AvidinâBiotin Cross-Linked Microgel Multilayers as Carriers for

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Avidin-biotin cross-linked microgel multilayers as carriers for antimicrobial peptides Lina Nyström, Noor Al-Rammahi, Sara Malekkhaiat Häffner, Adam A. Strömstedt, Kathryn L Browning, and Martin Malmsten Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01484 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018

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Avidin-biotin cross-linked microgel multilayers as carriers for antimicrobial peptides Lina Nyström1*; Noor Al-Rammahi1; Sara Malekkhaiat Häffner2; Adam A. Strömstedt3; Kathryn L. Browning2; Martin Malmsten1,2*

1. Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden 2. Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark 3. Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden

*Corresponding authors

Phone: +46 – 18 471 4368 Fax: +46 – 18 471 4377 Email: [email protected] [email protected]

Keywords: Antimicrobial peptide, Avidin, Biotin, Microgel, Multilayer, Surface coating

ACS Paragon Plus Environment

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Abstract Herein, we report on the formation of cross-linked antimicrobial peptide-loaded microgel multilayers. Poly(ethyl acrylate-co-methacrylic acid) microgels were synthesized and functionalized with biotin in order to enable the formation of microgel multilayers cross-linked with avidin. Microgel functionalization and avidin cross-linking were verified with infrared spectroscopy, dynamic light scattering, and z-potential measurements, while multilayer formation (up to four layers) was studied with null ellipsometry and quartz crystal microbalance with dissipation (QCM-D). Incorporation of the antimicrobial peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) into the microgel multilayers was achieved either in one shot after multilayer formation or through addition after each microgel layer deposition. The latter was found to strongly promote peptide incorporation. Further, antimicrobial properties of the peptide-loaded microgel multilayers against Escherichia coli were investigated and compared to those of a peptide-loaded microgel monolayer. Results showed a more pronounced suppression in bacterial viability in suspension for the microgel multilayers. Correspondingly, LIVE/DEAD staining showed promoted disruption of adhered bacteria for the KYE28-loaded multilayers. Taken together, cross-linked microgel multilayers thus show promise as high load surface coatings for antimicrobial peptides.


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Introduction Microgels are loosely cross-linked hydrogel particles with stimuli-responsive swelling capacities, triggered by a range of parameters, such as temperature, pH, ionic strength, oxidative/reducing conditions, light, and presence of specific analytes.1, 2 Microgel properties can easily be tuned due to the versatility of monomers, co-monomers, and cross-linkers incorporated in the polymer network during microgel synthesis, allowing for precise control over microgel size, functionality, and mechanical properties.3, 4 Due to the fine level of control in the design and synthesis, microgels have attracted interest as building blocks for stimuli-responsive functional surface coatings with predetermined properties.5, 6 When attached to an interface, either covalently or non-covalently, surface-bound microgels typically flatten to an extent depending on factors such as temperature, pH, ionic strength, and cross-linking density.7-9 Despite this distortion by surface attachment, they generally maintain their stimuli-responsive swelling properties. However, the presence of the surface may affect both the magnitude and the uniformity of their swelling transitions.8,

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Applications for surface-bound microgels has focused

primarily in the field of biosensors, where the predictability of their swelling transitions can generate high sensitivity for optical readings when combined with gold or other metal layers10, 11. However, microgel-based surface coatings offer much wider opportunities than this in biomedical applications, notably in drug delivery and as biomaterial coatings.5 Depending on microgel properties, surface-bound microgels have been shown to display low binding of serum proteins and human monocytes/macrophage adhesion,12 high biocompatibility, low adverse inflammation reactions and cytokine production,13, 14 low bacterial adhesion,15, 16 and drug depot properties.17

Due to their responsiveness, as well as their considerable capacity to incorporate cargo molecules, microgels provide an interesting alternative to commonly studied linear polyelectrolyte layer-by-layer (LbL) films, achieved by alternating oppositely charged polyelectrolytes into multilayers. Serpe et al. reported the first microgel multilayer deposited by a LbL deposition by alternating poly(Nisopropylacrylamide-co-acrylic acid) (NIPAm-co-AAc) microgels, serving as the polyanion, and linear poly(allylamine hydrochloride) (PAH) as the polycation.18 This system was subsequently developed by


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a spin-coating-assisted LbL approach and evaluated by quartz crystal impedance and surface plasmon resonance. Results showed that microgel thin films keep their responsiveness, displaying pH-dependent swelling responsiveness over several cycles. The swelling rate scales with the number of microgel layers, where an increasing number of layers resulted in a slower response of the film coatings.19 To increase the packing density of these type of LbL microgel layers, ‘active’ centrifugal deposition was later developed, layering anionic poly(NIPAm-PEGDA-AAc) microgels, covalently bound to (3‐aminopropyl)trimethoxysilane

(APTMS)-treated

substrates,

and

cationic

poly(diallyldimethylammonium chloride) into multilayers.20 Such microgel coatings show promising self-healing properties,21, 22 drug delivery applicability23, 24 and low cell adhesion.25

Dispersed microgels have been relatively extensively studied as drug delivery carriers for oppositely charged guest molecules.3,

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Their biocompatibility, water-rich environment, and large swelling

capabilities of dispersed microgels make them suitable as carriers of biomacromolecular drugs, e.g., peptides, proteins, nucleotides, and antibodies, to be exposed or released in a stimuli-responsive manner.27 For example, charged pH-sensitive dispersed microgels have been shown to display high loading capacities for oppositely charged peptides, depending on peptide length,28,

29

charge,30

hydrophobicity distribution,31 and peptide secondary structure32. Incorporation of the peptides into the polymer network has also been demonstrated to protect incorporated peptide from proteolytic degradation, although depending on factors such as microgel charge density.33, 34

