Nickel-Catcher-Doped Zwitterionic Hydrogel Coating on Nickel–Titanium Alloy Toward Capture and Detection of Nickel Ions

Nickel–titanium (NiTi) alloys show broad applicability in biomedical fields. However, the unexpected aggregation of bacteria and the corrosion of body fluid on NiTi-based medical devices often lead to the leakage of nickel ions, resulting in inevitable allergic and cytotoxic activities. Therefore, the capture and detection of nickel ions are important to avoid serious adverse reactions caused by NiTi-based medical devices. Herein, we presented a nickel ion capture strategy by the combination of zwitterionic hydrogels as anti-bacteria layers and carbon disulfide (CS2) components as nickel-catchers (Ni-catchers). On the one hand, the hydration layer of zwitterionic hydrogel can efficiently inhibit bacteria adhesion and reduce nickel ions leakage from NiTi corrosion. On the other hand, Ni-catchers can capture leaked nickel ions from NiTi alloy actively by chelation reaction. Therefore, this strategy shows great capabilities in resisting bacteria adhesion and capturing nickel ions, providing the potential possibility for the detection of nickel ion leakage for implantable biomedical materials and devices.


INTRODUCTION
With the rapid development of biomedicine, implantable materials have been widely used in diagnosis, treatment, and prosthesis (Sylvestre et al., 2020). As one of the most popular biomaterials, nickel-titanium (NiTi) alloys have been applied in orthodontic archwires , bone implants (Raphel et al., 2016), and vascular stents (Robertson and Ritchie, 2007), due to their unique superelasticity and good biocompatibility. However, the leakage of nickel ions (Ni 2+ ions) from NiTi alloys often leads to adverse reactions such as allergic signs (e.g., contact mucositis and eczematic and urticarial reactions; Huang et al., 2003;Kolokitha and Chatzistavrou, 2008) and cytotoxic responses (Jia et al., 1999). In addition, the unexpected adhesion of bacteria and acidic oral environment corrode NiTi alloys, aggravating the leakage of Ni 2+ ions (Huang et al., 2003). Therefore, a novel strategy for capturing and detecting leaked Ni 2+ ions is urgently needed for potential biomedical applications.
Herein, we present a Ni-catcher-doped zwitterionic hydrogel coating on the surface of NiTi alloy, showing robust anti-bacteria and long-term Ni 2+ ion capture (Figure 1). In brief, zwitterionic hydrogel was firstly prepared on the surface of NiTi alloy (H-NiTi) by the copolymerization of 2-hydroxyethyl methacrylate (HEMA), 3-dimethyl(methacryloyloxyethyl)ammonium propane-sulfonate (DMAPS), and methyl methacrylate (MMA; Su et al., 2020). Then, the CS 2 as a Ni-catcher was grafted into zwitterionic hydrogel by immersing H-NiTi in the mixture of CS 2 and triethylamine (TEA) to obtain CS-NiTi ( Figure 1A and Supplementary Figure 1). The zwitterionic hydrogel can prevent the adhesion of bacteria by the hydration layer on the surface of CS-NiTi. Moreover, the Ni-catcher endows this coating with Ni 2+ ion capture through chelation reaction. Therefore, this work provides a promising avenue to capture and detect Ni 2+ ions, which could be used for implantable biomedical materials and devices.

Preparation of CS-NiTi
The CS-NiTi was fabricated according to previous literature, with some modifications (Su et al., 2020). Firstly, pristine NiTi alloy was immersed in bis-HEMAP (5% ethanol solution) for 24 h and washed with deionized water for three times. Then, the 0.5 wt% of the photoinitiator DEAP was coated on the surface of NiTi alloy. The hydrogel precursor solution was obtained by mixing the crosslinker of PEGDMA (2 wt%) with the hydrogel monomers solution, including DMAPS (5 wt%), HEMA (40 wt%), and MMA (5 wt%). Then, it was degassed for 20 min by KQ5200DE ultrasonic cleaner (Kun Shan Ultrasonic Instruments Co. Ltd., Shanghai, China). The degassed precursor solution was poured on the surface of NiTi alloy and exposed to UV irradiation (wavelength ≈ 365 nm) in a dark chamber for 600 s by a UV LED curing system (Beijing NBET Technology Co. Ltd., Beijing, China), to obtain H-NiTi. To graft Ni-catchers on H-NiTi, H-NiTi was immersed into a mixture for 24 h containing CS 2 , TEA, and deionized water. LCS-NiTi, MCS-NiTi, and HCS-NiTi were obtained by regulating the concentration of CS 2 (0.2, 0.6, and 1.0 mol/L), respectively.

