**The Bioactive Polypyrrole**/**Polydopamine Nanowire Coating with Enhanced Osteogenic Di**ff**erentiation Ability with Electrical Stimulation**

## **Yuan He 1, Lingfeng Dai 1,2, Xiuming Zhang 1, Yanan Sun 1, Wei Shi 1,\* and Dongtao Ge 1,\***


Received: 11 November 2020; Accepted: 3 December 2020; Published: 5 December 2020

**Abstract:** Polypyrrole (PPy) is a promising conducting polymer in bone regeneration; however, due to the biological inertia of the PPy surface, it has poor cell affinity and bioactivity. Based on the excellent adhesion capacity, biocompatibility, and bioactivity of polydopamine (PDA), the PDA is used as a functional coating in tissue repair and regeneration. Herein, we used a two-step method to construct a functional conductive coating of polypyrrole/polydopamine (PPy/PDA) nanocomposite for bone regeneration. PPy nanowires (NWs) are used as the morphologic support layer, and a layer of highly bioactive PDA is introduced on the surface of PPy NWs by solution oxidation. By controlling the depositing time of PDA within 5 h, the damage of nano morphology and conductivity of the PPy NWs caused by the coverage of PDA deposition layer can be effectively avoided, and the thin PDA layer also significantly improve the hydrophilicity, adhesion, and biological activity of PPy NWs coating. The PPy/PDA NWs coating performs better biocombaitibility and bioactivity than pure PPy NWs and PDA, and has benefits for the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells cultured on the surface. In addition, PPy/PDA NWs can significantly promote the osteogenesis of MC3T3-E1 in combination with micro galvanostatic electrical stimulation (ES).

**Keywords:** PPy/PDA nanocomposite; nanowires; polydopamine; electrical stimulation

## **1. Introduction**

Conducting polymers (CPs) especially polypyrrole (PPy) with excellent conductivity, long-term stability and biocompatibility have been widely applied in many fields [1–3]. In particular, the conductivity of CPs enables their use as sensors [4], controlled drug release [5,6], and functional tissue repair materials [7,8]. Moreover, CPs with nano morphology, which mimics the natural morphology of the extracellular matrix (ECM), can be easily fabricated via electrochemical and chemical polymerization on various substrates, and makes it an outstanding coating used in the biomedical field [9,10]. Actually, many studies confirmed that the PPy applied with electrical stimulation (ES) could promote the expression of proliferation and differentiation for tissue regeneration such as bone and nerves [11–13]. However, PPy does not display functional groups to help it adhere to surfaces [14]; therefore, generating composite materials with PPy and other adhesive polymers can effectively enhance the functionality of PPy-containing coatings.

Polydopamine (PDA) could be synthesized by the self-oxidative polymerization of dopamine (DA), which is the most abundant catecholamine neurotransmitter in the brain. PDA has excellent biocompatibility and biological activity [15]. The rich functional groups, especially the catechol compounds, offers PDA astonishing adhesive ability on a variety of materials [16,17]. Many studies focusing on the use of PDA as a surface functional modification material to improve the biocompatibility and surface modifiability of other materials were reported [18]. However, the PDA has poor conductivity without electrical signal response. Moreover, the inescapable fact is that the oxidative polymerization of dopamine (DA) is uncontrollable, which means it is very difficult to control the morphology [19,20], and the condition for preparing the PDA coating with nano morphology is quite harsh.

At present, many researchers are attentive to the preparation of PPy/PDA composites, which can solve the drawbacks of the above-mentioned materials. This kind of composites can be fabricated via one-pot or two-step method to avoid the disadvantages of pure PPy and PDA [21–24]. By controlling reaction conditions, the pyrrole (PY) and DA composite material has both good morphology, conductivity, and bioactivity. Zhao et al. have demonstrated that the copolymer of DA-PPy performs improved water dispersibility, improved electrical conductivity and film adhesion than pure PPy [17,25]. Electrochemical method has more advantages than chemical polymerization in the preparation of nanostructured conducting coatings with better controllability and uniformity of thickness and morphology. By precise regulating the concentration of PY and DA, PDA/PPy composite coatings with nanowires and vesicular structure morphology with enhanced conductivity, adhesion and cell affinity have been successfully prepared via a one-pot electrochemical polymerization [26–28]. In the one-pot reaction system of PY and DA, the concentration of PY and DA both have obvious effects on the morphology and conductivity of the copolymer. The concentration of dopamine and the polymerization potential need to be precisely controlled in order to obtain the copolymer with nano morphology. Compared with the co-polymerization of PPy/PDA, the reaction condition for preparing PPy/PDA composite coatings by a two-step method is relatively simple, as for the reaction condition of preparation pure PPy coating with various nano structure and size morphology can be controlled more easily and precisely by electrochemical deposition [29,30]. However, in order to better control the morphology and conductivity of the products, the most reported study on preparation of PPy/PDA composites via a two-step method is always setting fabrication of PPy layer as the final step to ensure the excellent conductivity [23,31]. Taking the PDA as the outer layer is more beneficial to improve the biocompatibility of PPy/PDA composites, and the key point is to precisely control the thickness of PDA deposition. The best result is that the deposition of PDA on the surface of PPy NWs will not significantly change the nano morphology of the first layer, nor reduce the conductivity.

In this study, the PDA modified PPy/PDA nanowire (NW) coating is synthesized by a two-step reaction (Figure 1). The first layer is preparation PPy NWs coating by electrochemical polymerization as the template, and the PDA layer is following deposited on each PPy NWs surface by solution oxidation. By regulating the deposition time of PDA, the influence of different PDA depositing time on the conductivity of PPY/PDA NWs coating is studied, and the optimal PDA deposition time is determined. The morphology, chemical structure, hydrophilicity, adhesiveness are further characterized. The cell affinity and bioactivity of the PPy/PDA NWs coating on MC3T3-E1 cell are further evaluated. The PPy/PDA NWs coating provides enhanced hydrophilicity and adhesiveness. The introduced PDA layer indeed improves the cell adhesion, proliferation, and differentiation of MC3T3-E1. Combined with ES, the PPy/PDA NWs coating shows better osteogenic activity to accelerate maturation of MC3T3-E1 cells.

**Figure 1.** Illustration of preparation of PPy/PDA NWs coating and the synergistic effect with electrical stimulation on MC3T3-E1.

## **2. Materials and Methods**

## *2.1. Materials*

Pyrrole (PY, 98%) and sodium p-toluenesulfonate were purchased from J & K Chemical Ltd. (Beijing, China) Dopamine hydrochloride (98%), Vitamin C and β-glycerophosphate were bought from Sigma-Aldrich (Shanghai, China). Tris(dydroxymethyl) aminomethane (Tris-HCl, 99%) was purchased from Sangon Biotech Co. Ltd. (Shanghai, China). Other used inorganic salt reagents such as sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), hydrochloric acid (HCl) were bought from SINOPHARM (Beijing, China). Monocrystal silicon wafer (p-type, 0.5 mm) was taken from Xiamen Zhongli Technology Co. Ltd. (Xiamen, China).

Before used, pyrrole need to be purified by distillation under the protection of N2 gas and stored in the refrigerator at −20 ◦C for future use. Other reagents can be used directly without additional treatment. The water used in experiments was ultrapure water (18.2 MΩ).

Monocrystal silicon wafers were first cleaned via boiled in Piranha solution (H2SO4:H2O2 = 4:1) for 15 min and washed in hot water, then dried by nitrogen for further use. Next, a 500 nm thick of oxide layer was formed on the silicon surface by thermal diffusion oxidation. Finally, 50 nm titanium and 100 nm gold layer were fabricated in the silicon wafer surface via magnetron sputter. The Au/Ti/Si wafer was diced for further experiment.

## *2.2. Synthesis of PPy*/*PDA Nanowires (NWs) Coating*

The PPy/PDA NWs coating was fabricated via a two-step method. In brief, the first step was the polymerization of PPy NWs layer by electrochemical deposition, and the second step was the deposition of PDA layer on the surfaces of PPy NWs via solution oxidation.

First, Pyrrole monomers could be polymerized with proper oxidation potential to form PPy. In this manuscript, the PPy NWs coating was directly fabricated by electrochemical polymerized on the Au/Ti/Si wafer under a potential at 0.8 V for 200 s with CHI660D electrochemical workstation (Chen Hua Instrument Company, Shanghai, China). The electropolymerization process of pyrrole was carried out in a traditional three-electrode system, in which the Au/Ti/Si wafer was the working electrode, a platinum wire was the counter electrode and a saturated calomel electrode (SCE, INESA Scientific Instrument Co. Ltd., Shanghai, China) was the reference electrode. The pyrrole monomer was dissolved in the electrolyte of 0.2 M PBS solution (pH = 7.4) containing 0.14 M of pyrrole and 0.085 M sodium p-toluenesulfonate. After 200 s, a uniform layer of PPy NWs can be deposited on the surface of the Au/Ti/Si substrate.

Second, the PDA layer was directly deposited on the PPy NWs by immersing the PPy NWs substrate into the fresh 2 mg/mL DA alkaline Tris-HCl solution (10 mM, pH = 8.50). The whole reaction vessel was opened and standing at room temperature for 5 h. Finally, the PPy/PDA NWs coated substrate was fished out and cleaned with distilled water.

The pure PPy NWs coating was fabricated by electrochemical polymerized the same as the first step. The Pure PDA coating was obtained on Au/Ti/Si substrate by direct immersing the bare Au/Ti/Si substrate in the fresh 2 mg/mL DA alkaline Tris-HCl solution (10 mM, pH = 8.50) for 5 h. The fabricated PPy/PDA NWs, PPy NWs, and PDA coatings were carefully washed with distilled water and dried in vacuum at 50 ◦C for further use.

#### *2.3. Characterization*

The morphologies of PPy NWs and PPy/PDA NWs were observed by scanning electron microscopy (SEM, SU-70, Hitachi, Tokyo, Japan) at an accelerating voltage of 10.0 kV and transmission electron microscopy (TEM, JME-2100, JEOL, Tokyo, Japan) under 200 kV accelerating voltage. The chemical composition of pure PDA, PPy, and PPy/PDA were characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet IN10, Thermo Scientific, Waltham, MA, USA) with potassium bromide (KBr) tablet method. The static contact angle of different coating samples was measured by a contact angle meter (JC2000A, Shanghai Zhongchen digital technic apparatus Co. Ltd, Shanghai, China) with 2 μL water dropped at five different positions for each sample. Each sample randomly tested three parallel specimen. The electrical conductivity of the PPy and PPy/PDA NWs coatings was evaluated by a four-point probe apparatus (SZ83, Suzhou Baishen Technology Co. Ltd, Suzhou, China). Adhesion ability of PPy NWs and PPy/PDA NWs coating with the substrate was tested with ScotchTM MagicTM Tape 810 (3M, Saint Paul, MN, USA) and each sample was repeated three times in the same direction. Cyclic voltammetry (CV) was conducted in the 0.9% NaCl solution with a CHI660D electrochemical workstation. The voltage test ranging from −0.9–0.5 V at the scan rate of 10 mV/s.

#### *2.4. Cell Culture*

The mouse embryonic osteoblast precursor cells (MC3T3-E1) was bought at the Wuhan Cell Bank of Chinese Academy of Sciences Basic Science Cell Center. MC3T3-E1 cells were cultured in the alpha-modified minimum essential medium (α-MEM, Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin and streptomycin (Hyclone, Chicago, IL, USA) in the saturated humidity incubator with 5% CO2 at 37 ◦C. During the normal subculture of cells, the medium was changed every two days.

#### *2.5. E*ff*ect of Surface Morphology on MC3T3-E1 Adhesion and Proliferation and Di*ff*erentiation*

Three different morphology substrates including PPy NWs, PDA and PPy/PDA NWs coatings on Au/Ti/Si wafers and bare Au/Ti/Si wafers were first sterilized by soaking in 75% ethanol for 24 h and following immersing in distilled water for another 24 h. The bare Au/Ti/Si wafer was the control group. Before the experiment, all substrates were further irradiated under UV radiation for another 2 h.

The sterilized substrates with different morphology were placed in the 24-well plate and washed with PBS. Then 1 mL of MC3T3-E1 (2 <sup>×</sup> 104 cells/mL) suspension was seeded to each well and then cultured in cell incubator (5% CO2, 37 ◦C).

## 2.5.1. MTT Test

To test the adhesion and proliferation ability of MC3T3-E1 cell on different substrates, the MTT assay was used to evaluate the cell viability after cells were seeded and cultured for 4, 6, 8, 12, 24, and 48 h, respectively. In brief, at the tested time point, the cell culture was carefully removed and washed with PBS twice. 500 μL fresh culture medium containing 10% MTT working solution (5 mg/mL) was added to each well and continued to incubate for another 4 h. Next, the working solution was completely removed and incubated with 500 μL DMSO to dissolve the formazan crystal under 37 ◦C. Finally, the optical density was tested at 570 nm using a Microplate reader (Infinity 200 pro, Tecan, Männedorf, Switzerland). Each sample was set four-parallel experiments.

## 2.5.2. Cell Morphology

The cell morphology of adhesion and proliferation was observed via Fluorescence microscope (Leica DM2500, Leica Camera, Wetzlar, Germany). By staining the filamentous actin (F-actin) of the MC3T3-E1 cytoskeleton, the cell spreading morphology on substrates could be observed. Generally, after MC3T3-E1 cells were cultured for 12 and 24 h, cells were rinsed with PBS and then fixed with 4% paraformaldehyde solution for 15 min. After washed with PBS, cells were continued treated with 0.1% TritonX-100 solution to enhance the permeability of cell membrane. Next, the cell was incubated in 1% BSA solution for 20 min and washed with PBS. Followed above treatments, cells were finally stained with 5 U/mL Alexa Fluor 568 phalloidin solution for 30 min in the dark environment. Samples were observed by using a fluorescence microscope.

The morphology of MC3T3-E1 cell on different substrates was further watched with SEM. After cultured for 12 h, cells were fixed and dehydrated with gradient ethanol successively. After drying, samples were sputter-coated with gold before SEM testing.

#### 2.5.3. Total Protein Content and Alkaline Phosphatase Activity

The MC3T3-E1 cell proliferation and differentiation activities were further measured by testing the total protein content and alkaline phosphatase (ALP) activity in the period of cell proliferation and differentiation. After MC3T3-E1 cell was seeded and cultured on different substrates for 24 h, the culture medium was changed to induced medium, which additionally added 1% Vitamin C (5 mg/mL) and β-glycerophosphate (1 M). The cells were cultured normally and the fresh induced medium was changed every 2 days.

The total protein content and ALP activity were tested at 3, 7 and 14 d. At each time point, cells in each group were washed with PBS and lysed on ice by incubation with 0.1% TritonX-100-PMSF pyrolysis liquid for 3 min, then centrifuged at 4 ◦C for 10 min. The supernatant was collected for further protein content and ALP activity detections. Total protein content measured by using a commercial BCA protein Assay Reagen Kit (Beyotime Institute of Biotechnology, Shanghai, China) and tested absorption signal at 562 nm. The ALP activity was tested using the commercial ALP Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China), and measured the absorption intensity at 405 nm. Each sample was set four-parallel experiments.

## *2.6. E*ff*ect of Electrical Stimulation on MC3T3-E1 Adhesion and Proliferation and Di*ff*erentiation*

Micro constant current electrical stimulation signal (10 μA) was directly applied on the surface of different substrates (PPy NWs and PPy/PDA NWs) via an electrochemical workstation. ES was applied from the 12 h after cells inoculation and lasted for 2 h per day for 14 days. The cell proliferation and mineralization activity detection were the same as described in the above part.

## *2.7. Statistical Analysis*

Quantitative results were presented as mean ± standard deviation. Statistical differences among groups were analyzed by ANOVA followed by Turkey's posttest. *p* < 0.05 was considered a significant difference and represented by \*, and *p* < 0.01 was considered a highly significant difference and represented by #.

### **3. Results and Discussion**

## *3.1. Characterization of PPy*/*PDA NWs*

The PPy/PDA NWs was fabricated via a two-step method. First, PPy NWs coating was electrochemical polymerized on the surface of Au/Ti/Si wafer. Under the constant voltage of 0.8 V, after 200 s, the gold electrode surface could be covered with a layer of black PPy coating completely without revealing the color of the substrate, which mean the PPy coating could effectively cover the substrate with sufficient thickness. SEM images further showed the detailed morphology of obtained PPy NWs (Figure 2a). The PPy coating had nanowire morphology. PPy NWs were slender and uniform, and tightly wound with each other. The high magnification SEM and TEM images (Figure 2c,e) detailed showed the structure of PPy NWs, the surface of PPy NWs was smooth with a diameter of about 40–60 nm. Next, after immersing PPy NWs coating into the fresh dopamine (DA) alkaline solution for several hours, a layer of PDA could be formed on PPy NWs. The PDA coated PPy NWs composites retained the former nanowire morphology with uniform size (Figure 2b,d). While compared with pure PPy NWs, the surface roughness of PPy/PDA nanowires increased, and the PDA protrusion structure adhered to the surface of PPy nanowire could be observed clearly by TEM image (Figure 2f). At the same time, the diameter of PPy/PDA NWs also increased to about 80–100 nm, indicating that PDA could be successfully polymerized on the surface of each single PPy nanowire, and the PPy/PDA had core-shell structure.

PPy has excellent electrical conductivity and electrochemical activity. Many studies were conducted to prepare responsive coatings with electrical conductivity based on the electrical conductivity of PPy. As a bioactive material, PDA can significantly improve the bioactivity of the composite. However, PDA has poor conductivity. In alkaline solution, the thickness of PDA increases with the reaction, and after reacted for 24 h, the thickness of PDA could reach above 50 nm [32]. In addition, an excessively thick layer of PDA deposition covered on PPy NWs not only concealed the nano-scale morphology of PPy/PDA composite, but also significantly reduced the conductivity of the material. Therefore, it was very important to control the depositing thickness of PDA effectively on PPy NWs surface in order to ensure the maximum retention of excellent nano morphology and conductivity. As Figure 3 indicated, by controlling the PDA depositing time, the morphology and conductivity of the PPy/PDA nanocomposite performed regular changes. Within 5 h of PDA polymerization, the morphology of the PPy NWs could be well maintained and no obvious PDA aggregates were observed. With the prolongation of deposition time, a large number of PDA random aggregates were adhered on the surface of PPy NWs. When the deposition time prolonged to 15 h, PDA particles even filled into the gap of PPy NWs, and the nanostructure morphology was gradually disappeared. At 20 h, PDA almost completely covered the whole PPy NWs coating (Figure 3a). At the same time, the conductivity change of PPy/PDA coating also displayed cliff fall after deposition time above 5 h (Figure 3b). Therefore, the deposition time of PDA was chosen at 5 h. Under this condition, the PPy/PDA nanocomposite coating could retain both good nanowire morphology and conductivity (about 1 S·cm<sup>−</sup>1).

