Temperature-responsive properties of poly(4-vinylpyridine) coatings: influence of temperature on the wettability, morphology, and protein adsorption

Abstract

Although the pH-response of poly(vinylpyridine)-based systems is well-known and indeed used in several biomedical applications, the impact of temperature on the properties of this polymer has not been investigated in detail so far. Herein, we demonstrate the temperature-responsiveness and switchable wettability of two poly(4-vinylpyridine) coatings, mimicking the behavior of materials with lower critical solution temperature. The thermal response of P4VP spin-coated films, solvent cast on a glass, is weaker than that observed for P4VP-grafted brushes, fabricated via polymerization from an oligoperoxide grafted on an amino-silanized glass. Both the P4VP coatings exhibit a temperature dependence of the water contact angle with a well-defined transition at 13–14 °C. This transition is absent at acid pH levels wherein almost all pyridyl groups are protonated. The P4VP-grafted brushes were used to examine the impact of temperature on the surface morphology and protein adsorption. The coating surface, recorded with atomic force microscopy, evolved noticeably at alkaline pH, from being relatively smooth at 10 °C to structured and rough at 20 °C. In turn, at acid pH levels, flat surfaces with rare elevations were observed at both temperatures. The adsorption of bovine serum albumin and human fibrinogen was observed with fluorescence microscopy to be significantly more efficient for temperatures above the transition, indicating that P4VP coatings can act as a noteworthy switching material.

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1. Introduction

Due to their diverse properties, versatile polymers of the polyvinylpyridine (PVP) family, including in particular poly(4-vinylpyridine) (P4VP), are suitable for numerous applications. For instance, they are widely used to fabricate antibacterial surfaces,^1,2 pH-sensitive systems that can respond to local environment changes with unique sharp transitions from a hydrophobic to a hydrophilic state,^3,4 or three-dimensional (3D) molecular-level ordering systems.⁵ Coatings based on P4VP have been applied for corrosion inhibition,⁶ in humidity⁷ and nitroaromatic sensors,⁸ and in quasi-solid dye-sensitized solar cells.⁹ PVP polymers enable the immobilization of nanoparticles due to the strong affinity of the pyridyl group to metals and the ability to form hydrogen bonds with polar species.¹⁰ Moreover, quaternized or protonated forms of PVP can interact electrostatically with charged surfaces,^11,12 whereas pyridyl groups can interact with various nonmetallic polar surfaces, especially those terminated with amines, carboxyl, hydroxyl, and other groups capable of hydrogen bonding.¹⁰

The fabrication, properties and applications of the PVP-based pH-responsive systems have been widely described.^3,4,13,14,21 For instance, pH-responsive PVP particles, fabricated by Minko et al.¹³ and Fujii et al.,¹⁴ demonstrated excellent swelling transition at acidic pH. P4VP is a hydrophobic polymer that is insoluble in water until more than ca. 35% of the pyridine groups is charged, e.g., by protonation.¹⁵ However, pyridine molecules can form hydrogen bonds with water molecules.^16,17 Moreover, pyridyl groups were assumed to be responsive to moisture adsorption in shape memory polyurethanes containing pyridine moieties, as synthesized by Chen's group.^18,19 Similarly, the humidity and solvent effects on polythiophene and poly(vinyl pyridine), as investigated by Jaczewska et al.,²⁰ showed humidity absorption a few times higher for PVP, suggesting that the pyridine unit is responsive to the absorption of moisture.

Although PVP polymers have been widely studied, the influence of temperature on the P4VP properties has not been investigated in detail before. Therefore, in the present work, we demonstrate for the first time, the temperature-responsive properties of P4VP coatings, with them mimicking the typical behavior of materials with a lower critical solution temperature (LCST). For this purpose, grafted P4VP brushes were fabricated via polymerization from an oligoperoxide grafted to glass pre-modified with (3-aminopropyl)triethoxysilane (APTES). The influence of temperature on the wettability, morphology, and protein adsorption of the P4VP brushes were examined via water contact angle measurements (CA), atomic force microscopy (AFM), and fluorescence microscopy (FM), respectively, with the results revealing the well-expressed temperature-responsive properties of the P4VP coatings. Analogous wettability experiments were performed also for P4VP layers prepared conventionally, i.e., by spin-coating from a good solvent. They exhibit a similar temperature response, but this effect was significantly stronger for the P4VP brushes.

