Zwitterionic Polyurethanes with Tunable Surface and Bulk Properties

Oct 18, 2018 - To address the lack of blood compatibility and antifouling properties of polyurethanes (PUs), a novel zwitterionic poly(carboxybetaine ...
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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Zwitterionic Polyurethanes with Tunable Surface and Bulk Properties Huifeng Wang,† Yang Hu,† Dylan Lynch,† Megan Young,† Shengxi Li,‡ Hongbo Cong,‡ Fu-Jian Xu,§ and Gang Cheng*,† †

Department of Chemical Engineering, University of Illinois at Chicago, Chicago, Illinois 60607, United States Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio 44325, United States § State Key Laboratory of Chemical Resource Engineering, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China

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S Supporting Information *

ABSTRACT: To address the lack of blood compatibility and antifouling properties of polyurethanes (PUs), a novel zwitterionic poly(carboxybetaine urethane) (PCBHU) platform with excellent antifouling and tunable mechanical properties is presented. PCBHU was synthesized via the condensation polymerization of diisocyanate with carboxybetaine (CB)-based triols. Postpolymerization hydrolysis of triol segments at the interface generates zwitterionic CB functional groups that provide superior antifouling properties via the enhanced hydration capacities of CB groups. Thermogravimetric analysis and differential scanning calorimetry measurement show the high thermal stability of PCBHU with up to 305 °C degradation temperature. Tunable mechanical properties and water uptakes can be finely tuned by controlling the structure and ratio of CB-based triol cross-linkers. This study presents a new strategy to incorporate CB functional groups into PU without significantly changing the synthetic methods and conditions of PU. It also provides a deeper understanding on structure−property relationships of zwitterionic PUs. Because of its superior antifouling properties than existing PUs and similar cost, mechanical properties, stability, and processability, PCBHU has the great potential to replace current PUs and may open a new avenue to PUs for more challenging biomedical applications in which the existing PUs are limited by calcification and poor antifouling properties. KEYWORDS: zwitterionic, carboxybetaine, polyurethane, dual function, antifouling surface, elastomer molded devices.12,13 However, there are several challenges that will have to be overcome before the potential of PU can be fully realized.14 First, although protein absorption on PUs, the initial stage of the blood coagulation cascade, was found to be less than other polymeric materials because of the hydrophilicity/wettability attributed to hydrogen bond-forming groups,15 the antifouling properties of PU are still unsatisfactory for the applications in complex biological media (e.g., blood, body fluid, and cell lysate).16 Second, most PUs do not possess both antifouling properties and functionality to conjugate other moieties. Third, PUs can slightly reduce

1. INTRODUCTION Polyurethane (PU) comprises a large family of polymeric materials that carry urethane linkages along the polymer backbone.1−3 PU’s structure presents two distinct phases: alternated hard and soft segments.4,5 Hard segments are responsible for high mechanical resilience, whereas soft segments provide the PU an elastomeric behavior. Therefore, their singular molecular structure provides them good properties, such as high strength, ductility, chemical stability, and ease of processability.1,2,4,5 In addition, because of the high density of the hydrogen bond-forming (urethane) group, PUs carry good water wettability, high strength, and high elasticity.2,3 These properties are desired for a broad spectrum of medical applications, including catheters,4−6 drug delivery,7,8 tissue engineering,9−11 as well as a variety of injection© XXXX American Chemical Society