**Figure 2.** SEM images of PPy NWs (**a**) and PPy/PDA NWs (**b**). High resolution SEM images of PPy NWs (**c**) and PPy/PDA NWs (**d**). TEM images of PPy NWs (**e**) and PPy/PDA NWs (**f**).

**Figure 3.** (**a**) SEM images of PPy/PDA NWs with different deposition time of PDA. (**b**) The conductivity of PPy/PDA NWs with different deposition time of PDA.

Comparing the FTIR spectra of pure PDA, PPy and PPy/PDA nanocomposite, it could be seen that the PPy/PDA nanocomposite possessed characteristic peaks of both pure PDA and PPy (Figure 4a): the broad peak around 3400 cm−<sup>1</sup> was belonging to the N–H and O–H stretching vibrations, where PDA and PPy both have a broad peak. The peak at 1544 cm−<sup>1</sup> was the characteristic peak of PPy attributed to the symmetric stretching vibrations of pyrrole ring [33]; and the characteristic peaks of PDA at 1615 and 1488 cm−<sup>1</sup> ascribing to C=C and C=N/N–H stretching vibrations in aromatic amine species were also appeared [34,35], which further indicated the PDA was successfully deposited on the surface of PPy NWs to form a composite structure. Furthermore, the change of element proportion could also prove the successful modification of PDA by XPS analysis (Figure 4b). PY and DA monomers have

the same elemental composition, but there is a significant difference in the N/C ratio between PY and DA. PY (1:4) has higher theoretical N/C ratio than that of DA (1:8). After polymerization, it could be seen that the N/C ratio of PPy NWs coating (0.164) was higher than that of pure PDA coating (0.094). In addition, after PDA deposited, the N/C ratio in PPy/PDA NWs coating decreased to 0.14, which further demonstrated that PDA was successfully deposited on the surface of PPy NWs.

PDA had better hydrophilicity than PPy due to rich hydrophilic groups. The contact angle measurement displayed the change of surface hydrophilicity after PDA deposition (Figure 4c). For the bare Au surface, it had large hydrophobicity, which the average contact angle was more than 90◦. After the Au surface was covered by PPy NWs or pure PDA, the water contact angle decreased more than 30◦, and nanowire morphology was more conducive to the decrease of contact angle. The PPy/PDA NWs coating had the minimum water contact angle, which was only 16◦. The excellent hydrophilicity of PPy/PDA composite mainly came from both the rough porous nanostructure and a large number of exposed hydrophilic groups such as ammonia hydroxyl groups on the surface of PDA.

PDA have astonishing adhesion ability, which can adhere to a variety of materials. We used the 3M tape to test the adhesion ability of different coatings with the substrate by tear test (Figure 4d). The result demonstrated that the deposited layer of PDA could even enhance the adhesion property of the PPy/PDA NWs coating with the Au surface compared with pure PPy NWs. After several instances of the tear test, PPy/PDA NWs coating still adhered to the substrate firmly with almost no material loss. Therefore, the enhancement of surface hydrophilicity and the increase of adhesion stability with substrates made PPY/PDA NWs a better coating material than pure PPy NWs.

The PPy/PDA NWs had good conductivity. The electrochemical property was measured by cyclic voltammetry in 0.9% NaCl solution (Figure 4e). Pure PDA was hardly conductive and had no obvious electrochemical activity. After PDA deposited, compared with pure PPy NWs, the electrochemical activity of PPy/PDA NWs did not decrease significantly. However, compared with pure PPy NWs, the redox peak of PPy/PDA NWs was slightly shifted, indicating that PDA was not only successfully modified on the surface of PPy, but also interacted with PPy.

**Figure 4.** (**a**) FITR spectra of pure PPy NWs, PDA and PPy/PDA nanocomposite NWs. (**b**) XPS spectra of PPy NWs, PDA and PPy/PDA nanocomposite NWs. (**c**) The water contact angle of different material substrates. (**d**) The adhesion test of pure PPy NWs and PPy/PDA NWs. (**e**) Cyclic voltammogramms of different materials in 0.9% NaCl solution at the scan rate of 10 mV/s.

The above experiments demonstrated that via a two-step method, the PPy/PDA NWs nanocomposite coating was obtained. Compared with pure PPy NWs, PPy/PDA can effectively increase the hydrophilicity of the coating and the firmness of adhesion with the substrate materials while retaining the excellent nanostructure, conductivity, and electrochemical activity. PDA has excellent biocompatibility and bioactivity. It has been confirmed that PDA can promote osteoblast cell adhesion and biomineralization [14]. We speculated that this nano-structured PPy/PDA NWs might have excellent promoting effect on osteogenesis than pure PDA and PPy.

## *3.2. E*ff*ect of PPy*/*PDA NWs Coating on MC3T3-E1 Activity*

When contacting with materials, in the beginning 8–12 h, the main behavior of cells is to interact with the material and then adhere to the surface of material. The morphology, structure and chemical microenvironment of the materials all have significant effects on anchorage-dependent cell adhesion. SEM images showed that at the initial 12 h after cells were seeded, MC3T3-E1 cell could adhere and spread well on pure PPy NWs, PDA and PPy/PDA NWs coatings (Figure 5a). MTT test indicated that compared with bare Au, all modified coatings including pure PPy NWs, PDA and PPy/PDA NWs could promote the adhesion and proliferation of MC3T3-E1 cells, and the PPy/PDA NWs coating performed most effective facilitation (Figure 5b). SEM and fluorescence images detailed showed the morphology of MC3T3-E1 cells adhesion and spreading on the surface of different materials (Figure 5a). After 12 h, most cells on PPy NWs coating performed spindle and fibrous shape with some pseudo feet anchored on near PPy NWs. On the other hand, cells on the surface of PDA were flat and full spreading, and there were lots of pseudopods linked cells with each other, which proved that PDA can promote cell spreading better than PPy NWs, while MC3T3-E1 cells grew on PPy/PDA NWs coating were well spreading than pure PPy NWs. Most cells on PPy/PDA NWs surface were spreading flat, and pseudopodia filaments of cells were dispersed into surrounding nanowires and adhered closely to the substrate. Moreover, MC3T3-E1 cells were interconnected with each other through a large number of pseudopods. After 12 h of inoculation, the adherent cells entered the fast proliferative phase (Figure 5a,b). MC3T3-E1 cells on all substrates were grew well, and cells on PPy/PDA NWs coating had the fastest growth rate, which ensured that the PPy/PDA NWs coating had excellent biocompatibility and bioactivity and was more benefit for cell adhesion and proliferation than pure PPy NWs and PDA.

For the PPy/PDA NWs nanocomposite, the PDA deposition introduced abundant active functional groups on the inert surface of PPy, which facilitated early cell recognition and anchoring adhesion process. Moreover, the morphology of nanowires was closer to the natural extracellular matrix (ECM) morphology and the primary growth environment of osteoblasts, which significantly promoted the adhesion and proliferation of MC3T3-E1 cells on the surface. Therefore, the PPy/PDA NWs coating took the advantages of both nano morphology came from PPy NWs and excellent biological active inherited from PDA, and had the best value for cell adhesion and proliferation than pure PPy NWs and PDA.

In the procedure of culture osteoblasts for 14 days, it can be divided into three stages [36]. First, the adhesion between cells and matrix was mainly carried out within 12 h after cells were seeded. After 2–3 days, the cells entered the rapid proliferation stage. In this stage, large amounts of protein and DNA were synthesized. Finally, after 6–9 days, the cell proliferation basically stopped and began to enter the mineralization stage. At this stage, the metabolic activity of cells was still vigorous, and the activity of osteoblast markers represented by alkaline phosphatase (ALP) increased significantly, and osteoblasts began to enter differentiating and mature process.

The content of total protein in cells could be used to describe the proliferation and metabolic activity of cells. By testing the total protein content of MC3T3-E1 that cells grew on different coatings (Figure 5c), it could be found that the proliferation and metabolism activity of MC3T3-E1 cells growing on the surface of PPy NWs, PDA and PPy/PDA NWs coatings were all more vigorous than that of the control group (bare Au), and the cell on the PPy/PDA NWs coating maintained the highest protein content during the 14 days throughout, which indicated the PPy/PDA NWs coating could promote the cell rapid proliferation and metabolism activity than pure PPy NWs and PDA.

**Figure 5.** (**a**) SEM and fluorescence images of MC3T3-E1 cells on PPy NWs, PDA and PPy/PDA NWs cultured for 12 h and 24 h. (**b**) Proliferation of MC3T3-E1 cells on different coatings. (**c**) The protein content of MC3T3-E1 cells cultured on different coatings. (**d**) The ALP activity of MC3T3-E1 cells cultured on different coatings. \* *p* < 0.05, and # *p* < 0.01.

The ALP activity, the early marker of osteogenic differentiation, could evaluate the osteogenic activity of MC3T3-E1 cells cultured on different coatings. As the result indicated (Figure 5d), at the initial stage of three days, the ALP activity of cells grown on four kind of coatings were all at low level without obvious difference. At this time, t cells were mainly in the rapid proliferation phase with low osteogenic activity. After 7 days, cells gradually entered the mineralization stage and the ALP activity in all group increased, and the PPy/PDA NWs group had the highest ALP activity. After 14 days, the advantage of ALP activity in PPy/PDA NWs group continued to expand, indicating that PDA/PPy NWs could significantly promote the mineralization and osteogenesis of MC3T3-E1 cells.

Above results demonstrated that the PPy/PDA NWs nanocomposite coating was not only helpful to the rapid adhesion of cells on the surface of materials, but also conducive the osteogenic activity for new bone formation.

## *3.3. Synergistic E*ff*ect of PPy*/*PDA NWs Coating with Electrical Stimulation on MC3T3-E1 Activity*

In the process of bone formation and bone remodeling, electrical microenvironment around the bone cells occurs. Many studies confirmed that a small amount of electrical stimulation (ES) can significantly promote the proliferation and differentiation of osteoblasts at the cellular and tissue level [37,38]. PPy has good conductivity, and could be fabricated as a conductive coating to apply ES on cells sensitive to electrical signals. Our previous study have demonstrated that MC3T3-E1 cells shows better osteogenic activity on the surface of PPy coating with ES [39]. The PPy/PDA NWs was well maintained the excellent conductivity of PPy NWs layer, and performed better osteogenic differentiation activity than pure PPy NWs. Next, we used a self-made device to apply a constant current electrical stimulation (10 μA) to MC3T3-E1 cells through the PPy/PDA coating, and to study the synergistic effect of PPy/PDA NWs and ES working together on MC3T3-E1 cells.

After 12 h of the MC3T3-E1 cell culture, the constant ES was applied on cells for 2 h a day until 14 days. It could be seen that continuous ES can significantly increase the adhesion and proliferation of MC3T3-E1 cell (Figure 6a), the number of MC3T3-E1 cells on PPy/PDA NWs coatings was almost doubled higher after ES was applied. Additionally, the total protein content and ALP activity tests relating to the proliferation and osteogenesis activity of MC3T3-E1 cells both revealed that the ES could promote the osteogenic differentiation of MC3T3-E1 cells (Figure 6b,c). It was noticeable that the total protein content and ALP activity value of MC3T3-E1 cells with ES at 7 days was very close to that of the cells without ES at 14 days, which proved that the PPy/PDA NWs coating together with ES addition could accelerate the cells entering the mature stage of osteogenic differentiation.

**Figure 6.** (**a**) The effect of ES on the proliferation of MC3T3-E1 cells on PPy/PDA NWs coating. (**b**) The effect of ES on the total protein content of MC3T3-E1 cells cultured on PPy/PDA NWs coating. (**c**) The effect of ES on the ALP activity of MC3T3-E1 cells cultured on PPy/PDA NWs coating. \* *p* < 0.05, and # *p* < 0.01.

## **4. Conclusions**

We successfully fabricated the PPy/PDA NWs coating via a simple two-step method. By well controlling the depositing time of PDA within 5 h, the PPy/PDA NWs nanocomposite retained the nano morphology, conductivity, and electrochemical activity from the PPy NWs layer. Moreover, the introduction of the PDA deposition layer significantly improved the hydrophilicity and bioactivity of the material surface. In vitro experiments further proved that the PPy/PDA NWs composite coating had better biocompatibility and osteogenic differentiation ability than pure PPy NWs and PDA. MC3T3-E1 cells could quickly adhere and anchor on the surface of PPy/PDA NWs coating, and enter the proliferation and differentiation process. Furthermore, external applied ES with PPy/PDA NWs coating had the synergistic effect in stimulating the osteogenesis of MC3T3-E1 cell. Therefore, the PPy/PDA NWs composite is a potential coating with enhanced biocompatibility and biological activity in bone regeneration and repairing.

**Author Contributions:** L.D., Y.H., W.S. and D.G. designed the experiments; L.D. and Y.H. undertook the most experiments; Y.H. and L.D. completed the data and imaging preparation and edition; Y.H. wrote the manuscript; D.G., W.S., X.Z. and Y.S. gave valuable suggestions and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Nature Foundation of China (31870986), the National Nature Science Foundation of China (31271009, 81271689), the Fundamental Research Funds for the Central Universities (2011121001), the Natural Science Foundation of Fujian Province (2011J01331), the Program for New Century Excellent Talents in University, and the Program for New Century Excellent Talents in Fujian Province University.

**Acknowledgments:** We acknowledge the analysis and testing center of Xiamen University.

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

## **References**


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## *Article* **Corrosion Behavior and Biological Activity of Micro Arc Oxidation Coatings with Berberine on a Pure Magnesium Surface**

## **Liting Mu 1,2, Zhen Ma 1, Jingyan Wang 1, Shidan Yuan <sup>1</sup> and Muqin Li 1,\***


Received: 1 August 2020; Accepted: 22 August 2020; Published: 28 August 2020

**Abstract:** Bone tissue repair materials can cause problems such as inflammation around the implant, slow bone regeneration, and poor repair quality. In order to solve these problems, a coating was prepared by ultrasonic micro-arc oxidation and self-assembly technology on a pure magnesium substrate. We studied the effect of berberine on the performance of the ultrasonic micro-arc oxidation/polylactic acid and glycolic acid copolymer/berberine (UMAO/PLGA/BR) coating. The chemical and morphological character of the coating was analyzed using scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The corrosion properties were studied by potentiodynamic polarization and electrochemical impedance spectroscopy in a simulated body fluid. The cumulative release of drugs was tested by high-performance liquid chromatography. The results indicate that different amounts of BR can seal the corrosion channel to different extents. These coatings have a self-corrosion current density (Icorr) at least one order of magnitude lower than the UMAO coatings. When the BR content is 3.0 g/L, the self-corrosion current density of the UMAO/PLGA/BR coatings is the lowest (3.14 <sup>×</sup> <sup>10</sup>−<sup>8</sup> <sup>A</sup>/cm2) and the corrosion resistance is improved. UMAO/PLGA/BR coatings have excellent biological activity, which can effectively solve the clinical problem of rapid degradation of pure magnesium and easy infection.

**Keywords:** pure magnesium; ultrasonic micro-arc oxidation; berberine; corrosion resistance

## **1. Introduction**

Traumatic bone repair materials have become a research hotspot in the field of orthopedics. They are mainly derived from autologous and allogeneic bone grafts [1]. The source of autologous bone grafting materials is limited, complications are prone to occur after surgery, and the success rate is low. Allogeneic bone graft materials are expensive and prone to rejection. The degradation of allogeneic bone is slow, resulting in a smaller volume of new bone. In order to solve the above problems, material researchers are trying to find suitable human bone repair materials. With the development of biodegradable materials, metal bone repair biomaterials are undergoing a revolution. The properties of metal biomaterials have changed from being biologically inert to having biological activity and multiple biological functions [2,3]. Magnesium and magnesium alloys are characterized by good osteoinductivity, spontaneous degradability, and excellent biological safety. Their mechanical properties are similar to those of human cortical bone, and the biological properties of the graft are similar to those of natural bone, which has attracted a great amount of attention in the bone repair materials field [4,5]. The degradable properties of magnesium will eliminate the need for a second surgery to remove the implant. Thus, magnesium and magnesium alloy bone repair materials would

not only further reduce the pain and burden for patients, but also increase the application of these advanced materials [6]. However, a series of biological problems have been discovered during the clinical application of pure magnesium, such as osteolysis around the implant, easy loosening of the implant, rapid degradation, and hemolytic infection, which limit the application of pure medical magnesium and magnesium alloys [7–9].

Therefore, researchers should study composite coatings with antibacterial, anti-inflammatory, and bone growth promotion effects, and then adjust and control the degradation rate and biological characteristics of pure medical magnesium to solve the fundamental problem of magnesium implants and make magnesium more suitable as an implant material [10]. In recent years, ultrasonic micro-arc oxidation (UMAO) has been shown to be an effective surface modification technology that can effectively reduce the degradation rate of materials [11,12]. UMAO can effectively improve the corrosion resistance of magnesium alloys. However, the micropores and microcracks of the UMAO surface may cause undesirable rapid and unexpected degradation of magnesium and its alloys [13]. Therefore, scholars have tried to compound other surface modification technologies on the UMAO surface [14–16]. Li [17] reported using KH550 as a silane coupling agent to modify the surface of the ultrasonic micro-arc oxidation coating on pure magnesium. The organic film of the Si–O–Mg bond formed on the surface helps to reduce the pores in the UMAO coating and improve its corrosion resistance. In order to further improve the bone growth around the implant, as well as the antibacterial and anti-inflammatory effects, Peng [18] prepared a phytic acid puerarin solution on the UMAO coating by dip coating. The composite coating has better corrosion resistance than UMAO. It also accelerates the mineralization of apatite and improves the biological activity. Wang [19] prepared an UMAO/chitosan/citrin coating on a pure Mg substrate. They found that the Chinese herbal extract coating enhanced the corrosion resistance and biological activity of pure Mg, and also enhanced the adhesion and proliferation of osteoblasts. However, the coating drug released quickly and the concentration was low. Furthermore, its ability to promote bone growth, as well as its antibacterial and anti-inflammatory effects, did not reach the expected effect.

The most difficult problem with these coatings in bone injury is the low drug concentration at the lesion site. If the drug concentration around the local implant is maintained in a reasonable range, it will be slowly released around the implantation area at a certain rate within a certain period of time to achieve the purpose of the treatment. At present, long-acting sustained-release drug-loaded artificial bone is considered feasible [20,21]. The preparation of drug-loaded artificial bone usually includes methods such as the vacuum adsorption freeze-drying method and the compression molding method. The above methods can achieve the effect of sustained drug release. However, studies have found that the release of drugs is too fast or the drug-carrying materials cannot be degraded or they degrade very slowly, which limits the application [22–24]. Polylactic acid glycolic acid copolymer (PLGA) is a degradable functional polymer organic compound, usually used as a carrier for drug release [25]. PLGA is widely used in the fields of pharmaceutical and medical engineering materials, mainly because it is biocompatible, non-toxic, shows controllable degradation, and produces harmless metabolites in the body. It also has excellent coating-forming properties. At the same time, PLGA as a biomedical material has been certified by the U.S. Food and Drug Administration. Sevostyanov [26] studied the coating of PLGA-containing triple anti-tuberculosis patients' bones, which can stably and slowly release drugs and maintain the local drug concentration at a high level in the lesion. Qian [27] found that the degradation of PLGA in the human body is acidic, and magnesium is degraded to alkaline. After degradation, the environment is neutralized to a certain extent. Wu [28] prepared magnesium-enhanced PLGA copolymer composites by extraction and oil bath methods, and found that the pH of the environment during the degradation process is normal, does not affect bone growth, and can effectively regulate the strength and characteristics of the material according to the location and characteristics of the repaired bone degradation rate.

