Surface Modification by Polyzwitterions of the Sulfabetaine-Type, and Their Resistance to Biofouling

Films of zwitterionic polymers are increasingly explored for conferring fouling resistance to materials. Yet, the structural diversity of polyzwitterions is rather limited so far, and clear structure-property relationships are missing. Therefore, we synthesized a series of new polyzwitterions combining ammonium and sulfate groups in their betaine moieties, so-called poly(sulfabetaine)s. Their chemical structures were varied systematically, the monomers carrying methacrylate, methacrylamide, or styrene moieties as polymerizable groups. High molar mass homopolymers were obtained by free radical polymerization. Although their solubilities in most solvents were very low, brine and lower fluorinated alcohols were effective solvents in most cases. A set of sulfabetaine copolymers containing about 1 mol % (based on the repeat units) of reactive benzophenone methacrylate was prepared, spin-coated onto solid substrates, and photo-cured. The resistance of these films against the nonspecific adsorption by two model proteins (bovine serum albumin—BSA, fibrinogen) was explored, and directly compared with a set of references. The various polyzwitterions reduced protein adsorption strongly compared to films of poly(n-butyl methacrylate) that were used as a negative control. The poly(sulfabetaine)s showed generally even somewhat higher anti-fouling activity than their poly(sulfobetaine) analogues, though detailed efficacies depended on the individual polymer–protein pairs. Best samples approach the excellent performance of a poly(oligo(ethylene oxide) methacrylate) reference.

In an analogy to the synthesis of sulfobetaines by the ring-opening alkylation of tertiary amines with cyclic sulfonate esters, sulfabetaines can be readily synthesized without salt contaminants by the ring-opening alkylation of tertiary amines with cyclic sulfate esters. Despite the versatility of this approach, reports on poly(sulfabetaine)s have been scarce so far [43][44][45][46][47][48][49][50][51][52]. Most probably this apparent lack of interest is not due to the somewhat reduced hydrolytic stability of sulfabetaines compared to sulfobetaine analogues (which is still reasonably high [48,50]), but mostly due to the very low solubility of poly(sulfabetaine)s in water. In fact, the poly(sulfabetaine)s described so far exhibit typically upper critical solution temperatures (UCSTs) above 100 °C in pure water [43,47,49,50]. Large amounts of low molar mass salts must be added to induce their solubility at ambient temperature ("salting-in"). Although in special situations, e.g., in the case of thermo-responsive materials under physiologically relevant situations, this may be an advantage [51], the need of a strong salting-in scenario is mostly considered to be problematic.
Recently, we undertook an extended study on the structural variation of poly(sulfobetaine methacrylate)s (as exemplified in Figure 1) and its effect on the phase behavior in aqueous solution, in particular on the position of the aqueous UCST transition [38]. While empirically, the transition temperature could be effectively adjusted between 0 and 100 °C by straightforward variations of the chemical structure of the CRU, the results could not be rationalized by simple rules that enable a reliable prediction of the phase behavior of new variants Scheme 1. Generalized structure of major polyzwitterion classes. From left to right: Ammonio phosphates (I, specifically for R* = -CH 2 -CH 2 -: phosphatidylcholines), ammonio carboxylates (II, carboxybetaines), ammonio sulfonates (III, sulfobetaines), and ammonio sulfates (IV, sulfabetaines).
In an analogy to the synthesis of sulfobetaines by the ring-opening alkylation of tertiary amines with cyclic sulfonate esters, sulfabetaines can be readily synthesized without salt contaminants by the ring-opening alkylation of tertiary amines with cyclic sulfate esters. Despite the versatility of this approach, reports on poly(sulfabetaine)s have been scarce so far [43][44][45][46][47][48][49][50][51][52]. Most probably this apparent lack of interest is not due to the somewhat reduced hydrolytic stability of sulfabetaines compared to sulfobetaine analogues (which is still reasonably high [48,50]), but mostly due to the very low solubility of poly(sulfabetaine)s in water. In fact, the poly(sulfabetaine)s described so far exhibit typically upper critical solution temperatures (UCSTs) above 100 • C in pure water [43,47,49,50]. Large amounts of low molar mass salts must be added to induce their solubility at ambient temperature ("salting-in"). Although in special situations, e.g., in the case of thermo-responsive materials under physiologically relevant situations, this may be an advantage [51], the need of a strong salting-in scenario is mostly considered to be problematic.
Recently, we undertook an extended study on the structural variation of poly(sulfobetaine methacrylate)s (as exemplified in Figure 1) and its effect on the phase behavior in aqueous solution, in particular on the position of the aqueous UCST transition [38].
In an analogy to the synthesis of sulfobetaines by the ring-opening alkylation of tertiary amines with cyclic sulfonate esters, sulfabetaines can be readily synthesized without salt contaminants by the ring-opening alkylation of tertiary amines with cyclic sulfate esters. Despite the versatility of this approach, reports on poly(sulfabetaine)s have been scarce so far [43][44][45][46][47][48][49][50][51][52]. Most probably this apparent lack of interest is not due to the somewhat reduced hydrolytic stability of sulfabetaines compared to sulfobetaine analogues (which is still reasonably high [48,50]), but mostly due to the very low solubility of poly(sulfabetaine)s in water. In fact, the poly(sulfabetaine)s described so far exhibit typically upper critical solution temperatures (UCSTs) above 100 °C in pure water [43,47,49,50]. Large amounts of low molar mass salts must be added to induce their solubility at ambient temperature ("salting-in"). Although in special situations, e.g., in the case of thermo-responsive materials under physiologically relevant situations, this may be an advantage [51], the need of a strong salting-in scenario is mostly considered to be problematic.
