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Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Thermosensitive Hydrogel Interface Switching from Hydrophilic Lubrication to Infection Defense Kai Zhu,† Dayong Hou,§ Yue Fei,‡ Bo Peng,‡ Ziqi Wang,§ Wanhai Xu,§ Baoning Zhu,*,† Li-Li Li,*,‡ and Hao Wang*,‡
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†
Department of Environmental Science and Engineering, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China ‡ CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beiyitiao no. 11, Haidian District, Beijing 100190, China § Department of Urology, the Fourth Hospital of Harbin Medical University, Heilongjiang Key Laboratory of Scientific Research in Urology, Yiyuan Street no. 37, Nangang District, Harbin, 150001, China S Supporting Information *
ABSTRACT: An intervention-induced infection, such as a catheter-associated infection, is one of the most common nosocomialacquired infections, which causes huge healthcare threats and costs to clinical treatment. This work developed a thermosensitive hydrogel coating on polydimethylsiloxane (PDMS), which smartly switched from hydrophilic lubrication to antimicrobial and antifouling properties. Upon the optimization of the molar ratio of N-isopropylacrylamide versus N,N′-methylenebis(2propenamide) (NNMBA), the thermosensitive hydrogel coating exhibited hydrophilic lubrication and 2.5-fold and 4.4-fold contact angle hysteresis than those of silicone and PDMS at room temperature, respectively, which provided significant protection to prevent tissue injury during the intervention in vivo. Once reaching body temperature, the hydrogel coating collapsed into a rough morphology with a hydrophobic inlayer and an exposed antibacterial peptide outlayer, which was endowed with an excellent antibacterial adhesion ability, reducing 96.6% of bacterial adherence relative to bare PDMS. The in vivo implantation demonstrated that the coating significantly prevents the infection, which exhibited over 3 × 103 and 103 folds of the bacterial number of the surface and surrounding tissue lower than that of the bare implants, respectively. The hydrogel coating had a good biocompatibility with a rare cytotoxicity. This adapted hydrogel interface switching from hydrophilic lubrication to preventing infections offered a surface coating strategy for medical device implantation. KEYWORDS: thermosensitivity, hydrogel coating, antibacterial peptide, antifouling, infection
1. INTRODUCTION
and aldehyde-modified poly(ethylene glycol) hydrogel loaded colistin13 are reported with the antifouling capability and antimicrobial agent released manner.14,15 However, current antimicrobial coatings still face two challenges with extended use of implants in vivo: the antimicrobial sustainability and the prevention of bacterial colonization.16−18 Since Scarpa et al.19 first reported the thermal phase transition behavior of poly(N-isopropylacrylamide) (PNIPAM) in 1967, the temperature has become the most widely triggering
Intervention-induced infections, such as catheter-associated urinary tract infections,1 subcutaneous infections,2,3 prosthetic joint infections,4 etc. are the most common nosocomial infections among acquired infections in the hospital.5 The cases of implant infections have pervasive clinical incidence, associated mortality, and significant costs. In response, antimicrobial coatings have become an important approach for preventing infection without affection of the mechanical properties of implants,6−8 wherein the antimicrobial hydrogels emerging are associated with medical devices and wound healing.9−11 For instance, the chitosan hydrogels grafted with poly(ethylene glycol) (PEG),12 antimicrobial polycarbonate (APC) hydrogels,6 © XXXX American Chemical Society
Received: May 29, 2019 Accepted: July 3, 2019 Published: July 3, 2019 A
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials stimulus in response polymers.20−22 The PNIPAM displays a lower critical solution temperature (LCST) with a hydrophilicto-hydrophobic reversibly transition.23−25 PNIPAM-based hydrogels are not so susceptible to environmental influences that make LCST a big change, which guarantees the application of PNIPAM in complex biomicroenvironments.26−28 Moreover, antimicrobial peptides (AMPs) have been extensively considered as an antibacterial agent,29−31 which are not prone to cause drug resistance.32−34 The broad-spectrum antimicrobial activities of AMPs exhibit a commendable bactericidal effect by attaching onto the cell wall/membrane and inserting into the membrane bilayer of bacteria, causing the membrane to rupture by the formation of transmembrane pores, which are more effective against Gram-negative strains with thinner cell walls.35−37 In this work, we developed a thermosensitive hydrogel coating, which endowed thermo-stimuli-induced adapted switching from hydrophilic lubrication to antimicrobial and antifouling properties. The hydrogel coating was obtained by cross-linking of different molar ratios of N-isopropylacrylamide (NIPAM), N,N′-methylenebis(2-propenamide) (NNMBA), and synthetic antibacterial peptide (AMP) on the polydimethylsiloxane (PDMS) substrate through a free radical polymerization reaction (Scheme 1A). The free radicals were generated
a rough morphology with a hydrophobic inlayer for antifouling and an exposed antibacterial peptide outlayer for contact sterilization. This study provided an adapted hydrogel interface switching from hydrophilic lubrication to preventing infections for medical device implantation.
