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Competitive Adsorption of Polyelectrolytes onto and into Pellicle Coated Hydroxyapatite Investigated by QCM-D and Force Spectroscopy Hyun-Su Lee, Carl Myers, Lynette Zaidel, Prathima Chandra Nalam, Matthew Alexander Caporizzo, Carlo Daep, David M Eckmann, James G. Masters, and Russell J. Composto ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02774 • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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ACS Applied Materials & Interfaces

Competitive Adsorption of Polyelectrolytes onto and into Pellicle Coated Hydroxyapatite Investigated by QCM-D and Force Spectroscopy

Hyun-Su Lee,†,‡ Carl Myers,§ Lynette Zaidel,§ Prathima C. Nalam,∥ Matthew A. Caporizzo,† Carlo Daep,§ David M. Eckmann,‡ James G. Masters,§,* and Russell J. Composto†,*

†

Department of Materials Science and Engineering, ‡Department of Anesthesiology and Critical

Care, and ∥Department of Mechanical Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, United States §

Colgate-Palmolive Company, Piscataway, NJ 08855, United States

ACS Paragon Plus Environment

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ABSTRACT A current effort in preventive dentistry is to inhibit surface attachment of bacteria using antibacterial polymer coatings on the tooth surface.

For the antibacterial coatings, the

physisorption of anionic and cationic polymers directly onto hydroxyapatite (HA) and saliva treated HA surfaces was studied using quartz crystal microbalance, force spectroscopy and atomic force microscopy.

First, single species adsorption is shown to be stronger on HA

surfaces than on silicon oxide surfaces for all polymers (i.e., Gantrez, sodium hyaluronate (NaHa), and poly(allylamine-coallylguanidinium) (PAA-G75)).

It is observed through pH

dependence of Gantrez, NaHa, and PAA-G75 adsorption on HA surfaces that anionic polymers swell at high pH and collapse at low pH, whereas cationic polymers behave in the opposite fashion. Thicknesses of Gantrez, NaHa, and PAA-G75 are 52 nm (46 nm), 35 nm (11 nm), and 6 nm (54 nm) at pH 7 (3.5), respectively. Second, absorption of charged polymer is followed by absorption of the oppositely charged polymer. Upon exposure of the anionic polymer layers, Gantrez and NaHa, to the cationic polymer, PAA-G75, films collapse from 52 to 8 nm and 35 to 11 nm, respectively. This decrease in film thickness is attributed to the electrostatic crosslinking between anionic and cationic polymers. Third, for HA surfaces pretreated with artificial saliva (AS), the total thickness decreases from 25 nm to 16 nm upon exposure to PAA-G75. Force spectroscopy is used to further investigate the PAA-G75/AS coating. The results shows that the interaction between a negatively charged colloidal bead and the AS surface is strongly repulsive, whereas PAA-G75/AS is attractive but varies across the surface. Additionally, AFM studies show that AS/HA is smooth with a RMS roughness of 1.7 nm, and PAA-G75 treated AS/HA is rough (RMS roughness of 5.4 nm) with patches of polymer distributed across the surface with an underlying coating.

The high roughness of PAA-G75 treated AS/HA is


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attributed to the strong adsorption of the relatively small PAA-G75 onto the heterogeneouslydistributed negatively-charged AS surface. In addition, uptake of PAA-G75 by pellicle layer (saliva treated HA surface) is observed and the adsorbed amount of PAA-G75 on/into pellicle layer is ~2x more than that on/into AS layer. These studies show that polymer adsorption onto HA and saliva coated HA depends strongly on the polymer type and size and that there is an electrostatic interaction between polymer and saliva and/or oppositely charged polymers that stabilizes the coatings on HA. Lastly, assessing the viability of the adherent bacteria collected from the PAA-G75-coated surfaces showed a significant reduction (~93%) in bacterial viability when compared to bacteria collected from untreated and Gantrez-coated HA. These results suggest the potential antimicrobial activity of PAA-G75.

KEYWORDS: polyelectrolytes, physisorption, hydroxyapatite, pellicle, antibacterial coating, quartz crystal microbalance, force spectroscopy


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1. INTRODUCTION Biofilm formation on oral surfaces is contingent upon the successful attachment of bacteria onto the salivary pellicle. Over accumulation of these microbial communities within the mouth has been implicated in a variety of oral problems such as dental caries, gingivitis, periodontitis, and halitosis. Current efforts in preventive dentistry typically utilize a prophylactic approach either through the use of fluoride to prevent demineralization of the tooth enamel or standard scaling and planning to reduce the subgingival load of oral plaque.1-8 Despite these approaches, problems are often not alleviated due to the eventual repopulation of the oral cavity. Therefore, preventing oral diseases through the control of biofilms remains a major challenge. Currently numerous technologies have identified methods of disrupting biofilms9-12 through a variety of methods and technologies.13-16 Interestingly, one promising avenue for biofilm control is to inhibit surface attachment of bacteria using antibacterial surface coatings that resist adhesion of bacteria, kill bacteria on contact, and/or slowly release actives to kill bacteria.17-34 Polymeric surface coatings that impart antibacterial properties to surfaces and interfaces have received some attention. Recently, polyelectrolyte multilayers (PEMs) designed via layer-bylayer assemblies, alternately depositing oppositely charged polyelectrolytes on a charged surface using ionic cross-linking interaction have been developed for antibacterial applications.18-30 Neutral grafted polymer brushes which dictate protein interaction with surfaces also have potential as antibacterial adhesion surfaces.35-37

Highly swollen and hydrated multilayers that incorporate

polyelectrolytes that improve surface hydrophilicity and softness showed good anti-adhesive properties.20,21 The deposition of positively charged polymers onto the top surface of PEMs resulted in coatings with a net positive surface charge that effectively reduces the viability of bacteria.22,23 However, Zue et al. showed that surfaces with a net positive surface charge induce


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more bacteria adhesion due to the negatively charged bacterial cell wall.24

In addition,

antibiotics and antimicrobial peptides (cationic molecules), embedded in PEMs, indicated that the antimicrobial reagents released from the PEMs kill bacteria.25-30 To reduce or eliminate dental plaque in preventive dentistry, cationic antimicrobial molecules, such as benzethonium chloride (BTC) with a quatenary ammonium functional group, and anionic polymers, such as Gantrez, have been studied.38,39 Gaffar et al showed that a complex of BTC and Gantrez exhibits as much antibacterial activity as BTC alone while reducing calcified deposits on teeth.38 To predict performance and design new formulations for developing oral health-care products, a fundamental understanding of adsorption/desorption processes of polymeric systems to enamel surface is of critical importance. Lee et al showed adsorption processes of chitosan on/into artificial saliva (AS) layer on dental mimicking surface (hydroxyapatite-coated surface) and its role in preventing acid erosion using quartz-crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) analysis.40

Zong et al described adsorption/desorption

processes of Gantrez on hydroxyapatite surfaces and studied physical properties of the polymer layer using different solution environment.41 Recently, Delvar et al. showed that polyanions penetrate into the pellicle near the HA but polycations adsorb on top of the pellicle by crosslinking with mucins (anionic) in the outer layer.42 Several polycations have been developed for antibacterial applications due to their capability to penetrate cell membranes and induce cation exchange and cell lysis that disrupt the membrane integrity.18,19,32-34,43-48

Positively charged polymers with quaternary ammonium salts and

guanidine functionalized polymers have been synthesized which depict antibacterial properties.32-34,43-48 In this study, poly(allylguanidinium-co-allylamine) (PAA-G75, 75% guanidine functionalized polyallyamine, as shown in Figure 1) synthesized using polyallyamines


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and guanylating reagents (1H-pyrazole-1-carboximidamide hydrochloride),49 was used as a cationic polymer for polymeric surface coatings which may potentially impart antibacterial properties locally at the tooth surface. In this paper we studied the physisorption of cationic polymer PAA-G75, having a stable positive charge due to its guanidine group (pKa = 12.5), directly onto hydroxyapatite (HA, as a biomimetic system inspired by enamel) and HA pretreated with AS and human saliva using QCM-D, which is extremely sensitive to vary small changes in mass (ng). Moreover, in-situ QCM-D allows for sequential changes in local pH or introducing a second (or third) chemical component to the flow cell. As a comparison we also investigated the behavior of anionic polymers Gantrez, a well-known bio-adhesive polymer used in toothpastes and mouth rinses, and NaHa, an anionic biopolymer that is almost ubiquitous in biological tissues, as shown in Figure 1. First, these polymers were exposed directly to HA and physisorption of anionic and cationic polymers onto HA was evaluated.

In addition, the

physisorbed polymer layers were evaluated for their pH response. Second, HA surfaces were sequentially challenged by the charged polymer followed by the oppositely charged polymer. These studies showed that physisorption of individual polymers can be quite different when done in sequence. To complement the QCM studies, force spectroscopy is used to determine whether the surfaces repel or attract a negatively charged colloidal bead, whereas AFM gives the roughness and heterogeneity of polymer on the surfaces. In addition, uptake of PAA-G75 by pellicle layer (human saliva treated HA surface) as well as AS layer (artificial saliva treated HA) is observed and the adsorbed amount of PAA-G75 on/into both layers is estimated by QCM-D. These studies show that polymer adsorption onto HA and saliva coated HA depends strongly on the polymer type and size, that there is an electrostatic interaction between polymer and saliva and/or oppositely charged polymers that stabilize the coatings. Lastly, the antibacterial activity


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of the polymers is tested by pre-coating the surface of hydroxyapatite disks and performing a biofilm assay in the studies showing that PAA-G75 has potential utility in oral care as antimicrobial agent. 2. EXPERIMENTAL SECTION 2.1. Materials QCM-D sensor crystal, an AT-cut piezoelectric quartz crystal (14 mm in diameter and 0.3 mm thickness) coated with uniform nanocrystalline hydroxyl apatite (Ca10(PO4)6(OH)2, effective HA coating: 10 nm) and silicon oxide (SiO2, 50 nm) by Promimic AB, were purchased from Biolin Scientific, Inc., USA. Sodium periodate (⩾99.8%) and citric acid (⩾99.5%) were obtained from the Sigma-Aldrich Chemical Co., USA. All reagents for the AS solution (mucin from porcine stomach (type II), ammonium chloride (NH4Cl, ⩾99.5%), calcium chloride dihydrate (CaCl2·2H2O), magnesium chloride hexahydrate (MgCl2·6H2O), potassium chloride (KCl), potassium phosphate monobasic (KH2PO4), potassium thiocyanate (KCNS), sodium bicarbonate (NaHCO3), sodium phosphate dibasic (Na2HPO4), sodium citrate dihydrate (Na3C6H5O7·2H2O), albumin (BSA), urea, glycine, sodium azide (NaN3)) were also purchased from the Sigma-Aldrich Chemical Co., USA. Acetic acid, glacial (≥99.7%) was purchased from Fisher Scientific, USA. Sodium Hyaluronate (Mw = 397 kDa) was received from Shandong Topscience Biotech Co., Ltd. China. GantrezTM S-97 HSU SOLUTION (Mw = 1 ~ 1.5 MDa) and Poly(allylamine-co-allylguanidinium) (Mw = 8268 g/mol; Mw/Mn = 1.305) were received from Ashland and Colgate-Palmolive Co., USA, respectively. Polydimethylsiloxane (PDMS) (silicone fluid) and polyethylene glycol (PEG) (Mw = 8000 g/mol) were received from Dow Corning, USA.


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2.2. In-situ QCM-D A QCM instrument (Model E4, Q-Sense Inc., Gothenburg, Sweden) capable of dissipation monitoring was used to quantify polymer adsorption on both HA and SiO2 surfaces. QCM-D is a powerful tool for careful measurement of the mass deposited onto or released from a surface in contact with a liquid. QCM-D is based on the change in resonant frequency of a vibrating quartz crystal sensor, a piezoelectric material, according to the change in mass of the sensor. The QCMD instrument monitors real-time changes in the frequency of vibrational modes as well as changes in the vibrational energy dissipation. The Sauerbrey equation relates the change in the resonant frequency to the change in mass of the quartz crystal, 52-59

∆m = −C

∆f n n

where C is the mass sensitivity constant (C = 17.7 ng cm−2 Hz−1 for an AT-cut, 5 MHz crystal) and n is the vibrational mode number (n = 1, 3, 5,··). The dissipation change, ∆Dn, indicates physical characteristics of the deposited layer such as viscosity and elasticity. If ∆Dn is less than 2.0 × 10−6 and the plots of ∆fn/n over time for several modes superimpose, the adsorbed layer behaves like an elastic solid. As a result, mass of the elastic layer can be calculated using the Sauerbrey equation.

On the contrary, if ∆Dn is greater than 2.0 × 10−6, the adsorbed layer

behaves like a viscoelastic liquid. In this case, the physical properties (thickness, shear modulus, and viscosity) of the viscoelastic layer can be estimated by fitting the QCM-D experimental data (∆fn/n (n = 1, 3, 5,··· and ∆Dn) with a Voigt-based viscoelastic model.

For this study,

hydroxyapatite-coated QCM sensor crystals were cleaned by exposing to UV-Ozone for 20 minutes, followed by immersing in 95% ethanol for 30 minutes at room temperature, rinsing with ultrapure water (Millipore Direct-Q, 18 mΩ cm resistivity), drying with N2 (g), and last, re-


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exposing to UV-Ozone for 20 minutes to remove organic and biological impurities. The pH of the solutions was measured using a dual pH/conductivity meter (Denver Instru. Co. USA) and adjusted by using 1 N NaOH and 1 N HCl solutions. For QCM-D experiments, HA and SiO2 surfaces were exposed to 1.0 wt% polymer aqueous solutions (pH 7). All solutions were degassed by sonication. In addition, for QCM-D studies, we used AS solution as well as human saliva solution. AS solution was prepared according to an earlier publication.40 For human saliva collection, stimulated human whole saliva was collected from one healthy female adult donor by chewing parafilms to stimulate salivary flow and collecting saliva in a 50 ml sterile centrifuge tube. The collected saliva was chilled in ice and later centrifuged for 10 minutes at 10,000 rpm to remove cellular debris. Clarified saliva was immediately used without further treatment. The liquid medium was circulated by peristaltic pump at a rate of 100 µL/min through a flow cell with the sensor crystal. The temperature of the system was maintained at 21 °C and 37 °C, respectively. 2.3. Atomic force microscopy (AFM) Surface topography and roughness were captured by atomic force microscopy (Digital Instruments, Santa Barbara, CA: Dimension 3000 AFM). Tapping mode with a single crystal Si tip with a spring constant of 48 N/m, a radius of curvature of about 10 nm, and a resonance frequency of approximately 190 kHz were used. AFM images were obtained over scan sizes of 1 × 1 µm2 and 5 × 5 µm2. Technologies).

Images were analyzed using Picoview 1.6 software (Agilent

The root mean square roughness (Rrms) values were determined from five

separate 1 µm2 images for each substrate type. 2.4 Force spectroscopy using colloid-probe atomic force microscopy


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Force spectroscopy measurements were performed using AFM (Model MFP3D, Asylum Research, Santa Barbara, USA). Surface interactions between silica-colloid and polymer film were obtained by measuring force-distance curves (FD curves) in DI water. Silica micro-beads (Microspheres-Nanospheres, Corpuscular, NY, USA) were attached to a tip-less cantilever (CSC12, Mikromash, USA) using a micromanipulator. A two-part epoxy (JB Weld, Sulphur Springs, TX, USA) was used to glue the colloid at the end of the cantilever. The cantilevers were cleaned in ethanol (Sigma-Aldrich, USA) and treated in UV-Ozone (UVO Cleaner model 42, Jelight Co. Inc., Irvine, CA, USA) for at least 15 minutes before use. Colloids with the lowest available RMS roughness (~ 1.5 - 2 nm) were used for measurements. Pull-off forces were estimated from the retraction force curves.60 The normal stiffness of the cantilevers (before attaching the colloid) was calibrated with a thermal noise method using the MFP-3D controller and was obtained kn = 67 pN/m. The normal deflection sensitivities of the colloid-attached cantilevers were obtained by measuring the slope of the force-distance curves (deflection (d) vs. Z sensor position (Z)) on a clean silicon wafer in DI water. The approach speed for all the measurements was held constant at 0.25 Hz. Around 8-36 force curves were acquired in a scan area of 20 x 20 µm2 and across several locations on the polymer film. 2.5 Bacterial strains and culture. The following bacteria were used for this study: Fusobacterium nucleatum 10953, Streptococcus oralis 35037, Lactobacillus casei 334, Actinomyces viscosus 43146, and Veillonella parvula 17745. The bacteria were co-cultured in a growth medium consisting of 0.6

g/L proteose peptone (BD, Sparks, MD), 0.3 g/L trypticase peptone (BD, Sparks, MD), 0.15 g potassium chloride (Sigma-Aldrich, St. Louis, MO), 1.514 g/L dextrose (BD, Sparks, MD), 0.75


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g/L porcine gastric mucin (Sigma-Aldrich, St. Louis, MO), 0.3 g/L yeast extract (BD, Sparks, MD), 0.03 g/L L-cysteine HCl (Sigma-Aldrich, St. Louis, MO), 1.667 mg/L hemin (SigmaAldrich, St. Louis, MO), and 3.067 ml of 60% sodium DL-lactate (Spectrum Chemical MFC Corp, Gardena, CA). The bacterial suspension was maintained at 37 °C, pH = 6.02, and under constant agitation at a rate of 150 rpm using a Bioflo and Celligen 310 Fermentor/Bioreactor (Eppendorf, Hauppauge, NY). The bacterial suspension is adjusted to an optical density of 0.2 (λ = 610 nm) prior to each use in biofilm assays. 2.6 Biofilm assay. The antibacterial activity of the test polymers was tested by pre-coating the surface of hydroxyapatites disks (HAP; Himed, Old Bethpage, NY) with 1 wt% polymer solutions (~pH = 7.0) for 20 minutes at 37 °C. Unbound polymer was removed by dipping the treated disks 10 times in sterile deionized water. Each disk was then placed in a 1.5 mL of bacterial suspension for 3 hours at 37 °C under an environment containing 5% CO2. Unbound bacteria were washed out by dipping the inoculated HAP disks 10 times in sterile deionized water. Any remaining adherent bacteria were resuspended by sonicating each disk in 500 µl of sterile deionized water at 30 second intervals for 2 minutes per disk side. The relative amount of viable bacteria in the collected bacterial suspension was quantified using BacTiter-Glo Microbial Cell Viability Assay (Promega, Madison, WI) as described by the manufacturer. Total biomass accumulated on the treated disks was determined using Syto9 staining (Thermofisher, Waltham, MA). Briefly, the Syto9 working solution was prepared by adding 1µl of the fluorophore per 1 mL of sterile deionized water. Equal volumes of bacteria and Syto9 solution were combined and incubated for 7 minutes at room temperature. Statistical analysis of the resulting data was performed using the unpaired t-test with GraphPad Prism software (GraphPad software, Inc, La Jolla, CA).


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3. RESULTS AND DISCUSSION 3.1. Polymer adsorption on HA surfaces I. Polymer adsorption on surfaces a. Neutral, cationic and anionic polymer adsorption on HA and SiO2 surfaces. To study polymer adsorption on enamel surfaces by QCM-D, a HA coated QCM sensor was used as a biomimetic system inspired by enamel. A SiO2 coated QCM sensor was used as a control since silica particles, used to polish tooth surfaces for caries prevention, could affect polymer adsorption on oral surfaces. Two types of anionic polymers, Gantrez and NaHa, a cationic polymer, PAA-G75, a neutral hydrophobic polymer, polydimethylsiloxane (PDMS), and a neutral hydrophilic polymer, polyethylene glycol (PEG), were used in this study. As shown in Figure 2 (a), upon exposure of HA surfaces to Gantrez solution, frequency (∆f3/3) decreases from 0 to -15 while dissipation (∆D3) increases from 0 to 25. After rinsing with DI water, ∆f3/3 increases from -15 to -5 and does not return to its original value (0), whereas ∆D3 decreases from 25 to 16. This result shows that a viscoelastic layer of Gantrez is coating HA surfaces. After rinsing, weakly adsorbed Gantrez is released from the HA surfaces under the flow condition used in this study (flow rate = 100 µL/min; Temp = 21 °C; pH 7). A tightly adsorbed Gantrez layer remains on the HA surfaces and retains viscoelastic character (∆D3,5,7 = 16, 10, 7) at this flow condition. Upon exposure of SiO2 surface to Gantrez solution (Figure 2 (d)), traces of frequency and dissipation exhibit similar behaviors as upon exposure of HA surface (Figure 2 (a)). However, during the rinsing step, frequency and dissipation return to their original values (0), as shown in Figure 2 (d). This result means that Gantrez attaches weakly to


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SiO2 surface and is easily removed during the rinsing step. The adsorption of a second anionic polymer, NaHa, on HA crystal and SiO2 surface was investigated. As shown in Figure 2 (b), upon exposure of HA surfaces to NaHa solution, frequency (∆f3/3) decreases from 0 to -30 and dissipation (∆D3) increases from 0 to 20. After rinsing with DI water, ∆f3/3 increases from -30 to -12 and does not return to its original value of 0, whereas ∆D3 decreases from 20 to 2. This result indicates that NaHa adsorbs on HA surfaces. After rinsing, weakly adsorbed NaHa is released from the HA surface. The remaining adsorbed NaHa is tightly bound to the HA surface exhibiting more elastic (rigid) character (∆D3,5,7 = 2) at this flow condition when compared to Gantrez (∆D3,5,7 = 16, 10, 7). In the case of SiO2 surfaces, Figure 2 (e) shows that exposure to NaHa solution results in slight changes in frequency and dissipation. After rinsing, frequency and dissipation return to their original values (i.e. 0), as shown in Figure 2 (e). This result indicates that NaHa weakly interacts with the SiO2 surface. As shown in Figure 2 (c), upon exposure of HA surfaces to the cationic polymer, PAA-G75, frequency (∆f3/3) decreases from 0 to -14 and dissipation (∆D3) increases from 0 to 10. After rinsing with DI water, ∆f3/3 increases from -14 to -8 and ∆D3 decreases from 5 to 0. This result shows that PAA-G75 adsorbs on HA surfaces. During rinsing, weakly adsorbed polymers are released from surface leaving behind a tightly adsorbed PAA-G75 layer on HA surfaces behaving as the most elastic (rigid) film (∆D3,5,7 = ~0) when compared to Gantrez (∆D3,5,7 = 16, 10, 7) and NaHa (∆D3,5,7 = 2). For SiO2 surfaces, Figure 2 (f) shows that the PAA-G75 solution produces a slight change in frequency and dissipation. After rinsing, frequency and dissipation return to their original values (i.e., 0) as shown in Figure 2 (f), indicating that PAA-G75 weakly interacts with SiO2 surfaces.


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In contrast to SiO2 surfaces, the three ionic polymers (Gantrez, NaHa, PAA-G75) physically adsorb on HA surfaces and the polymer adsorbed layers are stable under the flow condition used in this study. This result suggests that weakly bound ionic polymers on SiO2 surfaces of silica particles in toothpaste could easily deposit to oral surface. The Gantrez layer on HA surface is the most viscoelastic, whereas the PAA-G75 layer is the most elastic (rigid). In addition, the dry areal masses of the strongly adsorbed Gantrez, NaHa, and PAA-G75 layers on HA surfaces are determined using a dry QCM-D analysis method (supporting information, Figure S1). For the Gantrez, NaHa, and PAA-G75 layers, the areal masses were 1200 ng/cm2, 399 ng/cm2, and 460 ng/cm2, respectively, listed in Table 1. This result shows that the anionic polymer, Gantrez, with the highest molecule weight, has the largest areal mass on the HA surfaces. The cationic polymer, PAA-G75, with the lowest molecular weight, has a greater areal mass than the anionic polymer, NaHa, due to stronger electrostatic interaction between the cationic polymer and the net negatively charged HA surfaces. Uskokovic et al. showed that HA has two types of crystal planes with significantly different net charges, positive charges on a and b planes, and negative charges on c planes, respectively.61 Positive and negative charged planes prefer to interact with anionic molecules and cationic molecules due to electrostatic interaction, respectively.61,62 Generally, HA crystals possess a hexagonal structure that grows along the c-axis, which represents that c-surface is predominant crystal growth facet compared to a and b surfaces.63 In this study, both cationic and anionic polymer adsorption on HA surfaces could result from HA surface properties that even though the predominant c planes of HA prefer to absorb cationic polymers, the a- and b- planes of HA surfaces could absorb anionic polymers. Thus, both molecular weight and electrostatic interaction play important roles in determining the adsorption of these polyelectrolytes on HA surfaces and physical properties of the adsorbed polymer layers.


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Additionally, the QCM-D results show that physical adsorption of PDMS (hydrophobic polymer) to the HA surfaces is negligible, whereas PEG (neutral hydrophilic polymer) very weakly adsorbs on the HA surfaces (supporting information, Figure S2). b. Effect of pH on swelling of anionic and cationic polymers adsorbed on HA Saliva maintains an oral mucosal pH value between 6.0 and 7.5. A drop of pH (below 5.5) is potentially harmful to human dentition. For example, formation of biofilm on the tooth surface, which makes more acidic microenvironments near/in biofilms, as well as a pH change due to environmental factors (e.g. acidic beverage and food) impact on tooth erosion.1,42,64-66. It is important to study pH-dependent morphology change of ionic polymer layers that physically absorb on the tooth surface, because cationic and anionic polymers have pH-dependent swelling properties.31,33,59 In order to evaluate pH-dependent swelling properties of physically adsorbed (not chemically grafted) polymer layers, it is required that physically adsorbed polymers do not release from the surface during the pH change. As shown above (Figure 2), formation and stability of cationic polymer (PAA-G75) and anionic polymer (Gantrez and NaHa) layers on HA were observed. In this study, swelling properties of physically adsorbed ionic polymer layers are observed in situ as a function of pH using a QCM-D under physiological pH and acidic environments outlined in Figure 3(a). Figure 3 (a), sequential QCM results of Figure 2 (C), are the QCM-D results for the PAA-G75 layer on the HA-coated sensor as a function of the pH of the solution. Upon decreasing pH from 7 to 5.5 (arrow 3 in Figure 3), ∆fn/n (n=3, 5, 7) and ∆Dn do not change. This means that there is no mass change or viscoelasticity change. Upon decreasing pH from 5.5 to 3.5 (arrow 4), ∆fn/n (n=3, 5, 7) decreases and correspondingly, ∆Dn increases. This change reflects an increase of protonated amine groups within the PAA-G75 layer. It leads to an increase in electrostatic repulsion and an increase in water content resulting


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in a swollen and viscous layer. Upon increasing pH from 3.5 back to 7 (arrow 5 in Figure 3), ∆fn/n (n=3, 5, 7) and ∆Dn return to their original values (pH 7 before arrow 3) indicating that the

mass of the PAA-G75 layer remains constant and that the PAA-G75 layer is stable for variation in pH from 3.5 to 7 at the flow condition used in this study. In summary, these observations show that at low pH (3.5), the physically adsorbed PAA-G75 layer swells and becomes more viscous, whereas at higher pH (pH 5.5 and 7), the PAA-G75 layer is elastic and rigid. In addition, the thickness, shear modulus, and viscosity of the PAA-G75 layer can be calculated by fitting the experimental results, namely ∆fn/n (n = 3, 5, 7) and ∆Dn, with the Voigtbased viscoelastic model. The PAA-G75 layer thicknesses obtained from best fit are plotted versus time (i.e., pH) in Figure 3(b). As pH decreases from 7 to 5.5 (arrow 3 in Figure 3), the layer thickness remains constant. At pH 7, the thickness of the PAA-G layer is 6 nm (Table 1), similar to the thickness at pH 5.5. As pH decreases from 5.5 to 3.5 (arrow 4 in Figure 3), the layer thickness increases to 54 nm. As pH increases from 3.5 back to 7 (arrow 5 in Figure 3), the layer thickness returns to the original value of 6 nm. In this flow condition, after change in pH at various values (arrows 3, 4, and 5), the layer thickness is recovered as expected due to its reversible behavior when pH of the solution returns to 7. The pH response of the PAA-G75 layer can be understood by protonation and deprotonation of -NH2 functional groups of PAAG75. As pH decreases, the amines become protonated resulting in electrostatic repulsion and swelling. This pH-dependent swelling and contraction of the PAA-G75 layer is similar to that observed for cationic polymer brushes grafted to surface.33,59 For anionic polymers (Gantrez, NaHa), swelling properties of physically adsorbed polymer layers are observed in situ as a function of pH using QCM-D in physiological pH and acidic environments shown in Figure S3 and S4. These observations show that at low pH (3.5), the


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physically adsorbed Gantrez and NaHa layers become less viscous, whereas at higher pH (7), the Gantrez and NaHa layers swell and become more viscous. The thicknesses of the Gantrez and NaHa layers are calculated by fitting the experimental results with the Voigt-based viscoelastic model (Figure S3 and S4). As shown in Table 1, at pH 3.5, the in situ thicknesses of the Gantrez and NaHa layers are 46 nm and 11 nm, respectively. Upon increasing pH from 3.5 back to 7, the in situ thicknesses of the Gantrez and NaHa layers return to their corresponding original values

(52 nm and 35 nm). The pH response of both anionic polymer layers can be understood by deprotonation and protonation of carboxylic acid (COOH) functional groups of Gantrez and NaHa. As pH increases, COOH becomes deprotonated resulting in electrostatic repulsion and swelling. This pH-dependent swelling and contraction of the Gantrez and NaHa layers are similar to that observed for surface grafted anionic polymer brushes.31 II. Sequential adsorption of polymers on HA In this QCM-D study, we evaluate the uptake of low molecular weight cationic polymer by anion polymer layers on HA using ionic cross-linking interaction. First, a Gantrez polymer layer with higher molecule weight, viscosity, and more flexible polymer chains than NaHa layer (in Figure 1, 2, and Table 1), was evaluated. Figure 4 shows the response of low molecular weight cationic polymer (PAA-G75) for Gantrez layer on HA surfaces. After establishing the Gantrez layer, PAA-G75 (pH 7) was introduced into the QCM-D flow cell (arrow 3 in Figure 4). Correspondingly, frequency, ∆f3/3, and dissipation, ∆D3, of the Gantrez layer immediately decreased (Figure 4a) indicating an increase in both mass and rigidity. The increase in mass is a direct result from the uptake of PAA-G75. The increase in stiffness of the Gantrez layer is consistent with that of the cationic PAA-G75 layer, indicating electrostatic cross-linking between the negatively charged carboxylate groups of Gantrez and positively charged guanidinium (or


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amine) groups of PAA-G75.

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The electrostatic cross-linking is similar to the behavior of an

anionic polyacrylic acid brush exposed to a multi-cationic small molecule, such as tobramycin.31 Upon rinsing with DI water (pH 7) (arrow 4 in Figure 4), ∆f3/3 increases to -20, whereas ∆D3 decreases to 4. The change in frequency and dissipation indicates that weakly adsorbed PAAG75 is rinsed off from the PAA-G75/Gantrez layer and the stable film retains rigid and elastic character due to strong electrostatic interaction between the anion polymer, Gantrez, and the cationic polymer, PAA-G75. Figure 4 (b) shows change in thickness after exposure of Gantrez layer to PAA-G75 solution. Thickness is obtained by fitting the experimental results with the Voigt-based viscoelastic model (Figure S5). As shown in Table 2, the thickness of the Gantrez layer at pH 7 is 52 nm; after exposure of PAA-G75 (pH 7), followed by exposure to DI water (pH 7), the layer thickness reduces to 8 nm, suggesting that the residual PAA-G75 retains strong electrostatic cross-linking with the anionic carboxylic groups of Gantrez. Correspondingly, the PAA-G75/Gantrez layer remains thin and rigid. In addition, according to the dry areal masses of PAA-G75 uptake by Gantrez layer, determined using dry QCM-D technique (supporting information, Figure S1), as shown in Table 2, the areal masses of Gantrez layer before and after cross-linking with PAAG75 are 1200 ng/cm2 and 1393 ng/cm2, respectively. As a result, the mass amount of PAA-G75 adsorbed on/into the Gantrez layer by electrostatic ionic cross-linking is 193 ng/cm2. Figure S4 shows that exposure of NaHa layer to PAA-G75 solution results in increases in layer stiffness and rigidity, similar to the behavior of Gantrez layer. Upon rinsing with DI water (pH 7) (arrow 7 in Figure S4), ∆D3 decreases to ~0, indicating that the film becomes rigid and elastic due to strong electrostatic interaction between the carboxylate anions and the cationic polymer, PAA-G75. Thickness of NaHa layer at pH 7 is 35 nm (Table 2, Figure S4 (b)). After


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exposure of NaHa layer to PAA-G75 (pH 7), followed by exposure to DI water (pH 7), the layer thickness is reduced to 11 nm. In addition, according to the dry areal masses of PAA-G75 uptake by NaHa layer (Table 2, Figure S1), the areal masses of NaHa layer before and after cross-linking with PAA-G75 are 399 ng/cm2 and 507 ng/cm2, respectively. Subsequently, the amount of PAA-G75 adsorbed on/into NaHa layer by electrostatic ionic cross-linking is 109 ng/cm2. In summary, because Gantrez has higher areal deposited mass (more COO- functional groups), flexibility, viscosity and thickness than NaHa (Figure 1, 2, and Table 1), Gantrez adsorbs a higher amount of PAA-G75 (cationic polymer) by forming ionic cross-linking resulting in 85% contraction of the thick Gantrez layer in comparison to 68% contraction of the NaHa layer (Table 2). In addition, in order to study the effect of order in which the cationic polymer (PAA-G75) and the anionic polymer (Gantrez) are exposed to the HA, on the formation and physical properties of polymer composite layers on HA, we evaluate the adsorption of Gantrez on PAA-G75 layer on HA (Figure S6). As shown in Figure S6, the thin, stable PAA-G75 layer of 9 nm first was established, and subsequent exposure to the Gantrez (arrow 3 in Figure S6 (a)), followed by rinsing step (arrow 4 in Figure S6 (a)), shows that the initially formed Gantrez/PAA-G75 layer is a viscoelastic layer of 57 nm. Thickness and dissipation values (∆Dn) of the Gantrez/PAA-G75 layer decrease to 19 nm and ~2 × 10-6 over a period of 1 hour, respectively, suggesting that the Gantrez/PAA-G75 layer becomes stable, thin, and stiff via slower electrostatic cross-linking process of the anionic carboxylic groups of Gantrez and cationic PAA-G75 compared to the PAA-G75/Gantrez layer. The results suggest that when low molecular weight PAA-G75 is


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utilized as the first polymer, there is a significant period of rearrangement before a stable polymer composite layer (Gantrez/PAA-G75) is formed. In addition, as shown in Table S1, PAA-G75/Gantrez layer on HA has lower thickness and higher shear modulus compared to Gantrez/PAA-G75 layer, indicating that PAA-G75/Gantrez layer is thinner and stiffer than Gantrez/PAA-G75 layer. This result suggests that the sequence in which the cationic polymer (PAA-G75) and the anionic polymer (Gantrez) were exposed to the HA surface could affect formation and physical properties of the polymer composite layers. 3.2 Adsorption of cationic and anionic polymers on AS treated HA Polymer adsorption on HA surfaces, pretreated with AS, was studied using in situ and dry QCM-D techniques. Exposing HA surfaces to AS solution at 21 °C (arrow 1 in Figure 5), frequency (n=3) decreases to -70 and dissipation (n=3) increases to ~10 × 10-6. These results are consistent with an adsorbed AS layer that behaves viscoelastically.40 After rinsing the AS layer with DI water, frequency slightly increases to -60 and dissipation decreases to ~ 4 × 10-6. The flow of DI water removes loosely bound species, whereas the AS layer remains stably attached to the HA surfaces. After exposing the AS layer to PAA-G75 solution (pH 7) (arrow 3 in Figure 5), followed by rinsing with DI water (pH 7) (arrow 4 in Figure 5), dissipation is similar to that of the initial stable AS layer but frequency increases to -45. As shown in Figure 5 (b), the in situ thickness of AS layer, determined by modeling (Figure S7), is 25 nm. The treatment of PAAG75 on AS layer results in a decrease in layer thickness (16 nm). In comparison, exposure of AS layer to Gantrez solution and DI water and has similar results (a decrease in layer thickness, not shown). In addition, the areal masses of AS layer before and after treating with PAA-G75 are 1249 ng/cm2 and 1091 ng/cm2, respectively (Figure S1 (c)). This result indicates that after


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treating with PAA-G75, the areal mass decreases -158 ng/cm2. This decrease in mass does not suggest “no PAA-G75 uptake by AS layer,” because during AS exposure to PAA-G75 solution and DI water, the mass change could result from mass change in relatively small components (such as proteins) in AS layer. Therefore, in order to measure the uptake of PAA-G75 by AS

layer, surface interactions (FD curves) of polymer treated AS layer on HA surfaces need to be evaluated by colloid-probe AFM techniques. 3.3 Interaction strength between negative colloidal bead and adsorbed polymers In order to evaluate the physisorption of PAA-G75 (cationic polymer) on/into AS layer, which is well-known to be negatively charged, colloid-probe atomic force microscope techniques were used. Force and distance (FD) curves were acquired between the bare, UV-O3 treated silica colloid and cleaned silicon wafer in DI water at low salt concentration and at pH = 7. Direct interactions show short-range repulsive interactions upon approach (not shown). Formation of structured water layers close to silica surface is known to generate repulsive hydration forces,60 and the presence of such short range forces in our measurements indicate the cleanliness of the colloids used. Further, in the absence of organic contamination, the isoelectric point for silica surfaces in water is measured as ~ 2 - 3.5,33,67 thus presenting high density negative charges on the surface of the silica colloid at pH = 7. Figure 6 shows the representative FD curves obtained on single films i.e., AS and PAA-G75, and combined films i.e., AS(top)/PAA-G75 and PAA-G75(top)/AS physisorbed on HA surfaces. The pull-off forces measured upon retracting of negatively-charged silica colloid probe from the film presents a qualitative comparison of the surface charges presented by different polymer modified-HA surfaces. A single film of the cationic polymer PAA-G75 showed high adhesion


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(~ 34.6 ± 9.7 nN), while the bio-polymer chains (mainly mucin) in AS predominantly presenting anionic groups resulted in negligible adhesion (~ 1.0 ± 0.3 nN) when probed with negativelycharged silica colloid at pH = 7. However, the combined films of AS/PAA-G75 (red) and PAAG75/AS (green) on HA films showed similar pull-off forces, i.e. 4.5 ± 1.7 nN and 6.7 + 17.5 nN, respectively. AS/PAA-G75 films showed slightly higher adhesion than single AS films with minimum spread in the measured forces, indicating a negative-charge dominated surface. On the other hand, the magnitude and the spread of the pull-off forces observed for PAA-G75/AS layer on HA (Figure 6 (C)) indicates the presence of both anionic and cationic groups on the surface. This result represents that during AS exposure to PAA-G75 solutions and DI water for rinsing, uptake of PAA-G75 by AS layer does occur; PAA-G75 polymers are non-homogenously adsorbed and distributed on/into the AS layer. 3.4 Surface morphology and roughness of adsorbed polymers To study morphological changes of HA surface after various polymers treatments, the surface morphology and roughness of each dry surface were characterized using tapping mode AFM. Figure 7 shows representative topography images (1 × 1 µm2 and 5 × 5 µm2 scan areas) of the polymer-treated crystals used in the QCM-D experiments that correspond to surfaces described in Figure 4 and 5. Figure 7 (I), (II), and (III) show the surface topography of the physically adsorbed AS, PAA-G75, and Gantrez layers on HA surfaces, respectively. The root mean square roughness (Rrms) values of the AS/HA, PAA-G75/HA, and Gantrez/HA surfaces are 1.7 ± 0.3 nm, 2.1 ± 0.3 nm, and 1.0 ± 0.1 nm, respectively. We reported previously on Rrms (2.2 ± 0.2 nm) of the as-received nanocrystalline hydroxyapatite, HA, on the QCM sensor and the nano-crystalline particle shapes (circular and flat domains) in the phase image.40 Figure 7 (I), (II), and (III) show the nanocrystalline domains of HA are still observed under the polymer


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layers. Relative to the roughness of the HA surface, the roughness of the adsorbed polymer surfaces do not changed significantly. The surface morphology and roughness of sequential adsorbed polymer layer on/into the first polymer adsorbed layer on HA-coated QCM sensor were studied. Figure 7 (IV) and (V) show the surface topography of the physically adsorbed PAA-G75 and Gantrez layers on/into the first AS adsorbed layer on HA-coated QCM sensor, respectively. Relative to the AS/HA, PAA-G75/HA, and Gantrez/HA surfaces, the Rrms values of PAA-G75/AS/HA and Gantrez/AS/HA increase from 1.7 ± 0.3 nm (AS/HA) to 5.4 ± 1.1 nm, and to 2.9 ± 0.4 nm, respectively. This result shows that the sequential polymer treatments (PAA-G75 and Gantrez) on AS layer changes the morphology and increase roughness of the layers. Specifically, the highest roughness value of PAA-G75/AS/HA is consistent with the magnitude and the spread of the pull-off forces observed for PAA-G75/AS/HA (Figure 6 (C)). This indicates that cationic PAA-G75 polymers, which have lower molecular weight, are non-homogenously adsorbed and distributed on/into AS layer which has negative charge. In addition, the roughness value of Gantrez/AS/HA is lower than that of PAA-G75/AS/HA and this suggests that anionic Gantrez polymers, which have the highest molecular weight, are more homogenously adsorbed and distributed on/into an AS layer which has negative charge. 3.5 PAA-G75 adsorption onto/into a pellicle compared to an AS layer. For studies of polymer adsorption onto enamel surface in human oral system, human saliva and temperature (37 °C) for a biomimetic system were used with HA-coated QCM-sensors. The polymer adsorption on the HA surfaces, pretreated with human saliva versus AS, respectively, was studied under the biomimetic condition using in situ and dry QCM-D techniques


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(Supporting information; Figure S8 and S9). First, in situ QCM-D traces of formation of AS layer on HA at 37 °C are similar to those of formation of AS layer on HA at 21 °C (Figure 5 and Figure S8-(I)).

As given in Table 3, the in situ thicknesses of AS layer at the different

temperatures (21 and 37 °C) are 25 and 28 nm, respectively, which are not significantly different. Deposited dry areal masses of at different temperatures (21 and 37 °C) are 1249 ng/cm2 and 792 ng/cm2, respectively. In in situ QCM-D study (Table 3), treatment of PAA-G75 on AS layer at 37 °C results in increase in layer thickness (33 nm) and small decrease in the layer viscosity (1.33 × 10-3 Ns/m2). In dry QCM study (Table 3), after treatment of PAA-G75 on AS layer at 37 °C, areal mass of adsorbed PAA-G75 on/into AS layer is 866 ng/cm2. Both results show that at 37 °C, uptake of PAA-G75 by AS layer is directly observed in contrast with the previous QCM results at 21 °C (Figure 5, Table 3 and Figure S1 (c)).

In situ QCM-D traces of formation of pellicle layer on HA at 37 °C are similar to those of

formation of AS layer on HA at 37 °C (Figure S8-(I) and S8-(II)). As shown in Figure S8-(II) (b) and Table 3, in situ thickness and viscosity of pellicle layer are 16 nm and 3.3 × 10-3 Ns/m2. A pellicle layer has lower thickness and higher viscosity than AS layer at 37 °C. Treatment of PAA-G75 on pellicle layer results in small increase in the layer thickness (17 nm). In addition, dry QCM study (Table 3 and Figure S9) shows that after the treatment of PAA-G75 on pellicle layer at 37 °C, areal mass of adsorbed PAA-G75 on/into pellicle layer is 1712 ng/cm2. Both results show that uptake of PAA-G75 by pellicle layer is observed and the adsorbed amount of PAA-G75 on/into pellicle layer is ~2x more than that on/into AS layer. 3.6 Anti-bacterial effect of PAA-G75.


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The current biofilm studies illustrate the antimicrobial impact of polymer coating on surfaces. Pre-treatment of HAP disks with Gantrez or PAA-G75 showed 40% and 65% reductions in bacterial accumulation when compared to an untreated surface, respectively (Figure 8A) with PAA-G75 showing significantly better anti-attachment properties in comparison to the control polymer. Approximately 25% less bacteria accumulated on the PAA-G75-coated surface when compared to the Gantrez-coated HAP disks. Interestingly, the co-deposition of PAA-G75 with Gantrez, regardless of the deposition order, attenuated the former polymer’s anti-attachment properties with no statistical difference in accumulated bacterial biomass between the Gantrezcoated and co-polymer-coated samples. Additional studies are required to further elucidate the exact mechanism that can be attributed to the observed biomass reduction on the PAA-G75coated HAP disk. However, the anti-bacterial activity of the guanidine-based polymer system may have a contributing role in the reduction of bacterial mass68 through the destabilization of the microbial cell wall leading to bacteriolysis.69

This effect can lead to a reduction in

accumulated bacterial biomass on the PAA-G75-treated surface over time. Indeed, assessing the viability of the adherent bacteria collected from the PAA-G75-treated surfaces showed a significant reduction (~93%) in bacterial viability when compared to bacteria collected from untreated and Gantrez-treated HAP disks (Figure 8B). These results suggest the potential antimicrobial activity of PAA-G75. The current bacterial tests on PAA-G75 suggest the polymer functions similarly with cationic antibacterial polymers; thus potentially sharing similar mechanisms of activity against bacteria.70-74 The anti-bacterial function of the PAA-G75 was contingent upon the order of polymer deposition, losing its antimicrobial activity when layered on a Gantrez-treated surface. The loss of functionality is primarily due to the difference of deposited area mass of PAA-G75 polymers following its complexation with Gantrez (Table 1


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and Table 2). As shown in Table 2, the deposited area mass amount of PAA-G75 of the polymer composite layer (PAA-G75/Gantrez), that PAA-G75 is adsorbed on/into the Gantrez layer by electrostatic ionic cross-linking, is 193 ng/cm2. In contrast, according to QCM-D studies (Figure S6), the deposited area mass of PAA-G75 of the polymer composite layer (Gantrez/PAA-G75) keeps the value of PAA-G75 adsorbed on the HA surface (deposited area mass of PAA-G75 on HA = 460 ng/cm2 in Table 1) since Gantrez adsorbed on the PAA-G75 layer forms a stable polymer composite layer via electrostatic ionic cross-linking.

The PAA-G75/Gantrez layer

appears to lack sufficient PAA-G75 for antimicrobial activity in this experimental condition compared to the Gantrez/PAA-G75 layer. Interestingly, the complexation of Gantrez on a PAAG75-coated surface appears to occur at a slower rate than the prompt adsorption of the cationic polymer to a Gantrez-coated surface (Figure S6). This putative slower rate of complexation would offer ample time for PAA-G75 to deliver its bactericidal activity to adhering bacteria. As a result, the anti-microbial function of Gantrez/PAA-G75 layer was still maintained (~84% reduction vs Gantrez-control), similar to PAA-G75 layer (~93% reduction vs Gantrez-control). In addition, according to force spectroscopy studies, both PAA-G75 polymers adsorbed on HA surface and anionic polymers adsorbed on PAA-G75 coated HA surface are homogeneously distributed on overall surface areas while PAA-G75 polymers absorbed on/into anionic polymer layers are non-homogenously distributed on overall surface area. The important observations in this experimental condition suggest that PAA-G75 and Gantrez/PAA-G75 has a uniform antimicrobial effect across the entire surface area while PAA-G75/Gantrez does not. 4. CONCLUSIONS In this paper, fundamental studies probed by QCM-D on surface physisorption of anionic polymers, Gantrez and NaHa, and cationic polymer, PAA-G75, have shown strong adsorption of


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polyelectrolytes on HA surfaces in comparison to silicon oxide surfaces. We have found that both anionic and cationic polymer layers that are physisorbed onto HA surfaces have pHdependent swelling properties and show opposite behaviors as a function of pH. Adsorption of anionic polymers (Gantrez and NaHa) followed by adsorption of the oppositely charged polymer (PAA-G75) results in decreased film thickness (film collapse) due to the electrostatic crosslinking between anionic and cationic polymers. Adsorption of low molecular weight cationic PAA-G75 polymer followed by adsorption of the oppositely charged polymer (Gantrez) shows decreased film thickness similar to the formation of PAA-G75/Gantrez layer but there is a significant period of rearrangement via electrostatic cross-linking between anionic and cationic polymers for formation of a stable Gantrez/PAA-G75 layer.

In addition, exposure of AS-

pretreated HA to PAA-G75 leads to a decrease in total thickness.

Furthermore, to study

interaction strength between negative colloidal bead and adsorbed polymers on HA surfaces as well as surface morphology of adsorbed polymers on HA surfaces, force spectroscopy and AFM were used. We have successfully observed that the relatively small PAA-G75 (cationic polymer) strongly adsorbs onto the heterogeneously-distributed negatively-charged AS surface. We have also observed that the adsorbed amount of PAA-G75 on/into the pellicle layer (human salivatreated HA surface) is ~2x more than that on/into AS layer. Lastly, biofilm studies suggest that the deposited guanidine-functionalized polymers, which could be used to stabilize polymer coatings on oral surfaces via electrostatic interaction between polymer and saliva and/or oppositely charged polymers, may have utility as potential antibacterial agents for protecting oral health by controlling bacterial accumulation on teeth.


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Table 1. pH-dependent swelling of physically adsorbed ionic polymers on HA surfaces

Gantrez

Area massa (ng/cm2) 1200 ± 94

NaHa

399 ± 26

6.05 × 10-3

166

11

35

PAA-G75

460 ± 21

4.37 × 10-1

2

54

6

Polymer

a

Grafting density Thickness (nm)b nm2/chain 2 (chains/nm ) pH 3.5 pH 7 -3 208 46 52 4.82 × 10

Areal mass measured by QCM-D based on dry layers (Supporting Information; Figure S1).

b

Thickness values were estimated using modeling simulated and experimental curves for ∆fn/n

(n=3, 5, 7) and ∆Dn vs. time, which showed a good fit between the viscoelastic model (Voigt) and the experimental data (c.f. Figure 3 and Supporting Information; Figure S3 and S4).


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Table 2. Thickness change of polymer layer by cross-linking and deposited areal mass of PAA-G75 cross-linking with anionic polymer layers on HA surface Before cross-linking After cross-linking Deposited areal mass of Polymer layer Thicknessa Areal massb Thicknessa Areal massb on HA PAA-G75 (ng/cm2) 2 2 (nm) (ng/cm ) (nm) (ng/cm ) 8 Gantrez 52 1393 ± 63 193 ± 31 1200 ± 94 NaHa 35 11 399 ± 26 507 ± 22 109 ± 43 a

Thickness determined using modeling simulated and experimental curves for ∆fn/n (n=3, 5, 7)

and ∆Dn vs. time, which showed a good fit between the viscoelastic model (Voigt) and the experimental data (c.f. Figure 4, Supporting Information; Figure S4 and S5).

b

Areal mass

measured by QCM-D based on dry layers (Supporting Information; Figure S1 (a) and (b)).


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Table 3. Adsorption of PAA-G75 on pellicle versus AS layer using in situ and dry QCM-D Before PAA-G75 treatment (I) After PAA-G75 treatment (II) Areal mass of Temp In-situ QCM Dry QCM In-situ QCM Dry QCM Absorbed (37 °C) Thickness Viscosity Areal mass Thickness Viscosity Areal mass PAA-G75 (ng/cm2) (nm) (10-3 Ns/m2) (ng/cm2) (nm) (10-3 Ns/m2) (ng/cm2) Pellicle 15.7 ± 0.1 3.38 ± 0.02 869 ± 26 17.2 ± 0.2 2.03 ± 0.01 2581 ± 40 1712 ± 56 AS 28.1 ± 0.5 1.36 ± 0.01 792 ± 10 32.7 ± 1.0 1.33 ± 0.01 1658 ± 75 866 ± 85 At 21 °C, thickness and areal deposited mass of AS layer forming on HA surface are 25 nm and 1249 ng/cm2, respectively; After PAA-G75 treatment, the thickness and areal mass of the treated layer are 16 nm and 1091 ng/cm2, respectively (Figure 5 (b), Figure S1 (c)).


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Figure 1. Chemical structures of (a) Gantrez, Mw = 1 ~ 1.5 MDa, Di-basic acid groups/polymer molecule = 5740 ~ 8600 (pKa1 = 3.5, pKa2 = 6.5), (b) Sodium hyaluronate (NaHa), Mw = 397 kDa, pKa = 3 ~ 4, carboxylate groups/polymer molecule = 990, radius of gyration (Rg) and persistence length = 59.7 nm and 7.5 nm, respectively,50 (c) Poly(allylamine-coallylguanidinium) (PAA-G75; 75% guanidine functionalized polyallyamine), Mw = 8268 g/mol, Mw/Mn = 1.305, pKa of polyallyamine = ~ 8.5 and pKa of guanidine = 12.5, persistence length of polyallyamine = 0.73 nm.51


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Figure 2. Response of frequency and dissipation in QCM-D to polymer adsorption and release on a surface. (a), (b), and (c) show traces of ∆fn/n (n=3, 5, 7) and ∆Dn vs. time for exposure of hydroxyapatite (HA) surfaces to 1 wt% Gantrez (pH 7), 1 wt% NaHa (pH 7), and 1 wt% PAAG75 (pH 7), respectively, followed by exposure to DI water (pH 7). (d), (e), and (f) show traces of ∆fn/n (n=3, 5, 7) and ∆Dn vs. time for exposure of silicon oxide (SiO2) surfaces to 1 wt% Gantrez (pH 7), 1 wt% NaHa (pH 7), and PAA-G75 (pH 7), respectively, followed by exposure to DI water (pH 7). Arrow * and ~ represent exposure of a surface to polymer solution and to DI water, respectively.


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Figure 3. (a) Traces of ∆fn/n (n = 3, 5, 7) and ∆Dn of PAA-G75 adsorbed layer on HA surface versus time as a function of sequential changes of solution pH; Simulated and experimental curves for ∆fn/n (n = 3, 5, 7) and ∆Dn vs. time (from 48 min to 130 min), showing the best fit between the experimental data and the viscoelastic model. (b)Thickness versus time determined from the fit shown in (a). As shown in Figure 2 (C), arrows 1 and 2 represent exposure of HA surface to 1 w% PAA-G75 (pH 7) and to DI water (pH 7) for rinsing, respectively. Arrows 3, 4, and 5 represent pH changes from 7 to 5.5 (DI water), 5.5 (DI water) to 3.5, 3.5 to 7, respectively.


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Figure 4. (a) Traces of ∆fn/n (n = 3) and ∆Dn vs time for formation of a Gantrez polymer layer and subsequent cross-linking by PAA-G75 polymers. (b) Thickness versus time determined using modeling simulated and experimental curves for ∆fn/n (n=3, 5) and ∆Dn vs. time, which


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showed a good fit between the viscoelastic model (Voigt) and the experimental data (c.f. Supporting Information; Figure S5). Arrow 1, 2, 3, and 4 represent the exposure of 1 w% Gantrez solution (pH 7) to HA surface, rinsing with DI water (pH 7), the exposure of 1 w% PAA-G75 solution (pH 7), rinsing with DI water (pH 7), respectively.


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Figure 5. (a) Traces of ∆fn/n (n = 3) and ∆Dn vs time for formation of an AS layer and subsequent exposure by PAA-G75 polymers. (b) Thickness versus time determined using modeling simulated and experimental curves for ∆fn/n (n=3, 5, 7) and ∆Dn vs. time, which showed a good fit between the viscoelastic model (Voigt) and the experimental data (c.f. Supporting Information; Figure S7). Arrow 1 and 2 represent the exposure of HA surface to AS solution and to DI water for rinsing (pH 7), respectively. Arrow 3 and 4 represent the exposure of HA surface to PAA-G75 solution and to DI water for rinsing (pH 7), respectively.


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Figure 6. Representative FD curves obtained on (A) single layers of AS (blue) and PAA-G75 (gray) and (B) combined layers of AS/PAA-G75 (red) and PAA-G75/AS (green) physicadsorbed on HA films. (C) Histogram representing the spread of the pull-forces measured for films is presented. An AFM-cantilever with a colloid probe of radius ~ 2.5 µm and stiffness kn = 66.96 pN/m was used for the measurements.


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Figure 7. AFM images of HA surfaces after various treatments. (I) AS layer on HA surface. (II) PAA-G75 layer on the HA surface. (III) Gantrez layer on the HA surface. (IV) Surface after exposure of AS/HA layer to PAA-G75. (V) Surface after exposure of AS/HA layer to Gantrez. All samples are prepared using HA-coated QCM sensors and the procedures in the QCM-D experiments. AFM images represent both 1 × 1 µm2 and 5 × 5 µm2. RMS represents the RMS roughness of the corresponding surface in nm.


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Figure 8. A) Syto9 staining shows significantly less bacterial accumulation on the PAA-G75coated HAP disks when compared to the Gantrez-coated surfaces (*P < 0.0079). No significant difference in accumulated bacterial mass was observed between the Gantrez-only or co-polymercoated HAP disks. B) Viability assays showed that PAA-G75-coated surfaces provided significant antibacterial activity in comparison to the Gantrez-treated HAP disks (*P < 0.006). The order of surface deposition for the cationic polymer impacted its antibacterial performance with the Gantrez/PAA-G75-coated surface possessing significant antibacterial activity in comparison to Gantrez-coated controls (**P < 0.0074).

No difference in antibacterial

performance was observed in the PAA-G75/Gantrez-coated surface versus untreated and Gantrez-coated controls.


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ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (J.G.M.) *E-mail: [email protected] (R.J.C.) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT R.J.C. and H.L. acknowledge funding from Colgate-Palmolive. D.M.E. acknowledges support from ONR Grant N000141612100. We also acknowledge support from NSF/Polymer DMR1507713 (RJC, CL), NSF/PIRE OISE-1545884 (RJC, DME), and facility assistance from NSF/MRSEC DMR-1120901. We also thank Danielle Fau and Dr. Matt Bruckmann for helping AFM studies and Sonya Kripe, Michael Minehan, and Han-Chang Yang for providing the saliva.

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