acrolein copolymer-based hydrogels as promising antimicrobial agents for effective bacteria therapy

Journal Pre-proofs Unusual allyl diazoacetate/acrolein copolymer-based hydrogels as promising antimicrobial agents for effective bacteria therapy Jueqin She, Yan Li, Chao Zhou, Shuguang Chen, Jianghua Li, Yue-Fei Zhang, Feifei Zhang, Bo Liu PII: DOI: Reference:

S1385-8947(20)30105-4 https://doi.org/10.1016/j.cej.2020.124114 CEJ 124114

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

14 October 2019 6 January 2020 11 January 2020

Please cite this article as: J. She, Y. Li, C. Zhou, S. Chen, J. Li, Y-F. Zhang, F. Zhang, B. Liu, Unusual allyl diazoacetate/acrolein copolymer-based hydrogels as promising antimicrobial agents for effective bacteria therapy, Chemical Engineering Journal (2020), doi: https://doi.org/10.1016/j.cej.2020.124114

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Unusual allyl diazoacetate/acrolein copolymer-based hydrogels as promising antimicrobial agents for effective bacteria therapy Jueqin She,a Yan Li, a Chao Zhou,c Shuguang Chen,a Jianghua Li,a Yue-Fei Zhang,a Feifei Zhang,*d Bo Liu*a a College

of Materials Science and Engineering, Changsha University of Science and Technology, Changsha, 410114, China

b Department c Institute

of Biomedical Engineering, Michigan Technological University, Houghton, Michigan 49931, United States

of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, 213164, China

d Department

of Materials Science & Engineering, National University of Singapore, 117585, Singapore

† Authors contributed equally to this work. *

Correspondence author: Bo Liu, E-mail: [email protected]; Feifei Zhang, E-mail: [email protected]

Abstract: The abuse of antibiotics has led to the enhancement of bacterial drug resistance, which has aroused widespread concern. In our study, an unusual polymer (PADAAC) with aldehyde, vinyl and azo groups was obtained by copolymerization of allyl diazoacetate (ADA) and acrolein (AC). We fabricated a series of hydrogels (PA0AM, PA5AM, PA10AM and PA20AM) by incorporation PADAAC into the acrylamide and N, N’-methylene bis(acrylamide) networks, which can suppress bacterial growth and kill the bacteria with > 95%. All the hydrogels possessed low cytotoxicity. More remarkably, the aldehyde groups were protected amino groups via a simple imine reaction. Protected PA20AM had no effect on the bacteria, while deprotected PA20AM recovered its antimicrobial property against Staphylococcus aureus (killing efficiency: 96.1%), Escherichia coli (killing efficiency: 98.3%) and methicillin-resistant Staphylococcus aureus (MRSA) (killing efficiency: 95.0%) after removing the amino groups. PA20AM and deprotected PA20AM also exhibited good antimicrobial property in vivo. Keywords: allyl diazoacetate; antimicrobial hydrogels; imine reaction; cytotoxicity; in vivo

1. Introduction Bacterial infections, especially those caused by drug-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)), have become a significant public health risk and can be a tremendous economic burden for the patients [1-3]. Additionally, bacteria will easily assemble to form biofilms that need hundreds dose of drugs to completely eradicate [4, 5]. In the meantime, bacteria are usually taking new drug resistant strategies, like restricting access of the drugs, getting rid of the drugs, decomposing the drugs and bypass the effects of the drugs, to avoid the effects of drugs and result the formation of drug resistance [6]. Researchers have found out some effective approaches to aim the widespread health problems caused by the bacteria and biofilm, like antibacterial peptides (AMPs) [7], photodynamic antibacterial method [8, 9], inorganic antibacterial material [10, 11], hydrophilic

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antibacterial adhesion material [12, 13], super hydrophobic or "slippery" antibacterial adhesion surface [14], biomimetic nanostructure antibacterial surface [15] and gas antibacterial method (CO [16], NO [17]) etc. Antimicrobial polymers as the new antimicrobial agents have been widely used to combat with drug resistant bacteria owing to the unique antimicrobial model [18-20]. Two main approaches are the (i) development of polymers or polymer surfaces with antifouling properties [21]and (ii) development of polymers with antimicrobial properties [22, 23]. The synthetic antimicrobial polymers mainly include polymers with quaternary nitrogen atoms [24, 25], guanidine containing polymers [26], polymers mimic natural peptides [27, 28], halogen polymers [29] and metal containing polymers [30]. However, there are some limitations in the existing approaches. For example, most investigated cationic polymers are prone to bind to the negatively charged bacterial cell surface, as such, bacteria will easily adhere on the polymers and retard the antimicrobial activities. Besides, the antimicrobial agents releasing from the substances (polymers, gels or surface) will be harmful to the environment and human health [31]. Hydrogels, which have unique three-dimensional network structure and can absorb large amounts of water, are widely used as antimicrobial materials because of their injectability, excellent mechanical properties and preferable biocompatibility [32]. There are two main preparation strategies for antibacterial hydrogels: one is to obtain antibacterial molecules through physical crosslinking or covalent crosslinking, and the other is to directly use antibacterial monomers to prepare antibacterial hydrogels, which show inherent antibacterial properties. Li and coworkers developed a new type of antimicrobial hydrogels using catechol and ε-poly-L-lysine, which exhibited excellent contact-active antimicrobial activities against Gram negative bacteria Escherichia coli (E. coli) and Gram positive methicillin-resistant Staphylococcus aureus (MRSA) [33]. Our group fabricated the antimicrobial hydrogels with a series of side-chain imidazolium salts and main-chain polyimidazolium salts. The hydrogels also exhibited strong antimicrobial activities against clinically isolated multidrug-resistant bacteria [34]. However, the antimicrobial property of cationic polymers and hydrogels is partially derived from its ability to interact with the negatively charged bacterial cell wall through electrostatic interactions [34]. This will result in bacteria adhering to the cationic polymers or surfaces, and subsequently retarding the antimicrobial properties. In this regard, we aim to address the above mentioned shortages of conventional polymers to design an intrinsically antimicrobial polymer. In this paper, we prepare poly(allyl diazoacetate-co-acrolein) (PADAAC) by copolymerization alkyl diazoacetates with α,β-unsaturated aldehydes in one pot. We fabricate PADAAC-containing hydrogels with acrylamide (AM) monomer as the backbone and N, N’-methylene bis(acrylamide) (MBAA) as the crosslinker. The antimicrobial activities and cytotoxicity of these hydrogels are examined. 2. Experimental section 2.1 Materials Unless otherwise specified, all the reagents were used as received. Acrylamide (AM) as a monomer, N, N′methylene bis(acrylamide) (MBAA) as a crosslinker, ammonium persulfate (APS) as an initiator, 2,2’(ethylenedioxy)bis(ethylamine) (EBEA), 3-[4, 5-dimethylthiazoyl-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), hydrogen chloride solution (37wt%), sodium chloride and dimethyl sulfoxide (DMSO) were all purchased from

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Sigma-Aldrich. Poly (ADA-co-Ac) (PADAAC) as a comonomer was synthesized by copolymerization of allyl diazoacetate (ADA) with acrolein (AC), as previously described. [35] Distilled water was taken from the Milli-Q Millipore system. All the bacteria (S. aureus ATCC 25923, E. coli DH5α and MRSA USA300) were bought from American Type Culture Collection (ATCC). Lysogeny Broth (LB) Agar medium, Mueller Hinton Broth medium (MHB) and Tryptic Soy Broth medium (TSB) and LIVE/DEAD Bac Light Bacterial Viability Kit were purchased from Thermo Fisher Scientific. 2.2 Methods 2.2.1 Preparation of poly (ADA-co-Ac) (PADAAC) ADA was fabricated according to a previously reported article. Simplicity, PADAAC was synthesized by copolymerization of ADA and acrolein (AC). Firstly, ADA (2.52g, 0.02 mol, 1.00 equiv) was dissolved in 20 mL chloroform in a 100-mL three-necked round-bottomed flask with blow of Ar, connected with a reflux condensation tube (the other end of the condensation tube was connected with a safety bottle containing silicone oil), and then dripped with 10 mL chloroform containing AC (1.34g, 0.024 mol, 1.20 equiv). The mixture was stirred and heated at 50 oC for 8 hours. After the reaction, the precipitates were precipitated with ether, and then centrifuged to collect the precipitates. Finally, the final product PADAAC (2.44g, 63.1%) was obtained by vacuum dried for 24 hours under vacuum conditions. 2.2.2 Preparation of the hydrogel We synthesized various AM-based hydrogels with different amounts of PADAAC comonomers ranging from 0 to 20 mol% (versus AM monomer) by the copolymerization of AM and MBAA. Firstly, the comonomer (PADAAC) which was varied from 0, 5, 10, and 20 mol% was dissolved in DMSO solution. Then the monomer (AM), the crosslinker (MBAA) (10 mol%, versus AM monomer), and the initiator (APS) (2 mol%, versus AM monomer) were added and completely dissolved in the above solutions under the ultrasonic at room temperature. Then the solution mixtures were separately added into the poly-(tetrafluoroethylene) (PTFE) molds and solidified within 10 min at 80 °C. The reactions were completed at 80 °C for 3 h. After the polymerization, the hydrogels were removed from the molds and immersed in a beaker containing DI water that replace the DMSO in the gels. The DI water was changed every 2h for 12 h. Finally, hydrogels were washed with ethanol and DI water several times to ensure that non-reacted initiators and monomers were totally removed from the hydrogels. 2.2.3 Preparation of EBEA protected PA20AM PADAAC (20 mol%, versus AM monomer), AM, the crosslinker (MBAA) (10 mol%, versus AM monomer), EBEA (1 mol%, versus AM monomer), and the initiator (APS) (2 mol%, versus AM monomer) were dissolved in DMSO under the ultrasonic at room temperature. Then the solution mixture was separately added into the poly(tetrafluoroethylene) (PTFE) molds and solidified within 10 min at 80 °C. The reactions were completed at 80 °C for 3 h. After the polymerization, the hydrogels were removed from the molds and immersed in a beaker containing DI water that replace the DMSO in the gels. The DI water was changed every 2h for 12 h. Finally, hydrogels were

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washed with ethanol and DI water several times to ensure that non-reacted initiators and monomers were totally removed from the hydrogels. Deprotection of PA20AM. EBEA protected PA20AM was deprotected by pH 3 solution, which was made from 1 N HCl and 0.1 N sodium chloride solution. The imide bond was cleavage after immersing in pH 3 solution for 12h. After that, the hydrogel was again immersed in DI water for another 4h to remove the remaining amine and salts. 2.2.4 Preparation of AM-PADAAC coating on the glass slides Glass slides (length*width*height = 0.5cm*0.5cm*0.1cm) were first treated with piranha solution for 1 min, and then washed with DI water for several times. Piranha treated Glass slides was dried at 50 oC overnight. The slides were immersed in the DMSO solution containing PADAAC (20 mol%, versus AM monomer), AM, and the APS (2 mol%, versus AM monomer). The orange coating was formed at 80 oC for 5h. Finally, the coating was washed with ethanol and DI water several times. 2.2.5 Polymer Characterization Flourier transformed infrared spectroscopy (FT-IR, Nicolet 5700, Thermo, USA) was used to analyze the chemical structure of materials. The morphology structures of the freeze-dried materials with conductive platinum were characterized by the scanning electron microscope (SEM, JEOL JSM 6700F, USA). Confocal microscopy was mainly used to determine the fluorescence properties of these samples. 1H NMR spectra were collected on a Bruker Avance 300 MHz instrument using CDCl3 as the solvent. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy was performed using a 4800 MALDI TOF/TOF analyzer (ABSciex) equipped with a Nd:YAG 355 nm laser. Single-photon fluorescence excitation and emission spectra were recorded on a RF-5301PC (Shimadzu) with a slit width settled as 5.0 nm for both emission and excitation. 2.2.6 Measurement of the swelling ratio The swelling behaviors of the hydrogels in PBS buffer were measured by the gravimetric method at room temperature. The swelling ratio was calculated according to the average values of the three measurements. The swelling ratio (SR) was calculated via the following equation: SR = (Ws−Wd)/Wd ×100%. Which Ws is the weight of the swollen hydrogel and Wd is the weight of the dried hydrogel. 2.2.7 Antimicrobial testing The antimicrobial activities of samples (hydrogels or coating) against bacteria were quantitatively evaluated using viable cell count method. Overnight bacteria cultures were prepared in LB broth medium at 37 oC, and sub-cultured to mid-log phase for further use. LB was remove and the bacteria were washed thrice with PBS (pH 7.4). 10 μL of 105 bacterial suspension in PBS was spread evenly onto the surface of samples, and these samples were incubated

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at 37 oC for 2, 4, 8, 24h (8h is for the coating). Samples were transferred to 2 mL centrifuge containing 1 mL PBS and the solutions were vortexed to obtain bacterial suspension. The bacterial suspension was diluted with PBS and plated on LB agar, and further incubated at 37 oC for 24h. The number of colony forming units (CFU) were determined. 2.2.8 Fluorescence Microscopy Bacteria suspensions were seeded onto confocal dish for 24 h. Then, the sterilized hydrogels (diameter = 1 cm, thickness = 2 mm) were put onto the dishes which seeded bacteria at 37 °C at a given time. The hydrogels then removed. Stain containing L13152 LIVE/DEAD

®

Bac Light TM Bacterial Viability Kit was consulted to the

manufacturer’s protocol. 40μL of stain solution (the final concentration of each dye will be 6 µM SYTO 9 stain and 30 µM propidium iodide) was added and spread on covered zone by hydrogel and incubated in the dark for 15 min before examination under a fluorescence microscope. Fluorescence imaging was done with 480/500 nm filter for SYTO 9 stain and 490/635 nm filter for propidium iodide in the microscope optical path by Zeiss LSM 710 for the LIVE/DEAD bacteria imaging. 2.2.9 Morphology of bacteria Bacteria suspensions were seeded onto glass dish for 24 h, respectively. Then, the sterilized hydrogels (diameter = 1 cm, thickness = 2 mm) were put onto glass dish which seeded bacteria at 37 °C at a given time. The hydrogels then removed. Each sample was immersed in 2 mL 2.5 wt% glutaraldehyde in PBS and refrigerated at 4 °C overnight. The samples were dehydrated with a graded concentration series of ethanol/water mixtures (25%, 50%, 75%, 100%). Dehydrated samples were further dried under nitrogen before coating with platinum for Field Emission Scanning Electron Microscopy (FE-SEM, Zeiss Sigma 500). 2.2.10 In vivo murine wound excision 5 weeks old female mice were used for the excisional wound experiment. Mice were anaesthetized, depilated followed by creating 6 mm diameter excisional wound on mice dorsal skin. MRSA (1×106 cfu in 10µL PBS) was then inoculated on the wound site and allowed to settle 10min to stimulate an infection. After that, PA20AM and deprotected PA20AM (diameter = 0.5 cm, thickness = 2 mm) were placed on the wound and further secured with Tegaderm (3M) dressing. Control hydrogel made of PA0AM (diameter = 0.5 cm, thickness = 2 mm) was used as control. After 24h post-infection, mice skin (1cm×1cm square) including wound and peripheral region were harvested, homogenized, followed by plating to determine bacterial CFU. One-way ANOVA analysis was used to calculate the p value. 2.2.11 Cell viability by contact MTT assays MTT assay was further used to determine the relative cell viability of 3T3-E1 exposed to the hydrogel extract. Sterilized hydrogel samples (diameter = 1 cm, thickness = 2 mm) were immersed in the PBS medium for 24h at room temperature. 100 μL of the hydrogel extract was added to 100 μL of 105 cells/cm2 of cell seeded in a 96-well cell culture plate and incubated for 24h at 37 °C in 5% CO2. 100 μL of PBS was used as the control instead of hydrogel

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extract. After 24 h incubation, 20 μL of MTT (5 mg/mL) was added to each well followed by another 4h of incubation. The supernatant was discarded, and 150 μL of DMSO was added to each well and shook for 15 min. OD values were measured at 570 nm using infinite F50 TECAN plate reader. Cell viability was calculated based on the following equation: Cell viability (%) = ODhydrogel well/ODcontrol well × 100% 2.2.12 Fluorescence of live/dead staining 3T3-E1 Firstly, each hydrogels sample (diameter = 0.5 cm, thickness=2mm, previously washed with water three times and UV sterilized for 1 h) were put into a 48-well plate; then 500 μL of 105 cells/ mL 3T3-E1 cells suspension in supplemented Dulbecco’s Modified Eagle’s Medium (DMEM with 10% FBS and 1% penicillin-streptomycin) were seeded in each well. The 48-well plate were incubated in Cell incubator for 24 h at 37 °C with 5% CO2; after 24 h, the solution in each well was discards then washed cells three times via added 500 μL 1X Assay Buffer to fully remove residual liquid. 200 μL of working solution (2 mM Calcein-AM and 1.5 mM PI) was added to each well followed by 30 min incubation. Photographs were captured via a polarizing optical microscope (Nikon, ECLIPSE E200) at 490 ±10 nm. 2.2.13 Statistical analysis Statistical analyses were performed using Student’s two-tailed unpaired t-test. In this mode of analysis, all measurements were presented in the form of mean values ± standard deviations (SD) of three or six groups of raw data collected in the experiment. For the test, a p-value < 0.05 was considered statistically significant.

3. Results and discussion

PADAAC is obtained by copolymerization of allyl diazoacetate (ADA) with acrolein (AC) in a one-pot reaction (Figure S1) [35]. The diradical of ADA firstly react with the double bond of AC self-initially to form a 1, 5-diradical intermediate. The polymer chain propagates by the insertion of ADA, AC and CAC (generated from ADA by the loss of N2), subsequently forming the 1, 5diradical. Finally, the chain terminates by the addition of hydrogen radical. Microstructure of PADAAC is investigated by MALDI-TOF (Figure S2). Taking m/z 913.3018 for example, it can be resolved as [H-(ADA)5-(AC)5-H]·H+ or [H-(ADA)6-(CAC)1(AC)1-H]·H+ with hydrogen atoms as the terminal groups (ADA 126Da, CAC 98 Da and AC 56 Da), in which the –N=N- group of ADA is inserted into the backbone. The copolymer PADAAC is a ternary copolymer, although it is obtained by the copolymerization of two monomers. As is shown in 1H NMR (Figure 1a), the signals of allyloxy group are located at δ 5.31 ppm (2H, CH=CH2), 5.91 ppm (1H, CH=CH2) and δ 4.66 (2H, –OCH2). The protons in the backbone composed of acrolein present broad peak from δ 2.8 ppm to 3.5 ppm. The ratio of the hydrogen in the aldehyde group (δ 9.4-9.8 ppm) to the hydrogen in the allyl group (δ 5.91 ppm) is about 1:2, which suggests that the number of aldehyde groups is half amount of allyl groups in the copolymer. More interestingly, azo groups are inserted into the backbone of the chain, which endow the copolymer with fluorescent properties. As is shown in Figure 1b, PADAAC can emit the green light of 500 nm excited by 395 nm. From the above, PADAAC is a fluorescent copolymer, but also an unusual copolymer that contains both allyl groups and aldehyde groups in the side chain, which makes it possible be further post-functionalized. Allyl groups can crosslink with

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unsaturated double-bond-contained monomers or polymers. Meanwhile, polymers with abundant aldehyde groups have rich chemical activities and bioactivities that can be widely used chemical engineering and biomedical fields. More importantly, they can undergo a wide variety of chemical reactions like reduction, oxidation and nucleophilic addition [36, 37].

Figure 1 (a) 1H NMR and the (b) fluorescence spectra of PADAAC.

We fabricate the hydrogels with the neutral PADAAC, hydrophilic acrylamide (AM) monomer, crosslinker N, N′-methylenebis (acrylamide) (MBAA) initiating by ammonium persulfate (APS). Hydrogels are named as PA0AM, PA5AM, PA10AM and PA20AM, representing the concentration of PADAAC is 0, 5, 10, 20 mol% respectively. All the hydrogels can be crosslinked in a short time (about 10 minutes) as determined visually using the vial inversion method (Figure 2a). The color of the hydrogels changed from white to orange and red when the concentration of PADAAC increases (Figure 2b). Morphology of lyophilized hydrogel exhibited typical porous structure of a hydrogel network (Figure 2c). FTIR spectra confirm the characteristics peaks for PADAAC (-CHO 1745 cm-1, -C-O-C- 1188 cm-1, -CH=CH2- 936 and 995 cm-1) in these PADAAC-contained hydrogels (Figure 2d). Meanwhile, these characteristics peaks grow as the component of PADAAC increases. When the hydrogels are immersed in deionized water, the PADAAC containing hydrogels to reach their maximum swelling ratios (around 238%) in PA10AM, which is lower than that of PA0AM (without PADAAC). Hydrogels can absorb huge amounts of water due to the hydrophilic AM and a richer network formed by the copolymerization of AM and PADAAC as shown in Figure 2e.

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Figure 2 (a) Schematic crosslink of PADAAC with AM and MBAA initiated by APS to fabricate hydrogels, (b) The color of different contents, (c) SEM, (d) FT-IR and (e) swelling ratio of hydrogels.

Antibacterial hydrogels, which can retain an abundance of water and apply under physiological conditions, are recognized as important biomaterials due to their effective inhibition of bacterial infections. Antimicrobial activities of PA0AM, PA5AM, PA10AM and PA20AM are evaluated by using contact killing protocol against pathogenic microbes Gram-positive S. aureus and Gram-negative E. coli. PA0AM without PADAAC exhibits no antimicrobial activity. In sharp contrast, PA5AM with 5 mol% PADAAC have 90.1~94.3% killing efficiency against S. aureus and E. coli from 2h to 24h, while PA10AM and PA20AM with the higher PADAAC contents can completely suppress bacterial growth and kill S. aureus and E. coli with 99.0% and 98.5% efficiency after 24h (Figures 3a and 3b). The antibiofilm ability of the gels is tested against E. coli and S. aureus. We seed two bacterial suspensions onto the confocal dishes to preform mature bacterial biofilms, then PA0AM and PA20AM are covered onto the surfaces of two biofilms. S. aureus and E. coli treated with PA0AM formed the dense and thick biofilms after 24h incubation (Figure 3b), indicating most of these bacteria are alive. In contrast, bacteria treated with PA20AM significantly reduce cellular density and the bacteria are mostly dead. Our results demonstrate that the PADAAC containing hydrogels are antimicrobial even after the bacteria are cultured for a longer time. The antimicrobial mechanism of the hydrogels is further confirmed by FE-SEM images against S. aureus and E. coli (Figure 4). Both spherical S. aureus and rodlike E. coli treated with PA0AM assemble and form the thick and dense bacterial films after inoculating for 24h (Figures 4a and 4c). In the case of PA20AM, the density of bacteria is sharply deceased after 24h (Figures 4b (i) and 4d (i)). More interestingly, cellular deformation and surface roughness are clearly found after exposure to PA20AM. Some concave holes are found on the

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surface of S. aureus, while debris and lysed cells are observed for E. coli (Figures 4b (ii) and 4d (ii)). This observation is consistent with antimicrobial study above (Figures 3a and 3b).

Figure 3 CFU of (a) S. aureus and (b) E. coli after treated with PA0AM, PA5AM, PA10AM, PA20AM after 2h, 4h, 8h and 24h; (c) Fluorescence images of LIVE/DEAD bacterial staining assay of S. aureus without and with PA20AM after 0h (c (i) and c (iii)) and 24h (c (i) and c (iv)); (d) Fluorescence images of LIVE/DEAD bacterial staining assay of E. coli without and with PA20AM after 0h (d (i) and d (iii)) and 24h (d (i) and d(iv)).

Figure 4 FE-SEM images of S. aureus with (a) PA0AM and (b) PA20AM with (i) low magnification and (ii) high magnification after 24h; FE-SEM images of E. coli with (a) PA0AM and (b) PA20AM with (i) low magnification and (ii) high magnification after 24h.

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Figure 5 (a) CFU of S. aureus, E. coli and MRSA after treated with EBEA protected PA20AM and deprotected PA20AM after 24h; Fluorescence images of LIVE/DEAD bacterial staining assay of (b) S. aureus with deprotected PA20AM after 0h and 24h (c (i) and c (ii)); FE-SEM images of (d) S. aureus and (e) E. coli with deprotected PA20AM after 24h.

We hypothesize that such strong antimicrobial activities can attribute the intrinsically high antimicrobial nature of aldehydes that can not only react with the amine groups of cell membrane, but also cleavage the peptide bond in cell wall peptidoglycan [38-40]. As such, we protect the aldehyde groups of PA20AM with 2,2’-(ethylenedioxy)bis(ethylamine) (EBEA). A new peak is appeared at 1607 cm-1 that ascribes to the stretching vibration of imine (-C=N) bond (Figure S1). The amineprotected PA20AM lose almost all the antimicrobial activities due to the hinder of aldehyde groups (Figure 5a). It is wellknown that the imine bond is pH responsive that can readily decompose into the amine and aldehyde in the acid pH [41]. The antimicrobial activities against S. aureus and E. coli are recovered when the protected PA20AM is incubated for 12 h at pH = 3 buffer. The deprotected PA20AM have 96.1, 98.3 and 95.0 % killing efficiency of S. aureus, E. coli and MRSA, respectively, which is also demonstrated by LIVE/DEAD assay and SEM (Figures 5b and 5c). As expected, this experiment confirms that the strong killing efficiency comes from the aldehyde groups of PADAAC. Researches are constantly eager to find out some small aldehyde molecules and aldehyde functional polymer with good antimicrobial activities. The antibacterial mechanism of aldehyde involves a strong association with the outer layers of bacterial cells, specifically with the unprotonated amines on the cell surface. It will irreversibly damage the cell wall and cytoplasmic membrane, resulting in decay and death of bacterial cells. For instance, glutaraldehyde [42], formaldehyde [43] and o-phthalaldehyde [44] are three main small aldehyde

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molecules that has found usage as disinfectant and sterilant in the biomedical and water treatment applications, but they can irritate people skin and respiratory tract. For the aldehyde functional polymers, the aldehydes usually come from the oxidation of alcohols. So, the grafting ratios of resulting polymers are typically not too high to have quite high bactericidal effect. Instead, the polymers usually are taken as the couple agents with the other amino group rich antimicrobial polymers (chitosan or polyethyleneimine) to fabricate the hydrogels or fibers [45]. Unlike the existing aldehyde functional monomers or polymers, we prepare a new multifunctional polymer with abundant aldehyde and polymerizable vinyl groups in a simple synthetic way, which can work as a “monomer” to incorporate into the polymer network or surface coating in any proportion. Additionally, to examine the antimicrobial activity of the surface coating, we fabricate a polymer coating with AM and PADAAC on a glass substrate using the dip-coating method. Specifically, AM-PADAAC coating obviously shows contact killing efficiency against the bacteria, and the log reduction is 1.72 (S. aureus killing ratio: 98.0%) and 1.64 (E. coli killing ratio: 97.7%) after 24h, respectively (Figure 6a). The in vivo would dressing experiments are performed on mice with MRSA to evaluate the antimicrobial ability of the hydrogels in a real bio-system. As can be seen from Figure 6b, the log reductions of PA20AM and deprotected PA20AM are 1.33 (killing ratio is 96.2%) and 1.13 (killing ratio is 91.3%) after 24 h, which is consistent with the in vitro results.

Figure 6 (a) CFU of S. aureus and E. coli with AM-PADAAC after 24h; (b) CFU of PA20AM and deprotected treated wounds after 24 h post-infection treatment model (n = 6).

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Figure 7 (a) Cell viability of 3T3-E1 when incubated with the hydrogels for 24 h with contact MTT assays; (b-f) Fluorescence images of live/dead staining 3T3-E1 with the hydrogels for 24 h.

The cytotoxicity of PA0AM, PA5AM, PA10AM, PA20AM, and EBEA protected PA20AM is further evaluated by MTT assay. The 3T3-E1 cell viability of PA0AM, PA5AM, PA10AM, PA20AM remained above 75% (Figure 7a), indicating good biocompatibility of the hydrogels. Interestingly, the amine protected hydrogel gets even better cell viability (> 85%). The fluorescence images show live 3T3-E1 spreading onto the hydrogels uniform and the fiber formed between the cells in Figures 7b-7f, which also indicate good biocompatibility of these hydrogels. Taken together, we open up a unique avenue to the antimicrobial hydrogels or coatings with the aldehyde functional polymer using a simple synthetic approach. In this paper, we just take advantage of basic polymerizable vinyl and intrinsic antimicrobial aldehyde groups of PADAAC in the antimicrobial application. However, the adjustable structure and versatile functional groups will broaden this polymer as a promising scaffold in the different applications. For example, PADAAC serves as a drug cargo by conjugating with peptides or drugs through amine and aldehyde coupling reaction. Simultaneously, due to the fluorescence azo groups, it also can used as a chemosensor or probe for bioimaging and heavy metal ions in the field of supramolecular, medicinal and environmental chemistry.

4. Conclusions

In conclusion, hydrogels were fabricated with aldehyde and vinyl functional poly(allyl diazoacetate-co-acrolein) and tested their effectiveness as antibacterial agents. The hydrogels exhibited broad-spectrum antimicrobial activities against S. aureus and E. coil, as well as multidrug resistant bacteria both in vitro and in vivo. Antimicrobial activity of amine protected PA10AM was pH responsive at pH 3 and pH 9 due to the formation of Schiff base. Moreover, PADAAC can be readily coated on the substrate to get an antimicrobial coating. The cytotoxicity of hydrogels was nontoxic towards 3T3-E1 cell. Combination of versatile polymer microstructure, strong antimicrobial activities and low cytotoxicity makes this biomaterial possible usage in the biomedical applications.

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Acknowledgements

Jueqin She, Yan Li, and Chao Zhou, contributed equally to this work. The authors would like to thank support from Hunan Provincial Natural Science Foundation of China (No. 2019JJ50641 and No. 2019JJ50652), Scientific Research Fund of Hunan Provincial Education Department (Grant No. 18C0197), the Natural Science Foundation of Jiangsu Province (No BK20180963), and Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

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Highlights 1. An oligomer with aldehyde and vinyl groups is introduced in a hydrogel with a simple synthetic way.

2. Hydrogels with aldehyde groups exhibit broad-spectrum antimicrobial actives in vitro and in vivo. 3. Via a simple imine reaction, and the resulting hydrogel can turn ON/OFF the antimicrobial property by pH adjustment.

4. The hydrogels are much less toxic than small aldehyde molecules.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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