* a

Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM),

Nanjing Tech University (NanjingTech), Nanjing 211800, P. R. China. E-mail: [email protected] b

School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), Nanjing 211800,

P. R. China. c

Department of Hepatobiliary and Pancreatic Surgery, Zhongda Hospital, Medical School,

Southeast University, Nanjing 210009, P. R. China d

School of Chemical and Biomedical Engineering, Nanyang Technological University, 62

Nanyang Drive, 637459, Singapore E-mail: [email protected] e

School of Physical and Mathematical Sciences, Nanjing Tech University (NanjingTech),

Nanjing 211800, P. R. China. E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Ti3C2 MXene is a new two-dimensional material exhibiting a variety of novel properties including good photothermal effect, and the capability of Ti3C2 for multimodal tumor therapy is in urgent need of development. Herein, ultrathin Ti3C2 MXene nanosheets (~100 nm) have been synthesized by supplying additive Al3+ to avoid Al loss and employed as a photothermal/photodynamic agent for cancer therapy. The as-obtained nanosheets exhibit outstanding mass extinction coefficient (28.6 Lg-1 cm-1 at 808 nm), superior photothermal conversion efficiency (~58.3%), and effective singlet oxygen generation (1O2) upon 808 nm laser irradiation. Based on these Ti3C2 nanosheets, a multifunctional nanoplatform (Ti3C2-DOX) is established via layer-by-layer surface modification with doxorubicin (DOX) and hyaluronic acid (HA). In vitro and in vivo experiments disclose that Ti3C2-DOX shows enhanced biocompatibility, tumor specific accumulation and stimuli-responsive drug release behavior, and achieve

effective

cancer

cell

killing

and

tumor

tissue

destruction

through

photothermal/photodynamic/chemo synergistic therapy.

KEYWORDS: Ti3C2 MXene, Photothermal therapy, Photodynamic therapy, Chemotherapy, Synergistic therapy


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1. INTRODUCTION Traditional cancer treatments, such as surgery ablation, radiotherapy and chemotherapy, are often unsatisfactory, due to the invasiveness, risk of tumor recurrence, and side-effects caused by the lack of tumor specific targeting.1 Therefore, photodynamic therapy (PDT) and photothermal therapy (PTT) emerge as promising non-invasive and light-triggered anti-cancer approaches.2,3 And various nanomaterials such as two-dimensional (2D) nanomaterials have been demonstrated unique potentials for tumor therapy.4-6 For instance, graphene,7-11 transition metal dichalcogenides (TMDs),12-14 and black phosphorus (BP)15-16 have been utilized as multimodal theranostic nanoplatforms for cancer therapy. Recently, 2D transition metal carbides/carbonitrides, MXene, have attracted great attention due to their interesting properties, including metallic conductivity and surface activity, etc.17,18 After A layers being selectively etched away from the original MAX phases (M refers to an early transition metal, A presents an element from group 13 or 14, and X is C or N), MXenes can be easily obtained.19,20 MXenes which exhibit metallic conductivity have been explored for catalysis, energy storage and conversion.21-25 In particular, benefitting from its specific surface activity and near infrared (NIR) light absorption, Ti3C2 MXenes have recently attracted interests in biomedical applications, such as cellular imaging, antibacterial activities, and photothermal therapy.26-29 Compared to other 2D nanomaterials, Ti3C2 MXenes distinguish themselves for readily controllable surface functionalities, which modulate the properties of Ti3C2 MXenes for various tasks. For example, Ti3C2 MXenes can be functionalized with hydroxyl groups (OH-),30 endowing them with the hydrophilicity and adsorption capability towards cations or positive