For surface-bound microgels much less is known about peptide incorporation and release. However, Nyström et al. showed that model peptide poly-L-lysine can be loaded into pH-sensitive poly(ethyl acrylate-co-methacrylic acid) microgels, and that the loading and release of the peptide depend on both media (pH and ionic strength), peptide (length and charge density), and microgel (charge density).35 With cationic peptide loading, microgel volume decreased and caused the polymer network to become more rigid. For higher Mw poly-L-lysine, peptide-induced deswelling was found to be lower for surfacebound microgels than for the corresponding microgels in suspension, inferred as being a result of surface-induced network deformation and partial peptide exclusion. From an application perspective,


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surface-bound microgels could be an interesting surface coating for implants, particularly if loaded with antimicrobial compounds. This approach could potentially overcome issues with reaching therapeutic concentrations at the vicinity of the implant after systemic administration, a key factor in relation to biofilm and fibrous capsule formation that characterize biomaterials-associated infections. Since such infections are frequently associated with antibiotics-resistant strains,36, 37 antimicrobial peptides (AMP) represent an interesting class of antimicrobial compounds for such applications, as these may be designed to be potent also against strains resistant to conventional antibiotics.38 AMPs are a heterogeneous class of peptides, typically 10-40 residues, net cationic, and containing a relatively large fraction of hydrophobic amino acids. As a result of the latter, AMPs are amphiphilic and surface active, and typically have a membrane disrupting mechanism of action. Recent developments have also reported on anti-inflammatory and anti-cancerous properties of (some of) these peptides.39, 40

We have previously investigated factors controlling the loading and release of KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), demonstrated to display potent antimicrobial and antiinflammatory properties,41,

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as well as a PEGylated variant, to/from surface-bound monolayers of

poly(ethyl acrylate-co-methacrylic acid) microgels. In addition, consequences of loading and release on antibacterial and anti-inflammatory effects were addressed.16 Results showed that antimicrobial effects of these peptide-loaded layers were controlled by ionic strength of the surrounding media. In low ionic strength (10 mM), bacteria were killed upon contact, whereas at physiological ionic strength (150 mM) peptide release was promoted, resulting in decreased contact killing and facilitated bulk antimicrobial effects. Quantitatively, however, the amount of peptide released from the layers was enough to kill planktonic bacteria in the bulk only at a high surface area-to-bulk volume ratio. Therefore, in the present investigation, the formation of peptide-loaded poly(ethyl acrylate-co-methacrylic acid) (MAA) microgel multilayers was investigated as a strategy to increase antimicrobial peptide load and released amount at physiological conditions (pH 7.4, ionic strength 150 mM). Apart from providing high peptide loading capacity and protection of microgel-incorporated peptides against infection-related proteolytic degradation,43 MAA microgels display low toxicity, as demonstrated by both low hemolysis43 and low degree of NF-kB induction in human monocytes16. For such microgels, the microgel multilayers were


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formed by alternating biotin functionalized microgels and avidin, in a cross-linked LbL deposition approach. Avidin-biotin interactions have been previously studied in a plethora of contexts and used in multiple bioassays and biological applications.44, 45 Avidin is known to bind extremely strong to biotin (binding constant KD = 10-15), coordinating up to four biotin molecules through its tetrameric structure.46 Hence, avidin-biotin complex formation was considered a suitable ‘cross-linking’ approach. Formation of the microgel-based multilayer was investigated by infrared spectroscopy, dynamic light scattering, zpotential measurements, ellipsometry, and quartz crystal microbalance with dissipation (QCM-D). The latter two techniques were used also for investigating loading and release of the antimicrobial peptide KYE28 into the formed microgel multilayers. Addressing functional effects, antimicrobial effect on Escherichia coli (E. coli) bacteria were monitored and compared to those of peptide-loaded microgel monolayers at physiological pH and ionic strength.


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Methods Materials KYE28 was synthesized by Biopeptide Co., Inc. (San Diego, CA, USA), and was of > 95% purity, as measured by HPLC. EZ-Link™ Amine-PEG3-Biotin (> 98%) and Pierce™ Avidin (> 98%), were obtained from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were of analytical grade and obtained from Sigma-Aldrich (Schnelldorf, Germany). Purified Milli-Q water was used throughout, and buffers used were either 10 mM MES (2-(N-Morpholino)ethanesulfonic acid), pH 5.0, or 10 mM Tris HCl, pH 7.4, henceforth referred to as MES and Tris buffer, respectively, unless otherwise stated.

Microorganisms A clinical strain of E. coli ATCC 25299 was obtained from the Department of Clinical Bacteriology at Lund University Hospital, Sweden.

Microgel synthesis Poly(ethyl acrylate/methacrylic acid/1,4-butandiol diacrylate) (EA/MAA/BDDA) microgels were synthesized by seed-feed (starved feed) emulsion polymerization, according to a protocol described previously.35, 47 (EA/MAA/BDDA) (66/33/1, w/w) were polymerized by ammonium persulfate initiator until the desired diameter of ~ 100 nm was reached and the reaction was stopped by cooling. Microgels are henceforth abbreviated according to the w/w MAA in the seed solution (MAA33). Titration showed the MAA content of the microgels to be 36.9 ± 0.4 % w/w.

