Nanomechanics of pH-Responsive, Drug-Loaded, Bilayered Polymer

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Nanomechanics of pH-Responsive, Drug-loaded, Bi-layered Polymer Grafts Prathima Chandra Nalam, Hyun-Su Lee, Nupur Bhatt, Robert W Carpick, David M Eckmann, and Russell J. Composto ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14116 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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ACS Applied Materials & Interfaces

Nanomechanics of pH-Responsive, Drug-loaded, Bilayered Polymer Grafts Prathima C. Nalam1,‡,†, Hyun-Su Lee3,4,‡, Nupur Bhatt5, Robert W. Carpick1,4, David M. Eckmann2,3 and Russell J. Composto2,4,* 1

Department of Mechanical Engineering and Applied Sciences, 2Department of Bioengineering,

3

Department of Anesthesiology and Critical Care, 4Department of Materials Science and

Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 5

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703.

* Corresponding Author, Tel.: +1 215 898 4451, E-mail: [email protected] ‡ Authors with equal contribution † Present address: Department of Civil and Environmental Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois, 61801, USA.

Keywords: Atomic force microscopy, pH responsive polymers, polyelectrolyte grafts, nano mechanics, antibacterial coatings, drug delivery

ACS Paragon Plus Environment

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ABSTRACT: Stimuli-responsive polymer films play an important role in the development of smart antibacterial coatings. In this study, we consider complementary architectures of polyelectrolyte films including a thin chitosan layer (CH), poly (acrylic acid) (PAA) brushes and a bilayer structure of CH grafted to PAA brushes (CH/PAA) as possible candidates for targeted drug delivery platforms. Atomic force microscopy (AFM) was employed to study the structuremechanical property relationship for these mono- and bi- layered polymer grafts at pH 7.4 and pH 4.0, corresponding to physiological and biofilm formation conditions, respectively. Herein, the surface interactions between polymer grafts and the negatively-charged silica colloid attached to an AFM lever are considered as representative interactions between the antibacterial coating and a bacteria/biofilm. The bi-layered structure of CH/PAA showed significantly reduced adhesive interactions in comparison to pure CH but slightly higher interactions in comparison to PAA films. Among PAA and CH/PAA films, upon grafting CH over the PAA brushes the normal stiffness increased by 10 fold at pH 7.4 and 20 fold at pH 4.0. Notably, the study also showed that the addition of an antibiotic drug such as multi-cationic Tobramycin (TOB) impacts the mechanical properties of the antibacterial coatings. Competition between TOB and water molecules for the PAA chains is shown to determine the structural properties of PAA and CH/PAA films loaded with TOB. At high pH (7.4), the TOB molecules, which remain multi-cationic, strongly interact with polyanionic PAA thereby reducing the film’s compressibility. On contrary at low pH (4.0), the water molecules preferentially interact with TOB in comparison to uncharged PAA chains and upon TOB release, results in a stronger film collapse together with an increase in adhesive interactions between the probe and the surface and the elastic modulus of the film. The bacterial proliferation on these platforms when compared to the measured mechanical properties shows a


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direct correlation and hence understanding nano-mechanical properties can provide insights into designing new antibacterial polymer coatings.

1. Introduction

The increasing usage of soft materials in advanced biomedical applications such as drug delivery and tissue engineering has led to the need for gaining a fundamental understanding of the interfacial properties of soft materials with their environment. The design and development of surface modifications for biomedical implants via end-grafted or self-adsorbed polymeric films to enhance their biocompatibility and for the controlled release of therapeutic drugs at the infected sites has been investigated recently1,2. The development of gradient or hierarchical structures using polymeric films has been shown to improve the mechanical compatibility of structure with its surrounding environment and also has enabled the generation of responsive structures with multifunctional properties3–5. Specifically, for preventing bacterial attachment and proliferation on medical devices, surface coatings are necessary for controlling the interfacial interactions and material stiffness. While there have been studies on several pH-responsive polyelectrolyte films such as poly (acrylic acid) (PAA)6,7, chitosan (CH)8–10, poly(ethylene imine)11 and poly (N,Ndimethylaminoethyl methacrylate) films as effective antibacterial coatings, these mono- layered polymeric films often do not present surfaces with tunable charge densities and mechanical stiffnesses in accordance with the external cues. Further, these structures are also required to be compliant enough to hold and release encapsulated drugs upon chemical or mechanical triggers. Hence, hierarchical structures such as layer-by-layer assemblies of polyelectrolytes12,13, mixed polymer films14, stratified polymer grafts15,16 or gel membranes17 are being explored for their mechanical properties to develop reliable and tunable structures. These structures consist of


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systematically assembled polymeric layers, in which each film individually or collectively provides a distinct functionality to the overall property of the soft structures. Such structures can also be responsive, i.e., the conformational properties of the films are sensitive to the external cues such as solvent quality, pH, ionic strength or temperature of the surrounding medium. Here, we present a systematic investigation of nano-mechanical properties of a novel pH responsive thin film with anti-infection capabilities to biomaterial surfaces. This film consists of a chitosan top layer covalently bound to a surface-grafted poly(acrylic acid) polymer brush (CH/PAA), which responds to local changes in pH to release antibiotics if an infection develops on the bilayer18,19. There exists few nano-mechanical studies for hydrogel-based20 and polyelectrolyte multilayered21,22 antibacterial films. Herein we additionally study the influence of the entrapped drug on the conformational and mechanical properties of the coatings as a function of solvent pH. Specifically, to resolve the influence of structural complexity of the bilayer films on their mechanical behavior, we compare the mechanical properties of the individual layers, i.e., PAA brushes and CH thin films, with those of the CH/PAA bilayers at pH 7.4 and pH 4.0, respectively. Weak polyelectrolyte PAA films undergo dissociation of carboxyl acid groups at high pH (above pKa ~4.5) generating ionized, highly swollen brushes with extended chain lengths. Chitosan, on the other hand, is a linear cationic polysaccharide biopolymer composed of glucosamine and N-acetyl glucosamine, and protonation of amino groups of CH generating positively charged polymer chains results in swollen films specifically in an acidic aqueous environment (below pKa ~ 6.3). The CH monolayer films are coatings where chains are free within the coating (i.e., not grafted). Tobramycin (TOB) exhibits bactericidal activity against a broad spectrum of bacteria. TOB contains five aminoglycosides resulting in at least five pKa values ranging from 6.2 – 8.623. Thus at pH 7.4, TOB is partially protonated and has an attraction towards


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the deprotonated PAA chains18 while at pH 4.0 TOB undergoes complete protonation. In the bilayer architecture of CH/PAA, the formation of a biofilm due to bacterial adhesion results in an acidic micro-environment24–26 (pH 4.0) which leads to release of the entrapped drug from the PAA under-layer and diffusion into its environment through the locally swollen chitosan film (Figure 1). In our prior work, the biocidal activity of TOB loaded PAA and CH/PAA films after overnight exposure to S. aureus (ATCC25923) colonies (pH ~ 3.8) showed a negligible or reduced bacterial attachment on both the films (18). In this study, we investigate the swelling, interfacial adhesion and the mechanical modulus of these mono- and bi- layered films using atomic force microscopy (AFM) and qualitatively attempt to compare the measured mechanical properties with previously observed bacterial proliferation. We observe that the lateral and elastic rigidity of the bi-layered structures can be systematically tuned by altering individual monolayers, thus presenting tunable platforms. However, the addition of multi-ionic antibiotics or drugs, such as Tobramycin, is shown to impact the surface interactions and compressibility of the antibacterial coatings. The competitive interactions between multicationic TOB and water molecules with PAA brushes alters the solvation state of PAA brushes in CH/PAA or PAA films at both pHs. This study hence also presents the importance of considering preferential solvation mechanism in the choice and design of the drug along with its antibacterial coating.