Characterizations
The morphology of pristine NiTi, H-NiTi, and CS-NiTi were observed with S-4800 scanning electron microscope (SEM; Hitachi, Tokyo, Japan) and Single Lens Reflex camera (SLR camera; Nikon D80, Tokyo, Japan). Water contact angles (WCAs) were measured by dropping 2 µl of water on the surface of pristine NiTi, H-NiTi, and CS-NiTi by the Dataphysics OCA 20 Contact-Angle System (Filderstadt, Germany). The S element analysis was performed on ESCALAB 250Xi X-ray photoelectron spectroscopy analysis (XPS; Thermo Fisher Scientific, Waltham, MA, United States). The transmittance was measured by UV-2600 UV-VIS spectrophotometer (SHIMADZU, Kyoto, Japan) with wavelength from 450 to 700 nm.

Cell Culture and Cellular Toxicity Test
Cell Counting Kit-8 (CCK-8) assay was performed to investigate the cell viability (Xiao et al., 2020). And the cytotoxicity of materials including pristine NiTi, H-NiTi, and CS-NiTi was evaluated with HepG2 cells. HepG2 cells with the density of 1 × 10 5 cells per well were seeded into the 24-well plates in DMEM containing 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin and streptomycin) and then cultured at 37 C in a humidified atmosphere with 5% CO 2 for 36 h. Pristine NiTi, H-NiTi, and CS-NiTi were added to each well, and the cells were incubated for 12 h. Subsequently, CCK-8 solutions were added to the wells followed by 2-h incubation at 37 • C. And then the supernatant was obtained and added to 96well plates. Finally, microplate reader was used to measure the absorbance values per well at a test wavelength of 450 nm and a reference wavelength of 690 nm to analyze the cell viability. Control experiments were done under the same condition with PBS. Eq. (1) was used to calculate the cell viability rate: where As represents the absorbance of the treatments of different groups and Ac represents the absorbance of the control treatments with PBS. All the experiments were performed in triplicate.

Bacteria Cultivation
Streptococcus mutans (UA159) was used as the model bacteria.
S. mutans was revived by incubating its freezing solution with 1 ml of BHI at 37 • C for 48 h. Then the suspension of S. mutans was centrifugated at 2,000 rpm for 10 min at room temperature to discard the supernatant. Then, the S. mutans aggregation was resuspended in another 1 ml of BHI and incubation at 37 • C for another 48 h. After the logarithmic phase was reached, the bacteria were harvested by centrifugation at 2,000 rpm for 10 min at room temperature and washed with Tris-HCl buffer three times. Subsequently, the bacteria were suspended in 50 ml of Tris-HCl buffer at a final concentration of 1 × 10 8 CFU/ml (Liu et al., 2018).

Anti-bacteria Property
To test anti-bacteria ability, the pristine NiTi, H-NiTi, and CS-NiTi were incubated in the as-prepared bacteria suspensions for 1, 3, 6, 12, 18, and 24 h at 37 • C. After the incubation, they were taken out and fixed in 2.5% glutaraldehyde for 1 h at room temperature, followed by immersing in a graded series of alcohol concentrations (30, 50, 70, 80, 90, and 100%) for 10 min to dehydrate the adhered bacteria. SEM was used to observe bacteria adhesion performance. The quantitative evaluation of the adhered bacteria was obtained by Cell Profiler 4.0.7. The antibacteria efficiency (E anti−bacteria ) could be calculated by Eq. (2): where D CS−NiTi and D NiTi refer to the bacteria density on the surface of CS-NiTi and pristine NiTi, respectively.