Received: June 22, 2018 Accepted: September 28, 2018

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DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces bacterial attachment compared to other materials17 but cannot resist long-term biofilm formation. Biofilm formation on catheters and implants results in the formation of persistent infections and microorganisms in the biofilm are up to 1000 times more resistant to conventional antibiotics than planktonic bacteria.18 To address these challenges, two strategies have been widely explored by incorporating either hydrolyzable/degradable or antifouling moieties into PUs to improve their antifouling properties. In the degradable PU approach, the hydrophobic degradable moieties, such as polycaprolactone19 and polylactic acid,20 still lead to the protein adsorption and blood coagulation because the hydrolysis rates of the degradable moieties are significantly slower than the blood adsorption/coagulation rate. In the antifouling approach, antifouling moieties, such as polyethylene glycol (PEG), have been incorporated into PUs.21 Although PEG has been used alone or combined with other components for a broad spectrum of applications for antifouling purposes almost 30 years, the critical challenges of PEG for in vivo applications, such as foreign body response, infection, and thrombosis, remain unsolved.22,23 Among antifouling materials, zwitterionic polymers are a group of emerging high-performance materials and they were inspired by the phospholipid bilayer of cell membranes and possess both anionic and cationic groups with overall charge neutrality.24 Because of their strong hydration layer via ionic solvation,25−30 zwitterionic materials have demonstrated superior antifouling properties resisting proteins,31−34 mammalian cells35 and microbes,36−38 and better biocompatibility39−42 compared to other antifouling materials.26,27 On the basis of the type of the anion, zwitterionic polymers can be classified into poly(phosphobetaine),27 poly(carboxybetaine) (PCB),29,33,34 and poly(sulfobetaine) (PSB).30 Among them, PCB-based materials showed better antifouling properties,1 structure flexibility,2 ionic conductivity,3 as well as capability of further functionalization.4 Although existing zwitterionic polymers have unique properties and superior performance at the interface, they suffer from weak mechanical properties and lack elastic property as polymeric structural materials. Several approaches have been explored to enhance the elastic and compressive properties of the zwitterionic materials by introducing more hydrogen bond-forming groups into the side chain or combine the zwitterionic material with other structural materials. Our previous study demonstrated that the elastic and compressive property of the PCB hydrogel can be improved by replacing the methyl group with the hydroxyl ethyl group on the quaternary ammonium of PCB side chains;5 however, the elasticity of zwitterionic materials needs to be further improved compared to existing elastomers. Because antifouling PUs not only provide an antifouling surface at the biointerfaces but also have the tunable mechanical properties, researchers have been focusing on conjugating zwitterionic side chains, such as sulfobetaine (SB),43 carboxybetaine (CB),44 and phosphobetaine (PB),45 onto PU backbones to address the drawbacks of both zwitterionic polymers and PUs. Wynne et al. reported that zwitterionic PU hydrogels derived from CB-functionalized diols showed great capability of resistance to bacterial colonization, but a strong base, sodium hydroxide, was needed to hydrolyze the protection group of CB.46 Shen et al. modified the PU membrane using a PSB polymer brush to enhance the antifouling property, which created an effective antifouling surface.47 However, this method requires postprocessing

functionalization of PU with a zwitterionic polymer, and it will significantly increase the cost and change the processability of PU. Because of the lack of chemistry, the potential of zwitterionic PUs cannot be fully realized for the most challenging biomedical applications. In this study, we developed a versatile methodology for the design and synthesis of zwitterionic PUs. To validate this method, a set of zwitterionic poly(carboxybetaine urethane)s (PCBHUs) were synthesized, where the CB content in the polymer could be readily tuned by altering the molar ratio of soft segments and hard segments employed in the synthesis. The dual function of both surface antifouling properties and tunable bulk properties can be achieved by the homopolymer. Chemical structure, thermal stability, thermal transition, and mechanical properties of resulting polymers with different component ratios were characterized. The hydrolysis kinetics of the PU in solutions with different pH values was recorded. The protein adsorption on PCBHUs was evaluated using bovine serum albumin (BSA) as a model protein via a fluorescence method. Mammalian cell attachment, bacterial adhesion, and biofilm formation on PCBHU surfaces were studied to evaluate their antifouling properties.

