Nitinol is a shape memory alloy made from nickel and titanium and has the ability to restore its original shape after deformation (Bhardwaj et al., 2019). Shape memory effects have been observed during austenite to martensite transformation, i.e., transformation from the face-centered cubic (FCC) structure to a body-centered tetragonal (BCT) structure (Parmar et al., 2021). Nitinol has gained popularity in the biomedical field, given its corrosion resistance, acceptable biocompatibility, super-elasticity, osseointegration, and fatigue and kink resistance properties (Hamann et al., 2020; Ulacia et al., 2020). The alloy has applications in the biomedical field as coronary stents, dental archwires, and actuators, although the concerns of nickel leaching limit the application (Fouladian et al., 2021).

To address the issue of possible nickel leaching from implants and reduce thrombogenicity, researchers have explored various surface treatments (passivation) viz. acid etching via HNO3, heat treatment, hydrogen peroxide treatment, mechanical rubbing, etc. (Norouzi and Nouri, 2021). It is believed that such surface treatments on nitinol result in changes in the surface composition of the alloy, where the percentage of nickel is reduced while that of Ti and TiO₂ becomes enhanced (Pérez et al., 2009; Norouzi and Nouri, 2021). Many reports in the literature have characterized the surface compositions of passivized nitinol and reported the presence of a titanium oxide layer (TiO₂), which can be explained in view of the favorable free energy for the formation of TiO₂. The presence of the oxide layer prevents corrosion of bulk materials and serves as a physicochemical barrier for Ni oxidation by modifying the oxidation pathways (Pulletikurthi et al., 2015). The effect of heat and electropolishing on the corrosion resistance in Hank’s physiological solutions was studied in different medical-grade titanium and nitinol. Ti6AI4V was found to be the most resistant to corrosion, while nitinol yielded the highest resistance to chemical breakdown (Chakraborty et al., 2019). In vitro and in vivo studies indicated that passivized nitinol exhibited good biocompatibility and does not promote toxic or genotoxic reactions when in contact with a physiological environment (Gill et al., 2015). Therefore, passivized nitinol can be considered a biologically safe implant material with unique mechanical properties. It is a well-characterized fact that material property and biological interaction drastically change between macro to the nanoscale, which emphasizes the detailed exploration of nanoparticles (NPs) of new materials (De Oliveira et al., 2017; Safranski et al., 2020). NPs for biomedical applications can be manufactured from both organic and inorganic materials. They have been explored for bio-sensing, purification of biomolecules, bioimaging, targeted delivery of biomolecules, anticancer agents, hypothermic agents, diagnostics, theranostics, and biosensing (McNamara and Tofail, 2017; Dembski et al., 2018; Pugazhendhi et al., 2018; Dahiya et al., 2021). All the applications of NPs have been made possible, thanks to the multi-dimensional properties, given its nanoscale size, high surface charge, tailorable surface properties, high agent loadings, controllable release patterns, and enhanced permeability and retention effect (Ealias and Saravanakumar, 2017; Loza et al., 2020; Zelepukin et al., 2021). Organic NPs such as liposomes, dendrimers, and micelles are less stable at high temperatures while inorganic NPs fabricated from metallic substrates (e.g., Al, Cd, Cu, Ti, Zn, Au, Ag) or metallic alloys (NiTi, FeNi, and FePt) or metallic oxides (Fe₂O₃ and Al₂O₃) are stable at high temperatures (Kashapov et al., 2021). Hence, inorganic NPs are more suitable for hypothermia-based cancer-killing and extendable to applications demanding a wide temperature range of operation. Inorganic NPs exhibit near-perpetual physical, chemical, and biological properties that have been exploited for industrial applications including additives, pigments, dyes, catalysts, biomaterials, and antimicrobial additives. (Jeevanandam et al., 2018; Cui et al., 2020). For the purpose of biomedical applications, it is important to prepare stable monodispersed solutions with NPs possessing less aggregation propensity (Phan and Haes, 2019). The exposure of NPs in a physiological environment leads to absorption of biological substances (proteins, lipids, peptides, nucleic acids, metabolites, and others) onto the surface of the NPs, leading to the formation of biocorona (Shannahan, 2017; Ulacia et al., 2020). The biocorona presents a unique challenge for the practical and safe biomedical application by affecting the toxicity, functionality, and characteristics of NPs and thereby altering thrombogenic properties as well (Chakraborty et al., 2019; Sena et al., 2020).

Even beyond the passivation aspect, researchers have raised some concerns about the safe handling of Ni-based NPs, compared to the bulk material. Crosera et al., 2016 also confirmed factors that affect the accumulation of Ni ions on the skin surface, especially in the case of NPs (Crosera et al., 2016). Ion et al. showed that surface nitriding through superficial treatment is an excellent method to enhance the biocompatibility of implantable devices (Ion et al., 2016). It was shown that passivized implants improved the interfacial bonding of human umbilical vein endothelial cells (HUVECs), cell spreading, and proliferation. Another group of researchers introduced a new one-step method, ultrasonic nanocrystal surface modification (UNSM), to fabricate hierarchical surface structures on the nitinol alloy to enhance the interaction between cells and implants and thus increase their biocompatibility (Hou et al., 2018; Osfouri and Rahmani, 2021). Anodization followed by chitosan coating of nitinol NPs was reported to result in improved biocompatibility and corrosion resistance (Mohammadi et al., 2019). Nitinol wire electropolishing before braiding enhanced in vitro behavior with lower activation of coagulation and a tendency toward reduced platelet adhesion (Cattaneo et al., 2019).

In this work, core-shell NPs of nitinol were prepared by nanosecond laser irradiation of differently passivized substrates. About 10 different surface treatments were employed in passivizing the nitinol targets for the fabrication of different NPs. The biocompatibility and cellular toxicity of these diversely prepared particles were assessed in detail by studying their effect on cellular pathways. The mechanism underlying the cellular toxicity of these nanoparticles was assessed through MTT and DCFDA assay (intracellular ROS) measurements while discussing the contributing alternative biological pathways. NPs fabricated using targets that were passivized using either chemical treatment (using HNO₃ and H₂O₂), anodization, or thermal heating were found to result in reduced nickel leaching in the media and improved biocompatibility. Given the obtained results and available literature studies, effective treatments for producing biocompatible NPs of nitinol are discussed along with its prospective application in the biomedical field.

Nitinol targets were ablated using a nanosecond laser at four different fluences (Table S1) in 1.2 ml of DI water. The size distribution profile suggested a more homogenous nanoparticle preparation at the average powers of 15 and 25 W in comparison with 5 and 40 W laser power, respectively (Figure 1A). Higher yields of ablated NPs were observed at 5 and 25 W laser power (Figure 1C). The prepared nitinol NPs showed a negative surface charge regime (Figure 1D) and characteristic phase peaks on an XRD analysis (Figure 1B). The resultant NPs were found to have a core-shell arrangement, as seen through HR-TEM imaging (Figure 2). The obtained characterizations on nitinol NP preparation suggested the use of 25-W laser power as the optimal operating power. The fabricated nanoparticles showed negligible aggregation propensity, as suggested by dynamic light scattering (DLS) measurement for 24 h (Supplementary Figure S2).

The effect of different passivation procedures (surface treatments) on enhancing the biocompatibility of nitinol NPs ablated from the substrates was studied. The study design for generating passivized NPs and to compare their effect on biocompatibility is summarized in Figure 3. Before either mechanical or chemical treatment, all the substrates were subjected to Kroll’s reagent, as described in the materials and methods section. This ensured the removal of the oxide layer on the substrates to minimize any variation between these. A total of 10 diverse NPs were studied from different surface treatments. FESEM-based imaging of the substrates post treatments showed different and distinct surface features (Figure 4A). Deep craters were observed post HNO₃ treatment with or without pre-polishing. Similarly, distinctly different surface cracks were found post H₂O₂ treatment with or without pre-polishing. A 3D topography map for all surfaces was observed by atomic force microscopy (AFM). The highest Z-axis (vertical) variation was observed post anodization of the samples (with or without pre-polishing) (Figure 4B). This observation was in line with the available literature on anodization, which suggests the growth of tubular TiO₂ structures on titanium and nitinol substrates during anodization [38]. Pre-polishing was found to reduce vertical anomalies, and the nitinol target subjected to pre-polishing yielded minimal z-axis variation.

Different nitinol substrates were subjected to ns laser ablation (25 W power), as described earlier for generating passivized NPs. The size distribution of these differently passivized NPs was then characterized with TEM imaging followed by ImageJ analysis, and the NPs ablated from HNO₃-etched surfaces (without pre-polishing) were found to have a comparatively narrow size distribution (20–60 nm) (Figure 5A). Narrow size distribution (20–60 nm) was also seen for particles ablated from PpH₂O₂ surfaces (Figure 5B). Interestingly, the particles ablated from PpHNO₃ surfaces showed wide size distribution.

NPs when used in biological applications can interrupt the typical structure and function of the tissue and organ and disrupt the normal physiology of the tissue. The particle size, shape, and composition are significant factors affecting toxicity (Canta and Cauda, 2020). Because of their small size, NPs permeate the cell membrane and spread to both the cardiovascular and lymphatic systems. The NPs accumulate in various tissues and interact with the cellular structure. The NPs also interact with proteins and may cause devastating consequences by creating protein corona on their surfaces, which causes deprivation of essential proteins and metabolites required for cell maintenance (Hoshyar et al., 2016; Curenton et al., 2021). Thus, it is important to understand the biocompatibility of NPs for a given application, by gaining insights into hemocompatibility, histocompatibility, and toxicity (Interactions et al., 2019). The size and polydispersity of nanoparticles also play an important factor in determining the cellular uptake and intracellular localization of nanoparticles, thereby associated toxicity pathways (Dahiya et al., 2019). Before extending any nanoparticles for putative biomedical application and related roles, it is imperative to understand the associated toxicity and the probable mechanistic alterations in the target tissues (Kyriakides et al., 2021).

The available literature on the biocompatibility and toxicity of nitinol portrays a well-divided opinion because of which its applicability and progress in the field are hampered (Sevost’yanov et al., 2018; Hamann et al., 2020). Hahn et al. reported higher cytotoxicity of nitinol NPs in comparison with other metal alloy NPs (Hahn et al., 2012; Puttaramaiah et al., 2021). To understand the bio-interaction and possible cytotoxicity, pathways related to nanoparticle-associated toxicity that can lead to cellular death and damage are identified and shown in Figure 6. To understand the mode of cellular toxicity of nitinol NPs and how surface passivation of these NPs impacts their cytocompatibility, cellular studies were performed with MG-63 osteoblast cells. The most challenging aspect of using nitinol in biological applications and as stents is the concern of nickel release. Characterization of nickel leaching from these passivized NPs was performed using the ICP-MS approach. The nitinol target was ablated for 30 min while being submerged in a thin layer (approximately 1 cm) of water above it, resulting in the formation of NPs in the solution. For an ICP-MS-based estimation of ion leaching, the prepared particles were harvested by centrifugation and resuspended in 1 ml of DMEM media (enriched with 10% FBS) followed by incubation for 24 h at 37°. The amount of leached ions was then measured in the supernatant.

Overall, nickel leaching was found to be more prominent and varying than titanium leaching across all sample types (Figure 7). A decade of extensive research on metallic NP-based cytotoxicity has suggested the involvement of multiple pathways including the classical extrinsic or intrinsic apoptotic pathway, an intrinsic mitochondrial pathway for ROS generation, or the simple mechanical disruption of the cell wall (Chakraborty et al., 2019).

As suggested by the ICP-MS data, polishing + hydrogen peroxide treatment resulted in the lowest nickel leaching among the tested samples. Mechanical polishing of the target was found to produce NPs with a slightly higher toxicity (7,300–8,344 ppb). As mechanical polishing was always the first treatment, it can be inferred from the ICP-MS data that heat and nitric acid treatments elevate nickel leaching, while hydrogen peroxide and anodization lead to lowering of nickel leaching. Even though the estimated values were not able to completely justify the corresponding cellular toxicity (largely owing to the involvement of other pathways), a significant correlation between the two can be observed (Table 2).

NPs ablated from the surface which is treated with HNO₃, H₂O₂, and anodization (with or without pre-polishing) were found to show very low intracellular ROS generation in the DCFDA assay. Yet another explanation of the observed discordance can be the presence of nanotubular structures (generated on anodization) on ablation, which can cause enhanced mechanical damage to the cell membrane and thus leading to toxicity. Both higher nickel leaching and intracellular ROS of heat-treated samples (with and without pre-polishing) justify the significant toxicity observed in these treatments.

We found that the cellular interaction of passivized and bare nanoparticles can be explained to a good extent with the help of ion leaching and intracellular toxicity. The possibility that these alloy NPs can also affect cellular proliferation through abstruse biological pathways persists (some of which have been discussed in Figure 6) and stresses the need for further studies. In essence, the present work showcases that the composition, ion leaching, and biocompatibility of nitinol NPs can be modulated by selective surface treatment prior to mechanical ablation.

Nitinol nanoparticles can be used in the biomedical field as pertaining to nano-actuators and in cell-specific applications; however, these particles are poorly explored in the domain of biological applications because of the associated concerns of cellular toxicity. On the contrary, bulk nitinol is widely used in medical implants and stents after surface modification (passivation). This work presents an attempt to prepare surface-modified nanoparticles of nitinol, by following a distinct approach of ablating the particles from passivized surfaces of nitinol. Cellular interaction studies on MG63 osteoblasts suggest intracellular ROS and nickel leaching as the major pathways of induced toxicity. The overall nanoparticles ablated from H₂O₂-treated nitinol targets were found to exhibit the highest biocompatibility with the cells, thus suggesting these treatments as effective in reducing nitinol nanoparticle toxicity. This study is a significant step toward preparing biocompatible nitinol for biomedical applications.