**Strong Biomimetic Immobilization of Pt-Particle Catalyst on ABS Substrate Using Polydopamine and Its Application for Contact-Lens Cleaning with H2O2**

**Yuji Ohkubo 1,\*, Tomonori Aoki 1, Daisuke Kaibara 1, Satoshi Seino 1, Osamu Mori 2, Rie Sasaki 2, Katsuyoshi Endo <sup>1</sup> and Kazuya Yamamura <sup>1</sup>**


Received: 13 November 2019; Accepted: 2 January 2020; Published: 7 January 2020

**Abstract:** Polydopamine (PDA)—a known adhesive coating material—was used herein to strongly immobilize a Pt-particle catalyst on an acrylonitrile–butadiene–styrene copolymer (ABS) substrate. Previous studies have shown that the poor adhesion between Pt particles and ABS surfaces is a considerable problem, leading to low catalytic durability for H2O2 decomposition during contact-lens cleaning. First, the ABS substrate was coated with PDA, and the PDA film was evaluated by X-ray photoelectron spectroscopy. Second, Pt particles were immobilized on the PDA-coated ABS substrate (ABS-PDA) using the electron-beam irradiation reduction method. The Pt particles immobilized on ABS-PDA (Pt/ABS-PDA) were observed using a scanning electron microscope. The Pt-loading weight was measured by inductively coupled plasma atomic emission spectroscopy. Third, the catalytic activity of the Pt/ABS-PDA was evaluated as the residual H2O2 concentration after immersing it in a 35,000-ppm H2O2 solution (the target value was less than 100 ppm). The catalytic durability was evaluated as the residual H2O2 concentration after repeated use. The PDA coating drastically improved both the catalytic activity and durability because of the high Pt-loading weight and strong adhesion among Pt particles, PDA, and the ABS substrate. Plasma treatment prior to PDA coating further improved the catalytic durability.

**Keywords:** catalytic durability; polydopamine (PDA); strong adhesion; supported catalyst; H2O2 decomposition

#### **1. Introduction**

Mussels can strongly adhere to several surfaces using their body fluid, regardless of whether the surfaces are dry or wet [1–3]. This phenomenon of adhesion to wet surfaces is unusual in the adhesives industry. The body fluid of mussels was examined, and it was found that 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine-enriched proteins contributed to its strong adhesion [4–6]. As a result, polydopamine (PDA) has attracted the attention of many scientists because its structure is similar to that of DOPA. Surface chemical composition affects adhesion properties. Both DOPA and PDA have hydroxyl and amino groups and a benzene ring, so they can interact with various materials such as metal oxides, metals, and polymers, not only through van der Waals forces, but also via hydrogen or coordinate bonding or π–π stack interaction. Since PDA has been reported as a novel adhesive coating for several materials such as Pt, Cu, TiO2, SiO2, and Al2O3 [7], it has received even more attention. For example, there are reports of PDA being utilized at sites for growing hydroxyapatite (HAp) [8]; PDA-coated polystyrene (PS)

particles have been used to prepare a structural-color-controlled ink [9]; a PDA-grafted hydrogel has been demonstrated to adhere to a wet mucous membrane [10]; and a PDA coating has been used as a seed layer for a TiO2–polytetrafluorethylene (PTFE) nanocomposite coating [11]. Recently, PDA was utilized to improve the adhesion between a polydimethylsiloxane (PDMS) nanosheet and a living body [12]. This research demonstrated that a PDA coating combines both high adhesion and biocompatibility.

The number of contact-lens wearers has increased in recent years and is currently estimated to be approximately 140 million [13]. There are two types of contact-lens wearers, namely, those who use one-day disposable lenses and those who prefer repeatable-use (e.g., monthly) contact lenses. Although one-day disposable contact lenses do not need cleaning and disinfecting, their high cost is a serious disadvantage. In contrast, repeatable-use contact lenses are less expensive in the long term, but it is essential to clean and disinfect them properly once per day to prevent eye infections. There are three types of methods for cleaning and disinfecting contact lenses: the first method is boil cleaning, the second is H2O2 cleaning, and the third is cleaning with a multipurpose solution (MPS). MPS cleaning has the advantage of being simple because only one solution is required for cleaning, disinfection, and storage. However, contact-lens wearers applying the MPS cleaning method are likely to have eye problems if they do not clean their contact lenses carefully enough. Thus, the number of contact-lens wearers using MPS cleaning has gradually decreased since 2009, while that of users applying the H2O2 cleaning method has increased [14]. The reason for this is that eye problems are unlikely to occur in the case of H2O2 cleaning because a 35,000-ppm H2O2 solution exhibits a high disinfecting performance. However, when H2O2 cleaning is applied, there is a risk of the eyes becoming bloodshot or painful—even of blindness—if the 35,000-ppm H2O2 solution enters the eyes without being decomposed to a concentration of 100 ppm [15]. Electroless platinum (Pt) plating has been performed to give an acrylonitrile–butadiene–styrene copolymer (ABS) substrate catalytic performance for accelerating the H2O2 decomposition process (Figure 1a). Pt is very expensive, so there is a strong need to decrease the amount of Pt used in contact-lens cleaners. We have suggested replacing the Pt film with Pt particles, which results in a drastic decrease in the amount of Pt required (Figure 1b) [16]. However, some problems remain regarding the catalytic durability, although we pretreated the surface by etching, electric charge control, or both [17]. As mentioned above, PDA has the potential to adhere metal particles to resin substrates. In this study, we used a PDA coating as a pre-treatment to strongly and safely immobilize Pt particles on an ABS substrate to improve the catalytic durability of the material (Figure 1c). The effects of the PDA coating on the properties of the ABS surface and the catalytic activity, Pt-loading weight, and catalytic durability of the system were investigated.

**Figure 1.** Schematic of the processes for preparing (**a**) a Pt-film/ABS sample by the electroless plating method, (**b**) a Pt-particle/ABS sample by EBIRM without pre-treatment, and (**c**) a Pt-particle/ABS-PDA sample by EBIRM adding a PDA coating as a pre-treatment.

#### **2. Results and Discussion**

#### *2.1. External Appearance*

The changes in the external appearances of the ABS samples were monitored to confirm that PDA coating had occurred. Figure 2 presents photographs of ABS samples with a masking using polyimide (PI) tape before and after PDA coating. The color of the PDA-coated area changed from cream to gray, and this gray color remained after ultrasonic cleaning, which confirmed that the PDA film was strongly attached to the ABS surface.

**Figure 2.** Photographs of ABS samples with a masking using polyimide (PI) tape (**a**) before PDA coating and (**b**) after PDA coating for 24 h.

#### *2.2. Confirmation of the Formation of a PDA Film by X-Ray Photoelectron Spectroscopy (XPS)*

To examine the effects of the PDA coating on the chemical composition of ABS substrates, we analyzed pretreated ABS surfaces that did not contain Pt particles by XPS. Figure 3 presents the XPS spectra of the surface of an ABS substrate before and after PDA coating at different immersion times in a dopamine (DA) solution. When the ABS substrates were immersed in a DA solution, the intensities of the peaks assigned to C–H and C–C (285 eV) decreased, whereas those of the peaks assigned to C–N and C–O (286.5 eV) increased, as illustrated in Figure 3a. The intensities of the signals in the N1s-XPS spectra did not increase because the ABS substrate originally contained a C≡N bond, as illustrated in Figure 3b. In addition, when the ABS substrates were immersed in a DA solution, the intensities of the signals in the O1s-XPS spectra also increased, as illustrated in Figure 3c. The calculated N/C atomic ratio is presented in Figure 3d, where it can be seen that it increased with increasing immersion time in the DA solution. When the ABS substrates were immersed in a DA solution for 3 and 24 h, the N/C ratios were 0.129 and 0.120, respectively. These ratios were roughly consistent with the theoretical value of N/C = 0.125. These results indicate that the ABS surfaces were coated with a PDA film.

