Orally administered titanium carbide nanosheets as anti-inflammatory therapy for colitis

Rationale: Oxidative stress, resulting from excessive reactive oxygen species (ROS), plays an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, developing novel strategies to target the disease location and treat inflammation is urgently needed. Methods: Herein, we designed and developed a novel and effective antioxidant orally-administered nanoplatform based on simulated gastric fluid (SGF)-stabilized titanium carbide MXene nanosheets (Ti3C2 NSs) with excellent biosafety and multiple ROS-scavenging abilities for IBD therapy. Results: This broad-spectrum and efficient ROS scavenging performance was mainly relied on the strong reducibility of Ti-C bound. Intracellular ROS levels confirmed that Ti3C2 NSs could efficiently eliminate excess ROS against oxidative stress-induced cell damage. Following oral administration, negatively-charged Ti3C2 NSs specifically adsorbed onto the positively-charged inflamed colon tissue via electrostatic interaction, leading to efficient therapy of dextran sulfate sodium salt (DSS)-induced colitis. The therapeutic mechanism mainly attributed to decreased ROS levels and pro-inflammatory cytokine secretion, and increased M2-phenotype macrophage infiltration and anti-inflammatory cytokine secretion, efficiently inhibiting inflammation and alleviating colitis symptoms. Due to their excellent ROS-scavenging performance, Ti3C2-based woundplast also promoted skin wound healing and functional vessel formation. Conclusions: Our study introduces redox-mediated antioxidant MXene nanoplatform as a novel type of orally administered nanoagents for treating IBD and other inflammatory diseases of the digestive tract.


Synthesis of Ti 3 C 2 NSs
The Ti 3 C 2 nanosheets (NSs) were synthesized via two-step exfoliation strategy, which was based on HF etching and TPAOH intercalation of Ti 3 AlC 2 powder 1 . In brief, 20 mL HF (20%) was added slowly into 1 g Ti 3 AlC 2 powder under stirring to remove the middle Al layer. After reaction for 4 h, the obtained Ti 3 C 2 powder was centrifuged and washed repeatedly with water to remove the excess HF. Subsequently, the Ti 3 C 2 powder was exfoliated in 20 mL TMPOH (~25%) and kept under stirring overnight. After collecting by centrifugation and washing with water and ethanol for three times, the final Ti 3 C 2 NSs was obtained.

Modification of Ti 3 C 2 NSs
The synthesized Ti 3 C 2 NSs were modified by polyvinylpyrrolidone (PVP, MW 10k).
Briefly, 50 mg Ti 3 C 2 NSs and 250 mg PVP were dissolved in 20 ml H 2 O and stirred for 4 h.
After dialysis (8-14.8 kDa), the PVP modified Ti 3 C 2 NSs were dispersed in water and stored in a -20 o C for future use (concentration, 1 mg/mL).

Characterization
Scanning electron microscopy (SEM) imaging was conducted by a ZEISS G500 SEM.
Transmission electron microscopy (TEM) imaging and elemental mapping were performed by a FEI Tecnai F20 TEM. Phase purity of the Ti 3 AlC 2 powder and its structural transformation were examined by powder X-ray diffraction (XRD) using a PANalytical and then incubated with Ti 3 C 2 NSs (2, 4, and 8 μg/mL). After reaction for 10 min, the absorption of oxTMB at 654 nm was monitored by UV-vis-NIR spectrophotometer.
TEMPO assay: TEMPO (20 μM) as intrinsic free radicals were examined by the electron spin resonance (ESR) spectra w/wo addition of Ti 3 C 2 NSs. The characteristic peaks intensity revealed the concentration of TEMPO and further verified the antioxidative properties of Ti 3 C 2 NSs.

Antioxidant efficiency and mechanism
According to the previous literatures, we selected four materials like cerium dioxide (CeO 2 ) 2-4 , Mo-based polyoxometalate (POM) cluster 5 , tungsten sulfide (WS 2 ) 6 , and gallic acid (GA) 7 to compare ROS scavenging performance of them with Ti 3 C 2 NSs. And ABTS+• radical were employed to evaluate the ROS scavenging ability of these materials. In addition, the fresh Ti 3 C 2 NSs, lightly oxidized Ti 3 C 2 NSs, and heavily oxidized Ti 3 C 2 NSs were characterized by XPS analysis and Raman spectrum to reveal the microstructure and the antioxidant mechanism of our Ti 3 C 2 NSs.