2. EXPERIMENTAL SECTION 2.1. Materials. All of Wang resins and amino acids for peptide synthesis were purchased from GL Biochem (Shanghai) Ltd. O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 4-methylmorpholine (NMM), triisopropylsilane (TIPS), trifluoroacetic acid (TFA), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), N,N′-methylenebis(acrylamide) N,N′-Methylenebis (2-propenamide) (NNMBA), N-isopropylacrylamide (NIPAM), and 3-(trimethoxysilyl) propyl methacrylate (TMSPMA) were purchased from Sigma-Aldrich Chemical Co. Polydimethylsiloxane (PDMS, Sylgard 184) was purchased from Dow Corning Co. Ltd. 2.2. Characterization. Fourier transform infrared spectrometer (Spectrum One, America) was used to measure functional groups of interfaces in the modification process. XPS data were obtained using an X-ray photoelectron spectrometer (ESCALAB250Xi, Britain). The water contact angle (WCA), advancing angle (θadv), and receding angle (θrec) were measured and analyzed by a standard type contact angle meter (DSA 100, Germany). The morphology of modified substrates was observed by field emission scanning electron microscopy (SEM, Hitachi-SU8220, Japan). The substrates modified with the hydrogel coating were treated by lyophilization and spray-gold before observation. The thickness of the hydrogel coating was measured by a surface profiler (Bruker-DektakXT, America). 2.3. Synthesis of Peptides. The peptide was synthesized by solid phase peptide synthesis using standard Fmoc-chemistry. Then, 0.35 mM Fmoc-Gly-OH-Wang resin (loading = 0.343 mmol/g) was swelled in anhydrous N,N-dimethylformamide (DMF) overnight. Then the deprotecting agent (piperidine/DMF = 1:4, v/v) was used to deprotect the N-terminal Fmoc group for 15 min. Qualitative Fmoc deprotection was confirmed by a ninhydrin test (ninhydrin/phenol/ ascorbic acid = 1:1:1, v/v). The next amino acid in the NMM/DMF (5:95, v/v) and HBTU mixture was added and oscillated for 1 h. The above steps were repeated until the threonine coupled successfully. Then acrylic acid in a mixture of NMM/DMF (5:95, v/v) and HBTU was added and oscillated for 1 h after the N-terminal Fmoc group of the threonine was deprotected. The peptide was cleaved from the beads, and all of the amino acid side chains were deprotected by reaction with a mixture of TFA (95%, v/v), H2O (2.5%, v/v), and TIPS (2.5%, v/v) for 3 h in an ice bath. After the mixture was filtered, the remaining liquid was dried with nitrogen. The peptide was precipitated in cold anhydrous diethyl ether and centrifuged three times to collect. Finally, the peptide was dried under a vacuum overnight and purified by preparative reversed-phase high-performance liquid chromatography. 2.4. Preparation of the Thermosensitive Hydrogel Interface. PDMS was hydroxylated by plasma cleaner for 10 min, and then the above substrates were reacted with TMSPMA (2%, v/v) in ethanol overnight at room temperature. After that, the substrates were rinsed with ultrapure water and dried with nitrogen. The different pore size thermosensitive hydrogel coatings without AMP (PDMShydrogel) were prepared by reacting with the mixture of APS (1 mg/mL), TEMED (1% v/v), different molar ratios of NIPAM versus NNMBA (HP1 = 50:1, HP2 = 100:1, HP3 = 200:1, HP4 = 400:1) in ultrapure water under nitrogen at room temperature for 2 h. The thermosensitive hydrogel coatings containing AMP (PDMS-hydrogel-AMP) were prepared in the same manner as above except antibacterial peptide was added. 2.5. Antimicrobial and Antifouling Assays. The substrates modified by hydrogel coatings formed by different molar ratios of NIPAM/NNMBA (50:1, 100:1, 200:1, 400:1) without AMP (HP1, HP2, HP3, and HP4) and containing AMP (n(AMP)/n(NIPAM) = 1:200, HP1-P, HP2-P, HP3-P, and HP4-P) were incubated in a