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charged molecules.31 Such Ti3C2 MXenes are useful for ion sieving and delivery of cationic drugs.32-34 The pioneering studies have suggested that the near infrared (NIR) light absorption and photothermal behavior of Ti3C2 MXene is attributable to the localized surface plasma resonance (LSPR) effect, similar to gold nanoparticles and cuprous sulfides.35,36 Such LSPR effect can be further enhanced by LSPR inducing Al oxoanion functional group Al(OH)4-. To functionalize the Ti3C2 nanosheets (~500 nm sized) with Al(OH)4-, the Al atoms (about 90%) in original MAX phase (Ti3AlC2) can be converted into Al(OH)4- in the presence of tetramethylammonium hydroxide (TMAOH).28 The Al oxoanion terminated MXene nanosheets exhibit excellent photothermal performance and light harvesting capability in NIR region. Alternatively, Ti3AlC2 can be etched with HF and intercalated with tetrapropylammonium hydroxide (TPAOH) for a long time to obtain Al oxoanion terminated Ti3C2 nanosheets with a small size of around 150 nm. Such a small size is desirable for tumor accumulation via enhanced permeability and retention (EPR) effect.29 However, the surface Al oxoanion groups on these Ti3C2 nanosheets lose over etching, thus leading to a decline of NIR absorbance and photothermal performance. Hence, there remains a great challenge to prepare Ti3C2 nanosheets with both small lateral size and stable surface Al oxoanion groups. Although Ti3C2 MXene disperses well in pure water, it tends to aggregate in physiological solutions. Therefore, surface modification of Ti3C2 nanosheets with polyethylene glycol (PEG) 28 or soybean phospholipid29 has been adopted to improve biocompatibility of Ti3C2 nanosheets. Herein, we develop a surface modification method by supplying additive Al3+ to avoid Al loss during the long-term etching, whereby both small lateral size and highly stable surface Al oxoanion Al(OH)4- can be achieved. The as-obtained Ti3C2 nanosheets exhibit a high mass extinction coefficient of 28.6 Lg-1cm-1, and a superior photothermal conversion efficiency of


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58.3% under 808 nm irradiation. In addition, to the best of our knowledge, the photodynamic behavior of Ti3C2 MXene nanosheets is reported for the first time. Furthermore, Ti3C2 nanosheets based multifunctional nanoplatform (denoted as Ti3C2-DOX) with a drug loading capacity as high as 84.2% is realized through a layer-by-layer adsorption of anti-cancer drug doxorubicin (DOX) and tumor-targeting hyaluronic acid (HA). This drug delivery system displays enhanced tumor specific accumulation owing to both EPR effect and active targeting to CD44+ over-expressed tumor cells. Benefitting from tumor specific accumulation, controlled multi-stimuli-responsive drug release and synergistic PTT/PDT/chemotherapy, the Ti3C2 MXene nanosheets based therapeutic nanoplatform acts as an effective multifunctional anti-cancer agent as evidenced through both in vitro and in vivo experiments. 2. EXPERIMENTAL SECTIONS 2.1. Materials. Ti3AlC2 (powder, 200-meshes) was purchased from Beijing Forsman Scientific (China). HCl (36%, w/w, purity>98%) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd (China). LiF, AlCl3 6H2O, TMAOH (25% wt in water), Doxorubicin hydrochloride (DOX), were purchased from Admas (USA). Hyaluronic acid (HA) (MW 6.4 kDa) was purchased from the Shandong Freda Biopharmaceutical Co., Ltd. Hyaluronidase (HAase) and IR-780 Iodide were purchased from Sigma-Aldrich (USA). All chemicals were used as received without further treatment unless otherwise stated. 2.2. Characterizations. X-ray diffraction (XRD) was performed on a Bruker diffractometer (D8 advance) with Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 40 mA. Characterizations of sample morphology were performed on an S-4800 field emission scanning electron microscope (FESEM, Hitachi, Japan) and transmission electron microscope (TEM, JEOL JEM-2010). X-Ray


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photoelectron spectra (XPS) of the samples were recorded by an X-ray photoelectron spectrometer (ESCALab 250Xi, Thermo Fisher Scientific US) with Al K α radiation. Atomic force microscope (AFM) measurement was performed on Bruker Multi Mode system. Particle size and zeta potential was measured on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). The Al/Ti atomic ratio was determined by inductively coupled plasma (ICP) analysis on an ICP OPTMA20000V (PerkinElmer, US). UV-vis-NIR absorption spectra were recorded by UV3600 Shimadzu UV-vis-NIR spectrometer (Shimadzu, Japan) and fluorescence spectra were recorded by an F-4600 spectrofluorophotometer (Hitachi, Japan). Thermal images were acquired by an E50 infrared camera (FLIR, Arlington, VA). The confocal fluorescence images were obtained by an Olympus IX 70 inverted microscope (Olympus, Japan). 2.3. Preparation of Ti3C2 nanosheets. Particularly, 1.000 g of Ti3AlC2 powders were etched in 10 mL 9 M HCl containing 1.000 g LiF and 0.300 g AlCl3.6H2O for 3 days at room temperature. After collecting by centrifugation, the etched materials were washed with water and ethanol for several times and then dispersed in 10 mL TMAOH (25% wt in water) for 3 days with stirring. The intercalated Ti3C2 were collected by centrifugation and washed. Eventually, the collected clay-like solid was re-dispersed into water under bath sonication for hours, and the supernatant at 7000 rpm centrifugation was obtained as the colloidal dispersion of Ti3C2 nanosheets. The concentration was determined to be 2.01 mg mL-1 by weighting freeze-dried samples. 2.4. Preparation of Ti3C2-DOX. 2.0 mL of Ti3C2 (2.0 mg mL-1) was mixed with 4.0 mL DOX in water (2.0 mg mL-1), stirring in dark overnight. Then, the mixture was centrifuged and resuspended with HA (4.0 mg mL-1). After a few hours of stirring, Ti3C2-DOX was collected by centrifuging to remove excessive HA. The obtained Ti3C2-DOX was re-dispersed into PBS and stored at 4 oC and kept from light for further use. DOX concentration in the supernatant was