**Figure 3.** *Cont*.

**Figure 3.** X-ray photoelectron spectroscopy (XPS) spectra of an ABS surface before and after PDA coating at different immersion times in a DA solution: (**a**) C1s-XPS, (**b**) N1s-XPS, (**c**) O1s-XPS, and (**d**) the N/C ratio.

#### *2.3. Observation of Pt Particles by SEM*

The Pt particles immobilized on the ABS surfaces were analyzed by SEM to confirm the deposition of Pt particles. Figure 4 presents SEM micrographs of the surfaces of Pt/ABS samples with or without PDA coating at a low magnification. The small white spots in the images are Pt particles. The high dispersibility of Pt particles was confirmed for all samples. The number of Pt particles clearly increased upon PDA coating. Moreover, the number of Pt particles increased with increasing DA immersion time. Some holes (with diameters of 50–200 nm) were observed in the Pt/ABS-untreated sample (Figure 4a), but they were absent in the Pt/ABS-PDA samples (Figure 4b–d). This indicates that the holes originally present on the ABS surface were successfully coated with the PDA film.

**Figure 4.** Scanning electron microscope (SEM) images of the surface of an ABS substrate before and after PDA coating and Pt immobilization: (**a**) Pt/ABS-untreated, (**b**) Pt/ABS-PDA(1h), (**c**) Pt/ABS-PDA(3h), and (**d**) Pt/ABS-PDA(24h).

#### *2.4. Pt-Loading Weight Determined by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES)*

The effects of the PDA coating on the Pt-loading weights of the Pt/ABS samples were also examined. Figure 5 illustrates the Pt-loading weights of substrates with or without PDA coating. It can be seen that the values were higher for the Pt/ABS-PDA samples than for the Pt/ABS-untreated ones, thus indicating that PDA coating increased the Pt-loading weight. In addition, the Pt-loading weights for the Pt/ABS-PDA samples increased with increasing DA immersion time. These results of Pt-loading weight are consistent with the SEM images illustrated in Figure 4. When an ABS substrate is coated with a PDA film, the number of sites for immobilizing Pt particles also increases, resulting in an increased Pt-loading weight. The Pt-loading weight of the Pt/ABS-PDA(24h) material (11.2 μg/substrate) was approximately twice that of the Pt/ABS-untreated sample, but at least 130 times lower than that of an ABS substrate coated with an electroless-plated Pt film (Pt-film/ABS) (1500 μg/substrate), which had been studied earlier [16].

**Figure 5.** Pt-loading weights of Pt/ABS samples with or without the PDA coating.

#### *2.5. Catalytic Activity for H2O2 Decomposition*

To evaluate the catalytic activity of the materials for H2O2 decomposition, the residual H2O2 concentration was measured in the system after immersing Pt/ABS samples with or without the PDA coating in a 35,000-ppm H2O2 solution for 360 min. Briefly, the lower the residual H2O2 concentration, the higher the catalytic activity. Figure 6 illustrates the catalytic activity of Pt/ABS samples with or without the PDA coating. The untreated ABS sample without Pt particles did not decompose H2O2 within 360 min, whereas all the samples with Pt immobilized on the ABS substrate significantly decreased the residual H2O2 concentration from 35,000 to less than 400 ppm. Moreover, the residual H2O2 concentrations for the Pt/ABS-PDA samples became lower than that for the Pt/ABS-untreated sample. It is clear that the PDA coating improved the catalytic activity for H2O2 decomposition, as well as that the residual H2O2 concentration decreased with increasing DA immersion time. This result is consistent with those obtained in the SEM (Figure 4) and ICP-AES (Figure 5) studies. In summary, an increase in the Pt-loading weight contributed to the improvement of the catalytic activity of the resulting material. The target value for the residual H2O2 concentration (i.e., <100 ppm) was successfully reached after 3 h of immersion in DA.

**Figure 6.** Catalytic activity of Pt/ABS samples with or without PDA coating: residual H2O2 concentration after immersion for 360 min. \* The data for the Pt/ABS-untreated are the same as in our previous report [17].

#### *2.6. Catalytic Durability during H2O2 Decomposition*

To examine the effect of the PDA coating on the catalytic durability of the system, the relation between the number of repeated uses and the residual H2O2 concentration was examined. Figure 7 illustrates the catalytic durability of Pt/ABS samples with or without the PDA coating. The residual H2O2 concentration for the Pt/ABS-untreated sample increased significantly with increasing usage, thus resulting in low catalytic durability. In the case of the Pt/ABS-PDA material, the residual H2O2 concentrations measured after using the samples 10 times were 935, 194, and 54 ppm for Pt/ABS-PDA(1h), Pt/ABS-PDA(3h), and Pt/ABS-PDA(24h), respectively; this indicates that the residual H2O2 concentrations decreased with increasing DA immersion time. This result demonstrates that the PDA coating effectively improved the catalytic durability of the material for H2O2 decomposition. Moreover, the residual H2O2 concentration for Pt/ABS-PDA(24h) was still below 100 ppm after the catalyst had been used 10 times.

**Figure 7.** Catalytic durability of Pt/ABS samples with or without PDA coating: the relationship between the number of usage cycles and the residual H2O2 concentration. \* The data for the Pt/ABS-untreated are the same as in our previous report [17].

#### *2.7. E*ff*ect of Plasma Treatment on Catalytic Durability*

Although Pt/ABS-PDA(24h) exhibited high catalytic durability, the residual H2O2 concentration mildly increased from 38 to 51 ppm with the number of usage cycles. Thus, to further improve the catalytic durability, plasma treatment was applied before the PDA coating. Figure 8 illustrates the catalytic durability of Pt/ABS-PDA(24h) samples with or without plasma treatment. Please note that the range of the vertical axis is from 0 to 100 ppm. The residual H2O2 concentration of the Pt/ABS-plasma-PDA(24h) sample after it had been used 10 times was 35 ppm, which indicates that the residual H2O2 concentration barely changed and that the plasma treatment before the PDA coating further improved the catalytic durability of the material. The catalytic durability of the Pt/ABS-plasma-PDA(24h) sample was sufficient for use in practical applications.

**Figure 8.** Catalytic durability of Pt/ABS-PDA(24h) samples with or without plasma treatment before PDA coating: the relationship between the number of usage cycles and the residual H2O2 concentration.

To investigate the effect of the plasma treatment on the morphology of the PDA coating, the surfaces of the ABS-PDA(24h) and ABS-plasma-PDA(24h) samples were observed and compared by SEM. Figure 9 presents SEM images of the surface of an ABS substrate before and after PDA coating, with or without plasma treatment. Although many cracks were observed on the as-received ABS surface, no cracks were observed on the PDA-coated one, regardless of the plasma treatment. This result indicates that the ABS surfaces were coated with a PDA film. A comparison between the ABS-plasma-PDA(24h) and ABS-PDA(24h) samples indicates that the surface roughness of the plasma-treated material was larger than that of the untreated one. The Pt-loading weights of Pt/ABS-plasma-PDA(24h) and Pt/ABS-PDA(24h) were 24.0 and 11.2 μg, respectively. As can be seen, the plasma treatment induced an increase in the Pt-loading weight, which is one of the reasons for the improved catalytic activity and durability. In addition, XPS measurements were also carried out for the ABS samples after Pt immobilization to investigate the effect of plasma treatment on the Pt state, such as Pt(0), Pt(II), and Pt(IV). Figure 10 presents the Pt4f-XPS spectra of the Pt/ABS samples with or without plasma treatment and with or without PDA coating. Two broad peaks indexed to Pt4f7/<sup>2</sup> and Pt4f5/<sup>2</sup> were observed for all the Pt4f-XPS spectra. The Pt4f7/<sup>2</sup> peak was resolved into three peaks indexed to Pt(0), Pt(II), and Pt(IV) at ca. 71.6, ca. 72.6, and 74.4 eV, respectively [18,19]. The ratios of Pt(0), Pt(II), and Pt(IV) are listed in Table 1 according to the references. Although the main state was Pt(0), minor states of Pt(II) and Pt(IV) also were detected. These results indicate two likelihoods: the first is a coordinate bond of Pt ions, and the second is an oxidation of Pt. In summary, it is possible that, firstly, Pt in an ionic state interacted with C–O groups on the electron-beam-irradiated ABS substrate in water or NH2 and N–H groups in the PDA film; secondly, a coordinate bond was formed; thirdly, Pt clusters grew to form Pt particles; and finally, the surface of the Pt particles were oxidized to change the surface to Pt oxide (PtO or PtO2). The PDA coating increased the ratio of the Pt metallic state to >80%, while plasma treatment mostly did not affect the ratio of the Pt metallic state.