Surface-bound microgels To enable covalent attachment of the initial microgel monolayer, the substrates (glass slips or sensors with outer silicon dioxide layer) were treated with 3-glycidoxypropyltrimethoxysilane (GOPS) to add epoxy functionality to the interface, as described in detail previously.35,


48

This allows for the

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carboxylates of the MAA-microgels (0.1 w/w) to bind covalently to the surface, as again described in detail previously.35

Biotin functionalization Microgels in dispersion To enable microgel multilayer formation, MAA33-microgels were functionalized with biotin, and subsequently cross-linked by avidin. For this, the primary amine of the amine-PEG3-Biotin cross-linker was covalently bound the carboxylic acid residues of MAA via an EDC/sulfo-NHS catalyzed amidecoupling (Figure 1a). In brief, MAA33-microgels (1 mL, 1 w/w in MES buffer) were activated with 2 mM N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 5 mM N-hydroxysulfosuccinimide (sulfo-NHS) under stirring for 20 min at room temperature. Subsequently, 3 mM amine-PEG3-Biotin was added and reaction left stirring for additional 3 h. Unbound fractions were thoroughly rinsed with water by centrifugation (Amicon Ultra-4 Centrifugal Filter Unit, 3 kDa Mw cutoff; Sigma-Aldrich, Schnelldorf, Germany). To verify the covalent coupling of amine-PEG3-Biotin to the carboxylic acids of the MAA33-microgels, IR spectra of freeze-dried un-functionalized MAA33 and functionalized (MAA33-Biotin) microgels were obtained using a Spectrum One IR-spectrometer (Perkin Elmer, Shelton, USA). For comparison, spectra were normalized at wavenumber 1814 cm-1.

Surface-bound microgels Biotin functionalization of surface-bound monolayers of MAA33 microgels, as described above, was performed with a slightly modified protocol, in order to ensure that biotin functionalization did not compete with the carboxylate groups needed to couple the microgels to the epoxy groups at the interface, and that a good coverage of the initial layer was reached. Thus, surface-bound MAA33 microgels in MES buffer were activated with 2 mM EDC and 5 mM sulfo-NHS for 20 min, after which 0.3 mM amine-PEG3-Biotin was added and allowed to react for 1 h before unbound reagents were rinsed off, first with MES buffer (15 min, 5 mL/min) and subsequently with Tris buffer (15 min, 5 mL/min). The


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surface-bound biotin-functionalized microgel monolayer thus obtained later served as the base for microgel multilayer formation, described below.

Biotin functionalization of already surface-bound MAA33-monolayers was investigated by in situ ATRFTIR using a Thermo Scientific™ Nicolet™ iS50 FTIR Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The sample environment consisted a Specac gateway ATR module fitted with a thermostabilised flow-through cell (Specac, Orpington, UK) allowing in situ measurements of the reaction on the same sample. For this, 45-degree trapezoid silicon ATR crystals were obtained from Crystran ltd. (Dorset, U.K.) and treated with GOPS, as described above, to introduce epoxide functionality and enable covalent attachment of MAA33-microgels. These microgel surfaces, immersed in MES buffer, were taken as background, and sequential IR spectra measured every 2 minutes during the activation step of the carboxylic acids of the MAA33-microgels with EDC (2 mM)/sulfo-NHS (5 mM) (20 min), and the introduction of amine-PEG3-Biotin (0.3 mM, 60 min). Unbound reagents were rinsed off in MES buffer (100 µL/min, 15 min).

Microgel size and zeta potential Cross-linking of biotin-functionalized MAA33-microgels by avidin in suspension was investigated by measuring microgel size and effective z-potential as a function of avidin concentration. Avidin were added at 0, 64, 128, 256, and 512 µg/mL to MAA33 and MAA33-Biotin microgels (0.01 w/w) in Tris buffer. Mean hydrodynamic diameter and effective z-potential were determined by dynamic light scattering at the standard non-invasive back-scattering angle of 173 ° (for minimization of dust contributions and achievement of good sensitivity to small particles), using a Zetasizer Nano ZSP (Malvern Instruments, Malvern UK). All samples were measured in triplicate at 25 °C, and effective zpotentials (disregarding the diffuse nature of microgel particles49) calculated using the Smoluchowski approximation.2


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Ellipsometry The formation of MAA-microgel multilayers was monitored in situ by null ellipsometry, using an Optrel Multiskop (Optrel, Kleinmachnow, Germany) at 532 nm and with an angle of incidence of 67.60 °. The principles and procedures of null ellipsometry have been described in detail previously.35, 50 Polished silicon wafers, oxidized to an oxide layer thickness of 30 nm (Semiconductor Wafer Inc., Hsinchu, Taiwan) were prepared with a monolayer of MAA33-microgels, subsequently functionalized with amine-PEG3-Biotin, as described above. Microgel multilayers were formed by alternating avidin (64 µg/mL) and MAA33-Biotin microgels (10 ppm) until equilibrium binding (reached after 20-30 min) followed by a 15 min rinse in Tris buffer (5 mL/min) to remove unbound fractions. Peptide was introduced either at the end, i.e., after the formation of four microgel layers (50 µM KYE28), or between each microgel layer, in the latter case alternating KYE28 (25 µM), avidin (64 µg/mL), and MAA33Biotin microgel (10 ppm) during multilayer build-up. The effect of ionic strength on the release of peptide was studied by rinsing the layers in Tris buffer and Tris buffer with additional 150 mM NaCl, 30 and 60 min at 5 mL/min flow, respectively. The adsorbed amount (Γ) was calculated using a refractive index increment of 0.154 cm3/g.