2. EXPERIMENTAL SECTION 2.1. Materials. Diethanolamine, 2-hydroxyethyl acrylate, ethyl acrylate, and fluorescein isothiocyanate (FITC) isomer 1 were purchased from Alfa Aesar (Haverhill, Massachusetts, USA). Phosphate-buffered saline (PBS), PEG 2000 and BSA were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 1,6-Diisocyanatohexane (HDI) was purchased from Acros Organics (Pittsburgh, Pennsylvania, USA). Dimethylformamide (DMF) was purchased from EMD Millipore (Burlington, Massachusetts, USA). Viability/ cytotoxicity assay kit for bacteria live and dead cells was purchased from Biotium (Fremont, California, USA). Medical grade PU was obtained from API (Mussolente, Vicenza, Italy). DMAO was purchased from Biotium (Fremont, California, USA). 2.2. Synthesis of Diethanoamino-N-hydroxyl Ethyl Acetate (DEAHA). DEAHA was synthesized using a Michael-type reaction.33 Diethanolamine (30 g, 0.28 mol) was added into 2-hydroxyethyl acrylate (36.4 g, 0.31 mol), and the reaction solution was stirred under the nitrogen gas overnight at 35 °C and kept away from the light during the reaction. The unreacted 2-hydroxyethyl acrylate was removed in a rotary evaporator, and then the product was purified by flash chromatography using dichloromethane and methanol (5:1) as the mobile phase to yield DEAHA as a colorless oil with a 96% yield. 1 H NMR (400 MHz, CDCl3, ppm): 4.03 (t, 2H), 3.59 (t, 2H), 2.67 (t, 2H), 2.35 (t, 2H), 3.42 (m, 4H), 2.44 (m, 4H). 2.3. Synthesis of PUs. In this study, we designed five polymers with different stoichiometric ratios of hydroxyl groups in DEAHA/ hydroxyl groups of PEG/isocyanate groups in HDI (2:8:10, 4:6:10, 6:4:10, 8:2:10, and 10:0:10), and the materials were named PCBHU2, PCBHU-4, PCBHU-6, PCBHU-8, and PCBHU-10, respectively. PGHU-8 with the ratios of hydroxyl groups in glycerol/hydroxyl groups of PEG/isocyanate groups in HDI (8:2:10) as a control was also synthesized. The PUs with different monomer ratios were synthesized via a one-pot reaction (Scheme 1). In a typical reaction, a PU prepolymer was synthesized in a three-necked round-bottom flask equipped with a mechanical stirrer, a temperature controller, and a nitrogen inlet. Before the reaction, PEG 2000 was placed into a vacuum oven at 110 °C for 2 h to remove moisture. HDI was then added into the flask dropwise. DMF was added once the viscosity of the reaction solution increased. The prepolymer solution was stirred for 2 h at 80 °C. DEAHA as the chain extender/cross-linker was added into the solution dropwise and stirred for another 30 min at 80 °C under nitrogen protection. The prepolymer solution was then poured into poly(tetrafluoroethylene) dishes and placed in an oven at 100 °C for 12 h. The resulting PU was dried at 100 °C in the vacuum B

DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

well. All samples were immersed in the solution for 30 min to allow protein adsorption on hydrogel surfaces. To remove loosely adsorbed proteins on sample surfaces, hydrogel samples were rinsed with PBS three times. Protein adsorption on the hydrogel surface was visualized with an Olympus IX81 fluorescence microscope (Olympus, Japan) with a 40× objective lens through the FITC filter at a fixed exposure time for all samples. The samples having no contact with FITC-BSA were used as the negative control to obtain a completely dark background. Commercially available PUs with adsorbed FITC-BSA were used as the positive control. The exposure time was chosen to make the images of the negative control completely dark while keeping the images of the positive control unsaturated. Then, ImageJ software was used to quantify the fluorescence intensity of each sample, and correlate with the amount of protein absorption. 2.11. Mammalian Cell Attachment. After all materials were equilibrated in DI water NIH-3T3 cells were seeded on different hydrogel substrates at 1 × 105 cells/well with a medium consisting of Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, and 1% penicillin−streptomycin. The samples were then kept in an incubator with 5% CO2 at 37 °C for 24 h. Cell density and cell morphology were visualized with an Olympus IX81 fluorescence microscope with a 60× objective lens through FITC filters. 2.12. Bacterial Attachment. Escherichia coli K12 was cultured at 37 °C in Luria−Bertani (LB) medium (20 g/L) to reach an optical density of 1.0 at 600 nm. After washing with PBS three times, cells were suspended in 0.85% NaCl aqueous solution to get a final concentration of 108 cells/mL. Fresh E. coli K12 suspension (50 μL) in PBS was pipetted onto samples (8 mm in diameter) and incubated at room temperature for 1 h. To analyze the density of bacteria accumulated on hydrogel surfaces, samples were gently rinsed with DI water and stained with the DMAO dye, which stains the nucleic acids of both live and dead cells. After staining, the number of total cells was determined with an Olympus IX81 fluorescence microscope with a 40× objective lens through FITC filters. 2.13. Biofilm Formation Assay. Pseudomonas aeruginosa PAO1 cells were inoculated in 15 mL of sterile LB medium in Petri dish with sterile samples and cultured at room temperature. Two-third of the culture solution was replaced by the sterile LB media daily. After 2 weeks, the accumulated bacteria were recorded in situ and stained with DMAO dyes. After staining, the number of total cells was determined using an Olympus IX81 fluorescence microscope with a 20× objective lens through FITC filters.

