Cross-Linking of a Hydrophilic, Antimicrobial Polycation toward a Fast

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Cross-linking of a hydrophilic, antimicrobial polycation towards a fast-swelling, antimicrobial superabsorber and interpenetrating hydrogel networks with long lasting antimicrobial properties Arne Strassburg, Johanna Petranowitsch, Florian Paetzold, Christian Krumm, Elvira Peter, Monika Meuris, Manfred Köller, and Joerg C Tiller ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10049 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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ACS Applied Materials & Interfaces

Cross-linking of a hydrophilic, antimicrobial polycation towards a fast-swelling, antimicrobial superabsorber and interpenetrating hydrogel networks with long lasting antimicrobial properties Arne Strassburg†, Johanna Petranowitsch†, Florian Paetzold†, Christian Krumm†, Elvira Peter‡, Monika Meuris†, Manfred Köller‡, Joerg C. Tiller*,† †Chair of Biomaterials and Polymer Science, Department of Biochemical and Chemical Engineering, TU Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany ‡Surgical Research, Bergmannsheil University Hospital, Ruhr-University Bochum, Bürklede-la-Camp-Platz 1, 44789 Bochum, Germany KEYWORDS: hydrogel, antimicrobial, ionene, superabsorber, interpenetrating network

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ABSTRACT: A hemocompatible, antimicrobial 3,4en-ionene (PBI) derived by polyaddition of trans-1,4-Dibromo-2-butene and N,N,N´,N´-Tetramethyl-1,3-propandiamine was crosslinked via its bromine end groups using tris(2-aminoethyl)amine (TREN) to form a fastswelling, antimicrobial superabsorber. This superabsorber is taking up the 30-fold of its weight in 60 s and the granulated material is taking up the 96-fold of its weight forming a hydrogel. It fully prevents growth of the bacterium Staphylococcus aureus. The PBI network was swollen with 2-hydroxyethyl acrylate and glycerol dimethacrylate followed by photopolymerization to form an interpenetrating hydrogel (IPH) with varying PBI content in the range of 2.0 to 7.8 wt.%. The nanophasic structure of the IPH was confirmed by atomic force microscopy and transmission electron microscopy. The bacterial cells of the nosocomial strains S. aureus, Escherichia coli, and Pseudomonas aeruginosa are killed on the IPH even at the lowest PBI concentration. The antimicrobial activity was retained after washing the hydrogels for up to 4 weeks. The IPHs show minor leaching of PBI far below its antimicrobial active concentration using a new quantitative test for PBI detection in solution. This leaching was shown to be not sufficient to form an inhibition zone and killing bacterial cells in the surroundings of the IPH.


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INTRODUCTION In each year, approximately 4 Mio. patients acquire a healthcare-associated infection (HAI) in the European Union (EU) and in a direct consequence of these infections approximately 37000 patients die.1 Therefore, biocompatible materials with long lasting intrinsic antimicrobial properties are important for modern medicine. Most antimicrobial materials, such as implants, catheters, and wound dressings contain biocides or antibiotics that are released into the surrounding and deplete eventually. Common coating systems contain silver or silver nanoparticles.2-4 Although typical tests suggest otherwise, they are based on releasing silver ions. Recent studies have shown that a coating that releases sufficient amounts of silver ions retains its antimicrobial activity for up to 6 weeks in an aqueous medium.5 Numerous other biocides and antibiotics are released from materials for medical use.6 Zumbuehl et. al. described an antifungal dextran hydrogel, loaded with amphotericin B, which kills fungi (Candida albicans) within 2 h of contact and remains biologically active for at least 53 days.7 Biodegradable antimicrobial implant materials are in current focus of research.8-10 They are considered for treatment of skin infections11 and as wound dressings12. Generating biocides, such as reactive oxygen species (ROS) that are formed from oxygen and/or water in the presence of light or other energy sources is an alternative to simple release systems, but always require an energy source, such as UV light.13-14 Systems that are antimicrobially active without leaching are contact-active antimicrobial coatings.15-19 They are mostly based on a mixture of hydrophobic and cationic groups in the right balance.20-22 Although effective, such coatings are thin and material imperfections might result in non-active surface areas. Materials with intrinsic antimicrobial properties might be an advantage over surface-modified devices. One way to achieve this is blending of a mass polymer with antimicrobial macromolecules.23 Another way is using polymer networks as contact-active antimicrobial



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materials for dental resins24-26, composite resins for clinical use27, fibers28, biocompatible polymer coatings29-30, catheters31, membranes32 and water disinfection33. Most contact-active materials are not highly swellable in water. However, numerous biomaterials applications require the use of hydrogels. The latter have unique qualities such as shock absorption, low sliding fraction, and stimuli-responsive swelling/deswelling.34 For these reasons, they are important in the several fields of medicine35, for example tissue engineering34,

36

, hygienic applications37, artificial cartilage38, cartilage regeneration38-39,

implantations into the subcutaneous tissue38, cornea repair material38, wound and surgical sealants40-42, drug delivery depots40, wound care materials40,

43

, artificial muscles40,

44

and

tissue repair45. The importance of antimicrobially active hydrogels is discussed in the recent reviews of Ng et. al.46 and Malmsten47. Only few examples of hydrogels that kill microbes only on their surface are known. Liu et. al. published an in situ formed antimicrobial and antifouling hydrogel, which can applied onto implants such as catheters as a coating, derived from polycarbonate containing quaternary ammonium groups and poly(ethylene glycol), which was active over 14 days, without showing an inhibition zone in the agar plate assay.48 A ß-hairpin peptide hydrogel was designed by Salick et. al. to inhibit potential infections on clean surfaces or can be delivered to an infected site where bacterial cells can be killed on contact.49-50 Giano et. al. reported an inherent antibacterial, syringe-injectable, bioadhesive hydrogel by mixing polydextran aldehyde and branched polyethylenimine solutions.51 This adhesive for wound-filling applications was sparing human erythrocytes and killing Gram-positive and Gram-negative bacteria. In all cases, the antimicrobial groups are part of the functional structure of the gel and defining its properties. In the present study, we aim towards a material that is an interpenetrating polymer network with one polymer mesh responsible for the material properties and another responsible for the antimicrobial properties.


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EXPERIMENTAL SECTION Materials: Methanol (VWR), acetone (Merck), tris(2-aminoethyl)amine (TREN; Acros), N,N.N´,N´-tetramethyl-1,3-propanediamine

(TMPDA;

Sigma

Aldrich),

2-hydroxyethylacrylate (HEA; TCI) were distilled prior use. Trans-1,4-dibromo-2-butene (DBB; Sigma Aldrich), glycerol dimethacrylate (GDMA; Sigma Aldrich), camphorquinone (Sigma

Aldrich),

ethyl

4-(dimethylamino)benzoate

(Sigma

Aldrich),

cetyltrimethylammonium chloride (CTAC, Sigma Aldrich), fluorescein sodium salt (Carl Roth), 2,3,5-triphenyltetrazolium chloride (TTC, AppliChem), nutrient broth (ISO, APHA; VWR), agar (AppliChem), RPMI 1640 Medium (GIBCO, Invitrogen GmbH), fetal bovine serum (FCS, GIBCO, Invitrogen GmbH), sodium chloride (Fischer), sodium dihydrogen phosphate dihydrate (Merck), sodium hydroxide (Merck), sodium citrate (Sigma Aldrich), citric acid monohydrate (Sigma Aldrich), and glucose monohydrate (Sigma Aldrich) were of analytical grade and used without further purification. The bacterial strains Staphylococcus aureus (strain ATCC 25923), Escherichia coli (strain ATCC 25922), and Pseudomonas aeruginosa (strain ATCC 17423), were provided by the German Resource Centre for Biological Material (DSMZ). Fresh porcine blood was provided by a local butcher shop and immediately processed to erythrocyte concentrates. Culture media, buffers, solutions: Nutrient broth: 25 g of standard 1 nutrient broth was dissolved in 1 L bidistilled water and autoclaved at 120 °C for 20 min. The pH was adjusted with aqueous 1 M NaOH or 1 M HCl solution.52 Nutrient agar: The nutrient agar (1.5 wt.% agar) was prepared by dissolving 3 g agar and 7 g nutrient broth (standard 1) in 200 mL bidistilled water. The nutrient agar was autoclaved at 120 °C for 20 min and stored at 4 °C. Phosphate buffered saline (PBS): 8.77 g NaCl, 1.56 g NaH2PO4*2H2O were dissolved in 1 L bidistilled water and autoclaved at 120 °C for 20 min. The buffer was adjusted to pH 7.0 with 0.1 M NaOH and stored at 4 °C. Citrate-phosphate-dextrose (CPD) buffer: Sodium citrate 5 ACS Paragon Plus Environment


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(26.30 g, 102 mmol), citric acid monohydrate (3.27 g, 16 mmol), glucose monohydrate (25.50 g, 129 mmol), and sodium dihydrogen phosphate dihydrate (2.51 g, 14 mmol) was dissolved in 1.00 L of bidistilled water, and was sterile filtered (0.2 µm). The pH was adjusted to 7.4 with

aqueous

1

M

NaOH.

TTC

solution

(2,3,5-triphenyltetrazolium

chloride):

2,3,5-triphenyltetrazolium chloride in a concentration of 1 mg/mL was dissolved in autoclaved bidistilled water. Bacteria stock solution: The stock pellet of the respective bacterial strain was suspended in 50 mL nutrient broth and shaken under bacterial strain specific conditions for 24 h.52 The confluent medium was centrifuged (3000 rpm, 10 min), and the supernatant was decanted. The residue was suspended in 50 mL of sterile PBS buffer and then centrifuged (3000 rpm, 10 min) again. This step was repeated three times. In the last step, the bacterial residue was suspended in 10 mL of sterile PBS buffer and mixed with 10 mL of a sterile-filtered 50% glycerin-solution. The stock solution was stored at −20 °C. Instrumentation: 1H NMR: 1H NMR spectra were recorded in D2O, respectively, using a DRX-400 spectrometer (Bruker Corp., Ettlingen, Germany) with a 5 mm sample head operating at 400.13 MHz for 1H. SEM-EDX: The samples were mounted on aluminum stubs with double-sided carbon tape and recorded using a Hitachi S-4500 scanning electron microscope with an Oxford Link ISIS System for the energy dispersive X-ray spectroscopy (EDX) measurements. The acceleration voltage was set to 10 kV. Bromine mapping of the sample was recorded with SEM-EDX in a standard element analysis procedure with a 300fold zoom, which corresponds to a surface area of 222*169 µm. AFM: Atomic-force microscopy (AFM) images were recorded with a Veeco Dimension Icon Scanning Probe Microscope (Veeco Instruments) equipped with a Nanoscope V Controller and an AVH-1000 Workstation. All measurements of the cross sections were performed in tapping mode using commercial tapping mode etched silicon probe (RTESP) cantilevers of various frequencies from 300 to 400 kHz. Phase images were recorded at 5% below the fundamental resonance frequency of the cantilever, with a typical scan speed of 1 Hz and a resolution of 512 samples 6 ACS Paragon Plus Environment

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per line for a 1000 nm scan size. TEM: Transmission electron microscopy (TEM) was performed using a FEI CM 200 microscope operating at an acceleration voltage of 200 kV. DMA: The Young´s moduli of the networks were investigated with the dynamic mechanical analyzer (DMA 2980, TA Instruments, Inc.) operating at room temperature in multifrequency-mode. To this end, samples of 7.3 mm, 2.7 mm, 1.2 mm (length x width x depth) were mounted to a film tension clamp of the DMA and using a frequency of 1 Hz, a preload force of 0.01 N, and an amplitude of 10 µm. Tensile test: The tensile test to investigate the maximal stretch/extension of the different networks were measured with tensile testing machine of Instron (Darmstadt, Germany) with a 1 kN capacity of the force transducer. The tensile speed was 5 mm/min with samples dimensions of 15 mm, 2.8 mm, 1.2 (length x width x depth). LS: Light scattering (LS) measurements were performed on a Malvern Zetasizer Nano S (ZEN 1600) in water/methanol/acetic acid (54/23/23 v/v/v) with 0.54 M sodium acetate at 25 °C with polymer concentrations varying from 0.5–5 wt.%. SEC: Size exclusion chromatography (SEC) was performed on water (0.1 M NaNO3, 0.01% TFA) based Agilent Technologies 1260 Infinity SEC with PSS GRAM analytical 1000 Å and 30 Å columns equipped with a Knauer RI detector Smartline 2300 using linear pullulan standards. UV/Vis: UV/Vis analysis was performed with a Specord 210 (Analytik Jena AG, Germany) UV/vis spectrometer at 25 °C. Synthesis of 3,4en-ionene (PBI): The procedure of the synthesis is described on the example of PBI7000 (0.97 TMPDA/DBB molar ratio). Trans-1,4-Dibromo-2-butene (DBB, 15.23 g, 71 mmol) was dissolved in 350 mL of acetone and cooled up to 0 °C under argon atmosphere. N,N,N´,N´-Tetramethyl-1,3-propandiamine (TMPDA, 9.04 g, 69 mmol) was added quickly (10 s) with a syringe to the DBB solution under vigorous magnetic stirring. A precipitate is formed within the first 5 min. The reaction mixture was stirred at 0 °C for 1 h and at 25 °C for another 2 h. Afterwards the precipitate was filtrated off, washed five times with 50 mL acetone and dried in vacuum. Then, the product (13.20 g) was dissolved in 120 7 ACS Paragon Plus Environment


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mL methanol and DBB (21.00 g, 98 mmol) was dissolved in 300 mL methanol. Both solutions were cooled to 0 °C under argon atmosphere. The polymer solution in methanol was added quickly to the DBB solution under vigorous magnetic stirring. The reaction mixture was stirred at 0 °C for 1.5 h and at 25 °C for further 16 h. The solvent was then removed on a rotary evaporator at 25 °C under reduced pressure. The resulting solid was dissolved in 300 mL bidistilled water and extracted five times with 300 mL diethylether. The aqueous phase was lyophilized. The white product was characterized by 1H NMR. 1H NMRPBI7000 (D2O, δ): 2.51 (bs, n·2H, N+(CH3)2CH2CH2CH2N+(CH3)2), 3.13-3.32 (bs n·12H, N+(CH3)2), 3.58 (bs, n·4H, N+(CH3)2CH2CH2CH2N(CH3)2), 4.13 (d, 4H, N+(CH3)2CH2CH=CH-CH2Br), 4.20 (d, 4H, N+(CH3)2CH2CH=CH-CH2Br), 4.30 (bs, n·4H, N+(CH3)2CH2CH=CH-CH2N+(CH3)2), 6.12 (m, 2H, N+(CH3)2CH2CH=CH-CH2Br), 6.49 (m, 2H, N+(CH3)2CH2CH=CH-CH2Br) and 6.57 (bs, n·2H, N+(CH3)2CH2CH=CH-CH2N+(CH3)2) ppm. PBI5700 and PBI3100 were synthesized with the same protocol only changing the TMPDA/DBB molar ratio to 1.01 (PBI5700) and 1.62 (PBI3100), respectively. Preparation of PBI networks (PBIN): Tris(2-aminoethyl)amine (TREN) was dissolved in bidistilled water to prepare a 5 wt.% or 10 wt.% stock solution, which was stored at -28 °C. The PBI was dissolved in bidistilled water to prepare a 40 wt.% PBI solution, which was used on the same day for the preparation of the networks. The two solutions were added in different ratios (detailed compositions are given in Table S1, in the supplements). Finally, bidistilled water was added to this solution to adjust a final PBI concentration of 30 wt.%. The mixture (300 µL) was pipetted in a plastic dish (diameter: 1.2 cm) and left at 20 °C for 24 h. A cloudy, brittle solid is formed. Preparation of interpenetrating hydrogels (IPH): A monomer mixture with 1 wt.% cross-linker was prepared by mixing the following components under exclusion of light in the given order: The monomer solutions (see Table S5 in the supplements) were prepared by 8 ACS Paragon Plus Environment

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homogeneous dimethacrylate

mixing

of

(GDMA),

2-hydroxyethylacrylate bidistilled

water,

(HEA),

ethyl

the

crosslinker

glycerol

4-(dimethylamino)benzoate

and

camphorquinone. The mixture was stored under exclusion of light at 4 °C. A PBIN sample (100 mg) was given to petri dish containing 25 mL of the monomer solution and stored under exclusion of light at 20 °C for 24 h. Then, the swollen network was taken out and the adhered liquid was quickly wiped off using a paper tissue. The swollen sample was irradiated with UV light for 3*180s in a Heraflash polymerization chamber (Heraeus, Germany) and air dried. Washing procedure of networks: The respective IPH was cut into pieces (surface ~50 mm², thickness 1-2 mm, mass 41-99 mg). In total, 1.6 - 1.7 g IPH1.50(1) or IPH1.74(1) were given into a petri dish (Ø= 90 mm) with 24-fold excess (40 mL) of bidistilled water. The IPHs were washed in an incubator at 37 °C under slight shaking on a compact shaker (50 rpm). The water was exchanged daily. Analysis of PBI content in the washing water: The washing water of each day was separately collected and freeze-dried. The resulting residues were weighed and dissolved in 2 mL of bidistilled water. Three glass slides were heated up to 200 °C on a heating plate (IKA RCT CL with digital temperature control unit) and subsequently 4*5 µL of the aqueous PBI solution was pipetted in the middle of the lower third of the glass slides. The dried glass slides were cooled to 20 °C on a metal plate. Then, the dried residue on the glass slide was covered with 90 µL aqueous fluorescein sodium salt solution (11 mg/mL). After 1 min, the fluorescein was rinsed off with bidistilled water leaving the insoluble PBI/fluorescein complex. The glass slides were dried on a heating plate at 200 °C and then cooled to 20 °C. The red residue of the glass slide was dissolved in 1 mL aqueous cetyltrimethylammonium chloride solution (CTAC, 1 mg/mL), transferred to an 1.5 mL Eppendorf cup and mixed with 0.1 mL PBS buffer with pH = 8.0. The absorbance of the resultant aqueous solution was measured at 501 nm by UV/Vis spectroscopy in reference to the CTAC solution with 9.1 vol.% PBS buffer at pH = 8.0. The test was calibrated with a concentration series of PBI5700, which showed a 9 ACS Paragon Plus Environment


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detection limit at a PBI concentration of 50 mg/mL. The whole protocol is explained with a graphical sketch in Scheme S1 in the supplements. Degree of swelling and gel content of networks: The respective network was cut into pieces of 5-20 mg. Each piece was soaked in 8 mL of the respective solvent at 20 °C for different periods of time. Then, the solvent was wiped off the surface with a paper tissue and the swollen network was weighed. The degree of swelling (S) is the ratio of the weight of the swollen sample (mswollen) to that of the dry sample (mdry initial). Afterwards, the networks were dried until constant mass (mdry after

swelling)

and were weighed again. The gel content was

determined after repeating the washing cycle for 5 times. degree of swelling = S = mswollen/mdry initial gel content = mdry after swelling/mdry initial Spray test with S. aureus: A sample of the IPH was pressed in liquid nutrient agar (40 °C) in a petri dish. The sample was allowed to solidify overnight and subsequently sprayed on with S. aureus cells according to a literature protocol53. The petri dish wash incubated at 37 °C overnight and the formed bacterial colonies were stained with TTC according to literature. Bacterial adhesion test: The bacterial cells S. aureus, E. coli, and P. pseudomonas were cultivated according to standard literature protocols50 and PBS suspensions with 107 cells/mL were prepared. Then IPH samples (surface area ~ 0.5 cm2, 1-2 mm in thickness) preswollen in sterile PBS overnight were rinsed with PBS three times and 1 mL of the respective bacterial cell suspension (~107 cells/mL) was added. After incubation at 37 °C for 3 h, the sample was taken out, washed three times with sterile PBS, and transferred into 2 mL of PBS. Then the sample was sonicated for 10 s in an ultrasonic bath. The number of surviving cells in the resulting PBS cell suspension was determined by agar plate counting of the colony forming units (CFU). The log reduction of the investigated IPH1.50(1) and IPH1.74(1) were calculated using the number of bacterial cells adhered to a PHEA(1) network as reference.


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Test in growth medium: A PBIN prepared from PBI5700 cross-linked with TREN using a NH2/Br ratio of 1.50 was sterilized with UV light in a clean bench for 25 min on each side. Then, 10 µL of the S. aureus suspension in growth medium (107 S. aureus cells/mL) were placed on the network and stored for 10 min. The still swollen network was transferred in a sterile 15 mL falcon tube, immersed in 4 mL nutrient broth and incubated at 37 °C for 24 h. Additionally, a negative control (4 mL nutrient broth) and a positive control (10 µL bacteria suspension pipetted directly into 4 mL nutrient broth) were incubated under the same conditions. After 24 h, 100 µL mL of a TTC solution (1 mg/mL) was added to all samples followed by 3 h of incubation. Hemocompatibility: The erythrocyte concentrate was prepared according to standard literature protocol52 in CPD buffer. 50 mg of the respective network networks were immersed separately in 1.6 mL CPD buffer for 48 h. The respective swollen network was immersed in 1.6 mL CPD buffer and 400 µL of the erythrocyte concentrate suspension was added. The samples were incubated at 37 °C for 3 h and then centrifuged (13500 rpm, 5 min). The supernatant liquid was further diluted with CPD buffer (1:20), and the absorbance of the released hemoglobin was measured with a UV/Vis spectrometer at 541 nm and 25 °C. Additionally, a positive control (100% hemolysis) that was treated with 2 µL Triton X and a negative buffer control containing no active substance were analyzed according to the same procedure. The percentage of the hemolysis was assessed by determining the amount of released hemoglobin as result of membrane rupture. The values are determined in at least duplicate showing full reproducibility in all cases.