2. Theoretical Background Hertz contact theory52 is usually employed to estimate the elastic modulus of polymer films attached to rigid substrates under axisymmetric loading. The force (F) applied by the indenter is related to the corresponding indentation depth by 3/2 power-law: F=

16 E sample R 0.5 d 1.5 9

(1)


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where R is the radius of the colloid, d the depth of indentation and Esample the elastic modulus of the soft polymer. A Poisson ratio of 0.5 was assumed for hydrated polymer films. One of the many assumptions of the Hertz model is that the contacting materials are homogenous, isotropic with small elastic deformations and no adhesion at the interface. Soft polymeric structures experience high strain/deformation even at the smallest applied loads, specifically when indented using sharp conical tips, resulting in a deviation from the Hertz’s 3/2 power-law rule35. Thus low contact pressures are employed to deform the polymer films elastically and within the linear stress-strain regime by using silica colloid probes (~ 5 µm), instead of sharp AFM tips. Robust Hertzian fits for soft and thick hydrogels (~ 5-40 kPa) are shown using a colloid probe with radius ~ 5 µm53. However, when the polymer film thickness (h) is comparable to the depth of indentation (d), the classical pressure distribution in the film produced by a spherical indenter deviates from the Hertz model as the contact zone no longer remains semi-infinite and substrate effects have to be included.54 Chadwick and co-workers developed appropriate pressure distributions to account for substrate effects while estimating the modulus for soft films55. The analytical results obtained at large indentation depths show that Hertzian mechanics overestimates the modulus for soft films on hard substrates and the underestimates it for hard films on soft substrates56. According to Dimitriadis et al.57, for highly incompressible films

F=

16 E sample 9

[

R 0.5 d 1.5 1 + 1.133χ + 1.283χ 2 + 0.769 χ 3 + 0.0975χ 4

] (2)

where χ = Rd h . However, for very thin samples with significant material deformation, i.e., when the contact area formed by the indenter (a), especially when using a colloid probe, becomes comparable to h, Eq. 3 loses its accuracy. For example, with a/h > 5 and with a colloid radius of


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~ 4 µm, the resultant limiting film thickness is only ~ 130 nm. Thus for films with smaller thicknesses, the Chadwick equation is shown to be more appropriate56

F = (2π 3)E sample R 0.5 d 1.5 χ 3 .

(3)

Figure S1 in the supporting information shows the validity of the three models as a function of indentation depth. For thick samples and at smaller deformations (aH 10057. Thus thin film elastic models developed by Chadwick et al. predict power-law exponents (n) ranging from 1.5 to 3.5. Materials with power indices n ~ 4.0 – 5.0 result from the non-linear cantilever deflections originating from hyper-elastic deformations of the films. Careful consideration has to be taken on the range of deformation in FD curves to fit an appropriate contact mechanical model and to accurately estimate the modulus of soft, heterogeneous thin polymer films.

3. Results and Discussion Swelling properties of pH responsive polymer thin films Table 1 summarizes the thicknesses of polymer films in their dry and wet state as estimated from AFM-based scratch measurements. In dry state, the film thicknesses measured from AFM scratch tests were compared with values obtained from ellipsometer measurements (not shown) and the difference between the two techniques was obtained to be < 10%. Representative topographical profiles of polymer films measured in their wet state along the edge of a scratch are given in the supporting information (Figure S2). All polymer films showed significant swelling when


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immersed in aqueous solutions. The percentage swelling, i.e., the swelling ratio (Sr) for the films is estimated from Sr =

(hwet − hdry ) hdry

× 100

(4)

where hwet, hdry are the wet and dry film thicknesses of the polymer films, respectively. At pH 7.4, the deprotonation of carboxylic acid groups in PAA chains results in highly extended polymer brushes. The highly-charged PAA brushes at low ionic strengths consist of higher concentrations of counter-ions inside the brush in comparison to salt concentration in the surrounding medium. Hence, the free energy of the unperturbed PAA chains is the balance between the entropic elasticity of the chain and the counter-ion osmotic pressure27–29. A swelling ratio (Sr) of ~ 1250% (Eq 4) was observed for PAA brushes at pH 7.4 and at low salt concentration (~ 0.001 mM) (Figure 1a). These values are similar to those reported in the literature16. However, when the pH of the aqueous medium is lowered from 7.4 to 4.0, the thickness for PAA brushes decreases by ~ 260%. At pH 4.0, the carboxylate anions of PAA protonate and the degree of ionization of the brushes are reduced. Unlike other studies6, PAA films in this study still showed significant swelling (Sr ~ 350%) at pH 4.0. At low pH, PAA films with higher grafting densities (0.78 - 0.85 chains/nm2)18 behave as neutral brushes in a good solvent resulting in higher excluded volume parameter and partial stretching of the brushes (Figure 1b). Further, the AFM topography of PAA films at pH 4.0 showed no surface aggregation of the polymer chains (usually seen for collapsed films15) representing only a partial collapse. The wet film thicknesses of cationic CH film in aqueous medium at both pHs were estimated from quartz crystal microbalance studies performed in detail elsewhere21. Protonation of amine groups along the CH chains at low pH results in a Sr of ~ 80%; however, at higher pH, i.e., pH > 6.5, the amine groups are neutral and are insoluble in water resulting in a strongly collapsed film (Figure 1d and 1c, respectively). The overall swelling


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of CH/PAA films, with CH chains attached onto the grafted PAA brushes, result from the complementary (i.e., opposing) swelling properties of the individual PAA and CH films at a given pH. Higher swelling ratio of Sr ~ 260% is observed for CH/PAA films at pH 4.0 (Figure 1f) in comparison to Sr ~ 102% at pH 7.4 (Figure 1e). Further, the overall wet film thickness for CH/PAA bilayer is less than the wet PAA monolayer films. The reason for this behavior is that the chitosan crosslinks with PAA at the interface of the CH/PAA which reduces the ability of the PAA chains to swell as much as in the case of end-grafted PAA monolayer films. The reduction in the wet film thickness for multi-layered films relative to individual polymer layers was observed elsewhere16. The aminoglycoside antibiotic TOB is electrostatically attracted to the polyanions (PAA in PAA monolayer and CH/PAA bilayer) resulting in film collapse and a decrease in film thicknesses at both pH values (Figure 1g and 1h, respectively). The wet thicknesses for PAA and CH/PAA films decreased by ~ 45% and ~ 14%, respectively, upon addition of TOB at pH 7.4. Upon releasing TOB, a larger collapse was observed at pH 4.0, namely, ~ 78% and ~ 51% for PAA and CH/PAA, respectively (Table. 1). Dry film thickness

Wet film thickness

Swelling ratio (Sr)

(nm)

(nm)

(-)

Sample pH 7.4

pH 4

pH 7.4

pH 4

PAA

126.6 ± 2.3

1150 ± 482**

595 ± 47

806

370

CH

-

~5.5*

~ 10*

0

82

CH/PAA

173.8 ± 5.2

351.8 ± 3.5

630.9 ± 14.6

102

263

CH/PAA with TOB

100.0 ± 1.0

302.1 ± 2.8

308.4 ± 3.5

202

208


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Table 1. Dry and wet thicknesses of mono- and bi- layered films with and without TOB loading are shown. The values are obtained from AFM scratch tests. * The wet film thicknesses for chitosan films are measured using quartz crystal microbalance8. ** Refer supporting information 1 on the PAA brush lengths at high pH.