Until now, PLGA has mostly been used as a carrier for the preparation of slow-release Western medicines. At present, the focus of attention has shifted from synthetic medicine to natural medicine (Chinese medicine), mainly because traditional Chinese medicine has the characteristics of stable action, low toxicity, and natural materials. Traditional Chinese medicine plays a positive role in promoting the growth of osteoblasts, has antibacterial and anti-inflammatory effects, and is able to regulate the differentiation and biological activity of osteoblasts. Berberine (BR), the main active component of the Chinese medicine coptis, has a variety of biological activities, such as inhibiting inflammation, promoting bone formation, and inhibiting osteoclasts [29,30]. However, few reports have focused on combining PLGA and BR to form a composite sustained-release drug coating to enhance the biological activity and corrosion resistance of magnesium alloys [31].

In this study, by modifying the surface of pure magnesium, we obtain a PLGA/BR multi-element composite functional coating that has antibacterial function, promotes bone growth, and can control the degradation rate, and the structural characteristics, corrosion resistance, and biological activity of the composite coating are systematically evaluated.

## **2. Materials and Methods**

## *2.1. Materials and Drugs*

Mg (Technology Co., Ltd., Yi'an, China) was cut into 10 × 10 × 1 mm sample cubes. The molecular weight of PLGA (polylactic acid/glycolic acid, 50:50) was 90,000 (Daigang Biological Engineering Co., Ltd., Jinan, China), and the berberine content was 99% (China Institute for Food and Drug Control).

## *2.2. Sample Preparation*

Sandpaper was used to polish the samples to a smooth surface; which were then soaked and degreased with ethanol for 30 min; and then cleaned with deionized water, dried, and sealed for later use. The following electrolytes were used in UMAO: Na2SiO3·9H2O (15 g/L), KOH (10 g/L), KF (8 g/L), and C10H14N2Na2O8 (1 g/L). The working parameters for the UMAO treatment were as follows: first, a pulse width of 50 μs, a pulse frequency of 500 Hz, an auxiliary ultrasonic frequency of 60 kHz, an ultrasonic power of 50 W, a voltage of 300 V, and an oxidation time of 7 min; and second, a regulated voltage of 260 V and an oxidation time of 3 min, cleaned with deionized water and dried in air.

The composite coating was prepared by immersion in 3 mol/L NaOH solution at 60 ◦C for 1 h, referred to as alkali treatment. Then, 500 mg of PLGA was dissolved in 10 mL of dichloromethane under ultrasound for 30 min. BR-loaded solutions in concentrations of 1.5 g/L, 3.0 g/L, and 6.0 g/L were prepared ultrasonically using the 50 g/L PLGA solution as the solvent for 30 min, giving a self-assembly solution. The sample was immersed in the self-assembly solution for 3 min. Then, the sample was removed from the solution in the vertical direction at a speed of 5 cm/min and dried in air. UMAO/PLGA/BR coatings were marked as 1.5, 3.0, and 6.0 g/L, respectively.

## *2.3. Coating Characterization*

The microstructure and elemental composition of the coatings were characterized by scanning electron microscopy (SEM, JSM-7800JJEOL, Tokyo, Japan) and energy dispersive spectroscopy (EDAX, FALCON60S, Mahwah, NJ, USA). The phases in the coatings were identified by X-ray diffraction (XRD, D8 ADVANCE, BRUKER, Karlsruhe, Germany) with Cu Kα radiation in the 2θ range of 10◦ to 90◦. The contact angle of the coatings was measured by a contact angle meter (JC2000C1, Zhongchen, Shanghai, China). The micro-roughness of the coatings was observed by German Bruker atomic force microscopy (AFM, BRUKER, Karlsruhe, Germany). X-ray photoelectron spectroscopy (XPS, ESCALAB250XI, Thermo Fisher Scientific, Waltham, MA, USA) was used to qualitatively analyze the presence of elements, carbon components, and chemical bonds on the sample surface. The excitation source was Al Kα, the test power was 300 W, and C1s (binding energy 284.8 eV) was used before the test to correct the charge displacement of each element in the test. For the XPS peak, software was used to fit the peaks of the high-resolution XPS spectra of the corrected elements.

## *2.4. Electrochemical Test*

The electrochemical impedance spectroscopy (EIS) and the Tafel curve measurements of the coating were performed by a VersaSTAT 3 electrochemical workstation in the simulated body fluid (SBF) solution at 37 ◦C to evaluate the corrosion behavior of the coating. SBF [32] composition is shown in Table 1. All reagents comply with American Chemical Society standards (ACS). The frequency range of EIS measurements was 10−1–104 Hz. ZsimpWin software was used to perform equivalent circuit fitting on the impedance results.

**Table 1.** Chemical composition and reagents grade (ACS, American Chemical Society standards) and purity used for preparation of simulated body fluid (SBF).


#### *2.5. Immersion Tests*

The temperature of the experiment was maintained at 37.0 ± 0.5 ◦C using a constant temperature water bath and soaked for 3, 7, and 14 days. The degradation and corrosion resistance of the composite bio-coating was identified by SEM. The morphology and composition of the sample before and after immersion were analyzed and studied.

## *2.6. Slow-Release Drug Measurement*

A high-performance liquid chromatography (HPLC) method was established for the qualitative analysis of berberine standard products. The chromatographic conditions were selected as follows: column: Hypersil GOLD C18 (100 × 2.1 mm, 1.9 μm); mobile phase: 0.1% formic acid–acetonitrile/0.1% formic acid water (A/B = 30:70); flow rate: 0.3 mL/min, temperature: 40 °C; injection volume: 10 μL; detection wavelength: 345 nm. A constant temperature method was used for the in vitro drug release test. The samples were sealed and placed in an eppendorf (EP) tube, added to a simulated body fluid with a pH of 7.4, and placed in a 37 °C incubator to simulate the growth environment. Samples were taken after 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31 d. Then, 40 μL of the sample was added to 360 μL methanol, and the same amount of simulated body fluid was added to the EP tube. The sample was injected into the HPLC, the peak area was recorded at 345 nm, and the cumulative release of BR was calculated using the peak area. The time was the abscissa and the cumulative release rate was the ordinate to draw the release curve, and analyze the results.

#### **3. Results and Discussion**

## *3.1. SEM Analysis*

The surface morphologies of the various coatings were characterized by SEM, as shown in Figure 1. The surface of the UMAO coating had a porous honeycomb structure typical of the micro-arc oxidation process [33,34], as shown in Figure 1a. The porous structure of the coating surface became a channel for body fluids to etch the substrate, which corroded the pure Mg substrate. The surface of the UMAO/PLGA/BR coatings was covered by PLGA/BR coating (Figure 1b–d). When the BR content was 1.5 g/L, the morphology of the micro-arc oxidation began to be obscured. When the content of BR was

3.0 g/L and 6.0 g/L, the surface morphology of the coating changed significantly. Most of the micro-arc oxidation morphology was filled by PLGA/BR, so the pore size and number were reduced, with better compactness. As the amount of the drug increased, the morphology of the micro-arc oxidation was more completely covered. As PLGA was acidic after degradation, the coating had some holes, and the degraded magnesium could be neutralized with PLGA degradation that was produced to avoid a local pH that was too low or too high [35].

**Figure 1.** Scanning electron microscopy (SEM) cross-sectional morphologies of various coatings: (**a**) ultrasonic micro-arc oxidation (UMAO); (**b**) UMAO/polylactic acid and glycolic acid copolymer (PLGA)/berberine (BR)1.5; (**c**) UMAO/PLGA/BR3.0; (**d**) UMAO/PLGA/BR6.0.

The cross-sectional morphologies of the coatings with different amounts of BR were characterized by SEM, as shown in Figure 2. Obvious through holes were seen in the UMAO coating section, and the coating thickness was 4.8 μm (Figure 2a). UMAO/PLGA/BR coatings had obvious double-layer superimposed structures, and the interface between the UMAO layer and the PLGA/BR layer was very clear. When the concentration of BR was 1.5 g/L, UMAO through holes were reduced, and the coating thickness was 5.4 μm (Figure 2b). At 3.0 g/L of BR, the UMAO through holes were significantly reduced, and the coating thickness was 7.7 μm (Figure 2c). Compared with the research of Peng [18], the coating thickness increases and the coating becomes denser. At 6.0 g/L of BR, the coating was dense and the coating thickness was 6.0 μm (Figure 2d). Comparative analysis of the cross-sectional morphology of the coating showed that, with the increase of the amount of BR, the thickness of the coating first increased and then decreased, but the overall coating thicknesses increased and the corrosion channels were better filled. When the BR drug content reached 3.0 and 6.0 g/L, the cross sections of the coatings were denser, and the bonding effect between the coatings was better.

**Figure 2.** SEM cross-sectional morphologies of various coatings: (**a**) UMAO; (**b**) UMAO/PLGA/BR1.5; (**c**) UMAO/PLGA/BR3.0; (**d**) UMAO/PLGA/BR6.0.

## *3.2. Phase Analysis of the Coatings*

All the samples were investigated from the phase structural point of view. The phase structures of the various coatings were characterized by XRD, as shown in Figure 3. Contrasted with international centre diffraction data (ICDD). The UMAO coating mainly consisted of MgO (ICDD file no. 65-0476) and Mg2SiO4 (ICDD file no. 83-1807) phase [36,37]. MgO and Mg2SiO4 phase were detected UMAO/PLGA/BR coatings. In addition, owing to the thin film, the Mg phase was detected in various coatings (ICDD file no. 35-0821).

**Figure 3.** X-ray diffraction (XRD) patterns of coatings with various BR contents: (**a**) UMAO; (**b**) UMAO/PLGA/BR1.5; (**c**) UMAO/PLGA/BR3.0; (**d**) UMAO/PLGA/BR6.0.

### *3.3. Contact Angle Analysis*

The wettability of the various coatings was determined by the static water contact angle, as shown in Figure 4. The five contact angles measured by various coatings are shown in Table 2. The water contact angle on the UMAO coating was 20.63 ± 0.56◦ (Figure 4a). Because the surface of the UMAO coating had higher porosity, the contact angle of the coating was smaller. The water contact angles of the UMAO/PLGA/BR (1.5 g/L), UMAO/PLGA/BR (3.0 g/L), and UMAO/PLGA/BR (6.0 g/L) coatings were 68.12 ± 0.96◦, 69.73 ± 0.83◦, and 70.46 ± 0.89◦, respectively (Figure 4b–d). After UMAO self-assembled BR treatment, the contact angle of the coating increased significantly, restricting the entry of corrosive media and improving the corrosion resistance of the coating [38]. However, the angle was less than 90◦, and the hydrophilic coating did not affect cell adhesion.

**Figure 4.** Contact angles of UMAO and coatings with BR on pure magnesium: (**a**) UMAO; (**b**) UMAO/PLGA/BR1.5; (**c**) UMAO/PLGA/BR3.0; (**d**) UMAO/PLGA/BR6.0.



### *3.4. Coating Roughness*

The micro-roughness of the coatings was observed by German Bruker atomic force microscopy (AFM). The roughnesses of the coatings with different amounts of BR are shown in Figure 5. The BR changed the micro-roughness of the various coatings. The surface of the UMAO membrane was densely populated with raised cells and hills, the size was obviously increased, and the roughness arithmetic (Ra) value was larger (Figure 5a). The surface roughness of BR (1.5 g/L) showed a downward trend, which effectively eliminated the bumps on the surface of the UMAO and reduced the surface roughness of the coating (Figure 5b). With the increase in BR content, the surface roughness value showed an increasing trend. At 3.0 g/L of BR, the bulges and depressions on the film surface increased, and the roughness of the coating became larger (Figure 5c). The surface roughness at 6.0 g/L of BR became smaller. This was because BR further filled the holes in the coating surface and the coating became flat (Figure 5d). It was seen that the amount of BR added greatly affected the Ra value of the UMAO/PLGA/BR coating. The specific roughness values are shown in Table 3.

**Table 3.** Roughness values of various coatings.


**Figure 5.** Atomic force microscopy (AFM) images of coatings with various BR contents: (**a**) UMAO; (**b**) UMAO/PLGA/BR1.5; (**c**) UMAO/PLGA/BR3.0; (**d**) UMAO/PLGA/BR6.0.

#### *3.5. XPS Analysis*

XPS measurements were carried out to characterize the composition of the coatings, as shown in Figure 6. Figure 6a illustrates the XPS survey spectra of the different coatings. The chemical composition and element state of the composite coatings were determined. The BR (1.5), BR (3.0), and BR (6.0) coatings all produced spectral peaks of Mg, C, O elements, and this result was consistent with the surface morphology of the coating.

Figure 6b–d illustrate the fine fitting spectra of the O, C, and N elements of the BR (3.0) coating. Using the BR (3.0) coating as an example, the peaks of the UMAO/PLGA/BR coating were analyzed. The fine fitting spectrum of the BR (3.0) coating is shown in Figure 6b–d. According to the further peak analysis of O 1*s* (shown in Figure 6b), the distinctive peak at 530.8 eV in its fine spectrum is the binding energy of the oxygen element in the Mg–O bond [39]. Figure 6b illustrates the O 1*s* XPS pattern for the UMAO/PLGA/BR coating, in which the Mg–O bond was present at a binding energy of 530.8 eV and the O element of the –OH was present at a binding energy of 531.4 eV. Figure 6c illustrates the fine spectrum of C 1*s*. There were two characteristic peaks at 286.3 eV and 284.6 eV in the fine spectrum of C 1*s*. The characteristic peak at 286.3 eV was attributed to the binding energy of C elements in –C–O– in PLGA. The characteristic peak at 284.6 eV was attributed to the binding energy of the C element in –C–H and –C–C– in PLGA and the binding energy of the C=C functional group C in BR [40,41]. Figure 6d illustrates the fine spectrum of N 1*s*. The characteristic peaks at 398.6 eV and 399.7 eV were attributed to C–N in BR [42]. It was further seen that, after changing the amount of BR, UMAO/PLGA/BR coating materials were self-assembled on the surface of the pure magnesium substrate.

**Figure 6.** X-ray photoelectron spectroscopy (XPS) spectra of coatings with various BR additions: (**a**) survey spectrum; (**b**)O1*s* spectra; (**c**)C1*s* spectra; (**d**)N1*s* spectra.

#### *3.6. Corrosion Resistance*

The polarization curves of the UMAO, 1.5 g/L, 3.0 g/L, and 6.0 g/L samples are shown in Figure 7. Relevant electrochemical values were obtained by fitting. The corrosion current density (Icorr) of the UMAO, UMAO/PLGA/BR (1.5 g/L), UMAO/PLGA/BR (3.0 g/L), and UMAO/PLGA/BR (6.0 g/L) coatings was 2.26 <sup>×</sup> 10−6, 3.33 <sup>×</sup> 10−7, 3.14 <sup>×</sup> 10−8, and 1.26 <sup>×</sup> 10−<sup>7</sup> A/cm2, respectively. The more positive corrosion voltage (Ecorr), smaller corrosion rate (CR) and Icorr demonstrate that BR (3.0 g/L) improves the corrosion resistance of Mg by forming the modified structure as described previously [43]. The corresponding fitting parameters are shown in Table 4.

**Figure 7.** Potentiodynamic polarization curves of various samples.

The Icorr of UMAO/PLGA/BR coatings with different BR content was at least one order of magnitude lower than that of UMAO. For the coating prepared by Zhang [44], the corrosion current of magnesium alloy in simulated body fluid was 2.05 <sup>×</sup> 10−<sup>6</sup> A/cm2. This illustrates that coatings with different amounts of BR protected magnesium substrates more effectively than UMAO coatings. In addition, among the drug-loaded coatings, the UMAO/PLGA/BR (3.0 g/L) coating had the lowest Icorr value. It is well known that the Ecorr can describe the thermodynamic property, so the corrosion resistance cannot be evaluated in terms of Ecorr [45]. It can be concluded that the UMAO/PLGA/BR (3.0 g/L) coating showed a lower corrosion rate and corrosion current density compared with other coatings. It was inferred that the corrosion resistance of the 3.0 g/L coating was the best.

**Table 4.** Corrosion current densities and corrosion potentials for different coatings in SBF at 37 ± 1 ◦C. Icorr, corrosion current density; Ecorr, corrosion voltage; CR, corrosion rate.


To further analyze the corrosion behavior of the various coatings, EIS measurements were performed in the SBF, as shown in Figure 8. The EIS measurements were analyzed using ZsimpWin software and fitted to the appropriate equivalent circuit. The symbols represent the experimental data, and the solid lines represent the fitted data. Figure 8a shows the Nyquist plots of the samples. The Nyquist plot of the UMAO coating is composed of a capacitive loop. The capacitive loop at the high frequency is attributed to the resistance and capacitance of the electrolyte penetrating through the UMAO layer [46]. The Nyquist plots of the UMAO/PLGA/BR coatings are composed of widened capacitive loops. The capacitive loop is attributed to the resistance and capacitance of the electrolyte penetrating through the PLGA/BR layer at the medium frequency [47]. The radii of the semicircles corresponding to the UMAO/PLGA/BR coating were visibly larger than those of the UMAO coating. These results reveal that the UMAO/PLGA/BR coating has a higher impedance value and higher corrosion resistance.

Figure 8b shows the impedance modulus <sup>|</sup>Z<sup>|</sup> of different coatings, which were 4.20 <sup>×</sup> <sup>10</sup><sup>4</sup> (UMAO), 5.24 <sup>×</sup> 10<sup>5</sup> (BR 1.5 g/L), 1.68 <sup>×</sup> 106 (BR 3.0 g/L), and 1.41 <sup>×</sup> 10<sup>6</sup> (BR 6.0 g/L) <sup>Ω</sup>·cm2. The impedance modulus of the drug-loaded coatings was an order of magnitude higher than that of the UMAO coating. However, after the addition of 6.0 g/L BR, the impedance modulus value of the UMAO/PLGA/BR coating did not continue to increase. Because the solution was a suspension when 6.0 g/L of BR was added, it was not conducive to the formation of self-assembled coatings. As we all know, good corrosion resistance between the coating and the electrolyte corresponds to a higher R value [48,49]. The results showed that, when the amount of BR was 3.0 g/L, the coating had higher resistance and better corrosion resistance.