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determined by UV-Vis, and DOX loading capacity can be calculated according to: Loading capacity = (weight of DOX in Ti3C2-DOX)/(weight of Ti3C2). The DOX loading capacity was determined to be 84.2%. Hydrodynamic diameters and Zeta-potentials of Ti3C2 and Ti3C2-DOX were measured. Alternatively, IR-780 was loaded for in vivo fluorescence imaging instead of DOX following the same procedure for in vivo real-time monitoring. 2.5. In Vitro Photothermal Effect and Photostability. 2.0 mL Ti3C2 aqueous dispersion of different concentrations in transparent quartz cells were irradiated by 808 nm laser with varied power densities for 10 min. The temperature changes were recorded by FLIR infrared camera. The absorbance of Ti3C2 (100 µg mL-1) before and after 30 min irradiation under laser irradiation (808 nm, 1.5 W cm-2) was recorded to validate the photostability of Ti3C2. 2.6. Singlet Oxygen Detection. 1,3-Diphenylisobenzofuran (DPBF) in ethanol (20 µg mL-1) was mixed with Ti3C2 and Ti3C2-DOX (equivalent Ti3C2 at 30 µg mL-1), respectively. And the UVvis spectra after irradiation (808 nm, 0.8 W cm-2) were recorded at different time points over 10 min. 2.7. Drug Release. Ti3C2-DOX (with equivalent loaded DOX dose of 0.25 mg mL-1, 2.0 mL) in different pH buffers (7.4, 6.0 and 4.5) at 37 oC was treated with/without laser irradiation (0.8 W cm-2) at different time intervals (0, 0.5, 1, 2, 3, 6, 9, 12, 24 and 48 h) and the temperature of irradiated solutions was heated to about 50 oC, eventually. For HAase-responsive drug release, Ti3C2-DOX of the same dose was incubated with HAase-containing pH buffers (pH 6.0 and 4.5, 0.5 mg mL-1, 2.0 mL). At time intervals mentioned above, the solutions were centrifuged and 0.15 mL of the supernatants were collected. The amount of released DOX from Ti3C2-DOX was determined by UV-vis.


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2.8. Hemolysis Assay. Erythrocytes were obtained from Nanjing Stomatological Hospital, Medical School of Nanjing University, and washed with PBS then collected at 3000 rpm. 4% erythrocytes (v/v, 1.0 mL) was mixed with 1.0 mL of PBS, water, Ti3C2 and Ti3C2-DOX solutions at different concentrations (200, 100, 50 and 0 µg mL-1). The mixture was incubated at 37 °C for 8 h. Then the mixture was spun down at 3000 rpm and the supernatants were collected and absorbance at 540 nm was measured. The percentage of hemolysis was calculated by following equation: Hemolysis (%) = I/I0 × 100%. Where I is the absorbance of supernatants, and I0 refers to totally hemolysis in pure water. 2.9. Cell Lines. HCT-116 cell line and A2780 cell line were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China), further cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. 2.10. Confocal Fluorescence Imaging. In cellular uptake study, HCT-116 cells and A2780 cells were incubated with Ti3C2-DOX (2.0 mL, 10 µg mL-1 of Ti3C2 with 8 µg mL-1 of DOX loaded) for 4 h. The images were viewed with Olympus IX 70 inverted microscope, and excited at 488 nm and collected around 570 nm. In cellular ROS study, HCT-116 cells were incubated with Ti3C2-DOX (2.0 mL, 20 µg mL-1) in dark for 4 h. Then the cells were treated with DCFH-DA, (2.0 mL, 10 µM) for another 30 min. Before imaging, the sample was irradiated with 808 nm laser (0.8 W cm-2, 10 min), with samples excited at 488 nm and images collected at 550 nm. All the cells were stained with 4',6-diamidino-2-phenylindole (DAPI, a nuclei-specific dye). 2.11. Cytotoxicity Assay. The cytotoxicity of Ti3C2-DOX and DOX were determined by MTT assay. HCT-116 Cells seeded in 96-well plates were divided into three groups. After 12 h


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incubation, the medium was replaced with 200 µL of DOX and Ti3C2-DOX at different concentrations (0, 6.25, 12.5, 25, 50 and 100 µg mL-1 of Ti3C2 with equivalent DOX at 0, 5.25, 10.5, 21, 42 and 84 µg mL-1). After being cultured for 4 h, the cells of phototherapy group were irradiated with 808 nm laser (0.8 W cm-2) for 10 min. Then the cells were incubated at 37 °C for 24 h. For MTT assay, the medium was replaced with MTT solution (0.2 mL, 0.5 mg mL-1) and kept for another 4 h. Subsequently, the supernatant was carefully removed and DMSO (0.2 mL) was added. The absorbance at 492 nm was measured by Bio-Tek microplate reader. 2.12. Flow Cytometry Study. HCT-116 cells were seeded on a 12-well plate and incubated for 24 h. The cells were then treated with bare Ti3C2 nanosheets (to exclude the fluorescence of DOX that interferes with the fluorescence signal of FITC) at varied concentrations (0, 25, 50, and 75 µg mL-1) for 6 h. After rinsed with PBS and refilled with 1 mL of culture medium, the cells were irradiated by 808 nm laser (0.8 W cm-2, 10 min). The cells were incubated for another 6 h, and then collected by centrifugation and re-suspended in binding buffer containing propidium iodide (PI, 5 µL) and annexin-V FITC (5 µL) for 15 min in darkness. The signal was collected by a BD FACSCalibur flow cytometer (Perkin Elmer) with 105 cells counted in each sample. The emitted fluorescence was collected at 525 nm for annexin-V and at 670 nm for PI. Data were analyzed with Cell QUEST Pro. 2.13. Animal and Tumor Model. Athymic nude mice (5~6 weeks old) were purchased from the Comparative Medicine Centre of Yangzhou University. All animal experiments were performed in compliance with the NIH guidelines for the care and use of laboratory animals. The School of Pharmaceutical Science, Nanjing Tech University has approved all the experiments. The tumor model was prepared by injecting HCT-116 cells at the left forelimb of the mice. When tumor volume reached about 100 mm3, mice were employed for imaging and therapeutic studies.


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2.14. In Vivo Thermal Imaging. The tumor bearing mice were intravenously injected with PBS and Ti3C2-DOX (dose of Ti3C2 at 2.0 mg kg-1 of body weight). At different time intervals (1, 3, 6, 9, 12 and 24 h post-injection) the mice were irradiated with 808 nm laser (0.8 W cm-2, 10 min) and the highest temperature at tumor site was recorded. 2.15. In Vivo Fluorescence Imaging. Tumor bearing mice was intravenously injected with Ti3C2-IR780 (2.0 mg kg-1), Ti3C2-DOX (loaded DOX does at 1.6 mg kg-1 and Ti3C2 at 2.0 mg kg1

) and DOX (1.6 mg kg-1), at different time intervals, the fluorescence images were recorded. At

24 h post injection, mice were sacrificed for tumors, hearts, livers, spleens, lungs and kidneys. All images were recorded by IVIS Lumina K Vivo Imaging System (PerkinElmer, USA). 2.16. In Vivo Tumor Therapy. The mice were randomly divided into five groups (n = 4 in each group, 2 of male and 2 of female) and intravenously injected with different doses of PBS solutions: control, DOX (1.6 mg kg-1) and Ti3C2-DOX (with equivalent loaded DOX dose at 1.6 mg kg-1 and Ti3C2 at 2.0 mg kg-1). The groups of Laser only and Ti3C2-DOX + Laser were irradiated by 808 nm laser (0.8 W cm-2, 10 min) at 9 h post-injection. The tumor site temperature was monitored by FLIR camera during irradiation. For Ti3C2-DOX + laser group, the tumor was irradiated for 1~2 times with a scar left and finally healed during the last days. The tumor volume and body weight were measured every other day and the tumor growth was monitored in the 16 days of treatment. The tumor volumes were calculated by the following equation: V = width2 × length/2. 2.17. Ex Vivo Histology Examination. After treatment, the mice were sacrificed and the mean organ tissues (heart, liver, spleen, lung, kidney and tumor) were dissected for histology analysis. After dehydration and stained with hematoxylin and eosin (H&E), all slices were embedded in


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paraffin cassettes. The H&E stained images were collected by microscope with the magnification of 400x. 3. RESULTS AND DISCUSSION When the formation of surface Al oxoanion groups of Ti3C2 MXene nanosheets in organic base TMAOH relies on Al atoms from the precursor Ti3AlC2 as previously reported, the long-term etching will inevitably induce Al loss. Therefore, Al3+ ions are added during etching as the extraneous source for the formation of Al oxoanion groups. As shown in Scheme 1, the ultrathin Ti3C2 nanosheets were prepared by etching for 3 days and subsequent intercalation by TMAOH for 3 days. Scanning electron microscopy (SEM) images (Figure 1a and Figure S1 in Supporting Information) show that the closely packed layer structures of original MAX phase was exfoliated into nanometer sized flakes, and the elemental mapping (Figure S2) of the etched samples confirms the surface modification by Al3+. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images reveal that Ti3C2 nanosheets have an average lateral size of ~100 nm (Figure 1b, 1c and Figure S3). The aqueous dispersion of Ti3C2 nanosheets appears dark green colored with the incident light scattered by colloidal nanosheets (Tyndall effect) (Figure 1b, inset). The formation of Ti3C2 is also confirmed by X-ray diffraction (XRD). After TMAOH intercalation, the most intense XRD peak of Ti3AlC2 (2θ ≈ 38°) disappears, instead, the characteristic periodic peaks originated from stacked Ti3C2 layers appear. After delamination, only the peak at 2θ ≈ 6° remains noticeable. In X-ray photoelectron spectroscopy (XPS), Ti-C (2p3) and Ti-O (2p3) doublets at 455.2 and 461.3 eV are identified, confirming the chemical composition of Ti3C2 nanosheets (Figure S4c). The fitting analysis of O 1s and Ti 2p XPS spectra implies the -Ti-O-Al-O- atom sequence, indicating the existence of Al(OH)4terminations (Figure S4).28 Furthermore, inductive plasma resonance (ICP) analysis shows that


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the Al/Ti atomic ratio of Ti3C2 nanosheets is 0.62:3 with extraneous Al3+, or 0.23:3 without the addition of Al3+, indicating that Al ratio is well preserved as the original ratio of 1:3 in the original MAX phase Ti3AlC2. And, Figure 1e shows that NIR absorption (600~900 nm) of Ti3C2 nanosheets is significantly enhanced by the addition of Al3+ during etching, attributable to the enhancement of LSPR effect by Al oxoanion. Conceivably, Al3+ ions are adsorbed onto the hydroxyl groups of Ti3C2 via electrostatic interaction30 during etching, and then converted into Al(OH)4- terminations by excessive TMAOH (Al3+ + 4OH- = Al(OH)4-). During the intercalation of TMAOH, the NIR absorption peak (~800 nm) of Ti3C2 nanosheets gradually grows (Figure 1f), indicating the forming of Al oxoanion. According to Lambert-Beer law (A = αCL, where α is the mass extinction coefficient), the extinction coefficient at 808 nm was determined to be 28.6 Lg-1cm-1 (Figure 1f, inset). As shown in Figure S6, the photothermal conversion efficiency of surface modified Ti3C2 nanosheets is calculated to be 58.3%, which is much higher than that of the reported Ti3C2 nanosheets (30.6%)29 etched without Al3+, 2D BP (36.8%)37 and Bi2Se3 nanodots (50.7%).38 Figure 2a shows the temperature elevation of Ti3C2 aqueous dispersion at different concentrations, under 808 nm laser irradiation (0.8 W cm-2). The temperature of Ti3C2 dispersion rapidly reached a plateau and increased by about 20 oC even at a low concentration of 25 µg mL-1 after 10 min irradiation. As seen from Figure 2b, with the increasing of laser power density, the temperature elevation of the Ti3C2 dispersion increases. And as shown in Figure 2c, the Ti3C2 nanosheets have good photothermal cycling stability. After irradiation for 30 min, no significant photobleaching of Ti3C2 nanosheets was observed (Figure S7). The superior photothermal performance and stability promise that Ti3C2 nanosheets could serve as an excellent agent for tumor photothermal therapy.