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Figure 1. Schematic representation of the swelling properties of polymer films anchored on the silica surface at two different pHs. PAA polymer brushes (orange chains), chitosan (green chains), CH/PAA and CH/PAA with Tobramycin (red blobs) at pH 7.4 are shown in (a), (c), (e) and (g)


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and at pH 4.0 are shown in (b), (d), (f) and (h), respectively. Note that the thicknesses for PAA and CH layers represented in the figure are not depicted to actual scale to clearly illustrate the swelling behavior of individual layers in a film at different values of pH. Adhesion measurements on pH responsive polymer thin films Figure 2 represents the average pull-off forces measured from the FD curves obtained by indenting the polymer films with bare silica colloid probes. Figure 2(a-c) gives comparison of load dependence of adhesion forces for PAA, CH and CH/PAA films, respectively at both pHs. Representative FD curves for the pH responsive films are given in the supporting information (Figure S3). The retraction curves on PAA brushes at pH 7.4 showed negligible pull-off forces. Upon compression, the interactions between the colloid probe and the PAA film are prevented by the osmotic repulsive forces generated by the mobile counter-ions within the highly solvated PAA film30. Additionally, the high dissociation degree of PAA chains (with ionization factor α ~ 0.57) at pH 7.4 renders a net negative charge to the film30. Thus, along with the reduced van der Waals interactions, the net electrostatic repulsion between the PAA chains and negatively-charged silica colloid results in negligible pull-off forces. At lower pH (pH 4), the surface of the silica colloid (isoelectric point ~ 1.7 – 3.5) becomes less negatively charged (i.e., Zeta potential of silicon oxide surface increases from -60.0 at pH 7.4 to -40.0 at pH 4)8 and the PAA chains become more protonated (predominantly neutral); as a result, the repulsive force between the polymer chains and the probe decreases resulting in higher pull-off forces (Figure 2a). Conversely, the CH films lack the ‘brush-type’ structure leading to an absence of osmotic repulsive forces under compression (Figure 1 c,d). Instead, strong electrostatic interactions between the poly-cationic CH chains and the negatively-charged silica colloid at pH 4.0 dominate the interfacial interactions. At pH 7.4, CH films form neutral and collapsed pancake-like structures at the surface resulting in


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higher van der Waals interactions between the probe and the film. Thus, CH films showed high and almost similar pull-off forces (~ 15- 20 nN) at both pHs. Pull-off force measurements as a function of applied load give an insight on the compressibility of the polymer films. Compressible, soft polymeric films, unlike rigid surfaces, show a contact areadependent adhesion when the number of interactions (either van der Waals or electrostatic) with the indenting probe depends on the applied normal load (contact area). PAA films at pH 4.0 showed a significant increase in the pull-off force as the applied normal load increases in comparison to the films at pH 7.4 (Figure 2a), indicating higher compressibility for PAA films at pH 4.0. PAA films at pH 7.4 on the other hand showed an increase in the measured pull-off force only when the films were highly compressed, i.e., when the applied normal load were higher than ~ 100 nN and the colloid probe overcomes the osmotic and electrostatic repulsive forces generated by the brush. Unlike PAA brushes, thin and rigid CH films showed negligible dependence of pulloff forces on the applied normal load (Figure 2b). CH/PAA bilayer films showed a ~ 97% reduction in the measured pull-off forces in comparison to CH monolayers at both pHs. Thus the relatively swollen, brush-like structure of the PAA underlayer in CH/PAA provides the osmotic repulsive force that opposes the compression. Further, when the CH chains crosslink with PAA brushes, electrostatic interactions between the two layers reduce the net charge on the top CH layer also resulting in reduced interactions. Higher adhesion was observed for CH/PAA films at pH 7.4 in comparison to pH 4.0, indicating that the hydration state of the top CH layer (CH film is swollen at pH 4.0) still dominates the measured pull-off forces. Further, in contrast to PAA films, the CH/PAA brushes at pH 7.4 showed slightly higher pull-off force (~ 95%) and as well a load-dependent adhesion behavior while at pH 4.0 the adhesion was independent of applied normal load (Figure 2c). Thus the partial swollen state of the


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underlying PAA layer and a swollen state of CH layer at pH 4.0 augments the incompressibility of the bilayer (Figure 1f). A comparison of the average pull-off forces measured on PAA, CH and CH/PAA films at pH values of 4.0 and 7.4, and at a normal load of 25 nN is represented in Figure 2d.

Figure 2. Pull-off forces measured between the bare silica colloid and polymer films as function of applied normal load for (a) PAA (b) CH and (c) CH/PAA films at pH 4.0 (red) and pH 7.4 (blue), (d) comparing adhesion forces for polymer films at a normal load of 25 nN. Stiffness of the cantilever kn = 1.84 N/m, radius of the colloid R = 5.6 µm and indentation rate = 0.25 Hz.


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Shear force measurements of pH responsive polymer thin films: Studies have shown that the cross-linking density, solvent quality and persistence length of the polymer films can influence the swelling properties, thereby altering the interface structure and its lubricious behavior31–33. Further, recent lateral force spectroscopy studies15,16 on stratified films showed that the shape of the friction loops obtained by laterally deforming the polymer films can be used to qualitatively estimate the swelling and conformational properties of individual layers within the overall structure. Figure 3a shows typical friction loops measured on the PAA monolayers at pH 4.0 (red) and pH 7.4 (blue), respectively. The friction loops for the more collapsed PAA brushes at pH 4.0 showed a “stick” (~ 4.5 µm) and a “slide” portion (~4.2 µm), while the swollen PAA brushes at pH 7.4 showed only the “stick” portion across the entire sliding distance (10 µm). When the slide distance of the friction loop was reduced, i.e., to 1 µm, and was comparable to the wet film thickness of PAA brushes at pH 4.0, the friction loop was dominated only by the “stick” portion and no interfacial sliding was observed (Figure 3b, orange data points). The “stick” or the tilted portion of the friction loop occurs at the outset of shearing or during the reversal of slide direction. The colloid in this state deforms the hydrated polymer grafts laterally before it begins to slide. The friction originates from the lateral resistance exerted by the polymer chains on the colloid probe and the length of the “stick” portion in the friction loop for polymer films indicates the hydration state and the thickness of the polymer film16. However, once the applied shear forces are larger than the lateral spring constant of the deformed chains a steadystate sliding is observed (insets in Figure 3a).