Recently, we undertook an extended study on the structural variation of poly(sulfobetaine methacrylate)s (as exemplified in Figure 1) and its effect on the phase behavior in aqueous solution, in particular on the position of the aqueous UCST transition [38]. While empirically, the transition temperature could be effectively adjusted between 0 and 100 °C by straightforward variations of the chemical structure of the CRU, the results could not be rationalized by simple rules that enable a reliable prediction of the phase behavior of new variants While empirically, the transition temperature could be effectively adjusted between 0 and 100 • C by straightforward variations of the chemical structure of the CRU, the results could not be rationalized by simple rules that enable a reliable prediction of the phase behavior of new variants for such stimuli-responsive polymers. The studies nevertheless demonstrated the strong effects of apparently small chemical variations on their water-solubility. In particular polymers of monomers 2a (P−2a), 4a (P−4a), and 5a (P−5a) were found to feature unusually low UCST transitions, and thus to exhibit only very small miscibility gaps in water, if at all [38]. Based on these findings, we synthesized two series of poly(sulfabetaine)s (Figure 1, series c and d) that are analogously designed to previously described series of poly(sulfobetaine)s ( Figure 1, series a and b), and explored them with respect to their water-solubility and possible improvements. Furthermore for selected examples, we explored their utility for low-fouling coatings, as initial studies on polymers P−1d and P−3d had indicated good resistance against the adsorption of proteins [12,52].

Instrumentation and Methods
1 H and 13 C NMR spectra, 1 H-1 H-correlation spectra (COSY) and 1 H-13 C-hetero-nuclear multiple quantum coherence spectra (HMQC) were recorded with an Avance 300 spectrometer (Bruker, Billerica, MA, USA, 300 MHz and 75 MHz, respectively) at ambient temperature in deuterated solvents. 13 C spectra were recorded in the 1 H-broad band decoupling mode and attached proton test (ATP) mode, respectively. Solvent signals or 3-(trimethylsilyl)propionic-2,2,3,3-d 4 acid sodium salt were used as internal shift references. High resolution mass spectra (HR-MS) were recorded with a Thermo Scientific ESI-Q-TOF micro (quadrupole-time of flight; Thermo Fisher Scientific, Waltham, MA, USA) with electrospray ionization (ESI) using water as the solvent. Element analysis was conducted by using a Vario ELII microanalyzer (Elentar Analysensysteme, Hanau, Germany). FT-IR spectra were recorded in a N 2 purged atmosphere with a Nicolet Nexus FT-IR spectrometer (Thermo Fisher Scientific) equipped with an attenuated total reflection (ATR) smart endurance element. Absorption spectra in solution were recorded with a UV/VIS/NIR two beam spectrometer (Lamda 19, Perkin Elmer, Waltham, MA, USA). Thermogravimetric analysis (TGA) was conducted under N 2 purged atmosphere with a TGA/SDTA 851e (Mettler-Toledo GmbH, Giessen, Germany) using a heating rate of 10 K min −1 . The occurrence of thermal transitions was verified by differential scanning calorimetry with an apparatus DSC 822e (Mettler-Toledo), using heating and cooling rates of 10 K min −1 . Temperature accuracy was calibrated with the onset of the melting peaks of indium (156.6 • C) and of zinc (419.6 • C). Size exclusion chromatography (SEC) for polyzwitterions was run with an apparatus SEC3010 (WGE-Dr. Bures, Dallgow-Döberitz, Germany) equipped with a refractive index detector and PL-HFIP gel columns (Agilent Technologies, Santa Clara, CA, USA), using hexafluoroisopropanol (HFIP) containing 50 mM of sodium trifluoroacetate as eluent (flow rate 0.8 mL min −1 ), and poly(methyl methacrylate) "PMMA" standards (500 to 520,000 Da, narrowly distributed, PSS Polymer Standard Service, Mainz, Germany) for calibration.