Performance of Ni 2+ Ion Capture
where C CS−NiTi and C H−NiTi refer to the concentration of Ni 2+ ions in the supernatant of remaining bacteria suspension incubated with CS-NiTi and H-NiTi, respectively.

Characterizations of CS-NiTi
To verify the introduction of Ni-catchers, we compared the pristine NiTi, H-NiTi, and CS-NiTi from surface morphology, WCAs, XPS spectra, transmittance, optical images, and biocompatibility. As shown in Figure 2A, the introduction of Ni-catchers resulted in the porous morphology of CS-NiTi, compared with the flat morphology of H-NiTi, probably because of the phase separation (Su et al., 2020). Furthermore, the WCAs decreased from 83.5 ± 3.7 • (NiTi) to 59.0 ± 3.4 • (CS-NiTi), indicating the hydrophilicity of CS-NiTi ( Figure 2B). The change of wettability might be attributed to the formation of hydrophilic xanthates in the presence of TEA (Weißl et al., 2018) and the porous morphology of CS-NiTi. More hydrophilic surface was beneficial to the formation of the hydration layer, enhancing the anti-bacteria adhesion ability of NiTi alloys (Lin et al., 2020). XPS was further employed to verify the presence of Ni-catchers in CS-NiTi. As shown in Figure 2C and Supplementary Figure 2, two S 2p peaks at 163 and 168 eV were observed in the XPS spectrum Frontiers in Bioengineering and Biotechnology | www.frontiersin.org of CS-NiTi, compared with the single S 2p peak at 168 eV for H-NiTi and no S 2p peak for NiTi. The S 2p peak at 168 eV shown in H-NiTi represented the S element in zwitterionic hydrogel network. The new S 2p peak at 163 eV indicated the successful introduction of Ni-catchers in CS-NiTi (Deng et al., 2013). Moreover, the appearance of zwitterionic hydrogels changed from colorless to yellow with lower transmittance after Ni-catcher (CS 2 ) graft ( Figure 2D), for xanthates always appear as a series of yellow compounds. The transmittance of Ni-catcher-doped zwitterionic hydrogel coating was higher than 60%, which has an insignificant impact on the appearance of NiTi alloys and would not affect the application of NiTi alloys in the implantable biomedical field. Furthermore, we tested the biocompatibility of CS-NiTi using CCK-8 test (Xiao et al., 2020). Higher HepG2 cell viability in Figure 2E indicated the excellent biocompatibility of CS-NiTi, showing promising applicability in biomedical field. Therefore, we successfully fabricated CS-NiTi with good biocompatibility.

Anti-bacteria Property of CS-NiTi
As a kind of widely used orthodontic material, NiTi alloys are often exposed to the oral environment. However, cleaning teeth is difficult during orthodontic treatment, leading to the adhesion of bacteria and causing caries and periodontitis. To investigate the anti-bacteria property in the oral environment, CS-NiTi was incubated in 1 × 10 8 CFU/ml bacteria suspension (S. mutans, the largest proportion of bacteria in oral environment) for different times, taking pristine NiTi and H-NiTi as controls. As shown in Figure 3A and Supplementary Figure 3, many bacteria adhered on the surface of pristine NiTi after being incubated for 1 h. In contrast, there were a few bacteria on the surfaces of H-NiTi and CS-NiTi, indicating their good anti-bacteria properties. By prolonging incubation time, a growing number of bacteria adhered on the surface of pristine NiTi and formed a biofilm at 12 h. In contrast, the bacteria density was still very low on the surfaces of H-NiTi and CS-NiTi. The quantitative analysis in Figure 3B revealed a good anti-bacteria efficiency in H-NiTi and CS-NiTi at 6 h, showing the highest efficiency of ca. 84% for H-NiTi and ca. 81% for CS-NiTi. By prolonging the incubation time to 24 h, the anti-bacteria efficiency maintained higher than 72% for H-NiTi and 66% for CS-NiTi. These results indicated that CS-NiTi had excellent long-term anti-bacteria property. Moreover, ICP was employed for exploring the performance of Ni 2+ ion capture by monitoring the concentration of Ni 2+ ions in bacteria suspension (1 × 10 8 CFU/ml). As shown in Figure 3C, the concentration of Ni 2+ ions in CS-NiTi was significantly lower than that of NiTi and H-NiTi. Even though it was incubated for 24 h, the concentration of Ni 2+ ions for CS-NiTi was still as low as 0.38 ± 0.07 mg/L, indicating the excellent ability of Ni 2+ ion capture of CS-NiTi. Therefore, CS-NiTi showed dual properties of anti-bacteria and Ni 2+ ion capture at the same time.