Scheme 1. Synthetic Route of PCBHUs

oven for 12 h to remove the residual solvent. After drying, PU films were peeled off and cut into disks with a biophysical punch (8 mm in diameter and 2 mm in thickness). PU hydrogels were formed after equilibration in water for 2 weeks. 2.4. Fourier Transform Infrared Spectroscopy (FT-IR). PU samples were analyzed by FT-IR using a Nexus 870 spectrometer (Thermo Nicolet, USA) with an attenuated total reflection module. The wavenumber ranges from 400 to 4000 cm−1 with 32 scans. 2.5. Thermogravimetric Analysis (TGA). A TGA/SDTA 851e thermogravimetric analyzer (Mettler Toledo, USA) was used to study the thermal stability of the PU materials. The temperature ranged from 50 to 700 °C with a heating rate of 10 °C/min with a continuous N2 flow of 50 mL/min. 2.6. Differential Scanning Calorimetry (DSC). The phase transition temperature of all polymers was measured using an 822e differential scanning calorimeter (Mettler Toledo, USA). A heating rate of 10 °C/min from −40 to 250 °C was employed with a continuous N2 flow of 50 mL/min. 2.7. Hydrolysis Kinetics in Water. Each sample (8 mm in diameter and 2 mm in thickness) was immersed in deionized (DI) water. The pH change of the solution was measured and recorded by a Mettler Toledo SevenExcellence pH meter every 5 min in first 30 min and then every day for 15 days. 2.8. Swelling Study. PU disks with 8 mm in diameter and 2 mm in height were prepared, and submerged in DI water. After the sample reached equilibrium in DI water for two weeks, the mass of PU hydrogels was recorded and then placed in a freeze-dryer and lyophilized prior to being measured again. The swelling ratio, Q, was calculated using the following equation:

3. RESULTS AND DISCUSSIONS 3.1. Synthesis of PU. Because of a wide range of molecular weight and its antifouling properties, PEG has been extensively studied and used as the soft segment in PUs to tune their mechanical properties and improve the biocompatibility. In our study, PEG with a molecular weight of 2000 Da was selected as polyol because it was found that the longer soft segments can improve the elasticity of PUs. However, if PEG soft segments are too long, the mechanical strength of PUs may be compromised. Although PEG has been used to improve the biocompatibility, blood compatibility, and antifouling properties of PUs, material-associated foreign body response, blood coagulation, and infection remain tough challenges for in vivo applications. To address these long-standing issues, we designed and synthesized a multifunctional building block, DEAHA, which combines crosslinkers, hard segments, and antifouling functions. DEAHA can be easily synthesized via a Michael-type reaction33 of diethanolamine and 2-hydroxyethyl acrylate. The reaction is highly efficient with >96% yield. The chemical structure of DEAHA was characterized and confirmed by 1H NMR spectroscopy (Figure S1). The PU materials with different monomer ratios were synthesized via a one-pot reaction (Scheme 1). In PU synthesis, DEAHA functions as both cross-

Q = (MS − MD)/MD where MS is the mass after swelling and MD is the mass after lyophilizing. 2.9. Compression Testing. The stress−strain behavior of PU samples was evaluated with a Shimadzu EZ-Test Compact Bench Testing Machine (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Each sample (8 mm in diameter and 2 mm in thickness for both before and after hydrolysis) was compressed to failure at the rate of 1 mm/min with a 500 N load cell. 2.10. Protein Adsorption Study. The adsorption of protein on PU samples was determined by a fluorescence method.35 All materials were equilibrated in DI water, cut into disks with a biophysical punch (8 mm in diameter and 2 mm thick), washed thoroughly with DI water, and transferred into a sterile 24-well plate. 1 mL of FITClabeled BSA (FITC-BSA) solution (0.1 mg/mL) was added into each C

DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

2863 cm−1, CO stretching at 1680 cm−1, and C−O stretching at 1188 cm−1. N−H deformation bands were observed in the range of 1500−1600 cm−1. The sharp peaks ranging from 1690 to 1700 cm−1 indicate CO stretching vibrations of urethane and carboxyl group on the cross-linker side chain. Compared to PCBHU-2, PCBHU-4, and PCBHU6, both PCBHU-8 and PCBHU-10 show stronger CO stretching peaks, indicating the higher ratios of DEAHA. Aliphatic C−H stretching mode of 2880−2929 cm−1 and carboxylic stretching absorption band at 3735−3770 cm−1 were also observed. Moreover, IR spectra showed the characteristic C−O−C stretching bands of soft segments at 1170, 1188, and 1193 cm−1. These signature stretching bands are a convincing evidence for the formation of PU. PCBHU-2, PCBHU-4, PCBHU-6, and PCBHU-8 show higher peaks of C−O stretching, which indicate higher ratios of the soft segment, compared to PCBHU-10. FT-IR results confirmed the formation of PU and the complete consumption of isocyanate groups. It should be noted that isocyanate is highly reactive and toxic; therefore, the unreacted isocyanates need to be quenched. 3.3. Thermal Stability and Thermal Transition. The thermal stability of PUs determines their processing method and condition. In this study, TGA was used to evaluate the thermal stability of all samples. The decomposition process usually undergoes three stages. In the first stage, the urethane bonds decompose to form alcohols and isocyanates. The resulting chain fragments are prevented from degradation by dimerization of isocyanates to carbodiimides, which subsequently react with the hydroxyl groups to produce relatively stable substituted ureas. The final step is the high-temperature degradation of these stabilized structures to yield volatile products and a small quantity of carbonaceous chain.48 Figure 2 shows the TGA and differential TGA (DTGA) profile observed for PCBHUs under nitrogen. All PCBHUs display two weight loss stages at 305 and 456 °C, respectively, and eventually turn into a small amount of high-temperature residue (7.0 wt %). The first TGA stage can be related to the urethane bond degradation and stabilized urea bond formation, whereas the second peak corresponds to the decomposition of the urea structures. The TGA study confirms that PCBHUs have the similar high thermal stability of PU (∼200−300 °C),48 which provides a wide processing window. The thermal transition of PU with different component ratios was characterized by DSC, and the results are shown in Figure 3. The melting temperatures for PCBHU-10, PCBHU8, PCBHU-6, PCBHU-4, and PCBHU-2 are 100.5, 79.3, 56.1, 47.1, and 24.7 °C, respectively. As the hard segment content increased, the melting temperature increased because of the higher cross-linking density. The glass-transition temperature was not observed within the studied temperature range. 3.4. Hydrolysis of PCBHUs. One important function of DEAHA in PCBHUs is to provide antifouling properties after the hydrolysis of the ester bonds that leads to the formation of zwitterionic CB at the material/water interface. Before hydrolysis, DEAHA is in the form of the tertiary amine. In DI water, the tertiary amines are protonated, and the resultant hydroxide ions cause an increase of pH. High local concentrations of hydroxide ions facilitate the hydrolysis of ester bonds and lead to the formation of carboxylate groups. The consumption of hydroxides during hydrolysis then leads to a decrease of pH, and eventually the pH value of the solution becomes stable after all esters are hydrolyzed. In this

linkers and antifouling precursors in PCBHU, and the mechanical, swelling, and antifouling properties can be easily tuned by adjusting the ratio of soft and hard domains. In addition, for all existing commercially available PU formulations, DEAHA can be used as either the additive or replacement for the existing cross-linkers without significantly changing the reaction and processing conditions. 3.2. Fourier Transform Infrared Spectroscopy Study. To confirm the completion of the condensation polymerization of PU, the obtained PCBHUs with different monomer ratios were characterized by FT-IR. Figure 1 shows the representative

Figure 1. FT-IR spectrum of PCBHU PU materials with different component ratios.

FT-IR spectra of PCBHU-2, PCBHU-4, PCBHU-6, PCBHU8, and PCBHU-10. FT-IR studies were carried out by focusing on principal regions such as −CH stretching (2700−2950 cm−1), CO stretching (1620−1740 cm−1), −NH stretching (3300−3500 cm−1), and C−O−C stretching (1050−1150 cm−1). In all PCBHUs, there was no signal at 2270 cm−1 (−NCO stretching) or 3590 cm−1 (O−H stretching), indicating that all isocyanate groups and hydroxyl groups were consumed. The absorption bands of N−H stretching vibrations were observed at 3326 cm−1, C−H stretching at D

DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. DSC profiles of PCBHUs with different component ratios. Figure 2. (a) TGA and (b) DTGA profiles of PCBHUs with different component ratios.

