Antibacterial and Biocompatible Cross-Linked Waterborne

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Antibacterial and Biocompatible Crosslinked Waterborne Polyurethanes Containing Gemini Quaternary Ammonium Salts Yi Zhang, Xueling He, Mingming Ding, Wei He, Jiehua Li, Jianshu Li, and Hong Tan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01016 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Antibacterial and Biocompatible Crosslinked Waterborne Polyurethanes Containing Gemini Quaternary Ammonium Salts ‡Yi Zhang a,b, ‡Xueling He a,c*, Mingming Ding a, Wei He a, Jiehua Lia , Jianshu Lia, Hong Tan a, * a

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials

Engineering, Sichuan University, Chengdu 610065, China b

High and New Technology Research Center of Henan Academy of Sciences, Zhengzhou

450002, China c

Laboratory Animal Center of Sichuan University, Huaxi Clinical College, Sichuan University,

Chengdu, 610040, China

ABSTRACT A crosslinked waterborne polyurethane (CPTMGPU) with long-term stability was developed from poly(ethylene glycol) (PEG), polyoxytetramethylene glycol (PTMG), isophorone diisocyanate (IPDI), L-lysine and its derivative diamine consisting of gemini quaternary ammonium salt (GQAS), using ethylene glycol diglycidyl ether (EGDE) as a crosslinker. Weight loss test, X-ray photoelectron spectroscopy (XPS) measurements and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) were performed to prove the surface structure and stability of these CPTMGPU films. Furthermore, the GQAS-bearing CPTMGPUs show repeatable contact-active antibacterial efficacy against both gram-positive Staphylococcus aureus (S.aureus) and gram-negative Escherichia coli (E.coli)

ACS Paragon Plus Environment 1

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bacteria, and do not show any inhibition effect against fibroblasts in vitro. After subcutaneous implantation in rats, the CPTMGPU films manifest good biocompatibility in vivo, despite the presence of a typical foreign body reaction toward surrounding tissues and mild systematic inflammation reaction that could be eliminated after a short implantation period, as demonstrated by histology and immunohistochemistry combined with interleukin (IL)-1β, IL-4, IL-6, IL-10 and TNF-α analysis though enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qRT-PCR). Therefore, these crosslinked waterborne polyurethanes hold great promise for antibacterial applications in vivo.

KEYWORDS Crosslink, waterborne polyurethane, gemini quaternary ammonium salt, antibacterial, inflammatory response

1. Introduction Bacterial infection has become a serious issue facing various biomedical and engineering devices.1 This problem is particularly severe for biomaterial implants required for short-term or long-term applications, such as urinary catheters,2 intravascular catheters,3 orthopaedic implants,4 and dental implants,5 etc., which may result in undesirable implant remotion and substitution, accompanied by inevitable medical risks and complications.4 It has been regarded that most infections are induced by the bacterial adhesion, multiplication and subsequent formation of biofims.6 The attached microorganisms on the surfaces of biomaterials may proliferate quickly in a moist physiological environment and tend to build up a biofilm. If a mature biofilm is formed, the bacteria can survive under harsh conditions and get highly resistant to both antibacterial compounds and host immune response.7 Therefore, the development of


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biomaterials capable of prohibiting bacterial adhesion and resisting biofilm formation are urgently needed.8, 9 To address these needs, various contact-active polymer coatings containing antimicrobial agents have been developed to first prevent the adhesion and spreading of microorganisms onto surfaces.10 Quaternary ammonium salt (QAS) is one kind of the most widely used antibacterial agents in contact-active surfaces,11,

12

owing to its broad antibacterial spectrum, good

environmental stability and biological activity, and low bacterial resistance.13, 14 However, QASdecorated antibacterial materials as implants can hardly meet the requirements of non-fouling property and biocompatibility, because biomacromolecules and killed microorganisms may easily accumulate at the biocidal layer and the surrounding bacteria can further adhere and proliferate on the carcasses, which eventually block the antimicrobial groups and induce other adverse effects.15 To suppress the accumulation of microorganisms, another commonly used strategy is to graft material surfaces with zwitterionic polymers or poly (ethylene glycol) (PEG). It has been demonstrated that surfaces containing PEG can defer the formation of biofilms over 24 h.16 A more promising approach is to kill microbes and repel them simultaneously. Chau et al. developed a surface consisting of an antibacterial sub-layer and an anti-adhesive upper-layer. The surface can repel bacteria and kill the anchored microbes that escape from the antifouling upper-layer.17 Alternatively, we have recently constructed a polyurethane-based reversed surface containing an biocidal upper-layer and an anti-adhesive sub-layer, which could kill bacteria on the upper-layer and release the attached organisms, showing a killing efficiency up to 99.99% against the attached S.aureus and E.coli.18 Nonetheless, these polymers are prone to release their



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active constituents due to high hydrophilicity and possible degradation by microbial biofilms,10 which limits their long-term application and raises safety concerns in vivo.

