Assembly of Poly(dopamine) Films Mixed with a Nonionic Polymer

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Letter pubs.acs.org/Langmuir

Assembly of Poly(dopamine) Films Mixed with a Nonionic Polymer Yan Zhang,†,‡,∥ Bo Thingholm,‡,∥ Kenneth N. Goldie,§ Ryosuke Ogaki,‡ and Brigitte Stad̈ ler*,‡ †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai, People’s Republic of China ‡ iNANO Interdisciplinary Nanoscience Centre, Aarhus University, Aarhus, Denmark § Center for Cellular Imaging & Nano Analytics, Biozentrum, University of Basel, Basel, Switzerland S Supporting Information *

ABSTRACT: Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications. Here, we investigate the film deposition of dopamine mixed with a nonionic polymer (i.e., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(N-vinyl pyrrolidone) (PVP)) onto silica substrates using X-ray photoelectron spectroscopy and quartz crystal microbalance. Furthermore, we assess the possibility of coating silica colloids to yield polymer capsules and liposomes with these mixtures. We found that mixed PDA/PEG and PDA/PVA films are deposited without the need for a covalent linker such as an amine or thiol. We also discovered the first material, namely, PVP, that can suppress PDA film assembly. These fundamental findings give further insight into PDA film properties and contribute to establish PDA as a widely applicable coating.

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INTRODUCTION Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications in different fields, including biomedicine. PDA films self-polymerize from a dopamine (DA) solution at slightly basic pH, a concept first introduced in 2007 by Lee et al.,1 but also oxidant-induced PDA deposition has been recently reported.2,3 Apart from numerous applications that benefited from this PDA coating reaching from biosensing to drug delivery,4,5 a renewed interest in catechols and their derived compounds has been sparked, as recently reviewed by Ye et al.6 Carbon nanotubes7,8 or liposomes9 have been coated with PDA to allow for secondary reactions or to develop advanced drug-delivery vehicles, respectively. Furthermore, PDA capsules10 have been assembled, and initial results of their use in vitro as drug-delivery vehicles11 or for encapsulated catalysis12 have been reported. PDA has also been considered for biosensing applications including the use of PDA as a matrix in the molecular imprinting of proteins,13−15 as a coating for electrodes,16 as a novel label,17 or to encapsulate entire cells.18 To date, PDA coatings probably had the largest impact in modifying surfaces for controlled interaction with mammalian cells and in some cases with bacteria.19,20 For mammalian cells, PDA can improve the interface between nonwetting materials and cells or tissues.21,22 PDA coatings can be further modified with biomolecules (e.g., hyaluronic acid,1 vascular endothelial growth factors,23 or bone morphogenetic protein 224) to control cell adhesion or with poly(ethylene glycol) (PEG)1,25,26 to prevent cell adhesion. We recently employed PDA in surface-mediated drug delivery using liposomes and polymersomes as drug deposits in PDA films27 or in PDA coated surface-adherent poly(vinyl alcohol) hydrogels,28 respectively. However, despite all of these promising © 2012 American Chemical Society

approaches, there is a fundamental lack of understanding about how PDA is formed and the details of its structure. The influence of pH or the initial DA concentration on the film formation and properties have been reported.29 Initially, a covalent model suggesting a structure where oxidized and cyclized dopamine monomers were covalently joined via aryl− aryl linkages has been proposed.1,12,30,31 Alternatively, the PDA structure has been suggested to consist of covalently bound monomers that aggregate into larger supramolecular complexes via noncovalent interactions (e.g., π−π stacking32). In the last few months, three different reports suggesting alternatives have emerged. The absence of any covalent binding has been proposed, and that PDA solely consists of monomers held together by strong noncovalent forces including charge transfer, π−π stacking, and hydrogen bonding.33 Also, a model has been proposed where it was suggested that PDA is formed through two different pathways occurring in parallel: (i) the noncovalent self-assembly of stable complexes of (DA)2/5,6dihydroxyindole (DHI) and (ii) covalent polymerization.34 Furthermore, the coexistence of structurally different components (uncyclized catecholamine/quinones, cyclized DHI units, and pyrrolecarboxylic acid moieties) within PDA that can be affected by the DA concentration has been demonstrated.35 In this letter, we aim to assess if it is feasible to incorporate a nonionic polymer within a PDA coating solely via noncovalent interactions. Therefore, we report the film assembly of a mixture of DA with nonionic polymers with different hydrogen acceptor/donor properties (i.e., poly(ethylene glycol) (PEG), a weak hydrogen acceptor, poly(N-vinyl pyrrolidone) (PVP), a Received: October 14, 2012 Revised: November 19, 2012 Published: December 3, 2012 17585