2. Experimental section

2.1. Materials

Pyridine and the other organic solvents were purified as reported by Weissenberger et al.²² Polyethylene glycol (PEG-9) was supplied by Merck Chem. Co. 4-Vinylpyridine (4VP), poly(4-vinylpyridine) M_w = 160.000 and 3-aminopropyltriethoxysilane (APTES) were supplied by Sigma-Aldrich. Bovine serum albumin, BSA, labeled with Alexa Fluor 555, was purchased from Invitrogen (USA).

tert-Butylhydroperoxide was obtained as described in ref. 23 and purified by vacuum rectification. The fraction boiling in the temperature range of 45–47 °C (at 1.6 kPa) was collected, and had a refractive index n_d²⁰ = 1.4002 ± 0.00002, which is in accord with previous reports (n_d²⁰ = 1.4010).²³

Pyromellitic acid chloride. In a 500 ml round-bottomed flask equipped with a thermometer and a reflux condenser, connected with a water scrubber, 43.6 g (0.2 mol) of pyromellitic dianhydride and 91.6 g (0.44 mol) of PCl₅ were mixed and boiled in the oil bath until the mixture became homogeneous. Afterwards, the mixture was additionally mixed for 15–16 h at 130–135 °C. The reflux condenser was then replaced by a Liebig condenser and approximately 60–63 g of POCl₃ was distilled off during 8 h. Then, the temperature of the mixture was increased to 180–185 °C. Then, the crude product was recrystallized from gasoline, yielding 51.2 g (78.1%) of a colorless crystalline product with a melting point of 67 °C (in accord with the literature value of 68 °C (ref. 24)) and acid number AN of 1373 mg KOH g⁻¹ (cf. the calculated value was 1368 mg KOH g⁻¹).

Oligoperoxide with residual acid chloride groups. 4.6 g (0.014 mol) of pyromellitic acid chloride was dissolved in 15 ml of anhydrous dichloroethane, placed in a three-necked flask equipped with a stirrer, and then 1.26 g (0.014 mol) of tert-butylhydroperoxide was added. The mixture was cooled down to 5 °C, and then 1.1 g (0.014 mol) of pyridine, dissolved in 10 ml of anhydrous dichloroethane, was added dropwise at 5 °C. Then, the suspension was mixed for 1 h. Subsequently, 5.6 g (0.014 mol) of PEG-9 was added, and again a solution of 2.2 g (0.028 mol) pyridine in 10 ml of anhydrous dichloroethane was admixed dropwise. The mixture was then stirred for another 3 h and the temperature increased gradually up to the room temperature. The precipitate of pyridinium chloride was filtered out. The solvent was distilled out and the pellet was dried in vacuum (100–200 Pa) at 40 °C for 3 h, yielding 8.2 g (81%) of oligoperoxide. The pellet had a yellowish resin-like appearance. Its characteristics are summarized as follows: content of active oxygen, 1.9% (calc. 2.2%); content of active chlorine, 5.4% (calc. 4.9%); AN = 163.1 mg KOH g⁻¹ (calculated value is 155.3 mg KOH g⁻¹); infrared spectra showed characteristic bands of ν(C=O) in Ar–C(O)Cl, ν(C=O) in ester group at 1760 and 1752 cm⁻¹; a doublet at 1390, 1365 cm⁻¹, referred to d(C(CH₃)₃), and a band of tert-butoxy group at 848 cm⁻¹.

2.2. Preparation of grafted P4VP brushes

Modification of glass surfaces with oligoperoxide. Glass plates (20 × 20 mm, marked as (1) in Scheme 1, of the ESI†), 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 dioxane in Soxhlet's apparatus over 4 h. As a result, oligoperoxides grafted to aminated surfaces (3) were obtained.²⁵

Polymerization of P4VP brushes. The glass plates with grafted oligoperoxide (3) were placed in a container with 0.1 M aqueous solution of the monomer P4VP, and heated under an argon atmosphere at 90 °C for a certain period (polymerization time from 20 to 48 h), resulting in the oligoperoxide-graft-P4VP coatings (4). Then, the plates coated with polymerized P4VP were washed with water in Soxhlet's apparatus for 4 h and dried. The procedure of modification of the peroxided glass surface by P4VP is illustrated in Scheme 1, ESI†.