The Bode plots of the coatings with different amounts of BR are shown in Figure 8c. The shapes were similar. Both coatings have two time constants distributed at a low frequency (10<sup>−</sup>1–102 Hz) and a high frequency (102–104 Hz). The low-frequency time constant is the response of the UMAO coating, and the high-frequency time constant is the response of the PLGA and PLGA/BR layers [46].

The time constant of the high frequency zone was ascribed to the PLGA/BR layer, and the time constant of the low frequency zone was ascribed to the UMAO layer. The phase angles of BR (1.5), BR (3.0), and BR (6.0) coatings were 65◦, 68◦, and 62◦, respectively. When the amount of BR added reached 3.0 g/L, the phase angle of the coating was the largest and the corrosion resistance was the highest. The Nyquist diagram of the composite coating was consistent with the results of the Bode diagram, indicating that the BR (3.0) coating had good corrosion resistance.

The equivalent circuit used to fit the electrochemical impedance plot of the coatings is shown in Figure 8d. The corresponding fitting parameters are shown in Table 5. In the circuit, Rs is the solution resistance, R1 is the resistance of the UMAO coating, and R2 is the resistance of the PLGA/BR coating. CPE1(capacitance of coating) and CPE2 are constant phase angle components of electric double layer capacitors, which represent the capacitive reactance of the UMAO dense layer coating and the PLGA/BR drug-loaded layer, respectively. In UMAO, R2 and CPE2 represent load transfer resistance and interface electric double layer capacitance, respectively. The impedance of the drug-loaded coating was greater than that of the UMAO coating, indicating that the drug-loaded coating could prevent corrosion more effectively [50].

**Figure 8.** Nyquist, Bode diagrams and equivalent circuits of different coatings: (**a**) Nyquist; (**b**) and (**c**) Bode plots; (**d**) equivalent circuit (CPE, capacitance of coating).

**Table 5.** Values of the equivalent circuit parameters for the various coatings extracted from the electrochemical impedance spectroscopy (EIS) plots. CPE, capacitance of coating.


Therefore, a conclusion can be drawn from the polarization curve and the EIS impedance; that is, the corrosion resistance of the coating was improved by doping BR in the PLGA solution to form a drug-loaded coating. The main reason was that the addition of BR to the PLGA solution was equivalent to a filler, which can fully block the defects, such as pores, of the ceramic layer in the drug-loaded coating and prevent the body fluid from penetrating the Mg matrix too quickly, thereby improving the corrosion resistance.

## *3.7. Surface Morphology and Phase Composition Analysis in SBF*

SEM images of UMAO and UMAO/PLGA/BR (3.0) coatings soaked in SBF solution for 0, 3, 7, and 14 days are shown in Figure 9. Figure 9a1,b1 shows the surface morphology of UMAO coating and UMAO/PLGA/BR coating without soaking. Compared with the UMAO coating, after 3 days of soaking, the surface of the coating was gray, and insect-like substances were deposited on the surface of the UMAO film around the pores (Figure 9a2,b2). After being immersed for 7 days, cracks appeared on the surface of the coating, and white deposits in the shape of flower clusters were attached to the

cracks. This deposit can effectively fill the cracks and form a new composite surface to prevent the corrosive medium from passing through the pores or cracks (Figure 9a3,b3), which was also reported in other studies [51,52]. After 14 days of soaking, the cracks deepened, the number increased, and the white deposits increased. The surface of the coating material became rough, and its surface area increased to provide an interface for ion adsorption (Figure 9a4,b4). Because of the extension in the immersion time, Ca and P elements were enriched and nucleated on the surface, and the mass increased with time. At the same time, Mg ions gradually degraded from the surface and were replaced by Ca ions, which'adsorb more CO3 <sup>2</sup><sup>−</sup> and PO4 <sup>3</sup><sup>−</sup> on the surface of the coating material; when the solubility product of forming bone-like apatite was reached, a new phase was formed on the surface [53,54], so the BR coating had excellent biological activity.

**Figure 9.** SEM patterns of different coatings after immersion in SBF. 0 days: (**a1**) UMAO; (**b1**) UMAO/PLGA/BR. 3 days: (**a2**) UMAO; (**b2**) UMAO/PLGA/BR. 7 days: (**a3**) UMAO; (**b3**) UMAO/PLGA/BR. 14 days: (**a4**) UMAO; (**b4**) UMAO/PLGA/BR.

## *3.8. Slow-Release Drug Measurements*

The drug-loaded coating on the metal surface gave it functional requirements [55,56]. According to the conditions and methods under Section 2.6, the UMAO/PLGA/BR (3.0) sample was subjected to an in vitro release test. The peak area at 345 nm was measured and used to calculate the cumulative release of BR. The results are shown in Figure 10. The cumulative release of BR from the UMAO/PLGA/BR coating reached 92.13% after soaking in the SBF solution for 28 days. Peng [18] and Wang [19] studied the drugs released for 2–3 days. This prolonged the action time of the drug, increased the concentration of the drug in the local lesion, and avoided the physical and mental harm to the patient caused by long-term oral administration or external topical administration.

**Figure 10.** Cumulative release curve of the UMAO/PLGA/BR coating in vitro.

#### **4. Conclusions**

The UMAO coating has through-pores and these were sealed by the capillarity of the PLGA/BR coatings to inhibit permeation of body fluid. The corrosion resistance of the UMAO coating was significantly improved by the coverage of PLGA/BR coatings with higher impedance and lower current density. This is because the PLGA/BR coating with high cross-link density and networks has a better capacity to inhibit the permeation of ions. In addition, after the BR-loaded coating was immersed in SBF for 14 days, a large amount of substance adhered to the surface, and the prepared composite coating had excellent in vitro biological activity. After 28 days, the cumulative amount of released BR was 98.12%. Thus, the inclusion of PLGA/BR on the UMAO coatings induces an antimicrobial effect.

**Author Contributions:** Data analysis, L.M. and Z.M.; manuscript writing, L.M. and S.Y.; literature search, chart making, Z.M. and S.Y.; research design, J.W. and Z.M.; data collection, J.W. and L.M.; writing—original draft preparation, L.M. and S.Y.; software, L.M. and Z.M.; revise the manuscript and finalize the version to be published, L.M. and M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was financially supported by the National Natural Science Foundation of China (No. 31370979). Fundamental Research Funds from the Provincial Education Department Fundamental Research Project, China (2019-KYYWF-1370). Jiamusi University PhD Special Scientific Research Fund Launch Project (JMSUBZ2019-10).

**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* **Mangrove Inspired Anti-Corrosion Coatings**

## **Miaomiao Cui 1,2, Peng-Yuan Wang 1, Zuankai Wang 2,\* and Bin Wang 1,\***


Received: 8 October 2019; Accepted: 28 October 2019; Published: 1 November 2019

**Abstract:** Marine corrosion accounts for one-third of the total corrosion cost and has been one of the greatest challenges for modern society. Organic coatings are known as the most widely used protective means. An effective control of the transport of corrosive substances is the key to the anti-corrosion performance. In nature, the mangrove survives and thrives in marine tidal zones despite high salinity and humidity. We first showed that the mangrove leaves have salt glands that can secrete excessive ions to control the ion transport in and out. Inspired by this, we proposed a design of bio-inspired, anti-corrosion coating that mimics this functional feature, and fabricated the bipolar, hydrophobic coatings by doping ion-selective resins and constructing surface structures, which restrict the transport of corrosive substances and the electrochemical corrosion at the coating/metal interface. Our results show that the bio-inspired coatings effectively block and control the transport of both the Na<sup>+</sup> and Cl−, and, together with the hydrophobic surface, the coating system exhibits significantly improved anti-corrosion properties, more than a three orders of magnitude decrease in corrosion current density when compared with the control group (epoxy varnish). Therefore, the mangrove-inspired coatings show a promising protective strategy for the ever-demanding corrosion issues plaguing modern industries.

**Keywords:** bio-inspiration; anti-corrosion coating; salt gland; mangrove

## **1. Introduction**

Corrosion, often metal corrosion, is the material destruction that leads to failure in function. It has been a major problem plaguing mankind dating back to ancient times. The first written description of corrosion appeared in the works of Plato (427–347 B.C.) [1] and the first patent of a protective paint appeared in 1625 [2]. Since a large number of metallic materials are developed and utilized in various fields such as marine oil and gas exploitation and transportation industries, the accompanying problem of corrosion becomes greater, which brings enormous economic loss and poses great threats to personal safety and the natural environment. According to the National Association of Corrosion Engineers International study 2016, the direct global corrosion cost was estimated to be \$2.5 trillion, which is equivalent to roughly 3.4% of the global Gross Domestic Product [3]. This study also reported that implementing corrosion control/prevention practices could result in savings of 15%–35% of the cost of damage. Among a variety of anti-corrosion techniques including the passive coatings and active methods [4], organic anti-corrosion coatings, which isolate and protect the substrate metal kinetically, are the most cost-effective and environmentally-friendly approach for the ever-pressing corrosion issue [5].

The fundamental principles of corrosion reveal that material corrosion is thermodynamically spontaneous and kinetically mediated by corrosive substances such as H2O, Na<sup>+</sup>, and Cl−. These substances invade and transport through the coating to the coating/substrate interface, which leads to an accelerated corrosion reaction [6] that consists of chemical corrosion and electrochemical corrosion. Moreover, the electrochemical corrosion processes at a high rate and it is a complex process that includes anodic and cathodic reactions. The anodic reaction transfers metal atoms into metal cations. This process can be accelerated by Cl−, especially the pitting corrosion process [7,8]. On the other hand, the cathodic reaction generates OH<sup>−</sup> followed by the aggregation of Na+, and this process could accelerate the accumulation of corrosion products, which leads to peeling-off of the coating [9,10]. These reactions, supplied by the penetrated corrosive substances, not only consume the metal substrate but also lead to the failure of the protective coatings. Therefore, the key to high-performance anti-corrosion coatings requires effective blocking of corrosive substances at the external surface and control of ion transportation within the coating [11–13].

Among the rapid research progress in protective coatings, learning from nature to design novel anti-corrosion materials is one of the best ways for creating these materials. Over the course of evolution, nature develops ingenious strategies that can be implemented to address the issues of surface blocking and ion control in anti-corrosion coatings. One representative dealing with the blocking is the superhydrophobic coating bio-inspired from self-cleaning lotus leaves, which protects metals from corrosion and has been studied extensively. The mechanism involves an isolative air layer formed between the external corrosive solution and the substrate, usually fulfilled by the hierarchical structure and the chemical constituents of the coating. Once the corrosive medium penetrates into the coating as time goes by, controlling the transportation of corrosive ions becomes significant to delay and, thus, prevent a corrosive reaction. Research efforts in controlling the transmission of corrosive agents can be passive, e.g., by adding fillers, such as zinc particles [14]. The mechanism involves the blocking and prolonging of the path of corrosive agents, and the preferred corrosion of zinc rather than steel due to a higher electrochemical activity. The other is an active approach that includes combining with conductive/reactive components to identify and interact with the ions selectively. A variety of ion-selective organic coatings doped with different ion exchange resins, first studied by Wang et al. [15–17], show effective control of the moving direction of ions such as Cl<sup>−</sup> and Na+. The ion-selective coating that interacts with one type of ions is called single polar coating. An anionic coating (cation-selective film) blocks the invasion of anions (e.g., Cl−) and allows the passage of cations (e.g., Na+), while a cationic coating blocks the transmission of cations. A bipolar coating composed of cationic and anionic layers, which can restrict both types of ions, shows promising application in metal protection. Besides, conductive polymers, such as polyaniline (PANI) and modified PANI [18], have also been used for ion selective anti-corrosion coatings [8]. However, the ion selective coatings can only adjust the transport of ions and cannot deal with the water infiltration, which deteriorate the protective function of the coatings greatly. Therefore, preventing the invasion of H2O into the coating layer is another important consideration of developing advanced anti-corrosion coatings.

Effective control of ion-containing fluids can also be observed in nature, e.g., the mangrove plants which survive and thrive in the marine intertidal environments featuring high humidity and high salinity through salt secretion [19]. The harsh habitat is close to the anti-corrosion coatings that protect the substrate metals in marine surroundings [20], and the strategies utilized by the mangrove through salt glands are vivid inspirations for developing ion-control coatings, formulated first in this work. In an aim to develop high-performance anti-corrosion coatings addressing the control of ion transport and external blocking, we explore the salt secretion of the mangrove, and, for the first time, fabricate mangrove-inspired anti-corrosion coatings employing ion-selective resins and hydrophobic surface construction. The structural and functional features of the salt glands on the mangrove (*Ceriops tagal (perr.) C. B. Rob*) are presented, and the bio-inspired, bipolar hydrophobic coatings were fabricated to exclude external H2O and corrosive ions (Cl<sup>−</sup> and Na+). Our results show that the bio-inspired anti-corrosion coatings exhibit excellent properties in restraining the corrosive ion intrusion and transport within the coating, which leads to significantly improved anti-corrosion performance.

## **2. Materials and Methods**

#### *2.1. Observation of the Mangrove Leaves*

The optical images of living mangrove were taken by a digital camera. Mature mangrove (*Ceriops tagal (perr.) C. B. Rob)* leaves and some branches of the mangrove were collected from Shenzhen Bay. The mangrove branches were cultured in nutrition solutions that were diluted by 100 mL tap water and 100 mL 3.5 wt % NaCl solution, respectively, for eight days.

For scanning electron microscopy, mangrove leaf samples were sputter-coated using a Leica EM ACE200 Automatic low vacuum coating apparatus (platinum, 30 s) (Leica, Wetzlar, Germany), and then observed by a ZEISS SUPRA55 Field emission sweep electron microscope (Carl Zeiss, Jena, Germany). The elemental mappings through energy-dispersive X-ray spectroscopy (EDS) of the samples were scanned by an Oxford X-Max 20 Electrically cooled X-ray spectrometer (Oxford, England).

#### *2.2. Fabrication of the Mangrove-Inspired Coatings*

Epoxy varnish (E-44 bisphenol A epoxy resin) was used as the film forming material. Single-polar coatings (two types) were obtained by doping different ion-selective resins, the 719 (202) strong base styrene anion exchange resin, and the 732 strong acid styrene cation exchange resin, respectively, into the epoxy varnish as paints. The ion exchange resins and epoxy varnish were diluted by xylene, and the doping concentrations were 0, 2.5 wt %, 5 wt %, 10 wt %, and 20 wt % for both groups. After the paints were evenly distributed in the epoxy varnish, TY-650 polyamide was mixed to cure the epoxy varnish. Then they were brush-coated on silica gel plate and also metal substrates (Q235) to obtain the single-polar coatings and the coated metal samples, respectively.

Q235 steel with the size of 10 mm × 10 mm × 5 mm was the metal substrate and the main elements are (wt %), C 0.127, Si 0.15, Mn 0.41, P 0.018, S 0.019, Fe balance. All metal substrates were polished with water on graded sandpapers (150#, 400#, 600#, 800#, and 1000#) step-by-step and were linked with copper wire by soldering. Metal samples were sealed by epoxy resin with a working surface of 10 mm × 10 mm exposed. Sealed samples were polished with water phase sandpapers (150#, 400#, 600#, 800#, and 1000#,) step-by-step, washed by anhydrous ethanol, dried, and kept in a dryer until utilization.

For the bipolar, hydrophobic coatings, a hydrophobic surface layer was fabricated using a template method. A superhydrophobic silicon plate was fabricated following the method in previous work [21,22]. The silicon nanowires and grooves were fabricated based on a 425-mm thick silicon wafer. A standard Micro-Electro-Mechanical System process technology was employed to fabricate rough structures on a silicon surface, which consists of two essential structural features, silicon micropillars, and silicon grooves. A photolithography process was first used to selectively cover a photoresist on a silicon wafer, which was followed by reactive ion etching (RIE) to etch the wafer areas that are not protected by the photoresist, and deep RIE was used to further etch the silicon substrate. This process formed silicon micropillars. The deep RIE process included cyclic passivation and etching modes in which C4F8 and SF6 were used. In the etching cycle, the SF6 flow rate was 130 sc·cm and platen power was set at 12 W. In the passivation cycle, the C4F8 flow rate was 85 sc·cm. Lastly, the photoresist was removed and deep RIE was used to further etch the silicon substrate covered by photoresist, which formed a silicon groove. Then, the surface fabrication process was completed. The prepared silicon wafer was taken as an original template, and PDMS was applied to copy the structure on the coating surface. The thicknesses of all fabricated coatings were measured by a micrometer. The thicknesses of bare metal substrates were monitored at three different points, and the average of all the measurements was taken as the thickness of metals (TM). The total thickness of coatings and metal substrate (TT) were measured by the same processes. Then the thickness of the coating is the difference of the TT and the TM. For the first type of single polar coatings, the coating thicknesses of the fabricated coatings and control groups were kept the same (45 ± 5 μm). For the second type of bipolar coatings, the total coating thicknesses of the fabricated bipolar coatings and control groups were kept the same (90 ± 5 μm), since those were fabricated layer-by-layer.

## *2.3. Electrochemical Measurements*

Electrochemical tests including open circuit potential (OCP), electrochemical impedance spectroscopy (EIS), and polarization curves were performed on CHI760E. The electrochemical experiments were carried out in 3.5 wt % NaCl aqueous solution and a three-electrode configuration was applied, including the as-prepared samples (Q235 steel coated with the epoxy varnish, the bioinspired single-polar coatings, and the bioinspired bipolar, hydrophobic coatings), platinum plate, and saturated calomel electrode (SCE) as working, counter, and reference electrodes, respectively. If there is no other specific indication, all potentials reported in this paper are taking SCE as the reference. EIS tests were performed in a frequency range of 105 through 0.01 Hz at the open circuit potential with an amplitude of 10 mV. Potentiodynamic polarization curves were obtained by setting the sweeping range of ±300 mV versus the rest potential value, and a rate of 1 mV/s was employed for scanning. The measured results were further analyzed by using software Cview.

## *2.4. Wettability and Ion-Resistant Property*

The surface wettability was measured using a contact angle meter (DSA-100, KRüSS Instruments, Hamburg, Germany) recording the contacting scenario of a water droplet to the surface. For the ion-selectivity analysis, the fabricated single-polar coatings were fixed in a custom-designed equipment, as shown in Figure 5a, between the 3.5 wt % NaCl solution (30 mL) on the left side and the ultrapure water (100 mL) on the right side. With increasing time at fixed intervals, 100 μL solution on the left side was taken out and the concentrations of Cl<sup>−</sup> and Na<sup>+</sup> were measured by ion chromatography (ICS-900, DIONEX, Sunnyvale, CA, USA) and inductively coupled the plasma mass spectrometer (iCAP Q, Thermo Scientific, Bremen, Germany).