Film Preparation and Protein Adsorption Studies
Pieces of 2.0 cm × 2.0 cm cut from silicon wafers (0.725 mm thickness, orientation <100> front polished, Silicon Materials, Kaufering, Germany). Silicon, quartz (Suprasil, 45 mm × 12 mm × 1 mm, Hellma Analytics, Müllheim, Germany) and glass substrates (2.5 cm × 2.5 cm, cut from microscope slides, VWR) were washed and hydrophilized for 6 h in a solution of 100 mg of potassium permanganate in 40 mL of concentrated sulfuric acid. The substrates were washed three times with water, then with ethanol and finally with toluene. Subsequently, their surfaces were modified via silanization to enable the photo-grafting of the copolymers. Samples were immersed in 1 vol % solutions of (3-aminopropyl) dimethylethoxysilane in toluene, for 12 h. Then, they were taken out of the solutions, washed with toluene and ethanol, and dried under nitrogen flow. Solutions of the copolymers in TFE were spin-cast onto the pre-treated substrates with a spin-coater (KL-SCV, Schaefer technologies, Langen, Germany). Exceptionally, reference P−xBMA was spin-cast from ethyl acetate solutions for experiments using fluorescence microscopy, as films cast from TFE solutions are rather rough [52]. One droplet of a 1-2.5 wt % solution was dispensed on the rotating wafer, and the copolymer was cast at 40-80 rotations per seconds for 10 s. Precise concentrations and rotation speeds were adjusted for each sample to produce equally thin coatings of 110 ± 10 nm thickness. Films were cured for 30 min under UV-light in a UVACUBE 100 radiation chamber (Hönle, Gräfelfing, Germany) at ambient temperature in air, using a 100 W iron doped mercury vapor lamp equipped with sheet glass as a filter (spectral cut off 310 nm). The distance of the sample from the light source was 20 cm. After photo-curing, the samples were immersed in three water washing baths, while being agitated, then removed, dried by a gentle nitrogen stream, and stored at ambient temperature in the dark.
Water-air contact angles were determined by the sessile drop method using an apparatus OCA 15EC equipped with the software module SCA 20 (Dataphysics Instruments, Filderstadt, Deutschland). Drops of 5 µL of Millipore water were placed on the sample, and pictures were taken with a USB-camera after 5 s. The given values were arithmetic averages of measurements of eight samples taken at two different positions.
The adsorption of fluorophore-labeled proteins was studied with a fluorescence microscope type CKX41 (Olympus Europa GmbH, Hamburg, Germany), using the filter set Cy5 ET (AHF Analysentechnik AG, Tübingen, Deutschland). Fluorescence micrographs were taken with 5 s of exposure, using tenfold magnification and signal amplification of 7.2 db. The relative fluorescence intensities were quantified using the software cellSens Dimension (Olympus). Experiments were performed applying a standard protocol. First, the fluorescence background of the uncontaminated glass support was determined. Then ca. 20 µL of protein solution (0.5 mg of fluorophore-labeled protein in 1 mL of freshly prepared phosphate buffered saline, PBS) were placed on the glass. After 2 min, the substrate was rinsed for 8 s with PBS, subsequently for 8 s with Millipore water, and finally dried in a gentle nitrogen stream. Then, the fluorescence intensity of the protein exposed sample was determined. Adsorption experiments were performed at least thrice for all proteins, with a minimum of six pictures taken at different positions of each sample. The relative amounts of adsorbed protein determined represent arithmetic averages of the fluorescence intensities after subtracting the background.

Monomer and Polymer Synthesis
As described for monomers 1c, 1d, 3c, 3d, 6c, and 6d [50], the new monomers 2c, 2d, 4c, 4d, 5c, and 5d were prepared by reaction of the respective tertiary amine functionalized methacrylates with cyclic sulfate diesters in acetonitrile, namely with ethylene sulfate or propylene sulfate. The methacrylate intermediates bearing tertiary amines were synthesized by transesterification of methyl methacrylate with the corresponding amino alcohols [38]. In all cases, a 10% molar excess of the tertiary amine intermediate with respect to the alkylating agent was employed, and rather long reaction times were used, in order to assure maximum consumption of the cancerogenic cyclic sulfates. As the zwitterionic products precipitate readily from the reaction solution, their separation and purification is straightforward. The analytical data (from elemental analysis, mass spectrometry, 1 H-NMR and the products. Via free radical polymerization, the various sulfabetaine monomers were smoothly converted into their homopolymers. Polymerizations were conducted in homogeneous solution in trifluoroethanol (TFE), which is one of the few effective solvents for most zwitterionic monomers and their polymers [30,38,54]. All polymers were modestly hygroscopic and colorless solids. Key analytical data of the homopolymers are summarized in Table 2. The apparent molar masses of the various polymers were rather high, being in the range of 130 to 420 kg mol −1 . Polymer dispersities Ð were in the range of 2 to~4, in agreement with a standard free radical polymerization process conducted up to high yields.