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Figure 3. (a) Friction loops for PAA film at pH 4.0 (red) and pH 7.4 (blue) acquired with a stroke length of 10 µm. The schematics of the polymer brush conformation during the “stick” and “slide” regime are indicated in the inset. (b) Friction loops for PAA brushes at pH 4.0 at two different stroke lengths 1µm (orange) and 10µm (red). Scan velocity of the probe = 1 µm/s and applied normal load = 10 nN. The enlarged scale for PAA brushes at pH 7.4 (10 µm) and at pH 4.0 (1 µm) is presented in Figure S4 to represent the “stick” regime clearly. During the onset of shearing, the highly hydrated and thick PAA brushes at pH 7.4 exert viscoelastic resistance and impedes the colloid sliding which results in the lateral deformation of chains as shown in the Figure 3a, Inset. The impeded sliding at pH 7.4 also results in smaller dissipations (area inside the friction loop) in comparison to PAA brushes at pH 4.0, i.e., dissipations from polymer inter-digitation or film-probe interactions are reduced due to the absence of sliding. Friction loops for CH/PAA bilayers (Figure 4b) in comparison to PAA brushes (Figure 4a) showed shorter a “stick” portion at both pHs, indicating a reduced resistance by the bilayer film against the interfacial sliding of the colloid. While the friction loops for CH/PAA film at both pHs showed a sharp transition from stick to steady-state sliding, the sliding portion of the loop especially at pH 7.4 showed a tilt (violet loop in Figure 4b), while this behavior was not


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observed for CH/PAA films at pH 4.0. Further, while similar stroke lengths (1 µm) were applied to films at both pHs, the scan length of the friction loop for CH/PAA film at pH 7.4 was observed to be smaller (~ 0.7 µm) in comparison to the friction loops acquired at pH 4.0 (~ 1 µm). This implies that the CH/PAA films impede the movement of colloid across its surface at pH 7.4. The hydration state of the individual layers is shown to affect the deformability of the film and hence this will affect the overall shape of the friction loop. While the collapsed CH top layer results in sharp transition from stick to the slide state, the highly hydrated state of the PAA bottom layer at pH 7.4 imparts resistance to the interfacial sliding, which results in a tilted friction loop. The friction forces measured as a function of load showed an approximately linear dependence for both PAA (Figure 4c) and CH/PAA (Figure 4d) films at higher loads. Friction forces if projected linearly according to Amonton’s law for CH/PAA brushes at pH 4.0 show a significant friction force at zero applied load. However, Figure 2c shows insignificant pull-off forces at smaller applied loads, thus predicting that at low loads the friction forces on heterogeneous bi-layered brushes need not necessarily be linear with the applied load. However, almost an order increase in the coefficient of friction (COF) was observed when pH of the solution was decreased from pH 7.4 (~ 0.005) to pH 4.0 (~ 0.045) for PAA films. CH/PAA bilayers on the contrary showed similar coefficients of friction (~ 2.7) at both pHs with higher shear forces at pH 4.0 in comparison to pH 7.4. The higher COF’s for CH/PAA bilayer films, in comparison to their equivalent PAA brushes at both pHs, indicate that by employing bilayer structures the structural rigidity of the films can be altered or even be amplified.


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Figure 4. Friction loops for (a) PAA brushes (b) CH/PAA films measured at pH 4.0 and pH 7.4 with a slide distance of 1µm are shown. Lateral force dependences on applied normal load for (c) PAA brushes and (d) CH/PAA films at pH 4.0 and pH 7.4 measured using a colloid probe of radius = 5.6 µm and a scan velocity = 1 µm/s are represented. Mechanical deformation of pH responsive polymer films under normal loads Figure 5 shows the force vs. depth of indentation curves measured on films during approach. The right axis in Figure 5 represents the power-law exponents (n, F α dn) as a function of indentation depth. The values for n were obtained from the slopes of log F – log d plots and were found to vary continuously between n ~ 0.5 to 3.0 for PAA brushes and n ~ 0.6 to 6.5 for CH/PAA bilayers,


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respectively. The deviation from the 3/2 power-law is observed more often for films with thicknesses comparable to the indentation depth34. The structural anisotropy resulting from varying polymer density or probe-film interactions along the film thickness also results in complex FD curves 34–36, as observed for biological systems such as bovine ovary37 or L. multiflorum cells35, for example. Such systems showed power-law exponents varying from n ~ 1.0 to n ~ 4.0 as a function of indentation depth. PAA and CH/PAA films in our current measurements have indentation depths comparable to their respective wet film thicknesses and especially for CH/PAA bilayers the film structure is shown to vary with the indentation depth. Further in Figure 5, the CH/PAA films at indentation depths higher than ~ 40-50 nm showed an almost vertical increase in force with indentation depth with values of n greater than 4.0. This regime was avoided in our analysis.


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Figure 5. FD curves (approach only) obtained on PAA brushes and CH/PAA bilayers at pH 4.0 and pH 7.4 using a bare silica colloid probe. Each curve is obtained by averaging at least 36 FD curves acquired across different locations on the sample. On the right axis, the power-law exponents (n) are represented as a function of indentation depth. Cantilever stiffness kn = 0.056 N/m, colloid radius R = 3.98 µm and approach speed = 1 µm/s. In order to measure the Young’s modulus of the film a commonly applied rule is to limit the maximum depth of indentation to 10% of the film’s thickness38. Figure S5 in the supporting information shows the indentation force as a function of the normalized indentation depth, i.e., d/h where d is the indentation depth and h is the wet film thickness, for PAA and CH/PAA films. The values for d/h were found to be higher than 0.1 at indentation forces as low as ~ 0.1 nN for PAA and ~ 0.4 nN for CH/PAA films. Thus the effect of substrate stresses on the polymer deformation should be included to accurately determine the mechanical modulus for these thin films. Further, the strong confinement of the counter-ions in densely-grafted polyelectrolyte brushes, especially at low salt concentrations will result in long-ranged double layer repulsion force, even before the probe comes in contact with the polymer film39. Upon coming in contact the osmotic repulsive forces from the entrapped counter ions and solvent molecules in the brushes result in additional repulsive forces30. These forces will lead to uncertainties in determining the accurate point of contact during approach and also result in power indices with values less than 1.0 (as seen in Figure 5)

40

. Thus, the evaluation of polymer deformation at higher indentation depths by

including substrate stresses is a more reliable approach to determine the elastic modulus of these thin hydrated films. Figures 6a and 6b compare Hertz, Chadwick, and Dimitriadis-Chadwick fits to the FD curves measured on PAA and CH/PAA films at pH 7.4, respectively. The corresponding residual error