Quartz crystal microbalance with dissipation (QCM-D) The formation of microgel multilayers was also monitored in situ by QCM-D using a Q-sense E4 microbalance (Q-sense, Gothenburg, Sweden) with Q-soft software (Q-sense, Gothenburg, Sweden). The theory describing the technique has been described elsewhere.51, 52 The Quartz sensors with 50 nm silicon dioxide layer, fundamental frequency of f0 ~ 5 MHz (Biolin Scientific AB, Västra Frölunda, Sweden) were cleaned using a Harrick Plasma (Ithaca, NY, USA) PDC-32G cleaner (5 min), placed in 2 % sodium dodecyl sulfate (30 min) followed by extensive rinsing in water before plasma cleaned for another 5 min. The samples were then prepared with a surface-bound monolayer of MAA33-microgels, and functionalized with amine-PEG3-Biotin, as described above. Baseline of covalently attached monolayer of MAA33 microgels was recorded overnight in water (5 µL/min). Changes in frequency (f) and dissipation (D) at overtones 3, 5, 7, 9, 11, 13 were monitored over time during multilayer formation


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by alternating avidin (64 µg/mL) and MAA33-Biotin (10 ppm), or KYE28 (25 µM), avidin (64 µg/mL), and MAA33-Biotin (10 ppm) until equilibrium had been reach (20-30 min in each step). Surfaces were rinsed with Tris buffer to remove unbound fractions between each addition (15 min, 100 µL/min). Furthermore, effects of ionic strength on the release of peptide were studied by rinsing the layers in Tris buffer (30 min) and Tris buffer with additional 150 mM NaCl (60 min) 100 µL/min (or, 12 h at 5 µL/min). Results of the latter experiments are expressed as fraction of mass released from the total mass of the layer after last addition.

Scanning electron microscopy (SEM) MAA33-microgel multilayers were visualized using a Zeiss LEO 1550 scanning electron microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an in-lens detector, operated at 3.0 kV electron beam accelerating voltage. Samples were dried in N2 (g) prior to visualization.

Antimicrobial effects Bacterial viability To investigate antimicrobial effects of peptide-loaded microgel multilayers, bacterial viability of Escherichia coli ATCC 25922 was measured using PrestoBlue® Cell Viability Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Microgel multilayer-modified surfaces (round glass coverslips, 14 mm in diameter) were prepared as described above, with 25 µM KYE28 equilibrated between each microgel layer. For comparison, microgel monolayers were incubated face down with 10 µL 25 µM KYE28 in Tris buffer overnight prior experiment. Bacteria were grown to mid-logarithmic phase in 50 mL Tryptic Soy Broth (Merck KGaA, Darmstadt, Germany) (3 %) at 37 °C, before they were washed twice by centrifugation in Tris buffer. Bacteria were then re-suspended in Tris buffer with additional 150 mM NaCl to yield a suspension of 108 colony forming units (CFU)/mL, verified by optical density (OD600 ≈ 0.6). Samples were placed faced-up in 24 well-plates with 500 µL bacteria suspension in each well and incubated at 37 °C for 4 h, before 50 µL PrestoBlue was added. After an additional 30 min incubation


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at 37 °C, the fluorescence intensity of each well was measured using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

LIVE/DEAD staining For visual representation of peptide-loaded, surface-bound, microgel mono- and multilayers antimicrobial effects, adhered E. coli was stained with LIVE/DEAD® Bacterial Viability Kit (BacLight™; Sigma-Aldrich, Schnelldorf, Germany) and imaged using confocal laser scanning microscopy (CLSM). The microgel multilayer surfaces were prepared as described above, and again incubated with 500 µL 108 CFU/mL E. coli in Tris buffer with additional 150 mM NaCl for 4 h. Loosely attached bacteria were then gently rinsed off and surfaces placed in new Tris NaCl buffer. The adhered bacteria were subsequently stained with 1:1 of the green-fluorescent SYTO9 and red propidium iodide (PI) 1.5 µL to 500 µL Tris buffer (15 min in room temperature) before visualization. Samples were moved to a holder and imaged using a Confocal Leica DM IRE2 laser scanning microscope (Leica Microsystems, Wetzlar, Germany), using a 100 x 1.40-0.70 oil objective and Leica TCS SL software (Leica Microsystems, Wetzlar, Germany). Samples were excited with a 488 nm argon laser, and emission span 515-545 nm for SYTO9 and 615-645 nm for PI was used. Measurements were performed in triplicate at 25 °C and further analyzed using software ImageJ.


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Results Microgel functionalization To enable cross-linking of MAA-microgels into multilayers, microgels were first functionalized with biotin. The amide coupling of amine-PEG3-Biotin to the carboxylic acid residues of MAA was performed in MES buffer, after which the functionalized microgels were thoroughly rinsed in water and unbound fractions of the cross-linker and reactants removed by a centrifugation filter. Conjugation of amine-PEG3-Biotin to the MAA microgels was confirmed by comparing IR spectra of freeze-dried nonfunctionalized MAA33 microgels and functionalized MAA33-Biotin microgels (Figure 1b-c). As can been seen in the subtraction spectrum in Figure 1c, functionalization via EDC/sulfo-NHS reaction results in small changes of the carbonyls in the ester (wavenumber ≈ 1730 cm-1) and carboxylic acid region (wavenumber ≈ 1700 cm-1), as well as an increased peak in the amide region (wavenumber ≈ 1550 cm-1). However, microgels still possess a significant amount of carboxyl groups, important for incorporation of the cationic KYE28 peptide. The latter was confirmed also with z-potential measurements, showing MAA33-Biotin to have similar effective z-potential as the non-modified MAA33 (Figure 2a) (Full IR spectra, 500 - 4000 cm-1, of the two microgels variants and reference spectrum of amine-PEG3-Biotin can be found in Figure S1, Supporting Information.)

To verify that the functionalization degree of biotin was sufficient to cross-link the MAA33-Biotin microgels, microgel size distribution and z-potential in the presence of avidin was measured by DLS. Results show that avidin is able to cross-link MAA33-Biotin (0.01 w/w) into large agglomerates at a concentration of 512 μg/mL (Figure 2b). In contrast, non-functionalized MAA33-microgels show only a slight increase in size, but an increased polydispersity index (PDI; Figure 2c). This indicates that avidin (znet = +20 at pH 7.4)53 binds also electrostatically to the anionic microgels, and not only specifically to the biotin moieties of MAA33-Biotin. This is further indicated by the decreased negative z-potential observed for the highest avidin concentration (Figure 2a). Importantly, however, the effective z-potential remains clearly negative also at the highest avidin concentration (zeff = -25 ± 2 mV and -28 ± 3 mV for


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MAA33-Biotin and MAA33, respectively) and similar for the two microgels. Hence, the large aggregation observed for MAA33-Biotin is primarily due to avidin-biotin coupling.

Biotin functionalization of a surface-bound monolayer of MAA33-microgels, already covalently bound to an epoxide-treated silica surface, was subsequently investigated. The functionalization was performed after microgel immobilization in order to not compete with the carboxylic acids needed for the epoxidecarboxyl coupling to the interface of the silica. This ensured a good coverage of the first microgel layer (Figure S2, Supporting Information).8 The amide coupling was followed in situ with ATR-FTIR, with surface-bound MAA33-microgels in MES as the background at the start of the experiment (Figure 3). The subtraction spectrum in Figure 3b again shows the loss of carboxyl and ester carbonyl groups and a gain of amide carbonyl vibrations, the same trend as for microgels in dispersion (Figure 1b,c). (Full ATR-FTIR spectra, 1500 - 4000 cm-1, are shown in Figure S3, Supporting Information.) A similar control to verify the efficiency of the biotin functionalization, as the one for microgel dispersions discussed above, was performed by in situ ellipsometry. Results show that more avidin binds to the MAA33-Biotin layer than to that formed by non-functionalized MAA33. The strong avidin-biotin interaction to surface-bound MAA33-Biotin also lowers the otherwise high release of electrostatically bound avidin at high ionic strength found for non-functionalized MAA33 layers. (Figure S4, Supporting Information)

Cross-linking microgel multilayers The formation of cross-linked microgel multilayers was next studied using time-resolved ellipsometry and QCM-D (Figure 4). Microgels layers were formed by alternating MAA33-Biotin microgels (10 ppm) and avidin (64 µg/mL). Figure 4b shows a representative kinetic curve, obtained by ellipsometry, of the formation of four cross-linked microgel layers. (The first covalently attached microgel monolayer was bound and functionalized with biotin prior the start of the experiment.) Quantitatively, avidin binds more extensively to the individual layers than microgels do (Figure 4c). This was also confirmed by QCM-D (Figure 4d-e), showing considerably larger frequency shifts and mass changes when avidin is


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added between the microgel layers. Nevertheless, QCM-D shows large dissipation shifts as microgel binds, demonstrating that the loosely cross-linked microgel particles are able to structurally change the interface to a much more diffuse one on binding, although the mass increases are relatively modest (Figure 4d, lower panel). The dissipation shifts from QCM-D also show an interesting behavior on avidin binding (Figure 4e, lower panel). Here, the introduction of avidin to the first covalently attached microgel monolayer results in a minor increase of the dissipation (1.4 ± 0.8 ·10-6 dissipation units). However, for subsequently added microgel layers a qualitatively different behavior is observed. Thus, avidin first results in an increased dissipation, which is likely due to loosely bound avidin, followed by a re-structuring of the layers, resulting in a pronounced lowering of the overall dissipation (after about 60-90 s). In parallel to this, the mass increases continuously during the binding process, as seen from the frequency drop in the top panel of Figure 4e. Taken together, these results show that avidin binding results in a rigidification of the microgel layers, an effects primarily caused by avidin-biotin crosslinking of the microgel layers, and possibly to a minor extent by the loss of some bound water due to the electrostatic nature of avidin binding.

Peptide incorporation Peptide loading into the cross-linked microgel multilayers was achieved in different ways; either by oneshot post-loading after multilayer formation, or step-wise between each microgel layer. Figure 5 shows ellipsometry and QCM-D results when 50 µM KYE28 peptide was added in Tris buffer to a microgel multilayer pre-formed by alternating avidin and MAA33-Biotin microgels. Both techniques show how the peptide adsorbs to the microgel layer. Shortly after peptide addition, the cationic peptide starts to compete electrostatically with the bound avidin, and possible also with loosely adhered microgels, leading to a mass drop. When comparing mass changes obtained from ellipsometry in Figure 5b with those obtained from frequency shift from QCM-D measurements (which measure the hydrated mass) in Figure 5c, one can notice that the frequency increases above its starting value (i.e., before peptide addition), indicating that the addition of peptide also induce removal of bound water and ions in the microgel layer, in line with microgel de-swelling of MAA microgels by cationic peptides observed


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previously.33, 35 From the magnitude of maximum peptide binding, being comparable to that of a single microgel layer,16 it can furthermore be concluded that the peptide is unable to diffuse through all the discrete layers of avidin and microgels and only binds to the outer regions of the multilayer.

In a second approach, the peptide was introduced step-wise by addition after each of the microgel layers during the buildup of microgel multilayers. For this, KYE28 (25 µM in Tris buffer) was added to the microgel layer before each avidin addition (Figure 6). Ellipsometry and QCM-D results show that crosslinked microgel multilayers were formed in this manner, and that this results in an increased amount of adsorbed peptide and microgel mass for each adsorbed layer (Figure 6b). QCM-D further showed that KYE28 incorporation between each microgel layer results in a reduced dissipation of the microgel film (Figure 6c-d). The latter effect is likely due to the release of bound water and ions as the oppositely charged peptide diffuses into the microgels and cause these to shrink, as shown previously for both microgels in dispersion54 and surface-bound microgel monolayers35. The final binding of KYE28 reaches 16.7 ± 2.0 mg/m2 (or ~ 5 µmol/m2), compared to only 1.1 ± 0.1 mg/m2 in the first MAA33microgel monolayer. Furthermore, the addition of KYE28 after each microgel deposition lowers the problem with electrostatic, unspecific binding of avidin, and results in a stable amount of approximately 1 mg/m2 of bound avidin after each addition (compared with an incrementing avidin binding 4-20 mg/m2 in the absence of guest peptide, Figure 4b). Correspondingly, avidin addition did not result in any change in dissipation (Figure 6e). Taken together, stepwise incorporation of KYE28 during multilayer build-up results in more compact microgel layers, containing a much higher loading of KYE28. SEM images of the peptide-loaded microgel multilayers are shown in Figure 7. As can be seen in the multilayer defect highlighted in the figure, three out of four well-defined distinct microgel layers can be clearly seen (indicated by the white arrows).

Peptide release After investigating the build-up of the MAA33-Biotin/KYE28/avidin multilayer in situ with ellipsometry and QCM-D, peptide release was investigated by rinsing the layer consecutive first in low


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(10 mM), and high (physiological) ionic strength Tris buffer. The results in Figure 8 show that only a small amount of the total mass are released in the two buffers over the time investigated. Only ~10 % of the material of the multilayers are released after continuously rinsing for 1.5 h, equivalent to 2.3 ± 0.8 mg/m2 of the original dry mass. Assuming the mass reduction from the covalently cross-linked multilayer to be due to peptide release only, this would correspond to 25 % and 58 % of the total amount of peptide incorporated being released for Tris and Tris NaCl buffer, respectively. While peptide release thus seems modest over the time-scale of these experiments, it should also be noted that release shown no sign of leveling off after 1.5 hours, meaning that considerably higher peptide release will likely be reached after longer release times, such as those (days to weeks) relevant for avoidance of biomaterialsassociated infections.55

Antibacterial effects Microgel monolayer compared to microgel multilayers Next, antibacterial properties of the peptide-loaded microgel multilayers were investigated and compared to those of single peptide-loaded MAA33-microgel monolayers. For this both antimicrobial effects in bulk, due to released antimicrobial peptide, and antimicrobial effects close to the interface using CLSM were monitored. Both mono- and multilayers were incubated in 25 µM KYE28 in Tris buffer, the monolayer overnight, and the multilayer first for 30 min per layer during buildup and lastly overnight after multilayer completion. The surfaces were then incubated with E. coli for 4 h before the bacterial viability was evaluated measuring bacterial metabolic activity of PrestoBlue by fluorescent intensity. Results show the peptide-loaded microgel multilayer are able to a greater extent lower the bacterial viability of the 108 CFU/mL planktonic bacteria in suspension, reflecting an increased amount of KYE28 released in physiological ionic strength from the loaded microgel multilayers compared to that from the monolayer (Figure 9a).

For visualization purposes, the same set of samples were prepared but the unbound bacteria instead gently rinsed off in Tris buffer, before the remaining adhered bacteria were imaged by confocal


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microscopy combined with LIVE/DEAD staining (Figure 9b,c). By quantifying the red-to-green fluorescence intensity of the CLSM images, it is clear that the peptide-loaded microgel multilayer significantly improve the antimicrobial effect of the surface compared to the untreated glass-slide and peptide-loaded microgel monolayer. As shown in the representative CLSM images of stained E. coli (Figure 9c) the amount of KYE28 peptide bound to the monolayer is not enough to kill all adhered bacteria. However, for the peptide-loaded microgel multilayer, more of the bacteria are stained by the red PI stain, reporting on membrane lysis. Images also show the anti-adhesive properties of the microgel layers compared to an untreated glass slide, as previously reported.16

Discussion As demonstrated in the present investigation, cross-linked microgel multilayers can be formed by taking advantage of the strong and specific interaction between biotin and avidin. Biotin-avidin binding has been widely investigated in literature, and its high affinity (KD =10-15) forms the basis for a range of bioassays.46, 56 Biotin-avidin binding has been used also in the formation of microgel coatings. For example, Kim et al. investigated biotinylated pNIPAm-co-AAC microgels as microlens detectors. As avidin bound to such surface-bound biotinylated microgels, the effective cross-linking obtained was demonstrated to be sufficient for the visual appearance of the microgels to change and be detectable by differential interference contrast (DIC) imaging.57 With regards to the use of microgel (multi)layers as drug delivery systems, avidin-biotin has been less investigated. Although not focusing specifically on avidin-biotin microgel multilayers, previous studies relating to the use of microgel-based (multi)layers include temperature-controlled release of FTIC-insulin24 and doxorubicin23. Furthermore, Wang et al. reported on the release of ibuprofen from LbL dextran-functionalized poly(allylamine hydrochloride) (PAHD) microgels and hyaluronic acid (HA)-coated sutures.58 Another interesting example of a similar approach was later implemented by Wang et al., who coated magnetic nanospheres with LbL-deposited luminescent FITC-PAHD microgels and chondroitin sulfate loaded with ibuprofen.59


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The results found in this study show that well-defined cross-linked microgel multilayers can be formed by a straightforward layer-by-layer deposition approach based on biotin-conjugated MAA microgels and avidin, incorporating KYE28 in a step-wise manner during the multilayer build-up. Although not investigated in the present study, such multilayers are not expected to dependent of substrate shape, thus representing an advantage compared to spin-coat assisted approaches previously discussed in literature.19 Although the present study was based on biotin-avidin interactions, the ease of making these microgel multilayers suggests that also other high-affinity ligand pairs, such as antibodies/antigens, might be able to be use to obtain stimuli-responsive triggered release in the presence of free competing analytes in solution, causing the ‘cross-links’ to break.60

