*Article* **Local and Systemic In Vivo Responses to Osseointegrative Titanium Nanotube Surfaces**

**Erin A. Baker 1,2,3,\* , Mackenzie M. Fleischer <sup>1</sup> , Alexander D. Vara <sup>1</sup> , Meagan R. Salisbury <sup>1</sup> , Kevin C. Baker 1,3 , Paul T. Fortin 1,3 and Craig R. Friedrich <sup>2</sup>**


**Abstract:** Orthopedic implants requiring osseointegration are often surface modified; however, implants may shed these coatings and generate wear debris leading to complications. Titanium nanotubes (TiNT), a new surface treatment, may promote osseointegration. In this study, in vitro (rat marrow-derived bone marrow cell attachment and morphology) and in vivo (rat model of intramedullary fixation) experiments characterized local and systemic responses of two TiNT surface morphologies, aligned and trabecular, via animal and remote organ weight, metal ion, hematologic, and nondecalcified histologic analyses. In vitro experiments showed total adherent cells on trabecular and aligned TiNT surfaces were greater than control at 30 min and 4 h, and cells were smaller in diameter and more eccentric. Control animals gained more weight, on average; however, no animals met the institutional trigger for weight loss. No hematologic parameters (complete blood count with differential) were significantly different for TiNT groups vs. control. Inductively coupled plasma mass spectrometry (ICP-MS) showed greater aluminum levels in the lungs of the trabecular TiNT group than in those of the controls. Histologic analysis demonstrated no inflammatory infiltrate, cytotoxic, or necrotic conditions in proximity of K-wires. There were significantly fewer eosinophils/basophils and neutrophils in the distal region of trabecular TiNT-implanted femora; and, in the midshaft of aligned TiNT-implanted femora, there were significantly fewer foreign body giant/multinucleated cells and neutrophils, indicating a decreased immune response in aligned TiNT-implanted femora compared to controls.

**Keywords:** orthopedic; nanomedicine; nanomodified surfaces; animal model; immune response

### **1. Introduction**

Titanium, both commercially pure and alloyed, has been used for decades in various biologic environments, including orthopedic implant designs [1–4]. With a combination of corrosion resistance, biocompatibility, mechanical properties approximating bone, and low cost, titanium continues to be a common material for fracture plates and screws as well as components requiring solid bone–implant fixation (e.g., knee arthroplasty tibial tray, hip arthroplasty femoral stem) [1–4]. To promote osseointegration, macroscale coatings have been applied to titanium implant surfaces via titanium plasma spray (TPS) as well as hydroxyapatite (HA) coating or sintering for powder- or bead-based coatings [5–8]. These coatings, however, are subjected to shear loads during surgical implantation, contact with surgical tools, and eventually micromotion at the bone–implant interface in vivo [5–8]. These coatings may then separate from the substrate, generating third-body wear debris

**Citation:** Baker, E.A.; Fleischer, M.M.; Vara, A.D.; Salisbury, M.R.; Baker, K.C.; Fortin, P.T.; Friedrich, C.R. Local and Systemic In Vivo Responses to Osseointegrative Titanium Nanotube Surfaces. *Nanomaterials* **2021**, *11*, 583. https:// doi.org/10.3390/nano11030583

Academic Editor: Angelo Ferraro

Received: 9 January 2021 Accepted: 15 February 2021 Published: 26 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

that increases mechanical wear of bearing surfaces. Additionally, local phagocytic cells encountering this debris may initiate a biologic cascade leading to periprosthetic osteolysis [9]. The body's immune response to wear debris is dependent on particulate composition, concentration, and morphology and may result in periprosthetic joint infection, bone fracture, catastrophic implant fracture, as well as osteoclastic bone resorption around the implant, component loosening, and, ultimately, revision surgery [5,10,11].

Titanium nanotube (TiNT) surfaces, which are electrochemically etched from the titanium implant substrate instead of applied as a coating, are an emerging technology that may enhance osseointegration of orthopedic implants [12–17], although literature is scarce regarding the in vivo immune response and toxicity to these nanostructured materials as implantable devices [18,19]. Due to improved interfacial strength, decreased thickness, and potential for more rapid osseointegration, TiNT surfaces may generate less third-body wear particulate compared to current coating technologies; however, thorough characterization of the immune and inflammatory response to new implantable materials is needed.

Two TiNT surface morphologies, termed aligned and trabecular, have been developed for orthopedic applications necessitating osseointegrative properties [17,20–22]. The aligned TiNT morphology comprises arrays of vertically oriented nanotubes, while the trabecular TiNT morphology exhibits a random surface resembling the three-dimensional porous network of trabecular bone. In this study, local and systemic responses to aligned and trabecular TiNT surfaces were assessed through in vitro cellular response on each material as well as in vivo performance in a clinically relevant, rodent model of long-term femoral intramedullary implant to simulate joint arthroplasty, including assessment of longitudinal animal weights, remote organ weights, metal ion levels in remote organs and whole blood, hematology, and nondecalcified histology [23,24]. Following on survivability testing and finite element modeling indicating that compression and shear loading to the TiNT surfaces were below yield failure strength, this study hypothesizes that TiNT surfaces will demonstrate equivalent local and systemic responses, equivalent to unmodified titanium alloy substrate surfaces [22]. Further, we hypothesize that aligned and trabecular TiNT surfaces yield comparable in vitro and in vivo results, as both morphologies provide additional surface area for cell attachment as well as resultant bony ongrowth and ingrowth.

### **2. Materials and Methods**

### *2.1. Implant Fabrication*

Samples for in vitro and in vivo studies were fabricated with titanium alloy (Ti-6Al-4V ELI) sheet and wire (wire: Custom Wire Technologies, Inc., Port Washington, WI, USA), respectively, using a well-established electrochemical anodization process (Appendix A— Supplemental Methods, Figure A1) [17,21]. Following the initial etching process, aligned TiNT surfaces were then ultrasonicated in deionized (DI) water for 2 minutes; however, trabecular TiNT surfaces received no additional treatment (Figure 1). Control samples were rinsed with DI water and air-dried. On average, inside diameter (ID) of individual nanotubes was 60 nm and 1 µm length on aligned TiNT surfaces. Similarly, trabecular TiNT surfaces contained a 1 µm layer of 60 nm ID aligned nanotubes, on average, covered with an additional 1 µm of over-etched nanotubes. The over-etched layer mimics the porous structure and topography of trabecular bone.

Bar: 1 μm.

**Figure 1.** Aligned titanium nanotubes (TiNT) (**A**) and trabecular TiNT (**B**) surfaces. (**A**,**B**) Scale **Figure 1.** Aligned titanium nanotubes (TiNT) (**A**) and trabecular TiNT (**B**) surfaces. (**A**,**B**) Scale Bar: 1 µm.

### *2.2. In Vitro Experimentation to Assess Cell Attachment and Morphology*

*2.2. In Vitro Experimentation to Assess Cell Attachment and Morphology*  Cells for all in vitro experiments were isolated from the intramedullary cavities of the femora of 14-week-old female Sprague Dawley rats (SD; Charles River Laboratories, Wilmington, MA, USA). Following isolation and culture using previously described methods, the plastic-adherent fraction of bone marrow cells (BMC) was collected and used for all subsequent experiments (Appendix A—Supplemental Methods, Figure A2) [17]. This cell population was selected for experimentation due to the potential of mesenchymal stem Cells for all in vitro experiments were isolated from the intramedullary cavities of the femora of 14-week-old female Sprague Dawley rats (SD; Charles River Laboratories, Wilmington, MA, USA). Following isolation and culture using previously described methods, the plastic-adherent fraction of bone marrow cells (BMC) was collected and used for all subsequent experiments (Appendix A—Supplemental Methods, Figure A2) [17]. This cell population was selected for experimentation due to the potential of mesenchymal stem cells (MSC) to differentiate toward numerous cell types, including osteoblasts.

cells (MSC) to differentiate toward numerous cell types, including osteoblasts. Early cell attachment and morphology were compared between aligned TiNT, trabecular TiNT, and control surfaces, using three samples per group per timepoint (coupons sectioned from sheet: 10 mm x 10 mm; timepoints: 30 min, 2 h, 4 h) [15,25]. To increase attachment potential, all sample coupons were soaked in fetal bovine serum (FBS) for 30 minutes prior to drop-seeding (density: 40,000 BMCs/coupon), followed by a 6 h incubation before adding the remaining volume of media and incubating overnight. At each timepoint, cells were fixed and stained with Actin Green (Actin Green 488 ReadyProbes Reagent, Life Technologies, Carlsbad, CA, USA) for cytoskeleton visualization and morphology (1 drop stain; 40 min incubation) as well as 4', 6-Diamidino-2-Phenylindole, dihydrochloride (DAPI; Life Technologies, Carlsbad, CA, USA) for nucleus visualization (0.5 mL stain; 20 min incubation). Following cell fixation, fluorescence imaging (IX71, Olympus America, Center Valley, PA, USA) was performed at thirteen standardized regions of interest to quantify the total number of adherent cells, cell equivalent diameter, and cell eccentricity. Because all non-adherent cells were removed prior to imaging, Actin Green and DAPI also demonstrated cell adhesion to the surfaces. Environmental scanning electron microscopy (SEM; Vega3XMU, Tescan USA, Warrendale, PA, USA) was used to further document cell morphology; specifically, cells were fixed in 4% glutaraldehyde on coupons, and then coupon was sputter-coated with a thin layer of gold–palladium alloy. Samples were imaged at 20.0 kV and a magnification of 300×. Total cell number, cell equivalent diameter, and cell eccentricity (measured using onboard microscope measurement Early cell attachment and morphology were compared between aligned TiNT, trabecular TiNT, and control surfaces, using three samples per group per timepoint (coupons sectioned from sheet: 10 mm × 10 mm; timepoints: 30 min, 2 h, 4 h) [15,25]. To increase attachment potential, all sample coupons were soaked in fetal bovine serum (FBS) for 30 min prior to drop-seeding (density: 40,000 BMCs/coupon), followed by a 6 h incubation before adding the remaining volume of media and incubating overnight. At each timepoint, cells were fixed and stained with Actin Green (Actin Green 488 ReadyProbes Reagent, Life Technologies, Carlsbad, CA, USA) for cytoskeleton visualization and morphology (1 drop stain; 40 min incubation) as well as 4', 6-Diamidino-2-Phenylindole, dihydrochloride (DAPI; Life Technologies, Carlsbad, CA, USA) for nucleus visualization (0.5 mL stain; 20 min incubation). Following cell fixation, fluorescence imaging (IX71, Olympus America, Center Valley, PA, USA) was performed at thirteen standardized regions of interest to quantify the total number of adherent cells, cell equivalent diameter, and cell eccentricity. Because all non-adherent cells were removed prior to imaging, Actin Green and DAPI also demonstrated cell adhesion to the surfaces. Environmental scanning electron microscopy (SEM; Vega3XMU, Tescan USA, Warrendale, PA, USA) was used to further document cell morphology; specifically, cells were fixed in 4% glutaraldehyde on coupons, and then coupon was sputter-coated with a thin layer of gold–palladium alloy. Samples were imaged at 20.0 kV and a magnification of 300×. Total cell number, cell equivalent diameter, and cell eccentricity (measured using onboard microscope measurement tool) were statistically compared using a one-way analysis of variance (ANOVA) model, with a Tukey post-hoc test and α = 0.05.

