Grafting-To - American Chemical Society

Chapter 8

Downloaded by UNIV OF ARIZONA on December 14, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch008

Control of Protein Adsorption and Cell Adhesion by Mixed Polymer Brushes Made by the “Grafting-To” Approach Eva Bittrich,1 Sina Burkert,1 Klaus-Jochen Eichhorn,1 Manfred Stamm,1,2 and Petra Uhlmann*,1 1Leibniz

Institute of Polymer Research Dresden, Hohe Strasse 6, D-01069 Dresden, Germany 2Technische Universität Dresden, Physical Chemistry of Polymer Materials, 01062 Dresden, Germany *E-mail: [email protected]

The capability of mixed polymer brush systems to modify and switch physico-chemical properties of biointerfaces is demonstrated in this paper. Mixed brushes were composed of the temperature sensitive polymer poly(N-isopropyl acrylamide) (PNIPAAM) and two polyelectrolytes, poly(2vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). Using these polymers binary brushes were prepared, the first one consisting of PNIPAAM – P2VP representing a system sensitive for temperature and pH changes, and the second one consisting of two polyelectrolytes (P2VP-PAA) showing sensitivity to pH changes of the surrounding fluid. Selected aspects concerning the environmental sensitivity as well as the protein adsorption affinity of these brushes are discussed. The stimuli-response towards temperature (PNIPAAm-P2VP) and pH (P2VP-PAA) of these binary polymer brushes was utilized to switch the adsorbed amount of protein and finally to regulate cell adhesion at brush coated surfaces.

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


1. Introduction Thin polymer layers have been proven to be able to modify physico-chemical interface properties to control the adsorption behaviour of proteins at artificial surfaces. Here two approaches are interesting for application. One strategy is to create biocompatible surfaces by modifying the interface of artificial material with protein resistant surface layers (1, 2), since the exposure of such foreign materials to blood without protein resistance may lead e.g. to the unwanted adsorption of plasma proteins and platelets resulting in surface-induced thrombosis (3). On the other hand the creation of functional interfaces that can be loaded/unloaded with proteins for drug delivery or sensor applications in a controllable way is promising (4). Such biocompatible or functional interfaces can be provided by thin polymer brush films that are grafted chemically to a substrate. Brush films and have been investigated theoretically and increasingly experimentally in recent years (5–7).

1.1. Polymer Brushes The term “brush” is often used as synonym for a dense layer of chain-like polymers with one end attached to a substrate. Such layers may appear in different conformations (Figure 1) depending on the grafting density as well as on interaction with the substrate and the surrounding medium (8).

Figure 1. Different conformations of tethered polymer chains at a surface.

Several grafting techniques (9), such as the “grafting to” method, enable to covalently bind polymer chains with only one reactive end-group towards a surface. Here, a very thin film of poly(glycidylmethacrylate) (PGMA) is used to enable the formation of chemical bonds with the oxidic substrate as well as with the in this case carboxy terminated polymer chains (Figure 2) (10, 11). Owing to better characterization possibilities silicon wafers are used as substrates but also other substrates like glass, metals or artificial tissues are possible (12–14). The covalent linkage of the polymer to the surface has several advantages, as easy and controllable introduction of polymer chains with high surface density, precise localization of the chain at the surface, and the possibility to graft different types of polymers onto the same substrate. 180 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. Schematic “grafting-to” mechanism of COOH-functionalized polymer chains. By using these polymer brushes it is possible to generate versatile surfaces with reproducible interface functionalization as well as stable homogeneous (with a certain order in z-direction) and adaptive smooth thin polymer films with a roughness of less than 1 nm (15–17). By grafting of two (or more) incompatible polymers mixed brushes can be formed that are characterized by switchable physico-chemical surface properties (18–20). These brushes represent polymer systems with remarkable responsive properties, i.e. they are able to adapt and respond to external fields and environmental conditions like temperature, pH or solvent (Figure 3) (21, 22). Mixed brushes are characterized by a collaborative response of the grafted chains of one brush component to environmental changes.