high water solubility and low cross-linker ratio, PCBHU-2 completely dissolved in DI water. PCBHU-4 and PCBHU-6 have a higher swelling ratio (Table 1) than that of PCBHU-8 and PCBHU-10. In addition, the size change was also measured after hydrolysis. The original diameter was 8 mm for each PCBHU sample. After hydrolysis, the diameter became 15, 14, and 13 mm for PCBHU-4, PCBHU-6, and PCBHU-8, respectively. There was negligible volume change for PCBHU-10. The size measurements corresponded to the swelling ratio studies. This study demonstrated that swelling of PCBHU materials can be controlled by adjusting the monomer ratio. 3.6. Mechanical Properties. Tunable mechanical properties for biomaterials are highly desired because the requirements for the mechanical properties of the materials may vary significantly for different applications. Figure 4a,b shows the stress−strain curves for the compression tests of PCBHUs before and after hydrolysis, respectively. Before hydrolysis, the compressive modulus of PCBHU increases when the hard segment, DEAHA, content increases. The compressive modulus increases from 174.1 kPa (PCBHU-4) to 1026.5 kPa (PCBHU-10), whereas the breaking strain decreases from 80.1% (PCBHU-4) to 50.1% (PCBHU-10) (Table 1). Before hydrolysis, the compressive modulus of PCBHUs increases when the hard segment, DEAHA, content increases. This result suggests a good interfacial adhesion between hard and soft domains and a higher cross-linking density. Meanwhile, the elasticity of the material was compromised with an increase of cross-linking density, which is probably due to the embrittlement caused by increasing the interfacial area between hard and soft segments. The compressive moduli

study, pH values were recorded to monitor the hydrolysis process of each PCBHU sample in DI water. As shown in Figure S2, all solutions containing PCBHUs are basic initially. It is caused by the protonation of tertiary amine from unhydrolyzed PCBHU. Then, the pH values of all solutions decreased as a function of time. The pH values of PCBHU-4 and PCBHU-6, which have lower DEAHA ratios, were lower than those of PCBHU-8 and PCBHU-10 with higher DEAHA ratios. The swelling of the hydrogel has a synergy effect with hydrolysis of the cross-linker component that also consumes hydroxide and causes the decrease of the solution pH. In addition, the higher PEG content leads to higher swelling and more water uptake, resulting in faster hydrolysis of DEAHA linkers. These results indicate that the hydrolysis of the DEAHA content has a significant impact on the tunable swelling behavior of the material, which can be effectively monitored by the pH change of the solution. 3.5. Swelling Study. Swelling experiments were also used to characterize the water uptake and penetration of PCBHUs with different compositions. It was expected that the swelling ratio of the PU hydrogel corresponds to the feed ratio of PEG and DEAHA. Theoretically, as the ratio of PEG increases, the spacing between the cross-linkers becomes larger, and therefore, the resulting materials will have a larger mesh size, and exhibit a higher swelling ratio. Table 1 shows the differences in swelling ratio for PU hydrogels. The results of this study, as expected, demonstrated a significant difference in swelling ratio between PU hydrogels with different ratios of soft segments and hard segments. Especially, because of the E

DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Table 1. Compressive Moduli, Breaking Strain, and Swelling Ratio of PCBHUs with Different Component Ratios before hydrolysis PCBHU-10 PCBHU-8 PCBHU-6 PCBHU-4

after hydrolysis

compressive modulus kPa

breaking strain %

compressive modulus kPa

breaking strain %

swelling ratio %

1026.5 465.2 390.9 174.1

50.1 62.4 67.2 80.1

294.1 133.3 14.5

86.5 100.4 120.4

37.6 335.3 1071.1 3644.4

nonspecific protein adsorption on PCBHUs. In this study, protein adsorption studies were conducted on the PCBHU surfaces and quantified by the fluorescence method. Four PCBHU samples were compared. Commercially available medical grade PU (API-PU) films were used as a control material. After reaching equilibrium in PBS, PCBHU samples were rinsed with DI water and submerged in the FITC-BSA solution. Materials having no contact with FITC-BSA were used as the control. All images of different samples were obtained from the fluorescence microscope at the same excitation light intensity and exposure time thereafter. As shown in Figure 5, API-PU shows the highest fluorescence

Figure 5. Fluorescence images for protein adsorption of the (a) commercially available API-PU, (b) PGHU-8 hydrogel, (c) PCBHU8 hydrogel, and (d) PCBHU-10 hydrogel. Based on 100% protein absorption of API-PU, the relative fluorescence intensities were 37.64%, 2.16%, and 0.68% for PGHU-8, PCBHU-8, and PCBHU-10 hydrogels, respectively.