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X-ray Photoelectron Spectroscopy (XPS). XPS data acquisition was performed using a Kratos Axis UltraDLD instrument (Kratos Analytical Ltd., Telford, U.K.) equipped with a monochromated Al Kα X-ray source (hν = 1486.6 eV) operating at 15 kV and 10 mA (150 W). Survey spectra (binding energy (BE) range of 0−1400 eV with a pass energy of 160 eV) were used for element identification and quantification. High-resolution C 1s, O 1s, and N 1s spectra were acquired with a pass energy of 20 eV. The acquired data were converted to VAMAS format and analyzed using CasaXPS (Casa Software Ltd., U.K.) software. Absorption Measurements. The glass slides were mounted in a UV/vis spectrometer (UV-3600, Shimadzu), and the absorbance was monitored from 350 to 1200 nm. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). QCM-D measurements (Q-Sense E4, Sweden) were used to analyze the assembly of the polymer films. Silica-coated crystals (QSX300, Q-Sense) were cleaned by immersion in a 2 wt % sodium dodecyl sulfate solution overnight and rinsing with Milli-Q water. Afterward, the crystals were blow-dried with nitrogen and treated with UV/ozone for 15 min before being mounted into the liquid-exchange chambers of the instrument. The frequency and dissipation measurements were monitored at 22 ± 0.02 °C. When a stable baseline in the TRIS buffer solution was achieved, DA (1 mg mL−1), nonionic polymer (5.3 mg mL−1 for PEG and PVP, 9.5 mg mL−1 for PVA), or a DA/nonionic polymer mixture (1 mg mL−1 DA, 5.3 mg mL−1 PEG and PVP, 9.5 mg mL−1 PVA; 10/1 DA/nonionic polymer mol/mol) was introduced into the measurement chamber and left to adsorb onto the crystal using a flow rate of 50 μL min−1 to minimize issues with the depletion of the monomers, mixing, or oxygen levels. After the surface was saturated, the chamber was rinsed with TRIS buffer solution to remove the excess polymer. The resulting polymer-coated surface was then exposed to a protein solution (DMEM plus 10% fetal bovine serum) or LZW (stock solution) until the surface was saturated. Then, the chamber was rinsed with TRIS buffer. Normalized frequencies using the third overtone are presented. The differences were statistically analyzed using an unpaired two-tailed t test. Capsule Assembly and Imaging. A suspension of 5 μm SiO2 particles (5 wt %) in TRIS buffer was incubated with PEG/DA (2 mg mL−1 DA, 10.6 mg mL−1 PEG) or PVA/DA (2 mg mL−1 DA, 19 mg mL−1 PVA) for 24 h under constant shaking and then washed three times (1060g, 30 s). Capsules were formed by dissolving the silica core particles using a 2 M HF/8 M NH4F solution for 2 min, followed by multiple centrifugation (4500g, 3 min)/MQ water washing cycles. The obtained capsules were visualized using a 1 × 81 motorized inverted Olympus microscope. Samples were prepared for electron microscopy by adsorbing 4 μL of the capsule suspension onto a carbon film mounted on 300 mesh copper grids (Quantifoil Micro Tools GmbH, Jena, Germany). Prior to adsorption, the grid surface was rendered hydrophilic by glowdischarge in a reduced atmosphere of air for 10 s. The specimen was applied as a droplet to the grid and left for 5 min of incubation on the surface to aid adsorption. The grid was blotted and left to air dry. No staining agents were applied. Transmission electron microscopy images of the capsules were acquired digitally using a Philips CM10 microscope (FEI, Eindhoven, The Netherlands) fitted with a 2k × 2k side-mounted CCD Camera (Olympus SIS, Münster, Germany) Liposome Coating. Zwitterionic liposomes were coated with PDA/nonionic polymer (PVP, PVA, and PEG) mixtures in different molar ratios. A dopamine stock solution (4 mg mL−1) in TRIS buffer was prepared and diluted to 2 mg mL−1 using TRIS buffer or a polymer solution. Typically, 200 μL of the liposome stock solution was mixed with 200 μL of the dopamine/nonionic polymer solution, and the final dopamine concentration was 1 mg mL−1. Also, as controls, liposomes were coated with PDA (1 mg mL−1) or mixed with nonionic polymer solution in the same concentration (9.5 mg mL−1 for PVA and 5.3 mg mL−1 for PEG and PVP). The samples were continuously agitated during the coating process. The size and polydispersity (PD) of the samples were determined at different time points by diluting 30−50 μL of the sample solution in 450 μL of TRIS prior to measuring in a dynamic light scattering (DLS) instrument