For microgel multilayers obtained by alternating MAA33-Biotin microgels and avidin (Figure 4), i.e., with no peptide added during multilayer build-up, the resulting multilayers are more similar to previously discussed electrostatically bound LbL of microgels, were avidin works as the polycation (having a + 20 charge at pH 7.453) and the microgels as the polyanion (zeff = -32 ± 1 mV at pH 7.4 from Figure 2a). From a drug delivery perspective, the large amount of electrostatically bound avidin in this case has no value in itself, and is undesirable. However, by incorporating the cationic KYE28 peptide between the microgel layers, this unspecific binding of avidin is screened by the peptide, which is most likely able to diffuse into the oppositely charged microgel layer (Figure 6). In a previous study by Nyström et al., it was found that surface-bound MAA microgel monolayers undergo deswelling, flattening, and stiffening, on incorporation of oppositely charge poly-L-lysine peptides of various length.35 This could explain the large decrease in dissipation, observed in the present investigation, of the microgel film after each KYE28 addition. Furthermore, it has been previously demonstrated that the surrounding media, e.g., pH and ionic strength, affects the degree of swelling and deformability of charged surface-bound microgels. On either ion-exchange, reduced charge density, or increased ionic strength, charged microgels de-swell and water is released, effects that may be straightforwardly monitored by following the dissipation of a surface-bound microgel layer with QCM-D.8, 19 Analogous loading of oppositely charged guest molecules to microgel layers has also been reported for various nonpeptide systems. Examples of this include, e.g., cationic microgel multilayers of poly(allylamine


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hydrochloride) PAH-dextran microgels and anionic poly(styrene sulfonate) LbL constructs loaded with negatively charged guest molecules, such as methyl orange, fluorescein sodium, and mercaptoacetic acid stabilized CdTe nanoparticles.61

Stepwise peptide loading after each of the four microgel depositions was found to result in a pronounced increase in loading capacity of KYE28 compared to post-loading of this peptide to the pre-formed microgel multilayer. This indicates that peptide diffusion through the multilayer from the outside is kinetically hindered, resulting from both a net positive microgel charge and microgel de-swelling on incorporation of an oppositely charged peptide. Analogously, it has been found previously that peptide diffusion fronts may be kinetically arrested by the microgel de-swelling, an effect particularly pronounced for microgels of lower charge density, low ionic strength, and large peptide size.29 Results from the present investigations furthermore indicate that the increased peptide loading capacity of these microgel multilayers significantly increases the antimicrobial effects. The microgel multilayers do not, however, keep the non-fouling properties of microgel monolayer, possibly be due to the increased surface roughness of the multilayers, and/or high concentration of the cationic peptide in the outer region of the multilayer. In this context, it is interesting to note previous findings by Wang et al., who investigated physisorbed monolayers of cationic L5 peptide-loaded poly(PEG-co-AAc) microgels against colonization of Staphylococcus epidermis.15 The antifouling properties displayed by this system were attributed to the net spacing between microgels, which was similar to the length of the bacterium. Although the L5 concentration in bulk solution was low, the local concentration at the interface was found to be higher than the minimum inhibitory concentration, hence efficiently suppressing bacterial growth. It was furthermore shown that these loaded microgels can be used for coating polycaprolactone/chitosan nanofibers in order to lower bacterial colonization while simultaneous promote osteoblast cell adhesion and proliferation in the scaffold.17

The present study focuses on factors affecting avidin-biotin-based multilayer formation by MAA microgels, how this affects peptide loading and release, and how this translates into functional performance of such systems as antimicrobial surface coatings. As such, focus has not been placed on


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issues related to the development of these systems towards biomaterials. Hence, studies on antimicrobial effects were kept at a basic level, focusing of demonstrating the principal effect only. Furthermore, no toxicity measurements were reported in the present study. However, it should be noted that MAA microgels have been previously demonstrated to display low toxicity against human cells, as shown by both low hemolysis, MTT results showing low toxicity against THP1 monocytes, as well as low activation of NF-kB in the latter,16, 33. From an antimicrobial perspective, ambient conditions determine the mechanisms underlying the antimicrobial effects. As shown in Figure 9, bacterial killing in bulk mirrors that at the interface in the present investigation, suggesting peptide release to be important for antimicrobial effects at the high (physiological) ionic strength used in the present investigation. In contrast, bacterial killing was previously demonstrated to be higher at the interface than to that of the bulk at low ionic strength conditions.16 Furthermore, it is clear that the increased loading of KYE28 by the stepwise incorporation of KYE28 results in increasingly efficient antimicrobial surface coatings, as judged both by effects on planktonic and adhered bacteria (Figure 9). Having said that, and keeping in mind that the bacteria load used was very high (108 CFU/mL), bacteria eradication is far from complete. A key mechanistic question is to what extent it is possible to reach even more efficiently antimicrobial coatings by further increasing the number of microgel layers in the multilayer assembly. Thus, while the total peptide load is expected to increase with the number of loaded microgel layers, diffusion and release of peptide molecules bound to the inner layers is expected to be suppressed. This is previously discussed in greater detail by Herman and Lyon, who investigated Alexa 488 fluorescently labeled polyL-lysine and poly(pNIPAm-co-AAc) microgel LbL constructs, and found little evidence of polyelectrolyte exchange and redistribution between the microgel layers compared to linear polyelectrolyte films.62 One way to get around the problem with low peptide release could be to combine microgel multilayers with disintegration of the multilayer structure to trigger a release of individual microgel particles, thereby increasing the effective surface area and facilitating peptide release. Hence, such disruptive microgel multilayers could offer opportunities for AMP-loaded surface coatings,63 particularly if the layer disruption can be made responsive to infection, e.g., through the use of antigen/antibody pairs64 or peptide linkers sensitive to bacterial proteases65, 66 are used as linkers.


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Conclusions The formation of cross-linked biotinylated MAA33 microgel multilayers was shown to be possible by alternating additions of microgels and avidin. Incorporation of the antimicrobial peptide KYE28 was achieved by either post-addition to pre-formed microgel multilayers or by successive addition between each layer during the multilayer build-up. The latter approach was found to substantially increase peptide loading. Peptide release was found to be slow, but promoted at high ionic strength. Mirroring the increased peptide loading in the microgel multilayers, the latter resulted in a decreased bacterial viability of E. coli in suspension compared to peptide-loaded microgel monolayers. Correspondingly, KYE28-loaded multilayers displayed considerably larger disruption of adhered bacteria, as observed by confocal microscopy for adhered bacteria. Although cross-linked microgel multilayers thus show some promise as versatile antimicrobial surface coatings due to their high loading capacity of antimicrobial peptides, peptide release needs to be accelerated for many applications, pointing in the direction of selfdisrupting multilayer structures as a way forward for microgel-based surface coatings of AMPs.

Supporting Information Supporting information is available free of charge on the ACS Publication website. Full IR and ATR-FTIR spectra of MAA33 and biotinylated MAA33-microgels in dispersion and when surface-bound, respectively, representative SEM image of a microgel monolayer, and ellipsometry results on avidin binding and release to surface-bound MAA33 and MAA33-Biotin microgel monolayers. Complete ellipsometry and QCM-D kinetic curves of MAA33-Biotin/avidin and MAA33Biotin/KYE28/avidin multilayer formation.

Acknowledgements The work was funded by the Swedish Research council (Grant 2016-05157), and by the LEO Foundation (Grant 2016-11-01).


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Figures

Figure 1. (A) Schematic illustration of EDC- and sulfo-NHS-assisted amide coupling of amine-PEG3Biotin to the carboxyl group of the methacrylic acid residues of the microgels. (B) IR spectra of MAA33 microgels before and after amide coupling of amine-PEG3-Biotin (MAA33-Biotin). (C) Subtraction spectrum of the microgels before and after amide coupling, showing the loss of carbonyl vibrations (1690-1730 cm-1) and the increase of amide vibrations (≈ 1550 cm-1).


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Figure 2. Differences in microgel effective z-potential (A), hydrodynamic diameter (B), and polydispersity index (PDI; C) in the presence of avidin. MAA33 and MAA33-Biotin microgels (100 ppm) in Tris buffer were mixed with increasing concentrations of avidin, the results showing crosslinking of MAA33-Biotin microgels into large clusters at high avidin concentration (n=3).


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Figure 3. (A) Representative ATR-FTIR spectra of the conjugation step involved when covalently binding amine-PEG3-Biotin to the carboxylic acids of surface-bound MAA33 microgels in MES buffer. (The subtracted background in the beginning of the experiment corresponds to surface-bound MAA33 microgels in MES buffer.) (B) Subtraction spectrum of surface-bound MAA33 microgels before and after amide coupling. A shift to lower wavenumbers seen as a reduction in the signal at from ~1730 cm-1 and the appearance of a peak between 1700-1720 cm-1 indicate a change in environment of the carbonyl stretch from carboxylic acid to amide. The peak is larger than the trough due to the addition of amide carbonyls from the biotin reactant (n=2).


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Biomacromolecules

Figure 4. (A) Schematic illustration of microgel multilayer build-up in absence of KYE28. (B) Representative kinetic curve of the formation of four microgel layers by alternating additions of avidin and MAA33-Biotin microgels in Tris buffer measured by null ellipsometry. (C) Quantified binding (adsorbed amount) after each addition (n=2). Multilayer formation was also investigated using QCMD, shown as the differences in frequency and dissipation for each addition (D) MAA33-Biotin (10 ppm) and (E) avidin (64 µg/mL) (n=3). (Complete kinetic curves for ellipsometry and QCM-D are shown in Figure S5, Supporting Information.)


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Figure 5. (A) Peptide addition to pre-formed MAA33-Biotin/avidin multilayers. (B) Representative kinetic curve of KYE28 binding (50 µM in Tris buffer) measured in situ by ellipsometry. (C) Frequency and dissipation shifts measured for the same system by QCM-D.


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Biomacromolecules

Figure 6. (A) Step-wise incorporation of KYE28 during build-up of the microgel multilayers by alternating biotin-functionalized microgels (MAA33-Biotin), KYE28, and avidin. (B) The adsorbed amount after each addition during the buildup quantified by ellipsometry (n=2), and by frequency and dissipation shifts measured with QCM-D for (C) MAA33-Biotin (10 ppm), (D) KYE28 (25 µM), and (E) avidin (64 µg/mL) (n=3) (Complete kinetic graphs for ellipsometry and QCM-D measurements are shown in Figure S6, Supporting Information.)


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Figure 7. Representative SEM images of MAA33-Biotin microgel multilayers loaded with KYE28 between each layer. (A) A multilayer defect is shown where three out of the four microgel layers are visible and (each marked with a white arrow). (B) Lower magnification of the same microgel multilayer, showing uniform microgel deposition over a larger area.


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Biomacromolecules

Figure 8. Representative relative mass release from a MAA33-Biotin/KYE28/avidin multilayer in Tris buffer (30 min) and Tris buffer with additional 150 mM NaCl (60 min) measured with ellipsometry.


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Figure 9. (A) Viability of E. coli bacteria in suspension (108 CFU/mL in Tris buffer, 150 mM NaCl) in the presence of unmodified glass-slides, peptide-loaded MAA33 monolayers (25 µM), or peptide-loaded MAA33-Biotin multilayers (25 µM KYE28 equilibrated between each layer) measured with PrestoBlue. (B) Viability of adhered E. coli quantified using Baclight LIVE/DEAD staining. Data normalized against E. coli killed in 70 % isopropanol (negative control) and compared to E.coli in Tris buffer, 150 mM NaCl (positive control). (n=3) (C) Representative CLSM images of LIVE/DEAD stained adhered E. coli. Images are presented as z-projections of eight images, with increased contrast done after data analysis for improved visualization. Scale bar 10 µm.


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Biomacromolecules

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AvidinâBiotin Cross-Linked Microgel Multilayers as Carriers for

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