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between the fit and the measured data in the fit region (appropriate indentation depth) is represented on the top graph. The fits for polymer films at pH 4.0 are given in the supporting information (Figure S6). Four different fits were employed to determine the appropriate model for measuring elastic modulus for polymer thin films. The Hertz fit (green, Eq. 1) was applied to the entire FD curve whereas a limited-Hertz fit (Eq. 1) was employed to the depths of indentation with n ~ 1.5-2.0. Chadwick (red, Eq. 3) and Dimitriadis-Chadwick (yellow, Eq. 2) fits were employed for indentation depths with n between 2.0-4.0, i.e., the last 50% of indentation depth for PAA films and between 10-20% of indentation depth for CH/PAA films. The Hertz fit to the entire curve showed the highest deviation from the measured FD curve in comparison to the fits from Chadwick and Dimitriadis-Chadwick models. Further, in the fit regions the Chadwick fits showed smaller residual errors (average error ~ 4.1 ± 4.8% for PAA ~ 6.2 ± 3.5% for CH/PAA) in comparison to the Dimitriadis-Chadwick fits (error ~ 20.0 ± 14.3% for PAA and ~ 28.6 ± 19.1% for CH/PAA) at pH 7.4. Similarly, the residual errors obtained with Chadwick fits (Eq. 3) were comparably smaller (> 10 % for PAA and > 25 % for CH/PAA) in comparison to Dimitriadis-Chadwick fits (Eq. 2) for PAA and CH/PAA films at pH 4.0 (Figure S6). Thin films when indented with larger probes (colloid probes) result in higher aH/h values (Figure S1 in supporting information) and thus the Chadwick model is more appropriate in comparison to the Dimitriadis-Chadwick model. Further, the limited-Hertz model, which is routinely employed in the literature, was employed to indentation depths with n ~ 1.5-2.0 (Figure 6, green solid). These fits showed smaller residual errors for both the films (< 5% for PAA and < 12% for CH/PAA). A comparison of the elastic moduli obtained from the Chadwick fit and the limited-Hertz fit for PAA and CH/PAA films at pH 4.0 and pH 7.4 is represented in Figure 6c.


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Figure 6. Force vs. depth of indentation curves for (a) PAA and (b) CH/PAA films at pH 7.4. Hertz (dotted green), limited-Hertz (solid green), Chadwick (dotted red) and Dimitriadis– Chadwick (dotted yellow) fits are shown. The estimated residual errors only in the fit regions are represented. (c) Young’s Modulus for PAA and CH/PAA films obtained from limited-Hertz and Chadwick fits are shown. The limited-Hertz fit for PAA films showed higher Young’s modulus at pH 4.0 in comparison to the films at pH 7.4. A trend reversal, i.e., higher modulus for PAA films at pH 7.4 in comparison to the films at pH 4.0 was observed when the indentation curves were fit using Chadwick model (Figure 6c). While the limited-Hertz fit showed a good fit with low residual error, the depths of


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indentation used for these fits are clearly greater than 10% of the original film thickness. Further, the load-dependent adhesion studies (Figure 2a) and friction loops (Figure 3a) showed PAA brushes at pH 7.4 to be highly hydrated and incompressible films. Thus the Chadwick fit, which includes the substrate effect, represents a more appropriate model for determining the mechanical modulus of these thin polymer films. Chadwick fits for PAA films at pH 7.4 (E ~ 1.32 kPa) shows an almost six times higher Young’s modulus in comparison to the films at pH 4.0 (E ~ 0.2 kPa). The protonated PAA chains at pH 4.0 are more compressible due to the reduced charge and loosely associated hydration shells along the backbone, while at pH 7.4, the highly hydrated and extended PAA chains result in higher modulus (Figure 1 a,b). A similar trend was measured by Liu et al. using quartz crystal microbalance, which showed a shear moduli that was three-times greater for PAA films at pH 6.6 compared to the films at pH 3.241. However, the magnitude of elastic moduli obtained for the polyelectrolyte brushes in our current measurement are smaller than those reported in the literature (~ kPa – MPa)

42–44

. This is expected as the magnitude of the modulus of

polyelectrolyte films depends on the anchoring method, ionic strength of the medium and the chain length. Further by neglecting the substrate stiffness effects on the measured modulus leads to an overestimation of elastic modulus values. Chadwick fits to the CH/PAA FD curves showed an order of magnitude increase in elastic modulus at pH 7.4 (E ~ 13.5 kPa) and a two-order increase at pH 4.0 (E ~ 24.1 kPa) in comparison to their equivalent PAA brushes. The CH chains cross-link with the underlying PAA brushes resulting in enhanced mechanical rigidity for the bilayers in comparison to PAA brushes. Further, CH/PAA films at pH 4.0 showed higher incompressibility (~ 2 times higher) in comparison to the films at pH 7.4. Higher Young’s modulus for CH/PAA films at pH 4.0 results from the deprotonation of CH chains (top layer). The charged CH chains are hydrated due to the presence of entrapped anions


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and solvent molecules. The slightly swollen underlying PAA layer at pH 4.0 can further assist in increasing the incompressibility through solvent-based osmotic repulsion. The higher incompressibility for CH/PAA layers at pH 4.0 was also observed in Figure 2c where a loadindependent adhesion was observed in comparison to the films at pH 7.4. Nano-mechanics of drug-loaded pH responsive polymer thin films Comparing CH/PAA films with and without TOB molecules: Approach and retraction curves for CH/PAA films in the presence of TOB at both pHs are represented in Figure 7a. CH/PAA films with TOB molecules at pH 7.4 initially showed repulsion upon approach. However at higher indentation depths a snap-in event (indicated by arrow in Figure 7a), i.e., a decrease in force with increase in indentation depth was observed. This snap-in was not observed for CH/PAA films without TOB. Snap-in events were observed in more than 95% of the measured FD curves (inset of Figure 7a) at indentation depths between ~ 80-110 nm and at a critical normal load of ~ 2.5-3.0 nN. The occurrence of these snap-in events at specific depths of indentation suggests an onset of interaction between the colloid probe and the TOB molecules only after compressing the top CH film. This behavior with the TOB location being in the PAA layer is represented in Figure 1g. At pH 4.0, the PAA layer protonates and collapses and the CH layer swells allowing TOB molecules to release and diffuse into the surrounding medium. TOB molecules are also highly protonated at pH 4.0 and thus the increased interactions between the negatively-charged colloid and the positively-charged TOB molecules results in snap-in event (in approach curve) already at the surface of the film (Figure 7a). A load-dependent adhesion analysis (from the retraction curves) for CH/PAA films in presence of TOB at pH 7.4 shows a sudden increase in the pull-off force from ~ 0.1 nN to ~ 1.2 nN (above the critical snap-in normal load > 3 nN) (Figure 7b). At low loads, an almost negligible adhesion


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between the CH/PAA film with the colloid probe was observed, similar to CH/PAA layers without TOB at pH 7.4, indicating that the top CH layer effectively shields TOB molecules within the PAA brushes (no interaction between the colloid probe and the TOB molecules). Load-dependent adhesion was not observed for CH/PAA films with TOB at pH 4.0; however, the pull-off forces measured at pH 4.0 were higher than those measured at pH 7.4 (Figure 7b) and for CH/PAA films without TOB at pH 4.0 (Figure 2c). These high pull-off forces indicate an increase in the electrostatic interactions between the diffused, protonated TOB molecules with the colloid probe at the film interface. The strong collapse of the film for the CH/PAA bilayer after the release of TOB (~ 300 nm) in comparison to the bilayer without TOB (~ 650 nm) at pH 4.0 (Table 1) also results in increased van der Waals interactions with the probe.

Figure 7. (a) Force vs. indentation curves on CH/PAA films loaded with TOB molecules. The scatter in the snap-in events occurred during approach at pH 7.4 is presented in the inset. A comparison between the (b) load-dependent pull-off forces and (c) elastic modulus for CH/PAA films with TOB at pH 7.4 and pH 4.0, respectively is shown. The fit residual error of < 5% was obtained when appropriate contact mechanics models were used. The elastic moduli for CH/PAA films with TOB were estimated by employing Chadwick fit to the FD curves measured at pH 7.4 (n below the critical snap-in load ~ 2.0 – 4.0) and with Hertz fit for


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FD curves at pH 4.0 (n ~ 1.2), resulting in a residual error of ~ 4.3% (at pH 7.4) and ~ 3.8% (at pH ~ 4.0). The measured moduli were compared to values obtained for CH/PAA films without TOB (Figure 7c). At pH 7.4, after TOB loading, the CH/PAA showed an 83% decrease in the Young’s modulus in comparison to CH/PAA films without antibiotic (CH/PAA ~ 14.4 kPa and CH/PAA with TOB ~ 2.5 kPa). The electrostatic cross-linking interactions between multi-cationic TOB molecules at pH 7.4 with COO- groups from the PAA chains could result in an increase in the film stiffness. On contrary, this increased compressibility suggests that the increased interactions of COO- groups (in PAA chains) with TOB reduce the net counter-ion charges within the film and as well alter the solvation of the PAA brushes, which together dominate the compressibility of the film. However, CH/PAA films with TOB at pH 4.0 showed a two order increase in modulus (E ~ 1.5 MPa) in comparison to CH/PAA film without TOB (E ~ 24.2 kPa). At pH 4.0, the TOB molecules diffuse into the surrounding media and therefore one would expect a similar elastic modulus as in the original CH/PAA films. However, this enhanced mechanical stiffness for CH/PAA films after the release of TOB molecules at pH 4.0 suggests that TOB molecules strongly alter the solvation of PAA brushes. At pH 4.0, multi-cationic TOB molecules, entrapped within the uncharged PAA brushes, can more strongly interact with surrounding water molecules resulting in the release of TOB into the surrounding medium. Such effects were observed previously for polymer brushes in the presence of solvent mixtures with intermediate solvent quality, which led to a co-nonsolvency effect accompanied by a strong collapse of the polymer brush45. Wang et al. have also observed larger structural transitions for polyelectrolyte brushes in the presence of multivalent cations (Ca2+ and La3+) in comparison to monovalent cations (Na+) in water-methanol solvent mixtures46. Thus the preferential solvation of charged TOB molecules compared to neutral PAA chains in CH/PAA, leads to reduced solvent-polymer


26

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interactions (i.e. a collapse of the polymer brush). This effect results in two contributions which enhances the overall increase in the modulus of CH/PAA films as observed at pH 4.0 (Figure 7c). First, the release of hydrated TOB molecules from CH/PAA at pH 4.0 leads to dehydration of PAA brushes resulting in a strong collapse (collapse of the PAA film in Figure 1h). Second, if any residual hydrated TOB molecules are retained inside the neutral PAA brushes (at pH 4.0), stiffens the film. Thus the strong collapse along with increased density within the PAA layer in CH/PAA film, in presence of TOB molecules at pH 4.0 (Table 1) contribute to the increased elastic modulus of the film (Figure 7b). Comparing PAA brushes versus CH/PAA bilayers with TOB molecules: The contribution of the CH top layer in designing an effective pH-responsive bi-layer film is determined by comparing the behavior of PAA brushes with the CH/PAA bilayers in presence of TOB molecules. In reference 18, the uptake and release of TOB by the PAA and CH/PAA films were quantified using quartz crystal microbalance. The areal mass for PAA brushes was estimated as 17.6 ± 0.5 μg/cm2 resulting in carboxylic acid areal density of 1475 ± 44 [COOH]/nm2. Upon incubating the films in TOB solution at pH 7.4, the PAA monolayers showed 10 fold higher TOB uptake in comparison to CH/PAA polymer grafts. As a portion of the carboxylic groups of PAA in CH/PAA film crosslink with the top CH layer, the number of carboxylic groups available for interacting with TOB molecules is reduced, resulting in lower TOB loading for CH/PAA films. However, after 30 minutes of exposure of PAA and CH/PAA films to pH 3.8, TOB release was measured as ~ 33% and ~ 36%, respectively (Supporting information section S7). Figure 8a shows the raw FD curves measured on PAA films loaded with TOB molecules at pH 7.4 (blue) and pH 4.0 (red). Similar to CH/PAA with TOB, the elastic modulus for PAA with TOB films were estimated using Chadwick fit (Eq. 3) at pH 7.4 (fit residual error ~ 0.4%) and with


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Hertz fit (Eq. 1) at pH 4.0 (fit residual error ~ 5.4%). Unlike the CH/PAA films (Figure 7a), PAA films with TOB showed no snap-in events with indentation. Even the load-dependent adhesion was not observed for PAA films with TOB at both pHs and thus was not reported. However, the pull-off forces (Figure 8b) for PAA brushes with TOB showed a ~ two-order decrease at pH 7.4 and an almost ~ 95% increase at pH 4.0 in comparison to CH/PAA films with TOB. Figure 8c compares the Young’s moduli for PAA and CH/PAA films with TOB. PAA brushes with TOB show lower elastic modulus in comparison to CH/PAA bilayers with TOB at both high and low pH values (~ 0.6 kPa for pH 7.4 and 1.46 MPa for pH 4.0). At higher pH, the TOB molecules interact with charged COO- groups in the PAA and are retained within the film. Hence, the reduced net counter-ion charges along the PAA chains result in compressible films. However, inspite of higher TOB loading in PAA brushes (supporting information 7) in comparison to CH/PAA films at pH 7.4, a larger decrease in elastic modulus was observed for CH/PAA brushes upon TOB loading (~ 75%) in comparison to PAA brushes (~ 53%) (Table 2). This indicates that TOB molecules also interfere with the interactions between CH chains and PAA brushes resulting in an altered hydration state of CH top layer. Further, CH/PAA and PAA films with TOB films showed a 3 order increase in the magnitude of the modulus as the pH of the solution was reduced from 7.4 to 4.0. At low pH, the highly-charged TOB molecules compete with the solvation of PAA or CH chains. Hence, the de-solvation of PAA brushes during the release of hydrated TOB molecules results in a significant film collapse and an increase in the elastic modulus. In accordance with higher TOB loading for PAA brushes, a larger reduction in osmotic pressure as well as increased adhesive interactions with the colloid probe were observed (in comparison to CH/PAA) at pH 4.0 (Figure 8c).


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Figure 8. (a) Force vs. indentation curves on PAA films loaded with TOB molecules are presented. Each FD curves is the average of 36 FD curves obtained across the sample. Comparison of (b) pull-off forces measured at 6nN and (c) elastic moduli for CH/PAA and PAA films loaded with TOB are presented. The fit residual error for PAA brushes with TOB loading was obtained to be < 5%, when appropriate contact mechanics models were employed. In summary, our surface interaction measurements show weaker pull-off strengths between colloid probes and PAA (~ 99% reduction) and CH/PAA (~ 97% reduction) in comparison to the “glassy” CH layer at pH 7.4. While CH layer can act as poly-cationic biocide at low pH, at high pH the collapsed state of CH favors bacterial adhesion18. Thus for retarding the initial bacterial attachment, the bilayer architecture of CH/PAA films in comparison to the CH monolayer film appears more effective. Among PAA and CH/PAA, the pull-off forces measured on CH/PAA layer are higher by ~ 90% in comparison to PAA films. Further, addition of TOB to CH/PAA films show a 2-order increase in pull-off force at pH 7.4. As the initial bacterial attachment occurs at neutral pH (7.4), the outer CH layer appears to maintain an environment favorable for strong bacterial adhesion and biofilm formation.


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The nano-mechanical studies show higher compressibility for PAA films in comparison to the CH/PAA bilayer (higher by ~ 90%) (Table 2). Similarly, lateral force spectroscopy studies also show that the bilayer structure amplifies the lateral rigidity of the coatings. This enhanced normal and shear rigidities for bi-layered structures in comparison to mono-layered films can be a useful property for bio-coatings to efficiently sustain flow forces. Biomaterials such as catheters etc. experience constant forces from the flow of blood and other fluids over them; hence enhanced rigidity of the structures can assist in developing reliable coatings. Further, our study also show that when multi-cationic drugs such as TOB is loaded into polyelectrolyte bilayer, there occurs significant changes in the structural conformation and the elastic modulus in comparison to drugfree bi-layered films (Figure 7).The competition between the TOB and water molecules for the solvation of PAA and CH chains leads to structural changes in the anti-bacterial films. Thus this study suggests a need for a systematic study of preferential solvation effects by other multivalent ionic drugs, especially when polyelectrolyte films are used to design antibacterial films with longterm stability even after the release of the biocides. Bacteria coverage (%) Pull-off forces (nN)

Young's modulus (kPa)

@ pH 3.8 [18] 4

pH 7.4

pH 4.0

5

pH 7.4

pH 4.0

~ 10 CFU/ml

~ 10 CFU/ml

PAA

0.03 ± 0.01 0.35 ± 0.01

1.3 ± 0.01

0.2 ± 0.01

48

77

CH/PAA

0.24 ± 0.15 0.04 ± 0.01

13.5 ± 0.10

24.1 ± 0.2

67

92


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PAA with TOB

0.02 ± 0.01 15.35 ± 1.31

CH/PAA 1.30 ± 0.30 4.73 ± 2.90 with TOB

0.61 ± 0.0

520.0 ± 6.3

3.37 ± 0.04 2730.0 ± 99.7

0

0

0

46

Table 2: Pull-off forces (at 6 nN), elastic moduli (measured using AFM) and the bacterial coverage (%) after overnight growth (measured by optical microscopy) for PAA and CH/PAA films with and without TOB loading at pH 7.4 and pH 4.0 are compared. The values for the bacterial adhesion study are adapted with the permission from Biomacromolecules 2015, 16, 650-659. Copyright 2015 American Chemical Society. The growth of S. aureus (ATCC25923) bacteria on PAA and CH/PAA films was measured at two different concentrations of bacterial colonies by Lee et al. in reference 18. Table 2 (more details in supporting information 8) shows the percentage coverages of the bacteria on polymer films after overnight growth (i.e. films at pH 3.8). High bacterial adhesion was observed for PAA and CH/PAA films without TOB loading at both concentrations. On the other hand, TOB-loaded PAA films act as an effective biocide platform showing negligible biofilm growth on both ∼104 or ∼105 CFU/mL incubated surfaces. S. aureus colonies did not grow on TOB-loaded CH/PAA films at 104 CFU/mL, however 46% surface coverage was observed at 105 CFU/mL. The outer layer (CH) appears to maintain an environment favorable for strong bacterial adhesion and biofilm formation. Further, the smaller TOB loading in CH/PAA films augments the inefficiency of bilayered structures to completely eradicate the adherent bacteria (at higher concentration) from the surface. The higher interaction forces and larger mechanical moduli measured in our studies


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especially for the bi-layered structures (in comparison to mono-layered films) give a likely explanation for the observed bacterial adhesion. However a direct correlation between the mechanical rigidity of the film and bacterial attachment is difficult to establish from our results. The current understanding of the influence of mechanical rigidity of the substrate on mechanoselectivity of bacteria varies widely in the literature. Lichter et al. have shown that the bacterial adhesion and colony growth for E. coli and S. epidermidis bacteria correlates positively with an increase in the elastic modulus of the polyelectrolyte multilayer (PEM) coating22. Similarly, higher adhesion for gram-negative Pseudoalteromonas sp. D41 bacteria was observed on stiffer agarose gels20. On the contrary, other studies with E. coli showed higher adhesion and growth on the softer PDMS substrates47 as well as on softer PEM coatings21 in comparison to their equivalent rigid interfaces. These studies thus suggest that the understanding of the influence of substrate stiffness on bacterial attachment is difficult, as the attachment process is simultaneously influenced by several factors such as substrate surface chemistry, roughness, and hydrophobicity as well as by the varying composition and structure of the bacterial cell wall (i.e., charge, rigidity, presence of appendices). A careful control of the film properties is required to establish the influence of mechanical rigidity of bilayer films on bacterial attachment such as the molecular weight of the grafted PAA underlying layer in CH/PAA should be systematically varied to alter the overall mechanical rigidity of the bilayer while keeping the same grafting density of the CH top layer (similar interactions)48. However, these studies are beyond the scope of the current work.

4. Conclusions The development of tunable and hierarchical structures using polymeric films as coatings has opened up new directions to explore robust and environmentally-sensitive interfaces. Since the structural and mechanical properties of the coatings can greatly affect and control biofouling, in


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this study we present pH- responsive bilayer architectures for antibacterial coatings: a polycationic biocide chitosan layer (at low pH) grafted on a dense, hydrated polyanionic poly(acrylic acid) brush (CH/PAA films). We mimic the interactions between the bacteria and the coating by probing the surface with a micrometer-scale colloid probe attached to an AFM cantilever. The pull-off and lateral force measurements as a function of applied load was used to characterize the surface interactions, compressibility and lateral rigidity of the films. The elastic moduli of the films were estimated by considering substrate effects on the measured polymer deformation. The bilayer structures in comparison to their respective individual monolayers showed an enhanced mechanical rigidity (incompressibility) while still providing a hydrated and viscoelastic structure at the surface. We have shown that the addition of biocidal drugs such as polycationic TOB alters the hydration of the CH/PAA films and hence influences the surface interactions and mechanical rigidity of the film. This is associated with the increased bacterial adhesion on the bilayer film as has been demonstrated previously18, which is revealed here to be due to the co-nonsolvency effect and the resultant enhanced mechanical rigidity of the bilayer. While, it has been shown that the enhanced stiffness of the bi-layered structures is desirable to build reliable soft platforms and the potential of the bilayer to swell and release biocidal drug in response to a pH reduction is ideal for preventing any localized infection, the correlation between the enhanced stiffness and bacterial adhesion needs further understanding.

5. Experimental Section Sample Preparation Silicon wafers used for surface preparation were cleaned by immersion in piranha solution (3: 1 (v/v) H2SO4/30% H2O2, Fisher Scientific, USA) for half an hour at 80oC. The wafers were then rinsed with excess of ultrapure water (Millipore Direct-Q, 18 MΩ cm resistivity), dried with N2


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gas and were exposed to UV-ozone to produce a homogeneous hydroxylated surface and to remove organic impurities. CH film on silicon wafers were prepared according to the procedure described elsewhere in the literature8. Epoxy-derivatized silicon oxide surfaces were prepared by immersion of the wafers into a 10% (v/v) 3-glycidoxypropyltrimethoxysilane (GPTMS) in anhydrous toluene at 80 °C for 12 hours under N2 condition. To immobilize CH on an epoxide-derivatized silicon oxide surface, the epoxide-derivatized surface was immersed in a 2 wt.% CH solution (pH ~4.5); water was allowed to slowly evaporate overnight (~12 h) until the polymer film made a direct contact with the epoxide-derivatized surface at 60 °C. The surface was later rinsed with ultrapure water and acidic solution (pH ~4.5) to remove physically adsorbed polymers and other impurities from the film. PAA and CH/PAA films on silicon wafers were prepared using procedures similar to those detailed in ref18. An alkyl halide initiator, 3-(chlorodimethylsilyl) propyl-2bromoisobutyrate, was prepared by chloroplatinic acid catalyzed hydrosilylation of allyl 2-bromo2-methylpropionate with chlorodimethysilane. Initiator immobilization on silicon oxide surfaces was prepared by immersing the wafers in the initiator solution (in anhydrous toluene) under N2 at 70 °C. After 12 hours, the wafers were rinsed in toluene to remove surface impurities. To graft PAA polymer brushes on silica surface, surface-initiated ATRP of tert-butyl acrylate (t-BA) was performed for ~ 6 hours. Freshly purified and deoxygenated tBA (350 mL, 2.38 mol), extra dry acetone (250 mL), PMDETA (612 µL, 3.0×10-3 mol), and the free initiator (moisture-quenched1(chlorodimethylsilyl) propyl-2-bromoisobutyrate) were added to the solids (purified CuBr (322 mg, 2.24 × 10-3 mol), CuBr2 (15 mg, 6.60×10-5 mol). The homogeneous mixture was transferred to the Schlenk flask containing the initiator-functionalized silicon oxide wafers. The flask was maintained at 60 °C under N2 with constant stirring. Molecular weight and molecular weight distribution of surface-grafted chains were determined by size exclusion chromatography (SEC)


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using free PtBA from SI-ATRP. For CH/PAA films, the PtBA polymer brush films were hydrolyzed using trifluoroacetic acid (TFA) solution for 12 hours. The CH layer was grafted onto the PAA brush via Sulfo-N-hydroxysuccinimide (Sulfo-NHS) ester functionalization of acrylic acid of the PAA brush, followed by quenching the reaction with basic solution (pH ~8, adjusted by using 1N NaOH solution). TOB-loaded CH/PAA films were prepared using previously published methods18. The pH of the aqueous medium was adjusted by the addition of 0.1 N HCl (for pH 4.0) and 0.1 N NaOH (for pH 7.4). No salt was added to the solution. pH 4.0 of the TOB solution was prepared by adding 10 μL of 0.1 N HCl in 0.3 mM TOB solution (40 mL). CH/PAA samples were first immersed in aqueous medium at pH 4.0 for 10 minutes and were then transferred to TOB solution at pH 4.0. The samples were incubated for 30 minutes and rinsed with aqueous medium at pH 7.4 before use. Film thickness measurements The dry and wet thicknesses of polymer films were measured using contact mode imaging (MFP3D, Asylum Research, Oxford Instruments). A wide scratch was made on the polymer samples using a blunt tweezer to gently remove the polymer film and to expose the underlying silicon substrate. A sharp Si3N4 tip with normal stiffness kn = 0.5 N/m was used for imaging the film along the scratch in both dry and wet conditions with a resolution of 256 scan lines and a scan rate of 0.75 Hz. Slope corrections for the images were made using Asylum Research Software (Version 13). Section profiles along the scratch were obtained and the average of 5 step heights was used to estimate film thickness. Force spectroscopy measurements Normal force-displacement curves (FD curves) were obtained on the polymers films at different pH using a colloid probe. Silica microbeads (Microspheres-Nanospheres, Corpuscular, NY, USA)


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were attached to tipless cantilevers (CSC37, Mikromash, USA) using a micromanipulator coupled to an optical microscope (40X, Alessi REL-4100A, NJ, USA). 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 then UV-ozone treated (UVO Cleaner model 42, Jelight Co. Inc., Irvine, CA, USA) for at least 15 minutes before use. Colloids with the lowest RMS roughness (~ 1.5 – 2.0 nm) were used for measurements (Figure S9). The roughness and the radii of the colloids were estimated by reverse imaging the colloid-attached cantilever against a clean test grating (MikroMasch, Spain). Image processing and deconvolution of the tip radius was performed using commercial software (Scanning Probe Image Processor (SPIP): Image Metrology, Horsholm, Denmark). An AFM cantilever with kn = 1.84 N/m and R = 5.6 µm was used to obtain the pull-off forces from the retraction curves. The approach speed was held at 0.25 Hz. A softer cantilever with kn = 0.056 N/m and R = 3.98 µm was used to obtain the FD curves for measuring elastic modulus. These indentation curves were measured at an approach speed of 1µm/s. The deflection from hydrodynamic drag was observed to be insignificant and was hence neglected in these measurements. At least 36 force curves were acquired across a scan area of 40 x 40 µm2. The normal stiffness of the cantilevers (before attaching the colloid) was calibrated using thermal noise method using the MFP-3D controller49. The normal deflection sensitivities of the colloid-attached cantilevers were obtained from the slope of force-distance curves (deflection (d) vs. Z sensor position (Z)) measured on a clean silicon wafer in DI water. Friction force measurements Lateral force measurements were performed by sliding a bare silica colloid (radius of curvature of ~ 5.6 µm) over polymer thin films. A gold-coated cantilever with kn = 1.84 N/m was used to measure the lateral forces. Friction loops were acquired at two different stroke lengths, 1 µm and


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10 µm, and friction forces were analyzed using custom developed IGOR 6.0 software. An average of 10 friction loops were used to measure the lateral forces on the polymer film at an applied normal load. The lateral forces were calibrated using the test-probe method where the torsional spring constant was estimated using Sader’s torsional calibration method50 and the lateral sensitivity of the photodetector was measured by obtaining a lateral force-distance curve using a test cantilever (with colloid diameter > 40 µm) against a hard silica wall. The details of the lateral calibration are reported elsewhere51. ASSOCIATED CONTENT: Supporting Information The Supporting Information is available for free of charge on the ACS Publications website. The supporting information includes AFM topographical images of the colloid probe and bilayer films. The details on the contact mechanical fits to the indentation curves obtained on bilayered polymer films are presented. Further, a summary of the drug loading and bacterial activity on mono- and bi-layered films has been presented. AUTHOR INFORMATION Corresponding Author *Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA Tel.: +1 215 898 4451, E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. The authors declare no competing financial interest. ‡These authors contributed equally.


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ACKNOWLEDGMENT The authors would like to thank the Nano/Bio Interface Centre at the University of Pennsylvania for use of instrumentation. This research was partially supported by the Nano/Bio Interface Centre through the National Science Foundation NSEC DMR08-32802 (RC, RJC, DME), Office of Naval Research grant N000141612100 (DME) and Swiss National Science Foundation (PBEZP2142930). RJC acknowledges partial support from NSF Polymers Program through DMR15-07713 and the NSF/PIRE program through OISE-1545884 (RJC, DME). REFERENCES (1)

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Nanomechanics of pH-Responsive, Drug-Loaded, Bilayered Polymer

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