strong hydrogen acceptor, and poly(vinyl alcohol) (PVA), which can be both a hydrogen acceptor and donor depending on the counterpart). Specifically, we (i) characterize the film assembly of DA, PEG, PVA, and PVP as well as that of DA mixed with PEG, PVA, and PVP on silica using X-ray photoelectron spectroscopy (XPS) and quartz crystal microbalance with dissipation monitoring (QCM-D), (ii) compare the amount of adsorbed proteins and liposomes to these six films using QCM-D, and (iii) deposit DA or DA mixed with PEG, PVA, or PVP onto silica colloids and zwitterionic liposomes (Scheme 1). Scheme 1. Schematic Illustration of Deposited Coatings onto Silica or Liposomes Using Three Different Nonionic Polymers (PEG, PVP, and PVA) and DA

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EXPERIMENTAL SECTION

Materials. Poly(vinyl alcohol) (PVA, MW 13 000−23 000 Da), poly(ethylene glycol) (PEG, MW 10 000 Da), poly(N-vinyl pyrrolidone) (PVP, MW 10 000 Da), dopamine hydrochloride (DA), tris(hydroxymethyl)aminomethane (TRIS), sodium chloride (NaCl), ethanol, hydrofluoric acid (HF), and chloroform (purity ≥99.5%) were purchased from Sigma-Aldrich. Zwitterionic 1hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (POPC, phase-transition temperature −2 °C) were purchased from Avanti Polar Lipids, USA. TRIS buffer consisting of 10 mM TRIS (pH 8.5) was used for all of the experiments. The buffer solutions were made with ultrapure water (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation, USA). Unilamellar liposome stock solutions (LZW) were prepared by the evaporation of the chloroform of the lipid solution (2.5 mg of POPC) under vacuum for 1 h, followed by hydration into 1 mL of TRIS buffer and extrusion through 100 nm filters (11 times). Coating of Planar Substrates. Pieces of silica wafer (1 × 1 cm2) and glass slides (18 × 18 mm2) were cleaned by sonication in ethanol for 10 min, followed by 10 min in MQ water for XPS and UV/vis absorption measurements, respectively. The samples were then blowdried under a stream of nitrogen and put into a UV/ozone cleaner for 15 min. The cleaned samples were then instantly coated with DA (1 mg mL−1 in TRIS buffer), nonionic polymer (6.5 mg mL−1 for PEG and PVP, 11.8 mg mL−1 for PVA), or a DA/polymer mixture (1 mg mL−1 DA, 6.5 mg mL−1 PEG and PVP, 11.8 mg mL−1 PVA) for 1 h with exchanging the solution after 30 min. The coated samples were rinsed under the Milli-Q water tap for several seconds, dried in a stream of nitrogen, and stored under vacuum for analysis. 17586

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Figure 1. High-resolution XPS C 1s spectra of (a) bare silica and silica exposed to PEG, PVP, and PVA, (b) silica coated with PDA or PDA/PEG, (c) silica coated with PDA or PDA/PVA, and (d) silica coated with PDA or PDA/PVP. The highlighted regions in C 1s are (i) the C−C/C−H region at BE = 285.0 eV, (ii) the C−N/C−O region at BE ≈ 286.5 eV, (iii) the C(O)−O region at BE ≈ 289.2 eV, and (iv) the N−CO region at BE ≈ 287.8 eV.