Scheme 1. Schematic structure of PTMGPU and the preparation of CPTMGPU films. In this work, we developed a crosslinked waterborne polyurethane with contact-active antibacterial and antifouling double layers, which possesses long-term stability and antibacterial


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activity while retaining non-fouling properties and good biocompatibility. The polyurethanes were prepared from PEG, polyoxytetramethylene glycol (PTMG), isophorone diisocyanate (IPDI), L-lysine and its derivative diamine consisting of gemini QAS (GQAS) and named as PTMGPU. Then the PTMGPUs were crosslinked using ethylene glycol diglycidyl ether (EGDE). The obtained crosslinked polyurethanes (CPTMGPU) were examined by X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The antibacterial activity of CPTMGPU films was evaluated by contact killing assay. In vitro cytotoxicity analysis studies and animal experiments were carefully performed to assess the biocompatibility and in vivo host inflammatory response of the polyurethanes.

2. Materials and Methods 2.1. Materials. Isophorone diisocyanate (IPDI, BASF) was distilled under vacuum before use. polyoxytetramethylene glycol (PTMG, molecular weight 2000, Dupont) and Poly (ethylene glycol) (PEG, molecular weight 1450, Dow Chemical) were dehydrated at 80-90 ºC under vacuum for 2 h before use. EGDE (IR) was obtained from TCI. 2,3,5-Triphenyltetrazolium chloride

(TTC,

>99.5%)

was

purchased

from

Sanland-Chem

International

Inc.

Methylthiazoletetrazolium (MTT, Ultra Pure Grade) was purchased from Beyotime Biotechnology. 2.2. Preparation of CPTMGPU Films. PTMGPU samples were synthesized from PTMG, PEG, IPDI, L-lysine and GQAS via a two-step polymerization method (Supporting information, Scheme S1).18,19, 20 The obtained PTMGPU samples were treated with EGDE at 50°C for 48 h to prepare crosslinked PTMGPU (CPTMGPU). The feed ratios of polyurethanes and crosslinkers are shown in Table S1. The emulsions as prepared were cast on siliconized glass plates, and dried under vacuum at 25 °C for 2 d and 60 °C for 2 d, followed by another 2 d at 60 °C. The



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obtained films with approximately 0.5 mm thickness were cut into 10 mm×10 mm. The films were immersed in water and placed in a horizontal laboratory shaker (37 ºC, 110 r/min) for 1 d to a constant weight. The films prepared were immersed in 50 mL water at room temperature for 6 h and 5 months respectively before measurements. The water was changed every 2 d in the first week, and every week in the following period. 2.3. ATR-FTIR. ATR-FTIR was recorded between 4000 and 600 cm−1, with a resolution of 4 cm−1 on a Nicolet 6700 spectrometer (Thermo Electron Corporation, U.S.A.). All spectra were attained by averaging 32 scans. 2.4. XPS. The X-ray gun worked at 10 mA current and 20 kV with a take-off angle of 30° (Kratos XSAM-800 Spectrometer, Mg KR.). The relative atomic percentage on the CPTMGPU film surface was obtained by using atomic sensitivity factors (XSAM-800). N1s, C1s and O1s spectra were deconvoluted into sub-peaks though spectrometer software (XPSPEAK4.0). 2.5. Long-Term Stability and Multiple Recycle Contact-Active Antibacterial Activities. The CPTMGPU emulsions (70 µL) were casted onto glass slides (15 ×15 mm2) and dried. The samples prepared were incubated in 50 mL water at room temperature for 6 h and 5 months. The water was changed every 2 d in the first week, and every week in the following period. At the predetermined time, the samples were moved out and dried under vacuum before antibacterial test. All the samples were sterilized by UV overnight, and sprayed with 1×106 CFU/mL S. aureus (ATCC 6538), dried in air for 10 min. Then, the inoculated glass slides were moved into a Petri dish and cultured within 0.8% nutrient broth agar at 37 ºC for 1d. Thereafter, 3 mL of 5% TTC were poured in to the Petri dish and stained for 30 min.21, 22 The glass slides were removed, washed with sterilized water and air dried. Then the samples were taken for contact-active