General Properties of the Homopolymers
The thermal behavior of the poly(sulfabetaine)s in bulk was studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA of the hygroscopic polymers under inert gas revealed three temperature regimes of weight loss (Figure 2). When the samples had been previously handled in air, bound water was lost at temperatures between 80 up to 150 • C, typically amounting to about 5-10 wt % for the poly(sulfabetaine)s. This weight loss is reversible, and corresponds to approximately one water molecule per constitutional repeat unit (CRU). This first weight loss seems somewhat less pronounced for the poly(sulfabetaine)s than for their sulfobetaine analogues, suggesting a lower hygroscopy of the former polyzwitterion class. Next, an irreversible step of weight loss is observed due to thermal decomposition starting at 250 • C, accounting for a mass loss of about 40-50 wt %, followed by a third step starting at about 330 • C. Accordingly, the thermal stability of the poly(sulfabetaine)s is comparable to the one of their sulfobetaine analogues [33,38], as illustrated in Figure 2. Putatively, the second step may be attributed to a dealkylation step at the nitrogen atom of the betaine moieties, the alkylsulfate groups being eliminated, as demonstrated for other polyzwitterions such as poly(carboxybetaine)s previously [22]. Thermograms of dried samples recorded by differential scanning calorimetry (DSC) did not exhibit thermal transitions for any of the poly(sulfabetaine)s before degradation starts, concordantly with reports on most poly(sulfobetaine)s [33,38,55]. Solubility of the poly(sulfabetaine)s was generally low. They were insoluble in most organic solvents, including highly polar ones such as dimethylsulfoxide, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylpyrrolidone. They were also insoluble in the protic polar solvent formamide HCONH2, which is known to dissolve many poly(sulfobetaine)s [32,33]. Most suited solvents were TFE, HFIP, and trifluoroacetic acid, which are typically considered to be good solvents for polyzwitterions in general [30,32,33,54,56]. Still, the solubilities of the polystyrenebased poly(sulfobetaine)s P−6c and P−6d were so low that the high molar mass samples produced swelled strongly in TFE or HFIP, but dissolved only sparingly.
The rather high molar masses of the synthesized poly(sulfabetaine)s aggravated the problem of the generally low solubility of most polyzwitterions in pure water. While the poly(sulfobetaine) analogues P−1a and P−3a that are used as references show a miscibility gap in pure aqueous solution at lower temperatures, i.e., UCST behavior, but are soluble in warm and hot water [38,57], none of the poly(sulfabetaine)s prepared could be dissolved in pure water up to the boiling point. Only when substantial amounts of inorganic salts were added, these polymers became water-soluble ( Table 2). The "salting-in" phenomenon is well known for many polyzwitterions [34,38,[58][59][60][61][62][63]. Still, in contrast to their sulfobetaine analogues, which all dissolve in normal saline solution ("physiological saline", 9.0 g/L NaCl) at ambient temperature [38], polymers P−1c to P−6c and P−1d to P−6d require much higher concentrations of added salt to form aqueous solutions, corroborating the few literature reports existing [43,47,49,51]. Consistently, none of the poly(sulfabetaine)s dissolved in PBS buffer. The strongly reduced water-solubility of poly(sulfabetaine)s compared to their sulfobetaine analogues is in agreement with molecular modeling studies deducing lower hydrophilicity for the ammoniosulfate in comparison to the ammoniosulfonate moiety [46,64]. The solubilities of the morpholine-derived poly(sulfabetaine)s P−5c and P−5d, and of the polystyrene-based poly-(sulfabetaine)s P−6c and P−6d were so low that the high molar mass samples used only swelled in brine.

Preparation of Thin Hydrogel Coatings
Similarly to homopolymer synthesis, free radical copolymerization of the sulfabetaine monomers together with 1 mol % of comonomer BPEMA (Figure 3) smoothly produced the corresponding copolymers in high yields. Copolymer P−x1d and reference copolymers P−x1a ("P−xSPP"), P−x3a ("P−xSPE"), P−xOEGMA, and P−xBMA (cf. Figures 1 and 3) were made analogously before, as reported elsewhere [52]. Solubility of the poly(sulfabetaine)s was generally low. They were insoluble in most organic solvents, including highly polar ones such as dimethylsulfoxide, acetonitrile, N,N-dimethyl-formamide, N,N-dimethylacetamide, or N-methylpyrrolidone. They were also insoluble in the protic polar solvent formamide HCONH 2 , which is known to dissolve many poly(sulfobetaine)s [32,33]. Most suited solvents were TFE, HFIP, and trifluoroacetic acid, which are typically considered to be good solvents for polyzwitterions in general [30,32,33,54,56]. Still, the solubilities of the polystyrene-based poly(sulfobetaine)s P−6c and P−6d were so low that the high molar mass samples produced swelled strongly in TFE or HFIP, but dissolved only sparingly.
The rather high molar masses of the synthesized poly(sulfabetaine)s aggravated the problem of the generally low solubility of most polyzwitterions in pure water. While the poly(sulfobetaine) analogues P−1a and P−3a that are used as references show a miscibility gap in pure aqueous solution at lower temperatures, i.e., UCST behavior, but are soluble in warm and hot water [38,57], none of the poly(sulfabetaine)s prepared could be dissolved in pure water up to the boiling point. Only when substantial amounts of inorganic salts were added, these polymers became water-soluble ( Table 2). The "salting-in" phenomenon is well known for many polyzwitterions [34,38,[58][59][60][61][62][63]. Still, in contrast to their sulfobetaine analogues, which all dissolve in normal saline solution ("physiological saline", 9.0 g/L NaCl) at ambient temperature [38], polymers P−1c to P−6c and P−1d to P−6d require much higher concentrations of added salt to form aqueous solutions, corroborating the few literature reports existing [43,47,49,51]. Consistently, none of the poly(sulfabetaine)s dissolved in PBS buffer. The strongly reduced water-solubility of poly(sulfabetaine)s compared to their sulfobetaine analogues is in agreement with molecular modeling studies deducing lower hydrophilicity for the ammoniosulfate in comparison to the ammoniosulfonate moiety [46,64]. The solubilities of the morpholine-derived poly(sulfabetaine)s P−5c and P−5d, and of the polystyrene-based poly-(sulfabetaine)s P−6c and P−6d were so low that the high molar mass samples used only swelled in brine.

Preparation of Thin Hydrogel Coatings
Similarly to homopolymer synthesis, free radical copolymerization of the sulfabetaine monomers together with 1 mol % of comonomer BPEMA (Figure 3) smoothly produced the corresponding copolymers in high yields. Copolymer P−x1d and reference copolymers P−x1a ("P−xSPP"), P−x3a ("P−xSPE"), P−xOEGMA, and P−xBMA (cf. Figures 1 and 3) were made analogously before, as reported elsewhere [52]. Key analytical data of the photo-crosslinkable copolymers are summarized in Table 3. The apparent number average molar masses Mn app of the various polymers were in the range of 100 to 250 kg mol −1 , i.e., they were similar, though slightly smaller than the molar masses of the homopolymers. Similarly, the copolymer dispersities Ð were in the range of 2.8-4. The photo-crosslinker content in the copolymers was determined to be about 1 mol % (by 1 H NMR, based on CRU), corresponding within the limits of precision to the content of monomer BPEMA in the copolymerization mixture. Considering that the molar masses of the zwitterionic monomers are in the range of 300 to 340 Da, these data suggest that the copolymer chains incorporate on average four to seven photo-reactive benzophenone moieties.  Key analytical data of the photo-crosslinkable copolymers are summarized in Table 3. The apparent number average molar masses M n app of the various polymers were in the range of 100 to 250 kg mol −1 , i.e., they were similar, though slightly smaller than the molar masses of the homopolymers. Similarly, the copolymer dispersities Ð were in the range of 2.8-4. The photo-crosslinker content in the copolymers was determined to be about 1 mol % (by 1 H NMR, based on CRU), corresponding within the limits of precision to the content of monomer BPEMA in the copolymerization mixture. Considering that the molar masses of the zwitterionic monomers are in the range of 300 to 340 Da, these data suggest that the copolymer chains incorporate on average four to seven photo-reactive benzophenone moieties. The solubility of the various poly(sulfabetaine)s in aqueous media did not virtually differ from the behavior of their homopolymer analogues, i.e., they required high concentrations of added salt, the concentration of 9 g L −1 of NaCl of normal saline not being sufficient. The copolymers seemed to dissolve somewhat more readily in TFE than the corresponding homopolymer samples. Yet, it is currently not clear whether this is due to the (at least apparently) slightly reduced molar masses, or due to the comonomer content. Still, the polystyrene derivatives P−6c and P−6d, and also samples P−4d and P−5c did not dissolve sufficiently in TFE and HFIP to allow for SEC analysis, or for the spin-coating preparations (see below).
The sufficiently soluble copolymers were spin-coated from TFE solutions onto glass slides, which beforehand had been modified by an aminosilane monolayer (water contact angle 59 ± 4 • ), to yield very smooth pinhole-free, coherent, and homogeneous films, with RMS roughnesses below 0.4 nm as exemplified for P−x1a, P−x1d, P−x3a, and P−xOEGA by AFM [52]. Exceptionally, reference P−xBMA was spin-coated from ethyl acetate solution, as films prepared from TFE solutions were quite rough [52]. The spin-coated films were stabilized by irradiation with near-UV light (Figure 4). This gives rise to simultaneous covalent cross-linking of the chains and covalent attachment to the support due to the specific photochemical reactivity of the benzophenone moiety [52,53,[65][66][67][68][69][70][71], overcoming the need for incorporating two different reactive groups in the hydrogel film forming polymer [72,73]. With its spectral cut-off at 310 nm, the irradiation system chosen excites only the flank of the intense absorbance band centered at about 295 nm of the 4-alkoyxy benzophenone chromophore (Figure 4a). This favors the in-depth homogeneous curing of the films, as the light intensities at the top and the bottom of the film are comparably high due to the weak absorbance.
the behavior of their homopolymer analogues, i.e., they required high concentrations of added salt, the concentration of 9 g L −1 of NaCl of normal saline not being sufficient. The copolymers seemed to dissolve somewhat more readily in TFE than the corresponding homopolymer samples. Yet, it is currently not clear whether this is due to the (at least apparently) slightly reduced molar masses, or due to the comonomer content. Still, the polystyrene derivatives P−6c and P−6d, and also samples P−4d and P−5c did not dissolve sufficiently in TFE and HFIP to allow for SEC analysis, or for the spin-coating preparations (see below).
The sufficiently soluble copolymers were spin-coated from TFE solutions onto glass slides, which beforehand had been modified by an aminosilane monolayer (water contact angle 59 ± 4°), to yield very smooth pinhole-free, coherent, and homogeneous films, with RMS roughnesses below 0.4 nm as exemplified for P−x1a, P−x1d, P−x3a, and P−xOEGA by AFM [52]. Exceptionally, reference P−xBMA was spin-coated from ethyl acetate solution, as films prepared from TFE solutions were quite rough [52]. The spin-coated films were stabilized by irradiation with near-UV light (Figure 4). This gives rise to simultaneous covalent cross-linking of the chains and covalent attachment to the support due to the specific photochemical reactivity of the benzophenone moiety [52,53,[65][66][67][68][69][70][71], overcoming the need for incorporating two different reactive groups in the hydrogel film forming polymer [72,73]. With its spectral cut-off at 310 nm, the irradiation system chosen excites only the flank of the intense absorbance band centered at about 295 nm of the 4-alkoyxy benzophenone chromophore (Figure 4a). This favors the in-depth homogeneous curing of the films, as the light intensities at the top and the bottom of the film are comparably high due to the weak absorbance.  Figure 4b illustrates that the photoreaction is fast, approaching its maximum after about 15 min. The decreasing intensity of the 295 nm band is attributed to the increasing consumption of the carbonyl group by H-abstraction, interrupting the conjugated π-system of the benzophenone ether chromophore. Further irradiation reduced the remaining absorbance only marginally. As prolonged irradiation risks to favor competing nonspecific photo-degradation reactions, an irradiation time of 30 min was considered to be optimal under the conditions employed, and thus chosen for the photo−curing of all samples.
The conditions for spin-coating were slightly adapted for each polymer to obtain consistently film thicknesses of about 100 nm ( Figure 5). This value appeared a reasonable choice, as very thin hydrogel coatings (<30 nm) seem prone to enhanced fouling [74,75]. Still, reports are somewhat conflicting with respect to the existence and range of an optimal thickness of thin hydrogel films for reducing protein fouling [76][77][78][79][80][81]. Hence in prior exploratory experiments with reference copolymer P−x3a (P−xSPE), we clarified that protein adsorption increased notably when the thickness of hydrogel films dropped below 80 nm, whereas the adsorbed amounts were about the same for thicknesses up to 120 nm at least (vide infra, see also    Figure 4b illustrates that the photoreaction is fast, approaching its maximum after about 15 min. The decreasing intensity of the 295 nm band is attributed to the increasing consumption of the carbonyl group by H-abstraction, interrupting the conjugated π-system of the benzophenone ether chromophore. Further irradiation reduced the remaining absorbance only marginally. As prolonged irradiation risks to favor competing nonspecific photo-degradation reactions, an irradiation time of 30 min was considered to be optimal under the conditions employed, and thus chosen for the photo−curing of all samples. The conditions for spin-coating were slightly adapted for each polymer to obtain consistently film thicknesses of about 100 nm ( Figure 5). This value appeared a reasonable choice, as very thin hydrogel coatings (<30 nm) seem prone to enhanced fouling [74,75]. Still, reports are somewhat conflicting with respect to the existence and range of an optimal thickness of thin hydrogel films for reducing protein fouling [76][77][78][79][80][81]. Hence in prior exploratory experiments with reference copolymer P−x3a (P−xSPE), we clarified that protein adsorption increased notably when the thickness of hydrogel films dropped below 80 nm, whereas the adsorbed amounts were about the same for thicknesses up to 120 nm at least (vide infra, see also  By the irradiation, the polymer films were effectively fixed on the supports. In most cases, even extended washing reduced film thickness hardly, if at all, within the precision of the measurements (Figure 5b). Comparatively high losses (ca. 25%) were exceptionally encountered for reference copolymer P−x1a (P−xSPP), which is in fact the most water-soluble polymer of the series studied next to reference copolymer P−xOEGMA. The latter however, may be more crosslinked than the zwitterionic polymers, as it is prone to photo-crosslinking under ambient conditions (air) even in the absence of benzophenone groups [53,82]. This is explained by the numerous α-methylene ether hydrogen atoms contained, which are most easily abstracted by oxygen centered radicals. Importantly, the findings demonstrate that the a priori low content of photo-reactive benzophenone moieties is sufficient to produce stable coatings from all polymers studied. Still, the extent of overall cross-linking must be assumed to be low. This implies that the hydrogel films produced are well swellable in aqueous media on the one hand, but mechanically very soft on the other hand.
The spin-coated films were analyzed by optical microscopy and water-air contact angle measurements ( Figure 6). Films were coherent, smooth, and free of defects within optical resolution. Water-air contact angle (WCA) measurements showed that only the coatings made from reference P−xBMA give rather hydrophobic surfaces (WCA~80°), as may have been anticipated. The WCA values of all other films are below the so-called "Berg limit" of about 60° for spontaneous hydrophobic fouling [83−85].  By the irradiation, the polymer films were effectively fixed on the supports. In most cases, even extended washing reduced film thickness hardly, if at all, within the precision of the measurements (Figure 5b). Comparatively high losses (ca. 25%) were exceptionally encountered for reference copolymer P−x1a (P−xSPP), which is in fact the most water-soluble polymer of the series studied next to reference copolymer P−xOEGMA. The latter however, may be more crosslinked than the zwitterionic polymers, as it is prone to photo-crosslinking under ambient conditions (air) even in the absence of benzophenone groups [53,82]. This is explained by the numerous α-methylene ether hydrogen atoms contained, which are most easily abstracted by oxygen centered radicals. Importantly, the findings demonstrate that the a priori low content of photo-reactive benzophenone moieties is sufficient to produce stable coatings from all polymers studied. Still, the extent of overall cross-linking must be assumed to be low. This implies that the hydrogel films produced are well swellable in aqueous media on the one hand, but mechanically very soft on the other hand.
The spin-coated films were analyzed by optical microscopy and water-air contact angle measurements ( Figure 6). Films were coherent, smooth, and free of defects within optical resolution. Water-air contact angle (WCA) measurements showed that only the coatings made from reference P−xBMA give rather hydrophobic surfaces (WCA~80 • ), as may have been anticipated. The WCA values of all other films are below the so-called "Berg limit" of about 60 • for spontaneous hydrophobic fouling [83][84][85]. By the irradiation, the polymer films were effectively fixed on the supports. In most cases, even extended washing reduced film thickness hardly, if at all, within the precision of the measurements (Figure 5b). Comparatively high losses (ca. 25%) were exceptionally encountered for reference copolymer P−x1a (P−xSPP), which is in fact the most water-soluble polymer of the series studied next to reference copolymer P−xOEGMA. The latter however, may be more crosslinked than the zwitterionic polymers, as it is prone to photo-crosslinking under ambient conditions (air) even in the absence of benzophenone groups [53,82]. This is explained by the numerous α-methylene ether hydrogen atoms contained, which are most easily abstracted by oxygen centered radicals. Importantly, the findings demonstrate that the a priori low content of photo-reactive benzophenone moieties is sufficient to produce stable coatings from all polymers studied. Still, the extent of overall cross-linking must be assumed to be low. This implies that the hydrogel films produced are well swellable in aqueous media on the one hand, but mechanically very soft on the other hand.
The spin-coated films were analyzed by optical microscopy and water-air contact angle measurements ( Figure 6). Films were coherent, smooth, and free of defects within optical resolution. Water-air contact angle (WCA) measurements showed that only the coatings made from reference P−xBMA give rather hydrophobic surfaces (WCA~80°), as may have been anticipated. The WCA values of all other films are below the so-called "Berg limit" of about 60° for spontaneous hydrophobic fouling [83−85].  Within the various polyzwitterion coatings, the films of the sulfobetaine polymers employed for comparison, namely of P−x1a (P−xSPP) and P−x3a (P−xSPE), showed WCAs of about 15−20 • in agreement with a previous study [52]. The new poly(sulfabetaine) coatings had also very hydrophilic surfaces, though they exhibited typically somewhat higher WCAs of around 25 • with only small differences between the samples. The higher WCA values of the poly(sulfabetaine) coatings may reflect the lower solubility of the underlying polymers in aqueous media in comparison to their sulfobetaine analogs (see above). In contrast, films of the nonionic reference P−xOEGMA exhibit a considerably higher contact angle of about 40 • , which is still well below the Berg limit.

Resistance Against Unspecific Adsorption of Proteins ("Fouling")
The effectiveness of the poly(sulfobetaine) films to reduce protein fouling was screened using two fluorophore-labeled model proteins, namely bovine serum albumin (BSA) and fibrinogen. For that, samples were exposed to solutions containing the protein in PBS buffer (pH = 7.4) for a specific time, then rinsed by PBS and water to remove the residual protein solution as well as loosely adsorbed protein, and dried, before the relative amount of irreversibly adsorbed protein was quantified by measuring the fluorescence intensity. Samples coated by the hydrophobic P−xBMA and hydrophilic P−xOEGMA polymers served as negative and, respectively, positive controls. Further, coatings of the often-used zwitterionic polymers P−x1a ("P−xSPP") and P−x3a ("P−xSPE") were included for comparison. Under these conditions, the amphiphilic BSA, which also tends to adsorb due to hydrophobic interactions, is markedly negatively charged (IEP~4.8), while fibrinogen is moderately negatively charged (IEP~5.9) [86,87]. The results of the experiments are displayed in Figure 7.
comparison, namely of P−x1a (P−xSPP) and P−x3a (P−xSPE), showed WCAs of about 15−20° in agreement with a previous study [52]. The new poly(sulfabetaine) coatings had also very hydrophilic surfaces, though they exhibited typically somewhat higher WCAs of around 25° with only small differences between the samples. The higher WCA values of the poly(sulfabetaine) coatings may reflect the lower solubility of the underlying polymers in aqueous media in comparison to their sulfobetaine analogs (see above). In contrast, films of the nonionic reference P−xOEGMA exhibit a considerably higher contact angle of about 40°, which is still well below the Berg limit.

Resistance Against Unspecific Adsorption of Proteins ("Fouling")
The effectiveness of the poly(sulfobetaine) films to reduce protein fouling was screened using two fluorophore-labeled model proteins, namely bovine serum albumin (BSA) and fibrinogen. For that, samples were exposed to solutions containing the protein in PBS buffer (pH = 7.4) for a specific time, then rinsed by PBS and water to remove the residual protein solution as well as loosely adsorbed protein, and dried, before the relative amount of irreversibly adsorbed protein was quantified by measuring the fluorescence intensity. Samples coated by the hydrophobic P−xBMA and hydrophilic P−xOEGMA polymers served as negative and, respectively, positive controls. Further, coatings of the often-used zwitterionic polymers P−x1a ("P−xSPP") and P−x3a ("P−xSPE") were included for comparison. Under these conditions, the amphiphilic BSA, which also tends to adsorb due to hydrophobic interactions, is markedly negatively charged (IEP~4.8), while fibrinogen is moderately negatively charged (IEP~5.9) [86,87]. The results of the experiments are displayed in Figure 7.
