Highly-Branched Poly(N-isopropylacrylamide) as a Component in

Aug 15, 2013 - Mixed films were confirmed. The protein adsorption at 24 °C was found to be reduced with increasing amount of pNiPAAm in the mixed ...
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Highly-Branched Poly(N‑isopropylacrylamide) as a Component in Poly(dopamine) Films Yan Zhang,†,‡ Boon M. Teo,‡ Almar Postma,§ Francesca Ercole,∥ Ryosuke Ogaki,‡ Meifang Zhu,*,† and Brigitte Stad̈ ler*,‡ †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai 201620, People’s Republic of China ‡ iNANO Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark § Ian Wark Laboratory, CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, Victoria 3168, Australia ∥ Department of Materials Engineering, Monash University, Clayton, Victoria 3800, Australia S Supporting Information *

ABSTRACT: Mixed one-step poly(dopamine) (PDA)/highly branched poly(Nisopropylacrylamide) (pNiPAAm) coatings have been assembled and characterized by X-ray photoelectron spectroscopy (XPS), UV−vis spectroscopy, atomic force microscopy, and quartz crystal microbalance with dissipation monitoring (QCM-D) depending on the deposition temperature below and above the lower critical solution temperature (LCST) of the pNiPAAm. Mixed films were confirmed. The protein adsorption at 24 °C was found to be reduced with increasing amount of pNiPAAm in the mixed coatings, while there was no difference observed for proteins deposition at 39 °C. Further, the ability of these mixed coatings in comparison to the pure PDA and pNiPAAm films to serve as capping layer for surface-immobilized zwitterionic or positively charged liposomes has been assessed by QCM-D. The adhesion of hepatocytes, macrophages, and myoblast to these liposomes-containing hybrid coatings and the uptake of fluorescent lipids from the surface by the adhering cells depending on the capping layers were compared. The latter aspect was found to be dependent on the used capping layer and the type of liposome as carrier for the fluorescent lipid, with the highest uptake found for positive liposomes and pure pNiPAAm as capping layer. Taken together, the assembled hybrid coatings have the potential to be used as functional coatings toward surfacemediated drug delivery.

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INTRODUCTION

temperature responsiveness or unknown degradation characteristics. Poly(N-isopropylacrylamide) (pNiPAAm) is a thermoresponsive polymer that has received recognition for biomedical applications because its lower critical solution temperature (LCST) is in the physiological relevant range.14 This polymer is a prominent candidate to be used as coating for temperatureresponsive cell culture substrates.15 pNiPAAm can be grafted to the substrates,16,17 or it has been considered as a building block in LbL thin films with the aim to render the coatings (partly) temperature responsive.18−20 Increasing the complexity of the macromolecule from linear or copolymer into (highly) branched forms has been achieved with reversible addition− fragmentation chain transfer (RAFT).21 In the latter case, pure pNiPAAm polymers22−24 or copolymers25−27 have been demonstrated. These (highly) branched pNiPAAm polymers have predominantly been used to assemble nanoparticles toward temperature-triggered drug delivery. However, to the

Polymer films are an integral part in biomedical research, e.g., as protective or active coatings of implantable devices or for tissue engineering substrates. The sequential deposition of interacting polymers (the layer-by-layer (LbL) technique) is among the most potent assembly approaches to deposit (drug eluting) films on virtually any substrate in an easy and reliable, but timeconsuming, manner.1,2 Catechol-based chemistry to assemble films for bioapplications is a versatile alternative.3 Poly(dopamine) (PDA) is a specifically prominent example in this context.4 PDA film assembly employs the “oxidative self-polymerization” of dopamine at slightly basic pH 5 and has attracted considerable interest. PDA coatings have multiple advantages, including their easy and fast preparation on many substrates, the option for postmodification via thiols and amines, or their biocompatibility. Further, the possibility of blending dopamine with other molecules of interest6−9 or of chemically modifying the reactants10−13 prior to assembly in particular improves the potential of PDA films and aims to address shortcomings such as limited inherent properties such as lack of pH and © 2013 American Chemical Society

Received: July 17, 2013 Revised: August 15, 2013 Published: August 15, 2013 10504