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antibacterial test again, and the process were replicated for many times to evaluate the multiple recycle antibacterial activity of CPTMGPU films. 2.6. Inhibition of Bacteria on the CPTMGPU Films Surface and in the Surrounding Environment. CPTMGPU films were washed with water at room temperature for 7 d to avoid the interference of water-soluble antibacterial components from CPTMGPUs on evaluation of their antibacterial activities. The films were immersed into nutrient broth (NB) culture at 37 °C for 1d to obtain the extracts of CPTMGPU films. The optical density (OD) value of the extracts after inoculated with bacteria (E. coli (ATCC 25922), S. aureus (ATCC 6538)) for 1 d was recorded on an ultraviolet spectrophotometer at 600 nm (Table S2). The result indicates that no noticeable antibacterial component was infiltrated into the surrounding environment. The antibacterial and antifouling properties of CPTMGPUs were tested against E. coli and S. aureus using a shaking-flask method.23-25 The CPTMGPU films were sterilized under UV overnight and placed in a 24-well plate. E. coli and S. aureus strains were cultivated at 37 °C for 16 h and diluted by NB broth to 107 CFU/mL. Each well containing a CPTMGPU film was added with 2 mL of the diluted bacteria strain culture. The plate was then incubated for 2 d (37 °C, 110 rpm). Afterward, CPTMGPU membranes was fetched, and rinsed with sterile water for three times, then transferred into a tube with 2mL sterile water. The tube was placed under ultrasonic for 5 min to detach bacteria attached on CPTMGPU film, and then counted with a flat colony counting method.25 Live bacteria in the surrounding culture were also counted using the same method. All the tests were repeated for three times. Antibacterial kinetics activity of CPTMGPU films was studied against S. aureus. The CPTMGPU films were sterilized under UV overnight and placed into a 24-well plate. S. aureus strains were cultivated at 37 °C for 16 h, and then diluted by NB broth to 106, 105, 104, 103



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CFU/mL separately. 2 mL of the bacteria liquid was added to the wells containing CPTMGPUs. CPTMGPU0 was used as a control. The plate was then incubated in a constant temperature incubator (37 °C, 110 r/min). At the predetermined times 0.5, 1, 2, 4, 6, 12, 24 and 48 h after incubation, 100 µL bacteria liquid was removed and counted, and 100 µL NB was added into the well. All the tests were repeated for three times. The percentage of bacterial viability was acquired according to the equation: Bacteria viability(%) =

Live bacteria Sample ×100 Live bacteria Control

2.7. Cytotoxicity of CPTMGPU Extracts. To determine the cytotoxicity of potential watersoluble substances released from CPTMGPU films. An extraction procedure was performed according to previous reports.26, 27 Briefly, the CPTMGPU films were sterilized with gamma irradiation and UV irradiation. 100 mg sample films were placed into 1 mL of culture medium for 1 d of incubation at 37 °C. Then the extracts were filtered by a membrane filter (0.2 µm) to remove bacteria and eliminate the interference of any insoluble fragments from broken films, and diluted with culture medium to 1-20 mg/mL. The extract of latex rubber film (Microflex Corp., Sparks, NV) was chosen as a positive control due to its inherent cytotoxicity.26 The cytotoxicity of the extracts was tested by methylthiazoletetrazolium (MTT) assay. Fibroblast cells were seeded in a 96-well plate (1000 cells/well) and cultured for 12 h. Polymer extracts with different concentrations were added into the wells (100 µL/well) containing cultured fibroblast cells The plates were then placed in a humidified 5% CO2 atmosphere at 37 °C for 24 h. Afterward, 20 µL of 5% MTT solution in PBS was added into each well. After the removal of the media, 150 µL of dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals. The OD value was recorded on a Microplate reader (Model 680, Bio Rad Corp.) at 492 nm. The viability of fibroblast cells cultured with samples was normalized to those incubated with negative control.