Figure 3. Mixed polymer brushes and their possible switching behaviour including the adsorption and desorption process. We used binary brushes of weak polyelectrolytes consisting of poly(2vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). Generally, the degree of dissociation of weak polyelectrolytes (annealed brush) depends on the pH value, and the thickness results from a balance of the osmotic pressure of the counterions inside the brush and the configurational elasticity of the chains (23). In the osmotic regime, with a low concentration of additional ions in the surrounding solution, the ion concentration inside the brush is higher than outside and most of the counterions condense inside the brush. One of the used polylelectrolytes (P2VP) was combined with poly(N-isopropyl acrylamide) (PNIPAAM), a polymer well known to be water soluble at room temperature. Besides it shows a phase transition to a water insoluble state when crossing its lower critical 181 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


solution temperature (LCST) of 32°C. It was recently shown that even in the brush conformation (at relatively low grafting density and in very homogeneous brushes made by the grafting-to approach) this polymer is showing the expected phase transition (30), however, also above the LCST those PNIPAAm brushes keep their protein resistance, apparently because the structure of the interfacial water layer remains intact in the sense to prevent the adsorption of proteins yet (24). Polymer brushes, as very thin homogeneous coatings turn out to be accordingly due to their molecular conformation (chain conformation), surface mobility, steric effects induces by the brush conformation, and their variability very promising for the design of tailored biointerfaces. 1.2. Protein Adsorption The adsorption of proteins to surfaces, as the initial event when a material comes into contact with a biosystem, is driven by various forces, including hydrophobic and electrostatic interaction, entropic driving forces as well as hydrogen bonding (25). For charged polymers, e.g. polyelectrolytes, electrostatic interaction with proteins and changes in the entropy of the system during adsorption can be identified as major driving forces for adsorption. Dependent on the pH of the environmental solution and on the isoelectric points (IEP) of the involved polyelectrolytes and proteins, equal or opposite surface charges of the grafted polymers and proteins can be adjusted respectively, leading to controlled adsorption and desorption processes (26). The influence of the amount of counter ions inside a mono brush on the entropic driving forces of the adsorption process was recently shown by investigating the adsorption of model proteins on spherical polyelectrolyte brushes (27). Here sodium or potassium ions in aqueous solution often act as counter ions which are responsible for local charge neutrality in polyelectrolyte brushes. The influence of buffer molecules on adsorption and desorption of a model protein at a PAA brush was recently shown by using in-situ a combination of a quartz microbalance (QMC-D) with spectroscopic ellipsometry (28). Non-charged water soluble polymers like the well investigated poly(ethylene glycol) (PEG) or the already mentioned poly(N-isopropyl acrylamide) are especially in a brush conformation very promising (29), because brush parameters as grafting density and molecular weight can be well adjusted to prevent primary and ternary protein adsorption by steric repulsion, additionally these polymers are likely to undergo stable interactions with water molecules making the formation of stable water layers at the surface likely, which also contributes to resistance to non-specific protein adsorption (30). Those protein resistant surfaces are important for applications in the field of bioprocessing, sensors, blood contacting devices, contact lenses etc. An interesting option to create advanced biomaterials are surfaces showing a controlled and/ or changing bioresponse triggered by stimuli as changing environmental conditions or signalling molecules. A first step in this direction is the creation of surfaces that induce protein adsorption/ desorption in a controlled manner, and are able to switch between activity and inertness in this sense. To achieve this it is essential to understand the mechanism of protein adsorption to be able to control the driving forces of adsorption 182 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

which are mainly enthalpic / entropic, van der Waals, electrical double layer and hydrophobic forces (29, 30). We discuss some selected aspects of how the environmental sensitivity of mixed brushes can be used to regulate protein adsorption affinity and cell adhesion.

2. Experimental


2.1. Materials All polymers (see Table 1) were purchased from Polymer Source, Inc. (Canada). Highly polished single-crystal silicon wafers of {100} orientation with a native SiO2 layer thickness of about 2 nm were used as substrates (Si-Mat Germany). An elaborate basic cleaning procedure of the silicon wafers enables the formation of homogeneous dense and adhering brush layers. Poly(glycidyl methacrylate) (PGMA) acted as adhesion promoter between substrate and the polymer layer. All dry layer thicknesses were determined by ellipsometry at λ=632.8 nm (SE-402 from Sentech Instruments GmbH) (see table 1) with fixed refractive indices (22).