Cellular experiments
Human colonic epithelial cells (HT29) and mouse macrophage cells (RAW 264.7) were cultured in a normal RPMI-1640 medium or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 o C under 5% CO 2 atmosphere, respectively.
In vitro cytotoxicity test: HT29 cells were seeded in 96-well plates were incubated with Axio-Imager LSM-800).
Macrophages polarization assays: RAW 264.7 were seeded and then incubated with lipopolysaccharide (LPS) or LPS/Ti 3 C 2 NSs (40 μg/mL). These above RAW 264.7 were stained by CD80 marker and then analyzed by CLSM and flow cytometric examination to evaluate the polarization of macrophages and the inhibiting effect of Ti 3 C 2 NSs for LPS-induced macrophages polarization.

Animal model
Six-week-old female C57BL/6 and BALB/c mice (18 ± 2 g) were purchased from Nanjing Sikerui Biological Technology Co. Ltd, and all animal experiments were carried out under the permission of Soochow University Laboratory Animal Center.

DSS-induced colitis model
Female C57BL/6 mice were grouped randomly and acclimatized for one week.
Subsequently, these mice were given 5% DSS supplemented in the drinking water for 5 days to induce inflammatory bowel disease (IBD), then returned to normal water.

IBD treatment by Ti 3 C 2 NSs
The healthy mice in the control group were provided with normal water during the whole time. And the DSS-induced IBD mice were orally administered by Ti 3 C 2 NSs (45 mg/kg) or PBS on the planned days. The body weights of these mice were monitored daily during the experimental period. On the last day over the experiment, the mice were sacrificed and their colons were collected. The colon lengths of these colons were collected, and then they were gently rinsed with physiological saline to remove the intestinal contents. Finally, two sections of the distal tissue with a length of 0.5 cm were taken for histological evaluation and fluorescence analysis. The remaining colon tissues were used for flow cytometric and cytokine analysis.

Histopathological evaluation
Histological analysis of colon tissue sections stained with hematoxylin and eosin (H&E).
In brief, the collected colon tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. The sections of distal colon tissues (5 µm) were made and stained by H&E for analysis.

Colon microenvironment evaluation
The collected colon tissues were placed in PBS buffer, and the intestinal contents were gently washed. Subsequently, these colon tissues were homogenized in FACS buffer, in these colon tissues were determined by enzyme-linked immunosorbent assay (ELISA).

Biosafety of Ti 3 C 2 NSs after oral administration
The DSS-induced IBD mice were orally administered by Ti 3 C 2 NSs (45 mg/kg) on predetermined days. As the protocol revealed, these mice were scarified at day 0, 1, and 14 after treatment. Then, the blood and major organs were collected to analyze and evaluate the biosafety of the synthesized Ti 3 C 2 NSs.

Preparation of Ti 3 C 2 -based woundplast by electrospinning
Ti 3 C 2 NSs with appropriate concentration was mixed with pre-prepared PVA solution to obtain Ti 3 C 2 -PVA spinning solution. Afterwards, the spinning solution was injected into the syringe with a 20-gauge needle (inner diameter 0.6 mm, outer diameter 0.9 mm) for electrospinning to prepare Ti 3 C 2 NSs-based woundplast (4 μg Ti 3 C 2 per piece). The parameters of electrospinning were as follow: voltage, 15 kV; distance between needle tip and collector, 12.0 cm; flow rate, 0.5 mL/h.

Wound closure accelerated by Ti 3 C 2 NSs-based woundplast
Healthy six-week-old female BALB/c mice were employed as experimental subjects.
Firstly, the back hair of the mice was shaved and disinfected, and then two full-thickness skin wounds in 8 mm diameter were made on the back of each mouse. These mice bearing wounds were randomly divided into three groups (six wounds per group): (1) Control; (2) PVA-based woundplast; (3) Ti 3 C 2 NSs-based woundplast. These treatments were repeated every two days and totally conducted four times. After various treatments, these wounds were monitored, measured, and analyzed by Image J software. On the last day of the treatment (day 8), these wounds were excised and stained by H&E and Masson to evaluate the wound healing by Ti 3 C 2 NSs-based woundplast. Lastly, these wounds also were stained with anti-CD31 and anti-integrin α3 antibody to reveal the generation of newborn blood vessels and collagen around the wounds.

Statistical Analysis
All the results in this work were presented as mean values ± SD. Statistical analysis was conducted via GraphPad Prism software 6. Two-tailed Student's t-test was used for two group comparison and One-way analysis of variance (ANOVA) with a Tukey post-hoc test was used for multiple comparisons. Statistical differences were calculated with two-tailed student's t-test, *p < 0.05, **p < 0.01, and ***p < 0.001. Figure S1. The stability of the Ti 3 C 2 NSs in various buffer solutions, including 0.9%NaCl, PBS, DMEM, SGF, and SIF.