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from Sigma-Aldrich. Zwitterionic lipids 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC) and 1,2-dimyristoyl-sn-glycero-3ethylphosphocholine (DMEPC) and fluorescent lipids 1oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids, USA. TRIS buffer consisting of 10 mM TRIS (pH 8.5) was used throughout all the experiments. The buffer solution was made with ultrapure water (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation, USA). Unilamellar liposome stock solutions were prepared by evaporation of the chloroform of 2.5 mg of lipid solution under vacuum for 1 h, followed by hydration into 1 mL of TRIS buffer and extrusion through 100 nm filters (11 times). Zwitterionic liposomes (Lzw) consisted of 2.5 mg of DOPC lipids and positively charged liposomes (L+) consisting of 2 mg of DOPC and 0.5 mg of DMEPC lipids, and for fluorescent liposomes (FLzw/+), 1 wt % NBD-PC was added to the lipid solution. Inimer RAFT Agent Synthesis. n-Butanethiol (5.4 g, 6.4 mL, 0.06 mol), carbon disulfide (9.1 g, 7.3 mL, 0.120 mol), and chloroform (50 mL) were added to a flask purged with argon. Triethylamine (12.5 g, 17.3 mL, 0.124 mol) was then added dropwise with stirring. The solution became orange as the addition proceeded with the formation of the intermediate triethylammonium S-n-butyl trithiocarbonate salt. The solution was left to stir at room temperature for 2.5 h. 1-Chloromethyl4-vinylbenzene (9.16 g, 8.45 mL, 0.06 mol) was then added dropwise, and the solution was left to stir under argon. Volatiles were removed under rotavaporation, poured into deionized water, and extracted with CHCl3. The organic layer was washed 2× with the following: deionized water, 1 M HCl, and brine. This was then dried over anhydrous MgSO4, filtered and the solvent removed to leave behind a yellow oil (16.96 g, yield ∼99%). The crude compound was further purified on silica column chromatography running a gradient from hexane to 5% DCM in hexane to obtain a pure fraction (14 g, yield = 83%, NMR purity of ∼95%). 1H NMR (400 MHz, CDCl3): δ 0.94 (tr, CH3, 3H), 1.43 (m, CH2, 2H), 1.67 (m, CH2, 2H), 3.37 (tr, CH2, 2H), 4.59 (s, CH3, 2H), 5.24 (d, CHCHH, 1H), 5.73 (d, CHCHH, 1H), 6.68 (q, CHCH2, 1H), 7.28−7.36 (q, aromatic CH, 4H). 13C NMR (400 MHz, CDCl3): δ 13.6, 22.1, 30.1, 36.8, 41.1, 114.2, 126.5, 129.5, 134.7, 136.3, 137.1, 223.3. Highly-Branched Poly(N-isopropylacrylamide) Synthesis. NiPAAm (1.012 g, 8.94 × 10−3 mol), inimer RAFT agent (0.0256 g, 9.06 × 10−5 mol), and AIBN (1.5 mg, 9.14 × 10−6 mol) were weighed in an ampule and dissolved in dioxane (2.945 mL) with a ratio of [NiPAAm]/[RAFT]/[AIBN] = 1000:10:1. The mixture was degassed with four freeze−pump− thaw cycles, sealed, and then heated at 60 °C in a thermostated oil bath for 19 h 11 min. The final polymers were purified by precipitation from the monomer/solvent mixture into hexane and dried under vacuum until constant weight to give 0.9792 g. Polymerization results for pNiPAAm were the following: conversion 90%; Mn(SEC) = 33 300 g mol−1, Đ = 1.56 (GPC− DMF, polystyrene calibration); Mn(SEC) = 44 100 g mol−1, Đ = 1.63 (GPC−DMAc, polystyrene calibration); Mn(Calc) = 10 500 g mol−1 (Supporting Information Figures S1 and S2). Turbidity Measurements. Cloud-point analysis was performed to estimate the LCST for the highly branched pNiPAAm using an EnSpire Perkin-Elmer multilabel plate reader equipped with a temperature control. Measurements were obtained using a wavelength of 547 nm, a temperature

best of our knowledge, these polymers have not yet been considered as a building block in polymer thin films. Thin polymer films can be loaded with active cargo by using them as building blocks in the assembly toward substrate mediated drug delivery (SMDD). This approach has proven particularly successful when assembling DNA containing coatings.28 Alternatively, the trapping of drug deposits in the form of cyclodextrins,29 micelles,30 or liposomes31−40 has been considered to facilitate the incorporation of small and/or fragile payload. Liposomes have the potential to encapsulate both hydrophilic and hydrophobic cargo and are therefore particularly promising for this purpose. Their incorporation into polymer thin films has been accomplished either using cholesterol-modified polymers34,37,40 or predominantly via electrostatic interactions.31−33,35,36,39 The interaction of these hybrid films with mammalian cells has been reported, and the cargo uptake by cells from these films with35 or without38−40 the use of an active trigger has been demonstrated. In the latter case, the access of the cells to the embedded payload can be steered by the thickness and type of polymer capping layers. Additionally, liposomes-containing polymer films with antimicrobial properties have been assembled.41 In a related approach, a catecholamine polymer has been used to immobilize adeno-associated virus on the surface in a spatially controlled manner, and successful gene delivery from these substrates has been reported.42 Here, we report on the assembly of mixed PDA films with a highly branched pNiPAAm deposited in one step and on the use of these coatings as capping layers to embed liposomal drug deposits into surface adherent thin films toward SMDD (Scheme 1). Specifically, we (i) confirmed the assembly of Scheme 1. Illustration of the Assembly of Highly-Branched pNiPAAm Containing PDA Coatings, the Protein Adsorption to Them, and Their Use as Capping Layer for Liposome-Containing Films in Substrate-Mediated Drug Delivery Using Adhering Cells

mixed PDA/pNiPAAm films using X-ray photoelectron spectroscopy, (ii) assessed the optical and (iii) topographical properties of these coatings, (iv) monitored the protein adsorption to films assembled with different ratios of DA/ pNiPAAm by quartz crystal microbalance with dissipation monitoring (QCM-D), (v) showed that PDA/pNiPAAm can be used as capping layers to entrap intact liposomes via QCMD, and (vi) evaluated the response of adhering myoblast, hepatocytes, and macrophages to these films in terms of cell adhesion and amount of internalized fluorescent lipids.

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EXPERIMENTAL SECTION Materials. Dopamine hydrochloride (DA), tris(hydroxymethyl)aminomethane (TRIS), sodium chloride (NaCl), poly(L-lysine) (PLL, MW of 40 000−60 000 Da), ethanol, and chloroform (purity of ≥99.5%) were purchased 10505

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range between 27 and 45 °C, and a polymer concentration of 1 mg mL−1 in TRIS 1. Substrate Preparation and Analysis. 1 × 1 cm2 pieces of silica wafer (XPS, AFM) or 1.8 × 1.8 cm2 glass slides (UV/vis) were cleaned by sonication in ethanol for 10 min followed by 10 min in Milli-Q water. The samples were then blow-dried under a stream of nitrogen and put in a UV/ozone cleaner for 15 min. The cleaned samples were then instantly coated with DA (1 mg mL−1 in TRIS buffer), pNiPAAm (1 mg mL−1 in TRIS buffer), or a DA/pNiPAAm mixture (1/1 wt % DA/ pNiPAAm, at a DA concentration of 1 mg mL−1 in TRIS buffer) for 1 h with exchange of the solution after 30 min. Samples with a coating temperature of 37 or 39 °C were prepared by putting the solution and substrate in a plastic tube and on a thermoshaker (Thermomixer comfort, Eppendorf). The coated samples were rinsed with Milli-Q water, dried under a stream of nitrogen, and stored under vacuum for further analysis. 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 Alkα 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. Note that the high resolution spectral data have been normalized to the total counts in order to compare the spectra between the samples. Three different areas per sample were analyzed to test for the homogeneity of the films. Atomic Force Microscopy (AFM). The coatings on the silica wafers were visualized in air using tapping mode AFM (Nanowizard 2, JPK Germany) using NCH cantilever (NanoWorld). The root mean squared (rms) roughness was assessed using at least three 5 × 5 μm2 images in the JPK software. Contact Angle Measurements (CA). The water CA of the different coatings was measured at room temperature (∼24 °C) (DSA100, Krüss) using the “tangent method 2” in the Drop Shape Analysis software. Absorbance 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 multilayers with or without embedded liposomes. Silica-coated crystals (QSX300, Q-Sense) were cleaned by immersion in a 2 wt % sodium dodecyl sulfate solution overnight and by rinsing with Milli-Q water. Afterward, the crystals were blow-dried with N2, treated with UV/ozone for 20 min, and mounted into the liquid exchange chambers of the instrument. The frequency and dissipation measurements were monitored at 24 ± 0.02 °C or 39 ± 0.02 °C. Normalized frequencies using the third harmonic are presented. Polymer Coatings. When a stable baseline in TRIS buffer solution was achieved, either pNiPAAm (1 mg mL−1 in TRIS buffer) or DA/pNiPAAm polymer mixture (1 mg mL−1 DA and pNiPAAm in different wt % in TRIS buffer) was introduced into the measurement chamber and left to adsorb onto the crystal until the surface was saturated. Then the

chamber was rinsed with TRIS buffer and the coatings were exposed to a protein solution (Dulbecco’s modified Eagle medium plus 10% fetal bovine serum) at 24 and 39 °C. Liposome-Containing Coatings. When a stable baseline in TRIS buffer solution was achieved, PLL (1 mg mL−1 in TRIS buffer) was introduced into the chamber and left to adsorb until the surface was saturated. Then the chamber was rinsed with TRIS buffer and Lzw/+ solution (no diluted stock solution) was added. Upon saturation of the surface, the chamber was rinsed with TRIS buffer and the polymer capping layers were deposited. As capping layers DA or pNiPAAm polymer (both 1 mg mL−1 in TRIS buffer) or DA/pNiPAAm polymer mixture (1 mg mL−1 DA and pNiPAAm in different wt % in TRIS buffer) was deposited at 39 °C. Alternatively, cholesterolmodified poly(methacrylic acid) (1 mg mL−1 in TRIS buffer, synthesized and dissolved as previously described38) was adsorbed prior to the pNiPAAm based capping layers. Cell Experiments. The C2C12 mouse myoblast cell line, the RAW264.7 mouse leukemic monocyte macrophage cell line, and the Hep G2 human hepatocellular carcinoma cell line (all from American Type Culture Collection) were used for the experiments. The myoblasts (175 000 cells/flask in 20 mL of medium) and the macrophages (1 100 000 cells/flask in 20 mL of medium) were cultured in 75 cm2 culture flasks in medium (Dulbecco’s modified Eagle medium (DMEM, 4500 mg L−1 glucose) supplemented with 10% fetal bovine serum (FBS), 50 μg mL−1 penicillin, 50 μg mL−1 streptomycin, and 1 mM sodium pyruvate (myoblasts only), all from Sigma) at 37 °C and 5% CO2. Hepatocytes (1 500 000 cells/flask in 20 mL of medium) were cultured in 75 cm2 culture flasks in minimum essential medium Eagle (MEME) supplemented with 10% FBS, 50 U mL−1 penicillin, 50 μg mL−1 streptomycin, 1% nonessential amino acids, and 2 mM L-glutamine (all from Sigma) at 37 °C and 5% CO2. Sample Preparation and Cell Analysis. The 9 mm diameter glass slides were cleaned via sonication in ethanol for 10 min, rinsed with ultrapure water, dried under nitrogen flow, and exposed to UV/ozone for 10 min. PLL (1 mg mL−1, 10 min) was adsorbed as a precursor layer and rinsed in buffer solution. The PLL coated glass slides were then exposed to the FLzw/+ solution (stock solution diluted 1/3 in TRIS buffer, 40 min), followed by rinsing in TRIS buffer solution and deposition of the capping layers: pNiPAAm (1 mg mL−1 in TRIS buffer, 30 min), PDA (1 mg mL−1 in TRIS buffer, 60 min), or DA/ pNiPAAm mixture (1/2 wt %, 1 mg DA in TRIS buffer, 60 min) at 39 °C. The coated substrates were UV-sterilized for 30 min submerged in sterile PBS buffer. The cells were seeded onto the substrates at a density of 100 000 cells/well in 1.5 mL of medium in 24-well plates and allowed to attach for 4 h at 37 °C and 5% CO2. The life cells were imaged on a 1X81 motorized inverted Olympus microscope. For fluorescent lipid uptake experiments, the cells were washed 2× with 3 mL of PBS. Then 300 μL of trypsin was used to detach the cells from the surface and further diluted in 600 μL of PBS for the analysis by flow cytometry using a C6 flow cytometer (Accuri Cytometers Inc.) and an excitation wavelength of λ = 488 nm. At least 3000 cells were analyzed. The autofluorescence of cells grown on PDA-coated glass slides has been subtracted, and the control cells have been gated out in all the presented results. All cell experiments were performed in at least three independent repeats. The statistical significance (P value) used to compare the distribution of samples was 10506

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Scheme 2. Inimer-Type RAFT Copolymerization of Butyl(4-vinylbenzyl)carbonotrithioate with N-Isopropylacrylamide

determined using a two-way ANOVA with a confidence level of 95% (α = 0.05), followed by a Tukey’s multiple comparison posthoc test (∗ = P < 0.05).

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RESULTS AND DISCUSSION

Synthesis and Characterization of Highly Branched pNiPAAm. Highly branched pNiPAAm was synthesized by the RAFT technique as shown in Scheme 2. The inimer (initiator monomer) RAFT agent that was used in this study was specifically chosen to contain the 4-vinylbenzyl reinitiating “R” group. This was based on the successful synthesis of hyperbranched pNiPAAm, reported by Carter and Rimmer,23,24,26 using a RAFT inimer with the same R group. For ease of synthesis, our RAFT inimer contained a butyl trithiocarbonate “Z” group. As seen in Supporting Information Figure S1, the characteristic peaks of pNiPAAm are evident in 1H NMR spectra, along with minor vinyl peaks, as a result of a small quantity of residual monomer. These were found to completely disappear after further purification. The refractive index size exclusion chromatography (SEC) trace of the highly branched pNiPAAm (Supporting Information Figure S2) comprises a low molecular weight fraction (Mp = 22 kDa) and high molecular weight multimodal peaks, representing linear and higher order branched polymer chains, respectively. This is indicative of a highly branched or hyperbranched polymer, and on the basis of the inimer RAFT copolymerization approach, we expect a dendritic or arborescent structure.43−46 The LCST of pNiPAAm was assessed via turbidity measurements (Supporting Information Figure S3). The transition of the highly branched polymer happened between 30 and 40 °C. Two temperatures, below and just above the transition, were chosen for the film assembly. These two temperatures were selected because we are aiming to use these coatings for biomedical applications. Assembly and Characterization of pNiPAAm Containing PDA Films. The assembly of mixed PDA-based films is an interesting approach to deposit functional coatings, e.g., thermoresponsive films by using pNiPAAm. In contrast to our previous report about embedded low molecular weight pNiPAAm,8 the highly branched pNiPAAm used here is expected to show increased response to temperature due to the larger volume change below and above the LCST. Further, our prior report only considered the film assembly below the LCST of the used pNiPAAm. First, we aimed to confirm that mixed pNiPAAm/PDA films can be deposited and how their composition depended on the adsorption temperature. Figure 1 represents high resolution C 1s spectra of PDA or PDA mixed with pNiPAAm films assembled on silica surface at either 24 or 37 °C. These

Figure 1. C 1s peak of different coatings as monitored by XPS: (a) deposition of PDA/24(37) onto SiO2 and (b) deposition of PDA/ pNiPAAm/24(37) onto SiO2. The highlighted regions in C 1s are (i) C−C/C−H region at BE = 285.0 eV, (ii) C−N/C−O region at BE ≈ 286.0−286.5 eV, and (iii) CO or N−CO region at BE ≈ 287.8 eV. The weight ratio of DA/pNiPAAm is 1/1. The high resolution spectral data have been normalized to the total counts in order to compare the spectra between the samples.

coatings are labeled PDA/24(37) and PDA/pNiPAAm/24(37) from now on. By comparing the C 1s spectra of PDA and PDA/pNiPAAm mixtures, the expected increase in the carbonyl (CO)/amide (C(O)−N) peak at ∼287.8 eV for PDA/pNiPAAm/24(37) compared to PDA/24(37) suggested the deposition of a PDA/pNiPAAm mixed film at both deposition temperature. This was further supported by the increase in normalized intensity for the aliphatic carbon (C−C, C−H) at 285 eV when comparing these coatings. Also, the bare and the mixed coatings exhibited more adsorbed polymer for 24 °C compared to 37 °C as deposition temperature. We note that there is an unexpected high ether component for PDA/24 10507

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Table 1. Atomic Composition of Different Coatings Assembled on Silica Wafers as Determined by XPSa elemental % C 24 °C 37 °C

PDA PDA/pNiPAAm PDA PDA/pNiPAAm

61.34 61.10 43.92 44.23

± ± ± ±

O 0.13 0.23 1.80 2.86

22.43 18.30 27.77 25.85

O/C 24 °C 37 °C a

PDA PDA/pNiPAAm PDA PDA/pNiPAAm

0.36 0.30 0.63 0.59

± ± ± ±

0.01 0.00 0.04 0.08

N/C 0.14 0.15 0.73 0.16

± ± ± ±

± ± ± ±

N 0.21 0.20 0.66 1.72 elemental ratio N/O

0.01 0.00 0.01 0.00

0.39 0.49 0.21 0.28

± ± ± ±

0.01 0.00 0.03 0.04

8.86 9.00 5.77 7.10

± ± ± ±

Si 7.36 ± 0.16 11.60 ± 0.19 22.54 ± 1.78 22.81 ± 1.67

0.07 0.14 0.64 0.50 N/Si

1.16 0.78 0.26 0.31

± ± ± ±

0.08 0.02 0.05 0.04

C/Si 8.30 5.27 1.96 1.95

± ± ± ±

0.13 0.08 0.23 0.26

DA/pNiPAAm = 1/1 wt %

pNiPAAm/24(39) films were compared to PDA/pNiPAAm/ 24(39) films (Figure 3). The small standard deviations in the

observed, not seen in typical C 1s spectra previously published by us6,39,47 and others.5,48,49 The results from the C 1s high resolution scans were further supported by the change in elemental composition by comparing PDA/24(37) to PDA/pNiPAAm/24(37) films (Table 1). The expected decrease in O/C ratio and increase in N/C and N/O ratio were observed for PDA vs PDA/ pNiPAAm coatings. The changes in the O/C and N/O ratios were more evident when the films were deposited at 24 °C compared to 37 °C. The elemental ratio N/C for PDA was close to the theoretical value of 0.125. Also, when the N/Si and C/Si ratios were compared, the higher amount of deposited polymer was confirmed for the lower adsorption temperature. With the aim to assess the optical properties of the coatings, the transparency of the coating was assessed by UV−vis spectroscopy (Figure 2). The absorbance of PDA/24(39)

Figure 3. Representative tapping mode AFM images (5 × 5 μm2) of PDA, PDA/pNiPAAm, and pNiPAAm deposited at 24 °C (top) or 39 °C (bottom). The (rms) roughness (standard deviation) is indicated at the bottom of the images. DA/pNiPAAm = 1/1 wt %.

roughness values hinted toward homogeneous films. For bare PDA and the mixed films the roughness decreased by ∼50% when comparing 24 °C vs 39 °C as the deposition temperature. This trend is in agreement with our previous report involving mixed films of PDA and low MW pNiPAAm.8 In contrast to those films, the mixed films reported here had an increased roughness compared to the bare PDA coatings. Further, pNiPAAm/24 films had low roughness, likely due to low amounts of adsorbed polymer. On the other hand, when pNiPAAm/39 was deposited, the topography became rougher and homogenously grainy, likely because of the adsorption of the collapsed pNiPAAm. The water CA of pNiPAAm/24(37) and PDA/pNiPAAm/ 24(37) was assessed at room temperature (Supporting Information Table S1). Bare pNiPAAm coatings exhibit a CA of ∼14° and ∼49° for a deposition temperature of 24 and 37 °C, respectively. In agreement with the XPS and AFM results, the former CA suggested very low amounts of deposited polymer. On the other hand, PDA/24(37) and PDA/ pNiPAAm/24(37) films have similar CA, indicating that the wettability of the films was not affected by the differences seen in XPS.

Figure 2. Representative UV/vis spectra of glass slides coated with PDA/24(39) or PDA/pNiPAAm/24(39). DA/pNiPAAm = 1/1 wt %.

coating was compared to that of PDA/pNiPAAm/24(39) films. The absorbance was found to be similar for PDA/24 and PDA/ pNiPAAm/24. As previously observed, the PDA/39 was found to be optically denser compared to PDA/24.8 Further, while there was a negligible difference in optical density between PDA/24 and PDA/pNiPAAm/24, when comparing the coatings assembled at 39 °C, the absorbance for PDA/39 was higher than for the mixtures, implying that the optical density was reduced by the presence of the pNiPAAm in the film. These findings suggest the deposition of mixed PDA/ pNiPAAm films. We visualized the different coatings using AFM and assessed their (rms) roughness from these images. PDA/24(39) and 10508

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mixed films led to lower protein adsorption at both tested temperatures of 24 and 39 °C. Therefore, the amount of proteins adsorbing onto mixed films consisting of PDA and the highly branched, higher molecular weight pNiPAAm was assessed at 24 and 39 °C in a next step (Figure 4b). Only polymer films assembled at 39 °C were tested, since they had higher polymer adsorption than films deposited at 24 °C. Unlike in our previous report where we used a linear, low molecular weight pNiPAAm, the amount of adsorbed proteins was unaffected by the amount of pNiPAAm in the film at 39 °C (above the LCST of pNiPAAm), shown by the similar changes in Δf and ΔD. On the other hand, when the protein adsorption was tested at 24 °C (below the LCST of pNiPAAm), the amount of deposited proteins decreased with increasing amount of pNiPAAm in the coating. This could be explained by the fact that pNiPAAm was in its hydrated state at this temperature and therefore possessed protein repelling properties. Surprisingly, pristine pNiPAAm films exhibited similar high protein adsorption properties at 24 and 39 °C. This hints toward a possible beneficial synergy between PDA and this type of pNiPAAm when deposited simultaneously. Capping Layer in SMDD. Surface-adhering hybrid films for SMDD rely on the entrapment of drug deposits, liposomes in our case, and the access of the adhering cells to the cargo. The access depends on the type and number of polymer capping layers. Here, we aim to compare PDA, PDA/pNiPAAm, and pNiPAAm in their ability to serve as capping layers for surfaceimmobilized liposomes. Importantly, good capping layers should trap the underlying liposomes without rupturing or displacing them. To this end, we performed QCM-D experiments by coating silica crystals first with PLL or PLL/ PMA, followed by the deposition of zwitterionic or positively charged liposomes (Lzw or L+, respectively) and capping with the three different polymer layers. Since a deposition temperature of 39 °C was found to allow a higher amount of polymer to be adsorbed onto silica, this temperature was also used for these experiments. Previously, we have demonstrated that cholesterol-modified poly(methacrylic acid) (PMAc) is a beneficial capping layer,34,38 since cholesterol51 can serve as linker between the lipids and the polymers. However, in this case it turned out to be disadvantageous (Supporting Information Figure S5). Although PMAc was deposited, the deposition of the capping layer pNiPAAm was low, and the layer was removed upon rinsing with buffer solution. Therefore, DA, DA/pNiPAAm, or pNiPAAm was directly deposited onto the PLL/Lzw (Figure 5a) or PLL/PMA/L+ (Figure 5b) precoated crystals at 39 °C. The expected changes for both Δf and ΔD (Supporting Information Figure S6) of PLL or PLL/PMA coated crystals were observed when exposed to a solution of Lzw or L+, respectively. First, all three capping layers were suitable for entrapping the liposomes, and higher amounts of capping layer were deposited onto PLL/PMA/L+. However, there was far less polymer adsorbed compared to bare silica (Figure 4a), demonstrated by the overall lower Δf and ΔD. Unexpectedly, pNiPAAm showed the lowest adsorption to PLL/Lzw precoated crystals, while the highest deposition was observed onto silica crystals. Although the amount of the adsorbed pNiPAAm capping layer to PLL/PMA/L+ precoated crystals was ∼20× higher, it was still ∼4× lower than onto silica crystals. PDA was deposited in a similar amount onto PLL/Lzw films than onto silica crystals,8 while PLL/PMA/L+ precoated crystals allowed ∼4× higher PDA deposition. The mixture DA/ pNiPAAm was adsorbed in similar amounts to PLL/Lzw and

QCM-D experiments were performed to monitor the amount of pNiPAAm or PDA/pNiPAAm deposited onto silica crystals depending on the adsorption temperature (Figure 4a).

Figure 4. (a) Frequency changes (Δf) of crystals upon exposure to pNiPAAm or DA/pNiPAAm (1/2 or 1/1 wt %) at 24 and 39 °C. (b) Frequency changes (Δf) of polymer precoated crystals upon protein adsorption at 24 and 39 °C.

The change in frequency (Δf) of crystals exposed to PDA/24 and PDA/39 has previously been assessed and was found to be Δf ≈ −20 Hz in both cases.8 The pNiPAAm/24 adsorption was very low, while pNiPAAm/39 exhibited a large Δf probably due to the physisorption of the collapsed polymer. PDA/ pNiPAAm/24(39) showed larger Δf than PDA/24(39), indicating the deposition of a mixed layer as previously observed by XPS. Δf (approximately −62 Hz) of PDA/ pNiPAAm/24 coated crystals was found to be independent of the tested wt % between DA and pNiPAAm. However, for PDA/pNiPAAm/39 decreasing the amount of pNiPAAm led to lower Δf of the crystals, while the change in dissipation (ΔD) was similar (Supporting Information Figure S4). This suggested similarly hydrated films but with lower mass content. (PDA film with higher pNiPAAm content could not be measured by QCM-D because of the sensitivity limit of the instrument.) Protein Adsorption. An important aspect to consider for polymer coatings with potential use in biomedicine is their protein adsorption characteristics. In particular, the temperature responsive pNiPAAm exhibits different ability to repel proteins depending on the temperature and the deposition protocols.50 We previously reported that the mixing of low molecular weight pNiPAAm-NH2 into PDA films affected the protein adsorption.8 In that case, increasing amounts of pNiPAAm in the 10509

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of hepatocytes (Figure 6a), macrophages (Figure 6b), and myoblasts (Figure 6c). None of the cell types were sensitive to the employed capping layer, and similar numbers of cells with the expected cell morphology were adhering to the different coatings. Finally, we assessed the ability of adhering hepatocytes, macrophages, and myoblasts to associated fluorescent lipids embedded in the liposomes in PLL/Lzw/PDA (1), PLL/Lzw/ (PDA/pNiPAAm) (2), and PLL/Lzw/pNiPAAm (3) films (Figure 7). Further, it was compared if there is a difference

Figure 5. Frequency changes (Δf) of crystals upon coating with PLL(/ PMA), Lzw (a) or L+ (b) and capped with PDA, PDA/pNiPAAm (DA/pNiPAAm = 1/2 wt %), or pNiPAAm. The deposition temperature was 39 °C for all the coatings.

Figure 7. Cell mean fluorescence of hepatocytes, macrophages, and myoblasts adhering to PLL/Lzw/PDA/39 (1), PLL/Lzw/(PDA/ pNiPAAm)/39 (2), or PLL/Lzw/pNiPAAm/39 (3), as well as the myoblast mean fluorescence when adhering to PLL/PMA/L+/PDA/ 39 (1*), PLL/PMA/L+/(PDA/pNiPAAm)/39 (2*), or PLL/PMA/ L+/pNiPAAm/39 (3*). The polymer deposition temperature was 39 °C for all the coatings, and all the cell adhesion times were 4 h (n = 3; ∗p < 0.05).

PLL/PMA/L+ films but ∼6× lower than on bare silica. These observations demonstrate the expected importance of the underlying substrate when depositing the capping layers. However, it also shows that the chosen substrate provides a handle to control/guide the DA/polymer assembly. In a next step, we aimed to compare the number of adhering cells after 24 h to PLL/(PMA/)Lzw/+/PDA/39, PLL/(PMA/ )Lzw/+/(PDA/pNiPAAm)/39, and PLL/(PMA/)Lzw/+/pNiPAAm/39. Figure 6 shows representative microscopy images

in myoblast response to these coatings when L+ (1*, 2*, and 3*) instead of Lzw was used as carrier for the fluorescent lipids. The cell mean fluorescence (CMF) of the cells adhering to these different coatings for 4 h was monitored by flow cytometry. A period of 4 h was chosen, since we have previously found that the lipid internalization by adhering cells was higher in the short term than after extended adhesion periods.38,39,52 When using PDA (1) as capping layer, there was no significant difference in CMF between the adhering cells. Although our QCM-D experiments showed a 1.5× higher Lzw than L+ adsorption and a 3× higher PDA deposition onto L+ (Figure 5), the difference in the coating did not lead to a different cell response, showing that the films behave equally in their interaction with the adhering cells in the tested period of time. PDA/pNiPAAm capping layers led to similar CMF for all the tested cells, hinting toward the fact that this polymer layer presents a similar barrier for all of the used cell types. Further, there was no significant difference in CMF when comparing the capping layers PDA vs PDA/pNiPAAm for all the cells. This was surprising, since the QCM-D results showed that more PDA/pNiPAAm was deposited because of the larger measured Δf (Figure 5) and similar ΔD (Supporting Information Figure S6) for films with Lzw and L+, and therefore, PDA/pNiPAAm was expected to provide a better barrier toward the access of the cells to the fluorescent lipids. This hints toward a difference in permeability of the two types of capping layers.

Figure 6. Representative microscopy images of hepatocytes (a), macrophages (b), and myoblasts (c) adhering to PLL/(PMA/)Lzw/+/ PDA/39 (top row), PLL/(PMA/)Lzw/+/(PDA/pNiPAAm)/39 (middle row), and PLL/(PMA/)Lzw/+/pNiPAAm/39 (bottom row) coatings for 24 h. The film deposition temperature was 39 °C for all the coatings. The scale bar is 100 μm. 10510

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from the Special Excellent Ph.D. International Visit Program by Donghua University, China. We thank Assistant Prof. Alexander Zelikin and Anton A. A. Smith (Department of Chemistry, Aarhus University) for their help assessing the MW of the pNiPAAm.

When considering pNiPAAm as capping layers, there was no significant difference in CMF for cells adhering to Lzwcontaining films. On the other hand, (PLL/PMA/L+/ pNiPAAm) films led to a significantly higher CMF for myoblasts adhering to this coating compared to all cell types adhering to PLL/Lzw/pNiPAAm (or any other film, significance not indicated in Figure 7). From the QCM-D results, there was less L+ deposited than Lzw and the highest amount of pNiPAAm capping layer was observed for deposition onto L+-containing films. These findings indicate the expected relevance of the capping layer not only in terms of amount but also in terms of type of deposited polymer.

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CONCLUSIONS We report on the assembly of mixed PDA/pNiPAAm films deposited in one step, the protein adsorption to them, and their use as capping layer for liposome-containing films. XPS experiments confirmed the deposition of mixed coatings. Visualizing the films with AFM revealed that the PDA and PDA/pNiPAAm coating looked similar independent of the deposition temperature, while pNiPAAm was grainier when adsorbed at 39 °C. The optical density was higher for PDA/39 and PDA/pNiPAAm/39 compared to PDA/24 and PDA/ pNiPAAm/24 measured by UV−vis, and more polymer was deposited in the former case as assessed by QCM-D. For mixed films deposited at 39 °C, reduced protein adsorption was observed with increasing amount of pNiPAAm in the mixture at 24 °C, while 39 °C did not show any difference in protein adsorption. PDA/39, PDA/pNiPAAm/39, and pNiPAAm/39 were found to be successful capping layers for Lzw- and L+containing films. Similar numbers of hepatocytes, macrophages, and myoblasts were adhering to these liposome-containing films. The internalization of fluorescent lipids from the surface by adhering cells was found to be dependent on the capping layer, the type of cells, and the type of carrier liposome, with PLL/PMA/L+/pNiPAAm and myoblast showing the overall highest CMF after 4 h of adhesion time. Taken together, our findings further promote the liposomecontaining PDA-based hybrid films as functional coatings toward controlled SMDD.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra, mole mass distribution trace, turbidity measurements, dissipation changes (ΔD) of the QCM-D experiments, and QCM-D sample curve comparing PLL/Lzw/ PMAc/pNiPAAm vs PLL/Lzw/ pNiPAAm. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*M.Z.: e-mail, [email protected]. *B.S.: e-mail, [email protected]. 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, and the National Natural Science Foundation for Distinguished Young Scholar of China (Y.Z., Grant 50925312). Y.Z. was supported by a scholarship 10511

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