Table 1. Used polymers with their corresponding dry layer thickness (d) and refractive index (n) in a homopolymer brush conformation polymer

notation

Mn [g/mol]

PDI

d [nm]

n (λ=632.8 nm)

poly(glycidyl methacrylate)

PGMA

17,500

1.7

2.3 ± 0.1

1.525

poly(2-vinylpyridine)a

P2VP

40,600

1.08

6 ± 0.2

1.595

poly-N-isopropylacrylamidea

PNIPAAM

66,000

1.35

14.5± 0.2

1.490

poly(tert-butyl acrylate)a

PtBA

42,000

1.12

7.4 ± 0.2

1.466

a

Those polymers are end-functionalized with one COOH group, which reacts with PGMA.

0.01M phosphate buffered saline solution (Aldrich, Germany) as high salt solutions and 0.01M acetate buffer (acid pH) as well as 0.01M trishydroxymethylaminomethane (TRIZMA) buffer (basic pH) as low salt solutions were used for adsorption measurements, simulating biological conditions. For the adsorption measurements human serum albumin (HSA), a plasma protein from human blood, and α-chymotrypsin, an enzyme of the bovine pancreas, were used (Sigma-Aldrich). The isoelectric points of these proteins are pH 4.7 and pH 8.1, respectively (22, 31). 183 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


2.2. Preparation of the Polymer Brushes The cleaned substrates were spin-coated with 0.02 wt% solution of PGMA in tetrahydrofuran (THF) and put in a vacuum oven for 20 min at 100 °C. To obtain mono polymer brush layers, 1 wt% for all polymers in THF was spin-coated on the PGMA layer. The adhesion was performed by annealing the coated substrate in a vacuum oven at 150 °C over night for all homopolymer brush layers. Noncovalently bound polymer was removed by extraction in the respective solvent. The mixed polymer brush system consisting of P2VP-PNIPAAM was prepared in a similar way. After deposition of PGMA on the substrate primarily 1 wt% of P2VP in THF solution was spin-coated. The grafting was performed at 150 °C for 5 minutes in vacuum. Secondly, after THF extraction, 1 wt% solution of PNIPAAM in THF was spin-coated and annealed in the similar way (170 °C, 3 h, extraction in THF). The PAA-P2VP brushes were prepared in a slightly different way. Firstly 1 wt% of PtBA in THF solution was spin-coated, grafted at 150°C for 12 min and non-covalently bound polymer removed by extraction. Secondly P2VP was spincoated, annealed over night at 150°C in an vacuum oven and the wafers extracted again afterwards. In a last step the PtBA was hydrolized to PAA in a benzene solution saturated with p-toluene sulfonic acid under reflux at 55°C for 1h. 2.3. Surface Analysis Methods Comprehensive analysis methods were used to carefully characterize the polymer brush layers and their behaviour towards bio-molecule adsorption. In Table 2 all techniques with their relevant parameters are mentioned. In-situ protein adsorption experiments were performed with null-ellipsometry using the experimental setup shown in Figure 4. The adsorbed amount of protein Γ was calculated from the ellipsometrically measured protein layer thickness by using an equation that was originally proposed by de Fejter:

In which dp is the layer thickness of the adsorbed protein layer, np is the refractive index of the protein layer, nl is the refractive index of the ambient solution, and dn/dc is the refractive index increment of the protein (33). This method is based on experiments showing that the refractive index of a protein in aqueous solution is a linear function of the protein concentration (34). It was desired to model the brush and the protein layer individually in a two layer model with nprotein=1.375 of an aqueous protein top layer (35). However if this model was not applicable a combined “composite” layer of swollen polymer brush and “dry” protein was assumed and both effective n and d values fitted for the combined layer leading to a total adsorbed amount of the combined layer of protein and polymer brush. 184 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

To evaluate the adsorbed amount of protein the amount of polymer was finally subtracted from the total amount, whereas the amount of polymer was calculated from the dry layer thickness. This model was mainly used for charged brushes, since proteins are most likely to penetrate into these brushes during the adsorption process (26).