2.3. Preparation of the P4VP spin-coated layers

0.1 ml of a 6 mg ml⁻¹ or 23 mg ml⁻¹ solution of P4VP in ethanol and a 23 mg ml⁻¹ solution of P4VP in alkali ethanol (pH = 9) or acid ethanol (pH = 2) was spin-coated (KW-4A, Chemat Technology) at 2000 rpm for 1 min onto the glass surface to prepare two samples of each kind. Then, the substrates were held prior to any usage at a room temperature for 24 h.

2.4. Characterization of the coatings: water contact angle measurements (CA)

Static contact angle experiments were performed by the sessile drop technique using a Kruss EasyDrop (DSA15) instrument with a Peltier temperature-controlled chamber. The measurements were carried out at temperatures ranging from 6 °C to 30 °C to determine the thermal response of the P4VP coatings. The temperature was measured by a thermocouple in contact with the sample surface. To determine the 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 temperature-controlled chamber of the EasyDrop instrument. Prior to the buffer exposure leading to the measurement at different pHs, the sample was immersed in deionized water for 60 min and dried under a nitrogen stream. Contact angles were expressed as the average of ten measurements at different spots. The relative humidity RH was evaluated and monitored by a humidity sensor “Kobold HND-Fx15”.

To determine the surface energies, the van Oss and Good equation^26,27 was used. Values from the literature²⁶ were used for the Lifshitz–van der Waals component λ^LW_S as well as the Lewis acid (λ_S⁺) and base (λ_S⁻) components: water (λ^LW_S = 21.8 mN m⁻¹ and λ_S⁺ = λ_S⁻ = 25.5 mN m⁻¹); diiodomethane (λ^LW_S = 50.8 mN m⁻¹ and λ_S⁺ = λ_S⁻ = 0 mN m⁻¹); and glycerol (λ^LW_S = 34.0 mN m⁻¹, λ_S⁺ = 3.9 mN m⁻¹, and λ_S⁻ = 57.4 mN m⁻¹).

2.5. Atomic force microscopy (AFM)

Topographic images of the oligoperoxide-graft-P4VP coatings were recorded in water at different temperatures using Atomic Force Microscopy (Agilent 5500) working in the MAC mode, equipped with type 6 MAC Levers. For each sample, images were recorded in three randomly chosen regions.

Representative AFM images were characterized by the root-mean-squared roughness RMS, which measures the spread (RMS) of the AFM height distribution. The topographical parameters were determined using the PicoImage software provided along with the AFM instrument.

2.6. Ellipsometry

Measurements were carried out with a serial null-ellipsometer LEF-3M, equipped with the “polarizer–compensator-specimen-analyzer” arrangement, enabling angular positions of the polarization elements to be determined within 0.01° precision. A He–Ne single-mode laser (light wavelength λ = 632.8 nm) was used as a light source. The polarization parameters of the light reflected from a sample (angles Ψ and Δ) were determined using the two-zone technique (in the third and fourth measuring zone) for an angle of incidence ϕ varying between 58° and 63° (with a 1° step). This ϕ-range, corresponding to the region of the pseudo-Brewster angle (where Δ ≈ π/2 or 3π/2), ensures maximal sensitivity. The iterative procedure using mono- and two-layer models was used to fit the (Ψ, Δ) data recorded at the optimal experimental conditions²⁸ and yielded an average thickness d (and refractive index n) for the APTES, oligoperoxide-graft-P4VP coatings, and spin-coated P4VP layer samples, respectively.