Figure 4. Compressive stress−strain curves of PCBHU PU materials with different component ratios (a) before and (b) after equilibration in DI water.

and breaking strains of the samples before and after hydrolysis are dramatically different. After hydrolysis, the compressive moduli of PCBHUs follow the similar trend as the materials before hydrolysis and range from 14.5 kPa (PCBHU-6) to 298.1 kPa (PCBHU-10). The breaking strain significantly increases to 120.4% for PCBHU-6, 100.4% for PCBHU-8, and 86.5% for PCBHU-10 after hydrolysis. After hydrolysis, the higher equilibrium water content and swelling ratio of samples with a lower cross-linking density lead to the lower compressive modulus. This study demonstrated that the cross-linking density can be adjusted to obtain a desired swelling ratio and suitable elasticity for different applications. 3.7. Protein Adsorption. Protein adsorption on the surface of an implanted medical device can cause foreign body response, blood coagulation, and/or inflammation,26,27 which affect the service life and performance of the material/ device. One major challenge for PU biomaterials is their unsatisfactory capability of resisting protein adsorption from blood, body fluid, or other complex media.16 It is hypothesized that zwitterionic CB moieties can dramatically reduce

intensity among all samples, which indicates the highest protein adsorption. The sample with the highest DEAHA ratio (PCBHU-10) showed the lowest amount of the absorbed protein, whereas the sample with a composition of 8:2:10 of glycerol/PEG/HDI (PGHU-8) showed a medium fluorescence density. Compared to API-PU, the protein adsorptions on PGHU-8, PCBHU-8, and PCBHU-10 are 37.64%, 2.16%, and 0.68%, respectively. The results demonstrated that PCBHU-8 and PCBHU-10 highly resist protein adsorption. The excellent antifouling property of PCBHU is due to the strong hydration of the CB groups that are generated via the hydrolysis of DEAHAs. Previous studies discovered that the strong hydration layer of the zwitterionic materials provides an effective barrier to prevent foulants to interact with the material surfaces.25 To determine their hydrophilicity, the water contact angle of PCBHUs and control surfaces were measured. Table S2 shows that compared to commercially F

DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces available PUs, incorporated zwitterionic groups render the PCBHU surfaces more hydrophilic. 3.8. Cell Attachment. For implantable biomedical devices, protein adsorption on surfaces from blood can trigger platelet49 and fibroblast/macrophage/monocyte50 attachment. The attachment of platelets leads to thrombosis, and the attachment of macrophages causes foreign body response and results in inflammation around implanted materials/devices. To further confirm the antifouling properties of PCBHU materials, cell adhesion studies were performed with NIH-3T3 fibroblast cells. After incubation at 37 °C for 24 h, the control tissue culture polystyrene (TCPS) surface demonstrated a full coverage of NIH-3T3 fibroblast cells. The cell densities on TCPS and PGHU-8 hydrogel surface were (195.12 ± 3.56) × 104 cells/cm2 and (19.51 ± 1.74) × 104 cells/cm2, respectively. There was minimal cell attachment on PCBHU-8 and PCBHU-10 surfaces (Figure 6). These results indicate that

Figure 7. Fluorescence images for E. coli K12 adhesion on the (a) commercially available API-PU, (b) PGHU-8 hydrogel, (c) PCBHU8 hydrogel, and (d) PCBHU-10 hydrogel. The cell densities on the commercial biomedical PU material and PHG-8 hydrogel surface were (5.21 ± 0.24) × 105 and (1.37 ± 0.05) × 105 cells/cm2, respectively. However, for the PCBHU-8 and PCBHU-10 surface, there was no observable E. coli K12 cell.