Scheme 1. Schematic Illustration of the (A) Fabrication Process and (B) Smart Switching from Hydrophilic Lubrication To Prevent Infections of the Thermosensitive Hydrogel Coating
from ammonium persulfate (APS) catalyzed by N,N,N′,N′tetramethylethylenediamine (TEMED). After the hydrogel coating under consideration of the thermosensitivity and bactericidal effect was optimized, the thermosensitive antibacterial hydrogel coating was obtained. As shown in Scheme 1B, the hydrogel coating performed a lubricative behavior at room temperature due to the hydrophilicity to reduce the risk of mechanical wound-induced infections. Once reaching body temperature, which was higher than the LCST of the PNIPAM-based coating, the hydrogel coating collapsed into B
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 1. Characterization of surface modifications. (A) Fourier transform infrared spectroscopy (FTIR) spectra of PDMS, hydroxylated PDMS (PDMS−OH), TMSPMA-modified PDMS (PDMS-TMSPMA) and hydrogel-coated PDMS (PDMS-hydrogel). (B) X-ray photoelectron spectroscopy (XPS) peak differentiating and fitting analysis of O 1s spectra of PDMS, PDMS-TMSPMA and the C 1s spectra of PDMS-TMSPMA, PDMS-hydrogel. 108 CFU/mL bacterial suspension at 37 °C for 3 days, rinsed with saline solution, and were placed in 1 mL of saline solution to sonicate for 15 min. Then the bacterial suspension was centrifuged 3 times after the substrates had been removed. One volume of DMAO and two volumes of EthD-III were mixed in a microcentrifuge tube, and 8 volumes of saline solution were added to obtain a 100× dye solution. For each 100 μL of the bacterial suspension, 1 μL of the staining solution was added. Then the mixture was incubated in the dark at room temperature for 15 min. Then 2 μL of the stained bacterial suspension was mounted on a slide with a coverslip. The live bacteria (green fluorescence) and dead bacteria (red fluorescence) were detected under excitation light at 488 and 543 nm, respectively, by a confocal laser scanning microscope. For each sample, 3−5 images of the field of view were taken along the length of the slide. The numbers of live and dead bacteria were enumerated per field of view by Software image J for each image taken. 2.6. In Vivo Implanted Infection. The animal experiment was approved by the Experimental Animal Unit of The National Center for Nanoscience and Technology. Two implant groups (PDMS and PDMS modified HP2-P) were subcutaneously implanted into 5 specific pathogen-free female mice ranging from 220 to 240 g (age 9−10 weeks). Five implants of each group were inoculated with 109 CFU/mL of E. coli prior to implantation. The mice were monitored daily after surgery. The mice were anesthetized on day 5, and all implants were removed. Three implants from each group were placed in 1 mL of sterile PBS and sonicated for 15 min to separate the adherent bacteria. The bacteria were counted after serial dilutions and plated on LB agar plates. The other two implants of each group were gently washed with PBS and fixed with 2.5% glutaraldehyde, dehydrated by an ethanol gradient, and dried for SEM observation. The tissues around the implant were observed by staining with hematoxylin and eosin (H&E) and grounded for colony counting. The entire experiment was repeated in parallel three times. 2.7. Catheterization. Two substrates of the indwelling catheter (TPU, 24G) and HP2-P-coated indwelling catheter were compared for demonstration of the lubrication in vivo. Upon the urethral catheterization repeated 3 times, the histopathological slices of the urethra were carried out. The slices were observed by staining with hematoxylin and eosin (H&E).