The molecular masses of the grafted polymer brushes were calculated concerning the ellipsometry data and kinetic parameters of the polymerization using equation:

where M is the molecular weight (g mol⁻¹), C_p is the concentration of P4VP on the surface (from the ellipsometry data) (g m⁻²), C₀ is the initial concentration of peroxide groups of the oligoperoxide on the surface (mol m⁻²) (C₀ = 2.38 × 10⁻⁶ mol m⁻²), 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) [our unpublished data].

2.7. Protein adsorption

Bovine serum albumin (BSA) labeled with Alexa Fluor 555, absorbing green light (λ_abs = 555 nm), and emitting red fluorescence (λ_emit = 562 nm) and fibrinogen from human plasma labeled with Alexa Fluor 488, absorbing blue light (λ_abs = 496 nm), and emitting green fluorescence (λ_emit = 520 nm) were taken as model proteins to examine the adsorption to the oligoperoxide-graft-P4VP coatings at different temperatures. The BSA and fibrinogen solutions, with constant concentrations equal to 125 μg ml⁻¹, were prepared using phosphate saline buffer (PBS, pH = 7.4). To examine protein adsorption, a 50 μl drop of protein solution was placed on the oligoperoxide-graft-P4VP substrates and kept at 10 °C and 20 °C for an incubation time of 15 min. Then, all the samples were rinsed with the buffer to remove non-adsorbed proteins and dried under a nitrogen stream.

2.8. Optical fluorescence microscopy

Protein adsorption to the oligoperoxide-graft-P4VP coatings was examined using an optical microscope Olympus BX51, equipped with a 100 W halogen lamphouse, a U-MWIG2 filter (λ_exit = 520–550 nm, λ_emit > 565 nm), and camera type DP72. All the images were recorded for dried samples using the Cell^F program.

Two series of experiments were performed to study the impact of temperature on the protein adsorption. The recording conditions were adjusted separately for both experimental series.

3. Results and discussion

In the present work, two types of the P4VP coatings, i.e., grafted brushes and spin-coated layers, were fabricated. However, the main attention was devoted to assessing the properties of the P4VP-grafted brushes, whereas investigations of the P4VP-spin-coated layers were carried out merely to confirm the conclusions about the temperature-responsive properties of the poly(4-vinylpyridine) coatings. The oligoperoxide-graft-P4VP coatings attached to glass were prepared in a three-step process (outlined in Scheme 1, ESI†). The hypothetical structure of the P4VP-grafted brushes is presented in Scheme 1a. For a good solvent, such as ethanol for P4VP, the interactions between the polymer segments and solvent molecules are energetically favorable, and promote the expansion of vertically arranged polymer brushes. In contrast, for polymer films spin-coated from a good solvent, the polymer forms a multilayer structure of laterally arranged macromolecules²⁹ (Scheme 1b).

Scheme 1 Hypothetical structures of the P4VP-grafted brushes (a) and P4VP spin-coated layers (b) on the glass surface.

The thicknesses of the grafted layers were examined using ellipsometry. The typical thickness of APTES, measured by ellipsometry for the conditions used for glass functionalization, was equal to 0.5–1 nm (in accord with the literature^25,30). The maximal thickness of oligoperoxide film, used as a substrate for P4VP polymerization, did not exceed 1.5 nm.²⁵ The average thickness and refractive index of the P4VP-grafted brushes in a dry state at room humidity (with a relative humidity RH ∼ 40%) were in the range from 17 to 40 nm and from 1.51 to 1.57, respectively. Analogous values for the P4VP-spin-coated layers were in the range from 38 to 180 nm and from 1.57 to 1.581, respectively. These measured values of the refractive indices are consistent with the literature (n = 1.58 (ref. 31–33)). The calculation of the grafting density of the P4VP chains on the peroxide glass surface was performed using the equation:

where σ is the grafting density (chains per nm²), h is the dry layer thickness measured by ellipsometry (nm), ρ is the P4VP bulk density, N_A is Avogadro's constant, and M (g mol⁻¹) is the molecular weight of the P4VP brushes grafted onto the surface. Here, we present the calculated molecular weights obtained for polymerization times of 12, 22, and 40 h (17, 26, and 40 nm) as 241 000, 230 700, and 223 000, respectively. The grafting densities for these polymerization times are 0.04, 0.07, and 0.1, respectively, which are very similar to the values previously described in similar works (0.1–0.4 chains per nm²).^40,41 The surface coverage with P4VP brushes polymerized from the oligoperoxide initiator was confirmed by X-ray photoelectron spectroscopy (see Fig. 1, ESI†).

3.1. Temperature-sensitive wettability of the P4VP coatings

To examine the temperature-sensitive properties of the P4VP coatings fabricated with two different methods, the contact angles of sessile water droplets were measured between 6 °C and 30 °C. The results, presented in Fig. 1a, show a weak thermal response of P4VP layers spin-coated from ethanol (38 and 180 nm), with the water contact angle changing from 62–65° to 70–75°. The wettability changes were significantly better expressed for the oligoperoxide-graft-P4VP brushes attached to glass with a thickness of 40 nm (the squares in Fig. 1b). In this case, the increasing temperature induces changes in the water contact angle of nearly 25°, from 50° to 72–76°. Although the grafted P4VP coating with a thickness of 40 nm is characterized by a sharp transition at 14 °C, the thinner grafted P4VP coatings (17 and 26 nm) show a weak thermal response similar to the spin-coated P4VP layers, with the transition point shifted down even to 9 °C. In contrast, there is no thermal response of the pure oligoperoxide layer (the stars in Fig. 1b). Interestingly, the increase in the grafting densities leads to elevation of the transition points from 9 °C to 14 °C. The thermal response of the wettability is manifested by a well-defined transition at 9–15 °C for both coatings, independently of the fabrication process. However, comparison of the temperature dependencies of the wettability measured for different types of the P4VP coatings indicated that the inner arrangement of P4VP molecules may play the key role in the intensity of the thermal response of the P4VP coating.

Fig. 1 Temperature dependence of the water contact angle determined for the as-prepared samples of P4VP layers spin-coated from ethanol solution with a thickness of (a) 38 nm (triangles), 180 nm (circles) and oligoperoxide-graft-P4VP with a thickness of (b) 0 nm (circles), 17 nm (triangles), 26 nm (stars), and 40 nm (squares). Solid lines were generated by sigmoidal function, with the exception of pure oligoperoxiode layer (b, circles).

To characterize better the P4VP-grafted brushes and the spin-coated P4VP layers, the surface energies of those coatings was calculated. For this purpose, in addition to water contact angle measurements, also diiodomethane and glycerin contact angles were determined at 10 °C, 15 °C, and 25 °C. The measured values were used to calculate the surface energy λ_S and its components (the Lifshitz–van der Waals apolar term λ^LW_S and the Lewis acid–base polar term λ^AB_S), using a method reported previously in the literature.^26,27 The results are summarized in Table 1. For the P4VP-grafted brushes coating (40 nm) at 10 °C, the values of both components, λ^LW_S and λ^AB_S, are high. In contrast, at 15 °C and 25 °C, the value of the λ^LW_S component is more than ten times higher than the λ^AB_S value (Table 1), indicating that with increasing temperature, the P4VP-grafted brushes change conformation and start to exhibit apolar surface characteristics. The spin-coated P4VP layer (38 nm) reveals similar tendencies. However, the value of the λ^LW_S component is more than ten times higher than the λ^AB_S value only at 25 °C. Interestingly, the total surface energy of the P4VP-grafted brushes is highest at 10 °C, in contrast to the spin-coated P4VP layer, where the highest total energy is observed at 25 °C.

Table 1 Contact angle and surface energy components of the P4VP coatings at different temperatures

Temperature of P4VP coating [°C]	Contact angle, °			Surface energy components, mN m^−1
	H2O	CH2I2	C3H6(OH)3
				λ^LWS	λS^−	λS^+	λ^ABS	λS
P4VP-grafted brush coatings (40 nm)
10	49.5 ± 1.2	51.9 ± 2.4	78.6 ± 3.5	33.2	64.6	2.1	23.7	56.9
15	63.6 ± 1.5	49.0 ± 1.8	70.5 ± 2.5	34.8	27.4	0.08	2.9	37.7
25	72.8 ± 3.1	46.3 ± 1.0	65.1 ± 2.9	36.3	10.6	0.26	3.3	39.6
P4VP layers spin-coated from ethanol solution (38 nm)
10	63.0 ± 3.0	53.6 ± 2.1	60.6 ± 2.2	32.2	20.0	0.5	6.7	39.1
15	62.9 ± 3.6	42.7 ± 1.6	62.4 ± 2.1	38.2	20.6	0.05	8.2	40.3
25	68.9 ± 2.0	46.6 ± 2.3	51.4 ± 2.0	36.2	7.4	2.3	2.1	44.4

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In the previous works,^18–20 the influence of pyridyl groups on the moisture-sensitive properties of shape memory polyurethanes was demonstrated. Therefore, we also studied the influence of humidity on the wettability of both P4VP coatings. The results, presented in Fig. 2, depict the influence of humidity on the water contact angle values around the wettability transition (at 8 °C and 25 °C) for the P4VP coatings examined under different conditions, i.e., at room humidity (RH ∼ 40%) after heating for 5 h at 40 °C (Fig. 2a) as well as after drying in ambient conditions (Fig. 2b) and at high humidity (RH > 80%) after drying in ambient conditions (Fig. 2c). Evidently, for higher humidity, the magnitude of the thermal response in the wettability is larger, with a more pronounced effect observed for the P4VP-grafted brushes. Interestingly, immersing the dry samples (Fig. 2a) in water for a short time resulted in an increased magnitude of the thermal response in the wettability, comparable to that marked in Fig. 2c.

Fig. 2 Temperature dependence of the water contact angle for the as-prepared samples of oligoperoxide-graft-P4VP at 40 nm (black circles) and P4VP film spin-coated from ethanol solution at 180 nm (red squares) layers, determined at: (a) room humidity (RH ∼ 40%) after heating for 5 h at 40 °C, (b) room humidity (RH ∼ 40%) after drying in ambient conditions, and (c) high humidity (RH > 80%) after drying in ambient conditions.

3.2. Thermal response of the wettability of P4VP coatings modified by pH

We assume that similar to pyridine, the pyridine motifs in PVPs are able to create hydrogen bonds with water^16,17,34,35 and to interact with each other via van der Waals or π–π interactions.^36,37 To identify the mechanism responsible for the thermal response of the P4VP coating, the temperature dependence of the water contact angle was examined at two pH values, i.e., 2 and 7, for both coatings. The results, presented in Fig. 3, do not exhibit any thermal response of the wettability at pH = 2 (red triangles), where almost all the pyridyl groups are transformed to protonated pyridyl groups.^{3,4,16,17,34,35} This indicates that the thermal transitions are not possible in protonated P4VP. The P4VP layers spin-coated from acid ethanol solution (pH = 2) (Fig. 3b, the red triangles) exhibit strong hydrophilicity, manifested by water contact angles in the range of 20–25°, which is in good agreement with the literature.^3,4 In contrast, the water contact angles recorded for P4VP-grafted brushes (Fig. 3a) are significantly higher and are equal to ∼45°. For neutral and alkaline conditions, i.e., pH = 7 or 9, the transition in the wettability is observed at 12–15 °C for both coatings. However, this effect is significantly better manifested for the P4VP brushes (cf. Fig. 3a and b). This feature, as well as the thermal response of the wettability at pH = 9, in general, mimics the behavior of both the as-prepared P4VP coatings (cf. Fig. 3 and 1). Surprisingly, the water contact angles values recorded for the P4VP brushes at pH = 9 were comparable at low temperatures to the values measured at pH = 2. As the temperature-responsive properties were noticeably better expressed for the P4VP-grafted brushes, they were chosen for the further investigations of the thermal response of the surface morphology and protein adsorption.

Fig. 3 Temperature dependence of the water contact angle, determined at pH values of 2 (red triangles) and 9 (black stars) for the oligoperoxide-graft-P4VP (a) and for the spin-coated P4VP layers (b) from acid ethanol (pH = 2) and alkali ethanol P4VP solution (pH = 9).

3.3. Thermal response of the surface morphology of P4PV-grafted brushes modified by pH

To test the impact of temperature and pH on the topography of the P4VP brushes, AFM measurements were performed in an aqueous environment (Fig. 4) using AFM working in the MAC mode, which enables greatly improved resolution in liquids.³⁶

Fig. 4 Topography of the oligoperoxide-graft-P4VP coatings at different temperatures (T = 10 °C (a) and (c); T = 20 °C (b) and (d)) and pH values (pH = 3 (a) and (b); pH = 7 (c) and (d)).

The resulting AFM images, presented in Fig. 4, show relatively flat surfaces with a rare elevation for low pH values (Fig. 4a and b) at both analyzed temperatures. In contrast, for an acid environment, the recorded topography changes noticeably with increasing temperature—from relatively smooth at 10 °C to structured and rough at 20 °C. These observations were qualified by root mean square (RMS) calculations, providing for low pH almost the same RMS values as the measurements performed at 10 °C and 20 °C, i.e., RMS = 5.3 ± 0.7 nm and 5.4 ± 0.9 nm, respectively. In contrast, at pH = 7, the RMS increases from 3.8 ± 0.6 nm at 10 °C to 7.4 ± 0.8 nm at 20 °C. These morphological changes accompany the transition from loose P4VP coils forming relatively homogeneous brushes to collapsed P4VP chains forming visible aggregates.

Based on the obtained results, a hypothetical scenario of van der Waals interactions and hydrogen bonding between the pyridyl groups of PVP, the protonated pyridyl groups of PVP, and the water molecules at various pH and temperatures for the P4VP coatings was proposed. For neutral and high pH values at high humidity, the thermal response of the wettability (Fig. 1, 2c, and 3 (the stars)) mimics the behavior of systems with a lower critical solution temperature, LCST, described for the other grafted brushes.^39–43 In this case, the properties of the coatings at T < T_c can be attributed mainly to the hydrogen bonds between the nitrogen of P4VP and hydrogen of the water^16,17,34,35 (Scheme 2c). In turn, at T > T_c, polymer–polymer interactions (van der Waals interactions)^37,38 are thermodynamically more favored than polymer–water interactions (Scheme 2d),^39–47 inducing P4VP transition from a relatively hydrated to a hydrophobic state. Besides the temperature dependence, the oligoperoxide-graft-P4VP coatings also show a strong response to pH, similar to the high pH-sensitivity of pure P4VP chains.¹⁵ At low pH, the pyridyl groups are transformed to protonated pyridyl groups, fostering repulsion between the positively charged pyridyl motifs in P4VP macromolecules. Moreover, the creation of hydrogen bonds with oxygen of the water is here strongly facilitated (Schemes 2a and 2b).

Scheme 2 Hypothetical conformations of the van der Waals interactions and hydrogen bonding among the pyridyl groups of the PVP, the protonated pyridyl groups of the PVP, and the water molecules at various pH values and temperatures.

3.4. Thermal response of protein adsorption to P4PV-grafted brushes

Novel materials, especially with switching properties, are much-desired nowadays for their potential use as medical implants, drug delivery carriers, biosensors, and membrane materials.^48–51 One of the most important properties of those materials is their ability to change protein adsorption strongly at different temperatures. It is well know that the non-fouling properties of materials prevent nonspecific protein adsorption and cell adhesion due to the hydration layer near their own surface,⁵² which forms a physical and energetic barrier for protein adsorption on the surface. In the present work, BSA and human fibrinogen labeled with Alexa Fluor were used as model proteins to evaluate protein adsorption to the oligoperoxide-graft-P4VP coating using fluorescence microscopy. The results of the adsorption experiments are presented in Fig. 5. The representative micrographs correspond to fluorescently labeled BSA and human fibrinogen adsorbed from phosphate saline buffer (pH = 7.4) to the as-prepared oligoperoxide-graft-P4VP coatings at two different temperatures of 10 °C and 20 °C (Fig. 5a and b). Direct examination of the micrographs reveals a significant change of the fluorescence intensity, proportional to the quantity of adsorbed proteins, from hardly visible at 10 °C for both proteins to very strong at 20 °C for BSA and strong for fibrinogen.

Fig. 5 Representative fluorescence micrographs of BSA (a) and human fibrinogen (b) proteins (labeled with Alexa Fluor) adsorbed to the as-prepared oligoperoxide-graft-P4VP coating at temperatures of 10 °C and 20 °C and the determined fluorescence intensities of proteins adsorbed to the oligoperoxide-graft-P4VP (columns) and glass reference samples (dashed line) (c).

The fluorescence intensities determined from the images⁵³ are presented in Fig. 5c. At 10 °C, BSA and fibrinogen adsorption to the grafted P4VP brushes is very weak. In contrast, when the temperature is elevated to 20 °C, protein adsorption increases almost 4 times for BSA, and is now more effective than for the control glass sample. A similar effect is observed for fibrinogen. However, for elevated temperatures, fibrinogen adsorption increases only twice, comparable to the control glass sample. The strong temperature sensitivity of BSA and fibrinogen adsorption is most probably related to the change in conformation of the P4VP brushes, induced by disruption of the hydrogen bond between the pyridyl groups and water.

4. Summary and conclusions

The influence of temperature on the properties of P4VP coatings, whereby they mimic the typical behavior of materials with a lower critical solution temperature (LCST), is presented here for the first time. To examine the temperature-dependent characteristic of P4VP, two types of coatings were prepared, namely, spin-coated thin P4VP films and P4VP brushes graft-polymerized “from the surface” of oligoperoxide, grafted to a native glass surface functionalized with APTES. Measurements of the water contact angle revealed temperature-induced changes in the wettability of both P4VP coatings, larger than 20° for the grafted P4VP brushes and nearly 10° for the spin-coated P4VP films, with a transition at T_c = 13–14 °C. The molecular arrangement of P4VP polymers, different for both types of coatings, plays the key role in influencing the magnitude of the thermal response. The influence of the humidity on the contact water contact angle values around the wettability transition was examined at different humidities. At higher humidity, the magnitude of the thermal response of the wettability was larger, with a more pronounced effect observed for the P4VP-grafted brushes. In addition, immersing the dry samples in water for a short time resulted in an increased magnitude.

The temperature dependence of the water contact angle was measured at two pH values, namely 2 and 9, for both coatings. The P4VP coatings did not exhibit any thermal response of wettability at pH = 2, where almost all the pyridyl groups were transformed to protonated pyridyl groups. This indicates that the thermal transition is not possible in protonated P4VP. For alkaline conditions, i.e., pH = 9, the thermal response of the wettability was observed at 12–15 °C, with a temperature-dependent behavior similar to the properties of the as-prepared P4VP coatings. The AFM images showed relatively flat surfaces with rare elevation for low pH values at 10 °C and 20 °C. In contrast, for an alkaline environment, the recorded topography changed noticeably with increasing temperature—from relatively smooth at 10 °C to structured and rough at 20 °C. A hypothetical scenario of van der Waals interactions and hydrogen bonding between the pyridyl groups of the P4VP, the protonated pyridyl groups of the P4VP, and the water molecules at various pH and temperatures for the P4VP coatings was proposed.

The reported experiments showed that BSA and human fibrinogen adsorption to the grafted P4VP brushes is very weak at 10 °C. In contrast, when the temperature is elevated to 20 °C, protein adsorption increases almost 4- and 2-fold, respectively. We, thus, have demonstrated here the strong temperature dependence of protein adsorption and of the thermal response of the wettability, which, in addition to the reported literature pH-response, quaternization, and protonation of pyridyl groups, makes the P4VP coatings a noteworthy switching material for biomedical and medical applications, such as in implants, drug delivery carriers, biosensors, and membrane materials.

Acknowledgements

The research reported in this work was partly funded by financial support in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08) and M. Smoluchowski Kraków Scientific Consortium in the framework of the KNOW (grant FOCUS). Dr Yurij Stetsyshyn also acknowledges for the financial support from the hetman Ivan Vyhovsky Foundation.

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Footnote

† Electronic supplementary information (ESI) available: Scheme of the procedure used to prepare P4VP grafted brushes, X-ray photoelectron spectroscopy data confirming surface coverage with P4VP grafted brushes. See DOI: 10.1039/c6ra07223b

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