Table 2. Characteristic parameters of used techniques Method

Company

Measuring principle

relevant information

In-situ spectroscopic ellipsometry (λ= 428 … 763 nm)

M44, J. A. Woollam Co., Inc., Lincoln, NE, USA

Changes of the polarized light upon specular reflection

Swollen layer thick-ness and refractive index of the brush

In-situ singlewavelengthnull-ellipsometry (Figure 4, λ=632.8 nm)

Multiscope, Optrel, Berlin, Germany

Streaming(Zeta)Potential

ElectroKinetic Analyser Anton Paar KG, Austria (32)

Electro kinetic surface properties

Zeta-potential, isoelectric point (IEP)

Contact Angle

OCA20, Dataphysics, Germany

Surface free energy and wettability

contact angles

Ellipsometric angles after protein adsorption

Figure 4. Null-ellipsometry setup: Brush coated silica wafers were inserted into a cuvette, fixed and immersed in buffer solution. Concentrated protein solution was added after the swelling process. 185 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

3. Results and Discussion


3.1. Physico-Chemical Surface Properties We are presenting two mixed brush systems: P2VP-PNIPAAM and PAAP2VP where changes in the physico-chemical surface properties could be achieved by the stimuli temperature or pH, respectively. For the first system we additionally investigated the wetting behaviour and the surface charge properties of the mono brushes PNIPAAM, and P2VP and compared them with PNIPAAM-P2VP mixed brushes. PNIPAAM containing brushes are interesting, because these brushes undergo a phase transition from a more hydrophilic to a more hydrophobic state at their lower critical solution temperature (LCST) at 32°C (36, 37). Since this switching temperature can be modified, for example by copolymerisation (38), these mixed brushes could be suitable for biomedical applications. In Figure 5 the temperature dependence of the respective contact angles is presented. For P2VP no changes in the advancing contact angle in the range of 20°C to 40°C could be measured whereas PNIPAAM mono brushes showed the expected temperature dependent increase of the advancing contact angle towards a more hydrophobic surface (Figure 5a).

Figure 5. (a) Temperature dependent advancing contact angles of PNIPAAM and P2VP mono brushes. (b) Temperature dependent advancing contact angles of P2VP-PNIPAAM mixed brushes with different compositions. This change in hydrophilicity was also observed for P2VP-PNIPAAM mixed brushes where the contact angle differences between 20°C and 40°C increased with the amount of PNIPAAM in the binary brush (Figure 5b). Surprisingly, the mixed brush surface was found to be more hydrophobic at higher temperatures than the PNIPAAm mono brushes, which could indicate an interaction between PNIPAAm and P2VP chains. Information about surface charge states of these P2VP-PNIPAAm brushes was gained by zeta (ζ) potential measurements (39), as from zeta potential curves the isoelectric point (IEP) of the brush coated surface at ζ = 0 was determined. In Figure 6 the pH at the IEP (pHIEP) is plotted for different compositions of P2VP-PNIPAAM. It was observed that PNIPAAM strongly influenced the surface properties even at low concentrations. 186 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 6. Isoelectrical point (IEP)for different compositions of P2VP-PNIPAAM mixed brushes. The second system we are presenting is a PAA-P2VP mixed brush consisting of two different polyelectrolytes. For the pH sensitive switching behaviour of this mixed brush and its corresponding mono brushes the dependency of the surface potential and the contact angle on the pH was reported in literature (40). Here swelling measurements are presented for PAA and P2VP mono brushes as well as a 50:50 mixed brush in Figure 7.

Figure 7. Swollen layer thicknesses of PAA and P2VP mono brushes as well as PAA-P2VP mixed brushes measured with spectroscopic ellipsometry. Since PAA is a weak acid and P2VP a weak base both polymers have dissociable groups and the degree of dissociation depends on the pH (25, 26). The amount of COO- groups in the PAA brush increases above its IEP of pH 2.1 whereas the amount of NH+ groups in the P2VP brush decreases until this polymer is neutral at its IEP at pH 6.0. Furthermore counterions from solution (e.g. Na+, Cl-) diffuse into these charged brushes to preserve local charge neutrality. Hence for a fixed ionic strength of the surrounding solution the thickness and the refractive index of the swollen polymer layers vary with pH because of different 187 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

degrees of stretching of the chains due to the repulsion of equally charged monomers as well as the gain of entropic energy by the separation of counterions (41).This leads to an increase of layer thickness with increasing pH for PAA and a decrease with increasing pH for P2VP mono brushes. For the mixed brush a qualitative combination of the swelling behaviour of the individual components is visible but the total swollen layer thickness is considerably lower than the one for the mono brushes. In the pH range from 3.2 to 6.7 the negatively charged PAA and positively P2VP chains interact leading to an uncharged surface at pH 4.9 (39).