## **3. Results and Discussion**

### *3.1. Salt Particles Deposited on Mangrove Leaves and Salt Gland*

Mangrove plants live along the marine coast sustaining the high salinity of seawater (living mangrove forest shown in Figure 1a). On the mangrove leaves, there are plenty of deposited particles (Figure 1b). To analyze the elements of the deposited particles, EDS was used and the results show that the main elements are Cl and Na with an atom ratio of approximately 1 (Figure 1c–e). Thus, the deposited particles are mainly NaCl particles. As reported in references [23–26], salt glands on the mangrove leaves can secrete salt solution to adapt to the harsh environment. However, the salt contained in the marine atmosphere can also be deposited on leaves. To further explore the source of the salt particles, we washed the fresh leaves (growing on branches) with distilled water to remove the deposited particles and cultured the collected branches in nutrition solutions diluted by tap water (NTW) and 3.5 wt % NaCl (NSW), respectively. After eight days, the cultured leaves were collected. The optical images of mangrove leaves cultured in NTW and NSW are shown in Figure 2a,b respectively. There are plenty of particles on leaves cultured in NSW, while no clear deposited particles on leaves cultured in NTW were observed. The same results can be seen in SEM images, as shown in Figure 2c,d with more particles observed in the sites of salt glands. EDS was applied and the results (Figure 2e,f) show that the deposited particles are NaCl. Thus, we conclude that, even though there may be two sources for the deposited salt particles on the fresh mangrove leaves, the salt glands of the mangrove leaves within the salted environment perform salt secretion well, which results in salt particles deposited on the leaf surfaces.

**Figure 1.** The mangrove and the deposited salt on the surface of fresh mangrove leaves. (**a**) The natural mangrove plants along the Shenzhen Bay. (**b**) SEM of the deposited salt on mangrove leaves, (**c**), (**d**) and (**e**) EDS of the deposited salt particles.

**Figure 2.** The deposited salt on the surface of cultured mangrove leaves. (**a**) and (**b**) are mangrove leaves cultured by nutrition solution and 3.5 wt. % NaCl nutrition solution, respectively. (**c**) and (**d**) are SEM images of mangrove leaves deposited with salt particles and the inserted images are the salt glands of mangrove leaves. (**e**) and (**f**) are EDS of the deposited salt particles.

Salt glands on mangrove leaves are multi-cellular tissues typically including cell types differentiated into basal collecting cells and distal secretory cells. The collecting cells are presumed to create a salt efflux gradient to collect salt from neighboring mesophyll cells and transport it to the secretory cells. The secretory cells are completely surrounded by a cuticle, with the exception of where they contact the subtending basal collecting cells, which is a feature that appears to channel the flow of salt through the secretory cells and prevent leakage back into the neighboring tissue via the apoplast [27]. We observe salt glands on both the top and lower surfaces of the mangrove leaves, as shown in Figure 3a,b. The magnified image (Figure 3d) of the salt gland shows that it is different from the stoma (breathing tissue). Based on the results we observed, we made a schematic illustration of salt gland distribution on the mangrove leaves. Salt glands function as a safety guard to control the ion transport into and out of the leaf to maintain healthy. From the perspective of bio-inspiration, this function is also necessary for an effective anti-corrosion coating, as well as needed to control the transport of corrosive ions and

water within the coating system. For this aim, we design and fabricate mangrove-inspired protective coatings that inhibit and control the transport of corrosive ions.

**Figure 3.** Salt glands distributed on the top and lower surfaces of mangrove leaves. (**a**) and (**b**) are morphologies of mangrove leaves on both surfaces. (**c**) Schematic illustration of the distribution of the salt glands on mangrove leaves. (**d**) SEM image of the salt gland on mangrove leaves.

## *3.2. Design and Properties of Mangrove-Inspired, Single-Polar and Bipolar Coatings*

As ions diffuse/transport from a high concentration to a low concentration without external energy input/interference, we employed doping pigment materials that react with ions to resist or reduce this tendency. Ion exchangeable pigments have been applied in organic coatings to improve the anti-corrosion performance by preventing the transport of corrosive ions. In this case, we chose two types of commercial ion-exchange resins (719 (202) strong-base styrene anion exchange resin and 732 strong acid styrene cation exchange resin) as pigments to modify the epoxy varnish, which can function as inhibiting corrosive ions.

Anion exchange resin has positive charges that exclude cationic ions. Thus, doping anion exchange resin into epoxy can obtain a coating that blocks cations and we call this coating as cationic coatings, as illustrated in Figure 4a. Similarly, doping cation exchange resin can obtain anionic coatings (Figure 4b). These bio-inspired, single-polar coatings, with different doping concentrations, were tested by performing potentiodynamic polarization curves to find the optimized percentage, as shown in Figure 4c,d and Tables 1 and 2. It is clear that Q235 covered with epoxy varnish has decreased corrosion current density compared with the bare substrate, while the anti-corrosion property was further improved by the single-polar, cationic coatings. The lowest current density of Q235 protected with the cationic coating was 1.0091 <sup>×</sup> <sup>10</sup>−<sup>7</sup> <sup>A</sup>/cm2, which corresponds to a doping concentration of 10 wt % of anion exchange resin. Similarly, the single-polar, anionic coatings also showed enhanced anti-corrosion properties compared with the epoxy varnish on the Q235 substrate, and the optimal doping amount was 5 wt % of cation exchange resin.

**Figure 4.** The single-polar, ion-selective coatings and the protective performance. (**a**) and (**b**) are the schematic illustrations of the repulsion function of the cationic coating and anionic coatings. (**c**) and (**d**) are potentiodynamic polarization curves of the cationic and anionic coatings with different doping concentrations of anion and cation exchange resins.

**Table 1.** Fitting results of Q235 steel coated with cationic coatings with different concentrations of anion exchange resin.


**Table 2.** Fitting results of Q235 steel coated with anionic coatings with different concentrations of cation exchange resin.


The different single-polar coatings (cationic and anionic) exhibit clear superior protective performance than the epoxy varnish coatings (e.g., the much lower corrosion current densities and higher corrosion resistance). This is due to the resisting/blocking ability of the coatings to corrosive ions (Na<sup>+</sup> and Cl−) resulting from electrostatic repulsion, which is investigated by the ion-selectivity measurements, as shown in Figure 5a. The single-polar, cationic/anionic coatings were placed and fixed between the 3.5 wt % NaCl aqueous solution and the ultrapure water. Measuring the concentration changes of Na<sup>+</sup> and Cl<sup>−</sup> of the ultrapure water with increasing time provides information about the ion-selectivity/ion-blocking properties of the single-polar coatings. For cationic coatings (Figure 5b), the concentration of Na<sup>+</sup> is much lower than that of Cl<sup>−</sup> throughout the testing time range, which illustrates a good blocking ability of Na+. This accounts for the lower corrosion current density of the cationic coatings covering Q235 than that of epoxy varnish. The anionic coatings clearly block Cl−, as the concentration of Cl<sup>−</sup> is much lower than Na<sup>+</sup> during the entire time range (Figure 5c), and this explains the improved anti-corrosion properties of Q235 steel coated with anionic coatings.

**Figure 5.** The ion-selectivity of the single-polar coatings. (**a**) Illustration of the custom-designed experimental equipment. (**b**) and (**c**) are the plots of the concentrations of Na<sup>+</sup> and Cl<sup>−</sup> in ultrapure water versus increasing time for the cationic and anionic coatings.

Another consideration of the anti-corrosion coatings is the water transmission. The ion-exchange resins could easily absorb water from surroundings, which is not favorable for corrosion inhibition. Our results of the single-polar, cationic, and anionic coatings immersed in 100 mL 3.5 wt % NaCl aqueous solution show significant water uptake (calculated via Equation (1)), which is much higher than the epoxy varnish (Figure 6a). For this, we include an external hydrophobic layer on the single-polar cationic/anionic coating systems in the design. In addition, a high-performance protective coating should control the transport of both the Na<sup>+</sup> and Cl<sup>−</sup> ions at the same time. Therefore, we present a design of bipolar, hydrophobic coating system (Figure 6b), which shows our top-down, bio-inspired approach that can block corrosive substances such as water and control transport of corrosive ions within the coating to enhance corrosion protection. The top hydrophobic surface, fabricated by copying the hierarchical structure of microgrooves and micropillars, is shown in Figure 6c, and the contact angle of the water droplet is about 139.1◦, which indicates the hydrophobicity.

$$\mathbf{w} = (\mathbf{w} - \mathbf{w}\_0) / \mathbf{w}\_0 \tag{1}$$

w- weight of coating at certain immersion times, w0- weight of the coating before immersion

**Figure 6.** Water absorption property of the coatings, design of bipolar, hydrophobic coatings, and the surface structure of the hydrophobic layer. (**a**) The water uptake with increasing immersing time of the single-polar and epoxy coatings. (**b**) The constructed bipolar, hydrophobic coating systems: hydrophobic, cationic/anionic/metal (H, Cationic/Anionic/M) and hydrophobic, anionic/cationic/metal (H, Anionic/Cationic/M). (**c**) The morphology of the hydrophobic surface with an insert showing the contact angle of a droplet about 139.1◦.

#### *3.3. Anti-Corrosion Performance of the Bipolar, Hydrophobic Coating*

The representative polarization curves of Q235 with different bipolar, hydrophobic coatings and epoxy varnish after being immersed in 3.5 wt % NaCl solution for 10 days are shown in Figure 7 and the corresponding fitting results are listed in Table 3. For Q235 coated with epoxy varnish, the corrosion current density is 6.1134 <sup>×</sup> 10−<sup>7</sup> A/cm2 (Table 3). For Q235 protected by bipolar coatings, the corrosion current density decreased significantly, which indicates a significant increase in anti-corrosion performance. The corrosion current density is only 8.3562 <sup>×</sup> 10−<sup>11</sup> A/cm<sup>2</sup> for the hydrophobic,anionic/cationic/metal system, and that for the hydrophobic,cationic/anionic/metal system is 1.6966 <sup>×</sup> 10−<sup>11</sup> A/cm2, which is more than three orders of magnitude of decrease in the corrosion current density when compared to the epoxy varnish. This demonstrates a substantial enhancement in corrosion protection. The change of polarization resistance is in accordance with the change of the corrosion current density, as shown in Table 3.

**Figure 7.** The potentiodynamic polarization curves of different coating systems after being immersed in 3.5 wt. % NaCl aqueous solution for 10 days.

**Table 3.** Fitting results of Q235 steel coated with the bipolar, hydrophobic coatings and the epoxy varnish after 10 days of immersion.


Electrochemical impedance spectroscopy was employed to investigate the protective performance of the bipolar, hydrophobic coatings. The impedance modulus |Z| decreased with the increase of immersion time, which indicates that the coating was gradually broken and, thus, the corrosion protection decreased (Figure 8a,b). The corrosion protection of the hydrophobic, cationic coating/anionic coating/metal (H, Cationic/Anionic/M) system was much better than that of the hydrophobic, anionic coating/cationic coating/metal (H, Anionic/Cationic/M) system. This could be attributed to the different degrees in changes of water absorption after applying the hydrophobic surface. The single-polar cationic coatings absorb much more water than the anionic coatings (Figure 6a), and an external hydrophobic surface could lead to a more significant decrease in water uptake, and, thus, a higher increase in corrosion inhibition and anti-corrosion performance enhancement for the H, Cationic/Anionic/M system. Additionally, the impedance modulus |Z| of the H, Cationic/Anionic/M system still remained a high value

after immersion for 10 days (the low frequency range in Figure 8a). For the H, Anionic/Cationic/M system, the impedance modulus |Z| clearly decreased after 1-day immersion, and this decrease was substantial after 10 days, which suggests that the surface was broken. These results indicate that the H, Cationic/Anionic/M system has better anti-corrosion performance than the H, Anionic /Cationic/M system.

**Figure 8.** The anti-corrosion capability of the bipolar, hydrophobic coatings during immersion from one to ten days. (**a**) and (**b**) are Bode plots of the H, Cationic/Anionic/M system and H, Anionic/Cationic/M system, respectively. (**c**) is the Nyquist plot of the H, Cationic/Anionic/M system, and (**d**), (**e**), and (**f**) are those of the H, Anionic/Cationic/M system.

The same results can be observed through the Nyquist plots. The results include the impedance change of the different bipolar, hydrophobic coating systems during immersion in 3.5 wt % NaCl solution for 10 days (Figure 8c–f). A large radius of the impedance represents a higher corrosion resistance, and, thus, better anti-corrosion property. Since the radius of the impedance becomes smaller, the anti-corrosion performance of the coatings decreases. Moreover, it is observed that the H, Anionic/Cationic/M system loses protective ability at a faster rate than the H, Cationic/Anionic/M system when comparing the changes of the radius of the impedances. Therefore, the mangrove-inspired, bipolar hydrophobic coatings (H, Cationic/Anionic) can protect the substrate Q235 steel more significantly and for a much longer time.

## **4. Conclusions**

To control the corrosive substances, e.g., water, Na+, and Cl−, is the key for a high-performance anti-corrosion coating to inhibit the corrosion reaction. Strategies developed by nature could provide numerous ingenious designs for dealing with that issue. In this study, we investigated the mangrove salt glands, which are distributed on both surfaces of the leaves (*Ceriops tagal (perr.) C. B. Rob*), and the salt secretion of the salt glands. Inspired by the function of controlling transport of ions into and out of the plant, we designed single-polar and bipolar coatings that have different ion-selective abilities and, thus, control of transport of Na<sup>+</sup> and Cl−. We further fabricated mangrove-inspired, bipolar hydrophobic coatings that have a top-down protective ability. Our electrochemical evaluations show that, among the manufactured mangrove-inspired protective coatings, the bipolar, hydrophobic coatings (H, Cationic/Anionic) possess significant outstanding and long-term anti-corrosion performance.

**Author Contributions:** Conceptualization, M.C., Z.W., and B.W. Data curation, M.C., Z.W., and B.W. Investigation and methodology, M.C., Z.W., and B.W. Writing-original draft, M.C. Writing-review and editing, M.C., B.W., Z.W., and P.-Y.W.

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

**Acknowledgments:** B.W. appreciated the financial supports from the Natural Science Foundation of China (No. 51703240) and the Shenzhen Peacock Technology Innovation Fund (No. KQJSCX20180330170430100). Z.W. is grateful for financial support from Shenzhen Science and Technology Innovation Council (JCYJ20170413141208098), Innovation Technology Fund (9440175), Research Grants Council of Hong Kong (No. C1018-17G, No. 11275216), and the City University of Hong Kong (No. 9360140, No. 9667139). P.-Y.W. thanks the supports from the National Key Research and Development Program of China (2018YFC1105201), the general program of National Natural and Science Foundation of China (31870988), the CAS-ITRI cooperation program (CAS-ITRI201902), and the International cooperative research project of the Shenzhen collaborative innovation program (20180921173048123).

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

## **References**


© 2019 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* **Guiding Stem Cell Differentiation and Proliferation Activities Based on Nanometer-Thick Functionalized Poly-***p***-Xylylene Coatings**

**Chih-Yu Wu 1,2,3,†, Yu-Chih Chiang 3,4,†, Jane Christy 1, Abel Po-Hao Huang 4,5, Nai-Yun Chang 1, Wenny 1, Yu-Chih Chiu 6, Yen-Ching Yang 1, Po-Chun Chen 6,\*, Peng-Yuan Wang 7,8,\* and Hsien-Yeh Chen 1,2,3,\***


**Abstract:** Modifications of biomaterials based on the combination of physical, chemical, and biological cues for manipulating stem cell growth are needed for modern regenerative medicine. The exploitation of these sophisticated modifications remains a challenge, including substrate limitation, biocompatibility, and versatile and general cues for stem cell activities. In this report, a vapor-phase coating technique based on the functionalization of poly-*p*-xylylene (PPX) was used to generate a surface modification for use with stem cells in culture. The coating provided the ability for covalent conjugation that immobilized bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2), and the modified coating surfaces enabled direct stem cell differentiation and controlled proliferation because of the specific activities. The ligations were realized between the growth factors and the maleimide-modified surface, and the conjugation reactions proceeded with high specificity and rapid kinetics under mild conditions. The conjugation densities were approximately 140 ng·cm−<sup>2</sup> for BMP-2 and 155 ng·cm−<sup>2</sup> for FGF-2. Guiding the activities of the human adipose-derived stem cells (hADSCs) was achieved by modifying surfaces to promote the hADSC differentiation capacity and proliferation rate. The reported coating system demonstrated biocompatibility, substrate-independent conformity, and stability, and it could provide an effective and versatile interface platform for further use in biomedical applications.

**Keywords:** growth factor; surface modification; CVD polymerization; biointerface; stem cells

## **1. Introduction**

Surface properties are fundamental to advanced biomaterials that are designed to induce biological functions, and they include tuning surface wettability; the ability to enhance or to resist protein adsorption, cell adhesion or resistance; antibacterial action; and engineered niches for stem cell proliferation, migration, and differentiation [1–5]. In

**Citation:** Wu, C.-Y.; Chiang, Y.-C.; Christy, J.; Huang, A.P.-H.; Chang, N.-Y.; Wenny; Chiu, Y.-C.; Yang, Y.-C.; Chen, P.-C.; Wang, P.-Y.; et al. Guiding Stem Cell Differentiation and Proliferation Activities Based on Nanometer-Thick Functionalized Poly-*p*-Xylylene Coatings. *Coatings* **2021**, *11*, 582. https://doi.org/10.3390/ coatings11050582

Academic Editor: Alenka Vesel

Received: 23 March 2021 Accepted: 12 May 2021 Published: 17 May 2021

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**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/).

particular, sophisticated applications of these modification techniques that make stem cells renew themselves and retain their potency (i.e., the capability to differentiate into specialized cell types) [6,7] are under development. Promising results of unlimited cell expansion or lineage-specific differentiation in a reproducible and controlled manner and efficient and controlled differentiation commitment of specific cells and tissues were achieved in vitro and in vivo with combinations of mimicked physical, chemical, and/or biological cues [8–10]. Emerging therapeutic uses, encouraged by the advances in modern regenerative medicine, have also benefited from these promising results from research on stem cells used for tissue transplantation and repair. The current obstacles to achieving sophisticated material modifications include the potential harm from substances used during the process, the need for multiple steps to achieve the required sophistication, and techniques that are based on previous performance in a case-by-case manner and with a limited selection of materials.

The study herein aims to demonstrate an effective and general interface modification method for use in defining stem cell fate and enhancing the functions related to proliferation rate and multilineage differentiation capacity. The proposed modification technique was realized by the functionalization of poly-*p*-xylylene that includes a maleimide side group. The functional polymer was synthesized as an interface layer generated in one vapor-phase coating process and applied to the material surface through the exploitation of a chemical vapor deposition (CVD) polymerization process used to prepare maleimide-functionalized poly-*p*-xylylene (hereafter referred to as maleimide-PPX) coatings. Compared to other similar poly-*p*-xylylene systems, this technology, free of solvents, initiators, and catalysts, produced a coating that exhibited good conformability with the topology and geometry of substrates [11–13] with excellent cohesive properties and thermal stability on various substrates, including metals, oxides, polymers, nonvolatile liquids, silicon, and glass [12]. Most importantly, in the current study, conjugation-immobilized (i) bone morphogenetic protein 2 (BMP-2) and (ii) fibroblast growth factor 2 (FGF-2) enabled the modified coating surfaces to direct stem cell differentiation and controlled proliferation driven by the functions specified by (i) and (ii), respectively. The resulting BMP-2- and FGF-2-modified substrates were expected to be sustainable and multifunctional, so that they could provide an effective and flexible interface platform for biomedical applications.

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

## *2.1. CVD Surface Modifications*

The synthesis of the coating, poly[(4-N-maleimidomethyl-*p*-xylylene)-co(*p*-xylylene)] (maleimide-PPX), was conducted by a custom-built CVD system (Kao Duen Technology Co., Ltd., Taipei, Taiwan) consisting of a sublimation zone, pyrolysis furnace, and deposition chamber. During the CVD process, the starting material (dimer) of 4-N-maleimidomethyl- [2.2]paracyclophane, which was prepared following procedures reported elsewhere [14], was first sublimated at approximately 120 ◦C in the sublimation zone. The sublimated species were then transferred to the pyrolysis furnace (560 ◦C) with a constant flow (30–50 sccm) of argon carrier gas. Following pyrolysis, the resulting highly reactive monomers were transferred into the deposition chamber and polymerized onto a rotating holder at 20 ◦C to form a uniformly deposited film of maleimide-PPX. To optimize the deposition, a pressure of 75 mTorr was maintained throughout the CVD process, and deposition rates were controlled at approximately 0.5 Å·s−1, which was monitored via an in-situ quartz crystal microbalance (STM-100/MF, Sycon Instruments, Syracuse, NY, USA). The thickness of the polymer layer was measured as approximately 100–150 nm by using spectroscopic ellipsometry (M2000, Woollam Co., Inc., Lincoln, NE, USA) after retrieving the coated samples from the CVD system. The resultant polymer coating was also characterized by using Fourier transform infrared (FT-IR, PerkinElmer, Waltham, MA, USA) and X-ray photoelectron spectroscopy (XPS, Thermo Scientific, Leicestershire, UK) to verify the anticipated chemical compositions.

## *2.2. Growth Factor Immobilization*

Selected human recombinant growth factors, including BMP-2 and FGF-2, were obtained commercially from R&D Systems Inc. (Minneapolis, MN, USA) and reconstituted into a 100 <sup>μ</sup>g·mL−<sup>1</sup> solution following the manufacturer's instructions. These two growth factors were then conjugated to the maleimide-PPX coated substrates via a coupling reaction between the maleimide and the thiol (sulfhydryl) groups under conditions of 4 ◦C and pH 6.5–7.5 for 6 h. Dithiothreitol (DTT, Sigma Aldrich, St. Louis, MO, USA) (5 mM) was used as a reducing agent for the BMP-2 and FGF-2 immobilization. A rinsing process was performed three times with PBS to remove any unbound molecules.

## *2.3. Characterizations*

FT-IR spectra were recorded using a spectrum 100 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) equipped with an advanced grazing angle specular reflectance accessory (AGA, PIKE Technologies, Fitchburg, WI, USA) and a liquid nitrogen-cooled MCT detector. The samples were mounted in a nitrogen-purged chamber to eliminate noise from the CO2 and H2O, and the recorded spectra were corrected for any residual baseline drift. The XPS data were recorded using a Theta Probe X-ray photoelectron spectrometer (Thermal Scientific, Leicestershire, UK) with a monochromatized AlKα X-ray source at an X-ray power of 150 kW. The pass energies were 200.0 eV and 20.0 eV for the survey scan and the high-resolution C1s elemental scan, respectively. The XPS spectrum atomic analysis was reported based on atomic concentrations (%), and the results were compared to theoretical values calculated based on the structure. The binding capacities of the modified surfaces for the growth factor protein were measured using a standard QCM instrument (ANT Technologies, Taipei, Taiwan) with a quiescent mode under sealed conditions, and the measuring of the deposited protein mass was based on the fundamental frequency shift as compared with unbinding or unmodified surfaces. For the antibody affinity experiments performed by flow mode, the QCM instrument was further equipped with a flow injection analysis (FIA) device (MasterFlex, Cole-Parmer Instrument Co., Chicago, IL, USA), and a continuous frequency variation recording device was used for the characterization. The flow rate was controlled using a peristaltic pump connected to the FIA device, and the pumping process was temporarily stopped for 25 min (10 min after injection) to allow anti-BMP-2 or anti-FGF-2 antibodies to bind to the proteins. All experiments were carried out at 25 ◦C, and each sample was measured in triplicate. The frequency shift ΔF resulting of a deposited mass Δm is described by the Sauerbrey equation [15]:

$$
\Delta \mathcal{F} = \frac{-2\Delta m f^2}{A\sqrt{\mu \mathcal{P}\_\emptyset}} = -C\_f \Delta m
$$

where *f* is the intrinsic crystal frequency (9 MHz), *A* is the piezo-electrically active area (0.091 cm2), *ρ<sup>q</sup>* is the quartz crystal density (2.648 g/cm3), *μ* is the shear modulus of the quartz crystals (2.947 × 1011 dyn/cm2), *Cf* is the mass sensitivity (2.013 Hz/ng for a 9 MHz crystal), and Δm is the adding mass on the crystal surface due to specific binding, respectively.

#### *2.4. μCP and Immunofluorescence*

Confinement of the reactions using μCP was performed using two different poly- (dimethylsiloxane) (PDMS) stamps consisting of squares with 30 μm × 30 μm sides (for BMP-2) and 50 μm × 50 μm sides (for FGF-2) and arrays with 100 μm center–center spacing. Solutions with the growth factor proteins (BMP-2 or FGF-2) were used as the inking solutions and allowed to react on top of the maleimide-PPX-modified substrates for 6 h. The μCP process was performed at 4 ◦C and 55% humidity. The resulting sample was washed twice with PBS to remove any unbound molecules. For the self-assembled binding of the primary and secondary antibodies, the BMP-2- and FGF-2-modified samples were incubated in the corresponding antibody solution, anti-BMP-2 antibody (50 <sup>μ</sup>g·mL−<sup>1</sup> in PBS, R&D Systems, Minneapolis, MN, USA) or anti-FGF-2 antibody (50 <sup>μ</sup>g·mL−<sup>1</sup> in PBS, R&D Systems, Minneapolis, MN, USA), for 2 h, and the samples were washed twice with PBS to remove any unbound antibodies. Subsequently, fluorescently labeled secondary antibodies were incubated with the samples for 1 h and washed twice with PBS to remove any unbound antibodies. The samples with fluorescence signals were analyzed under a LEICA fluorescence microscope (DMI3000B, Leica Microsystems, Wetzlar, Germany).

#### *2.5. Induced Cellular Activities*

Tissue culture polystyrene (TCPS) plate substrates (24 well, Corning) were modified with a CVD copolymer coating and conjugated with BMP-2 or FGF-2 growth factor proteins following the procedures described above, and the modified TCPS plates were used for cell culture experiments. The pure maleimide-PPX coating surface and the untreated TCPS plate surface served as controls for the comparison. Normal human adipose-derived stem cells (hADSCs) were isolated from subcutaneous adipose tissue following the reported procedures [13], and the protocols were approved by the Internal Ethical Committee of National Taiwan University Hospital. To evaluate differentiation and proliferation activities, the hADSC cultures were initially seeded at a density of 1 × <sup>10</sup><sup>5</sup> cells·cm−<sup>2</sup> and <sup>2</sup> × 104 cells·cm−2, respectively, for the following characterization. Each experiment was conducted in triplicate.

#### *2.6. Osteogenesis*

Osteogenic differentiation-induced activity was examined by culturing hADSCs in osteogenic differentiation medium [16]. The osteogenic activity of early stage alkaline phosphatase (ALP) expression was analyzed using a 5-Bromo-4-Chloro-3-Indolyl Phosphate / Nitroblue Tetrazolium (BCIP/NBT) liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA) at day 10, whereas the late stage osteogenic activity, as indicated by calcium deposition, was confirmed by staining with a 2% alizarin red S solution (ARS, Sigma Aldrich, St. Louis, MO, USA) at day 21. The resulting ALP and ARS signals were quantitatively measured using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) with an absorbance wavelength of 405 nm following the manufacturer's instructions.

## *2.7. Chondrogenesis*

Chondrogenic differentiation-induced activity was examined in chondrogenic differentiation medium [16] for 14 days. The cultured cells were fixed in 10% formalin (Macron Fine Chemicals, Center Valley, PA, USA), and the proteoglycan present in the cartilage matrix was detected by staining with 0.4% (*w*/*v*) toluidine blue O (Sigma-Aldrich) in 0.1 M sodium acetate buffer (pH 4.0). The sulfated glycosaminoglycan (sGAG) content was further analyzed by using a 1,9-dimethylmethylene blue (DMMB, Sigma-Aldrich) dye-binding spectrophotometric assay, and quantification was based on the absorbance difference between 525 and 595 nm [17]. The expression profiles of the chondrogenesis-related gene encoding collagen type II were also analyzed.

#### *2.8. Adipogenesis*

For the analysis of adipogenic differentiation induced activity, hADSCs were cultured in adipogenic differentiation medium [16]. On day 10, the cells were fixed with 10% formalin and then stained with 0.25% (*w*/*v*) oil red O (Sigma-Aldrich) to observe lipid droplets. The resulting oil red O signals were further quantified using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) with an absorbance wavelength of 510 nm following the protocol reported [18]. The expression profiles of the adipogenic gene Fatty Acid-Binding Protein 4 (FABP-4) were also analyzed.

#### *2.9. Proliferation Activities*

To judge proliferation activities, a cell growth medium recipe and culture conditions were used as previously described [19]. After culturing with cells for 1 day and 5 days, the resulting cultured sample surfaces were observed and photographed using an optical microscope to determine cell proliferation levels. The cell numbers for the studied surfaces were further measured quantitatively using a 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium Bromide (MTT) assay (Sigma-Aldrich) according to the manufacturer's instructions. The normalized ratio of cells on day 5 to cells on day 1 was used to evaluate the cell proliferation-induced capacities of the modified surfaces.

#### *2.10. Gene Expression Profiles*

The gene expression levels were determined by quantitative real-time polymerase chain reaction (qPCR) analysis following the manufacturer's protocols. Briefly, total RNA from the hADSCs and differentiated cells was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and then the RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized subsequently from the RNA template using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Gene expression level was analyzed using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific) with gene-specific primers (as indicated in Table 1), and the β-actin was used as a loading control to normalize the expression of the target gene(s) between different samples. The thermal program for PCR was as follows: 35 cycles of 94 ◦C for 30 s; 60 ◦C for 30 s; and 72 ◦C for 1 min followed by incubation at 72 ◦C for 5 min. The fold changes in gene expression were calculated with the delta-delta Ct method [20].

**Table 1.** PCR primer sets used in this study.


## *2.11. Statistical Analysis*

All data are reported as the mean ± standard deviation (S.D.) and are representative of three or more independent experiments. According to the unpaired *t*-test, GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) was used to test the statistical differences between the experimental group and the control group, and a two-tailed *p*-value less than 0.05 was considered significant.

## **3. Results**

The synthesis of the maleimide-PPX coating was conducted similarly to that of other poly-*p*-xylylene (PPX) systems; that is, a precursor of a maleimide-substituted [2.2]paracyclophane (prepared in-house) [21] was sublimated at approximately 120 ◦C and pyrolyzed at approximately 560 ◦C to generate reactive monomers of quinodimethane. The entire process involved vapor-phase deposition and polymerization (chemical vapor deposition, CVD) under a reduced pressure of 75 mTorr, and a deposition rate of approximately 0.5–1.0 Å·s−<sup>1</sup> was regulated during the CVD process with a linear-dependent time theoretical parameter [22,23] that was controlled to ensure that the final coating thickness was in the range of 100–150 nm. As shown in Figure 1a, the rational maleimide functionality enabled the resultant coating to readily undergo a maleimide-thiol coupling reaction to the target molecules that contain thiols through a Michael-type nucleophilic addition at pH 6.5–7.5 [14], and successful conjugation created through this reaction to attach protein molecules without compromising protein structures and functions has been shown [24]. The important application of using maleimide-PPX to conjugate FGF-2 and BMP-2 growth factor proteins was therefore exploited under mild reaction conditions. The successful

conjugation was confirmed by comparing the Fourier transform infrared (FT-IR) spectra with those of a pure maleimide-PPX coating, and the characteristic absorption values of N–H peaks in the range from 3200 to 3600 cm−1, which represent the immobilized FGF-2 and BMP-2 proteins, were detected (Figure 1b). The additional FT-IR spectra that comparing maleimide-PPX surface with parylene-C surface combined with the X-ray photoelectron spectroscopy (XPS) survey to verify the anticipated chemical compositions of the maleimide-PPX coating are included in Supplementary Figure S1. Most importantly, the FT-IR spectra showing the thiol groups of BMP-2 and FGF-2 proteins (i.e., the S-H band at 2634 cm<sup>−</sup>1) were highlighted in Supplementary Figure S2. After conjugating BMP-2 and FGF-2 to the coating surface, the S-H peak disappeared (see Figure 1). This result proved that the two adhesion cues BMP-2 and FGF-2 are covalently bonded to the coating surface.

**Figure 1.** (**a**) Schematic illustration of the created cell differentiation-induced surfaces using the maleimide-PPX coating to immobilize the BMP-2 and FGF-2 growth factor proteins. (**b**) FT-IR spectra show the immobilized BMP-2 and FGF-2 on the maleimide-PPX surface. The peaks in the range from 3200 cm−<sup>1</sup> to 3600 cm−<sup>1</sup> indicate the characteristic N–H band absorption of BMP-2 and FGF-2. FT-IR spectra of the pure maleimide-PPX surfaces are also presented for comparison. The peaks located at 1396 cm−<sup>1</sup> (C–N) and 1706 cm−<sup>1</sup> (C=O) correspond to the characteristic stretching bands for the maleimide groups.

Furthermore, the generation of the maleimide-PPX coating and the subsequent conjugation of FGF-2 and BMP-2 were seamlessly performed on a quartz crystal microbalance (QCM) sensor chip, and the recorded frequencies before and after conjugation were compared to estimate the density of conjugated FGF-2 and BMP-2, which was thus determined as approximately 140 ng·cm−<sup>2</sup> for BMP-2 and 155 ng·cm−<sup>2</sup> for FGF-2 (Figure 2a). These results showed agreement with other similar PPX systems that have been used to attach biomolecules. Theoretically, the conjugation density is tunable, and the effectiveness of the resultant biological activities is predictable [25]. On the other hand, as presented in Figure 2b, a dynamic QCM analysis in which anti-BMP-2 or anti-FGF-2 antibodies could bind to the modified surfaces was performed in the elapsed time, and the results indicated significantly decreasing frequencies due to the binding of anti-BMP-2 antibody and anti-FGF-2 antibody on the BMP-2-modified and the FGF-2-modified coatings, respectively, compared to the nearly unchanged frequencies on the pure coating surfaces. Notably, the small frequency drop in the pure coating study cannot be considered as expected antibody-antigen specific bonding, because this reduction level of frequency may be due to the limitations of the QCM equipment and the measurement errors caused by random fluctuations in the experimental phase [26]. The results not only confirmed the identity and activity of the immobilized FGF-2 and BMP-2 proteins but also indicated a firm attachment of these proteins by covalent conjugation, which was also found by the abovementioned FT-IR data. Other supporting data were used to verify the reactivity and specificity of the immobilized FGF-2 and BMP-2 surfaces. As shown in Figure 2c, they were derived from the use of a microcontact printing (μCP) technique with a PDMS stamp to induce a selective reaction with BMP-2 and FGF-2 at confined locations of the maleimide-PPX coating surfaces. Subsequently, solutions with a primary antibody and a fluorescently labeled secondary antibody (fluorescein for the BMP-2 secondary antibody and Alexa Fluor® 555 for the FGF-2 secondary antibody) were incubated with these selectively modified samples, and the combined self-assembled bindings between the growth factor proteins and these antibodies showed strong, distinguishable fluorescence signals in the green channel with fluorescein on the BMP-2-modified regions and in the red channel by Alexa Fluor® 555 on the FGF-2 regions. These patterns were consistent with those from the μCP stamps. The specific binding affinities of the two growth factor proteins were unambiguously verified.

**Figure 2.** (**a**) Quantification of the conjugation densities of BMP-2 and FGF-2 by using QCM analysis. (**b**) QCM dynamic analysis of the binding affinity of the studied surfaces toward anti-BMP-2 and anti-FGF-2 antibodies. (**c**) The activity and specificity of the immobilized growth factors were verified by exploiting a μCP technique to spatially confine the conjugation of BMP-2 and FGF-2 to defined locations. Combinations of self-assembled binding affinities by first antibodies (anti-BMP-2 and anti-FGF-2) and by secondary antibodies (fluorescently labeled secondary antibodies) show that the fluorescence patterns are consistent with the μCP patterns.

The induction of these hADSCs toward osteogenic differentiation was studied by examining their alkaline phosphatase (ALP) expression in the early stage and alizarin red staining (ARS) in the mature stage of osteogenesis, and the results in Figure 3a indicated the enhanced ALP expression at day 10 on the modified surfaces of BMP-2 compared to the expression levels on the control surfaces consisting of pure maleimide-PPX and unmodified tissue culture polystyrene (TCPS) plates. Similar results from experiments on enriched calcium deposition, as evaluated by ARS signals at day 21, were also observed for the BMP-2 surfaces. Statistical analysis of these ALP and ARS signals, shown in Figure 3b,c, further confirm the enhanced osteogenesis on the growth factor-modified surface (\*\*\* *p* < 0.001 relative to the control surfaces). The BMP-2-modified surface had a greater level of osteogenesis activity signal expression, with a 276.9% higher intensity compared to the lowest level, which was for the TCPS control surface. Furthermore, the hADSC activity toward chondrogenic differentiation was investigated on those surfaces by detecting the known mechanism and characteristic indications of chondrogenesis, as determined at day 14 by (i) staining and characterizing the color and intensity changes caused by the metachromatic binding between toluidine blue and cartilage polysaccharides, (ii) sulfate glycosaminoglycan (sGAG) deposition, and (iii) collagen type II expression. The results presented in Figure 4a–c reveal that the intensities and signals for (i), (ii), and (iii) were all consistently greater on the BMP-2-modified surface than they were on the other surfaces (\*\*\* *p* < 0.001), indicating that chondrogenesis was best enhanced on this modified surface among those studied.