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Considering the unique electronic structure and photoelectronic property of Ti3C2 nanosheets, the capability of Ti3C2 nanosheets to generate reactive oxygen species (ROS) under irradiation and their potential to be a novel photosensitizer for photodynamic therapy were investigated. Using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen (1O2, most prominent one of ROS) detector, the decrease of the absorbance of DPBF suggests that 1O2 generated by Ti3C2 nanosheets was captured by DPBF, verifying the generation of 1O2 of Ti3C2 nanosheets under NIR light irradiation (Figure 3d and S8). Conceivably, the generation of 1O2 is due to the energy transfer of photo-excited electrons from Ti3C2 to triplet oxygen (ground state oxygen, 3O2), perhaps similar to the photodynamic behavior of BP and graphene quantum dot.39,40 The LSPR effect of Ti3C2 may also play a role in 1O2 generation. According to the previous report, the ability of metal, especially gold nanoparticles, to generate ROS under the irradiation of visible light relies on its LSPR effect.41-43 And the efficiency of energy transfer is especially high for low-energy surface state and aggregated particles. The large surface area of Ti3C2 nanosheets might also be favorable for LSPR. PDT triggered by NIR laser, is highly desired for deep tissue penetration. Hence, Ti3C2 nanosheets are revealed to be a novel photosensitizer for effective photodynamic therapy. Making use of Ti3C2 to interact with cationic molecules, it can serve as the carrier for cationic anti-cancer drugs.31 Ti3C2 nanosheets based nanoplatform (Ti3C2-DOX) was prepared through a layer-by-layer adsorption method. DOX was used as cationic model drug and loaded onto Ti3C2 nanosheets with a loading capacity as high as 84.2%. Then the nanosheets were coated by HA to improve the biocompatibility and endow the system with active targeting to CD44+ overexpressed tumor cells.34,44 The UV-vis spectra indicates the successful loading of DOX, with the absorption peak of DOX slightly red-shifted from 487 nm to 505 nm (Figure 3a), which is


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agreeable to a previous study.16 Moreover, the fluorescence of DOX is quenched by Ti3C2 due to fluorescence resonance energy transfer (FRET) resulting from the overlap between the Ti3C2 absorption band and DOX emission band (Figure S5c). This further verifies the molecular interaction between DOX and Ti3C2 nanosheets. The zeta potentials and hydrodynamic diameters of Ti3C2, Ti3C2@DOX and Ti3C2@DOX@HA (Ti3C2-DOX) were also measured. As expected, the negative charge of Ti3C2 (-23.18 mV) was increased to +13.25 mV after DOX loading, and then decreased back to -20.71 mV after HA capping (Figure 3b, Table S1). The mean hydrodynamic diameter of Ti3C2-DOX was measured to be 178 nm (Figure S5d), which enables these nanosheets to accumulate at tumor sites via enhanced permeability and retention (EPR) effect.45 The characteristic absorbance at about 200 nm validates the existence of HA shell on Ti3C2-DOX (Figure S5b). The photothermal effect was not obviously changed after DOX and HA modification (Figure 2d). However, a decrease of 1O2 generation after HA capping was observed, probably because the polymeric HA shell hinders the energy transfer between Ti3C2 and oxygen.46 The as-prepared Ti3C2-DOX is stable during the storage at 4 oC, without any decay in UV-VIS and aggregation, indicating the potential of clinical transition as a storable anticancer drug (Figure S9b). The biocompatibility of Ti3C2 nanosheets are evaluated. Firstly, as shown in Figure 3c, no significant hemolysis (< 5%) was caused by both unmodified Ti3C2 nanosheets and Ti3C2-DOX even at a high concentration of 200 µg mL-1. Notably, after being capped with HA, the biocompatibility of Ti3C2-DOX was remarkably improved in biological conditions (Figure S9a). To minimize the side effects and maximize the therapeutic efficacy, it is preferable for a drug delivery system to be responsive to tumor specific stimuli. Here, we utilize the acidic tumor microenvironment47 and the photothermal effect as the stimuli to achieve the tumor specific turn-


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on therapy. As Ti3C2-DOX is designed based on the electrostatic interaction which is highly sensitive to pH and temperature, it is expected to be responsive to pH, laser induced hyperthermia and hyaluronidase (HAase) in cancer cells. Figure 3e and 3f demonstrate the stimuli-responsive drug release behavior of Ti3C2-DOX under different pH, with/without laser irradiation or HAase. As shown, HA shell can effectively prevent DOX release in neutral conditions (e.g., in blood and interstitial fluids in normal tissues). While under the laser-induced photothermal effect of Ti3C2 (to ~50 °C), DOX release is stimulated because of the disruption of electrostatic interaction in Ti3C2-DOX system. The pH responsiveness implies enhanced drug delivery in the acidic microenvironment of tumors. Furthermore, in the presence of HAase, up to 70% of the drug can be released at pH 4.5 within 48 h. All these results suggest that Ti3C2-DOX is capable of targeted release because of its high responsiveness to photothermal, pH and enzyme stimuli. The therapeutic efficacy of Ti3C2 nanosheets were investigated through flow cytomerty study and MTT cytotoxicity assay against human colon carcinoma cell line (HCT-116 cells). The flow cytomerty study (Figure 4a-4f) suggests that cell death caused by dose-dependent phototherapy of Ti3C2 nanosheets is mainly due to the late apoptosis mechanism, and Ti3C2 nanosheets showed a low dark toxicity. As bare Ti3C2 nanosheets heavily aggregate in physiological conditions, their cytotoxicity cannot be assessed. There is no significant difference between the cytotoxicity of DOX and Ti3C2-DOX (at the same dosage of DOX), without illumination. Upon 808-nm laser irradiation (0.8 W cm-2), dramatic increase of cell death was observed, manifesting the efficient synergistic killing of cancer cells in vitro (Figure 4g, Figure S10). Figure 4h and 4i present the fluorescence images of HCT-116 cells treated with Ti3C2-DOX for 4 h. The bright red fluorescence represents DOX molecules dispersed in the cytoplasm and nucleus after


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endocytosis. In contrast, quite low signal of DOX was detected in A2780 cells (cell line with low expression of CD44+) incubated with Ti3C2-DOX at the same dose for 4 h (Figure S11), indicating the high selectivity of HA modified Ti3C2-DOX towards CD44+ overexpressed tumor tissues. To validate the intracellular ROS generation of Ti3C2-DOX, 2’,7’-dichlorofluorescein diacetate (DCFH-DA, form fluorescent 2’,7’-dichlorofluorescein, DCF upon reacting with ROS), was used as the ROS probe. As shown in Figure 4i, irradiated by 808 nm laser, bright green fluorescence of DCF in the cells suggests that Ti3C2-DOX can intracellularly produce high level ROS, once HA shell is degraded by intracellular HAase to expose the active surface of Ti3C2 nanosheets after endocytosis.48 To examine the specific accumulation of Ti3C2-DOX at tumor site, HCT-116 tumor-bearing mice were intravenously injected with Ti3C2-DOX and DOX, and at 24 h post-injection, tumors and main organ tissues were dissected out. As shown in Figure 5e, for Ti3C2-DOX injected mice, intense fluorescent signal of DOX was detected in the tumor, but much lesser signal in liver and kidney (suggesting the potential excretion process of DOX), and very low levels in heart, spleen and lung (Figure S12), suggesting the efficient and selective accumulation of Ti3C2-DOX at tumor site owing to both CD44+ targeting and EPR effect of the nanoplatform.49 In contrast, the ex vivo fluorescence of free-DOX injected mice exhibit no significant accumulation in tumor. Furthermore, loading NIR dye (IR-780) onto Ti3C2 for real time monitoring, it was found that tumor accumulation of Ti3C2 occurred only a few hours after injection (Figure S13). As shown in Figure 5a, 5b and Figure S14, the temperature of Ti3C2-DOX accumulated tumor was elevated from 34.0 °C to 53.1 °C within 5 min, sufficient for tumor ablation,50-52 while the PBS injected mice only showed slight temperature increase. Finally, in vivo therapeutic effects of


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Ti3C2-DOX were evaluated in HCT-116 tumor-bearing mice. Treatment with free DOX or Ti3C2-DOX without laser irradiation only partially inhibited tumor growth. In comparison, synergistic PDT/PTT/chemotherapy of Ti3C2-DOX triggered by laser irradiation were completely eliminated tumor without reoccurrence (Figure 5c, 5d, Figure S15). Over the period of 16 days treatment, except for the free DOX treated group with a minor weight loss, the body weight of all mouse groups remained stable (Figure 5f) and all mice of all groups survived after 16 days treatment. Compared to Ti3C2 nanosheets previous reported for PTT only,29 the Ti3C2DOX nanoplatform with synergistic therapy has exhibited its superiority in a dosage (DOX dose at 1.6 mg kg-1 and Ti3C2 at 2.0 mg kg-1) and lower laser power density of 0.8 W cm-2 compared to that of 1.5 W cm-2 due to the enhanced photothermal performance via the specific surface modification by Al(OH)4- in this work. After the synergistic treatment, tumor and main organ tissues were dissected out, and the tissue slices were stained with hematoxylin and eosin (H&E) for histopathological examination (Figure 5g). In the tumors of DOX and Ti3C2-DOX treated mice, broken and small nuclei were observed, indicating cell apoptosis caused by the combined therapies. No significant damage was noticed in all the organs (Figure S16), benefiting from the low dosage of DOX and the stimuliresponsive drug release. 4. CONCLUSION In summary, a surface modification method has been developed to synthesize Ti3C2 MXene nanosheets with a small lateral size (~100 nm) and stable surface functional group Al(OH)4-. A high mass extinction coefficient at 808 nm (28.6 Lg-1cm-1) and superior photothermal conversion efficiency (58.3%) are attained presumably owing to Al(OH)4- enhanced localized surface


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plasmon resonance effect. Moreover, the reactive oxygen species generation ability of Ti3C2 nanosheets is reported for the first time here, suggesting the potential as a photosensitizer for cancer photodynamic therapy. Taking advantages of the negatively charged surface of Ti3C2 nanosheets, doxorubicin as the chemotherapy agent and hyaluronic acid as the active tumor targeting agent are composited with Ti3C2 nanosheets through layer-by-layer adsorption. The resulting multifunctional nanoplatform displays enhanced biocompatibility, stimuli-responsive drug release, tumor specific accumulation, and outstanding tumor ablation efficacy at a low dose (Ti3C2 at 2 mg kg-1 with DOX loaded at 1.6 mg kg-1) under a low power density of NIR laser (808 nm, 0.8 W cm-2). This work not only offers a new effective strategy for cancer therapy based on surface modified Ti3C2 nanosheets, but also suggests the theranostic potentials of the emerging MXenes. ACKNOWLEDGMENT The work was supported by the NNSF of China (61525402, 61775095, 61604071), Jiangsu Provincial key research and development plan (BE2017741), Key University Science Research Project of Jiangsu Province (15KJA430006), Natural Science Foundation of Jiangsu Province (BK20161012). Supporting Information. Additional characterization results (SEM, SEM-EDX, AFM size analysis, XPS, UV-VIS, FL) of Ti3C2, calculation information of photothermal conversion efficiency of Ti3C2, singlet oxygen detection of Ti3C2 and Ti3C2-DOX by DPBF, photos of dispersion of Ti3C2 and Ti3C2-DOX in water and PBS, MTT assay of Ti3C2-DOX, Confocal fluorescence images of A2780 cells treated with Ti3C2-DOX, relative intensity of ex vivo fluorescence imaging of DOX and Ti3C2-DOX injected mice, real time fluorescence imaging of


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Scheme 1. Schematic illustration of the preparation of Ti3C2-based nanoplatform, and the synergistic photodynamic/photothermal/chemotherapy of tumor.


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Figure 1. (a) SEM image of layered Ti3C2 after etching for 1 day. (b) TEM image of delaminated Ti3C2 nanosheets. Inset: the colloidal dispersion of Ti3C2 nanosheets with Tyndall effect with a green laser beam. (c) AFM-measured thickness of obtained Ti3C2 nanosheets. Inset: AFM image of Ti3C2 nanosheets. (d) XRD patterns of raw Ti3AlC2 and Ti3C2 after intercalation and further delamination. (e) Normalized UV-vis absorbance of Ti3C2 nanosheets with/without Al3+ addition during etching. (f) UV-vis spectra of Ti3C2 nanosheets dispersed in water at various concentrations. Inset: Lambert-Beer law absorbance plot for absorption at 808 nm.


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Figure 2. (a) Photothermal profile of Ti3C2 nanosheets in water at varied concentrations (808 nm, 1.0 W cm-2). (b) Photothermal profile of Ti3C2 nanosheets in water (50 µg mL-1) under different laser power densities. (c) Photostability test of Ti3C2 nanosheets in water under 808 nm irradiation (1.5 W cm-2). (d) Photothermal performance of Ti3C2, Ti3C2-DOX and free DOX solutions (808 nm, 1.0 W cm-2).


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Figure 3. (a) UV-vis absorption spectra of Ti3C2, DOX and Ti3C2-DOX. (b) Zeta potentials of Ti3C2, Ti3C2@DOX and Ti3C2@DOX@HA (Ti3C2-DOX). (c) Hemolysis assay of Ti3C2 and Ti3C2-DOX at different concentrations, both of them will not cause hemolysis (

Surface Modified Ti3C2 MXene Nanosheets for Tumor Targeting

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