Figure 6. Images for NIH-3T3 cell attachment of the (a) commercially available API-PU, (b) PGHU-8 hydrogel, (c) PCBHU-8 hydrogel, and (d) PCBHU-10 hydrogel. The cell densities on TCPS and PGHU-8 hydrogel surface were (195.12 ± 3.56) × 104 and (19.51 ± 1.74) × 104 cells/cm2, respectively. However, for the PCBHU-8 and PCBHU-10 surfaces, there was no observable cell.

surfaces. The cell densities on the surface of API-PU and PGHU-8 materials were (5.21 ± 0.24) × 105 and (1.37 ± 0.05) × 105 cells/cm2, respectively. Nevertheless, there was almost no observable bacterial cell attached to PCBHU surfaces. The ability to resist bacterial attachment of the PCBHU surface is attributed to the hydration layer formation induced by the ionic solvation between the zwitterionic moiety and water molecules. Furthermore, the surface coverage, molecular weight, and density of zwitterionic components also play essential roles to inhibit the bacterial adhesion.51 The long-term biofilm formation of P. aeruginosa was also studied on PCBHU surfaces. As shown in Figure 8, almost no P. aeruginosa PAO1 bacterial cell was observed on PCBHU-8 and PCBHU-10 surfaces as compared to the PGHU-8 and API-PU surfaces after 2 weeks. The API-PU surface was

we have successfully created zwitterionic PUs that highly resist cell adhesion. PCBHUs potentially can be applied to be implanted devices to solve long-standing device-induced thrombosis and inflammation. We plan to further evaluate the biocompatibility of PCBHU with in vivo studies in the future. 3.9. Bacterial Attachment and Biofilm Formation. Infection and biofilm formation on implants are a major cause of the implant failure.17 Formation of a biofilm starts with the initial attachment of microorganisms. Once the biofilm is established, the microorganisms in the biofilm are no longer sensitive to antimicrobial agents. Biomaterials with superior properties to resist bacterial attachment and biofilm formation are urgently needed for implantable medical devices. Because of the zwitterionic moieties at the material/water interface, PCBHUs were expected to effectively resist the attachment of bacterial cells. Bacterial cell attachment onto PCBHU surfaces was investigated using E. coli K12 as a model strain. Figure 7 shows that minimal bacterial cells were observed on the PCBHU-8 and PCBHU-10 surfaces after 1 h in the solution. In contrast, much more E. coli K12 cells attached on API-PU

Figure 8. Fluorescence images for Pseudomonas aeruginosa PAO1 biofilm formation of the (a) commercially available API-PU, (b) PGHU-8 hydrogel, (c) PCBHU-8 hydrogel, and (d) PCBHU-10 hydrogel. G

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ACS Applied Materials & Interfaces completely covered by P. aeruginosa at 25 °C after 2 weeks. The cell density on the PGHU-8 surface is about 34.8% compared to that on API-PU. We discovered that PEG alone cannot effectively resist biofilm formation, and our results show that zwitterionic PCBHU surfaces possess a great capability to resist bacterial adhesion and biofilm formation.

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4. CONCLUSIONS In summary, we developed a facile strategy to synthesize antifouling PUs. A series of zwitterionic PCBHUs were synthesized with CBs as part of the PU backbone, where the CB content in the polymer could be finely tuned by altering the molar ratio of PEG and DEAHA triol. This study provides new insights into the relationships between structure, function, and stability of zwitterionic PUs. The mechanical properties and swelling ratio were sensitive to the content of CB precursor, DEAHA. TGA and DSC results show that the PCBHU materials are thermally stable. PCBHUs, such as PCBHU-8 and PCBHU-10 with higher DEAHA cross-linker ratios, markedly reduced protein adsorption, cell attachment, bacterial attachment, and biofilm formation. Thus, both tunable surface and bulk properties have been achieved in one single PU material. Through this study, a better understanding of the structure−function relationship of zwitterionic PUs was achieved, which will enable us to design suitable materials for specific applications. The present work provides effective and promising methods to prepare stable zwitterionic PU materials for a broad spectrum of biomedical applications in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b10450. NMR spectrum for DEAHA cross-linkers; hydrolysis process of PU; and fluorescence intensity of protein adsorption and contact angles for the commercial biomedical PU material, PGHU-2 hydrogel, PCBHU-6 hydrogel, and PCBHU-8 hydrogel (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. URL: https://che.uic.edu/kteacher/gang-cheng-ph-d/. ORCID

Hongbo Cong: 0000-0001-5263-6623 Fu-Jian Xu: 0000-0002-1838-8811 Gang Cheng: 0000-0002-7170-8968 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the US National Science Foundation (DMR-1454837). REFERENCES

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DOI: 10.1021/acsami.8b10450 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX