Temperature-Controlled Three-Stage Switching of Wetting

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Temperature-Controlled Three-Stage Switching of Wetting, Morphology, and Protein Adsorption Yurij Stetsyshyn,*,† Joanna Raczkowska,*,‡ Ostap Lishchynskyi,† Andrzej Bernasik,§,⊥ Andrij Kostruba,∥ Khrystyna Harhay,† Halyna Ohar,† Mateusz M. Marzec,⊥ and Andrzej Budkowski‡ †

Lviv Polytechnic National University, S. Bandery 12, 79013 Lviv, Ukraine Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland § Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Al. Mickiewicza 30, 30-049 Kraków, Poland ∥ Lviv Academy of Commerce, Samtshuk 9, 79011 Lviv, Ukraine ⊥ Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Al. Mickiewicza 30, 30-059 Kraków, Poland ‡

S Supporting Information *

ABSTRACT: The novel polymeric coatings of oligoperoxidegraf t-poly(4-vinylpyridine-co-oligo(ethylene glycol)ethyl ether methacrylate246) [oligoperoxide-graf t-P(4VP-coOEGMA246)] attached to glass were successfully fabricated. The composition, thickness, morphology, and wettability of resulting coatings were analyzed using X-ray photoelectron spectroscopy, ellipsometry, atomic force microscopy, and contact angle measurements, respectively. In addition, adsorption of the bovine serum albumin was examined with fluorescence microscopy. The thermal response of wettability and morphology of the coatings followed by that of protein adsorption revealed two distinct transitions at 10 and 23 °C. For the first time, three stage switching was observed not only for surface wetting but also for morphology and protein adsorption. Moreover, the influence of the pH on thermo-sensitivity of modified surfaces was shown. KEYWORDS: stimuli-responsive polymer coatings, POEGMA, poly(4-vinylpyridine), copolymer, LCST, wettability, protein adsorption

1. INTRODUCTION Temperature-responsive polymer brushes consist of surface grafted polymer chains which are able to change their properties sharply upon relatively small temperature changes. This effect may be driven by different mechanisms, including the transition from the glassy to the rubbery state1,2 or from the nematic to the isotropic state3,4 or the coil−globule transition in aqueous solutions in the case of the low critical solution temperature (LCST).5,6 In the latter case, the properties of coatings may be attributed mainly to the hydrogen bonds between the polymer and water6 at T < LCST. These bonds are disrupted at T > LCST, when polymer−polymer interactions (van der Waals interactions) are thermodynamically favored in comparison to polymer−water interactions. This induces the polymer transition from the hydrated to hydrophobic state.6 Temperature-responsive grafted polymer brushes including poly(N-isopropylacrylamide),7−9 poly(oligo(ethylene glycol)methacrylates),10−13 and others14 are well-known. The temperature-responsive grafted copolymer brushes, such as poly(Nisopropylacrylamide-co-N-butyl methacrylate),15 poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide),16 poly(N-isopropylacrylamide-co-N,N-dimethylaminopropylacrylamide-co-N-butyl methacrylate),17 poly(N-isopropylacryla© XXXX American Chemical Society

mide-co-N,N-dimethylaminopropylacrylamide-co-N-tert-butylacrylamide),18 various oligo(ethylene glycol)methacrylates,19 and poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide), modified with cell adhesive peptide20,21 have been synthesized and very successfully used for the separation of molecules or as thermoresponsive surfaces for cell cultures. The composition of the copolymer brushes has a huge influence on their temperature-responsive properties tuning the low critical solution temperature22 or blocking temperature-responsive properties.23,24 Almost all previously reported works demonstrated two-stage temperature-controlled switching where, in general, only two phases can be displayed with a short transition between them. A dual thermally responsive wettability transition was described only for grafted brushes of poly(N-isopropylacrylamide-co-Nisopropylmethylacrylamide).25 Similarly, three-stage pH-controlled switching of surface wetting was shown by Zhou and Huck26 using phosphate-bearing polymer brushes. Received: January 4, 2017 Accepted: March 14, 2017 Published: March 14, 2017 A

DOI: 10.1021/acsami.7b00136 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Fabrication of P4VP (4), P(4VP-co-OEGMA246) (5), and POEGMA246 (6) Brushes Using Polymerization from Peroxide Groups of Oligoperoxides (3) Covalently Immobilized on Amino-Functionalized (2) Glass Surfaces (1)

In our previous paper,27 we demonstrated for the first time that even nearly water insoluble polymers that are able to create hydrogen bonds, such as poly(4-vinylpyridine), exhibit LCST in coatings. The temperature-responsive properties of the wellknown pH-responsive poly(4-vinylpyridine) coatings were reported. Motivated by the strong progress in responsive polymeric materials,1−6,27−30 we decided to synthesize grafted copolymer brushes with two different polymer motifs of (4vinylpyridine) and (oligo(ethylene glycol)ethyl ether methacrylate246), characterized by different LCST values for homopolymer, equal to 14 and 26 °C, respectively. As a result, the novel polymeric coatings of oligoperoxide-graf t-poly(4vinylpyridine-co-oligo(ethylene glycol)ethyl ether methacrylate246) [oligoperoxide-graf t-P(4VP-co-OEGMA246)] attached to glass with temperature-controlled three-stage switching are presented here. The composition, thickness, morphology, and wettability of the resulting coatings were analyzed using X-ray photoelectron spectroscopy (XPS), ellipsometry, atomic force microscopy (AFM), and contact angle measurements, respectively. Moreover, molar composition of the P(4VP-co-OEGMA246) coatings were calculated. Here, we demonstrated the thermal-response of the oligoperoxide-graf t-P(4VP-co-OEGMA) coatings with two temperature-induced transitions (at 10 and 23 °C) for the first time. Moreover, the influence of pH on the thermosensitivity of modified surfaces was shown. Changes of surface roughness and protein adsorption, induced by these transitions, were observed with AFM and fluorescence microscopy, respectively. Surprisingly, for low pH values, only the transition typical for POEGMA246 is blocked, whereas the transition typical for P4VP remains well expressed. In addition, the complex temperature dependence of bovine serum albumin adsorption is observed for P(4VP-co-OEGMA246) coatings.

Fluorescence intensity, corresponding to the amount of adsorbed proteins, grows from an extremely low level at 10 °C to high values at 32 °C, through the intermediate values recorded at 15 and 20 °C.

2. EXPERIMENTAL SECTION 2.1. Materials. Pyridine and other organic solvents were purified as reported by Weissberger and co-workers.31 Polyethylene glycol (PEG9) was supplied by Merck Chem. Co. 4-Vinylpyridine (4VP), 3aminopropyltriethoxysilane (APTES), and oligo(ethylene glycol)ethyl ether methacrylate (OEGMA246) were supplied by Sigma-Aldrich. Bovine serum albumin, BSA, labeled with Alexa Fluor 555 was purchased from Thermo Fisher Scientific. Tert-butyl hydroperoxide was synthesized as described in ref 32. Oligoperoxide with residual acid chloride groups was synthesized according to ref 33. The chemical structure of the oligoperoxide is presented in Scheme S1. 2.2. Preparation of Grafted Brushes and Modification of Glass Surfaces with Oligoperoxide. The modification procedure of the peroxide glass surface by grafted polymer brushes is sketched in detail in Scheme 1. Glass plates 20 × 20 mm, marked as (1) in Scheme 1, were dipped into a 0.2% (w/w) methanolic solution of APTES for 24 h. After the incubation, loosely attached silane molecules were removed with methanol in Soxhlet’s apparatus. Then, the plates functionalized with APTES (2) were dipped into a 1% solution of oligoperoxide in arid dioxane for a certain period (grafting time of 24 h). Similarly, loosely attached oligoperoxide was removed with dioxin during 4 h in a Soxhlet’s apparatus. As a result, oligoperoxides grafted to aminated surfaces, marked in Scheme 1 as (3), were obtained.33 2.3. Polymerization of Grafted Polymer Brushes. Glass plates with grafted oligoperoxides (3) were placed in a container with a 0.1 M aqueous solution of the monomers 4VP or OEGMA246 or with their mixture containing a different ratio of the 4VP and OEGMA246 fragments (see Table 1) and heated under an argon atmosphere at 90 °C for a 48 h, resulting in oligoperoxide-graf t-polymer brush coatings marked in Scheme 1 as (4), (5), and (6). Then, the plates coated with B


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oligoperoxide layer and the oligoperoxide-graf t-polymer brushes, respectively. The molecular masses of the grafted polymer brushes were calculated taking into account the ellipsometry data and the kinetic parameters of the polymerization using eq 1:

Table 1. Calculated Composition of the P(4VP-coOEGMA246) Grafted Copolymer Brushes mole fractions of the segments in P(4VPco-OEGMA246) copolymer brushes

M=

sample

monomer molar ratios in reaction mixture, 4VP/OEGMA246

4VP

OEGMA246

P4VP P(4VP-co-OEGMA246)1 P(4VP-co-OEGMA246)2 P(4VP-co-OEGMA246)3 P(4VP-co-OEGMA246)4 POEGMA246

0.1/0 0.09/0.01 0.08/0.02 0.05/0.05 0.03/0.097 0/0.1

1 0.91 0.83 0.63 0.11 0

0 0.09 0.17 0.37 0.89 1

Cp Co(1 − e−kτ ) × n

(1)

where M is the molecular weight (g/mol), Cp is the concentration of the grafted polymer brushes on the surface (from ellipsometry date) (g/m2), C0 is the initial concentration of peroxide groups of the oligoperoxide on the surface (mol/m2) (C0 = 2.38 × 10−6 mol/m2), k is the first order constant of the initiation at 90 °C (k = 8.1 × 10−6 s−1), τ is the polymerization time (s), and n is the polymerization effectiveness on the surface (n = 0.1).27 The grafting density of grafted polymer chains on the peroxide glass surface was calculated using eq 2:

polymerized brushes were washed with ethanol and water in Soxhlet’s apparatus for 4 h and dried. 2.4. Characterization of Coatings and Water Contact Angle Measurements (CA). Static contact angle experiments were performed by the sessile drop technique using a KrussEasyDrop (DSA15) instrument with the Peltier temperature-controlled chamber. The measurements were carried out at temperatures ranging from 6 to 38 °C to determine the thermal response of the coatings. The temperature was measured by thermocouple in contact with the sample surface. To determine wettability corresponding to a given pH, the sample was immersed for 60 min in a solution with the required pH, then dried carefully, and finally placed in the temperaturecontrolled chamber of the EasyDrop instrument. Prior to the buffer exposure leading to measurements at different pH values, the sample was immersed in deionized water for 60 min and dried under a stream of nitrogen. Contact angles were expressed as the average of ten measurements at different spots. 2.5. AFM. The commercially available Agilent 5500 system (Keysight) equipped with a temperature controlled sample plate was used to examine the surface topography for temperatures equal to 10, 20, and 30 °C and pH ranging from 3 to 9. The measurements in air were performed in the noncontact mode with noncoated super sharp silicon probes. The measurements in water were carried out by AFM working in the MAC mode, using type VI MAC Levers with the nominal parameters, provided by the supplier (Keysight Technologies Inc., USA), equal to 100 μm length, 18 μm width, 0.6 μm thickness, 0.2 N/m force constant, 66 kHz resonant frequency in air, 22 kHz resonant frequency in water, and 10 nm tip radius. Representative AFM images, recorded in three randomly chosen areas on the sample, were characterized by their root-mean-square (RMS) value, determined using the PicoImage software provided along with the AFM instrument. 2.6. XPS Analysis. X-ray photoelectron spectroscopy measurements were performed using a PHI 5000 VersaProbeII (ULVAC-PHI) spectrometer equipped with a monochromatic Al Kα radiation source (1486.6 eV). A dual-beam charge neutralizer was used to compensate the charge-up effect. Spectra were recorded with an analyzer pass energy of 29.35 eV and charge corrected to the main line of the C 1s spectrum set to 284.8 eV. 2.7. Ellipsometry. Measurements were carried out with a serial null-ellipsometer LEF-3M, equipped with a “polarizer−compensator− specimen−analyzer” arrangement, enabling angular positions of polarization elements to be determined within a 0.01° precision. A He−Ne single-mode laser (light wavelength λ = 632.8 nm) was used as a light source. Polarization parameters of light reflected from a sample (angles Ψ and Δ) were determined using the two-zone technique (in the third and fourth measuring zone) with the angle of incidence ϕ varied between 55° and 58° (with a 0.25° step). This ϕrange, corresponding to the region of the pseudo-Brewster angle (where Δ ≈ π/2 or 3π/2), ensures maximal sensitivity. The iterative procedure using single- and two-layer models was used to fit the (Ψ, Δ) data recorded at optimal experimental conditions33−36 and yield an average thickness d and refractive index n for the ultrathin

σ=

hρNA M

(2)

where σ is the grafting density (chains/nm ), h is the dry layer thickness measured by ellipsometry (nm), ρ is the polymer bulk density, NA is Avogadro’s constant, and M (g/mol) is the molecular mass of the polymer brushes grafted onto the surface. 2.8. Protein Adsorption. Bovine serum albumin (BSA) labeled with Alexa Fluor 555, absorbing green light (λabs = 555 nm) and emitting red fluorescence (λemit = 562 nm), was taken as a model protein to examine adsorption to the oligoperoxide-graft-P4VP, the oligoperoxide-graft-POEGMA246, and the oligoperoxide-graf t-(4VPco-POEGMA246) coatings at different temperatures. A BSA solution, with a constant concentration equal to 125 μg/mL, was prepared using a phosphate buffer saline (PBS, pH = 7.4). To examine protein adsorption, a 50 μL drop of protein solution was placed on the substrates and kept at 10, 15, 20, and 32 °C for an incubation time of 15 min. Then, all samples were rinsed with the buffer to remove nonadsorbed proteins and dried under a nitrogen stream. 2.9. Optical Fluorescence Microscopy. Protein adsorption to the oligoperoxide-graf t-polymer brush coatings was examined using the Olympus BX51 optical microscope, equipped with a 100 W halogen lamphouse, a U-MWIG2 (λexit = 520−550 nm, λemit > 565 nm) filter, and a DP72 camera type. All images were recorded for dried samples using the Cell^F software with the 10× objective (Universal Plan Fluorite, magnification of 100). 2

3. RESULTS AND DISCUSSION In the present work, three types of grafted brush coatings (P4VP, POEGMA246, and P(4VP-co-OEGMA246)) were fabricated and characterized. In each case, the coatings attached to glass were prepared in a three-step process (details are outlined in Scheme 1). The compositions of the temperaturesensitive grafted brush coatings, fabricated with a different ratio of the monomers, are described in Section 3.1. The temperature-sensitive wettability and morphology of the grafted brush coatings are shown in Section 3.2. The impact of pH on the thermal response of wettability and surface morphology in P(4VP-co-OEGMA246) brushes is discussed in Section 3.3. In turn, adsorption of the bovine serum albumin to the P4VP, P(4VP-co-OEGMA246), and POEGMA246 coatings is presented in Section 3.4. 3.1. Composition and Wettability of P4VP, P(4VP-coOEGMA246), and POEGMA246 Coatings (XPS, Ellipsometry, and Calculated Parameters). The copolymerization process of the 2-vinylpyridine (2VP) with oligo(ethylene glycol) methyl ether methacrylates with molecular weights of 300 g/mol (OEGMA300) was described in ref 37. The calculated reactivity ratios were rVP = 1.08 and rOEGMA300 = 0.23 C


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consists of three peaks, corresponding to the C−C, C−O (286.37 eV), and O−C = O (288.37 eV) bonds. In the case of XPS spectra recorded for copolymer coatings, their shapes depend strongly on the brush composition. For a highly asymmetric reaction mixture, resulting in a P(4VP-coOEGMA246)4 coating, with excessive POEGMA246 segments (Figure 1, orange line), the observed spectrum resembles the one measured for the “pure” POEGMA246 layer. However, for the P(4VP-co-OEGMA246)3 copolymer coating with a more symmetric composition, the shape of the spectrum is substantially modified (Figure 1, blue line). The intensity of the peak visible at 286.37 eV grows noticeably, due to the high contribution of C−N bonding from the P4VP fragments. To confirm these observations quantitatively, the C 1s spectra were resolved into three contributions related to the neutral carbon (C−C), C−O (and C−N), and O−CO (CO) and their relative intensities were analyzed (Table 2). The relative intensity of the C−C signal increases from 35% for the P4VP to 72% for the POEGMA246 coating. In contrast, the peak observed for a binding energy equal to 286.05 eV slightly shifts to a higher energy (286.37 4 V) and its intensity drastically decreases from 65% for the C−N bonding in P4VP to 20% for the C−O bonding in POEGMA246, through an intermediate value, corresponding to a combined contribution of C−O and C−N bondings of both monomers building a nearly symmetric copolymer brush. Additionally, the relative intensity of the C−O (C−N) peak recorded for the asymmetric copolymer is comparable to “pure” POEGMA246. The intensity of the CO peak, characteristic for POEGMA246 monomers with a high abundance of oxygen, remains constant for all POEGMA246 coating fragments. The obtained results of XPS analysis validate their chemical composition and confirm formation of copolymer brushes. In addition, grafted polymer coatings were characterized using ellipsometry. The typical thickness of oligoperoxide film, used as a substrate for polymer polymerization, did not exceed 1.5 nm.33 By changing the ratio between monomers (4VP and OEGMA246) in the reaction mixture, different compositions of the grafted polymer brushes with modified properties were obtained. The average thicknesses, refractive indexes, molecular weights, grafting densities, ratio between fragments in grafted brushes, and contact angles of the wetting determined at 20 °C in a dry state for grafted brush coatings are shown in Table 3. The measured values of the average thickness are in the range from 32 to 54 nm. The refractive index lies between 1.480 and 1.508, and the molecular weight of grafted brushes in a dry state after fabrication is calculated to be between 174 000 and 301 000. Grafting densities for all grafted brushes are comparable and equal to approximately 0.1 chains per nm2, similar to the values previously described.27 The ratios between 4VP and OEGMA fragments in grafted coatings were calculated using refractive indexes (for details, see the Supporting Information). The obtained results demonstrate good correlation with XPS data and theoretical calculations. It is interesting to compare CA values measured for different types of grafted brushes at 20 °C. The measured water contact angle exceeds 65°, which is the border value defining hydrophobic (CA > 65°) and hydrophilic (CA < 65°) surfaces, only for P4VP brushes.38,39 In contrast, POEGMA246 and copolymers with high content of the OEGMA246 chains are the most hydrophilic ones. However, there is no linear dependence between the amount of the monomer motifs of both copolymers and the CA value.

for the statistical copolymers P(VP-co-OEGMA300) and suggest that the homopolymerization of VP is favored over the homopolymerization of the corresponding methacrylate monomers. In the present study, similar monomers (4vinylpyridine (4VP) and oligo(ethylene glycol)ethyl ether methacrylate with molecular weights of 246 g/mol (OEGMA246)) are used. Therefore, the same reactivity ratios may be assumed for estimating the molar composition of the P(4VP-co-OEGMA246) grafted brushes. The molar composition of the P(4VP-co-OEGMA246) coatings was controlled by changing the ratio of the monomers in the reaction mixture and calculated according to eqs 3 and 4:37 F4VP = r4VP × f4VP 2 + f4VP × fOEGMA246 r4VP × f4VP 2 + 2f4VP × fOEGMA246 + rOEGMA246 × fOEGMA246 2 (3)

FOEGMA246 = 1 − F4VP

(4)

where F4VP and FOEGMA246 are mole fractions of the 4VP and OEGMA246 in the copolymer, respectively; r is the reactivity ratio, rVP = 1.08 and rOEGMA246 = 0.23; f4VP and f OEGMA246 are concentrations of the 4VP and OEGMA246 in the reaction mixture, respectively. Results are presented in Table 1. The chemical composition of the synthesized coatings was verified using X-ray photoelectron spectroscopy (XPS). Representative C 1s spectra, recorded for the coatings of “pure” P4VP, POEGMA246, and their two copolymers, with asymmetric and symmetric composition (Figure 1) qualitatively confirm calculated compositions.

Figure 1. C 1s XPS spectra of P4VP, P(4VP-co-OEGMA246)3, P(4VP-co-OEGMA246)4, and POEGMA246 coatings.

For the P4VP brush (Figure 1, red line), an asymmetric, wide peak, comprising two components corresponding to the C−C (284.80 eV) and C−N (286.05 eV) bonds, is observed, accompanied by π−π* shake-up at 292.0 eV characteristic for an aromatic ring present in the P4VP structure. In contrast, the XPS spectrum determined for the oxygen rich POEGMA246 brush (Figure 1, green line) is significantly narrower and D


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ACS Applied Materials & Interfaces Table 2. Relative Intensities (INT) of C−C, C−O (C−N), and O−CO Signalsa sample P4VP P(4VP-co-OEGMA246)3

P(4VP-co-OEGMA246)4

POEGMA246

a

type of the bond

BE [eV]

fwhm [eV]

INT [%]

C−C C−N (C−O) C−C C−O (C−N) O−CO (CO) C−C C−O (C−N) O−CO (CO) C−C C−O O−CO (CO)

284.80 286.05 284.80 286.25 288.25 284.80 286.37 288.27 284.80 286.37 288.37

1.76 1.76 1.54 1.54 1.54 1.37 1.37 1.37 1.39 1.39 1.39

35.0 65.0 58.0 34.5 7.5 75.1 17.8 7.1 72.4 20.8 6.8

BE, binding energy; FWHM, full width at half maximum.

Table 3. Average Thicknesses, Refractive Indexes, and Contact Angles of the Wetting (CA) at 20 °C in a Dry State for Different Types of the Grafted Polymer Brushes type of the of the grafted brush coatings P(4VP-co-OEGMA246) characteristic of the grafted polymer brushes

P4VP

1

2

3

4

POEGMA246

thickness [nm] refractive index molecular weight grafting density chains per nm2 ratio between fragments 4VP OEGMA CA

32 1.508 174 000 0.11 1 0 73.0 ± 3.0

40 1.506 219 000 0.11 0.906 0.094 59.8 ± 3.3

42 1.502 232 000 0.11 0.801 0.199 60.6 ± 3.8

54 1.494 301 000 0.1 0.556 0.444 49.5 ± 1.2

40 1.48 223 000 0.1 0.054 0.946 45.9 ± 2.3

40 1.48 245 000 0.1 0 1 46.0 ± 3.2

Figure 2. Temperature dependences of water contact angles determined for the grafted brush coatings fabricated with different polymerization mixtures, polymerization time of 48 h.

3.2. Temperature-Induced Changes in Wetting and Morphology of P4VP, P(4VP-co-OEGMA246), and POEGMA246 As Prepared Coatings (CA and AFM). To investigate the switching of wetting at different temperatures, the contact angles of sessile water droplets on the grafted brush coatings were measured between 6 and 38 °C. Although this

experimental method has some limitations, especially for small contact angles (below 20°),40 it can be adequately applied for the measured P(4VP-co-OEGMA246) coatings, characterized by the water contact angles of much larger than 20°. The temperature dependences of water contact angles determined for the grafted brush coatings fabricated with different E


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Table 4. Transition Temperatures Tc1 and Tc2 and Lower (LCA1 and LCA2) and Upper Contact Angles (UCA1 and UCA2) Determined for Grafted Brush Coatingsa sample P4VP P(4VP-co-OEGMA246)1 P(4VP-co-OEGMA246)2 P(4VP-co-OEGMA246)3 P(4VP-co-OEGMA246)4 POEGMA246 a

transition model one one one two two one

transition transition transition transition transition transition

model model model model model model

LCA1 45 47 52.5 41.3 35.9 39.7

± ± ± ± ±

1.7 1.7 0.5 0.4 0.5

UCA1 73.2 59.5 61.9 49.5 42.8 55.5

± ± ± ± ± ±

0.7 0.6 0.4 0.3 0.5 0.5

LCA2

49.5 ± 0.3 42.9 ± 0.2

UCA2

58.6 ± 0.3 51.0 ± 0.4

Tc1 13 13.1 11.9 10.9 9 23

± ± ± ± ± ±

Tc2 0.4 0.9 1.2 0.4 0.2 0.5

23 ± 0.3 24 ± 0.7

See Figure 2.

Figure 3. Topography of P4VP (a, b, and c), P(4VP-co-OEGMA246)3 (d, e, and f), and POEGMA246 (g, h, and i) grafted brush coatings, recorded at temperatures of 10 °C (a, d, and g), 20 °C (b, e, and h) and 30 °C (c, f, and i).

polymerization mixtures are shown in Figure 2. All coatings demonstrate significant changes of wettability, from hydrophilic to almost hydrophobic (CA ∼ 60°) in the investigated temperature interval. However, the status of this transition depends on the composition of the coatings. Transition temperatures, determined for all performed experiments, are summarized in Table 4. “Pure” P4VP and POEGMA246 grafted brush coatings were characterized by a sharp transition temperature at 13 and 23 °C, respectively, similarly to literature data.10,27 In contrast, P(4VP-co-OEGMA246)3 and P(4VP-coOEGMA246)4 obtained from reaction mixtures with the 4VP to OEGMA246 ratio equal to 50 to 50 or 97 to 3 mol %, respectively, show contact angle curves with two transitions well-expressed at 9−11 and 23−24 °C (see Table 4). Finally, grafted brush coatings P(4VP-co-OEGMA246)1 and P(4VP-co-OEGMA246)2 fabricated from a reaction mixture

with a low OEGMA246 content (10 and 20 mol %) demonstrate only one transition at 12−13 °C. These results of wetting measurements imply that the samples that were fabricated using a reaction mixture with a 50:50 mol % 4VP to OEGMA246 ratio are the most prospective material for further studies and potential applications. This type of grafted brush coating (P(4VP-co-OEGMA246)3) was studied in detail. Although the P(4VP-co-OEGMA246)4 coating exhibits double transitions, it has a limited range of wetting angles and for this reason was not considered further. The topography of the surface is an important factor strongly affecting its interactions with biomolecules. Nanostructured materials can influence the conformation and orientation of attached proteins41−43 as well as the cellular adhesion and proliferation44−48 or bacterial surface fouling.49 Therefore, the impact of temperature on the topography of POEGMA246, F


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Table 5. Transition Temperatures Tc1 and Tc2 and Lower (LCA1 and LCA2) and Upper Contact Angles (UCA1 and UCA2) Determined for P(4VP-co-OEGMA246)3 Grafted Brush Coatings at Different pH Valuesa pH 3 5 7 9 a

transition model one two two two

transition transition transition transition

model model model model

LCA1 45.6 38.1 46.1 46.9

± ± ± ±

2.1 1.4 1.8 3.8

UCA1 65.6 54.4 65.1 69.4

± ± ± ±

LCA2

1 1.9 1.7 2.1

53.3 ± 1.6 64.8 ± 2.1 66.2 ± 1.2

UCA2 67.3 ± 0.7 78.9 ± 1.5 76.3 ± 0.8

Tc1 13.8 9.1 12.2 13.9

± ± ± ±

Tc2 0.6 1.1 0.8 0.5

17.4 ± 0.5 22.4 ± 0.5 24.4 ± 0.7

See Figure 4.

Figure 4. Temperature dependence of the water contact angle, determined at pH values of 3, 5, 7, and 9 on oligoperoxide-graf t-P(4VP-coOEGMA246)3 coating.

3.3. Influence of pH on the Temperature Response of the P(4VP-co-OEGMA246)3 Coating (CA and AFM). In our previous works,10,27 we have shown the huge influence of pH on homopolymer grafted brushes. The temperatureresponsive properties of the grafted P4VP were eliminated at low pH due to the transformation of the pyridyl groups to protonated pyridyl groups, where creation of hydrogen bonds with water’s oxygen is strongly facilitated. Moreover, it was demonstrated that temperature-responsive properties of the grafted POEGMA brushes at low and midrange concentrations of POEGMA in coatings were blocked at low pH,10 due to interaction of the protonated (−COOH) groups with ether oxygens of POEGMA. On the basis of these results, in the present work, we expected to find similar dependencies, i.e., temperatureresponsive properties blocked at low pH. Surprisingly, for the investigated coatings, a very well expressed temperature transition (Tc) of 13.8 °C, i.e., in a temperature range that corresponds to LCST of P4VP, appears at pH = 3. However, in a temperature range corresponding to LCST of the POEGMA246, no transition is observed. For other pH values, the curves resemble the one obtained for “as prepared” samples and can be separated into two independent parts, described by two Boltzmann curves. Both transition temperatures tend to grow with increasing pH (see Table 5). For the first transition, the observed shift is relatively small (∼4 °C), whereas for second transition (Tc2) it increases by 7 °C, from 17 °C at pH = 5 to 24 °C at pH = 9.

P4VP, and P(4VP-co-OEGMA246)3 coatings, as promising materials for numerous biomedical applications, was examined using AFM working in air. Recorded topographies, presented in Figure 3, show relatively smooth P4VP surfaces (Figure 3a−c), characterized by root-mean-square (RMS) values increasing slightly from 3.8 to 5.7 nm for temperatures rising from 10 to 30 °C. Similarly, the roughness of POEGMA246 coatings (Figure 3g−i) increases with growing temperature. However, in this case, this effect is significantly more pronounced and the RMS value grows more than twice, from 4.9 at 10 °C up to 10.4 nm at 30 °C. In contrast, for P(4VP-co-OEGMA246)3 brushes (Figure 3d−f), a strong increase of surface roughness from 6.5 nm at 10 °C to 11.8 nm at 20 °C is followed by a rapid decrease of RMS value, to 2.1 nm at 30 °C. These changes of surface topography may be related to the brush transition from the hydrated state with loose coils (at lower temperatures) to the almost hydrophobic state with collapsed chains (at higher temperatures),10,29 which tend to aggregate and form the large globular structures observed in the AFM images. The unusual behavior of the P(4VP-co-OEGMA246)3 coating suggests temperature-controlled three-stage switching of morphology, reflecting the semicollapsed state of the coating at 20 °C. Interactions of 4VP fragments with water molecules are destroyed, whereas OEGMA246 fragments continue to hold water molecules. At 30 °C, the coating evolves to a completely collapsed state. However, in contrast to the P4VP and POEGMA246 homopolymer brushes, the observed surface is relatively smooth. This effect may be related to an earlier intermediate transformation to semicollapsed state. G


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Figure 5. Topography of the oligoperoxide-graf t-P(4VP-co-OEGMA246)3 coating, recorded for pH values of 3, 5, 7, and 9 at 10, 20, and 30 °C.

complex process, depending on both size and conformation of the protein as well as physicochemical properties of the surface. However, the most important role in protein adsorption is played by hydrophobic interactions.56−58 As investigated polymer brush coatings demonstrate significant temperaturedependent wetting changes from hydrophilic to almost hydrophobic, protein adsorption was examined for two model proteins, BSA and fibrinogen, at four different temperatures, corresponding to values below the first (10 °C) and above the second transition (32 °C) as well as between them (15 and 20 °C). Representative fluorescence micrographs, recorded for BSA from a phosphate saline buffer (pH = 7.4) adsorbed onto coatings composed of P4VP, POEGMA246, and their copolymer, are presented in Figure 6a. For the P4VP coating (upper row), the observed intensities are very high, especially for temperatures higher than 10 °C, i.e., above the transition (cf. Figure 2), indicating a slightly temperature dependent, extremely effective protein adsorption. In contrast, micrographs recorded for POEGMA246 coatings (bottom row) remain black for all investigated temperatures, confirming the wellknown antifouling properties of POEGMA.10 A significantly more complex temperature dependence of protein adsorption is observed for P(4VP-co-OEGMA246)3 coatings. The observed intensity grows from an extremely low level at 10 °C to slightly higher values at 15 °C and then increases significantly at 20 and 32 °C. These observations were verified quantitatively using the integral geometry approach,59 providing an average measure of the fluorescence intensity (Figure 6b). The results obtained

To examine the impact of pH and temperature on the topography of the oligoperoxide-graf t-P(4VP-co-OEGMA246) 3 coatings, AFM measurements were performed. There are several approaches, using AFM working in specific modes (e.g., peak force tapping,50,51 quantitative imaging,52,53 magnetic AC54), to enable precise determination of various physicochemical properties of the examined surface. As the mechanisms of pH responsivity are strongly related with the possibility to create hydrogen bonds between brush molecules and water, the experiments were carried out in an aqueous environment using the AFM working in the MAC mode, which enables a greatly improved resolution in liquids.54 For pH values equal to 5, 7, and 9, recorded topographies, presented in Figure 5, show a similar temperature-dependent behavior. Structured and rough surfaces visible at 10 and 20 °C transform to significantly smoother ones at 30 °C. However, at pH = 3, this effect is slightly modified and a rapid increase of surface roughness is observed at 20 °C. This distinct behavior may be related to the temperature-responsive properties of the oligoperoxide-graf t-P(4VP-co-OEGMA246)3 coating, strongly affected for low pH values (see Figure 4) where only one temperature transition is observed. 3.4. Proteins Adsorption to Oligoperoxide-graft-P(4VP-co-OEGMA246)3 Coatings. “Smart” surfaces that are able to change the surface properties with temperature variations are required for applications in regenerative medicine, bioengineering, and cell biology.55 One of the most important properties of these materials is their ability to sharply change protein adsorption at different temperatures.55 Nonspecific protein adsorption to polymer surfaces is a very H


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3 brushes, a strong increase of surface roughness from 6.5 nm at 10 °C to 11.8 nm at 20 °C is followed by a rapid decrease of the RMS value, to 2.1 nm at 30 °C. The unusual behavior of the P(4VP-co-OEGMA246)3 coating suggests temperature-controlled three-stage switching of morphology, reflecting a semicollapsed state of the coating at 20 °C. Interactions of 4VP fragments with water molecules are destroyed, whereas OEGMA246 fragments continue to hold water molecules. At 30 °C, the coating evolves to a completely collapsed state; however, in contrast to P4VP and POEGMA246 homopolymer grafted brushes, the observed surface is relatively smooth. This effect may be related to an earlier intermediate transformation of the coating to semicollapsed state. Additionally, the influence of pH on thermo-sensitivity of P(4VP-co-OEGMA246)3 brushes was shown. At pH = 3, a well expressed temperature transition (Tc) was observed at 13.8 °C, i.e., in the temperature range that corresponds to LCST of the P4VP. However, no transition was observed in the temperature range that corresponds to LCST of the POEGMA246. The AFM measurements also revealed a different behavior for low pH values. Recorded topographies showed structured and rough surfaces visible at 10 and 20 °C, transforming to significantly smoother ones at 30 °C. However, for low pH, this effect is slightly modified and a rapid increase of surface roughness is observed at 20 °C. This distinct behavior may be related to temperature-responsive properties of the oligoperoxide-graf t-P(4VP-co-OEGMA246)3 coating, which are strongly affected for low pH values where only one temperature transition is observed. Moreover, complex temperature dependence of bovine serum albumin adsorption was observed for P(4VP-coOEGMA246)3 coatings. Fluorescence intensity, corresponding to the amount of adsorbed proteins, grows from an extremely low level at 10 °C to higher values at 15 and 20 °C and then increases significantly at 32 °C. Research on the P(4VP-co-OEGMA246) copolymer brushes as temperature-controlled three-stage switching platforms offers at least two advantages. First, they can be prospectively used for numerous applications in sensor technologies and regenerative medicine. Second, properties of the grafted brushes can be easily tuned by modification of the pyridine groups. Moreover, adsorption of the proteins on the P(4VP-co-OEGMA246) coatings is relatively low suggesting low fouling properties.

Figure 6. Representative fluorescence micrographs of BSA (labeled with Alexa Fluor) adsorbed to the P4VP, P(4VP-co-OEGMA246)3, and POEGMA246 coatings at 10, 15, 20, and 32 °C (a) and the determined fluorescence intensities of BSA adsorbed to the P4VP, P(4VP-co-OEGMA246)3, and POEGMA246 (columns) and glass reference samples (dashed line) (b).

confirm different characteristics of protein adsorption onto investigated coatings, i.e., antifouling properties of POEGMA246, very good protein adsorption onto P4VP, and slightly less effective but strongly temperature-dependent BSA adsorption to the P(4VP-co-OEGMA246)3 copolymer brush. At 10 °C, BSA adsorption is very weak, resembling the adsorption onto POEGMA246. When the temperature is elevated to 15 °C, the BSA adsorption increases almost 3 times. A further increase of the temperature, up to 20 °C, leads to the BSA adsorption nearly doubled as compared to 15 °C. At 30 °C, BSA adsorption increases again, approximately 1.5 times as compared to 20 °C.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00136.

4. SUMMARY AND CONCLUSIONS In the present work, novel polymeric coatings of oligoperoxidegraf t-P(4VP-co-OEGMA246) attached to glass were presented. The overall composition of the coatings was confirmed by XPS and ellipsometry. The molar compositions of the P(4VP-coOEGMA246) coatings were calculated. Measurements of water contact angle revealed temperature-induced changes in wettability of the P(4VP-co-OEGMA246) grafted brush coatings with two temperature-induced transitions at 10 and 23 °C. They were explained in terms of the retention of individual properties of the two different polymer motifs in grafted copolymer brushes (4-vinylpyridine and oligo(ethylene glycol)ethyl ether methacrylate246), characterized by different LCST values, equal to 14 and 26 °C, respectively. In contrast to P4VP and POEGMA246 coatings, for P(4VP-co-OEGMA246)

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Chemical structure of the oligoperoxide; calculation of the polymer volume fraction using ellipsometry (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.S.). *E-mail: [email protected] (J.R.). ORCID

Yurij Stetsyshyn: 0000-0002-6498-2619 Notes

The authors declare no competing financial interest. I


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Temperature-Controlled Three-Stage Switching of Wetting

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