**Figure 3.** The osteogenesis activities of hADSCs were examined on the modified surfaces with immobilized BMP-2 growth factor protein. Pure maleimide-PPX coating surfaces and unmodified tissue culture polystyrene (TCPS) surfaces were used for the comparison. (**a**) The early stage osteogenesis marker, ALP expression, was analyzed at day 10, and the mature stage marker, calcium deposition, was observed at day 21 using alizarin red staining (ARS). The results of statistical analyses of the quantified (**b**) ALP signals and (**c**) ARS signals were also compared for the studied surfaces. The data bars represent the mean value (n = 3) and the standard deviation (±SD) based on three independent experiments. The significance level \*\*\* stands for \*\*\* *p* < 0.001, and is shown by the results of the unpaired *t*-test.

**Figure 4.** Chondrogenic activities of hADSCs were examined on the studied surfaces. (**a**) Toluidine blue staining was performed on day 14 to trigger the metachromatic color change (yellow arrows). The detected intensities from (**b**) sulfated glycosaminoglycan (sGAG) by using a spectrophotometric 1,9-dimethylmethylene blue (DMMB) dye binding assay and (**c**) the chondrogenic marker collagen type II gene expression by quantitative PCR analysis were statistically analyzed and compared for the studied surfaces. The data bars represent the mean value (n = 3) and the standard deviation (±SD) based on three independent experiments. The significance level \*\*\* stands for \*\*\* *p* < 0.001, and is shown by the results of the unpaired *t*-test.

Furthermore, the adipogenic induction of hADSCs on the modified coating surfaces was analyzed by observing the light-refraction of lipid droplets, which accumulate during adipogenesis, within the differentiated cells at day 10. A characteristic staining technique in which oil red O dye binds to lipids was used to visualize the lipid droplets (Figure 5a). In addition, 172.3% increases in adipogenesis activity based on quantitative oil red O staining were confirmed for the FGF-2-modified surfaces, significant difference (\*\*\* *p* < 0.001) compared to that for other surfaces (Figure 5b), and the expression of the FABP-4 gene was detected (Figure 5c), further supporting the quantitative results. Finally, in another demonstration, the functions that determine stem cell action, as based on the use of the growth factor-modified coating, were examined by culturing normal human adipose-derived stem cells (hADSCs) on the modified surfaces. As displayed in Figure 6, the analysis by using phase-contrast microscopy and MTT assay revealed high biocompatibility for cell growth (data are included in Supplementary Figure S3) and reflected a consistent level of cell adhesion for all the studied surfaces on day 1 (Figure 6a,b). Notably, significant variation was discovered between these surfaces over an extended culture time frame such that, at day 5, the FGF-2-modified surfaces specifically exhibited profound proliferation of hADSCs compared to that of the pure maleimide-PPX coating surface and unmodified TCPS surface (Figure 6a,b). Statistically, the proliferation ratio of cell number that was normalized by day 5 to day 1 unambiguously revealed the anticipated results by showing an approximate 377.8% higher ratio for the FGF-2-modified surfaces, a significant difference (\*\*\* *p* < 0.001) compared to that for other surfaces, and the enhanced hADSC proliferation activity from using the growth factor-modified coating technique was also evident (Figure 6c).

**Figure 5.** The adipogenic activities of the hADSCs were examined on the studied surfaces. (**a**) Recorded oil-red O staining images of differentiated cells on these surfaces at day 10. Statistical analyses of the (**b**) oil red O signal intensities obtained by spectrophotometry and the (**c**) FABP-4 marker gene expression obtained by quantitative PCR was performed and compared for these sample surfaces. The data bars represent the mean value (n = 3) and the standard deviation (±SD) based on three independent experiments. The significance level \*\*\* stands for \*\*\* *p* < 0.001, and is shown by the results of the unpaired *t*-test.

The conjugation was attributed to the maleimide-equipped functionality, which was readily accessible to site-specific and covalent binding to thiol moieties through Michaeltype nucleophilic addition with high specificity and rapid kinetics under mild conditions at room temperature in aqueous solutions of pH 7.4. Such conjugation reactions have been shown to be successful without perturbing the protein structures or functions [13,24,27]. In addition, the covalent immobilization of the growth factors to biomaterial interfaces addresses challenges associated with delivering freely diffusible growth factors and has thus emerged as a promising method of achieving localized and sustained growth factor delivery based on a phenomenon called juxtracrine signaling (or contact-dependent signaling) [3,28,29]. Immobilized growth factors (i.e., BMP-2 and FGF-2) provide chemical cues for the guidance of stem cell behaviors and are therefore applicable to the material surface, where they demonstrate the concept. More importantly, both BMP-2 and FGF-2-functionalized surfaces can promote multilineage differentiation. However, only the FGF-2-functionalized surface had an enhanced effect on the normal growth of cultures, and this result is consistent with other publications. BMP-2, which belongs to the transforming growth factor beta (TGF-β) family, is now recognized as a multipurpose cytokine. When the chemical composition of the modified coatings and the conjugation reactions were confirmed, the current coating technique was also demonstrated to direct cellular activities for mesenchymal stem cells (MSCs) and induce BMP-2 function via activation of the SMAD signaling mechanism [30,31], which leads to MSC multilineage differentiation that includes not only osteogenesis and chondrogenesis [32] but also adipogenesis [33–35]. On the other hand, FGF-2 has been shown to be part of a signaling pathway distinct from that of BMP-2 that enhances the self-renewal of MSCs such that they maintain their multilineage differentiation potential [36–38]. The coating material, poly-*p*-xylylene, is a highly biocompatible polymer of Class VI in the United States Pharmacopeia (USP), and the biocompatibility (or cell viability) examinations associated with the polymer system (thin films) or the same

produced devices were well-reported during the past including biocompatibility against various types of cells (including hADSCs). The convincing results of stem cell differentiation in this report also clearly support the argument that the cells are alive, especially in the final stage of cell differentiation. Compared with committed or differentiated cells, the most fascinating feature of undifferentiated stem cells is their strong plasticity [39,40], and the coating technology based on the proposed growth factor modifications by using vapor-phase maleimide-PPX can give full play to these characteristics of stem cells. The statistical quantification of these guided cellular activities and, for comparison, that of the pure maleimide-PPX coating surfaces and the unmodified TCPS control surfaces, is further analyzed and summarized in Table 2. These results clearly support that the surfacemodified products can promote the differentiation and proliferation of stem cells under various induction conditions, so as to achieve the expected effects for potential regenerative medicine applications.

**Figure 6.** The proliferation activities of hADSCs were examined on the studied surfaces. (**a**) Phase-contrast micrographs of cell growth patterns recorded on day 1 and day 5. (**b**) Estimated cell numbers on day 1 and day 5 were compared for these surfaces. (**c**) The proliferation ratio that statistically normalized the cell numbers on day 5 to day 1 was compared for these studied surfaces. The data bars represent the mean value (n = 3) and the standard deviation (±SD) based on three independent experiments. The significance levels n.s. and \*\*\* stand for nonsignificant difference and \*\*\* *p* < 0.001, respectively, and are shown by the results of the unpaired *t*-test.


**Table 2.** Enhanced induction properties of growth factor-modified surfaces based on the maleimide-functionalized poly-*p*xylylene (maleimide-PPX) coating a.

<sup>a</sup> Data were normalized and compared against data from a control surface of commercially available tissue culture polystyrene (TCPS) plates. <sup>b</sup> Data were compared and shown based on results from alizarin red staining, 1,9-dimethylmethylene blue staining, oil-red O staining, and cellular expansion rates.

#### **4. Conclusions**

In this study, an effective and general interface modification method for use in defining stem cell fate and enhancing the functions related to proliferation rate and multilineage differentiation capacity was demonstrated. The introduced surface modification was synthesized based on a clean and dry vapor deposition process that rendered a functional polymer coating. Most importantly, the facile integration of the maleimide functionality into the coating enabled the covalent conjugation of BMP-2 and FGF-2 growth factor proteins in mild conditions, which enabled guided differentiation and proliferation of the MSCs. With the proven concept that coating technology can be used not only on a variety of substrate materials, but also for conformity and stability on devices with sophisticated topology and geometry, we foresee that these findings have significant potential for further use in biomedical applications because of the various controllable parameters in favorable conditions offered by this coating technology.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/coatings11050582/s1, Figure S1: Maleimide-functionalized poly-p-xylylene (maleimide-PPX) coating. (a) Infrared reflec-tion-absorption spectroscopy (IRRAS) spectra for the maleimide-PPX coating. The peaks located at 1396 cm−<sup>1</sup> (C–N), 1706 and 1767 cm−<sup>1</sup> (C=O) correspond to the characteristic band stretches for the maleimide groups. Infrared reflection absorption spectroscopy (IRRAS) spectra for the Parylene-C coating which has no maleimide was also shown for the better comparison. (b) X-ray photoelectron spectroscopy (XPS) characterization of the maleimide-PPX coating. The table com-pares the experimental values of the XPS survey high-resolution C1 s spectra with the theoretical predictions. The signal at 285.0 eV is attributed to the aliphatic and aromatic carbons (C–C, C–H), and the intensity at 84.5 at% compares well with the theoretical concentration of 84.2 at%. The C–N bond was detected with 5.6 at%, which compares well with the theoretical value of 5.3 at%. The peak at 288.6 eV was assigned to the O=C–N group of the maleimide (7.1 at%) and agrees with the theoretical value of 10.5 at%. The signal at 291.4 eV (2.8 at%) indicates π→π\* transitions, Figure S2: FT-IR spectra of BMP-2 (bone morphogenetic protein 2) and FGF-2 (fibroblast growth factor 2) alone. The thiol groups of the BMP-2 and FGF-2 proteins (i.e., S-H band at 2634 cm−1) in the spec-tra were highlighted, Figure S3: Biocompatibility analysis of the maleimide-PPX coatings. The human adipose-derived stem cells (hADSCs) were cultured on each surface for 1 day and the cell viabilities were then quantified by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data bars represent the mean value and the standard deviation (±SD) based on three inde-pendent samples. The p-value large than 0.05 indicates the studied surface has a good biocom-patibility with no significant difference (n.s.) as compared to the unmodified TCPS surfaces.

**Author Contributions:** Conceptualization, C.-Y.W., Y.-C.C. (Yu-Chih Chiang), Y.-C.Y., P.-C.C., P.-Y.W., and H.-Y.C.; methodology, C.-Y.W., Y.-C.C. (Yu-Chih Chiang), Y.-C.Y., P.-C.C., P.-Y.W. and H.-Y.C.; software, C.-Y.W., Y.-C.C. (Yu-Chih Chiang), Y.-C.C. (Yu-Chih Chiu), and Y.-C.Y.; validation, C.-Y.W., A.P.-H.H., and W.; formal analysis, C.-Y.W., J.C. and Y.-C.C. (Yu-Chih Chiu); investigation, Y.-C.C. (Yu-Chih Chiu), N.-Y.C. and Y.-C.Y., ; resources, P.-C.C., P.-Y.W., and H.-Y.C.; data curation, Y.-C.C. (Yu-Chih Chiang), A.P.-H.H. and W.; writing—original draft preparation, C.-Y.W., Y.-C.C. (Yu-Chih Chiang), P.-C.C., P.-Y.W. and H.-Y.C.; writing—review and editing, C.-Y.W., Y.-C.C. (Yu-Chih Chiang), P.-C.C., P.-Y.W. and H.-Y.C.; visualization, C.-Y.W., N.-Y.C. and J.C.; supervision, P.-C.C., P.-Y.W. and H.-Y.C.; project administration, P.-C.C., P.-Y.W. and H.-Y.C.; funding acquisition, P.-C.C., P.-Y.W. and H.-Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** H.-Y.C. gratefully acknowledges support from the Ministry of Science and Technology of Taiwan (MOST 108-2221-E-002-169-MY3). P.-C.C. gratefully acknowledges support from the Ministry of Science and Technology of Taiwan (MOST 107-2221-E-027-009-MY2; 108-2321-B-010-008-MY2). P.-Y.W. acknowledges the support from the National Key Research and Development Program of China (2018YFC1105201), the National Natural Science Foundation of China. This work was further supported by the "Advanced Research Center For Green Materials Science and Technology" from The Featured Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (107L9006) and the Ministry of Science and Technology in Taiwan (MOST 107-3017-F-002-001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in this published article and its supplementary information files.

**Conflicts of Interest:** The authors declare no competing financial interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**


## **References**


## *Article* **Vapor-Phase Fabrication of a Maleimide-Functionalized Poly-***p***-xylylene with a Three-Dimensional Structure**

**Shu-Man Hu 1,†, Chin-Yun Lee 1,†, Yu-Ming Chang 1, Jia-Qi Xiao 1, Tatsuya Kusanagi 1, Ting-Ying Wu 1, Nai-Yun Chang 1, Jane Christy 1, Ya-Ru Chiu 1, Chao-Wei Huang 2, Yen-Ching Yang 1,\*, Yu-Chih Chiang 3,4,\* and Hsien-Yeh Chen 1,4,5,\***


**Abstract:** A vapor-phase process, involving the sublimation of an ice substrate/template and the vapor deposition of a maleimide-functionalized poly-*p*-xylylene, has been reported to synthesize an advanced porous material, with readily clickable chemical interface properties, to perform a Michael-type addition of a maleimide functionality for conjugation with a thiol group. In contrast to the conventional chemical vapor deposition of poly-*p*-xylylenes on a solid surface that forms thin film coatings, the process reported herein additionally results in deposition on a dynamic and sublimating ice surface (template), rendering the construction of a three-dimensional, porous, maleimidefunctionalized poly-*p*-xylylene. The process seamlessly exploits the refined chemical vapor deposition polymerization from maleimide-substituted [2,2]paracyclophane and ensures the preservation and transformation of the maleimide functionality to the final porous poly-*p*-xylylene products. The functionalization and production of a porous maleimide-functionalized poly-*p*-xylylene were completed in a single step, thus avoiding complicated steps or post-functionalization procedures that are commonly seen in conventional approaches to produce functional materials. More importantly, the equipped maleimide functionality provides a rapid and efficient route for click conjugation toward thiol-terminated molecules, and the reaction can be performed under mild conditions at room temperature in a water solution without the need for a catalyst, an initiator, or other energy sources. The introduced vapor-based process enables a straightforward synthesis approach to produce not only a pore-forming structure of a three-dimensional material, but also an in situ-derived maleimide functional group, to conduct a covalent click reaction with thiol-terminal molecules, which are abundant in biological environments. These advanced materials are expected to have a wide variety of new applications.

**Keywords:** vapor sublimation and deposition; functional poly-*p*-xylylene; maleimide; porous material; orthogonal conjugation

## **1. Introduction**

Porous materials, owing to their remarkable interface properties, have been widely applied in sensing, catalysis, biomedical, drug delivery, adsorption/desorption applica-

**Citation:** Hu, S.-M.; Lee, C.-Y.; Chang, Y.-M.; Xiao, J.-Q.; Kusanagi, T.; Wu, T.-Y.; Chang, N.-Y.; Christy, J.; Chiu, Y.-R.; Huang, C.-W.; et al. Vapor-Phase Fabrication of a Maleimide-Functionalized Poly-*p*-xylylene with a Three-Dimensional Structure. *Coatings* **2021**, *11*, 466. https://doi.org/10.3390/ coatings11040466

Academic Editor: Alessandro Patelli

Received: 12 March 2021 Accepted: 13 April 2021 Published: 16 April 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/).

tions, etc. [1–4]. These applications can especially be highlighted in their drug delivery and emerging biomedical applications due to the multiple premium interface properties that are required [5,6]. The benefits of porous structures are attributed to their large surface area, the adjustable number of pores, and a functionalized surface, which can provide multifunctional active sites [7]. In addition to porous properties, bioconjugation functionalities that provide selectivity and orthogonal reactivity, while avoiding any side reactions in the vast and diverse conditions of biological microenvironments, are key to achieving successful biointerface construction. For example, thiol–maleimide "click" chemistry and maleimide functional polymers selectively target thiol-containing cysteine residues in proteins and enzymes, with excellent levels of selective conjugation being achieved. Cysteine-reactive polymers can be applied to prevent the degradation of sensitive poly(ester) backbones [8]. In addition, the prototypical click chemistry reaction, Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), involves a modification to the triazole-forming 1,3-dipolar cycloaddition. The application of CuAAC to produce fluorogenic compounds would be a powerful way to track biomolecules in the cell [9]. Specific reactions can also be applied for protein modification. The ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine enables protein modification at low concentrations. This reaction can tolerate multiple functional groups and proceed in high-yield organic solvents, water, or cell media [10]. Recently, many bio-orthogonal reactions have been developed that enable the efficient formation of a specific product in complex environments, which is particularly beneficial for biological research [11].

These fascinating chemistries, however, have seldom been exploited during the fabrication of porous materials and are sporadically seen using complicated synthetic approaches [12] or by post-modification attempts, which involve using harsh solvents or potentially harmful chemicals during the modification process [13,14].

In the current study, we introduced a simple and versatile approach, based on a vapor-phase fabrication process, to produce a functionalized porous polymer material. Fabrication occurred by the vapor deposition of a poly-*p*-xylylene polymer on an iced substrate (vapor sublimation), which was discovered in our previous reports, and was compared to the conventional vapor deposition of poly-*p*-xylylene on a still and nonsublimated substrate to produce a thin, dense coating [15]. This sublimation mechanism resulted in the formation of a porous poly-*p*-xylylene monolith [16,17]. Such a unique fabrication process, with the use of doped ice templates, has also been exploited to fabricate tissue repair membranes [18] and scaffolds for stem cell cultures [19]; however, only composites combined with other functional materials have been used for these works in the past. On the other hand, the functionalization of the poly-*p*-xylylene polymers can be prepared by modifying the substituents of [2,2]paracyclophane precursors, and a variety of such substitutions have been produced for functionalized poly-*p*-xylylene polymer and copolymer coatings and applications [20–32]. Therefore, in the current work, we hypothesized that maleimide-substituted [2,2]paracyclophane could be used during vapor sublimation on an iced substrate, and that vapor deposition by such a precursor can produce a maleimide-functionalized poly-*p*-xylylene in porous and monolith forms (Figure 1). Compared to the maleimide-functionalized coating prepared previously [33], the current monolith poly-*p*-xylylene comprised (i) porous and structural information in three dimensions and (ii) a maleimide functionality that exhibits specific reactivity toward thiol-terminated biomolecules. This unique fabrication process, and the resultant polymer product, combines interface properties from its surface porosity, topology, and specific chemical reactivity and provides a robust tool with enhanced synergistic ability for biointerface engineering.

**Figure 1.** A schematic illustration of vapor-phase fabrication by chemical vapor deposition polymerization of 4-N-maleimidomethyl-[2,2]paracyclophane on a sublimating ice substrate/template to construct a porous and maleimidefunctionalized poly-*p*-xylylene material. The resultant functional material exhibits interface chemical properties and is readily able to perform the maleimide-thiol click reaction under mild conditions.