3.2. Stimuli Responsive Adsorption of Proteins Having studied the surface properties of the two mixed brushes, aspects of their influence on protein adsorption will now be presented. We firstly present temperature sensitive adsorption experiments at P2VP-PNIPAAM brushes for two different pH in Figure 8 and will show the pH-dependent reversibility of adsorption at P2VP-PAA brushes (Figure 9), giving two examples of tunable adsorption processes at mixed brushes. For the P2VP-PNIPAAM mixed brush the adsorbed amount of human serum albumin (HSA) dependent on brush composition and two different temperatures (22 °C, 40 °C) is displayed in Figure 8a for measurements at pH 4 and in Figure 8b at pH 7.4. For both pH values a higher adsorbed amount of protein was observed at 40 °C, above the LCST of PNIPAAm, as compared to the adsorption at room temperature. Additionally the protein amount increased with increasing P2VP content. Comparing the measurements at pH 4 and pH 7.4 a higher adsorbed amount was found at pH 4 for both temperatures and all brush compositions, most pronounced for high P2VP content. For a comprehensive explanation of these findings more information on the brush-solution interface in-situ would be needed. However a deswelling with temperature of the P2VP-PNIPAAm brushes with the ratios 20:80 and 50:50 could be shown by in situ spectroscopic ellipsometry at pH 7.4 (42). Thus, the collapse of PNIPAAm chains could be hold responsible for the increase of adsorbed amount with temperature. This is highly interesting since comparable PNIPAAm mono brushes proved to be protein resistant below and above the LCST (24). In general the resistance of PNIPAAm brushes above the LCST was found to depend on molecular weight and grafting density (43). Thus the low density of PNIPAAm chains in the mixed brushes could be the reason for the observed effect. Also an effect of the P2VP is probable. At pH 4 both P2VP and HSA are positively charged and adsorption takes place under repulsive conditions, whereas at pH 7.4 P2VP is neutral and hydrophobic forces are most likely to drive the adsorption process. Additionally, the IEP of the mixed brush shifts with the ratio of components (see Figure 6), indicating a change in the charge conditions inside the brush with composition. Thus electrostatically driven protein adsorption should be affected. An interaction of PNIPAAm and P2VP chains is possible as well and was indicated by a higher hydrophobicity of the surfaces as measured by the contact angle (Figure 5). 188 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 8. a,b: Adsorbed amount of HSA on various P2VP-PNIPAAM mixed brushes at two pH values, different surface composition and temperature.

In summary tuning of the adsorbed amount of 1 mg/ml HSA in 0.01 M PBS solution on P2VP-PNIPAAM mixed brushes by brush composition, pH and temperature could be shown, and possible mechanisms for the observed adsorption behavior were discussed. A detailed explanation of the effects leading to the observed protein amounts is desired, but needs more characterization of this rather complex behaving soft matter film. The reversibility of switching the adsorbed amount of protein sensitive to pH changes is presented for a PAA-P2VP mixed brush (Figure 9). Adsorption of α-chymotrypsin (IEP: 8.1) was carried out at pH 7.8 in 0.01M TRIZMA buffer and very high amounts of protein could be adsorbed. The counter ion release mechanism is discussed in literature to explain this high adsorption affinity (25, 26). In the presented experiments adsorption takes place at electrostatic attractive conditions between the positively charged protein and the negatively charged surface due to COO- groups at the swollen PAA chains. Upon adsorption protein molecules replace counter- and co-ions in the brush layer and at the brush solution interface. Thus, this mixed brush system serves as a support for ion exchange, in the presented case due to the charged PAA chains. After adsorption at pH 7.8 the buffer was exchanged to pH 4 (0.01M acetate buffer). At this pH positively charged P2VP is dominating the surface of the PAA-P2VP mixed brush and the electrostatic conditions become repulsive, thus leading to desorption of the protein. With this approach the adsorbed amount could be switched between 26 mg/m2 and 0.5 mg/m2 which is equivalent to a release of 98% of the adsorbed amount of protein into the surrounding solution. Also the reversibility of this adsorptiondesorption process was tested and after three cycles the adsorbed amount was still 95% of that of the initial adsorption. Nevertheless long-time reversibility measurements were not done so far but will be considered in the future. 189 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 9. Reversible switching of the adsorbed amount of a-Chymotrypsin at PAA-P2VP brushes shown for three switching cycles.

