Bioinspired from Salivary Acquired Pellicle: A Multifunctional Coating

Jun 9, 2017 - ... using a salivary acquired pellicle (SAP) inspired dendrimer. ... The coating can also provide a general platform for secondary ... C...
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Bioinspired from Salivary Acquired Pellicle: A Multifunctional Coating for Biominerals Xiao Yang,† Fuhui Huang,† Xinyuan Xu,† Yanpeng Liu,† Chunmei Ding,† Kefeng Wang,‡ Anran Guo,§ Wei Li,§ and Jianshu Li*,† †

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People’s Republic of China ‡ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610065, People’s Republic of China § School of Optoelectronic Information, University of Electronic Science and Technology of China, Chengdu 610054, People’s Republic of China S Supporting Information *

ABSTRACT: Manipulation of the surface properties of biominerals is very important for their biomedical applications. However, the straightforward preparation of a multifunctional and stable coating on biominerals remains a challenge. Herein we report a rapid and universal method for the preparation of multifunctional coatings on various biominerals using a salivary acquired pellicle (SAP) inspired dendrimer. The dendrimer has a highly branched structure and an external surface modified with DDDEEKC peptide. It mimics the adsorption function of statherin, which is one of the main components of SAP, to endow the coating with a universal capability for adhesion on various biominerals such as hydroxyapatite, tertiary calcium phosphate, calcium carbonate, pearls, enamel, dentin, and bone. The coating can be formed by a simple dip-coating method on the surface of biominerals within 10 min, and is stable for more than 1 month. The coating can also provide a general platform for secondary modifications. For example, we use pregrafting or postgrafting methods to introduce functional molecules such as fluorescein isothiocyanate, heptadecafluoroundecanoyl chloride, and collagen to the surface of the coatings, thus these biomineral surfaces can be applied for different applications such as for protein crystallization by forming superhydrophobic surface, or promoting cell adhesion and proliferation by immobilizing collagen.

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INTRODUCTION Biominerals exist in the bodies of most living organisms, to provide skeletal support and protection. Among all types of biominerals, Ca-based minerals are the most common.1,2 Surface modification of biominerals by functional coatings can provide an effective method to manipulate their surface features, leading to enhanced bioactivity or biocompatibility, which are important for their biomedical applications.3 Generally, there are two methods utilized to modify the surface of biominerals. The first method is surface grafting, through which functional groups can form covalent bonds with specific groups on the surface of biominerals such as hydroxyl groups.4 For example, zwitterionic polymer brushes were designed on material surfaces by atom transfer radical polymerization (ATRP) and the surface could selectively control the directional migration of Schwann cells.5 ATRP initiator and substrate monomer were also copolymerized to form an initiator-embedded substrate, which could be used for the next polymer grafting.6 The second method is to adsorb functional molecules by interaction with inorganic ions of biomineral surfaces.7 Even though this interaction is ionic rather than covalent, it is postulated that this method can provide a © 2017 American Chemical Society

universal modification technique for the biomineral surface. For instance, mussel-inspired8−10 or tea stain-inspired11 materials are widely used for surface modification, since they can form surface-adherent films on an arbitrary material surface.12 Two mussel-derived peptides ((DOPA)4-G4-GRGDS and (DOPA)4G4-YGFGG) were prepared on Ti implants. The coated implants can induce specific cell adhesion, and also enhance synergism of osteogenicity, osseointegration and finally the mechanical stability of the Ti implants.13 Some peptide-/ protein-based materials such as amyloid14 or lysozyme15 also can be utilized to modify material surfaces. For instance, a lysozyme phase transition was reported recently. While lysozyme was added in HEPES buffer with Tris (2carboxyethyl)phosphine (TCEP), a rapid phase transition was initiated to obtain microfiber aggregates. The amyloidcontaining microfiber could stably attach to the surfaces of metals, oxides, semiconductors, and polymers.16 Received: April 10, 2017 Revised: June 7, 2017 Published: June 9, 2017 5663