## **2. Materials and Methods**

## *2.1. Fabrication Process*

For the production of poly-*p*-xylylene three-dimensional porous materials, cubeshaped ice templates, with the dimensions of 300 μm × 300 μm × 300 μm, were prepared in order to prove the concept of the reported fabrication process. Using a negative mold of polydimethylsiloxane (PDMS) with the same dimensions to prepare this ice template, it was created as previously reported for the PDMS mold [34]. For the preparation of the ice templates, deionized water was held in the PDMS mold, and then the solution underwent a solidification process using a liquid nitrogen bath to transform the water into ice, which was then retrieved from the PDMS mold after a few minutes to obtain the ice templates. The obtained ice templates were then used for further vapor sublimation and deposition processes. During the vapor deposition, 4-N-maleimidomethyl-[2,2]paracyclophane was used as the starting material, and the monomer was polymerized and deposited to form poly[(4-N-maleimidomethyl-*p*-xylylene)-co-(p-xylylene)] via chemical vapor deposition (CVD) polymerization. The starting materials were vaporized under reduced pressure at 100 mTorr and approximately 90 ◦C, followed by pyrolysis at approximately 670 ◦C, for conversion to highly reactive monomers. During the vapor sublimation, the prepared ice templates were placed in the chamber with an operating environment of approximately 4 ◦C and 100 mTorr, which is below the triple point for ice. From a thermodynamic point of view, ice naturally undergoes a sublimation process from the solid phase to the vapor phase. Under the same conditions, the vapor-phase monomer of the maleimide-functionalized poly-*p*-xylylene underwent polymerization and was then simultaneously deposited onto the ice surface. During the above fabrication process, argon gas, with a mass flow rate of 20 sccm (standard cubic centimeter per minute), was used as a carrier gas for transporting the starting material for the sublimation of the maleimide-substituted [2,2]paracyclophane. Depending on the feed amount of the starting material, the deposition rate was adjusted to approximately 0.5 to 1.0 Å/s and monitored by a real-time quartz crystal microbalance (QCM) sensor (STM-100/MF, East Syracuse, NY, USA) mounted in the deposition chamber.

#### *2.2. Characterizations*

Ice substrate/template images were recorded by using a cryo-SEM (scanning electron microscope) instrument (Tabletop TM-3000, Hitachi, Tokyo, Japan), and the samples were examined at the sample stage by cooling with a continuous supply of liquid nitrogen. SEM was performed with an electron energy of 15 keV and a pressure of 100 mTorr. The detection of poly-*p*-xylylene bulks was performed with a Nova NanoSEM 230 scanning electron microscope (FEI, Hillsboro, OR, USA) that was operated at room temperature under a reduced pressure of 4 × <sup>10</sup>−<sup>6</sup> Torr. A micro-CT (micro computed tomography) X-ray imaging system (SkyScan 1176, Bruker, Billerica, MA, USA) was used to examine the three-dimensional images of the porous structures that scanned the sample at 40 kV. The scanning resolution was 9 μm voxels with an integration time of 2000 ms per projection. The acquired projection images were converted into three dimensional (3D) images using CTvox software (Bruker, Billerica, MA, USA). The FTIR spectra were recorded with a Spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, MA, USA). During spectral acquisition, a liquid nitrogen-cooled mercury–cadmium–telluride (MCT) detector and an advanced grazing angle specular reflectance accessory (PIKE Technologies, Fitchburg, WI, USA) were applied. The scanning range was from 500 cm−<sup>1</sup> to 4000 cm−<sup>1</sup> with 64 acquisitions each time. The X-ray photoelectron spectroscopy (XPS) was completed using monochromatic Al K-alpha radiation as the X-ray source with 150 kW of power. The process was performed with a Theta Probe X-ray photoelectron spectrometer (Thermo Scientific, Leicestershire, UK). Elemental analysis of high-resolution C1s was performed at a flux energy level of 20 eV, and the experimental results were compared with the theoretical (calculated) values based on the proposed chemical structures. Analysis of the real-time mass spectra was carried out with a residual gas analyzer (RGA, Hiden Analytical, Warrington, UK) mounted on the deposition chamber. The RGA was operated at 10−<sup>9</sup> Torr in an ultrahigh vacuum with an ionizing emission current of 20 μA and ionizing electron energy of 70 eV. The fluorescence images were obtained with a TCS SP5 CLSM confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) with an Ar/ArKr laser light source (wavelength: 488 nm) to detect the fluorescein (FITC)-labeled cysteine (emission wavelength: 505–525 nm).

#### *2.3. Conjugations*

The conjugation of FITC-labeled cysteine (Thermo Fisher Scientific, Waltham, MA, USA) was carried out by the reaction of a 5 mM molecular solution of FITC-labeled cysteine with the thiol groups of a manufactured sample of porous material for 4 h at 20 ◦C. To remove excess and unreacted reagents from the conjugated samples, a washing process was performed using the following procedure: wash three times with phosphate buffered saline (PBS, pH = 7.4, containing Tween 20, Sigma-Aldrich, St. Louis, MO, USA), once with PBS (without Tween 20) and finally rinse with deionized water.

#### **3. Results and Discussion**

To fabricate the proposed maleimide-functionalized poly-*p*-xylylene materials, ice templates were first constructed, and a reported mechanism showed that the vapor deposition of polychloro-*p*-xylylene on a dynamic substrate of the sublimating ice can result in a final porous and three-dimensional polychloro-*p*-xylylene material [17]. Theoretically, other poly-*p*-xylylene systems, such as its functionalized derivatives [15] and the maleimide-functionalized poly-*p*-xylylene proposed herein, which have been deposited using the same conventional chemical vapor deposition (CVD) polymerization, should be extendable and applied on an ice substrate. Thus, in this experiment, the preparation of the ice substrates/templates was enabled by using a polydimethylsiloxane (PDMS) mold, with the dimensions of 300 μm × 300 μm × 300 μm, and resulted in ice cubes

with the same dimensions, analogous to how ice cubes are made. For vapor deposition, a maleimide-substituted [2,2]paracyclophane starting material was synthesized via the reported routes [33]; theoretically, in the conventional CVD polymerization, these starting materials were sublimated at approximately 90 ◦C under a reduced pressure of 100 mTorr and pyrolyzed at 540 ◦C, forming highly reactive monomer quinodimethanes species. Finally, these monomers underwent radical polymerization upon condensation at a low temperature (40 ◦C or below) and solid substrate formation of thin-film coatings [27,33,35]. However, in the experiments herein, the vapor deposition and polymerization occurred on the prepared ice substrates/templates and under the devised thermodynamic conditions (10 ◦C and under 100 mTorr). The ice substrate transformed from its solid phase to a vapor phase by sublimation, and the deposition and polymerization of quinodimethane occurred on the dynamic ice substrate instead of a conventional solid substrate. The vapor composition during the process was measured by a mass spectrometric residual gas analyzer (RGA). The recorded mass spectra in Figure 2 indicated the presence of sublimating H2O molecules at 18 amu, the Ar carrier gas at 40 amu, and pyrolyzed quinodimethane monomers and derivatives, including N-ethylmaleimide and maleimide-substituted quinodimethane, at 104 amu, 125 amu, and 213 amu, respectively. These data support the hypothesis that the proposed vapor-phase species existed and underwent the sublimation and deposition processes at the substrate interface. The two processes finally resulted in a construction mechanism [16,17] to build a porous, three-dimensional, maleimide-functionalized poly-*p*-xylylene material. As shown in Figure 3a, the scanning electron microscopy (SEM) images revealed that the resultant porous materials represented replica structures as ice substrates/templates with the same measured dimensions (300 μm × 300 μm × 300 μm). Moreover, the formed porous structures were measured to be in the range of 3 μm to 45 μm, which was confirmed with an average of 20 μm based on micro-computed tomography (micro-CT) analysis (Figure 3b). In addition, the expected interconnection of the pores due to the voids of vapor sublimation and the nucleated polymerization of the poly-*p*-xylylene was confirmed by the reconstructed micro-CT scanning images in Figure 3b, and a video is also included in the Supplementary Materials. From the pore distribution data, the total porosity was 53.8%, of which open porosity accounts for the majority (99.8%). There is an advantage to this structure when combining cell therapy or drug delivery. This structure is beneficial for cell growth, as open pores will not stop drugs from moving, and the drug can also be released smoothly to work in the open pore. Moreover, with closed pores, the materials will be blocked from the outside. However, closed pores only account for 0.2% of the total pores. Various types of porosity and pore sizes were computed during the micro-CT analysis (Figure 3c). Hence, in this study, we demonstrate that micro-CT is versatile in evaluating scaffolds and is able to characterize them from multiple aspects.

**Figure 2.** The characterizations of the vapor compositions, based on mass spectrometric analysis using a (residual gas analyzer) RGA during the fabrication process. The recorded mass spectra in (**a**) show the presence of 5 expected molecules: (1) the sublimating water molecules at 18 amu, (2) the carrier gas of argon at 40 amu, (3) the depositing quinodimethane at 104 amu, (4) a fraction of N-ethylmaleimide at 125 amu, and (5) the maleimide-substituted quinodimethane at 213 amu. (**b**) A table summarizing the details of the detected molecules in the vapor compositions.

**Figure 3.** *Cont*.

**Figure 3.** The characterization of the porous structures and porosity. (**a**) Scanning electron microscopy (SEM) images showed that cube-shaped ice templates, with the dimensions of 300 μm × 300 μm × 300 μm, were used to form porous maleimide-functionalized poly-*p*-xylylene materials by the proposed fabrication method. (**b**) A reconstructed micro-CT image shows the three-dimensional structure and the pore structures. (**c**) The calculated pore size distribution based on a micro-CT analysis suggests an interconnected pore structure (high open porosity) with an average porosity of 53.8 ± 0.12% and an average pore size of 20 ± 2 μm.

Furthermore, the chemical structures of the resulting porous materials were verified by using Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and mass spectrometry analysis. As shown in Figure 4, the FT-IR spectra compared the characteristic peaks of the porous materials of the maleimide-functionalized poly-*p*-xylylene with the thin-film form of this polymer that was deposited on a conventional, gold-coated silicon wafer solid substrate. The verification of the maleimide functionality was found from the peaks between 1704 to 1775 cm−<sup>1</sup> that were attributed to C=O bands, while the peak at 1345 cm−<sup>1</sup> was from the C-N bond of maleimide. Additionally, the characteristic peaks widely seen for poly-*p*-xylylenes and their derivatives were also observed from 2920 to 2854 cm−<sup>1</sup> [26,27,30,32,33,36]. Compared to the pure poly-*p*-xylylene, the spectra of the maleimide-functionalized poly-*p*-xylylene were found to be significantly higher with the peaks of C=O and C-N bonds. In addition, the comparison with pure (non-functionalized) poly-*p*-xylylene spectra showed an absence of C=O and C-N bonds for the pure poly-*p*-xylylene, and strong aromatic C=C bands (1530 to 1480 cm<sup>−</sup>1) were discovered for the pure poly-*p*-xylylene but weak bands were discovered for the maleimide-functionalized poly-*p*-xylylene groups. The variations in the aromatic C=C bands, and in other fingerprint regions for different poly-p-xylylene derivatives, were also previously reported [30,33,37,38]. On the other hand, XPS analyses confirmed the molecular compositions and chemical structures. As shown in the XPS high-resolution C1s spectrum (Figure 5), the signal at 285.0 eV belonged to the aliphatic and aromatic carbons (C–C, C–H). In addition, the 76.7 atom% intensity was consistent with the theoretical concentration of 76.2 atom%, and the anticipated binding states, including C-C=O, C-N, O=C-N, and π→π\* transition bonds (indicated in the enlarged image), were also analyzed. The characteristic signals at 286.0 eV and 286.7 eV, whose calculated values were 9.6 atom% and 4.6 atom%, respectively, corresponded to the C–C=O and C–N bonds matching the theoretical values of 9.5 atom% and 4.8 atom%, respectively. The signal at 289.2 eV had a comparably low value from O=C–N bonds (6.7 atom% compared to the theoretical value of 9.5 atom%), and the signal at 291.5 eV (2.4 atom%) indicated π→π\* transitions that are characteristic of aromatic polymers and have been reported for other functionalized poly-*p*-xylylenes [38]. Collectively, the combined FT-IR and XPS data confirm the chemical structures and support the assumption of the synthesized porous maleimide-functionalized poly-*p*-xylylene materials.

**Figure 4.** Fourier transform infrared (FT-IR) analysis showed consistent spectra. Characteristic peaks of the maleimide-functionalized poly-*p*-xylylene were detected for C-H, C=O, and C-N on the fabricated porous materials. The results were compared to a thin-film maleimide-functionalized poly-*p*-xylylene and pure (non-functionalized) poly-*p*-xylylenes.

To further verify the maleimide functionality of the fabricated 3D porous materials that would be available to perform bio-orthogonal reactions toward thiol-terminated molecules, we devised a series of conjugation experiments for the fabricated samples. The maleimide functionality enabled a Michael-type addition that crossed the unsaturated carbon–carbon bonds of the maleimide moiety and specifically reacted with a thiol group. The reaction represented a class of mild, but specific, reactions that were able to conjugate thiol-terminated molecules (widely available in biological systems) at room temperature and in water solvents without the need for metal catalysts, a high temperature, or UV irradiation. Analogous to a series of click reactions, including thiol-ene or thiol-yne reactions and azide–alkyne 1,3-dipolar cycloaddition, the maleimide-thiol coupling reaction has also been described due to its click characteristics and benign reaction conditions [39]. In these experiments, fluorescein (FITC)-labeled cysteines were selected as a model molecule for demonstration, and via the aforementioned reaction mechanism, maleimide specifically clicked to the thiol terminus of a cysteine residue (i.e., the distributed maleimide functionality at the material interface readily reacted with cysteine-FITC, and green-channeled FITC fluorescence signals were therefore detected in the expected and registered areas). As indicated in Figure 6, the FITC signals detected by the confocal laser scanning microscopy (CLSM) showed the same 300 μm × 300 μm × 300 μm array patterns that were consistent in both the ice substrates/templates and the fabricated maleimide-functionalized poly-*p*-xylylene porous materials. The coupling reaction ensured firm covalent bonding between the maleimide and thiol, and precise interface chemistry could be realized compared to approaches using physical adsorption and desorption, which comprise loosely bound interactions that are usually uncontrollable [40]. Therefore, in control experiments, a nonfunctionalized poly-*p*-xylylene porous material was fabricated from unsubstituted [2,2]paracyclophane, and the same ice substrates/templates were used to attempt the

same conjugation reaction by applying cysteine-FITC molecules to the fabricated samples. However, due to the lack of the required maleimide functionality, only the physically and loosely bound cysteine-FITC molecules were attached to the sample surfaces; a regular wash process with detergent removed these physically adsorbed molecules from the samples, and only suppressed fluorescence signals were detected [25,30]. In addition, the CLSM images were collected along the z-axis with a devised distance interval (Z= 10 μm, 50 μm, 90 μm, 130 μm, 170 μm, 210 μm, 250 μm, and 290 μm). These images, in the specific positions shown in Figure 6, indicated distinct patterns of the attached fluorescence signals and are believed to be due to conjugation occurring on the anisotropic porous structure of a maleimide-functionalized poly-*p*-xylylene within the material's 3D architecture. The sustained intensity of the fluorescence signals after the wash process also confirmed the firm attachment of cysteine-FITC via the conjugation reaction. The fabricated maleimidefunctionalized poly-*p*-xylylene materials provided a porous structure and additionally exhibited clickable functional conduct from the maleimide available on the material interface for specific targeting of thiol-terminated molecules, which are widely seen in biological systems. Such porous and functional products are expected to have unlimited applicability.

**Figure 5.** (**a**) X-ray photoelectron spectroscopy (XPS) high-resolution analysis shows the recorded spectra of the fabricated porous maleimide-functionalized poly-*p*-xylylene. Details of the deconvolution spectra showing components with respect to a specific binding energy, including C-C=O, C-N, O=C-N, and π→π\* transition bonds, are indicated in the enlarged image. (**b**) Detailed chemical compositions were confirmed by comparing the binding energies of the expected chemical states for the experimental values with the theoretical values (in brackets).

**Figure 6.** An analysis of the reactivity of the fabricated porous maleimide-functionalized poly-*p*-xylylenes. The fluorescence images recorded by confocal laser scanning microscopy (CLSM) showed consistent 300 μm × 300 μm arrayed patterns that confirmed the conjugation between the fluorescein (FITC)-labeled cysteine and the maleimide group on the porous materials, and the images at various Z-axes (with a 40 μm spacing interval from 10 μm to 290 μm) further verified the reactivity on the interface of the porous materials in three dimensions.

#### **4. Conclusions**

A unique fabrication process by vapor sublimation and vapor deposition to produce a porous and functionalized poly-*p*-xylylene monolith material was introduced in the current work. The monolith comprised structural information in terms of porosity and topology and, more importantly, a maleimide functional moiety for specific conjugation reactivity via a maleimide–thiol coupling reaction. The vapor-phase fabrication process was simple, clean, and free of harsh chemical agents, and the poly-*p*-xylylene used is a USP (United States Pharmacopoeia) Class VI biocompatible material, which is favorable for sensitive applications. The enhanced and synergistic interface properties, combining the porosity and chemical functionality of the fabricated porous monolith, represent robust bioengineering materials and will be useful for unlimited applications in scaffolding for tissue engineering, biosensors, bioimaging, targeted drug-eluting devices, and regenerative materials.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/coatings11040466/s1, Video S1: Reconstruction images of the three-dimensional pore structure by micro-CT.

**Author Contributions:** Conceptualization, S.-M.H., C.-Y.L., Y.-C.Y., Y.-C.C. and H.-Y.C.; methodology, S.-M.H., C.-Y.L., Y.-C.Y. and H.-Y.C.; precursor synthesis, T.K., T.-Y.W., N.-Y.C., J.C., Y.-R.C.; CVD polymerization and characterizations, S.-M.H., C.-Y.L., T.-Y.W., Y.-R.C.; micro-CT software analysis, J.-Q.X., and Y.-M.C.; validation, S.-M.H., C.-Y.L., T.K. and T.-Y.W.; fluorescence visualization and analysis, C.-Y.L. and C.-W.H.; XPS analysis, S.-M.H., J.C., and Y.-R.C.; FT-IR analysis S.-M.H., C.-Y.L., and Y.-R.C.; data curation, S.-M.H., C.-Y.L., Y.-M.C. and Y.-R.C.; writing—original draft preparation, S.-M.H., C.-Y.L., Y.-C.Y. and H.-Y.C.; writing—review and editing, S.-M.H., C.-Y.L., Y.-C.C. and H.- Y.C.; supervision, Y.-C.Y., Y.-C.C. and H.-Y.C.; project administration, Y.-C.Y., Y.-C.C. and H.-Y.C.; funding acquisition, Y.-C.C. and H.-Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Technology of Taiwan (MOST 108-2221-E-002-169-MY3; 108-2218-E-007-045; 109-2314-B-002-041-MY3; 109-2634-F-002-042). In addition, this work was further supported by the "Advanced Research Center for Green Materials Science and Technology" from The Featured Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (109L9006).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in this published article and its supplementary information files.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **References**


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