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2.8. Animal Experiments. Sixty male, specific-pathogen free Sprague-Dawley rats weighed 220 ± 20 g were supplied by the Animal Center of Sichuan University (Sichuan, China), and acclimated for 7 d in an animal laboratory before experiment. All the animals were housed at 24 ±1°C and 55±5% relative humidity in a room, where they had free access to rat standard diet and water under a 12-h (7 a.m. to 7 p.m.) light/dark cycle. Prior to surgery, the rats were anesthetized through intraperitoneal injection with 10% chloral hydrate. Thereafter, 2 parallel incisions (8 mm long) were made through the shaved and cleaned back skin of each rat. Various CPTMGPU films were implanted subcutaneously in the pockets made in the left/right flank of SD rats. Rats receiving surgical operation without implantation of sample films were choosed as negative controls. The animal experimental procedures were in agreement with the “Principles of Laboratory Animal Care” of the National Institutes of Health and allowed by the Ethics Committee of Sichuan University. 2.9. Hematoxylin and eosin (H&E) and Immunohistochemistry. After the experiment, SD rats were anesthetized using chloral hydrate and the blood samples were collected from carotid artery. The skin soft tissues previously in contact with the samples was removed and divide tissue into two parts, one was kept at -80 °C, and the other was fixed in 10% neutral buffered formalin (NBF). Skin soft tissues were embedded in paraffin after dehydrating through a graded, then sectioned at 5 µm thickness. All the samples were stained by H&E and applied with OX6, R73 and ED-1 immunohistochemical staining. Immunohistochemistry protocol was described previously.28 The primary antibody against rat OX6, R73 and ED-1 (abcam; dilution: 1:100) was applied in the procedure. Positive staining was subjectively classified as weak, moderate, or strong.



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2.10. Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). The extraction of RNA was carried out by a Trizol RNA extraction method. One microgram of RNA was reversely transcribed using RNA PCR kit (Takara). Complementary cDNA was synthesized from RNA using reverse transcription kit (Bio-Rad, USA). One microliter of cDNA was then used to perform PCR using primers for TNF-α IL-1β, IL-4, IL-6, IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences used were listed in Table S3. PCR program was initiated for 3 min at 95 °C, and then for 40 cycles (15 s per cycle), 10 s at primer-specific annealing, 72 °C for 15 s, and a melt curve analysis at 65–96 °C. The PCR product was taken for melting curve measurement to study if there was any non-speciﬁc substance produced. The relative expression was determined by a comparative cycle threshold (Ct) methods. The expression of GAPDH mRNA was used for normalization, and standardized on a dilution curve from cDNA samples. 2.11. Enzyme-Linked Immunosorbent Assay (ELISA). Rat blood was placed at 4 °C overnight and centrifuged at 3500 r/min for 15 min. All serum was removed and frozen at -80 °C before assay. The TNF-α, IL-1β and IL-6 levels were assayed by an ELISA kit (Abiocode, USA) based on the instruction from the manufacturers. 2.12. Statistical Analysis. Statistical Package for the Social Sciences (SPSS, version 17.0) software was applied for statistical comparisons though a one-way analysis of variance (ANOVA). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.005.

3. Results and Discussion 3.1. Preparation of Crosslinked CPTMGPU Films To prepare waterborne polyurethane with long-term stability and antibacterial activities, a class of PTMGPUs were successfully synthesized from PTMG, PEG, IPDI, lysine and GQAS


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(Figure S1). PTMG was chosen to control the hydrophilicity of PTMGPU, while PEG and GQAS were used to construct antifouling and antibacterial layered surface structures. The biocidal GQAS content in the polymers can be well controlled by using lysine as a chain extender. The feed ratios of the polyurethanes are shown in Table S1, and the preparation of CPTMGPUs is illustrated in Scheme 1. The obtained PTMGPUs were further crosslinked with EGDE to attain CPTMGPUs with high stability. The structures of crosslinked CPTMGPUs were first characterized with ATR-FTIR and 1HNMR. As shown in Figure S3a, the stretching vibration of carboxyl groups in PTMGPU (3500 cm-1) and those of epoxy groups in EGDE (845 cm-1) are observed in the spectra of PTMGPU/EGED blends (MPTMGPU), while these signals are much weaker for the corresponding crosslinked CPTMGPU samples, indicating successful crosslinking by EGDE. Moreover, the characteristic peaks of GQAS, IPDI, PTMG and EGDE were observed in the 1HNMR spectra of CPTMGPUs (Figure S2). However, the degree of crosslinking cannot be determined from the 1HNMR spectra due to the overlap of methylene protons from EGDE, PEG and PTMG. Alternatively, we estimated the crosslinking degree using a solvent extraction method.29, 30 We found that PTMGPU20 was totally dissolved in chloroform, while CPTMGPU20 was partially soluble with a crosslinking degree of 60.2% after EGDE treatment (Supporting Information). The effect of crosslinking was also analyzed by a weight loss test. It was found that the weight loss of CPTMGPU0, CPTMGPU20 and CPTMGPU30 are sharply reduced compared with uncrosslinked PTMGPU samples (Figure S3b, c), demonstrating a higher stability of CPTMGPU films in an aqueous environment. 3.2. Surface Properties of CPTMGPU Films It is known that the surface structure of materials plays an important role in antibacterial and antifouling properties, especially for contact-active systems.10 To study the surface structures of



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CPTMGPU films, the membranes were washed in water for 6 h or 150 d, and characterized by ATR-FTIR (Figure 1 and Figure S4). It was found that the adsorption of urea groups (~1650 cm-1) and urethane groups (~1720 cm-1) change little in CPTMGPU films after washing. Considering that GQAS was introduced into the polyurethanes by urea bonds, the result suggests that GQAS groups in CPTMGPU are non-leachable and these polymers possess high stability and potential for long-term applications.

Figure 1. (a, b) ATR-FTIR spectra (enlarged region, 1760 ~ 1600 cm-1) and (c, d) high resolution N 1s spectra of XPS at 30o take-off angle of CPTMGPU films after washing for 6 h (a, c) and 150 d (b, d) in water.


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To further investigate whether washing in water leads to the change of antibacterial contents on CPTMGPU film surfaces, the atomic percentages of nitrogen, oxygen and carbon on the film surface were determined by XPS. The atomic percentages of N+ reflected the content of GQAS, which could be observed at around 402.5 eV for CPTMGPU20 and CPTMGPU30 (Figure 1c, d). The calculated atomic percentages of N+ are listed in Table S4. Although the content of N+ on the surfaces of CPTMGPU films are relatively lower compared with uncrosslinked PTMGPU films due to the restricted mobility of hard segment by crosslinking,18 there is no further decrease of N+ concentration after washing in water for 6 h and 150 d (Table S4). The result agrees with ATR-FTIR results and suggests that the GQAS groups on CPTMGPU surfaces are stable with washing for a long period. Interestingly, the N+ content on the surfaces were observed even higher after washing for 150 d, probably due to slight migration of GQAS groups onto the surfaces when the films were immersed in water for a long time. The high surface activity of GQAS moieties enables them to move to the liquid/gas or solid/liquid interface gradually, and the longer the films immersed in water, the more the GQAS groups on the membrane surfaces. As a consequence, the atomic contents of N+ on CPTMGPU surface (CPTMGPU20: 6 h - 0.57%, 150 d - 0.62%; CPTMGPU30: 6 h - 0.61%, 150 d - 0.65%) are higher than those in bulk (CPTMGPU20: 0.33%, CPTMGPU30: 0.47%) (Table S4). In addition, the oxygen content on CPTMGPU0 surface (6h- 23.06%, 150d-26.23%;) are also higher than those in bulk (19.85%), because of the movement of PEG segments to the film surfaces after contacting with water (Table S4).28 On the other hand, the GQAS-containing films (CPTMGPU20 and CPTMGPU30) do not show significantly increased oxygen concentration on their surfaces compared with CPTMGPU0 films, suggesting a limited migration of PEG segments to the surfaces. These results demonstrate that the crosslinked CPTMGPU film were constructed with a GQAS



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antibacterial upper layer and an antifouling PEG sublayer (Scheme 1), which is similar to that reported for uncrosslinked polyurethane films.18 Table 1. The number of live bacteria in surrounding culture and film surface. Samples

Surrounding culture

Film surface

E.coli (CFU/mL)

S.aureus (CFU/mL)

E.coli (CFU/mL)

S.aureus (CFU/mL)

CPTMGPU0

1.3×109

1.3×109

3.4×106

4.4×106

CPTMGPU20

2.3×103

4.4×103

0

0

CPTMGPU30

1.1×103

5.6×103

0

0

3.3 Antibacterial Activity of CPTMGPU Films. The PTMGPU emulsions exhibit good antibacterial efficacy,20 and the crosslinking does not exert significant impact on the minimal inhibitory concentration (MIC) of CPTMGPU emulsions (Table S5). To investigate the effect of crosslinking on the antibacterial activity of CPTMGPU films, the film samples were sterilized and analyzed with a shaking-flask method. As shown in Table 1, a great number of S. aureus (4.4×106 CFU/cm2) and E. coli (3.4×106 CFU/cm2) cells were found on the surfaces of CPTMGPU0 films, while no live bacteria adhered to GQAScontaining CPTMGPU films. Nonspecific protein bovine serum albumin (BSA) adsorption and water contact angle of CPTMGPU films were also analyzed and the results indicate good hydrophilicity and antifouling capacity of CPTMGPU films (Supporting information, Table S6, Figure S5).19 Additionally, the amount of surviving bacteria in the surrounding culture of GQAS-based CPTMGPU was much lower than that of CPTMGPU0 (Table 1), showing potential inhibition effect of CPTMGPU on the proliferation of surrounding bacteria.


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To further explore the inhibition effect of CPTMGPU films on surrounding bacteria, the study of antibacterial kinetics were performed against different concentrations of S. aureus (103, 104, 105, 106 CFU/mL). The results showed that more than 99.99% bacteria in the local culture were killed in the surrounding environment of CPTMGPU films after 6 h of incubation (Figure 2), verifying that CPTMGPU films can not only kill the bacteria effectively on contact, but also inhibit the bacteria proliferation in the surrounding environment. This phenomenon may be associated with the characteristic of the grow curve of the bacteria. When the bacteria enters a new environment, there is an adaptive phase during which the growth rate is pretty.31 Thus, when the bacteria suspension was incubated with CPTMGPU films, the killing rates were higher than the growth rates of bacteria.

Figure 2. Antibacterial kinetics of CPTMGPU films against S. aureus in the surrounding culture. a): 103 CFU/mL, b): 104 CFU/mL, c): 105 CFU/mL, d): 106 CFU/mL



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Figure 3. Photographs of CPTMGPU films (washing in water for 150 d before test) after repeating the contact-killing experiment for (a) 1 time, (b) 6 times and (c) 10 times. Each black dot represents a colony of S.aureus grown from a single live S.aureus cell. The long-term stability and antibacterial property of biomaterials are of great importance for their applications in vivo. Herein, to determine long-term stability and multiple recycle contactactive antibacterial property of CPTMGPU membranes, the crosslinked CPTMGPU films were first immersed in water for 150 d to mimic the humid environment where CPTMGPU films may be applied, and then evaluated with a glass slide spreading method. As shown in Figure 3a, CPTMGPU0 without GQAS biocidal components shows no antibacterial effect, and both the glass slide and CPTMGPU0 surfaces are covered with S. aureus colonies. In contrast, the GQAS-containing CPTMGPU films exhibit excellent biocidal efficiency, with no S. aureus colony observed on the membranes even after 150 days’ washing, suggesting that the CPTMGPU films could potentially maintain their antibacterial capacity for long-term applications in physiological environment. Meanwhile, inhibition zone could not be observed around all the samples, indicating that no antibacterial agents were released (Figure 3). Moreover, the CPTMGPU films could be repeatedly used for contact active bacterial killing, and the antibacterial function can be fully restored even recycled for 10 times (Figure 3c). All these


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results indicate that the crosslinked CPTMGPU films show repeatable and stable antibacterial activity. To further support this conclusion, commercial nano silver (AgNP)/CPTMGPU0 composites were also tested as controls. Although AgNP/CPTMGPU0 composites containing more than 2.5wt% AgNP show good antibacterial activities (Figure S6a), the antibacterial activities were completely lost after washing in water for 21 d (Figure S6b), further demonstrating that CPTMGPUs possess stable, antibacterial activity and excellent contact-killing property compared with traditional antibacterial agents. Furthermore, the antibacterial property of GQAS containing polyurethane films were preliminarily investigated in vivo. As seen in Figure S7, the CPTMGPU films show more than 95% killing efficiency at day 2 and day 7 after implantation. (Figure S7), suggesting a good antibacterial activity in vivo. More intensive study on the antibacterial activity of CPTMGPU films in vivo is ongoing in our laboratory. In addition, since antibacterial materials with biofilm growth retardation property is crucial for their biomedical utility,32 the anti-biofilm capacity of CPTMGPU films will be evaluated in our follow up work.

Figure 4. Cell viability of L929 mouse fibroblasts after 24 h of incubation with the extracts of CPTMGPU films with different concentrations.



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3.4 Cytotoxicity of CPTMGPU Films The cytocompatibility of both PTMGPU and CPTMGPU films in vitro was investigated by determining the cytotoxicity of their extracts against L929 mouse fibroblasts using an MTT assay. As seen from Figure 4 and S8, the viability of L929 cells is higher than 89% after incubation with various extract dilutions for 24 h. Latex rubber extract as a positive control shows much lower cell viability than all the polyurethane samples even after 100 times of dilution. Consequently, these antibacterial CPTMGPU samples could meet safety requirements for wide range applications, such as water purification systems, food storage and packaging, textiles and medical implants and devices.

Figure 5. Light micrographs of H&E stained skin soft tissues previously in contact with the samples after implantation for 2 d a), 7 d b), 30 d c) and 90 d d). Normal cells and inflammationmediating cells were stained pink and purple, respectively. Magnification 200×. Black arrows indicate the thickness of fibrotic capsule, and * shows the site of implantation (for CPTMGPU0 and CPTMGPU20) or incisions (for blank).


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3.5. Biocompatibility of CPTMGPU Films in vivo Implantation of a foreign material can interrupt the regular series of events that result in resolutions of inflammation and wound healing, leading to the so-called foreign body response (FBR).33 The FBR commonly contains a phase of acute inflammation followed by a chronic stage that eventually activates healing and tissue repair,34 which ultimately resulting in the formation of reactive fibrotic capsules around the implant.34-36 Since the knowledge of inflammatory in response

Figure 6. Micrographs of OX6-positive, R73-positive and ED-1-positive stained sections for PE and CPTMGPU films after 2 days’ (a, c, e) and 90 days’ (b, d, f) implantation. * indicates the removed implants (for CPTMGPU0 and CPTMGPU20) or incisions (for blank). The OX6positive, R73-positive and ED-1-positive cells are denoted in brown and the counterstained cells are shown in blue. Magnification 200×. to material implantation in vivo is essential to decide the survival of implants, herein, we investigate the FBR and tissue changes caused by subcutaneous implantation of CPTMGPU in



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SD rats. Representative sections of surrounding tissues with H&E staining are presented in Figure 5. Evidently, fibrous capsules was found to be deposited around all the implants at 2 d and 7 d post-implantation, characterized by a large amount of purple color ascribed to the presence of acidic structures (e.g., eosinophil).

37

The infiltrating inflammatory cells were

observed at the interface between the fibrous capsule and implants (Figure 5). These phenomena have also been reported for the implantation of other polymeric biomaterials such as polyester and polyethylene,38-41 suggesting an acute phase of FBR caused by implants.35 The average capsule thicknesses of CPTMGPU20 at days 2 and 7 are 378 and 583 µm, respectively, which are relatively higher than those of CPTMGPU0 (49 and 114 µm) (Figure S9). The result indicates that the GQAS-bearing polyurethane membranes trigger a more acute response compared with inactive polyurethanes (CTPMGPU0). As the implantation time is up to 30-90 d, the inflammatory response was found to be alleviated considerably, as evidenced by the disappearance of capsules and infiltrating inflammatory cells around the CPTMGPU films (Figure 5c, d). According to the ISO 10993-6 standard, these polyurethane films could be considered to be biocompatible. To investigate the mechanism of fibrotic capsule formation, the reactive capsules containing some inflammatory cells such as macrophages were assessed by immunohistochemical staining. As shown in Figure 6, MHC-class-II cells (OX6-positive cells), T-lymphocytes (R73-positive cells) and total monocytes/macrophages (ED1-positive cells) are observed in the fibrotic capsules elicited by all implants. ED1-positive cells are blood-derived macrophages or monocytes, which are highly phagocytic.42 MHC class II (OX6) over-expression in fibrotic capsules confirms the recruitment and activation of inflammatory and immune cells.43 Tlymphocytes are usually detected in infection cell components with potential for T-cell mediated


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Biomacromolecules

responses, e.g., production of cytokine.44 In 2-days’ group, severe inflammatory responses were observed with all the samples, due to the acute response stage caused by surgical implantation (Figure 6). The phenomenon is in good agreement with H&E-staining assay (Figure 5). However, in both the 30-days’ and 90-days’ groups (Figure 6 and Figure S10), OX6-positive, R73-positive and ED1-positive cells were found diminished sharply with time and disappeared eventually, further indicating that CPTMGPU films exhibit good biocompatibility even after a long implantation period.

Figure 7. The production of IL-1β a), IL-4 b), IL-6 c), IL-10 d) and TNF-α proteins e) measured by PCR in the surrounding tissues obtained from the SD rats after implantation for 2, 7, 30 or 90 d. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.005. To investigate the effect of CPTMGPU implantation on the overall cellular responses in surrounding tissues during the foreign body response, genes associated with inflammation and immunoregulation, including IL-1β, IL-4, IL-6, IL-10 and TNF-α, were measured by real-time



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qRT-PCR analysis. It was found that the expressions of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) tend to increase in tissues surrounding CPTMGPU films and reach their highest levels on 7 d after implantation (Figure 7a, b). These levels drop over the implantation period of 90 d, but remain detectable and relatively higher than those for control group without sample implanted (P < 0.05). The result suggests an acute inflammation phase followed by a chronic stage involved in the FBR upon implantation of PU films.34 Moreover, the production of IL-4, an antiinflammatory/pro-wound healing cytokine, was also noticed (Figure 7b), indicating the participation of T cells in immune response.34 Of interest, the anti-inflammatory/anti-wound healing cytokine IL-10 was expressed albeit at relatively lower level (Figure 7c). The low concentration of IL-10 combined with a high level of TNF-α imply an overall acute inflammatory response after implantation.45 Subsequently, the decrease of IL-10 and TNF-α expression over the experiment period may reveal a gradual transformation from an early inflammatory response to a later wound healing process.33, 46 In addition, CPTMGPU20 containing GQAS exhibits higher levels of IL-1β, IL-6 and TNFα expressions compared with CPTMGPU0 (P < 0.05). The result indicates that the contact-active CPTMGPU20 surface may induce greater inflammatory response than normal polyurethane (CPTMGPU0), which is in agreement with histological assay (Figure 5 and 6). Nevertheless, the expression levels of these genes were found diminished with time, and there was no significant difference in the expressions between CPTMGPU20 and CPTMGPU0 groups after 90 d post-implantation, demonstrating that the inflammatory response caused by GQAS could be eliminated over implantation period and the antibacterial polyurethane are potentially biocompatible in vivo.


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Biomacromolecules

Figure 8. The production of IL-6 (a), IL-1β (b) and TNF-α (c) determined by ELISA in the serum obtained from the SD rats after implantation for 2, 7, 30 or 90 d. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.005. Since the determination of cytokines in body fluids such as serum appears as a feasible method to evaluate the inflammatory and immunological process of the body,47 serum IL-6, TNF-α and IL-1β levels as markers were measured by ELISA to further study the global effect of inflammation after implantation of CPTMGPU films.48 It was found that all the cytokines in serum reach considerably high levels at 2 d after surgical implantation (Figure 8), owning to the acute response stage caused by surgery trauma.49 Moreover, the animals receiving CPTMGPU implantation exhibit moderately higher levels of cytokines than empty control group. The highest cytokine level was observed for TNF-α (250 pg/mL), followed by IL-6 (120 pg/mL), and finally IL-1β (40 pg/mL). These levels decrease over the implantation period, and eventually to a normal range at 90 d in comparison with control group (P > 0.05), indicating that CPTMGPU films evoke mild systematic inflammation reaction, which could be eliminated after 90 d of implantation. Interestingly, there is no much difference in the serum IL-6, TNF-α and IL-1β levels between CPTMGPU20 and CPTMGPU0 groups at all-time points investigated (Figure 8), despite a more acute local response caused by CPTMGPU20 over CPTMGPU0 toward surrounding tissues before 30 d (Figure 7). This phenomenon suggests that the antibacterial



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polyurethanes with contact-active surface do not arouse global inflammatory response after a long period of implantation (> 90 d), and the CPTMGPUs are safe for long-term applications in the body. Further work is clearly needed and ongoing in our laboratory to demonstrate the potential anti-infection effect of CPTMGPUs in vivo. 4. Conclusion In conclusion, a series of crosslinked waterborne polyurethanes containing quaternary ammonium salts with long-term stability, excellent antibacterial activity and good biocompatibility were successfully fabricated by the crosslinking of PTMGPU with EGDE through a facile method. The CPTMGPU films possess a GQAS antibacterial upper layer and an antifouling PEG sublayer, with largely improved stability by crosslinking. As a result, these films exhibit recyclable contact-active bactericidal activity. In addition, the CPTMGPUs are potentially biocompatible both in vitro and in vivo, without significant cytotoxicity against healthy cells and acute irritation on their surrounding tissues and general immune system. Hence, the crosslinked waterborne polyurethane systems are potentially suited for using as coatings of implants and biomedical devices to combat microbial infections. Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.xxxxx. Additional experimental details; Figure S1-S10; Table S1-S6.

Corresponding Author * Tel.: +86-28-85460961; fax: +86-28-85405402. Email addresses: [email protected] (H. Tan), [email protected] (X. He)


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Author Contributions ‡These authors contributed equally. Acknowledgement We acknowledge the financial support from National Natural Science Foundation of China (51573112 and 51573114), the National Science Fund for Distinguished Young Scholars of China (51425305) and the Project of State Key Laboratory of Polymer Materials Engineering (Sichuan University) (SKLPME 2016-2-04).

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For Table of Contents use only

Antibacterial and Biocompatible Crosslinked Waterborne Polyurethanes Containing Gemini Quaternary Ammonium Salts ‡Yi Zhang a,b, ‡Xueling He a,c*, Mingming Ding a, Wei He a, Jiehua Lia , Jianshu Lia, Hong Tan a, *


Antibacterial and Biocompatible Cross-Linked Waterborne

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