#### with a Tukey post-hoc test and α = 0.05. *2.3. In Vivo Experimentation to Assess Biologic Response to Nanotube Surfaces*

tool) were statistically compared using a one-way analysis of variance (ANOVA) model,

*2.3. In Vivo Experimentation to Assess Biologic Response to Nanotube Surfaces*  After preparing titanium alloy Kirschner wires (K-wire; Custom Wire Technologies, Inc., Port Washington, WI; USA, material: Ti6Al4V ELI Hard, diameter: 1.25 mm, single trocar tip for insertion), aligned TiNT-etched, trabecular TiNT-etched, or unetched titanium (control) K-wire implants (n = 6 per group) were inserted retrograde into the femoral intramedullary canals of SD rats for a single, long-term endpoint of 12 weeks [24,26,27]. Three naïve/nonoperative animals were housed for the same duration, in order to estab-After preparing titanium alloy Kirschner wires (K-wire; Custom Wire Technologies, Inc., Port Washington, WI; USA, material: Ti6Al4V ELI Hard, diameter: 1.25 mm, single trocar tip for insertion), aligned TiNT-etched, trabecular TiNT-etched, or unetched titanium (control) K-wire implants (n = 6 per group) were inserted retrograde into the femoral intramedullary canals of SD rats for a single, long-term endpoint of 12 weeks [24,26,27]. Three naïve/nonoperative animals were housed for the same duration, in order to establish baseline characteristics. Treatment groups were randomized just prior to surgery by nonoperative staff. Rats received daily veterinary care to identify complications and

lish baseline characteristics. Treatment groups were randomized just prior to surgery by

were weighed weekly; at endpoint, hematologic, metal ion, and histologic analyses were performed.

### *2.4. Surgical Procedure for Unilateral Intramedullary Implantation*

Under an Institutional Animal Care and Use Committee (IACUC)-approved protocol, 14-week-old, female SD rats underwent unilateral femoral implants via retrograde insertion. After anesthetization, rats were placed supine on a sterile, heated operating table with the knee in maximum flexion. Following final sterile preparation, a lateral–patellar skin incision allowed access to the distal femur. After drilling a shallow, pilot hole in the intercondylar groove, a K-wire was inserted into the intramedullary canal to the greater trochanter (proximal femur). Placement of the implant was confirmed via fluoroscopy. The K-wire was then clipped and recessed at the insertion site, followed by closure of arthrotomy and skin incisions with appropriate suture materials. Animals were then recovered and allowed ad libitum activity under endpoint.

### *2.5. Postoperative Care and Health Assessments*

All rats were evaluated daily for clinical signs of complications and overall health, including pain level, activity level, and food/water consumption. Rats were weighed preoperatively and weekly throughout the experiment. At endpoint, remote organs were collected and weighed, as another assessment of animal health. A one-way ANOVA model was used to compare longitudinal animal weights and endpoint organ weights between treatment groups, with α = 0.05.

### *2.6. Hematologic Analysis*

At endpoint, each anesthetized rat underwent antemortem cardiac puncture to collect blood for hematologic analyses (CBC; complete blood count with differential) to quantify the following thirteen parameters associated with either systemic inflammation/infection or anemia status: white blood cell count (WBC), lymphocyte concentration (Lymph), monocyte concentration (Mono), granulocyte concentration (Gran), hematocrit (HCT), mean cell volume of red blood cells (MCV), red blood cell distribution width (RDWa), hemoglobin concentration (Hgb), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), red blood cell count (RBC), total platelet count (PLT), and mean and platelet volume (MPV) (HemaTrue Hematology Analyzer, Heska, Loveland, CO, USA). Blood was collected in vacutainer tubes with Ethylenediaminetetraacetic acid (EDTA) and immediately processed. A one-way ANOVA model was used to compare hematologic parameters between treatment groups, with α = 0.05.

### *2.7. Metal Ion Analysis of Whole Blood and Remote Organs*

Inductively coupled plasma mass spectrometry (ICP-MS; HP 4500, Agilent Technologies, Santa Clara, CA, USA) was used to measure the titanium, aluminum, and vanadium concentrations in each remote organ and a whole blood sample from each animal (Appendix A—Supplemental Methods). ICP-MS calibration was performed using both ASTM standards of each target element as well as non-implanted samples of the rods from each testing condition. At endpoint, remote organs (i.e., spleen, liver, lungs, kidneys, brain) were harvested, weighed, and stored (4 ◦C in ultra-low leachable sample tubes) until analysis. Samples were analyzed in tandem with a laboratory reagent blank and duplicate laboratory fortified blanks to quantify background metal levels as well as process accuracy and precision, respectively. A one-way analysis of variance model was used to compare metal ion levels between treatment groups, with α = 0.05.

### *2.8. Nondecalcified Histologic Analysis*

At study endpoint, implanted femora were also harvested for nondecalcified histologic analysis. Femora from each animal were stripped of soft tissues and 10% zinc-buffered formalin for 96 h, followed by three rinses in phosphate buffered saline, and 70% ethanol

storage to prepare for histologic processing. Femora were subsequently embedded in methyl methacrylate, then ground and polished longitudinally to the approximate center of the implant, followed by cutting and applying one 5 µm thick section to a slide. Each slide was stained with Stevenel's Blue and van Gieson picrofuchsin (SBVG) for visualization of presence of immune-related cellular activity. SBVG is a widely-used stain to assess bone formation, due to visualization of both mature bone and osteoid as well as fibrous tissue, which may signify inferior osseointegration and/or increased inflammatory response, especially at the bone–implant interface [28–30]. Stained sections were manually scanned at a magnification of 20× to facilitate both high-magnification and whole-mount analyses (90i, Nikon Instruments, Inc., Melville, NY, USA). Three 20× regions of interest (ROI) per location (i.e., distal, midshaft, proximal) per section were captured. Within each ROI, five cell types were identified: foreign body giant (FBG)/multinucleated cell, granulocyte (nonneutrophilic, including eosinophils and basophils), neutrophil, monocyte, and lymphocyte. Cell constituents within each ROI were then graded on a scale of 0 to 2, with 0 = cells comprising 0–25% of the field, 1 = cells comprising 25–50% of field, and 2 = cells comprising greater than 50% of field. For FBG/multinucleated or granulocyte, a Grade 2 was defined as three or more cells per field. Grades were statistically compared between TiNT groups and control, using a Mann–Whitney rank sum test, with α = 0.05. ethanol storage to prepare for histologic processing. Femora were subsequently embedded in methyl methacrylate, then ground and polished longitudinally to the approximate center of the implant, followed by cutting and applying one 5 μm thick section to a slide. Each slide was stained with Stevenel's Blue and van Gieson picrofuchsin (SBVG) for visualization of presence of immune-related cellular activity. SBVG is a widely-used stain to assess bone formation, due to visualization of both mature bone and osteoid as well as fibrous tissue, which may signify inferior osseointegration and/or increased inflammatory response, especially at the bone–implant interface [28–30]. Stained sections were manually scanned at a magnification of 20× to facilitate both high-magnification and whole-mount analyses (90i, Nikon Instruments, Inc., Melville, NY, USA). Three 20× regions of interest (ROI) per location (i.e., distal, midshaft, proximal) per section were captured. Within each ROI, five cell types were identified: foreign body giant (FBG)/multinucleated cell, granulocyte (non-neutrophilic, including eosinophils and basophils), neutrophil, monocyte, and lymphocyte. Cell constituents within each ROI were then graded on a scale of 0 to 2, with 0 = cells comprising 0–25% of the field, 1 = cells comprising 25–50% of field, and 2 = cells comprising greater than 50% of field. For FBG/multinucleated or granulocyte, a Grade 2 was defined as three or more cells per field. Grades were statistically compared between TiNT groups and control, using a Mann–Whitney rank sum test, with α = 0.05.

At study endpoint, implanted femora were also harvested for nondecalcified histologic analysis. Femora from each animal were stripped of soft tissues and 10% zinc-buffered formalin for 96 h, followed by three rinses in phosphate buffered saline, and 70%

#### **3. Results 3. Results**

#### *3.1. In Vitro Experimentation to Assess Cell Attachment and Morphology 3.1. In Vitro Experimentation to Assess Cell Attachment and Morphology*

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 5 of 17

DAPI staining indicated that cells were viable and adhered on both the TiNT and control surfaces at the three early timepoints of 0.5, 2, and 4 h (Figure 2). The total number of adherent cells was significantly greater on the TiNT surfaces than on control surfaces, demonstrating more rapid cell attachment on both TiNT surfaces compared to control (Figure 3); specifically, total adherent cells on trabecular TiNT and aligned TiNT surfaces were significantly greater than control at 0.5 h (*p* = 0.014 and *p* = 0.018, respectively) and 4 h (*p* = 0.008 and *p* = 0.044). Over the 3.5-hour period, the number of cells increased on all surfaces, and the total number of adherent cells was equivalent or slightly greater on trabecular TiNT surfaces compared to aligned TiNT surfaces. Analysis of total cell count was unfeasible at later timepoints (3, 7, 14, 21 days) due to cell coalescence or superimposition. Actin Green staining showed active cell spreading on TiNT and control surfaces at all timepoints (Figure 2). DAPI staining indicated that cells were viable and adhered on both the TiNT and control surfaces at the three early timepoints of 0.5, 2, and 4 h (Figure 2). The total number of adherent cells was significantly greater on the TiNT surfaces than on control surfaces, demonstrating more rapid cell attachment on both TiNT surfaces compared to control (Figure 3); specifically, total adherent cells on trabecular TiNT and aligned TiNT surfaces were significantly greater than control at 0.5 h (*p* = 0.014 and *p* = 0.018, respectively) and 4 h (*p* = 0.008 and *p* = 0.044). Over the 3.5-hour period, the number of cells increased on all surfaces, and the total number of adherent cells was equivalent or slightly greater on trabecular TiNT surfaces compared to aligned TiNT surfaces. Analysis of total cell count was unfeasible at later timepoints (3, 7, 14, 21 days) due to cell coalescence or superimposition. Actin Green staining showed active cell spreading on TiNT and control surfaces at all timepoints (Figure 2).

**Figure 2.** Representative fluorescent images of dihydrochloride (DAPI)-stained (**A**) and Actin Green-stained (**B**) aligned TiNT, trabecular TiNT, and control surfaces at three early timepoints, 0.5 h, 2 h, and 4 h. **Figure 2.** Representative fluorescent images of dihydrochloride (DAPI)-stained (**A**) and Actin Green-stained (**B**) aligned TiNT, trabecular TiNT, and control surfaces at three early timepoints, 0.5 h, 2 h, and 4 h.

At the 2- and 4-h timepoints, increased spreading was observed on TiNT surfaces compared to control surfaces. Additional imaging via SEM showed differential cell mor-

phology patterns as a function of sample topography (Figure 4). On the TiNT surfaces, cells exhibited a globular shape, compared to the elongated, fibrillar cell morphology on control surfaces. control surfaces. Additional imaging via SEM showed differential cell morphology patterns as a function of sample topography (Figure 4). On the TiNT surfaces, cells exhibited a globular shape, compared to the elongated, fibrillar cell morphology on control surfaces.

At the 2- and 4-hour timepoints, increased spreading was observed on TiNT surfaces compared to

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 6 of 17

**Figure 3.** Average total cell number (**A**), cell equivalent diameter (**B**), and cell eccentricity (**C**) on TiNT and control surfaces at 0.5, 2, and 4 h. For Cell Number, significant comparisons were: aligned TiNT vs. control, *p* = 0.018; trabecular TiNT vs. control, *p* = 0.008; aligned TiNT vs. control, *p*= 0.044. For Cell Diameter, significant comparisons were: aligned TiNT vs. control, *p* = 0.048; trabecular TiNT vs. control, *p* = 0.031; aligned TiNT vs. control, *p* = 0.008; aligned TiNT vs. control, *p* = 0.003; trabecular TiNT vs. control, *p* = 0.003. For Cell Eccentricity, significant comparisons were: aligned TiNT vs. control, *p* = 0.004; trabecular TiNT vs. control, *p* = 0.007; aligned TiNT vs. control, *p* < 0.001; trabecular TiNT vs. control, *p* = 0.003. **Figure 3.** Average total cell number (**A**), cell equivalent diameter (**B**), and cell eccentricity (**C**) on TiNT and control surfaces at 0.5, 2, and 4 h. For Cell Number, significant comparisons were: aligned TiNT vs. control, *p* = 0.018; trabecular TiNT vs. control, *p* = 0.008; aligned TiNT vs. control, *p* = 0.044. For Cell Diameter, significant comparisons were: aligned TiNT vs. control, *p* = 0.048; trabecular TiNT vs. control, *p* = 0.031; aligned TiNT vs. control, *p* = 0.008; aligned TiNT vs. control, *p* = 0.003; trabecular TiNT vs. control, *p* = 0.003. For Cell Eccentricity, significant comparisons were: aligned TiNT vs. control, *p* = 0.004; trabecular TiNT vs. control, *p* = 0.007; aligned TiNT vs. control, *p* < 0.001; trabecular TiNT vs. control, *p* = 0.003.

Subsequent analysis of cell morphology images yielded cell equivalent diameter and eccentricity (Figure 3). Quantification of the cell equivalent diameter indicated that BMC on the TiNT surfaces were smaller (in diameter) than BMC on control surfaces. The discrepancy in cell diameter was significant between trabecular TiNT and aligned TiNT surfaces versus control at 0.5 h (*p* = 0.031 and *p* = 0.048, respectively). At the 2-hour timepoint, there was a significant difference in diameter between only the aligned TiNT and control surfaces (*p* = 0.008). At 4 h, the difference diameter was significant for between both trabecular TiNT and aligned TiNT, compared to control (both *p* = 0.003). Cells on the TiNT surfaces had significantly greater eccentricity than on the control surfaces, with cells on aligned TiNT demonstrating the greatest eccentricity (Figure 3). At 0.5 h, there was a significant difference in eccentricity between the trabecular TiNT and aligned TiNT, compared to control (*p* = 0.007 and *p* = 0.004, respectively). There was a significant difference between both the trabecular TiNT and aligned TiNT groups versus control again at 2 h (*p* = 0.003 and *p* < 0.001, respectively). There were no significant differences in eccentricity between groups at the 4-hour timepoint. Subsequent analysis of cell morphology images yielded cell equivalent diameter and eccentricity (Figure 3). Quantification of the cell equivalent diameter indicated that BMC on the TiNT surfaces were smaller (in diameter) than BMC on control surfaces. The discrepancy in cell diameter was significant between trabecular TiNT and aligned TiNT surfaces versus control at 0.5 h (*p* = 0.031 and *p* = 0.048, respectively). At the 2-hour timepoint, there was a significant difference in diameter between only the aligned TiNT and control surfaces (*p* = 0.008). At 4 h, the difference diameter was significant for between both trabecular TiNT and aligned TiNT, compared to control (both *p* = 0.003). Cells on the TiNT surfaces had significantly greater eccentricity than on the control surfaces, with cells on aligned TiNT demonstrating the greatest eccentricity (Figure 3). At 0.5 h, there was a significant difference in eccentricity between the trabecular TiNT and aligned TiNT, compared to control (*p* = 0.007 and *p* = 0.004, respectively). There was a significant difference between both the trabecular TiNT and aligned TiNT groups versus control again at 2 h (*p* = 0.003 and *p* < 0.001, respectively). There were no significant differences in eccentricity between groups at the 4-hour timepoint.

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 7 of 17

### *3.2. In Vivo Experimentation to Assess Biologic Response to Nanotube Surfaces*

*3.2. In Vivo Experimentation to Assess Biologic Response to Nanotube Surfaces*  Following the in vitro study, which demonstrated cell viability on the TiNT surfaces, the planned in vivo experiment was performed. Over the 12-week study, animals gained an average of 148 g, with control animals gaining the most weight on average (179 g) and trabecular TiNT animals gaining the least on average (118 g) (Figure 5). No animals lost more the 10% body weight, our institutional trigger for intervention. In week 9, the aligned TiNT group weighed significantly more than the trabecular TiNT group (*p* = 0.013), and in week 11, the control group weighed significant more than the trabecular TiNT group (*p* = 0.023). There were no significant differences at any other weekly time points. Mass of the spleen, brain, kidneys, lungs, and liver were obtained, and there were Following the in vitro study, which demonstrated cell viability on the TiNT surfaces, the planned in vivo experiment was performed. Over the 12-week study, animals gained an average of 148 g, with control animals gaining the most weight on average (179 g) and trabecular TiNT animals gaining the least on average (118 g) (Figure 5). No animals lost more the 10% body weight, our institutional trigger for intervention. In week 9, the aligned TiNT group weighed significantly more than the trabecular TiNT group (*p* = 0.013), and in week 11, the control group weighed significant more than the trabecular TiNT group (*p* = 0.023). There were no significant differences at any other weekly time points. Mass of the spleen, brain, kidneys, lungs, and liver were obtained, and there were no significant differences between any groups for the specimens (Figure 5).

no significant differences between any groups for the specimens (Figure 5). Hematologic analysis was performed, and no significant associations were found between the three groups for the thirteen parameters, and there were no significant differences between any groups for any parameters (Tables 1 and 2). Additionally, titanium, aluminum, and vanadium levels in each implant were analyzed to quantify the effect of the etching process on chemistry. Compared to control, aligned TiNT had 2.0 times the concentration of aluminum, approximately 1.8 times the titanium, and 2.9 times the vanadium; similarly, trabecular TiNT had 2.5 times the aluminum, 2.2 times the titanium, and Hematologic analysis was performed, and no significant associations were found between the three groups for the thirteen parameters, and there were no significant differences between any groups for any parameters (Tables 1 and 2). Additionally, titanium, aluminum, and vanadium levels in each implant were analyzed to quantify the effect of the etching process on chemistry. Compared to control, aligned TiNT had 2.0 times the concentration of aluminum, approximately 1.8 times the titanium, and 2.9 times the vanadium; similarly, trabecular TiNT had 2.5 times the aluminum, 2.2 times the titanium, and 3.4 times the vanadium.

3.4 times the vanadium.

**Figure 5.** Average animal body weight over 12-week study (**A**) and organ weight at endpoint (**B**) per group. For Week 9, significant comparison: aligned TiNT vs. trabecular TiNT, *p* = 0.013; For Week 11, significant comparison: trabecular TiNT vs. control, *p* = 0.023. **Figure 5.** Average animal body weight over 12-week study (**A**) and organ weight at endpoint (**B**) per group. For Week 9, significant comparison: aligned TiNT vs. trabecular TiNT, *p* = 0.013; For Week 11, significant comparison: trabecular TiNT vs. control, *p* = 0.023.


**Table 1.** Hematologic analysis of white blood cell function of each treatment group at endpoint.\* **Table 1.** Hematologic analysis of white blood cell function of each treatment group at endpoint \*.

\* Standard deviation listed in parentheses. \* Standard deviation listed in parentheses.

**Table 2.** Hematologic analysis of red blood cell function of each treatment group at endpoint.\* **Table 2.** Hematologic analysis of red blood cell function of each treatment group at endpoint \*.


\* Standard deviation listed in parentheses. \* Standard deviation listed in parentheses.

After weighing each organ, metal concentrations were assessed (Figure 6). Aluminum levels in the lungs were significantly greater in the trabecular TiNT group compared to control (*p* = 0.022). No other organs exhibited significantly increased titanium, aluminum, or vanadium levels compared to control. Histologic analysis demonstrated a lack of inflammatory infiltrate in proximity to the nails within the intramedullary space, for all groups (Table 3; Appendix A—Supplemental Results, Table A1). There were significantly fewer granulocytes and neutrophils in the distal ROI of the femora implanted with trabecular TiNT-etched implants (*p* = 0.040 and *p* = 0.019, respectively). In the midshaft ROI, there were significantly fewer foreign body giant/multinucleated cells and neutrophils in After weighing each organ, metal concentrations were assessed (Figure 6). Aluminum levels in the lungs were significantly greater in the trabecular TiNT group compared to control (*p* = 0.022). No other organs exhibited significantly increased titanium, aluminum, or vanadium levels compared to control. Histologic analysis demonstrated a lack of inflammatory infiltrate in proximity to the nails within the intramedullary space, for all groups (Table 3; Appendix A—Supplemental Results, Table A1). There were significantly fewer granulocytes and neutrophils in the distal ROI of the femora implanted with trabecular TiNT-etched implants (*p* = 0.040 and *p* = 0.019, respectively). In the midshaft ROI, there were significantly fewer foreign body giant/multinucleated cells and neutrophils in the

the aligned TiNT group (*p* = 0.039 and *p* = 0.019, respectively). There were no observed

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 9 of 17

**Figure 6.** Concentration of aluminum (**A**), titanium (**B**), and vanadium (**C**) in remote organs and whole blood for each group. (WB = whole blood). For aluminum concentration, significant comparison: trabecular TiNT vs. control, *p* = 0.022. **Figure 6.** Concentration of aluminum (**A**), titanium (**B**), and vanadium (**C**) in remote organs and whole blood for each group. (WB = whole blood). For aluminum concentration, significant comparison: trabecular TiNT vs. control, *p* = 0.022.


**Table 3.** Statistical comparisons (*p*-values) between implant groups with control of average histologic grade for three regions of interest \*. **Table 3.** Statistical comparisons (*p*-values) between implant groups with control of average histologic grade for three regions of interest \*.

\* Bolded *p*-values indicate significant results; ROI = region of interest.

**Figure 7.** Longitudinal histologic sections and regions of interest of representative femora implanted with aligned TiNT (**A**), trabecular TiNT (**B**), and (**C**) control K-wires. **Figure 7.** Longitudinal histologic sections and regions of interest of representative femora implanted with aligned TiNT (**A**), trabecular TiNT (**B**), and (**C**) control K-wires.

### **4. Discussion**

**4. Discussion**  Local and systemic responses to aligned and trabecular TiNT surfaces were evaluated via an in vitro study focused on cell morphological and attachment behavior, followed by a clinically relevant in vivo study of biologic response to implant materials. The in vivo study included multiple characterization methods, including a general health as-Local and systemic responses to aligned and trabecular TiNT surfaces were evaluated via an in vitro study focused on cell morphological and attachment behavior, followed by a clinically relevant in vivo study of biologic response to implant materials. The in vivo study included multiple characterization methods, including a general health assessment (e.g., body weight) as well as hematologic, metal ion, and histologic analyses.

sessment (e.g., body weight) as well as hematologic, metal ion, and histologic analyses. DAPI and Actin Green staining demonstrated cell attachment and spreading from 0.5 h through 21 days on all surfaces, an indication of the suitable environment of both TiNT surface es and control. Total cell counts were greater on TiNT surfaces compared to unetched controls at the three early timepoints, which corresponds with current reports of more rapid cell attachment on nanotube surfaces [31–33]. Additionally, the diameter of BMC on TiNT surfaces was smaller compared to cells seeded onto controls, and cells on TiNT surfaces were more eccentric than cells on controls. These data correspond with SEM imaging of attached BMC, which showed a rounded cell morphology on TiNT surfaces and fibrillar-shaped cells on control surfaces. The diameter, eccentricity, and imaging findings may be indicative of cell development in different planes on the TiNT surfaces compared to controls. Shokuhfar et al. sectioned TiNT arrays seeded with osteoblasts (MC3T3-E1; mouse osteoblasts) and observed cell attachment on nanotube arrays as well as cell filopodia stretching downward into the hollow portion of individual nanotubes;[20] therefore, differences in cell morphologic behavior between groups may be explained by the BMC interaction with TiNT surfaces, as the BMC extend into nanotubes and voids within the array, instead of across the top of the surface. Rangamani et al. performed modeling of cell shapes and determined that greater cell eccentricity was associated with enhanced signal modulation due to temporary collections of activated receptors in sections of greater curvature of cells, specifically in growth factor receptor pathways; therefore, DAPI and Actin Green staining demonstrated cell attachment and spreading from 0.5 h through 21 days on all surfaces, an indication of the suitable environment of both TiNT surfaces and control. Total cell counts were greater on TiNT surfaces compared to unetched controls at the three early timepoints, which corresponds with current reports of more rapid cell attachment on nanotube surfaces [31–33]. Additionally, the diameter of BMC on TiNT surfaces was smaller compared to cells seeded onto controls, and cells on TiNT surfaces were more eccentric than cells on controls. These data correspond with SEM imaging of attached BMC, which showed a rounded cell morphology on TiNT surfaces and fibrillar-shaped cells on control surfaces. The diameter, eccentricity, and imaging findings may be indicative of cell development in different planes on the TiNT surfaces compared to controls. Shokuhfar et al. sectioned TiNT arrays seeded with osteoblasts (MC3T3-E1; mouse osteoblasts) and observed cell attachment on nanotube arrays as well as cell filopodia stretching downward into the hollow portion of individual nanotubes [20]; therefore, differences in cell morphologic behavior between groups may be explained by the BMC interaction with TiNT surfaces, as the BMC extend into nanotubes and voids within the array, instead of across the top of the surface. Rangamani et al. performed modeling of cell shapes and determined that greater cell eccentricity was associated with enhanced signal modulation due to temporary collections of activated receptors in sections of greater curvature of cells, specifically in growth factor receptor pathways; therefore, greater cell eccentricity on nanotube surfaces may demonstrate amplified signals resulting in downstream effects, such as bone formation [34].

In the in vivo study, there were several significant differences in body weight between groups at two weeks during the experiment; however, the body weights recovered were not an established trend, and no interventions were required at any time for weight loss. At endpoint, remote organ masses were compared between groups, and these also showed no significant difference. Despite increased concentrations of aluminum, titanium, and vanadium in the aligned TiNT and trabecular TiNT wires versus control wires, only aluminum levels were significantly increased in the lungs of the rats implanted with trabecular TiNT-etched wires compared to controls. No other organs or whole blood samples showed significantly elevated metal levels, when the experimental groups were compared to control. A study of intraarticular injection of TiO<sup>2</sup> nanoparticles (45 nm average diameter; 0.2, 2, 20 mg/kg TiO<sup>2</sup> in suspension) into rat knee joints showed nanoparticle migration to remote organs as well as pathologic changes in the heart, lung, liver, and knee at 7 days post-injection [35,36]. Although our study showed metal ion concentration in remote organs via ICP-MS and did not histologically assess remote organs, we theorize that the amount of TiO<sup>2</sup> particulate debris shed in the rat femora was likely less than 0.2 mg/kg. At study endpoint, no hematologic markers of white and red blood cell function, which would signal systemic inflammation-, infection-, or anemia-related complications, were significantly elevated in the experimental groups compared to control. Histologic analysis showed several significant differences in cell populations between the TiNT groups and control, with less foreign body giant/multinucleated, eosinophil/basophil, and neutrophil cell activity in TiNT-implanted femora than control. There were no significant differences between monocyte and lymphocyte activity.

In in vitro and in vivo studies of vascular toxicity, Bayat et al. established that titanium nanotubes with 30 nm diameter as well as ultra-small TiO<sup>2</sup> nanoparticles (1−3 nm) were not cytotoxic, and nanoparticles did not possess oxidative potential [37]. In vivo studies in rat models have also shown that TiO<sup>2</sup> nanoparticles are not genotoxic [38,39]. Neacsu et al. showed that after seeding murine macrophages onto TiNT and unetched titanium surfaces under pro-inflammatory and standard conditions, inflammatory activity related to cytokine and chemokine gene expression, foreign body giant cell products, and nitric oxide release all decreased on TiNT surfaces but not on unetched controls; therefore, the authors suggested that the TiNT surfaces may regulate macrophage response, thereby diminishing the overall inflammatory cascade [40]. Studies by Ostberg et al. and Latha et al., using models of leukocyte-seeded TiNT surface and mixed lymphocyte reaction (MLR), respectively, concurred with these findings [41,42]; specifically, Latha et al. showed a 30–35% suppression of splenocyte proliferation of titanium nanotubes (H2Ti3O7) [42]. Assessing hemocompatbility, Smith et al. showed increased adsorption of blood serum protein, platelet adhesion and activation, and clotting of whole blood as well as no evidence of monocyte activation and cytokine secretion on TiNT surfaces versus control [43]. Radizun et al. showed that aluminum nanoparticles, in concentrations up to 400 µg/mL, have no significant toxic effect on mammalian cell viability, and Alshatwi et al. found human MSC cytotoxicity via Al2O<sup>3</sup> nanoparticles at 40 µg/mL; in this study, aluminum content in all specimens was substantially below this threshold (maximum aluminum content within range of standard deviation; Figure 6: 4.24 µg/mL) [44,45]. In a clinical, in vivo study, Swiatkowska et al. described titanium levels ranging between 2.20 and 2.56 µg/L in blood and plasma, measured via ICP-MS, in well-functioning unilateral hip implants, indicating some level of titanium may be tolerable by patients, without eliciting a whole-body response [46].

Study limitations included difficulties with staining TiNT surfaces, especially at timepoints greater than 4 h. Because of the nanotopography and voids, which were ideal for cell attachment, stain penetration of the cells was complicated and incomplete. While three naïve animals were included to assess background metal levels, inclusion of additional animals may have been beneficial to further study and quantify these levels. Additionally, a full assessment of metal levels of animal food, bedding, enrichment materials, etc. may have allowed further reduction and elimination of background aluminum, titanium,

and vanadium levels in all animals [47,48]. Based on the results of this study, a full systematic characterization of this material's biocompatibility and toxicity properties should be conducted in accordance with applicable standards, such as ISO 10993-11 (Biological Evaluation of Medical Devices—Part 11: Tests for Systemic Toxicity).

### **5. Conclusions**

Previous studies have demonstrated the biocompatibility of TiNT surfaces and correspond with the findings of this study showing TiNT surfaces support cell attachment and proliferation and do not initiate systemic effects in an in vivo model of intramedullary implantation. The presented in vitro data demonstrated that cells cultured on bare TI6Al4V surfaces were spindle shaped, while those cultured on TiNT surfaces demonstrated a more rounded appearance, which may confer benefit with respect to in vivo bone formation. In vivo data demonstrated that the accumulation of metal ions in filtering organs was largely similar between the two morphologies of TiNT surfaces and control bare Ti6Al4V surfaces, aside from an increased aluminum concentration in the lungs of rats with aligned TiNT implants. This increased aluminum concentration in lung specimens was not associated with alterations in overall health of the animal or observed pathology. As such, it can be concluded that at the time points studied, metal accumulation associated with TiNT surfaces was similar to control surfaces, and no systemic or local complications were observed in TiNT- or control-implanted animals.

**Author Contributions:** Conceptualization, E.A.B., K.C.B., P.T.F. and C.R.F.; methodology, E.A.B., M.M.F., A.D.V., K.C.B., M.R.S., P.T.F. and C.R.F.; formal analysis, E.A.B., K.C.B., A.D.V. and M.R.S.; investigation, E.A.B., M.M.F., A.D.V., M.R.S., K.C.B., P.T.F. and C.R.F.; resources, E.A.B., K.C.B., P.T.F. and C.R.F.; writing—original draft preparation, E.A.B. and M.R.S.; writing—review and editing, E.A.B., M.M.F., A.D.V., K.C.B., P.T.F. and C.R.F.; supervision, P.T.F. and C.R.F.; project administration, E.A.B. and K.C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to institutional policies.

**Conflicts of Interest:** The authors declare no conflict of interest specific to this work.

### **Appendix A. Supplemental Methods and Results**

### *Appendix A.1. Titanium Nanotube-Etched Specimen Fabrication*

Titanium nanotube-etched specimens were prepared for in vitro and in vivo experiments, based on previous methods developed and tested by our group [17,20–22]. Titanium alloy (Ti-6Al-4V ELI) sheet (in vitro experiments) or wire (in vivo study) were polished with 600 grit abrasive sheet and deionized (DI) water, then rinsed in DI water and air-dried. All sample materials were cleaned with acetone just prior to etching, followed by air drying. On wire samples, the trocar tip was coated to prevent nanotube formation on the site of insertion. To etch, samples (sheet or wire) were suspended vertically toward one side of a glass beaker and then connected as the anode to a variable DC power supply. Additionally, in the beaker, a small diameter graphite rod was suspended, diametrically opposed to the sample, and connected to ground (cathode). An electrolyte solution (98 vol.% ethylene glycol, 2 vol.% deionized water, and 0.6 wt.% NH4F) was added to the beaker, with a final volume just below the electrical connections to the sample and graphite. Before adding NH4F to the electrolyte solution, it was first completely dissolved in DI water; following this addition, the electrolyte solution was mixed to ensure all constituents were fully combined. Then, the power supply was engaged (+60 VDC) and etching was allowed to proceed for 40 minutes. After etching, the power supply was disengaged and sample(s) were immediately removed from the beaker, followed by a 1-minute rinse under DI water and air drying. After drying, the coating on each trocar tip was removed after etching and sonication (aligned TiNT samples were sonicated for 2 min; trabecular TiNT samples

were not sonicated). Sheet samples were then sectioned into coupons (10 mm × 10 mm); wires were net-shape and did not require sectioning. To convert the amorphous titania oxide layer to the crystalline anatase phase, which increases the hydrophilicity of the TiNT surfaces, samples were placed in a programmable annealing oven. The temperature was increased by 7.5 ◦C per minute to a steady state of 450◦C for a total heating time of three hours. Samples were removed after the oven was completely cooled, which was approximately 5 h. Specimens were stored in cushioned, sealed storage cases until use (Figure A1). 2 min; trabecular TiNT samples were not sonicated). Sheet samples were then sectioned into coupons (10 mm × 10 mm); wires were net-shape and did not require sectioning. To convert the amorphous titania oxide layer to the crystalline anatase phase, which increases the hydrophilicity of the TiNT surfaces, samples were placed in a programmable annealing oven. The temperature was increased by 7.5 °C per minute to a steady state of 450°C for a total heating time of three hours. Samples were removed after the oven was completely cooled, which was approximately 5 h. Specimens

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 13 of 17

were stored in cushioned, sealed storage cases until use (Figure A1).

**Figure A1.** Digital photographs showing the final appearance of 10 mm × 10 mm coupon samples (top) and Kirschner wires (bottom). TiNT-etched surfaces appear golden and smooth, compared to unmodified control surfaces. **Figure A1.** Digital photographs showing the final appearance of 10 mm × 10 mm coupon samples (top) and Kirschner wires (bottom). TiNT-etched surfaces appear golden and smooth, compared to unmodified control surfaces.

#### *Appendix A.2. In Vitro Experimentation to Assess Adherent Cell Count, Cell Equivalent Diameter, and Cell Eccentricity Appendix A.2. In Vitro Experimentation to Assess Adherent Cell Count, Cell Equivalent Diameter, and Cell Eccentricity*

Rat bone marrow cells (BMCs) were used for all in vitro experimentation, with cell harvest and preparation conducted based on previously developed laboratory protocols by our group [49]. To obtain BMCs, rats were euthanized via CO2 asphyxiation, and then each femur and tibia were aseptically harvested. After removing both the proximal and distal ends of each bone, marrow cavities were flushed with warm, sterile phosphatebuffered saline. Whole bone marrow was plated (T-25 culture flasks) and cultured (37oC and 5% CO2) in a sterile, copper-lined CO2 incubator. Non-adherent cells were removed after 24 h of incubation via thorough rinsing with warm, sterile saline. Then, the plasticadherent fraction of bone marrow cells (BMCs), an enriched source of mesenchymal stem cells (MSC) capable of differentiating toward numerous cell types, was obtained. Rat bone marrow cells (BMCs) were used for all in vitro experimentation, with cell harvest and preparation conducted based on previously developed laboratory protocols by our group [49]. To obtain BMCs, rats were euthanized via CO2 asphyxiation, and then each femur and tibia were aseptically harvested. After removing both the proximal and distal ends of each bone, marrow cavities were flushed with warm, sterile phosphate-buffered saline. Whole bone marrow was plated (T-25 culture flasks) and cultured (37 ◦C and 5% CO2) in a sterile, copper-lined CO2 incubator. Non-adherent cells were removed after 24 h of incubation via thorough rinsing with warm, sterile saline. Then, the plastic-adherent fraction of bone marrow cells (BMCs), an enriched source of mesenchymal stem cells (MSC) capable of differentiating toward numerous cell types, was obtained.

For in vitro experimentation, sample coupons were placed into individual wells of 12-well polystyrene culture plates and soaked in fetal bovine serum (FBS) for 30 min prior to seeding to increase attachment. Rat BMCs (P2-3; density = 40,000 cells per coupon; suspended in 50 μL of media) were drop-seeded on each sample and incubated (37 °C and 5% CO2) for 6 h before adding the remaining volume of media (DMEM; Dulbecco's Modified Eagle Medium; supplementation = 10% fetal bovine serum, and 1% penicillin-streptomycin). Incubation (same conditions) continued for an additional 20 h to ensure attachment. Following incubation, fluorescence imaging at 13 standardized regions of interest per coupon was performed, approximately 70% of the specimen surface, followed by subsequent quantification of (Figure A2). For in vitro experimentation, sample coupons were placed into individual wells of 12-well polystyrene culture plates and soaked in fetal bovine serum (FBS) for 30 min prior to seeding to increase attachment. Rat BMCs (P2-3; density = 40,000 cells per coupon; suspended in 50 µL of media) were drop-seeded on each sample and incubated (37 ◦C and 5% CO2) for 6 h before adding the remaining volume of media (DMEM; Dulbecco's Modified Eagle Medium; supplementation = 10% fetal bovine serum, and 1% penicillinstreptomycin). Incubation (same conditions) continued for an additional 20 h to ensure attachment. Following incubation, fluorescence imaging at 13 standardized regions of interest per coupon was performed, approximately 70% of the specimen surface, followed by subsequent quantification of (Figure A2).

**Figure A2.** Imaging convention for each coupon in the experiment. Regions were chosen to reflect the corners (1–4), edges (5–8), central area (9–12), and absolute center (13) of each sample. **Figure A2.** Imaging convention for each coupon in the experiment. Regions were chosen to reflect the corners (1–4), edges (5–8), central area (9–12), and absolute center (13) of each sample.

#### *Appendix A.3. Metal Ion Analysis of Remote Organs and Whole Blood Appendix A.3. Metal Ion Analysis of Remote Organs and Whole Blood*

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 14 of 17

Inductively coupled plasma mass spectrometry (ICP-MS; HP 4500, Agilent Technologies, Santa Clara, CA) was performed to quantify aluminum, titanium, and vanadium content in whole-blood and remote organ samples. Sample tubes, made of ultra-low leachable material to avoid metal contamination, were labeled and weighed to three significant figures before sample collection. At endpoint, whole blood (≥2 mL) was collected, before proceeding with dissection of remote organs (i.e., spleen, liver, lungs, kidneys, brain). Organs were weighed before being placed into tubes, and each filled sample tube was weighed again to determine the actual/net weight of the collected specimen. Inductively coupled plasma mass spectrometry (ICP-MS; HP 4500, Agilent Technologies, Santa Clara, CA) was performed to quantify aluminum, titanium, and vanadium content in whole-blood and remote organ samples. Sample tubes, made of ultra-low leachable material to avoid metal contamination, were labeled and weighed to three significant figures before sample collection. At endpoint, whole blood (≥2 mL) was collected, before proceeding with dissection of remote organs (i.e., spleen, liver, lungs, kidneys, brain). Organs were weighed before being placed into tubes, and each filled sample tube was weighed again to determine the actual/net weight of the collected specimen.

All samples were transported and held on ice (~4 °C) until analysis. Samples were digested to dissolve the metals into an aqueous solution by placing a combination of highly pure solutions of nitric acid, hydrochloric acid, hydrogen peroxide, and water into the tubes. The tubes were heated at 95 °C for 2 h per cycle. Some of the samples went through multiple digestion cycles for thorough digestion. For ICP-MS analysis, samples were included in analytical batches of 20 samples, which includes a laboratory reagent blank (LRB) to establish a reference to the laboratory background value, a laboratory fortified blank (LFB) to measure accuracy of the analytical procedure, and a duplicate LFB to measure the precision of the analytical procedure. The final volume of digestion was set to 40 mL per specimen digested, and the digested solutions were further diluted by 10 times and analyzed for aluminum, titanium, and vanadium contents using a standard operating procedure (UL SOP# 114) based on the EPA guidelines (Method 200.8). All samples were transported and held on ice (~4 ◦C) until analysis. Samples were digested to dissolve the metals into an aqueous solution by placing a combination of highly pure solutions of nitric acid, hydrochloric acid, hydrogen peroxide, and water into the tubes. The tubes were heated at 95 ◦C for 2 h per cycle. Some of the samples went through multiple digestion cycles for thorough digestion. For ICP-MS analysis, samples were included in analytical batches of 20 samples, which includes a laboratory reagent blank (LRB) to establish a reference to the laboratory background value, a laboratory fortified blank (LFB) to measure accuracy of the analytical procedure, and a duplicate LFB to measure the precision of the analytical procedure. The final volume of digestion was set to 40 mL per specimen digested, and the digested solutions were further diluted by 10 times and analyzed for aluminum, titanium, and vanadium contents using a standard operating procedure (UL SOP# 114) based on the EPA guidelines (Method 200.8).

The total weight of each metal per specimen and concentration of each metal in a specimen were recorded; weights and concentrations were reported to three significant figures. Accuracy and precision data as part of the quality control carried through the analytical batches were also documented. Chain of custody forms containing sample descriptions, sampling dates, etc. were completed and retained. The total weight of each metal per specimen and concentration of each metal in a specimen were recorded; weights and concentrations were reported to three significant figures. Accuracy and precision data as part of the quality control carried through the analytical batches were also documented. Chain of custody forms containing sample descriptions, sampling dates, etc. were completed and retained.

#### *Appendix A.4. Histologic Analysis of Bone-Implant Interface Appendix A.4. Histologic Analysis of Bone-Implant Interface*

Average cell count data of five cell types (foreign body giant/multinucleated, granulocyte, neutrophil, monocyte, lymphocyte) for the three implant groups (trabecular TiNT, aligned TiNT, control) in the three regions of interest (ROI; distal, midshaft, and proximal femora) have been provided (Table A1). Average cell count data of five cell types (foreign body giant/multinucleated, granulocyte, neutrophil, monocyte, lymphocyte) for the three implant groups (trabecular TiNT, aligned TiNT, control) in the three regions of interest (ROI; distal, midshaft, and proximal femora) have been provided (Table A1).


**Table A1.** Average histologic grade for three regions of interest in each treatment group \*.

\* Standard deviation listed in parentheses.

### **References**


### *Article* **Potential Production of Theranostic Boron Nitride Nanotubes ( <sup>64</sup>Cu-BNNTs) Radiolabeled by Neutron Capture**

**Wellington Marcos Silva <sup>1</sup> , Helio Ribeiro <sup>2</sup> and Jose Jaime Taha-Tijerina 3,4,\***


**Abstract:** In this work, the radioisotope <sup>64</sup>Cu was obtained from copper (II) chloride dihydrate in a nuclear research reactor by neutron capture, (63Cu(n,γ) <sup>64</sup>Cu), and incorporated into boron nitride nanotubes (BNNTs) using a solvothermal process. The produced <sup>64</sup>Cu-BNNTs were analyzed by TEM, MEV, FTIR, XDR, XPS and gamma spectrometry, with which it was possible to observe the formation of64Cu nanoparticles, with sizes of up to 16 nm, distributed through nanotubes. The synthesized of <sup>64</sup>Cu nanostructures showed a pure photoemission peak of 511 keV, which is characteristic of gamma radiation. This type of emission is desirable for Photon Emission Tomography (PET scan) image acquisition, as well as its use in several cancer treatments. Thus, <sup>64</sup>Cu-BNNTs present an excellent alternative as theranostic nanomaterials that can be used in diagnosis and therapy by different techniques used in nuclear medicine.

**Keywords:** theranostic nanomaterials; boron nitride; neutron capture reaction; nuclear medicine

### **1. Introduction**

The discovery and development of new materials are often the catalysts for technological advances, particularly when they can be applied to various areas of research. A historic milestone in the search for new materials occurred in 1995 when boron nitride nanotubes (BNNTs) emerged as a key material in nanotechnology science [1]. BNNTs have cylindrical structures (one-dimensional—1D), formed only by atoms of boron (B) and nitrogen (N), with diameters in the order of nanometers and lengths in the order of microns [2]. Similar to single-wall carbon nanotubes (SWCNT), depending on the angle *θ* in which the sheet is rolled, nanotube structures are formed with armchair (*θ* = 30◦ ), zig-zag (*θ* = 0◦ ) and chiral (0 < /*θ*/ < 30◦ ) forms [3]. Otherwise, the multi-walled BNNTs are formed by several sheets of hexagonal boron nitride (h-BN) concentrically wrapped. Both BNNTs have excellent chemical, physical and mechanical properties [4–8] and a band gap of approximately 5.5 eV [3]. Several methods have been used for the synthesis of BNNTs [5]; however, chemical vapor deposition (CVD) is the most used method and requires a simple apparatus which produces BNNTs with excellent structural quality [9].

The interest in BNNT-based technologies in recent years can be measured by the large number of scientific works that have been published, in addition to the increase in large-scale production [10]. Within the class of nanostructured materials, the BNNTs have great potential for several biomedical applications. Some early studies demonstrated that nanotubes did not show toxicity at concentrations below 50 µg/mL [11]. Furthermore, they favor the reuptake of molecules into the cell interior and can be functionalized with different biological protein epitopes [12]. Recently, BNNTs have been used as nanovectors for DNA,

**Citation:** Silva, W.M.; Ribeiro, H.; Taha-Tijerina, J.J. Potential Production of Theranostic Boron Nitride Nanotubes (64Cu-BNNTs) Radiolabeled by Neutron Capture. *Nanomaterials* **2021**, *11*, 2907. https:// doi.org/10.3390/nano11112907

Academic Editors: Félix Zamora and Angelo Ferraro

Received: 1 September 2021 Accepted: 17 October 2021 Published: 30 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

drugs and radioisotopes, and as boosters for biomaterials. In 2012, Soares et al. [13] used BNNTs radiolabeled with 99mTc to investigate the cell-distribution behavior *in vivo* through a process of passive accumulation in solid tumors. Diverse studies applying BNNTs to cancer treatment have been reported. For example, when linked to target molecules, BNNTs could be used as therapeutic agents capable of killing cancer cells by boron neutron capture therapy. This medical approach is generally applied in brain cancer treatments, and it is based on the capture of the neutron reaction <sup>10</sup>B (n,α) <sup>7</sup>Li, where a <sup>10</sup>B atom captures a low-energy thermal neutron and then decays to produce <sup>4</sup>He (alpha particles) and <sup>7</sup>Li, resulting in a dense ionizing radiation which is capable of destroying the cells where the reaction takes place [12]. Another potential application of BNNTs is in diagnostic medicine. In this sense, BNNTs doped with rare earth beta-emitters with short half-lives, such as <sup>153</sup>Sm and <sup>159</sup>Gd, can also be used as radioisotopes for imaging [14].

In this context, nanotechnology has revolutionized so-called traditional medicine by introducing novel concepts and methods that had never been imagined. Thus, nanomedicine has improved the diagnosis of various diseases through techniques based on magnetism or nuclear reactions with different electronic devices, using biosensors or radioisotope-doped nanomaterials. In this way, the study of a more accurate diagnostic method using novel technologies is as relevant a goal as the prevention and treatment of oncological diseases. Therefore, a class of new nanomaterials, in which boron nitride nanotubes (BNNTs) stand out, has been the target of studies that have led to an understanding of the correlation between their structure and properties, which enables their use in diagnostic medicine. Due to their empty internal spaces, BNNTs can be filled by different chemical species, such as enzymes, noble metals, rare earths, and radioisotopes, especially copper-64 (64-Cu), which allows this type of material to be applied as a biological marker and in diagnostic medicine. For instance, copper-64 (T1/2 = 12.7 h; β+, 0.653 MeV (17.8%); β - , 0.579 MeV (38.4%)) has decay characteristics that allow it to be applied to obtain images of positron emission tomography (PET-scan) and in cancer-directed radiotherapy. Copper, for instance, has already well-established coordination chemistry that allows its reaction with an extensive variety of chelating systems that could potentially be linked to peptides and other interesting biological molecules such as antibodies, proteins, and nanoparticles. Its specific half-life expands the ability to image molecules of various dimensions, mainly including the slower compensating proteins and nanoparticles. Due to the versatility of applications of 64-Cu, a significant increase in scientific and technical publications has been seen over the last 2 decades, mainly in PET-scan imaging, but also in targeted cancer radiotherapy.

Thus, this work aimed to synthesize and characterize <sup>64</sup>Cu-BNNTs with appreciable properties that suggest numerous multifunctional applications, with advantages for cancer diagnosis and therapy, such as: (i) increased bioavailability; (ii) reduction in systemic adverse effects, thereby increasing patient comfort and adherence to treatment; (iii) improved osteogenic differentiation response promoted by the <sup>64</sup>Cu-BNNTs system and targeting of tumor cells, among others. It is also important to mention that the combination of <sup>64</sup>Cu-BNNTs has not yet been reported in the literature.

### **2. Experiment**

### *2.1. Raw Materials*

Copper (II) chloride dihydrate (99.999%), iron (III) oxide nano powder (<50 nm particle size) and amorphous boron powder (≥95%) were obtained from Sigma Aldrich Brazil-Ltda, Sao Paulo, Brazil (CAS Number 10125-13-0) and used as received.

### *2.2. Synthesis and Purification of Boron Nitride Nanotubes*

BNNTs were processed from mixing amorphous boron and iron (III) oxide powder (ratio 0.02) in a horizontal tubular reactor. This reactor consisted of an alumina with an inlet and outlet for the flow of ammonia and nitrogen gases. The synthesis was carried out under a NH3/N<sup>2</sup> atmosphere at a 150/20 sccm (standard cubic centimeters per minute) flow rate with a heating rate of 10 ◦C min−<sup>1</sup> from room temperature up to 1200 ◦C. An

isotherm was maintained for 2 h. After this step was completed, the reactor was cooled down to room temperature under a N<sup>2</sup> atmosphere.

The synthesized BNNTs were purified using sulfuric and nitric acids in the ratio of 3:1, respectively. The reaction mixture was kept under stirring and reflux conditions at 80 ◦C for 2 h, followed by the filtration process. The resulting solid was washed with deionized water and oven-dried for 4 h at 110 ◦C. In this process, hydroxyl groups (-OH) were introduced into the structure of the tubes.

### 2.2.1. Activation Process of <sup>64</sup>Cu Radioisotope

The radioisotope <sup>64</sup>Cu was obtained by neutron activation of the copper (II) chloride dihydrate sample in a nuclear research reactor (TRIGA Mark-1) at CDTN (Belo Horizonte, Brazil) by the neutron capture reaction <sup>63</sup>Cu(n,γ) <sup>64</sup>Cu. The irradiation was performed on 20 mg samples over 8 h under a thermal neutron flux of 6.6 <sup>×</sup> 1011 cm−<sup>2</sup> s −1 . The theoretical induced activities were estimated according to the research of Zangirolami et al. [15]. The calculations were carried out while considering the amount of Cu in the sample and using the thermal neutron capture cross-sections as a reference, in accordance with an IAEA (International Atomic Energy Agency) publication [16].

### 2.2.2. Incorporation of Cu and <sup>64</sup>Cu to the BNNT Samples

The BNNT (100 mg) sample was dispersed in anhydrous ethanol. With the aid of an autoclave with a polytetrafluoroethylene (PTFE) vessel, the Cu and <sup>64</sup>Cu radioisotope were incorporated into the BNNTs. The incorporation reaction was carried out in an oven at a temperature of 180 ◦C for two hours. After this period, the material was cooled to room temperature and filtered. The radiochemical purity of the sample was assessed by gamma spectroscopy, using an HP-Ge detector (Ortec Ametek, Oak Ridge, TN, USA) with 25% efficiency, and analyzed using the Canberra Genie 2000 software, Meriden, CT, USA [17]. The evaluation of the specific activity was carried out using a CRC 15R activimeter that had been previously calibrated for copper-64 emission. Figure 1a,d schematically show all stages of <sup>64</sup>Cu-BNNT production. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 4 of 14

**Figure 1.** *Cont*.

BNNTs samples (**d**).

**3. Characterization** 

**Figure 1.** Schematic representation of synthesis of the BNNTS (**a**), purification process (**b**), activation process of CuCl2·2H2O to obtainment of 64Cu radioisotope (**c**) and incorporation of Cu and 64Cu into

FTIR measurements of the BNNT and Cu-BNNT samples were performed with a Bruker model Vertex 70v instrument (Belo Horizonte, Brazil). The spectra were collected in ATR mode with 64 accumulations, a resolution of 4 cm−1, and in the 4500–300 cm−1 region in transmission mode and then were systematically adjusted; baseline corrections were considered for this analysis. An ultima IV Rigaku Diffractometer with Cu-Kα radiation was employed to study the main crystalline phases in the synthesized BNNT and Cu-BNNT samples (Belo Horizonte, Brazil). The Bragg's angle values were measured in the 10–80° range, with a scanning rate of 0.02° min−1. XPS spectra were obtained using monochromatic Al Kα radiation (1486.6 eV) with an electron energy analyzer (Specs, Phoibos-

**Figure 1.** Schematic representation of synthesis of the BNNTS (**a**), purification process (**b**), activation process of CuCl2·2H2O to obtainment of 64Cu radioisotope (**c**) and incorporation of Cu and 64Cu into **Figure 1.** Schematic representation of synthesis of the BNNTS (**a**), purification process (**b**), activation process of CuCl<sup>2</sup> ·2H2O to obtainment of <sup>64</sup>Cu radioisotope (**c**) and incorporation of Cu and <sup>64</sup>Cu into BNNTs samples (**d**).

#### BNNTs samples (**d**). **3. Characterization**

**3. Characterization**  FTIR measurements of the BNNT and Cu-BNNT samples were performed with a Bruker model Vertex 70v instrument (Belo Horizonte, Brazil). The spectra were collected in ATR mode with 64 accumulations, a resolution of 4 cm−1, and in the 4500–300 cm−1 region in transmission mode and then were systematically adjusted; baseline corrections were considered for this analysis. An ultima IV Rigaku Diffractometer with Cu-Kα radiation was employed to study the main crystalline phases in the synthesized BNNT and Cu-BNNT samples (Belo Horizonte, Brazil). The Bragg's angle values were measured in the 10–80° range, with a scanning rate of 0.02° min−1. XPS spectra were obtained using monochromatic Al Kα radiation (1486.6 eV) with an electron energy analyzer (Specs, Phoibos-FTIR measurements of the BNNT and Cu-BNNT samples were performed with a Bruker model Vertex 70v instrument (Belo Horizonte, Brazil). The spectra were collected in ATR mode with 64 accumulations, a resolution of 4 cm−<sup>1</sup> , and in the 4500–300 cm−<sup>1</sup> region in transmission mode and then were systematically adjusted; baseline corrections were considered for this analysis. An ultima IV Rigaku Diffractometer with Cu-Kα radiation was employed to study the main crystalline phases in the synthesized BNNT and Cu-BNNT samples (Belo Horizonte, Brazil). The Bragg's angle values were measured in the 10–80◦ range, with a scanning rate of 0.02◦ min−<sup>1</sup> . XPS spectra were obtained using monochromatic Al Kα radiation (1486.6 eV) with an electron energy analyzer (Specs, Phoibos-150) that enabled high-energy resolution and an excellent signal-to-noise ratio (Belo Horizonte, Brazil). The signal of adventitious carbon (C 1 s at 284.6 eV) was used to correct the binding-energy scale of the survey and the high-resolution spectra. Highresolution spectra in the regions of interest were fitted assuming its shape as a convolution of Lorentzian and Gaussian functions of different components, and the background contribution was removed by the Shirley method [18,19]. SEM analysis was performed with a Carl Zeiss Field Emission Scanning Electron Microscope, model sigma VP (Belo Horizonte, Brazil), operating in vacuum with an electron-beam-acceleration voltage between 5 and 30 kV. The BNNT and Cu-BNNT powders were deposited directly onto the carbon tape. The Transmission Electron Microscopy (TEM) images were obtained on a FEI TEM-LaB6 TECNAI G2 microscope (Belo Horizonte, Brazil), with a tungsten-filament electron gun operating at 200 kV. Samples were dispersed in acetone for 30 min using a water bath sonicator and one drop was deposited onto a 200-mesh holey carbon–copper grid. The activity of the <sup>64</sup>Cu-BNNTs after irradiation was obtained from the gamma spectrum, using an HP-Ge detector (Belo Horizonte, Brazil), with a nominal efficiency of 25%, and the Canberra Genie 2000 software.

### **4. Results and Discussion**

The FTIR spectrum was obtained in order to identify the vibrational modes in the BNNT samples (Figure 2). The absorption peaks between 3400 and 3200 cm−<sup>1</sup> could be attributed to the vibrational modes of the hydroxyl groups (-OH) from water molecules adsorbed on

the sample surface [20,21]. However, it could also be attributed to the presence of copper hydroxide. The region between 2000 and 60 cm−<sup>1</sup> has several peaks (Figure 2b,c). The well-known longitudinal (LO) vibrations along the axis resonate sharply around 1369 cm−<sup>1</sup> , and a second signal (1545 cm−<sup>1</sup> ) appears for tangential (T) circumferential in-plane modes (νB-N). These T modes should be dependent on the diameter (curvature) but seem to only be visible in highly pure, crystalline BNNTs [22]. Another typical absorption peak for BNNTs is located around 790 cm−<sup>1</sup> and is related to out-of-plane B-N-B bending (δB-N-B) vibrations [20,21,23]. In both spectra, the peaks between the 1100 and 880 cm−<sup>1</sup> regions give an account of the anti-symmetrical and symmetrical stretching vibrations of B-O bonds in BO<sup>3</sup> and BO<sup>4</sup> groups formed from B-OH, and peaks at 701.3, 685.8 and 453.3 cm−<sup>1</sup> are assigned to the bend vibrations of B-O bonds in BO<sup>3</sup> and BO<sup>4</sup> groups [24]. The peak at 426.0 cm−<sup>1</sup> is assigned to the stretching vibrations νCu(II)-O of copper oxide CuO [25]. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 6 of 14

**Figure 2.** Infrared spectra of (**a**) BNNTs and Cu-BNNTs in the region between 4500 and 60 cm<sup>−</sup>1. (**a**) Highlighted regions between 2000 and 60 cm<sup>−</sup>1 for (**b**) BNNTs and (**c**) Cu-BNNTs. **Figure 2.** Infrared spectra of (**a**) BNNTs and Cu-BNNTs in the region between 4500 and 60 cm−<sup>1</sup> . (**a**) Highlighted regions between 2000 and 60 cm−<sup>1</sup> for (**b**) BNNTs and (**c**) Cu-BNNTs.

, which correspond to the (100), (101), (102), (004), (103) and (110)

The XRD of BNNTs and Cu-BNNTs is shown in Figure 3. An intense peak close to 2*θ*

planes, respectively [14,23,26]. After the introduction of Cu nanoparticles to the BNNTs, new diffraction peaks were observed (Figure 3b),so the region between 30° and 80° was highlighted. The presence of CuO and Cu2O nanoparticles were identified at 36.89°, 39.71° and 65.3°, which may have occurred due to the exposure of the nanoparticles to the surrounding environment during characterization [27]. The characteristic diffraction peaks

55.09°, 59.40° and 76.05◦

The XRD of BNNTs and Cu-BNNTs is shown in Figure 3. An intense peak close to 2*θ* = 26.65◦ (Figure 3a) corresponds to the plane (002) and is attributed to the main peak of the h-BN structure. Peaks assigned to h-BN are also observed at 2*θ* = 41.78◦ , 42.81◦ , 50.16◦ , 55.09◦ , 59.40◦ and 76.05◦ , which correspond to the (100), (101), (102), (004), (103) and (110) planes, respectively [14,23,26]. After the introduction of Cu nanoparticles to the BNNTs, new diffraction peaks were observed (Figure 3b),so the region between 30◦ and 80◦ was highlighted. The presence of CuO and Cu2O nanoparticles were identified at 36.89◦ , 39.71◦ and 65.3◦ , which may have occurred due to the exposure of the nanoparticles to the surrounding environment during characterization [27]. The characteristic diffraction peaks of copper nanoparticles located at 32.42◦ and 44.81◦ were observed. They correspond to the (110) and (200) crystallographic planes of face-center cubic (fcc), respectively [27,28]. Debye–Scherrer's equation, i.e., D = 0.9 × λ/(β × cos*θ*), was used to calculate the size of copper nanoparticles, where D represents crystalline size, 0.9 is Scherrer's constant, λ is the wavelength of the X-ray, β is the full width at the half-maximum of the diffraction peak (FWHM) and *θ* represents Bragg's angle [27,29]. The calculations were performed using the mean values of the FWHM of the peaks, with 2*θ* of 32.4◦ and 44.8◦ . The average size of the Cu nanoparticles was 16 nm. This is a dimension in which nanoparticles can penetrate tumor cells through the Increased Permeability and Retention Effect (EPR), thus they can be used as a theranostic nanomaterials. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 7 of 14 of copper nanoparticles located at 32.42° and 44.81° were observed. They correspond to the (110) and (200) crystallographic planes of face-center cubic (fcc), respectively [27,28]. Debye–Scherrer's equation, i.e., D = 0.9 × λ/(β × cos*θ)*, was used to calculate the size of copper nanoparticles, where D represents crystalline size, 0.9 is Scherrer's constant, λ is the wavelength of the X-ray, β is the full width at the half-maximum of the diffraction peak (FWHM) and *θ* represents Bragg's angle [27,29]. The calculations were performed using the mean values of the FWHM of the peaks, with 2*θ* of 32.4° and 44.8°. The average size of the Cu nanoparticles was 16 nm. This is a dimension in which nanoparticles can penetrate tumor cells through the Increased Permeability and Retention Effect (EPR), thus they can be used as a theranostic nanomaterials.

**Figure 3.** XRD of BNNTs and Cu-BNNTs in the region between 10° and 80° (**a**). Highlighted regions between 30° and 80° (**b**). **Figure 3.** XRD of BNNTs and Cu-BNNTs in the region between 10◦ and 80◦ (**a**). Highlighted regions between 30◦ and 80◦ (**b**).

Figure 4 shows the XPS survey of the samples. In all the survey spectra, only the presence of B (190.10 eV), N (398.12 eV), C (284.49 eV) and O (532.51 eV) was identified. The C and O signals were also identified on the BNNT surface. The presence of C is related to the surface contamination that usually occurs during the preparation process and to the exposure of the specimens to air, and the presence of O is due to the purification process Figure 4 shows the XPS survey of the samples. In all the survey spectra, only the presence of B (190.10 eV), N (398.12 eV), C (284.49 eV) and O (532.51 eV) was identified. The C and O signals were also identified on the BNNT surface. The presence of C is related to the surface contamination that usually occurs during the preparation process and to the exposure of the specimens to air, and the presence of O is due to the purification

particles.

process and is commonly observed in XPS measurements. The presence of F (689.51 eV) and Si (102.53 eV) is due to residues from the vessel that was used for synthesis and the Cl (198.53 eV) comes from the reagent CuCl2·2H2O that was used for the synthesis of copper nanoparticles. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 8 of 14

**Figure 4.** Survey XPS spectra for BNNTs and Cu-BNNTs. **Figure 4.** Survey XPS spectra for BNNTs and Cu-BNNTs.

Table 1 shows the data obtained from the XPS survey. The stoichiometry rate of boron and nitrogen atoms (B:N) is confirmed by the peak areas of the XPS survey spectra. The obtained value for the B:N ratio is 1.09. A similar result was obtained by Silva et al. and Juan Li et al. [21,30]. According to these studies, the non-stoichiometric BN nanotubes present an excess of B atoms, and oxygen-doping in the h-BN network leads to the formation of ternary BNxOy species [31]. Hydroxyl groups increase in modulus to negative charges on the tubes' surfaces. As copper nanoparticles have positively charged surfaces, we believe that an electrostatic interaction occurs between the two nanostructures. Table 1 shows the data obtained from the XPS survey. The stoichiometry rate of boron and nitrogen atoms (B:N) is confirmed by the peak areas of the XPS survey spectra. The obtained value for the B:N ratio is 1.09. A similar result was obtained by Silva et al. and Juan Li et al. [21,30]. According to these studies, the non-stoichiometric BN nanotubes present an excess of B atoms, and oxygen-doping in the h-BN network leads to the formation of ternary BNxO<sup>y</sup> species [31]. Hydroxyl groups increase in modulus to negative charges on the tubes' surfaces. As copper nanoparticles have positively charged surfaces, we believe that an electrostatic interaction occurs between the two nanostructures.

**Table 1.** Surface composition (at.%) and B:N ratio, as determined by XPS for Cu-BNNT sample. **Table 1.** Surface composition (at.%) and B:N ratio, as determined by XPS for Cu-BNNT sample.


B:N - 1.090 The B 1s, N 1s and O 1s core-level photoemission spectra for all samples are shown in Figure 5. The B 1s peak (Figure 5a,b) at 190.1 eV and N 1s peak (Figure 5c,d) at 398.0 eV correspond to the B–N bonding, matching the BE values reported for bulk h-BN [21]. The component at 188.3 eV (Figure 5a,b) is attributed to B bonded to Fe from the catalyst (Fe2B) [32]. A minor contribution of boron oxide (B2O3) was also identified at 192.4 eV (Figure 5a). After the synthesis of Cu nanoparticles, two new contributions were observed at 200.4 and 197.0 eV (Figure 5b); the first is attributed to Cl II and the second to the presence of metallic copper (Cu 0). In addition, the binding energies at 396.6 eV and 400.0 eV are attributed to B–N–B bonding [33] and O-B-N bonding[14], respectively. The O 1s spectra The B 1s, N 1s and O 1s core-level photoemission spectra for all samples are shown in Figure 5. The B 1s peak (Figure 5a,b) at 190.1 eV and N 1s peak (Figure 5c,d) at 398.0 eV correspond to the B–N bonding, matching the BE values reported for bulk h-BN [21]. The component at 188.3 eV (Figure 5a,b) is attributed to B bonded to Fe from the catalyst (Fe2B) [32]. A minor contribution of boron oxide (B2O3) was also identified at 192.4 eV (Figure 5a). After the synthesis of Cu nanoparticles, two new contributions were observed at 200.4 and 197.0 eV (Figure 5b); the first is attributed to Cl II and the second to the presence of metallic copper (Cu 0). In addition, the binding energies at 396.6 eV and 400.0 eV are attributed to B–N–B bonding [33] and O-B-N bonding [14], respectively. The O 1s spectra are also shown in Figure 5e,f. Both samples show peaks at 535.5, 532.9 and 530.2 eV, respectively. The first is associated with oxygen atoms bonded to O-H from

oxygen atoms bonded to Fe-O (Fe2O3), which is related to the synthesis process [26].

Si 2p 102.53 2.138

the purification process, the second is characteristic of B–O bonds (B2O3) and the third is associated with oxygen atoms bonded to Fe-O (Fe2O3), which is related to the synthesis process [26]. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 9 of 14

**Figure 5.** B 1s, N 1s and O 1s high-resolution XPS spectra for (**a**,**c**,**e**) BNNTs, (**b**,**d**,**f**) Cu-BNNTs. **Figure 5.** B 1s, N 1s and O 1s high-resolution XPS spectra for (**a**,**c**,**e**) BNNTs, (**b**,**d**,**f**) Cu-BNNTs.

Figure 6 depicts the high-resolution spectrum XPS for Cu-BNNTs. The dominant Cu 2p3/2 peaks at 933.8 and Cu 2p1/2 at 953.6 eV, which demonstrate the successful reduction of CuCl2, can be assigned to metallic Cu(0), whereas the small peak at 936.2 eV can be assigned to Cu(II). Meanwhile, the peaks of Cu 2p1/2 at 955.9 eV, in combination with the satellite peak at 944.3 eV, are typical characteristics of CuO, implying the uniform surface oxidation of Cu nanoclusters that were exposed to air under ambient conditions [34,35]. Cu(0) and Cu(I) are hard to differentiate since they have a ~0.3 eV separation in binding energy, whereas Cu(0) and Cu(II) have more than a 2eV separation. The Cu 2p5/2 peak around 958.1 eV indicates a Cu(II) oxidation state [36,37]. Figure 6 depicts the high-resolution spectrum XPS for Cu-BNNTs. The dominant Cu 2p3/2 peaks at 933.8 and Cu 2p1/2 at 953.6 eV, which demonstrate the successful reduction of CuCl2, can be assigned to metallic Cu(0), whereas the small peak at 936.2 eV can be assigned to Cu(II). Meanwhile, the peaks of Cu 2p1/2 at 955.9 eV, in combination with the satellite peak at 944.3 eV, are typical characteristics of CuO, implying the uniform surface oxidation of Cu nanoclusters that were exposed to air under ambient conditions [34,35]. Cu(0) and Cu(I) are hard to differentiate since they have a ~0.3 eV separation in binding energy, whereas Cu(0) and Cu(II) have more than a 2eV separation. The Cu 2p5/2 peak around 958.1 eV indicates a Cu(II) oxidation state [36,37].

**Figure 6.** High-resolution spectrum XPS for Cu-BNNTs. **Figure 6.** High-resolution spectrum XPS for Cu-BNNTs. **Figure 6.** High-resolution spectrum XPS for Cu-BNNTs.

The morphological characteristics of the produced samples were studied using SEM and TEM microscopy, shown in Figures 7 and 8, respectively. It was observed in both samples that the produced BNNTs have non-uniform lengths and diameters. This feature is due to the nanotubes entanglement during their growth process, which is very common during the synthesis process. This nanotube characteristic was also observed in our previous work [23]. The morphological characteristics of the produced samples were studied using SEM and TEM microscopy, shown in Figures 7 and 8, respectively. It was observed in both samples that the produced BNNTs have non-uniform lengths and diameters. This feature is due to the nanotubes entanglement during their growth process, which is very common during the synthesis process. This nanotube characteristic was also observed in our previous work [23]. The morphological characteristics of the produced samples were studied using SEM and TEM microscopy, shown in Figures 7 and 8, respectively. It was observed in both samples that the produced BNNTs have non-uniform lengths and diameters. This feature is due to the nanotubes entanglement during their growth process, which is very common during the synthesis process. This nanotube characteristic was also observed in our previous work [23].

**Figure 7.** SEM images of BNNTs (**a**) and Cu-BNNTs (**b**). **Figure 7. Figure 7.** SEM images of BNNTs (**a**) and Cu-BNNTs (**b**). SEM images of BNNTs (**a**) and Cu-BNNTs (**b**).

TEM images are shown in Figure 8a–f. The highlighted regions in Figure 8a,c,e are shown in high-resolution images in Figure 8b,d,f. All images represent typical nanotubes with defined internal channels and external walls that are structurally well-organized [14].

When comparing Figure 8a,b with Figure 8c–f, particles with higher electron absorptions, appearing as darker sites isolated from each other, can be associated with the presence of an electron-conducting nanostructure, indicating the presence of metallic Cu [33]. It is expected that combining these metal nanostructures with BNNTs can improve the stability of the nanoparticles within the solution, allowing their application as theranostic nanomaterials. After increasing image resolution (Figure 8d–f) it was observed that Cu nanoparticles are present in great numbers on the tube surfaces and inside the channels. Referring to the scale bar, it is important to note that the size of nanoparticles is close to 16 nm, as determined by the Debye–Scherrer´s equation.

**Figure 8.** TEM images of BNNTs (**a**,**b**) and Cu-BNNTs (**c**–**f**). **Figure 8.** TEM images of BNNTs (**a**,**b**) and Cu-BNNTs (**c**–**f**).

TEM images are shown in Figure 8a–f. The highlighted regions in Figure 8a,c,e are shown in high-resolution images in Figure 8b,d,f. All images represent typical nanotubes with defined internal channels and external walls that are structurally well-organized [14]. The <sup>64</sup>Cu nanoparticles, synthesized from the high-purity CuCl2·H20 by using asolvothermal method, produced stable and contaminant-free radioisotopes, as shown in the gamma spectrum in Figure 9.

When comparing Figure 8a,b with Figure 8c–f, particles with higher electron absorptions, appearing as darker sites isolated from each other, can be associated with the presence of an electron-conducting nanostructure, indicating the presence of metallic Cu [33]. It is expected that combining these metal nanostructures with BNNTs can improve the stability of the nanoparticles within the solution, allowing their application as theranostic The pure photopeak of the <sup>64</sup>Cu-BNNTs is 511 keV [38,39]; this energy is compatible with gamma rays for the obtention of images by Photon Emission Tomography (PET-scan), and for cancer treatment due to its β-emissions, with an energy of 579 keV. This result illustrates that the <sup>64</sup>Cu-BNNTs can be used as a potential nanomaterial that is able to produce images as well as promote several cancer treatments.

nanomaterials. After increasing image resolution (Figure 8d–f) it was observed that Cu nanoparticles are present in great numbers on the tube surfaces and inside the channels.

Referring to the scale bar, it is important to note that the size of nanoparticles is close to

vothermal method, produced stable and contaminant-free radioisotopes, as shown in the

16 nm, as determined by the Debye–Scherrer´s equation.

gamma spectrum in Figure 9.

**Figure 9.** Gamma spectrum of 64Cu-BNNTs. **Figure 9.** Gamma spectrum of <sup>64</sup>Cu-BNNTs.

#### The pure photopeak of the 64Cu-BNNTs is 511 keV [38,39]; this energy is compatible **5. Conclusions**

with gamma rays for the obtention of images by Photon Emission Tomography (PETscan), and for cancer treatment due to its β-emissions, with an energy of 579 keV. This result illustrates that the 64Cu-BNNTs can be used as a potential nanomaterial that is able to produce images as well as promote several cancer treatments. **5. Conclusions**  The 64Cu nanostructures were incorporated within the BNNTs structure by a solvothermal method which produces stable and contaminant-free radioisotopes. Using XDR data and Debye–Scherrer's equation, it was possible to determine that metallic Cu The <sup>64</sup>Cu nanostructures were incorporated within the BNNTs structure by a solvothermal method which produces stable and contaminant-free radioisotopes. Using XDR data and Debye–Scherrer's equation, it was possible to determine that metallic Cu nanoparticles have sizes of about 16 nm. The TEM images showed that the BNNTs are structurally wellorganized, presenting Cu nanoparticles in their internal channels with an even distribution on their surfaces. The <sup>64</sup>Cu nanoparticles in the BNNTs also showed a pure photoemission peak of 511 keV, which is characteristic of gamma radiation. These results corroborate the fact that the studied system has high potential to be used in nuclear medicine as a theranostic material. However, this subject needs to be further explored.

nanoparticles have sizes of about 16 nm. The TEM images showed that the BNNTs are structurally well-organized, presenting Cu nanoparticles in their internal channels with an even distribution on their surfaces. The 64Cu nanoparticles in the BNNTs also showed a pure photoemission peak of 511 keV, which is characteristic of gamma radiation. These results corroborate the fact that the studied system has high potential to be used in nuclear **Author Contributions:** W.M.S. and H.R. contributed to the conceptualization, methodology, measuring campaign, literature research, project administration, data interpretation, data analysis, validation, formal analysis, resources, investigation, figures, study design, supervision and writing. J.J.T.-T., contributed to the methodology, resources, data interpretation, validation, formal analysis, investigation, figures, and writing. All authors have read and agreed to the published version of the manuscript.

medicine as a theranostic material. However, this subject needs to be further explored. **Author Contributions:** W.M.S. and H.R. contributed to the conceptualization, methodology, measuring campaign, literature research, project administration, data interpretation, data analysis, validation, formal analysis, resources, investigation, figures, study design, supervision and writing. **Funding:** This research was funded by Mackenzie Research Fund (MackPesquisa, ProjectNo. 181009). Supported by the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES), and the Universidad de Monterrey.

J.J.T.-T., contributed to the methodology, resources, data interpretation, validation, formal analysis, **Institutional Review Board Statement:** Not applicable.

investigation, figures, and writing. All authors have read and agreed to the published version of the **Informed Consent Statement:** Not applicable.

**Funding:** This research was funded by Mackenzie Research Fund (MackPesquisa, ProjectNo. **Data Availability Statement:** Not applicable.

181009). Supported by the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES), and the Universidad de Monterrey. **Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable. **Acknowledgments:** The authors would like to thank Universidad de Monterrey, Mexico, the Centro de Microscopia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, and the Centro de Desenvolvimento da Tecnologia Nuclear—CDTN, Belo Horizonte, Brazil National Council for Scientific and Technological Development (CNPq), and Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES).

**Data Availability Statement:** Not applicable. **Conflicts of Interest:** The authors declare no conflict of interest or financial intention.

### **References**

manuscript.