Already on a first view, Figure 7 shows that all polyzwitterions coatings strongly reduced the adsorption of both BSA and fibrinogen compared to the negative control P−xBMA. It was also evident from Figure 7b that their anti-fouling capabilities were somewhat lower than that of the positive control of P−xOEGMA. Nevertheless, the results demonstrate that not only the well-established polyzwitterions of the sulfobetaine type (as references P−xSPP and P−xSPE) but also such of the sulfabetaine type can confer distinct low-fouling behavior to surfaces. Figure 7a illustrates for polymer P−x3a (P−xSPE) the influence of the hydrogel film thickness on the fouling resistance. While the thinnest film of about 50 nm thickness is somewhat less resistant to protein fouling, in particular to fibrinogen, than the thicker ones, the latter hydrogel films appear be Already on a first view, Figure 7 shows that all polyzwitterions coatings strongly reduced the adsorption of both BSA and fibrinogen compared to the negative control P−xBMA. It was also evident from Figure 7b that their anti-fouling capabilities were somewhat lower than that of the positive control of P−xOEGMA. Nevertheless, the results demonstrate that not only the well-established polyzwitterions of the sulfobetaine type (as references P−xSPP and P−xSPE) but also such of the sulfabetaine type can confer distinct low-fouling behavior to surfaces. Figure 7a illustrates for polymer P−x3a (P−xSPE) the influence of the hydrogel film thickness on the fouling resistance. While the thinnest film of about 50 nm thickness is somewhat less resistant to protein fouling, in particular to fibrinogen, than the thicker ones, the latter hydrogel films appear be comparatively effective in repelling proteins. The data might suggest a shallow minimum of fouling for films that are approximately 100 nm thin, but due to the limited experimental statistics of this explorative study, this observation cannot be considered significant. In any case, when studying the effect of structure variation on fouling efficacy, we used uniformly coatings of~100 nm thickness for all polymers (Figure 7b).
Analyzing Figure 7b in more detail, additional features can be derived. We note that in agreement with a previous study using surface plasmon resonance SPR [52], the anti-fouling effectivity of the poly(sulfobetaine)s P−x1a and P−x3a (P−xSPP and P−xSPE) used as references were equal against fibrinogen, while P−x1a was more efficient than P−x3a in repelling BSA.
In comparison to the references, all poly(sulfabetaine) coatings tested were similarly or even more effective in reducing protein fouling. In particular the adsorption of BSA was notably lower for the poly(sulfabetaine) films than for the sulfobetaine references, which are already highly fouling-resistant. This finding was unexpected, as under the test conditions (namely in PBS buffer), the poly(sulfabetaine)s are not water-soluble in contrast to their sulfobetaine analogues (see Table 3), and are thus less hydrated. Moreover, theoretical studies predicted the contrary performance [64]. When comparing the protein fouling resistance of the poly(sulfabetaine) series c and d, efficacies were about equal. This implies that the precise length of the spacer group separating the cationic and the anionic moieties, namely a dimethylene or a trimethylene spacer, is of little importance for their anti-fouling effect. Furthermore, the findings corroborate that the relative anti-fouling efficiencies of the individual polymers differ for individual proteins [12]. There is no absolute "champion", but the detailed performance depends on the specific pair of polyzwitterion and protein. For instance, highest fouling resistance against BSA was encountered for coatings of P−x1c, which gives nearly the same excellent result as the control P−xOEGMA. In contrast, highest fouling resistance against fibrinogen was observed for films of P−x3c. The best combination of fouling resistance against both proteins was found for P−x3c and P−x3d. Still, one should not overstress these rankings, as the anti-fouling effects of all the polyzwitterions were generally high and the observed differences were comparatively small.

Conclusions
A large variety of poly(sulfabetaine) methacrylic monomers is easily accessible by ring-opening alkylation of polymerizable tertiary amines with cyclic sulfates. Their straightforward free radical polymerization provides high molar mass polymers in good yields. These polyzwitterions are insoluble in most solvents, including pure water, physiological saline, and PBS buffer. Still, solubility in aqueous media can be achieved in many cases by the addition of high concentrations of inorganic salts such as NaCl. When copolymerized with small amounts of benzophenone bearing monomers, the copolymers formed can be spin-coated and photo-crosslinked to yield stable thin hydrogel coatings. An exploratory screening demonstrated that such coatings are highly resistant against the nonspecific adsorption of proteins. While their detailed anti-fouling performance depends on the precise couple protein−polyzwitterion, it tends to surpass even the one of films prepared from poly(sulfobetaine) analogues, which have been established as effective fouling protection coatings. As previous studies have also shown good long-term stability of polysulfabetaines against hydrolysis in aqueous media, except for very low or high pH values where polysulfobetaines excel [50], such polymers may be useful components for implementing low-fouling behavior to surfaces.
Author Contributions: Experiments were conceived by all authors, and performed by E.S., E.W., and J.K. All authors analyzed the data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.