DOI: 10.1021/acs.chemmater.7b01465 Chem. Mater. 2017, 29, 5663−5670

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Chemistry of Materials Salivary acquired pellicle (SAP)-based surface chemistry is expected to provide a new strategy to solve this problem. SAP is made up of proteins and glycoproteins of salivary origin that tightly coat on the tooth surface.17 Even after completely cleaning the teeth, SAP can form on a tooth surface within several minutes, and can grow up to a thickness of 100 μm within 2 h and 400 μm within 24−48 h.18 Statherin, one of the salivary proteins, is an essential part of SAP. It is composed of 43 amino acid residues, with a well-defined structure and charge asymmetry, and exhibits strong adsorption and immobilization on a hydroxyapatite (HA) surface.19,20 Previous work on the Nterminus of statherin found that the sequence of the first 15 peptides (SN15) played a significant role in recognizing HA.21,22 Later, it was demonstrated that the sequence of the first six amino acids (DpSpSEEK) had an α-helix conformation and the ability to recognize HA.23 After that, the peptide sequence of DDDEEK was shown to be similar to DpSpSEEK, as it also has the ability to adsorb strongly on the HA surface.24 Dendritic polymers include dendrimers, dendrons, and dendronized polymers. They have a highly branched structure and abundant external groups, and have been widely studied as “artificial proteins” because of their dimensional length scaling, biomimetic properties, and well-defined/easily tailorable structures in terms of generations, functional peripheral groups, and spatial structures.25,26 Up to now, various dendritic polymers have been produced, such as polyphenylenes, polysiloxanes, polyethers, polyesters, polyamides, polyimides, and polyurethanes.27−29 Among them, poly(amido amine) (PAMAM) dendrimers were the first complete dendrimer family to be synthesized, characterized and commercialized. They are usually prepared by the “divergent” method.30−36 PAMAM have been widely studied for biomedical applications.37 For example, peptide sequence R5 (SSKKSGSYSGSKGSKRRIL) was used to modify PAMAM as a template to produce highly active catalytic Pd nanoparticles.38 A structurally nanoengineered antimicrobial peptide polymer (SNAPP) was also prepared by initiating lysine and valine N-carboxyanhydrides (NCAs) from the terminal amines of PAMAM via ring-opening polymerization. The SNAPP shows great potential as a low-cost and effective antimicrobial agent and may be used as a tool to combat the growing threat of multiple drug resistance (MDR) Gram-negative bacteria.39 Thus, PAMAM could also provide an ideal platform to introduce a very dense SAP-bioinspired peptide sequence for biomineral coatings. Here we report a DDDEEKC-functionalized dendrimer that mimics the adsorption function of statherin for a universal surface coating of biominerals, both by electrostatic interactions between the dendrimer and substrates, and noncovalent intermolecular cross-linking among dendrimers (Scheme 1). The integration of the properties of dendrimers and functional peptides may achieve important synergistic effects, such as amplification of peptide functions by enabling simultaneous interactions with multiple receptors.40 In this work, cysteine labeled (Fmoc) DDDEEK (Fmoc) was introduced to a PAMAM dendrimer through previously conjugated acryloyl groups (60%, Figure S1 and S2). Then, the product was deprotected under alkaline conditions to obtain the DDDEEKC-PAMAM (Figure S3, S4, S5, and S6). After that, the SAP-bioinspired PAMAM was verified to exhibit universal adsorption on Ca-based biominerals such as HA, tertiary calcium phosphate (TCP), and calcium carbonate (CC).

Scheme 1. Schematic Illustration of the Structure of SAPInspired Dendrimer, and Its Coating on Biomineral Surfacesa

a

The coating is formed both by electrostatic interactions and noncovalent intermolecular cross-linking.

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EXPERIMENTAL SECTION

Materials. The peptide sequence (Fmoc) DDDEEK (Fmoc) C was purchased from Shanghai Biotech BioScience &Technology Company. The HA powder/slice was obtained from the National Engineering Research Center for Biomaterials, Sichuan University (medical grade, M30, the size of the slice was Φ8*2). Tricalcium phosphate and calcium carbonate powders were obtained from Tianjin Bodi Chemical Holding Company, and the slices (Φ8*2) were prepared by a powder compressing machine (FW-A, China). Collagen was purchased from Baoxin Biotechnology Co., Ltd. (Chengdu, China). Dialysis was performed in benzoylated cellulose tubes (width: 32 mm, molecular weight cutoff (MWCO) 2000 g·mol−1, USA). All other chemicals and solvents were purchased from Sigma (HPLC grade). The deionized water used was purified using a Millipore water purification system with a minimum resistivity of 18.25 MΩ·cm. Syntheses. Amine-terminated PAMAM with Mn = 6900 g·mol−1 was prepared by a divergent approach as described in previous work.41−43 Using an acylation reaction, acryloyl chloride was grafted to the amine-terminated PAMAM to obtain acryloyl-PAMAM. The peptide sequence (Fmoc) DDDEEK (Fmoc) C was clicked to the acryloyl-PAMAM by Michael addition reaction, and deprotected under alkaline condition to obtain DDDEEKC-PAMAM. The 1H NMR were recorded on a Bruker AV II operating at 400 MHz, at a concentration of 5 mg·mL−1. Acryloyl Chloride Grafted Amine-Terminated PAMAM (acryloyl-PAMAM). Amine-terminated PAMAM (1.00 g) was dissolved in anhydrous DMSO (100 mL), to which TEA (22.64 mL, 1.1 equiv for amine group) and acryl chloride (13.27 mL, 1.1 eqvui for amine group) were added dropwise. The mixture was stirred at 25 °C for 24 h under nitrogen. Then the crude product was dialyzed in deionized water for 3 days. Finally, the product was collected by lyophilization (yield: 88%). Fmoc Protected DDDEEKC-PAMAM. Acryloyl-PAMAM (0.05 g) and dimethylphosphine (20 μL) were dissolved in deionized water (20 mL). A solution of (Fmoc) DDDEEK (Fmoc) C (0.24 g, 1.1 equiv for acryloyl group) in deionized water (5 mL), was introduced dropwise into the reaction solution. The reaction mixture was stirred at room temperature for 24 h. Then the crude product was dialyzed against deionized water to remove the impurity for 3 days and was freezedried to obtain the product (yield: 75%). DDDEEKC-PAMAM. Fmoc protected DDDEEKC-PAMAM was dissolved in mixed solvent (40 mL), which contained NH3·H2O, C4H8O2 and NaOH (4 M) (the volume ratio was 30:9:1). The mixture was stirred at room temperature for 24 h. Then the crude product was dialyzed in deionized water for 3 days. Finally, the product was collected by lyophilization of the solution using a freeze-dryer (Yield: 90%). Fluorescein Isothiocyanate Labeled DDDEEKC-PAMAM. DDDEEKC-PAMAM was labeled with fluorescein isothiocyanate 5664

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Chemistry of Materials (FITC) using a procedure reported previously.4 Briefly, FITC (5 mg/ mL) was dissolved in acetone and slowly added to the solution of SAPPAMAM. The reaction mixture was incubated at room temperature for 24 h, and dialyzed in deionized water for 3 days. Finally, the conjugate was lyophilized. UV. We then evaluated the binding capability of SAP-PAMAM on HA, TCP, and CC. HA, TCP or CC powder (50 mg) was added to 1 mL of DDDEEKC-PAMAM aqueous solution of different concentrations ranging from 0.25 to 2.75 mg/mL, respectively. The mixture was stirred at 37 °C for 12 h and then centrifuged at 10000 rpm for 3 min. The supernatant was taken out for UV absorbance analysis at a wavelength of 282 nm (MAPADA 1800PC, China). The amount of DDDEEKC-PAMAM adsorbed on the mineral powder was calculated by measuring the decrease of DDDEEKC-PAMAM in solution. Coatings. The HA, TCP, and CC slices were immersed in 5 mg/ mL DDDEEKC-PAMAM aqueous solution, respectively. After a specific time point, the coated slides were thoroughly rinsed with deionized water and dried by an N2 stream. ATR-IR. ATR-IR spectra were measured on a Thermo Scientific NICOLET iS10 spectrometer. The coatings of DDDEEKC-PAMAM were prepared as described previously. After sufficient washing and drying, and the slice was directly measured. For comparison, bare slices were also measured. QCM-D. The amount of DDDEEKC-PAMAM that immobilized on the HA surface was also evaluated by qCell T Q2 (3T analytik, 3T GmbH & Co. KG, Germany). The dynamic tests were carried out at a constant speed of 60 μL/min. The mass of the immobilized DDDEEKC-PAMAM was calculated by the Kelvin−Voigt model. The HA-coated gold slices were used for measurement. CLSM. The bare HA, TCP, and CC slides were immersed in 5 mg/ mL FITC labeled DDDEEKC-PAMAM solution for 4 h. The coated slides were thoroughly rinsed and dried by an N2 stream, and then observed by confocal laser scanning microscopy (CLSM) (ZEISS LSM 700, Germany). Collagen Immobilization. The DDDEEKC-PAMAM coated HA slices were immersed in the mixed solution of 0.1 mg/mL collagen, 0.01 mg/mL N-hydroxysuccinimide (NHS), and 0.01 mg/mL 1-ethyl3-(3-(dimethylamino)propyl) carbodiimide (EDC) in PBS for 1 day. Then the slides were thoroughly rinsed with PBS and water, respectively, and dried by an N2 stream. For comparison, the collagen adsorbed HA slices were prepared by incubating dishes in 0.1 mg/mL collagen for 5 min before removing the solution. The freshly prepared slices were immediately used for cell culture without further rinsing. Superhydrophobic Coatings. The DDDEEKC-PAMAM coated HA, TCP, and CC slices were immersed in 1 mg/mL 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoyl chloride with 10 equiv of triethylamine dissolved in diethyl ether, after drying under vacuum for 1 h. After 4 h, the perfluorinated coatings were thoroughly rinsed with diethyl ether and dried with an N2 stream. SEM. The surface morphology of the coatings was observed with a field emission scanning electron microscope (Hitachi S-450, 20 kV, Japan). The samples were dried under high vacuum and coated with an 8−10 nm gold layer by using a sputter coater. The EDS spectra was performed using a HITACHI S3400. Contact Angle. The measurements were performed with a Kruss DSC100 (Germany) and images were analyzed with the DSC100 software. Ellipsometry. Ellipsometry measurements were performed in the spectral range of 380 to 850 nm at an incidence of 50° on HA samples with an ellipsometer (SENTECH SE850, Germany). Each data point resulted from an average of at least five measurements, and the obtained sensorgrams were fitted with a three-layer model (HA, organic layer, and air) using analysis software SpectraRay/2. The HA layer was measured before being coated with organic layers as a Cauchy layer. The organic layers were fitted by the Cauchy model. AFM. The AFM measurements were performed using a Nanoscope MultiMode 8 equipped with a Nanoscope V controller. The microscope was operated in the tapping mode (TM) using SNL-C probes with a radius of approximately 2 nm and a spring constant of around 0.24 N/m (Shimadzu SPM-9700) under ambient conditions at

room temperature. The cantilever was forced to oscillate near its resonance frequency. DLS. The DLS measurements were performed using a Zetasizer Nano ZSP (Malvern, UK) with an He−Ne laser (wavelength = 532 nm) at a scattering angle of 90°. Preparation of Enamel, Dentin, and Bone Slices. The human teeth were obtained, with approval, from the West China Hospital of Stomatology (Sichuan University). They were stored at 4 °C in saturated thymol aqueous solution. A diamond-coated band saw was utilized to separate the root from the crowns and to cut sections longitudinally, leading to an approximately 5 × 5 mm square plate. These samples were ground flat and polished with water-cooled carborundum discs. Acrylic resin was used to proctect the surfaces of the sections except the polished enamel surface. Using a similar method, we also prepared dentin and bone samples. Preparation of Dimethyl Yellow Loaded DDDEEKC-PAMAM. To a 10 mg/mL aqueous solution of DDDEEKC-PAMAM was added excessive dimethyl yellow (DY), and the solution was stirred for 24 h. The solution was centrifuged at 4000 rpm twice, and the supernatant was lyophilized to obtain DY-loaded DDDEEKC-PAMAM. Fifty milliliters of 1000 ppm DY-loaded DDDEEKC-PAMAM solution was pipetted on the surface of the enamel, dentin, and bone samples, respectively. Then, the samples were washed using deionized water three times and air-dried before observation. Protein Crystallization. Lysozyme (100 mg/mL) was dissolved in NaOAc (0.1 M, pH 4.5) to obtain a protein solution with a determined protein concentration. Then, 50 μL portions of protein solution were pipetted onto the superhydrophobic HA slice. The slice was placed in a closed and determined humid environment. Within 4 h at 4 °C, lysozyme crystals appeared. Cell Adhesion and Cytotoxicity Assay. Cell culture experiments were performed using MG63 cells. The modified and unmodified HA slides were disinfected within 75% ethanol for 1 h and sterilized by UV irradiation for 1 h. The MG63 cells were allowed to adhere for 1 and 3 days onto the HA slides in 24-well plates at a density of 5 × 104 cells/ cm2 in DMEM at 37 °C with the atmosphere of 5% CO2 and 95% relative humidity. After 1 and 3 days, the slide surfaces were rinsed three times with PBS to remove the loosely attached cells. The slides were transferred into fresh 24-well plates and incubated in 2.5 mL of DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C for 24−48 h in an atmosphere of 5% CO2 and 95% relative humidity. For CLSM imaging, at predetermined time points, the culture medium was removed and the cells were washed three times with PBS. Then, the cells were fixed using 4% paraformaldehyde at 4 °C for 20 min followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min and rinsed with PBS three times. Subsequently, cells were incubated with 1 μg/ mL phalloidin-TRITC in PBS at 25 °C for 30 min to label the filamentous actins (F-actins), and rinsed using PBS three times. Finally, the HA slides were put on a glass microscope slide for fluorescence imaging by CLSM. MTT assays were performed to measure the cytotoxicity of the dendrimer. MG63 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% heatinactivated fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 mg/mL streptomycin at 37 °C, with 5% CO2 and 95% relative humidity. The cells were seeded in a 96-well microtiter plate at a density of 104 cells/well and incubated in 100 μL of DMEM/well for 24 h. The culture medium was replaced with fresh culture medium containing serial dilutions of DDDEEKC-PAMAM, and the cells were incubated for 24 h. Then, 20 μL of sterile-filtered MTT stock solution in 5 mg/mL PBS was added. The unreacted dye was removed by aspiration after 5 h. The formed formazan crystals were solubilized in DMSO (150 μL/well). The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at a wavelength of 570 nm. The cell viability (%) was calculated using the following equation: 100 × ([A]test − [A]zero)/([A]control − [A]zero), where [A]test, [A]zero, and [A]control represent the absorbance values of the wells with DDDEEKC-PAMAM, without cells (zero set) and without DDDEEKC-PAMAM (negative control), respectively. The 5665

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Chemistry of Materials absorbance was the average value measured from six wells in parallel for each sample.

that the saturated adsorption amount of DDDEEKC-PAMAM was 2.2 mg on 50 mg of TCP powder, and 1.8 mg on 50 mg of HA powder, respectively. However, DDDEEKC-PAMAM had a much weaker adsorption capability on CC. The reason for this phenomenon was that the density of Ca2+ on the CC surface was lower than that on HA and TCP (Figure S8). Meanwhile, the biomineral surfaces treated with DDDEEKCPAMAM coatings were analyzed by energy dispersive spectroscopy (EDS). Compared to the bare surfaces, there was a dramatic change in signals for these coated substrates (Table S1). The Ca, P, and O compositions of the coated surfaces changed, and the elemental C, N appeared after coating. This change further proved that DDDEEKC-PAMAM was adsorbed on the biomineral surface. We also found that the adsorption of DDDEEKC-PAMAM on biominerals slices is strong, as it can resist washing (ATR-IR data, Figure S9). As shown, the characteristic peaks of HA, TCP, and CC (1450 cm−1 for CO32− and 1000 cm−1 for PO43−) can be clearly observed. Then, after the coating of DDDEEK-PAMAM, the bands for the deformational vibration of amide bond at about 1535 and 1650 cm−1 indicated the adsorption of DDDEEKC-PAMAM. Further, there was almost no change in amide vibration after washing with deionized water, indicating that it can strongly bind to the biomineral surface. After the adsorption of the first DDDEEKC-PAMAM layer onto the biomineral surface, other DDDEEKC-PAMAM molecules could further immobilize and be cross-linked by noncovalent interactions among each other (Figure 1b, Figure S10). Hydrogen bonding is the primary interaction in this step.44 To measure the thickness of the DDDEEKC-PAMAM coating, we immerse HA slices with DDDEEKC-PAMAM solution at different time points. As illustrated in Figure 1b, the ellipsometry result shows that the thickness of the DDDEEKCPAMAM coating increased rapidly during incubation, was 25 nm after 10 min, and reached a maximum value of 140 nm after 2 h on the HA surface. The thickness growth of this coating is similar to the cross-linking of a mussel-inspired dendritic polyglycerol coating.8 Owing to the adsorption and cross-linking interactions, DDDEEKC-PAMAM can spontaneously and uniformly deposit onto the surfaces of different biominerals (Figure 2, Figure S11). For example, the surfaces of the HA slices were completely covered with a layer of DDDEEKC-PAMAM coating. The porous surface of HA is effectively hidden (Figure 2b), which confirms the formation of the DDDEEKC-PAMAM coating. The high-resolution AFM images of the HA surface before and after DDDEEKC-PAMAM treatment are shown in

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RESULTS AND DISCUSSION The DDDEEKC-PAMAM coating on the biominerals was prepared in a neutral condition. To evaluate its adsorption on HA, we used a quartz crystal microbalance with dissipation (QCM-D). Using this technique, we may correlate a change in the mass loading of DDDEEKC-PAMAM over time on an HA coated crystal. The adsorbed mass was collected using the same concentrations of DDDEEKC-PAMAM, PAMAM, and DDDEEKC. As shown in Figure 1a, it is apparent that

Figure 1. (a) QCM-D results of the adsorption of DDDEEKCPAMAM, DDDEEKC, and PAMAM on the HA surface. (b) Timedependent thickness growth of the DDDEEKC-PAMAM coating on the HA surface measured by ellipsometry.

DDDEEKC-PAMAM displays strong affinity toward the HAcoated crystal surface in comparison with PAMAM and DDDEEKC. The maximal adsorptive capacity on the HAcoated crystal surface was calculated as 280 ng/cm2. Whereas the maximal adsorptive amounts of DDDEEKC and PAMAM were 160 and 60 ng/cm2, respectively. The above results demonstrate the successful synergistic effect of dendrimer and DDDEEKC peptide on the specific adsorption capability on the biomineral surface. After flushing with deionized water, the amount of adsorbed mass was slightly decreased. The part washed off by the deionized water was due to nonspecific adsorption. In order to quantify the adsorption behaviors of DDDEEKC-PAMAM on HA, TCP, and CC, the DDDEEKCPAMAM was dissolved in deionized water at different concentrations, fully stirred with HA, TCP, and CC powder and then centrifuged. The amount of the DDDEEKC-PAMAM adsorbed on HA, TCP, and CC powder was calculated by the decrease of DDDEEKC-PAMAM in the solution. Analysis by ultraviolet/visible (UV/vis) spectroscopy (Figure S7) indicated

Figure 2. (a) SEM image of bare HA slice. (b) SEM image of DDDEEKC-PAMAM coated HA slice. (c) Rhodamine B loaded DDDEEKC-PAMAM was dropped on the pearls with the pre-etched word “love” and washed with PBS three times. 5666

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decreased. This result clearly illustrated that the surface chemistry of the coating, i.e., the hydrophilic groups of DDDEEKC-PAMAM coatings, such as carboxyl groups, have changed the original wetting properties of these substrates. Moreover, after being incubated in PBS for 1 month, the contact angles of DDDEEKC-PAMAM coated HA, TCP, and CC had hardly changed (Figure 3b), which demonstrates that the coating has a high stability under physiological conditions. This SAP-inspired coating exhibits similar hydrophilic property and stability compared to previously reported coatings prepared using other methods.8 The DDDEEKC-PAMAM coating on biominerals can be applied as a universal modifiable platform for many purposes. Functional molecules can be introduced to the surface of the coatings by using a pregrafting or postgrafting method. For instance, fluorescein isothiocyanate (FITC) was employed as a model to test the surface immobilization of functional molecules by a pregrafting method (Figure 4). FITC was

Figure S12. It can be seen that the roughness of the bare HA slice was high (Ra 277.557 nm; Rz 200.316 nm), and after the coating with DDDEEKC-PAMAM, the roughness of the coated HA decreased to Ra 62.98 nm and Rz 2.185 nm. There was a much smoother surface after coating, as indicated by the Ra and Rz data from the AFM analysis. Further, the DDDEEKCPAMAM coating is shown to be universal on natural biominerals, e.g., it can tightly stick two irregular pearls (8 mm in diameter, Figure S13). Quite a few hydrophobic reagents could be incorporated into PAMAM, and their solubilities in aqueous solution could be significantly improved. In order to directly visualize the adsorption behavior of DDDEEKC-PAMAM on biominerals, a hydrophobic dye (DY) was loaded into the cavity of DDDEEKC-PAMAM and then dissolved in deionized water to form a saturated solution. Then, the solution was dropped onto the surfaces of enamel, dentin, and bone slices. After being thoroughly rinsed with deionized water, the surfaces of these biominerals remained yellow. This phenomenon further indicated that DDDEEKC-PAMAM has a strong adsorption capability on biominerals (Figure S14). The wetting properties of the HA, TCP, and CC slices were measured in the coating experiments (Figure 3a). At

Figure 4. CLSM and fluorescence intensity of FITC-DDDEEKCPAMAM coated HA, TCP, and CC, and the coated slices of HA, TCP, and CC after being washed with deionized water, separately. Data are expressed as mean ± SD, n = 6.

pregrafted to the DDDEEKC-PAMAM molecule to obtain FITC-DDDEEKC-PAMAM, which was then directly used for surface coating. FITC-DDDEEKC-PAMAM coated HA, TCP, and CC slices were visualized by confocal laser scanning microscopy (CLSM). After rinsing with deionized water, a strong fluorescence signal could be detected on the coated surface, which reflected the successful immobilization of FITC molecules. Quantitatively, the fluorescence intensity of FITCDDDEEKC-PAMAM coated HA and TCP were quite similar. Both were higher than that of FITC-DDDEEKC-PAMAM coated CC. This result also demonstrates the strong adsorption capacity of FITC-DDDEEKC-PAMAM on HA, TCP, and CC slices. Using this method, other functional molecules, e.g., initiators,6 may also be directly pregrafted to a single DDDEEKC-PAMAM molecule to form multifunctional coatings. Heptadecafluoroundecanoyl chloride was used as a model to test the immobilization of functional molecules on the surfaces by a postgrafting method (Figure 5). After perfluoroalkylfunctionalization, the DDDEEKC-PAMAM coating exhibits a self-assembled morphology with hierarchical roughness. As shown in Figure 5b3, some micrometer-sized aggregates appeared on the perfluoroalkyl-functionalized coating as compared with the DDDEEKC-PAMAM coating (Figure 5b2), which is due to the hydrophilic−hydrophobic interaction. The hierarchical roughness (in micro/nanoscale) on the

Figure 3. (a) Static water contact angles of bare HA, TCP, and CC, and DDDEEKC-PAMAM coated HA, TCP, and CC, separately. (b) Static water contact angles of the DDDEEKC-PAMAM coat-ed HA, TCP, and CC surfaces before (black) and after (white) being incubated in physiological PBS for 1 month.

equilibrium conditions, the water contact angles were 25.9°, 13.6°, and 19.5° for DDDEEKC-PAMAM coated HA, TCP, and CC, respectively. The water contact angles were decreased as compared to those of bare slices (59°, 44°, and 38.5° for HA, TCP, and CC, respectively). Generally, with the decrease in surface roughness, the water contact angle should increase. However, in this system, the surface roughness was decreased after coating (Figure S12), but the water contact angle was 5667

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Figure 6. Amount of immobilized collagen on the DDDEEKCPAMAM coated HA surfaces. The mass was calculated from the QCM-D measurements. Events: (1) 0.1 mg/mL collagen with 0.01 mg/mL NHS and 0.01 mg/mL EDC in deionized water; (2) deionized water; (3) 1% (w/w) SDS; (4) deionized water. Figure 5. (a) Scheme of the DDDEEKC-PAMAM constructed perfluoroalkyl-functionalized superhydrophobic coatings; the surface morphology of bare HA (b1), DDDEEKC-PAMAM coated HA (b2), and the superhydrophobic coatings on HA (b3) by SEM imaging; the static water contact angle of the superhydrophobic surfaces (c1) and the lysozyme crystal formed on the surface (c2).

DDEEKC-PAMAM coating was an important factor to obtain a possible superhydrophobicity. As a result, the water contact angle increased to about 150.6° (Figure 5c1). Different from the “coffee ring” during the evaporation of a solution droplet on a normal surface, the superhydrophobic surface could have a convergent concentration effect of solutes in the droplet during the droplet compact process.14 Because of the introduced superhydrophobic property, this surface can be applied for protein crystallization. In this work, lysozyme was used because it is a classical model protein for crystallization. The spotted lysozyme droplets could be stably isolated and settled, and it was shown that the lysozyme can be effectively crystallized and clearly observed on the modified surface bacause of the concentration effect within each liquid droplet (Figure 5c2).14 We also introduced collagen as a model of biomolecules to the DDDEEKC-PAMAM coated biomineral surface to explore its further biomedical applications. Collagen can react with DDDEEKC-PAMAM by forming amide bonds through NHS and EDC. The QCM-D measurement (Figure 6) was employed to quantify the amount of immobilized collagen on the DDDEEKC-PAMAM coating. At the beginning, the mass loading of collagen on bare and DDDEEKC coated HA increased over time until it became balanced. When deionized water was introduced, the mass loading began to decrease. At this point, the nonspecific absorption portion was washed off. After being rinsed with SDS solution, collagen on the bare HA coated crystal surface was completely wiped out, whereas the collagen on the DDDEEKC-PAMAM coated crystal surface remained plentiful. Compared with the bare HA-coated crystal surface (almost no adsorption after washing), there was 42 ng/ cm2 collagen immobilized on the DDDEEKC-PAMAM modified HA-coated crystal surface, even after being washed with 1% (w/w) sodium dodecyl sulfate (SDS) aqueous solution and water. To illustrate the growth behavior of cells on biomineral surfaces, the morphology and density of MG63 cells after growth for 1 and 3 days were studied (Figure 7 and Figure S15). A significant enhancement in MG63 proliferation was

Figure 7. (a) Scheme of collagen-functionalization on DDDEEKCPAMAM coated surface; MG63 cell adhesion on bare (b1) and collagen-modified DDDEEKC-PAMAM coated HA surfaces (b2) after 3 days; (c) MG63 cell numbers on the bare and different HA surfaces after 3 days. Data are presented as mean ± SD, n = 6, *: p < 0.05.

observed on collagen−DDDEEKC-PAMAM coated HA slices as compared with other samples. After culturing with MG63 cells for 3 days, only a few cells could be found on the bare HA surface (200 ± 50 cells mm−2). As for the collagen-adsorbed HA surfaces, there were more cells (400 ± 80 cells mm−2) after 3 days of culturing, but only 240 ± 50 mm−2 cells could be found from the rinsed slices, which was similar to the quantity of cells on the bare HA. This is because the adsorbed collagen was not stable on the HA in the cell culture medium, thus it could be washed off by PBS. DDDEEKC-PAMAM coated HA without immobilized collagen improved the attachment of MG63 cells (360 ± 50 cells mm−2), and much more cells (560 ± 100 cells mm −2 ) adhered to collagen-immobilized DDDEEKC-PAMAM coated HA surfaces. Thus, the DDDEEKC-PAMAM coating can promote the proliferation of MG63 cells, and the well-retained collagen that was 5668

DOI: 10.1021/acs.chemmater.7b01465 Chem. Mater. 2017, 29, 5663−5670

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Chemistry of Materials ORCID

immobilized on the DDDEEKC-PAMAM can further enhance the growth of MG63 cells. This is because collagen served as a recognition site for cell adhesion, leading to the enhancement of adhesion and proliferation of MG63 cells on the collagen− DDDEEKC-PAMAM coating. Thus, we can conclude that the collagen-functionalized DDDEEKC-PAMAM coated HA surface is suitable for cell adhesion and proliferation. Cytotoxicity tests were also performed by the MTT method. DDDEEKCPAMAM displayed good cell viability in the range of 80−100%, demonstrating that the DDDEEKC-PAMAM coating has good cytocompatibility even at a high concentration of 250 μg/mL (Figure S16).

Jianshu Li: 0000-0002-1522-7326 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Key Research and Development Program of China (2016YFC1100404) and the National Natural Science Foundation of China (51573110 and 51322303) are gratefully acknowledged.

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CONCLUSION In summary, we have designed a novel biomineral coating material, i.e., DDDEEKC-PAMAM, which is bioinspired from the adsorption function of statherin in the salivary acquired pellicle. The DDDEEKC-PAMAM dendrimer was prepared by acylation reaction and Michael addition reaction. The coating could be prepared in a neutral condition. It could effectively coat on various biominerals such as hydroxyapatite, tertiary calcium phosphate, calcium carbonate, pearls, enamel, dentin, and bone slices, both by electrostatic interactions and noncovalent intermolecular cross-linking. The formation of a DDDEEKC-PAMAM coating is fast and stable. It can also provide a universal platform for secondary modifications for different applications. We demonstrated pregrafting and postgrafting methods to introduce new functional molecules to the coated biomineral surface. FITC was introduced by the pregrafting method, and a strong fluorescence signal could be detected on the coated surface, which reflected the successful immobilization of FITC−DDDEEKC-PAMAM molecules. We also used a postgrafting method to add heptadecafluoroundecanoyl chloride to the surface of the coatings. After the modification, the surface presented a superhydrophobicity property. The superhydrophobic surface can be applied to induce the crystallization of proteins such as the lysozyme. Collagen was also added to the coated surface by the postgrafting method. The density of adhered MG63 cells was 560 ± 100 cells mm−2 on the collagen−DDDEEKC-PAMAM surface, demonstrating that it was a suitable substrate for cell adhesion and proliferation in tissue engineering applications. Using the above methods, other functional molecules such as initiators, biotin, and enzymes may also be directly grafted to a single DDDEEKC-PAMAM molecule to form multifunctional coatings on biominerals.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b01465. Synthetic route and 1H NMR data; adsorption isotherm; ATR-IR data; size distribution data by DLS; SEM spectra; AFM images; pearls sticked by this coating; adsorption on the surface of enamel, dentin and bone; MG63 cells adhesion on bare and different HA surfaces; cytotoxicity data; EDS data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Jianshu Li. E-mail: [email protected]. 5669

DOI: 10.1021/acs.chemmater.7b01465 Chem. Mater. 2017, 29, 5663−5670

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