Mechanism of Ni 2+ Ion Capture
To further investigate the performance of Ni 2+ ion capture, a series of CS-NiTi were fabricated by regulating the CS 2 concentration in the mixed solution, that is, 0.2 mol/L of CS 2 for low concentration (denoted as LCS-NiTi), 0.6 mol/L of CS 2 for medium concentration (MCS-NiTi), and 1.0 mol/L of CS 2 for high concentration (HCS-NiTi). With the increasing concentration of Ni-catchers, the surface became more hydrophilic, revealing by the WCAs changing from 73.0 ± 2.8 • to 59.0 ± 3.4 • , along with the increased pore size (Supplementary  Figures 4A,B). Furthermore, the appearance of CS-hydrogel changed from colorless with high transparency to yellow with reduced transparency (Supplementary Figure 4C). To verify the long-term ability of Ni 2+ ion capture, we incubated H-NiTi, LCS-NiTi, MCS-NiTi, and HCS-NiTi with S. mutans suspensions for different days. After the incubation, the concentration of leaked Ni 2+ ions from NiTi alloy was reduced from 0.91 ± 0.01 mg/L (H-NiTi) to 0.65 ± 0.02 mg/L (HCS-NiTi) with the incubation time of 1 day (Figures 4A-D). By prolonging the incubation time to 5 days, the concentration of leaked Ni 2+ ions increased to 1.12 ± 0.03 mg/L for H-NiTi gradually. On the contrary, the concentration of leaked Ni 2+ ions in HCS-NiTi reduced from 0.65 ± 0.02 to 0.48 ± 0.07 mg/L dramatically, indicating its excellent long-term property of Ni 2+ ion capture. The possible mechanism of Ni 2+ ion capture is proposed in Figures 4E-H. Due to the corrosion of the adhered bacteria and acidic oral microenvironment, Ni 2+ ions are released slowly and persistently from NiTi alloy (Huang et al., 2003). Briefly, the leaked Ni 2+ ions can pass through the zwitterionic hydrogel coating on the surface of NiTi alloy and be released to oral environment easily (Figure 4E), as the diffusion of Ni 2+ ions is not efficiently restricted in single zwitterionic hydrogel. In contrast, the concentration of Ni 2+ ions is dramatically decreased after introducing Ni-catchers because grafted CS 2 can capture free Ni 2+ ions and restrain their diffusion to oral environment (Figures 4F-H). Moreover, the long-term capture efficiency of Ni 2+ ions is improved with the increasing concentration of Ni-catchers. Therefore, the introduction of Ni-catchers endows CS-NiTi with robust Ni 2+ ion capture property.

DISCUSSION
In summary, we demonstrate a long-term Ni 2+ ion capture coating on the surface of NiTi alloy. The zwitterionic hydrogels and Ni-catchers endow CS-NiTi with excellent anti-bacteria and Ni 2+ ion capture properties. The CS-NiTi exhibited long-term anti-bacteria efficiency of ca. 66% at 24 h and Ni 2+ ion capture efficiency of ca. 57% at 5 days. Furthermore, by integrating chelation reaction and introducing color change, this coating may apply to Ni 2+ ion capture and detection. Therefore, this coating provides a new clue to design the next generation of biomaterials and devices with capture and detection of Ni 2+ ions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
QN and JM conceived the idea. DH and XF contributed the fabrication process. XF and WX performed the experiments. XF and XL analyzed the results. XF, XL, QN, and JM wrote the manuscript. All authors contributed to the article and approved the submitted version.