Figure 2. (A) The SEM images of the hydrogel coatings in different molar ratios of NIPAM/NNMBA. HP1 = 50:1, HP2 = 100:1, HP3 = 200:1, HP4 = 400:1. (B) The thickness and pore size of hydrogel coatings of HP1, HP2, HP3, and HP4. Data are presented as mean ± standard deviation (sd) (n = 3). C
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Figure 3. (A) Water contact angles of PDMS, PDMS−OH, PDMS-TMSPMA, and PDMS-hydrogel. (B) Cyclic thermosensitivity between 4 and 37 °C of HP1, HP2, HP3, and HP4 by water contact angles. (C) The lubricity of HP2, silicone, and PDMS by contact angle hysteresis (Δθ), which was calculated by subtraction of the advancing contact angle (θadv) with the receding contact angle (θrec). (D) The calculated contact angle hysteresis (Δθ). Data are presented as mean ± standard deviation (sd) (n = 3), analyzed by a two-pair sample t test. **p < 0.01, and ***p < 0.001.
3. RESULTS AND DISCUSSION 3.1. Characterizations of Surface Modification. The surface modification of the thermosensitive hydrogel onto a PDMS substrate was characterized and analyzed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angles (WCAs), respectively. As shown in Figure 1A, the hydroxylated PDMS (PDMS−OH) produced an absorption peak at ∼3368 cm−1 due to the stretching vibration of OH group (zoom in with the black dotted box in Figure 1A).38 When the TMSPMA was successfully grafted onto the PDMS substrate, the reaction weakened the intensity of the OH group absorption peak, while two new absorption peaks at ∼1710 cm−1 and ∼1640 cm−1 were generated, which were respectively assigned to CO and CC groups of TMSPMA. After the free radical initiated the double bond cross-linking of the precursors, the peak of the CC group disappeared and strong absorption peaks at ∼1635 cm−1 and ∼1545 cm−1 were formed, which were representative of the stretching vibrations of CO and NH groups of the amide bond, respectively. In the meantime, a broad absorption peak was newly produced at ∼3270 cm−1, which was seen as a marker of the NH stretching vibration in the associated secondary amide. For further quantitative demonstration of the surface modification, the XPS spectra and detailed peak differentiating and fitting analysis of the chemical bond were carried out (Figure 1B). By contrast with the O 1s spectra of PDMS and PDMS-TMSPMA, it was found that PDMS mainly
contained OSi bonds, and the content of OSi bonds reduced from 98.2% to 81.9% after grafting TMSPMA onto the surface. Compared with the C 1s spectra of PDMSTMSPMA, the NCO bond from the PDMS-hydrogel was newly found with a ratio of 12.9%. Meanwhile, the generated signal of N 1s around the binding energy of 399.91 eV with a relative N atomic percent of 4.43% both confirmed the hydrogel modification (Figure S1 and Table S1). In this regard, the hydrogel coating was successfully covalent modified onto the PDMS substrate step by step. We adjusted the molar ratio of precursors (NIPAM and NNMBA) from 50:1 to 400:1 and observed the morphologies of hydrogel coatings by SEM. Dependent on the molar ratio of NIPAM/NNMBA, the hydrogel coatings were respectively named as HP1 (50:1), HP2 (100:1), HP3 (200:1), and HP4 (400:1) (Table S2). The morphologies of the dried hydrogel coatings presented differently in the networks in Figure 2A. The quantitative calculations of the thickness and the average pore size of the networks (Figure 2B), provided us with a regular change that the higher the molar ratio of NIPAM/ NNMBA, the bigger the average pore size and the thinner the coating thickness was. As presented, the thickness of HP1 to HP4 was gradually decreased from 61.5 to 0.7 μm, while the pore size increased synchronously from 1.1 ± 0.2 μm to 25.0 ± 5.1 μm. The observation of morphologies of the dried hydrogel coatings further demonstrates the successful modification of the hydrogel coating on PDMS. D
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 4. Antibacterial and antifouling properties of the hydrogel coatings. (A) Fluorescence microscopy images of E. coli on PDMS and PDMS modified HP2 with different molar ratios of AMP/NIPAM after 72 h incubation. Bars: 5 μm. (B) Fluorescence microscopy images of E. coli on PDMS, PDMS-hydrogel, and PDMS-hydrogel-AMP (n(AMP)/n(NIPAM) = 1:200) after 72 h incubation. Bars: 10 μm. (C) The bacterial adherence (PDMS as 100%) of the different coatings. Data are presented as mean ± standard deviation (sd) (n = 3), analyzed by a two-pair sample t test. Statistical significance is indicated as **p < 0.01, and ***p < 0.001. (D) The rough network architectures of HP2 and HP2-P dried at 37 °C were observed by SEM.
3.2. Thermosensitivity and Lubricity of Hydrogel Coatings. As we know, the thickness and pore size both play important roles in regulation of thermosensitivity. First, the wettability of the interface exhibited dramatic change during the modification procedure (Figure 3A). The pristine PDMS was hydrophobic with a water contact angle (CA) of 96.0 ± 8.7°. After the fresh hydroxylation with plasma cleaner, the CA was reduced to 35.3 ± 4.2°, which acted the hydrophilicity temporarily in atmosphere. Upon grafting of TMSPMA, the contact angle slightly increased to 42.1 ± 3.5°. Then, the hydrogel coating stabilized as the hydrophilic surface with a contact angle of 28.1 ± 7.4° at room temperature. When tested the water contact angles of the coatings formed by different molar ratios of NIPAM/NNMBA, the temperature changing from 4 to 37 °C dramatically induced the transformation of the surface from hydrophilicity to hydrophobicity, which can be cyclically repeated (Figure 3B). The HP2 with the molar ratio of NIPAM/NNMBA of 100:1 was the chosen candidate due to the most pronounced change of hydrophilicity/hydrophobicity. Next, the lubricity of the hydrogel coating below LCST was verified by water contact angle hysteresis (CAH),39 which was
defined as the difference between two relatively stable angles, the advancing contact angle and the receding contact angle (Figure 3C).40 The phenomenon called contact angle hysteresis was proposed due to the true solid surface is somewhat uneven or chemically heterogeneous, which makes the contact angle on the surface of the actual object not unique as predicted by the Young equation.41 CAH directly characterized resistance to mobility and the low values therefore confirm smooth surface, i.e., lubricity.42 Compared HP2 modified PDMS to silicone and PDMS (Figure 3D), it is revealed that the average CAH value of HP2 was 2.5-fold and 4.4-fold lower than that of traditionally used silicone and PDMS respectively, which contributed to a better lubricity. 3.3. Antibacterial and Antifouling Properties of the Network Architectures. When the addition ratios of the antibacterial peptide were adjusted (Figure 4A, Figure S2 and Table S3), a high efficiency antibacterial hydrogel coating was obtained by covalence cross-linking with 0.5% molar ratio of AMP/NIPAM. Meanwhile, the antifouling properties of the network coated substrates were much different in performance observed by CLSM (Figure 4B, Figure S3 and Figure S4). E
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 5. In vitro biocompatibility of the hydrogel coatings. (A) L929 cells cultured in the presence of the medium soaked with PDMS and hydrogel-coating-modified PDMS for 1, 3, and 5 days, expressed as the percentage of fluorescence intensity of the TCPS control group on the first day. Data are presented as mean ± standard deviation (sd) (n = 6) (B) Fluorescence microscopy images of L929 cells on PDMS and hydrogelcoating-modified PDMS. Bars: 10 μm.
After 108 CFU/mL bacterial suspension incubation for 3 days and the free bacterial cells were removed, the quantitative calculation of the bacterial adherence compared with PDMS (set as the control with 100% bacterial adherence) revealed that the surface morphology difference of the coatings optimized the antibacterial adherence property, which provided us with the best HP2-P candidate with as little as 3.4% adherence percentage with almost 100% bacteria killing capability (Figure 4C). Along with thermosensitive hydrogel coating switching from hydrophilicity to hydrophobicity at 37 °C, the rough micronano network architecture was also formed at the same time (Figure 4D). On the one hand, the network architecture was not conducive to the maximize bacteria− surface interaction, hindering the adhesion of bacteria;43 on the other hand, the rough surface structure on PDMS reduced the surface free energy with the hydrophobic inlayer, which contributed to produce the self-cleaning ability.44−46 In addition, the synthesized AMPs formed a hydrophilic outlayer of the coating, exhibiting antibacterial ability and further contact killing the adhered bacteria. Thus, the collapsed hydrogel coating with a rough morphology of the hydrophobic inlayer and AMPs outlayer endowed the surface coating, a good antifouling, and antibacterial property simultaneously.
3.4. Biocompatibility of the Network Architectures. To study the biocompatibility of the hydrogel coating, the cell viability was well considered. When the addition ratios of the antibacterial peptide were adjusted, the cell viability well maintained over 80% with a 0.5% AMPs addition (Figure S5). Moreover, the biocompatibility of the hydrogels (HP1, HP2, HP3, and HP4) and the hydrogel with AMPs (HP1-P, HP2-P, HP3-P, and HP4-P) were systematically studied. The viability of murine fibroblast cells (L929) cultured in the presence of the medium soaked with PDMS and hydrogel-modified PDMS for 1, 3, and 5 days. Once the cell viability by a cell counting kit-8 (CCK-8) assay was quantitatively determined, there was no significant statistical decrease in cell viability of the hydrogelcoated PDMS after 5 days of coincubation with cells at 37 °C compared with bare PDMS, except for HP4-P, which showed around 70−80% cell viability (Figure 5A). The cytotoxicity of the hydrogel coating was also confirmed by CLSM with a Live/Dead staining assay. As presented, the groups of PDMS and hydrogel-coated PDMS were well adhered to alive cells except HP4-P, which was well in accordance with the cell viability above (Figure 5B). Finally, the hemolysis property of the HP2-P from 8 to 24 h was carried out (Figure S6). As observed, up to 24 h incubation with HP2-P, there rarely F
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 6. (A) The histopathological slices of the urethra structure and the urethra after catheterization of the catheter or the HP2-P-coated catheter for 3 times. Red arrows pointed at the shedding of the endothelial cell of mice urethra. All the histopathological slices were stained by hematoxylin and eosin (H&E). Bars: 50 μm. (B) Illustrations and photographs of the subcutaneous implantation and infection of untreated PDMS (right, blue circle) and HP2-P-coated PDMS (left, red circle) slices for 5 days. (C) Photograph of the inflammation of the surrounding tissues after the implants were removed. (D) SEM images of the bacterial adherence on the removed implants (PDMS and HP2-P-coated PDMS). The insets were the colony count corresponding to the implants. Bars: 1 μm. (E) The histopathological slices of the surrounding tissues corresponding to the implants (PDMS and HP2-P-coated PDMS). The yellow arrows indicated the bacterial-infection-induced leukocyte (deep purple nuclear) accumulation. The insets show the colony count corresponding to the tissues. Bars: 10 μm. (F) Statistical calculation of the number of E. coli on the surface of the implants and in the surrounding tissues after 5 days of implantation. Statistical significance is assessed by the one-way analysis of variance (ANOVA), ***p < 0.001. (F).
hemolysis happened. All of the results provided us with a good biocompatibility hydrogel coating strategy. 3.5. Lubrication Properties and Antibacterial Activity of the Hydrogel Coating in Vivo. First, the lubrication during catheterization of the different substrates, e.g., catheter (TPU, 24G) and HP2-P coated catheters, was compared (Figure 6A). Upon the urethral catheterization repeated 3 times, the histopathological slices of mice urethra were carried out. Due to the hydrophilic lubrication of the hydrogel coating at room temperature, the reduced mechanical friction to urethral endothelial cells contributed no obvious damage to the cells, while the bare catheter exhibited a urethral injury and shedding of the endothelial cell (red arrows). For further demonstrations of the infection defense property of the hydrogel coating in vivo, a protocol of an implanted infection mice model was proposed (Figure 6B). First, the PDMS group (right, blue circle) and HP2-P-modified PDMS group (left, red circle)
were subcutaneously implanted into 5 specific pathogen-free female mice with a body weight of 220−240 g (age 9−10 weeks) after inoculation with 109 CFU/mL E. coli at 37 °C for 3 days. After 4 days of observation without any operations, the implants and surrounding tissues were removed by surgery on the fifth day. As shown in Figure 6B, the PDMS group showed an obvious symptom of infection from the first to the third days and putrefied on the fourth day after infection, while the HP2-P group has no observable inflammation. After the implants were removed by surgery, the surrounding tissues in the PDMS group exhibited a serious inflamed symptom, neither observed on the HP2-P group (Figure 6C). Next, the two removed implants of each group were gently washed with PBS and fixed with 2.5% glutaraldehyde, dehydrated by an ethanol gradient, and dried for SEM observation. As shown in Figure 6D, abundant E. coli cells were adhered to the surface of PDMS, while the HP2-P surface exhibited a clear hydrogel G
DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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network without bacterial adhesion. The plate colony counting further provided the antibacterial and antifouling properties of HP2-P in vivo (inserted photos in Figure 6D). Meanwhile, we also investigated the tissues around the implants through histopathological observation by staining with hematoxylin and eosin (H&E) and statistically calculated the bacterial infection by plate cultivation (Figure 6E). The results indicated that the PDMS group suffered serious inflammation, but the HP2-P group was healthy and cured the preinoculated infection, which acted as a good antibacterial capability. The statistical analysis of plate colony counting of the implants and the corresponding tissues was shown in Figure 6F. The bacteria number adhered to the surface of implants was around 2.5 × 104 CFU in the PDMS group and lower than 10 CFU in the HP2-P group, respectively. The bacterial infection in tissues showed significant inhibition in the HP2-P group (7.1 × 104 CFU). Both results confirmed that the optimized hydrogel coating with AMPs covalence showed dramatical inhibition of bacterial infection and adhesion in vivo than the pristine substrate.
Li-Li Li: 0000-0002-9793-3995 Hao Wang: 0000-0002-1961-0787 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51873045, 31671028, and 21838001), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), and CAS Key Research Program for Frontier Sciences (QYZDJ-SSWSLH022), Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541), and the CAS Interdisciplinary Innovation Team. Dr. L.-L.L. thanks the Youth Innovation Promotion of CAS (2017053). We are very grateful to Yuhan Wei (University Grenoble-Alpes) for helping revise the manuscript.
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4. CONCLUSIONS In summary, we have developed a thermosensitive hydrogel coating switching from hydrophilic lubrication to antimicrobial and antifouling properties for preventing infection during implantation. The hydrogel coating was optimized under consideration of the thermosensitivity and bactericidal effect, and the best molar ratio of the precursors of NIPAM/NNMBA named as HP2 was obtained, which was 100:1, and the appropriated AMP covalent cross-linking onto the hydrogels with 0.5% in molar ratios with NIPAM named as HP2-P. Compared to the HP2-modified PDMS to silicone and PDMS, the average CAH value of HP2 was 2.5-fold and 4.4-fold lower than that of traditionally used silicone and PDMS, respectively, contributing to a better lubricity. In addition, according to the rough morphology of the hydrogel coating collapsed on the surface, we speculated that the rough structures with a hydrophobic inlayer and AMPs outlayer provided a reduced bacteria−surface interaction and the surface free energy, resulting in hindering the adhesion of bacteria with a good antibacterial capability. We prospected that the thermosensitive hydrogel coating strategy will provide a desirable reference based on united the hydrophilic lubrication and the preventing infection capability for medical device implantation.
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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/acsabm.9b00457. Materials and instruments, characterization of lubricity, in vitro biocompatibility assays, XPS survey spectra, characterizations of MALDI-TOF and HPLC for an antibacterial peptide, fluorescence microscopy images, cell viability, hemolysis assay, the list of surface modifications with different chemical components, and bactericidal performance of an antibacterial peptide (PDF)
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DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsabm.9b00457 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