3.3. Cell Adhesion onto P2VP-PNIPAAM Brushes L929 mouse fibroblasts were obtained from DSMZ, Braunschweig, Germany and cultivated in RPMI medium (RPMI 1640; Pan Biotech GmbH, Aidenbach, Germany) containing 10% fetal calf serum and antibiotics. Further details of cell cultivation are described herein (44). Thin glass slides were used as substrates. Polymer brushes were grafted on those slides as described in section 2.2. The brushes were subsequently conditioned at 37°C in PBS solution. In the next step PBS was exchanged with the tempered cell culture medium. After a period of 30 min L929 mouse fibroblasts with a density of 7.7·104 cells/cm2 were seeded while keeping the temperature at 37°C. The cultivation of the cells at 37°C was proceeded until confluence of the cells was reached. Images at room temperature were made after a cooling time of six hours. L929 mouse fibroblasts were found to become adherent on P2VP mono brushes (see Figure 10). A confluent monolayer was reached after 24h. By contrast no proliferation and adherence was seen for PNIPAAM mono brushes. The cells seem to agglomerate in small groups. Regardless, all three polymer systems showed no cell toxicity during the measuring time of three days. The following images were made using a common light microscope; brightness and contrast depend on manual adjustment.

Figure 10. L929 mouse fibroblasts cell growth images at 37 °C on a) P2VP and b) PNIPAAM mono brushes, similar images were made at 22°C. 190 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Taking a mixed brush system we wanted to combine cell adhesion and no adhesion by temperature sensitive switching. P2VP-PNIPAAM showed very promising results (Figure 11).

Figure 11. L929 mouse fibroblasts cell growth images on a-c) P2VP-PNIPAAM (80:20) mixed brushes at different temperatures. We were able to induce a reversible switching of the cell sheet by changing the surrounding conditions. In Figure 11a typical spindle-shaped cell morphology is seen for P2VP-PNIPAAM at 37°C. The good proliferation enables the formation of a confluent monolayer. When switching to 22°C PNIPAAM is dominating the surface and the cells start to roll together and agglomerate. The reverse switching to 37°C is clearly shown in Figure 11c, where a confluent monolayer of cells is formed again. This resembles the results we obtained for the protein adsorption measurements. A switching was also seen for mixed brushes with a composition of 50:50 and 20:80. However in the case of 80:20 brushes the switching effect is the greatest.

4. Conclusions We demonstrated the tuning and switching of the physico-chemical surface properties and hence the affinity to adsorb biological components, i.e. proteins and cells, for selected combinations of polymers into binary mixed brushes. In the first system, we presented a temperature sensitive mixed brush, P2VP-PNIPAAM, with its surface properties tunable from hydrophilic to more hydrophobic. This dewetting behaviour was accompanied with an increased affinity for protein adsorption and cell adhesion for higher temperatures, in for example switching the adsorbed amount of HSA from 0.5 mg/m2 to 2 mg/m2 for a (80:20) P2VP-PNIPAAM mixed brush. The surface potential and adsorption measurements show, that PNIPAAM strongly influenced the physico-chemical surface properties of the mixed brush, which could be used to design a switchable surface in the desired manner. Furthermore the reversibility of the pH sensitive swelling due to changes in the electrostatic charges along the polymer chains for a fixed ionic strength of the buffer solution was demonstrated for P2VP-PAA mixed brushes. Very high adsorbed amounts of α-chymotrypsin of 26 mg/m2 could be detected around the IEP of the protein at pH 7.8. This adsorption behaviour was used to release high 191 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

amounts of protein (25 mg/m2) into the buffer solution when switching the PAAP2VP brush in changing the pH to electrostatic repulsive conditions at pH 4. The reversibility of the adsorption and desorption was proven for three cycles with only marginal decreases in the adsorption affinity and a remaining adsorbed amount of 95% of the initial adsorption before the third release process.


Acknowledgments The work was kindly supported by the Deutsche Forschungsgemeinschaft (DFG) in the frame of the Sonderforschungsbereich 287 “Reaktive Polymere in nichthomogenen Systemen, in Schmelzen und an Grenzflächen” within the particular project B10 and in the DFG-NFS cooperation project “Design of new responsive materials based on functional polymer brushes for smart tuning and sensoring of proteins and particles adsorption” in the frame of “Materials World Network”. For performing the cell adhesion experiments we thank T. Götze and C. Werner, Max-Bergmann Center, Leibniz IPF Dresden.

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