*Nanomaterials* **2020**, *10*, 114

(**a**) (**b**)

(**c**)

**Figure 9.** Scanning electron microscope (SEM) images of the surface of an ABS substrate before and after PDA coating with or without plasma treatment: (**a**) ABS-untreated, (**b**) ABS-PDA(24h), and (**c**) Pt/ABS-plasma-PDA(24h).

**Figure 10.** Pt4f-XPS spectra of the Pt/ABS samples with or without plasma treatment and with or without PDA coating.

**Table 1.** Ratios of Pt(0), Pt(II), and Pt(IV) calculated from peak resolution of the Pt4f7/<sup>2</sup> in Figure 10.


#### **3. Materials and Methods**

#### *3.1. PDA Coating and Immobilization of Pt Particles on ABS*

Pt particles were immobilized on an ABS substrate by the electron-beam-irradiation reduction method (EBIRM) because of the advantages of this procedure, which include a low processing temperature, highly uniform deposition, and a high throughput [20]. Although there are many methods for preparing and immobilizing particles (e.g., sonolytic [21–24], polyol [25–27], and impregnation [28–30] methods), they all have disadvantages such as high processing temperatures, nonuniform deposition of the metal particles, and low throughput. Therefore, EBIRM was selected in this study. The mechanism of metal ion reduction by a radiochemical approach has been described in previous reports [31,32], and the methods applied for washing the ABS substrate and depositing the Pt particles on its surface were the same here as those reported previously [16,17]. The differences between this report and previous ones are the plasma pre-treatment and the use of a PDA coating to improve the catalytic durability during H2O2 decomposition.

A commercially available ABS sheet (thickness *t* = 1 mm, 2-9229-01, AS-ONE, Nishi-ku, Osaka, Japan) was cut into substrates with a size of 20 <sup>×</sup> 15 mm2. The ABS substrates were first washed with ethanol (99.5%, Kishida Chemical, Chuo-ku, Osaka, Japan) and pure water for 10 min each in an ultrasonic cleaner (USK-1R, AS-ONE) and then dried through blowing N2 gas (99.99%, Iwatani Fine Gas, Amagasaki, Hyogo, Japan). Prior to immobilizing the Pt particles, the washed substrates were pretreated either by only PDA coating or both plasma treatment and PDA coating. Table 2 presents the sample conditions and IDs.

Low-pressure plasma treatment was applied at 100 Pa using a plasma chamber (PR-501A, Yamato Scientific, Chuo-ku, Tokyo, Japan) with a radio frequency power source of 13.56 MHz. Before the plasma treatment, the washed ABS substrates were placed in the plasma chamber; subsequently, the pressure in the chamber was decreased to 5 Pa using a rotary vacuum pump (2012AC, Alcatel Vacuum Technology, Annecy, France). Then, helium gas (99.99%, Iwatani Fine Gas) flowed into the chamber until its pressure reached 100 Pa. The applied power for plasma generation was 100 W, and the standing wave ratio was controlled at less than 1.1 through impedance matching. The plasma treatment time was 60 s. XPS measurements were performed to confirm that the surface of the ABS substrate was modified by the plasma treatment. The results confirmed that oxygen-containing functional groups (–O–C=O and –C–O) were generated by the plasma treatment, as illustrated in Figure S1.

Aqueous solutions (20 mL) containing 2 mg/mL of DA were prepared using 2-(3,4 dihydroxyphenyl) ethylamine hydrochloride (C8H12NO2·HCl; 98%, Fujifilm Wako Pure Chemical, Chuo-ku, Osaka, Japan) as the dopamine precursor and Tris hydrochloride acid buffer, controlled at pH = 8.5 (1 mol/L, Fujifilm Wako Pure Chemical), as the solvent. The ABS substrates were immersed in the DA solution for different times (i.e., 1, 3, or 24 h), and the DA solutions were neither stirred nor bubbled with oxygen gas during the immersion. After PDA coating, the substrates were removed from the DA solution and washed with pure water for 10 min using an ultrasonic cleaner to remove the unreacted DA and PDA. Finally, they were dried with N2 gas. Precleaned slide glasses (S7213, Matsunami Glass, Kishiwada, Osaka, Japan) were also used and coated with a PDA film to confirm that the PDA coating was completed because ABS substrates originally contain carbon and nitrogen atoms. The results demonstrated that the intensity of the peaks in the Si2p-XPS spectra decreased with increasing DA immersion time and eventually disappeared when the glass slide was immersed for 24 h, as illustrated in Figure S2. This result indicates that immersion in a DA solution for 24 h is enough to uniformly cover the substrate's surface.

Aqueous solutions (5 mL) containing 4 mM of Pt ions were prepared using hexachloroplatinic acid hexahydrate (H2PtCl6·6H2O; 98.5%, Fujifilm Wako Pure Chemical) in cylindrical PS containers (diameter ø = 33 mm and height *h* = 16 mm). Then, 2-propanol (IPA; 99.7%, Kishida Chemical) was added to the Pt ion solution (to be controlled at 1 vol %), and the pretreated ABS substrate was immersed in the Pt precursor solutions. A high-energy electron beam (of 4.8 MeV) was irradiated on these Pt precursor solutions containing the pretreated ABS substrate for 7 s using a Dynamitron® accelerator from SHI-ATEX Co. Ltd. (Izumiotsu, Osaka, Japan). During electron-beam irradiation, H radicals and hydrated electrons were generated by the radiolysis of water. The reductive species reduced Pt4<sup>+</sup> ions to Pt3<sup>+</sup>, Pt2+, Pt1+, and Pt0, as described in a previous report [31]. Subsequently, clusters of Pt atoms were formed, which grew to produce Pt particles. The Pt particles were formed not only on the substrate, but also in the solution. When the Pt particles grew from Pt clusters on the substrate, they were immobilized on it. When the Pt particles grew from Pt clusters in the solution, they fell down and were deposited on the substrate, but were not immobilized on it. To remove the unimmobilized Pt particles, the ABS substrates were taken out of the solution, washed with pure water for 10 min using an ultrasonic cleaner, and finally dried with N2 gas. In a previous report [16], XPS and water contact angle (WCA) measurements were performed for untreated ABS substrates before and after electron beam irradiation to investigate the immobilization mechanism; however, this mechanism is not yet clear. Those XPS and WCA results suggest two models: the first model assumes the chemical adhesion of Pt nanoparticles through a chemical reaction of functional groups (C–O) and/or carbon radicals with Pt ions and/or Pt0, and the second model assumes the unreactive immobilization of Pt nanoparticles through a C–C crosslinking network under EB irradiation. To comprehensively clarify the immobilization mechanism, further experiments using a simplex polymer such as polyethylene should be conducted.



"—" indicates no operation and "-" indicates operation.

#### *3.2. Characterization*

To confirm that the ABS surface was coated with a PDA film, its chemical composition was determined by XPS using Quantum 2000 equipment (Ulvac-Phi, Chigasaki, Kanagawa, Japan) attached to an Al-*K*α source at 15 kV. The diameter of X-ray irradiation was ø = 100 μm; the pass energy and step size were 23.50 and 0.05 eV, respectively; and the take-off angle was 45◦. To neutralize the electric charges on the surfaces, the measured samples were irradiated with a low-speed electron beam and an Ar ion beam during the XPS measurements.

Secondary electron images using a field-emission scanning electron microscope (FE-SEM; S-4800, Hitachi High-Technologies Corporation, Minato-ku, Tokyo, Japan) at 5 kV of accelerated voltage were obtained to monitor the deposition behavior of the Pt particles on the ABS and/or ABS-PDA surfaces. Prior to the observations, osmium (Os) was coated on the Pt/ABS surfaces by plasma chemical vapor deposition using an osmium plasma coater (OPC60AL, Filgen, Nagoya, Aichi, Japan) to prevent the generation of electrostatic charges during the measurements. The same FE-SEM instrument was also used to investigate the effect of the plasma treatment on the film-forming state of PDA.

To measure the Pt-loading weight, the Pt particles on the ABS and/or PDA surfaces were dissolved in aqua regia, which was prepared by mixing hydrochloric acid (HCl; 35%, Sigma-Aldrich Japan, Meguro-ku, Tokyo, Japan) and nitric acid (HNO3; 69%, Sigma-Aldrich Japan) at a ratio of 3:1. Then, the Pt concentrations were measured by ICP-AES (ICPE-9000, Shimadzu, Chukyo-ku, Kyoto, Japan) using the diluted aqua regia solutions containing Pt ions. A calibration curve prepared with a standard Pt solution (1000 ppm, Fujifilm Wako Pure Chemical) was used to calculate the amount of Pt in the Pt/ABS samples, as illustrated in Figure S3.

Figure 11 is a schematic of the process for evaluating the catalytic activity and durability of the system using representative H2O2 decomposition curves. First, the Pt/ABS samples were immersed in 5 mL of a 35,000-ppm solution of H2O2 (30 wt%, Kishida Chemical) at 25 ◦C for 360 min in an incubator (i-CUBE FCI-280, AS-ONE, Nishi-ku, Osaka, Japan). H2O2 decomposition occurred with increasing immersion time. The residual H2O2 concentrations were measured after immersion times of 1, 2, 5, 10, 30, 60, 120, 240, and 360 min to obtain the decomposition curves. The method for measuring the H2O2 concentration was the same as that reported in a previous article [16]. The optical absorbance of a H2O2 solution, colored using diluted (5 wt%) titanium sulfate (Ti(SO4)2; 30 wt%, Fujifilm Wako Pure Chemical), was measured using a deuterium–halogen and tungsten lamp (DH-2000, Ocean Optics, Largo, FL, USA), a fiber multichannel spectrometer (HR-4000, Ocean Optics), and optical fiber (P600-1-UV/VIS, Ocean Optics). The absorbance at 407 nm was used to calculate the residual H2O2 concentration from the calibration curve, as presented in Figure S4. The catalytic activity for H2O2 decomposition was evaluated from the value of the residual H2O2 concentration after immersing the Pt/ABS samples in the H2O2 solution for 360 min. This process of immersing the catalyst in the H2O2 solution for 360 min and drying it was repeated 10 times. The residual H2O2 concentrations were measured after one, three, five, and 10 immersions. The catalytic durability for H2O2 decomposition was evaluated from the value of the residual H2O2 concentration after immersing the Pt/ABS samples in the H2O2 solution 10 times (for 360 min each). The target value was less than 100 ppm, which means that if the residual H2O2 concentration was below 100 ppm after repeated use (i.e., after 10 uses), the Pt/ABS sample had long catalytic durability.

**Figure 11.** Schematic of the process for evaluating the catalytic activity and durability of the system using typical H2O2 decomposition curves. \*<sup>1</sup> Value of the residual H2O2 concentration for evaluating the catalytic activity. \*<sup>2</sup> Value of the residual H2O2 concentration for evaluating the catalytic durability.

#### **4. Conclusions**

We introduced PDA coating as a pre-treatment for the strong immobilization of Pt-particle catalysts on ABS substrates and investigated the effect of the PDA coating on the deposition behavior of the Pt particles, the Pt-loading weight, and the catalytic activity and durability of the material. We found that the PDA coating improved both the catalytic activity and durability of the Pt-based material. Moreover, introducing a plasma treatment before the PDA coating was effective for further improving the catalytic durability. Finally, in the case of the Pt/ABS-plasma-PDA(24h) catalyst, the residual H2O2 concentrations were 30, 33, 33, and 35 ppm after using the material 1, 3, 5, and 10 times, respectively. Although the PDA coating also increased the Pt-loading weight (from 5.9 to 24.0 μg/substrate), the value measured for the Pt-particle/ABS-plasma-PDA(24h) catalyst (i.e., 24.0 μg/substrate) was significantly below that determined for a Pt-film/ABS catalyst (1500 μg/substrate) prepared by electroless plating. In summary, we successfully achieved a decrease in Pt usage while maintaining the high catalytic activity and durability. In addition, the developed process, which includes a combination of plasma treatment, PDA coating, and EBIRM, does not require etching of the ABS surface using dangerous chemical solutions, as is the case for electroless plating, where previous etching is necessary to obtain high adhesion between the ABS substrate and the Pt film. Therefore, the developed process is more ecofriendly. Although the PDA coating was used to improve the catalytic durability of a Pt-based catalyst in this study, this type of coating is useful as a pre-treatment for the strong immobilization of metal particles on several substrates or microparticles.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2079-4991/10/1/114/s1. Figure S1: XPS spectra of the ABS surface before and after plasma treatment, Figure S2: Results of XPS analysis of the glass surface before and after PDA coating, Figure S3: Calibration curve for calculating the amount of Pt on Pt/ABS samples, and Figure S4: Calibration curve for calculating the H2O2 concentration.

**Author Contributions:** Y.O., K.E., and K.Y. supervised the work. T.A. and S.S. prepared the Pt particle/ABS samples. Y.O., T.A., and D.K. performed the XPS analysis and SEM observation. Y.O., T.A., and S.S. measured and calculated the Pt-loading weights of the samples using ICP-AES. T.A. evaluated the catalytic activity and durability. O.M. and R.S. helped with the evaluations. All authors contributed to the scientific discussion and manuscript preparation. Y.O. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Japan Science and Technology Agency, grant numbers JST No. MP27215667957 and JST No. VP29117941540.

**Acknowledgments:** We thank the staff of the SHI-ATEX Co., Ltd. for their assistance with the electron-beam irradiation experiments. We also thank Tohru Sekino, Sunghun Cho, and project researcher Hideki Hashimoto for their assistance with the Os coating prior to SEM observations.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Photoluminescent Hydroxylapatite: Eu3**<sup>+</sup> **Doping E**ff**ect on Biological Behaviour**

**Ecaterina Andronescu 1,2,3, Daniela Predoi 4, Ionela Andreea Neacsu 1,2, Andrei Viorel Paduraru 1, Adina Magdalena Musuc 1,5, Roxana Trusca 2,3, Ovidiu Oprea 1,2, Eugenia Tanasa 2,3, Otilia Ruxandra Vasile 2,3, Adrian Ionut Nicoara 1,2, Adrian Vasile Surdu 1,2, Florin Iordache 6, Alexandra Catalina Birca 1,2, Simona Liliana Iconaru <sup>4</sup> and Bogdan Stefan Vasile 1,2,3,\***


Received: 20 July 2019; Accepted: 18 August 2019; Published: 22 August 2019

**Abstract:** Luminescent europium-doped hydroxylapatite (EuXHAp) nanomaterials were successfully obtained by co-precipitation method at low temperature. The morphological, structural and optical properties were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), *X*-ray diffraction (XRD), Fourier Transform Infrared (FT-IR), UV-Vis and photoluminescence (PL) spectroscopy. The cytotoxicity and biocompatibility of EuXHAp were also evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay, oxidative stress assessment and fluorescent microscopy. The results reveal that the Eu3<sup>+</sup> has successfully doped the hexagonal lattice of hydroxylapatite. By enhancing the optical features, these EuXHAp materials demonstrated superior efficiency to become fluorescent labelling materials for bioimaging applications.

**Keywords:** europium doped hydroxylapatite; photoluminescence; MTT assay; oxidative stress assessment; fluorescent microscopy

#### **1. Introduction**

Bioceramics can be defined as the category of ceramics used in repairing and replacing processes of damaged and diseased parts of skeletal system [1,2]. Their biocompatibility varies from inert ceramic oxides to bioresorbable materials. One of the most used bioresorbable ceramics for biomedical applications are calcium orthophosphates (CaP's) [3,4]. The CaP's give hardness and stability to the tissues and can be found in teeth, bones, and tendons. Starting with Ca/P molar ratio equal to 0.5 and finishing with 2.0, there are 11 known non-ion substituted calcium orthophosphates, among which the most used one is hydroxylapatite (HAp) [5,6].

HAp synthesis, with its diverse morphologies, structures and textures, has attracted much interest in academic and industrial research for many heterogeneous catalysis applications [7–9]. Numerous synthetic routes for obtaining hydroxylapatite were developed over time, and can be divided in four main categories: (1) wet methods [10–12], (2) dry methods [13], (3) microwave-assisted methods [14–16], ball-milling [17–19] or ultrasound methods [20,21], and (4) miscellaneous methods [22]. Depending on the reagents and conditions, each category offers several variations [23,24].

Stoichiometric hydroxylapatite (Ca10(PO4)6(OH)2) is the most similar material to the mineral component of human hard tissues and therefore it is considered the ideal substance for bone defects restorations [25–27]. Keeping the same geometry while accepting a big variety of anions and cations is one of the most important structural characteristics of hydroxylapatite [28,29]. Synthetic HAp can also be doped with several metal ions in order to improve its properties, like bioactivity, degradation rate, antibacterial characteristics, luminescence and magnetic properties [30–34].

A photoluminescent material is the most promising candidate for clinical applications and implantation. The biocompatibility is not the only important feature, a longer lifetime of luminescence being also an significant benefit in practical applications [35,36].

Photoluminescence is a very important and useful mechanism in in situ investigations for tissue engineering, surgery, tissue restoration, etc. Using of organic fluorescent molecules for labelling it was a popular practice in clinical trials for many years. Recent studies use inorganic components, even in form of nanoparticles, to replace photoluminescent organic compounds. Because of the toxicity and the nano-size of this particles, the usage of these materials represents a challenge yet [37,38].

Due to high values of the Stokes shift and long lifetime of the excited state, lanthanide coordination compounds are the perfect materials for bioimaging. Europium complexes possess this unique luminescent properties and beside this Europium combine high photoluminescence quantum yields (PLQYs) with the emission in the long wavelength range, which can easily penetrate through the tissues [39–41].

The luminescence of the europium (III) and terbium (III) complexes is especially sensitive to changes in the structure and coordination environment of ions and depends substantially on the interaction with the analyte. The intensive luminescence and characteristic Stark structure of Eu3<sup>+</sup> luminescence spectra allow registering fine changes in the structure of the coordination sphere of a rare-earth ion upon surrounding impact. Luminescent lanthanide-containing complex compounds can be applied as optical chemosensors in detection of anions, cations, gases, etc. [42].

The new generation of biomaterials with multifunctional europium (III)-doped HAp scaffolds has shown remarkable development. The luminescent multifunctional biomaterials show potential for use in various biomedical applications such as smart drug delivery, bioimaging, and photothermal therapy. In this generation, the Eu3<sup>+</sup> ion has been widely used as traceable fluorescence probe due to well-known dopant narrow emission spectral lines by visible-light excitation caused by shielding by the 5 s and 5 p orbitals. The spectral shapes depend on the local ion symmetry and forbidden f–f transitions. Thus, Eu3<sup>+</sup> is a sensitive optical probe for the dopant site environment because of its characteristic luminescence properties [43].

#### **2. Experimental**

#### *2.1. Materials*

All the reagents for synthesis, including calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, 99.0%, Sigma-Aldrich, St. Louis, MI, USA), europium-(III) nitrate hexahydrate (Eu(NO3)3·6H2O, 99.9%, Alfa Aesar, Haverhill, MA, USA), ammonium phosphate dibasic (NH4)2HPO4, 99.0%, Alfa Aesar, Haverhill, MA, USA), sodium hydroxide (NaOH, 25% solution, Alfa Aesar, Haverhill, MA, USA) were used as received, without further purification. Deionised water was used for the experiment.

#### *2.2. Synthesis*

The biocompatible photoluminescent europium-doped hydroxylapatite (EuXHAp) nanomaterials have been synthesized by co-precipitation method. In order to obtain EuXHAp powders, appropriate amounts of calcium nitrate tetrahydrate and europium-(III) nitrate hexahydrate were dissolved in deionized water, under vigorous stirring at room temperature, thus obtaining solution A. Meanwhile, a solution B was prepared by dissolving an appropriate amount of ammonium phosphate dibasic in deionized water, under vigorous stirring at room temperature. The atomic ratio Ca/P and [Ca+Eu]/P

was 1.67, while the atomic ratio Eu/(Eu+Ca) was varied between 0 and 50%. Solution B was added dropwise to solution A, at 80 ◦C, under vigorous stirring, while adjusting and maintaining the pH of the resulting suspension at 10, by adding NH4OH (25%) solution, for 2 h. Pure HAp was synthesized following the same methodology, except the Eu3<sup>+</sup> precursor addition. The resulting suspensions were matured for 24 h. The precipitate was then filtered and washed several times with deionized water, until the pH values were close to 7. Finally, the resulting precipitates were dried at 80 ◦C for 50 h in an air oven.

#### *2.3. Samples Notation*

EuXHAp represents Eu–doped hydroxylapatite, where X is equal to the used europium content (XEu = 0.05, 0.1, 0.15, 0.2, 1.0, 5.0). The correspondent Eu-free hydroxylapatite is noted HAp (XEu = 0).

#### *2.4. Morphological and Structural Characterization*

X-ray diffraction (XRD) studies were carried out using a PANalytical Empyrean diffractometer at room temperature, with a characteristic Cu X-ray tube (λ Cu Kα<sup>1</sup> = 1.541874 Ǻ) with in-line focusing, programmable divergent slit on the incident side and a programmable anti-scatter slit mounted on the PIXcel3D detector on the diffracted side. The samples were scanned in a Bragg - Brentano geometry with a scan step increment of 0.02◦ and a counting time of 255 s/step. The XRD patterns were recorded in the 2θ angle range of 20◦–80◦. Lattice parameters were refined by the Rietveld method, using the HighScore Plus 3.0 e software. The morphology of the samples was analyzed using a Quanta Inspect F50 FEG (field emission gun) scanning electron microscope with 1.2 nm resolution, equipped with an energy-dispersive X-ray (EDX) analyzer (resolution of 133 eV at MnKα, Thermo Fisher, Waltham, MA, USA) on sample covered with a thin gold layer. The high-resolution TEM images of the samples were obtained on finely powdered samples using a Tecnai G2 F30 S-Twin high-resolution transmission electron microscope from Thermo Fisher (former FEI) (Waltham, MA, USA).The microscope operated in transmission mode at 300 kV acceleration voltage with a TEM resolution of 1.0 Å. FTIR spectra were recorded with a Nicolet iS50R spectrometer (Thermo Fisher Waltham, MA, USA), at room temperature, in the measurement range 4000–400 cm−1. Spectral collection was carried out in ATR mode at 4 cm−<sup>1</sup> resolution. For each spectrum, 32 scans were co-added and converted to absorbance using OmincPicta software (Thermo Scientific, Waltham, MA, USA). Raman spectra were recorded at room temperature on a Horiba Jobin-Yvon LabRam HR spectrometer equipped with nitrogen cooled detector. The near–infrared (NIR) line of a 785 nm laser was employed for excitation and the spectral range went from 500 to 1200 cm<sup>−</sup>1. UV-Vis diffused reflectance spectra were obtained using an Able Jasco V-560 spectrophotometer (PW de Meern, Netherlands,) with a scan speed of 200 nm/s, between 200 and 850 nm. The fluorescence spectra were measured by using a Perkin Elmer LS 55 fluorescence spectrophotometer (Arkon, OH, USA). Spectra were recorded with a scan speed of 200 nm/s between 350 and 800 nm, and with excitation and emission slits widths of 7 and 5 nm, respectively. An excitation wavelength of 320 nm was used.

#### *2.5. Cellular Viability Assays*

#### 2.5.1. Quantitative In-Vitro Evaluation of Biocompatibility—MTT Assay

MTT assay is a quantitative colorimetric method, which allows evaluation of cell viability and proliferation, and cytotoxicity of different compounds. The method is based on reduction of MTT tetrazolium salt (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) to dark blue formazan. Reduction by mitochondrial enzymes (especially succinate dehydrogenase) is an indication of cell/mitochondrial integrity. Formazan, insoluble in water, can be solubilized with isopropanol, dimethylsulfoxide or other organic solvent. The optical density (DO) of solubilized formazan is evaluated spectrophotometrically, resulting in a color-absorbent-color-counting function of the number of metabolic active cells in the culture.

The human mesenchymal amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of EuXHAp nanoparticles. The cells were cultured in DMEM medium (Sigma-Aldrich, Saint Luis, MI, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma-Aldrich, Saint Luis, MI, USA). To maintain optimal culture conditions, medium was changed twice a week. The biocompatibility was assessed using MTT assay (Vybrant®MTT Cell Proliferation Assay Kit, Thermo Fischer Scientific, Waltham, MA, USA). Briefly, the AFSC were grown in 96-well plates, with a seeding density of 3000 cells/well in the presence of EuXHAp for 72 h. Then 15 mL Solution I (12 mM MTT) was added and incubated at 37 ◦C for 4 h. Solution II (1 mg Sodium Dodecyl Sulphate + 10 ml HCl, 0,01M) was added and pipettes vigorously to solubilize formazan crystals. After 1 h the absorbance was read using spectrophotometer at 570 nm (TECAN Infinite M200, Männedorf, Switzerland).

#### 2.5.2. Oxidative Stress Assessment—GSH-Glo Glutathione Assay

The GSH-Glo Assay is a luminescent-based assay for the detection and quantification of glutathione (GSH) in cells or in various biological samples. A change in GSH levels is important in the assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death. The assay is based on the conversion of a luciferin derivative into luciferin in the presence of GSH. The reaction is catalyzed by a glutathione S-transferase (GST) enzyme supplied in the kit. The luciferin formed is detected in a coupled reaction using Ultra-Glo Recombinant Luciferase that generates a glow type luminescence that is proportional to the amount of glutathione present in cells. The assay provides a simple, fast and sensitive alternative to colorimetric and fluorescent methods and can be adapted easily to high-throughput applications.

AFSC were seeded at a density of 3000 cells in 300 μL of Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin/neomycin) in 96-well plates. Twenty-four hours after seeding, cells are treated with EuXHAp and incubated for 72 h.

The working protocol consisted of adding 100 μL 1X GSH-Glo Reagent and incubating at 37 ◦C for 30 min. Then, 100 μL Luciferin DeectionReagent was added and incubated at 37 ◦C for an additional 15 min. At the end of the time, the wells were well homogenized and then the plate was read on the luminometer (MicroplateLuminometerCentro LB 960, Berthold, Germany).

#### 2.5.3. Qualitative In-Vitro Evaluation of Biocompatibility—Fluorescent Microscopy

The biocompatibility of the EuXHAp was also evaluated by fluorescent microscopy, using RED CMTPX fluorophore (Thermo Fischer Scientific, Waltham, MA, USA), a cell tracker for long-term tracing of living cells. The CMTPX tracker was added in cell culture treated with EuXHAp nanomaterials and the viability and morphology of the AFSC was evaluated after 5 days. The CMTPX fluorophore was added in the culture medium at a final concentration of 5 μM, incubated for 30 min in order to allow the dye penetration into the cells. Next, the AFSC were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken with Olympus CKX 41 digital camera driven by CellSense Entry software (Olympus, Tokyo, Japan).

#### **3. Results and Discussions**

#### *3.1. X-ray Di*ff*raction*

The *X*-ray diffraction patterns of Eu-doped hydroxylapatite, with different concentration of europium and with pure HAp are shown in Figure 1.

**Figure 1.** *X*-ray diffraction patterns of hydroxylapatite (HAp) and EuXHAp.

Pristine HAp (Figure 1) is shown as a single phase calcium phosphate material (ICDD PDF4+ no.04-021-1904 [44]), with a crystallinity of 33.9% and an average crystallite size of 10.84 nm (Table 1). The XRD patterns of all EuXHAp samples indicate only the pure crystalline hexagonal HAp phase (according to ICDD PDF4+ no.04-021-1904 [44] of the space group P63/m, in consistence with literature [45] up to a substitution degree of 10%. For 50% substitution, Eu(OH)3 secondary phase in a mass ratio of 27.6% has occurred (according to ICDD PDF4+ no. 01-083-2305 [46]). This means that for 50% substitution, the limit of solubility of Eu in HAp lattice was exceeded. The intensities of *X*-ray peaks decrease when the Eu-doping level increases up to 2%, indicating an interference of Eu3<sup>+</sup> with HAp crystal structure. Also, the peak position is influenced by Eu for Ca substitution as they shift to higher angle values which suggest decreasing of unit cell parameters. Table 2 and Figure 2 illustrate the values of unit cell parameters *a*, *c* and *V* and the agreement indices of the Rietveld analysis (*Rexp*, *Rp*, *Rwp* and χ2), which indicate the quality of the fit.


**Table 1.** Calculated crystallite size (*D*) values and degree of crystallinity (Xc) of pure HAp and europium doped hydroxylapatite with various amount of Eu.

**Table 2.** Unit cell parameters *a*, *c*, *V* and agreement indices for hydroxylapatite HAp and Eu-doped HAp (with concentration of Eu3<sup>+</sup> of 0.5, 1, 1.5, 2, 10 and 50%).


**Figure 2.** Unit cell parameters versus substitution degree for HAp and EuXHAp.

Hydroxylapatite crystallizes in a hexagonal symmetry with lattice parameters *a* = *b c* and space group *P63*/*m*. In this structure, PO4 tetrahedral form basic structural units, while the coordination around the distinct Ca sites defines the Ca1O13O23 metaprism and the distorted Ca2O1O2O34(OH) polyhedron. Thus, the formula per unit cell may be expressed as Ca14Ca26(PO1O2O32)6(OHH)2 [47]. Taking into consideration that the ionic radius of Eu3<sup>+</sup> (0.947 Å) is smaller than that of Ca2<sup>+</sup> (1 Å) and that small ions are preferentially substituted at Ca1, in this case Eu3<sup>+</sup> most probably substitutes Ca2<sup>+</sup> from site 2 [48,49]. While a low concentration does not induce significant changes (Figure 2), when looking at higher substitution degree (10% and 50%) it may be concluded that there is an increase of *a*-axis and a decrease of *c*-axis parameters which is consistent with the results obtained for Al3<sup>+</sup> substitution by Fahami et al. [49]. Moreover, the decrease of unit cell volume proves the incorporation of Eu3<sup>+</sup> in hydroxylapatite lattice.

The estimation of crystallite size, lattice microstrain and degree of crystallinity are shown in Table 2 and Figure 3.

Up to 1.5%, the introduction of Eu3<sup>+</sup> in HAp lattice induces an increase of crystallite size from 9.62 nm to 17.3 nm and a decrease of lattice microstrain from 0.95% to 0.65%. For a substitution degree of more than 1.5%, there may be observed a decrease of the crystallite size to 3.55 nm and an increase of lattice microstrain to 2.66% in the case of maximum substitution degree. In what concerns the crystallinity degree (Table 2), as it may be appreciated qualitatively from the profile of the peaks, the crystallinity decreases when the substitution degree increases.

**Figure 3.** The estimated crystallite size, lattice microstrain and degree of crystallinity.

#### *3.2. SEM Analysis*

Figure 4 shows the SEM images (column A) and EDX spectra (column B) of pure HAp (Figure 4a) and EuXHAp samples (Figure 4b).

It can be seen that the doping with Eu3<sup>+</sup> has little influence on the morphology of substituted HAp compared to the pure HAp. SEM images (Figure 4, column A) reveal quasi-spherical (at low dopant concentration) and acicular (mostly after 10% Eu) particles with width in the range of 6–16 nm. Due to high surface area, agglomerates are present in all samples. The EDX spectra of studied samples (Figure 4, column B) confirm the presence of all elements specific to Eu-doped HAp powders: calcium (Ca), phosphor (P), oxygen (O) and europium (Eu).

(**a**)

**Figure 4.** *Cont*.

**Figure 4.** The SEM images (column **A**) and EDX spectra (column **B**) of pure HAp and EuXHAp samples (**a**) HAp, (**b**) Eu0.5HAp, (**c**) Eu1HAp, (**d**) Eu1.5HAp, (**e**) Eu2HAp, (**f**) Eu10HAp, (**g**) Eu50HAp.

#### *3.3. TEM Analysis*

Figure 5 shows the bright field TEM, HRTEM images, SAED patterns and particle size distribution of pure HAp and EuXHAp at different Eu3<sup>+</sup> concentrations.

**Figure 5.** *Cont*.

(**m**) (**n**)

**Figure 5.** *Cont*.

**Figure 5.** The TEM and HRTEM images, SAED patterns and particle size distribution of pure HAp and EuXHAp samples (**a**–**c**) HAp, (**d**–**f**) Eu0.5HAp, (**g**–**i**) Eu1HAp, (**j**–**l**) Eu1.5HAp, (**m**–**o**) Eu2HAp, (**p**–**r**) Eu10HAp, (**s**–**u**) Eu50HAp.

From the bright field TEM images presented in Figure 5a,d,g,j,m,p,s it can be observed that the doping level does not affect the the sample morphologies from 0 to 10% Eu3<sup>+</sup> doping, the only visible modification if for the 50% Eu3<sup>+</sup> doping and it is related to the width (smaller) and length (longer) of the acicular particles. The SEM micrographs together with TEM images confirmed the tendency to form agglomerate due to probably the nanometric dimensions of particles. The size distribution presented in Figure 5c,f,i,l,o,r,u is depended on the dopant concentration. The HRTEM images of the Eu-doped HAp powders are also presented in Figure 5 which allows us to see that the particles are well crystalize and the measured distance of the Miller indices correspond to the hexagonal hydroxylapatite.

#### *3.4. FTIR Spectra*

The FTIR spectra of EuXHAp samples with various europium concentrations are shown in Figure 6. The broad band in the region 3200–3400 cm−<sup>1</sup> corresponds to [OH]- bands of adsorbed water. The strong band at 632 cm-1 (presented in all FTIR spectra) corresponds to [OH]- arising from stretching vibrational mode [50,51]. The typical bands attributed to [PO4] <sup>3</sup><sup>−</sup> can be also found. The bands at around 1090 cm−<sup>1</sup> and about 1040 cm−<sup>1</sup> may be assigned to the antisymetric stretching ν3[PO4] <sup>3</sup><sup>−</sup> of P-O bond, while the band at 962 can be due to the symmetric stretching ν1[PO4] <sup>3</sup>−. The 602 cm−<sup>1</sup> and 564 cm−<sup>1</sup> bands appear from ν4[PO4] <sup>3</sup><sup>−</sup> of P-O bond. The band at 475 cm−<sup>1</sup> can be attributed to the ν2[PO4] <sup>3</sup><sup>−</sup> [52]. In all spectra of EuXHAp a band at 875 cm−<sup>1</sup> was detected, and is due to [HPO4] <sup>2</sup><sup>−</sup> ions [53]. The

intensity of the phosphate bands decreases with increase of europium concentration. The bands at 475 and 962 cm−<sup>1</sup> progressively reduced their intensities with the increase of europium concentration, and disappear in Eu50HAp sample, as already suggested also by XRD results. The different possible mechanisms of Ca substitution with Eu are not fully comprehended and need further studies

**Figure 6.** FTIR spectra of EuXHAp samples.

#### *3.5. Raman Spectroscopy*

The Raman spectra of EuXHAp samples are shown in Figure 7.

**Figure 7.** Raman spectra of EuXHAp samples.

As it can be seen from Figure 7 all analyzed powders present bands attributed to PO4 <sup>3</sup><sup>−</sup> group [54]. Raman spectrum of pure HAp shows a very intense band at 959 cm−<sup>1</sup> attributed to symmetric stretching mode of PO4 <sup>3</sup>−, which is the characteristic peak of HAp. The asymmetric stretching and symmetric bending modes of PO4 <sup>3</sup><sup>−</sup> are also observed at 580 cm−<sup>1</sup> and 1044 cm−<sup>1</sup> [53]. Raman spectra of EuXHAp powders show the internal modes of the frequency ν<sup>1</sup> PO4 <sup>3</sup><sup>−</sup> tetrahedral, which appears at 962 cm−<sup>1</sup> and it corresponds to the symmetric stretching of P–O bonds [55]. There are observed several other bands at 580 cm−<sup>1</sup> and 610 cm−<sup>1</sup> attributed to ν<sup>4</sup> PO4 <sup>3</sup>−, and 1048 cm−<sup>1</sup> and 1080 cm−1, respectively, attributed to ν<sup>3</sup> PO4 <sup>3</sup>−. The intensity of the characteristic peak of HAp, compared to the other secondary peaks, decreases with increasing of the europium content.

#### *3.6. UV-Vis and PL Spectra*

The electronic spectra of EuXHAp samples (Figure 8) contain several bands, which increase in their intensity with increasing of europium content.

**Figure 8.** (**A**) UV-Vis absorption spectra of EuXHAp at different Eu-concentrations (**B**) Room-temperature photoluminescence spectra of EuXHAp at different Eu-concentrations.

The pure HAp has an absorption peak in UV at 218 nm. The 242 nm (41322 cm−1) absorption peak is absent in HAp, but is increasing in intensity from a weak shoulder in Eu0.5HAp to a strong, broad peak in Eu50HAp. This is a charge-transfer band characteristic to Eu3<sup>+</sup> in oxides [56]. The band which appears at 395 nm, close to the visible light domain, corresponds to the 7F0 <sup>→</sup> 5L6 transition, and is the most intense transition of europium (III) in UV-Vis absorption spectra [57]. Another three bands appear at 465, 526 and 535 nm, assignable to 7F0 <sup>→</sup> 5D2; 7F0 <sup>→</sup> 5D1; and 7F1 <sup>→</sup> 5D1, transitions respectively [56]. The last peak, at 535 nm, is belonging to a group called "hot" bands because it can be observed only at room temperatures or higher since it requires the thermal population of 7F1 level (at room temperature ~35% of ions are populating this level, rest being on 7F0 ground state) [56].

Figure 8B presents the photoluminescence emission spectra of EuXHAp samples excited with 320 nm wavelength. Usually, HAp sample shows three strong emission peaks at 458 nm with two shoulders (433 nm and 446 nm), 403 nm and 482 nm, respectively, associated with various oxygen defects. The intensity of luminescence for HAp is enhanced by the presence of small quantities of Eu3<sup>+</sup> ions. The effect is stronger for small numbers of dopant ions and is decreasing as the quantity of Eu3<sup>+</sup> ions is increasing.

Over the fluorescence maxima of HAp, the emission spectra of europium are superposed. The peak at 389 nm is due to the 7F0 <sup>→</sup> 5L6 electronic transition, the peak at 512 nm is due to the 7F0 <sup>→</sup> 5D1 electronic transition and the peak at 526 nm is due to the 7F1 <sup>→</sup> 5D1 electronic transition. The characteristic emission peaks at 589 nm and 613 nm are due to the 5D0 <sup>→</sup> 7F1 and 5D0 <sup>→</sup> 7F2

electronic transition, indicating a red fluorescence [58]. For higher Eu3<sup>+</sup> concentration even the 648 nm emission peak of 5D0<sup>→</sup> 7F3 becomes visible. As expected, when the europium doping concentration increased, the characteristic emission intensity at 589 and 613 nm are also increased.

Beside the two dominant peaks, the weak 5D0 <sup>→</sup> 7F0 transition from 579 nm (which can be observed in emission spectra of Eu50HAp) is related with Eu3<sup>+</sup> ions distributed on Ca2<sup>+</sup> sites of the apatitic structure [59]. The dominant emission peaks present no shift due to modification of Eu3<sup>+</sup> concentration, the most intense being the hypersensitive 5D0 <sup>→</sup> 7F2 transition from 613 nm. This feature shows the potential application of the EuXHAp compounds to be tracked or monitored by the characteristic luminescence.

#### **4. Biocompatibility and Cytotoxicity of EuXHAp Photoluminescent Ceramic Materials**

Cytotoxic effect of EuXHAp was evaluated by measuring the metabolic activity of AFSC using MTT assay. EuXHAp biomaterials did not have cytotoxic effect, the absorption values being near or higher compared to the control sample. Furthermore, the EuXHAp increases cellular metabolism, suggesting that it stimulates cell proliferation. The proliferation increases between 12%–58%, depending on the sample, highest increase being observed for Eu2HAp (58%) (Figure 9). The results are similar to other research groups that showed the viability of HGF-1 fibroblast was decreased in the Eu10Hap after 48 h. Naderi et al. 2012 [60] tested different concentrations of nanohydroxyapatite from 2 to 0.002 mg/mL on gingiva-derived fibroblast cell line (HGF-2) at 24, 48, and 72 h, and concluded that after 24 h high doses of nanohydroxyapatite have cytotoxic effect on gingival-derived fibroblasts suggesting that cytotoxicity is dependent on the cell line [60,61].

**Figure 9.** MTT assay showing the viability of AFSC in the presence of the EuXHAp ceramic materials: HAp, Eu05HAp, Eu1HAp, Eu1.5HAp, Eu2HAp, Eu10HAp, Eu50HAp, and control (cell only).

Glutathione, an oxidative stress marker, is capable of preventing cellular damage caused by reactive oxygen species, such as free radicals, peroxides, lipid peroxides and heavy metals. In the presence of EuXHAp biomaterials, AFSC responded similarly to control cells, indicating that the analysed materials did not induce cellular stress (Figure 10). Furthermore, the morphology of AFSC was investigated by fluorescence microscopy using CMTPX cell tracker for long-term tracing of living cells. Cellular metabolism is active, as shown in microscopy images, cells absorbing CMTPX fluorophore in the cytoplasm, suggesting that they are viable.

**Figure 10.** GSH assay showing the oxidative stress of AFSC in the presence of the EuXHAp ceramic materials: HAp, Eu05HAp, Eu1HAp, Eu1.5HAp, Eu2HAp, Eu10HAp, Eu50HAp and control (cell only).

After 5 days in the presence of EuXHAp, AFSCs presents a normal morphology with fibroblastic-like characteristic appearance (Figure 11). Fluorescent images show that AFSC cells are viable, no dead cells or cell fragments are observed, more the cells spread filopodia to move and establish contacts with neighboring cells, suggesting that AFSC have an active phenotype.

**Figure 11.** *Cont*.

**Figure 11.** Fluorescence images of EuXHAp samples coloured with CMTPX fluorophore (**a**) Control sample, (**b**) Eu0.5HAp, (**c**) Eu1HAp, (**d**) Eu1.5HAp, (**e**) Eu2HAp, (**f**) Eu10HAp, (**g**) Eu50HAp.

#### **5. Conclusions**

In recent years, a special attention has been created regarding to multimodal imaging. This technique represents various imaging modalities in the manner of photoluminescence and magnetic resonance imaging that are generally connected in an individual diagnostic step. If we talk about multimodal contrast agents that can be used or marker for biomedical imaging with luminescence, literature data provides information on the use of doped hydroxyapatite with rare earth elements like europium of nanometric dimensions.

The aim of the present study was to obtain europium-doped nano hydroxylapatite by using a simple method of synthesis (co-precipitation method) and, thereafter characterize by physico-chemical techniques and also by biological point of view because of the fact that the obtained material has medical application.

The results after the XRD analysis shows that the obtained material is represented by pure hexagonal phase of hydroxylapatite, when using up to 10% dopant Eu3<sup>+</sup> ions concentration, but there is a correlation between HAp and Eu3<sup>+</sup> by the interference of Eu3<sup>+</sup> ions with HAp crystal structure. About the morphology of HAp doped with Eu3<sup>+</sup> it can be concluded that there is a limited modification after the addition of Eu3<sup>+</sup> and also by the EDS spectra is confirmed the presence of Eu3<sup>+</sup> in all obtained materials. Typically, the HAp sample shows three strong emission peaks, but the photoluminescence intensities is enhanced by the characteristic Eu3<sup>+</sup> peaks with the presence of Eu3<sup>+</sup> ions. This feature is required and necessary to have a good action in medical imaging.

From the biological point of view, it can be seen by the MTT assay results that the obtained material supports the proliferation process of the amniotic fluid stem cells. Also, the best results about the viability characteristic is offered by the Eu2HAp material which can be a standard for the doped HAp, because after the addition of more Eu3<sup>+</sup> ions there is a small evidence about the cellular viability that can decrease. As a complete evaluation, the qualitative information by the fluorescence microscopy gives characteristic about the biocompatibility of the material in light of the fact that after 5 days of incubation with amniotic fluid stem cells, no dead cells are observed.

Therefore, there is a fine line about the using doped HAp with Eu3<sup>+</sup> as fluorescent or multimodal contrast agent, but the results can be a promising start due to its characteristics but there is a need for the further investigation.

**Author Contributions:** The authors have participated to the paper as follows; conceptualization, B.S.V., D.P.; methodology, E.A., B.S.V., D.P., S.L.I.; validation, E.A., B.S.V.; formal analysis, I.A.N., A.P., R.T., O.O., E.T., O.R.V., A.I.N., A.V.S., F.I., A.C.B., S.L.I.; investigation, I.A.N., R.T., O.O., E.T., O.R.V., A.I.N., A.V.S., F.I., A.C.B.; data curation, A.V.P., A.M.M.; writing—original draft preparation, A.M.M, A.V.P.; writing—review and editing, I.A.N., B.S.V., A.M.M.; visualization, E.A.; supervision, E.A., B.S.V.; project administration, E.A.

**Funding:** This research was funded by project "Innovative biomaterials for treatment and diagnosis" BIONANOINOV—P3 grant number PN-IIIP1-1.2-PCCD-I2017-0629, and the support of the EU-funding project POSCCE-A2-O2.2.1-2013-1/Priority Axe 2, Project No. 638/12.03.2014, ID 1970, SMIS-CSNR code 48652 is gratefully acknowledged for the equipment's purchased from this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

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