Table 1. Elemental Composition of Different Coatings Assembled on Silica Wafers as Determined by XPS elemental % SiO2 PDA PEG PDA/PEG PVA PDA/PVA PVP PDA/PVP

C 4.09 35.87 9.61 41.26 8.26 35.08 20.21 23.85

± ± ± ± ± ± ± ±

O 0.78 10.60 3.51 15.62 3.8 6.84 1.20 2.59

42.68 30.34 36.65 27.81 36.54 31.52 32.71 31.02

± ± ± ± ± ± ± ±

N 1.06 3.38 1.33 3.50 1.30 0.35 2.01 0.78

0.00 3.99 0.00 3.92 0.00 2.05 2.61 3.29

± ± ± ± ± ± ± ±

Si 0.00 1.62 0.00 2.02 0.00 0.46 0.30 0.4

53.24 29.80 53.75 27.02 55.21 31.36 44.47 41.84

± ± ± ± ± ± ± ±

O/C 1.82 8.84 4.79 14.12 5.12 7.00 2.57 3.54

(Zetasizer Nano, Malvern Instruments) using a material refractive index of 1.590 and a dispersant (water at 25 °C) refractive index of 1.330. Within this article, samples with PD > 0.4 were considered to be aggregated and were discarded. Absorbance Measurements. Solutions consisting of 100 μL (4 mg mL−1) of DA and 100 μL of PVP in different concentrations with or without 200 μL of Lzw were allowed to react for 100 min or 24 h. In the latter case, the 200 μL Lzw was replaced with 200 μL of TRIS buffer. The absorbance of 100 μL of these solutions diluted with 600 μL of TRIS buffer was measured using a spectrophotometer (λ = 298 nm) (Nanodrop 2000c).

10.72 0.94 5.53 1.15 7.60 1.08 1.59 1.41

± ± ± ± ± ± ± ±

1.82 0.37 0.05 0.03 1.43 0.02 0.08 0.08

N/C 10.72 0.11 0.00 0.08 0.00 0.06 0.15 0.14

± ± ± ± ± ± ± ±

1.77 0.01 0.00 0.01 0.00 0.01 0.01 0.01

N/O 0.00 0.14 0.00 0.07 0.00 0.05 0.09 0.10

± ± ± ± ± ± ± ±

0.00 0.07 0.00 0.01 0.00 0.01 0.00 0.01

N/Si 0.00 0.16 0.00 0.05 0.00 0.04 0.06 0.07

± ± ± ± ± ± ± ±

0.00 0.10 0.00 0.01 0.00 0.01 0.00 0.01

C/Si 0.08 1.40 0.11 0.68 0.08 0.79 0.42 0.48

± ± ± ± ± ± ± ±

0.02 0.80 0.00 0.03 0.01 0.01 0.02 0.02

PVA only or DA mixed with polymers on the silica surface. Because PVP, PEG, or PVA contain the same type of elements as PDA (i.e., C, N, and O), it is difficult to distinguish them from PDA and quantitatively monitor the level of nonionic polymers present at the surface. Therefore, we discuss only qualitatively the changes in specific chemical species from the high-resolution scans as well as the changes in the elemental ratios in the wide scans. First, there was only minor binding for PEG and PVA to the silica surface observed, characterized by the slight increase in the ether (C−O) component at a BE of ∼286.5 eV (Figure 1aii), and the elemental percentage remained relatively unchanged. However, PVP was adsorbed onto silica more extensively, as indicated by the rise in the amide peak at ∼278.8 eV (Figure 1aiv) and further confirmed by the larger increase and decrease in C 1s and Si 2p, respectively (Table 1).

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RESULTS AND DISCUSSION With the aim of understanding if it is possible to assemble PDA films containing a nonionic polymer without using thiols or amines, we chose three polymers with different hydrogen acceptor/donor properties: PEG, PVA and PVP. Figure 1 represents high-resolution C 1s spectra of PDA, PVP, PEG, and 17587

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PDA/PEG films, suggesting a more transparent coating in the former case and that the film properties depend on the type of incorporated nonionic polymer. With the aim of further investigating the assembly of PDA films mixed with a nonionic polymer (i.e., PEG, PVP, or PVA) and to assess the amount of protein and liposome adsorption to these films, QCM-D was used. Figure 2a shows the frequency change (Δf) of QCM silica crystals upon exposure to different polymers and polymer mixtures. PEG and PVA were adsorbing to only a minor amount whereas more PVP was deposited, both in good agreement with the XPS results. DA was also

By comparing the C 1s spectra of PDA and PDA/PEG, the expected increase in the ether (C−O) component at ∼286.5 eV for PDA/PEG (Figure 1bii) and the low level of PEG adsorbed onto silica suggest that the deposition of a PDA/PEG mixed film is likely. This was also the case for PDA compared to PDA/PVA (Figure 1c). The peak at ∼289.2 eV (Figure 1ciii) for PDA/PVA is attributed to the carboxylate (C(O)−O) species originating from the acetate impurities in the PVA powder.36 The results from the C 1s high-resolution scans are further supported by the change in elemental composition by comparing PDA to PDA/nonionic polymer. The expected increase in the O/C ratio was observed for PDA versus PDA/ PEG and PDA/PVA films. Additionally, the elemental percentage of Si 2p was found to be lower for PDA/PEG and PDA/PVA than for PEG or PVA only, indicating that the mixtures have been deposited on the surface. Taken together, the XPS results support the conclusion that mixed PDA/PEG and PDA/PVA coatings were deposited on silica surfaces. These findings are quite unexpected because neither PEG nor PVA has any chemical group (i.e., amines or thiols known to interact with PDA films). Although the trapping of poly(ethylene imine)-graf t-PEG by mixing it into the DA solution prior to the coating deposition has been reported,25 to the best of our knowledge there is no prior work demonstrating the assembly of mixed PDA films solely via noncovalent interactions or physical entrapment. Interestingly, the C 1s high-resolution scans of PVP to PDA/ PVP are identical to one another, with the characteristic amide peak at ∼287.8 eV (Figure 1div) indicating that only PVP but not PDA was deposited onto the silica surfaces in the latter case (Supporting Information Figure S1). This is further supported by the same elemental composition obtained for PVP and PDA/PVP (within the experimental error of ±10%37). In theory, the O/C ratio for PDA is higher than that for PVP (O/ C ratios of 0.25 and 0.17, respectively), thus upon mixing of PDA with PVP, if PDA is incorporated and present on the surface of the film, an increase in the O/C ratio would be expected. However, our results showed a slight decrease in the O/C ratio, suggesting that PDA is absent from the film. (See Supporting Information Table S1 for the list of theoretical elemental ratios.) To the best of our knowledge, this is the first material reported that was found to hinder PDA formation. We attribute this effect to the strong hydrogen acceptor nature of PVP that is likely to interfere with PDA formation, which is considered to occur via hydrogen bonding, among others. We hypothesize that PVP may bind to the catechol groups of DHI, hindering the subsequent PDA formation. These findings support two different models proposed for PDA formation: the notion that the chemical structure of PDA is a supramolecular aggregate of monomers held together by a combination of charge transfer, π−π stacking, and hydrogen bonding interactions33 and the presence of physical self-assembled complexes of (DA) 2/DHI.34 The self-assembly of the complexes in the latter case might be hindered when PVP is interacting with DHI. In the next step, we aimed to substantiate the XPS findings by assessing the optical differences among PDA, PDA/PEG, and PDA/PVA coatings using UV/vis absorption measurements (Supporting Information Figure S2). The PDA/PVA and PDA/PEG films exhibited a lower absorbance than the PDA coating, showing that the film was more transparent when the nonionic polymers were present. Furthermore, the absorbance measured for PDA/PVA films was lower than for

Figure 2. (a) Frequency change in QCM crystals upon the deposition of polymers or polymer mixtures. Frequency change Δf of coated QCM crystals upon exposure to (b) a protein solution and (c) a solution of zwitterionic liposomes (* represents p < 0.05, n = 3). 17588

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deposited to a greater extent when DA was mixed with one of the nonionic polymers. When comparing the three mixtures, the largest Δf was observed for crystals exposed to PEG/DA. There was only a nonsignificant increase in Δf measured for crystals exposed to PVP/DA and PVA/DA as compared to those exposed to PVP and PVA, respectively. In the next step, we exposed coated crystals to a protein and liposome solution. Both of these compounds are important in biorelated applications and will provide a good measure of to what extent the nonionic polymers is affecting the PDA-based coatings (i.e., if it is possible to add low-fouling properties to these films). Figure 2b shows Δf of the (polymer-coated) crystals upon exposure to a protein solution. The highest protein was observed to bare silica and crystals exposed to PVA. This shows that the amounts of adsorbed PEG and PVP were sufficient to reduce the protein deposition, although that was not the case for PVA. Furthermore, it is not surprising that proteins were adsorbed to PDA coatings because of the ability of the quinones in the film to interact with amines and thiols of the proteins. However, when comparing PDA films to the mixed films, the protein adsorption was significantly reduced when PVA was present in the PDA films, but not for PEG. Although the highest Δf for crystals upon deposition of PEG/ PDA was observed, the protein adsorption remains high, suggesting that the coating largely consisted of PDA with only very little PEG trapped or that the PEG was probably predominantly located inside the film and therefore was not affecting the interface in its ability to repel proteins. We concluded from the XPS results that no PDA film was formed in the presence of PVP on silica but that the significant decrease in Δf was observed for the protein adsorption of crystals pre-exposed to PVP/PDA as compared to PVP. Although we currently cannot explain the details, it suggests that the DA in solution was affecting the PVP coating. Figure 2c depicts the adsorption of zwitterionic liposomes to these films. Bare silica surfaces were neglected in this context because these liposomes are known to rupture and form a supported lipid bilayer on this type of surface,38 which is not relevant in the context of this report. We compared the liposome binding to crystals coated with PDA, and the polymer mixtures showed significantly lower liposome adsorption to the mixed films, suggesting that the amount of nonionic polymer in the film was sufficient to affect the liposome adsorption in all cases. The dissipation changes ΔD indicated that intact liposomes were deposited in all cases (Supporting Information Figure S3). In an attempt to understand to what extent the incorporated nonionic polymer is affecting the structural integrity of the PDA film, we assembled PDA/PVA and PDA/PEG films on 5 μm silica particles, followed by the removal of the templates to yield polymer capsules. The assembly of PDA capsules has previously been reported,10 and to yield capsules, the polymer membrane needs to be sufficiently structurally stable (Figure 3a). It was possible to obtain intact (slightly aggregated) PDA/PEG capsules (Figure 3b). However, the PDA/PVA coating of the silica colloids disintegrated upon core removal; therefore, no capsules were obtained. This result indicates that either the amount of PEG incorporated into the film was not affecting the assembly of the polymer membrane or that the interaction between PEG and DA was stronger than that between PVA and DA. This result is further substantiated by the optical properties of the films as assessed by UV/vis, suggesting that less PDA was present in the coating with PVA than with PEG (Supporting

Figure 3. Representative (i) bright-field and (ii) TEM images of capsules assembled using (a) DA or (b) DA/PEG. The scale bars are (i) 20 μm and (ii) 1 μm.

Information Figure S2). TEM images of the capsules revealed that there is a difference in morphology in the polymer membrane (Figure 3aii,bii). While PDA capsules have a scaly/ grainy appearance, the PDA/PEG capsules demonstrate a smoother membrane structure. This supports the previous findings that a mixed film has been assembled in the latter case and also points toward the possibility of controlling the properties of the coating using a blend of DA and another polymer during assembly. We previously reported the coating of liposomes with PDA for drug-delivery applications.9 In this context, we assessed the possibility to coat zwitterionic liposomes with PDA in the presence of PEG, PVA, and PVP (Figure 4). We aimed to identify if the presence of the nonionic polymer is affecting the PDA deposition in terms of the growth rate and tendency to aggregate. Furthermore, we wanted to understand if PVP only prevents PDA formation on silica or if the same is also observed on a different substrate (i.e., the hindering of the PDA coating of liposomes). Figure 4a,b shows the changes in the diameter of liposomes as measured by DLS for mixtures with PEG and PVA, respectively. Neither PEG nor PVA were affecting the liposomes as shown by the absence of change in the measured diameter. Mixtures of DA with small amounts of PEG or PVA allowed for the coating of the liposomes without affecting the PD of the liposomes. However, increasing amounts of PEG and PVA in the mixture with DA led to the aggregation of the samples. Control samples using just PEG (or PVA) and DA were neglected because we previously showed that DA solutions in the absence of liposomes were prone to form large aggregates.9 Although we currently cannot provide proof of the presence of PVA or PEG in the films assembled on the liposomes, we have strong indications that they are incorporated because we have previously shown that these liposomes could be coated with PDA,9 and from our XPS and QCM-D results, we can determine the deposition of mixed coatings. Figure 4c shows the change in liposome diameter when exposed to DA and PVP. First, PVP itself was not 17589

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Figure 5. (a) Absorbance measurements of DA solutions (1 mg mL−1) mixed with different concentrations of PVP with and without Lzw present after 100 min and 24 h of incubation. (b) Representative photograph of tubes containing solutions with 1 mg mL−1 DA only (left) or with 1 mg mL−1 DA and 78 mg mL−1 PVP (right) after 24 h of incubation.

longer incubation times as expected. In the presence and absence of Lzw, the absorbance of the solution decreased with increasing PVP concentration for both incubation times. Figure 5b shows representative photographs of a tube containing solutions with 1 mg mL−1 DA only (left) or with 1 mg mL−1 DA and 78 mg mL−1 PVP (right) after 24 h of incubation. In the presence of PVP, the darkening of the solution can no longer be visually observed. The suppression of PDA formation in solution further supports the fact that PVP is interfering with PDA assembly.

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CONCLUSIONS Herein, we demonstrated that mixed films of PDA/PEG and PDA/PVA could be deposited by simply mixing DA with the nonionic polymer. Surprisingly, there was no requirement for thiol or amine moieties on the polymer to be trapped in the PDA coating, but noncovalent interactions were sufficient for deposition. Furthermore, we report PVP as the first material that suppressed PDA formation, probably because of the strong hydrogen acceptor characteristics of PVP. These findings support the hypothesis that PDA is formed via noncovalent interactions. Taken together, the possibility of depositing PDA coatings with a polymer blended in without covalent coupling opens up an entirely new avenue of opportunities for assembling functional coatings. Because we have identified PVP as a material that prevents PDA formation, this could in turn be useful for patterning purposes and will further broaden the use and application of PDA.

Figure 4. Change in the diameter of zwitterionic liposome coated with (a) DA and PEG, (b) DA and PVA, and (c) DA and PVP as assessed by DLS. * indicates the aggregation of the sample.

affecting the liposomes. However, the presence of PVP in the DA solution hindered the film deposition on the liposomes. By keeping the DA concentration at 1 mg mL−1, we systematically reduced the amount of PVP until film deposition around the liposomes was observed again. Very small amounts of PVP (0.1 mg mL−1) affected the coating of the liposomes. These results confirmed that PVP hindered PDA formation. With the aim of investigating to what extent PVP is preventing PDA formation, absorbance measurements of solutions with or without Lzw and 1 mg mL−1 DA and different amounts of PVP were performed (Figure 5a) after 100 min and 24 h of incubation time. First, the absorbance increased with 17590

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ASSOCIATED CONTENT

S Supporting Information *

High resolution XPS C 1s spectra of a silica substrate exposed to PVP and PDA/PVP. Theoretical elemental ratios for the different polymers. Representative UV/Vis spectra of glass slides coated with PDA, PDA/PEG, or PDA/PVA. Dissipation change of precoated QCM crystals upon deposition of zwitterionic liposomes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45 8715 6668. E-mail: [email protected]. Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Sapere Aude Starting Grant from the Danish Council for Independent Research, Technology and Production Sciences, Denmark.

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REFERENCES

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