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RESULTS AND DISCUSSION Goal of this work is the preparation of a contact-active long-lasting antimicrobial hydrogel with a minimal content of antimicrobial functions and adjustable in its properties. The concept to achieve this, is the preparation of an interpenetrating hydrogel (IPH) that is composed of a highly swellable polymer network of the hemocompatible, hydrophilic antimicrobial polycation PBI, interpenetrated by a polymer network based on the hydrogel forming 2hydroxylethyl acrylate (HEA) (see Scheme 1).

Scheme 1. Scheme of the preparation of an antimicrobial IPH. First, a polymer network was prepared by cross-linking the previously reported polymer PBI52. Cross-linking of ionenes has been achieved by modifying the polymers with various double bonds, such as styrene54, acrylates55, and cinnamate56, followed by UV radiation. In this work, we have decided to chemically cross-link the end groups to achieve a maximal possible degree of swelling, which is required to get highest possible water uptake. To this end, three different polymers were prepared by polyaddition reaction of trans-1,4-Dibromo-2butene (DBB) and N,N,N´,N´-Tetramethyl-1,3-propandiamine (TMPDA) and subsequent treatment with DBB in the final step to ensure bromine groups at all terminals of the polymers. The polymers were characterized using 1H NMR, light scattering (LS), and size exclusion chromatography (SEC) to determine molecular weight, molecular weight distribution and end group modification (Table 1).


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Table 1. Analytical data of synthesized PBIs.

Mn NMR a)

Mw LS b)

Mn SEC c)

[g/mol]

[g/mol]

[g/mol]

PBI3100

3100

2400

5500

1.67

77

PBI5700

5700

3500

8800

1.87

61

PBI7000

7000

4400

8900

1.85

63

polymer

PDI SEC c)

functionality (Mw LS/Mn NMR) [%]

a) The molecular weight (Mn) was calculated from the integrals of the CH3 groups of the quaternary ammonium groups at 3.13-3.32 ppm, after normalizing to the end groups signal, in the 1H NMR spectra. b) The molecular weight (Mw) was calculated from a CONTIN fit using mixture of acetic acid/methanol (1:1, v/v) in the light scattering (LS) measurement. c) The molecular weight (Mn) and the polydispersity index (PDI) were calculated from size exclusion chromatography (SEC) measurements.

According to these data, all polymers show molecular weight distributions typical for polyaddition reactions. Comparison of the Mn values, determined by 1H NMR and SEC, shows that the latter values are higher, because 1H NMR can only overestimate molecular weight, due to the fact that it references the bromine end groups. Further, the used pullulan standard for calibrating the SEC might have less interaction with the column material, which will then lead to overestimated molecular weights. The 1H NMR and LS molecular weight values are closer, suggesting that the majority of the PBI end groups are indeed modified by bromine groups. Thus all synthesized PBIs should be cross-linkable. The polymers were then reacted with tris(2-aminoethyl)amine (TREN) in different molar ratios of NH2/bromine end groups (Details see Table S1 in the supplements).57 Ideally, the cross-linker TREN should be working best, when added in a 1:1 ratio NH2/bromine, because then all end groups can react. This should then lead to the lowest degree of swelling (S) in water. As seen in Figure 1, S increases expectedly with increasing molecular weight of PBI. The minimal swelling is not reached at a 1:1 ratio but at an excess of NH2 groups. 13 ACS Paragon Plus Environment


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This can be explained by side reactions of the primary amino groups of TREN with the polymer backbone. Such a side reaction could be a chain scission by transamination as observed previously.52 Consequently, the side reaction would generate non-cross-linked fragments or dangling ends in the volume of the IPN (Details see Scheme S2 in the supplements). However, PBI networks could be achieved in all cases.

Figure 1. Dependency of the degree of swelling (S) in bidistilled water for PBI networks on the molar ratio of NH2/bromine (Br) end groups for TREN-cross-linked PBI3100, PBI5700 and PBI7000. The networks were swollen at room temperature for 2 h. (Compare to Table S2 in the supplements)

The gel content of the PBI networks (PBIN) was determined by swelling a sample repeatedly with high excess of water and following the mass loss of the respective dried specimen. After 5 washing cycles, no further weight loss could be detected. The gel content was found to be between 61 and 75 wt%. The comparatively low level of the gel content might be due to the chain scission side reaction.


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The cross-linking experiments also show that variation of the NH2/bromine ratio and the molecular weight of the PBI can be used to control the degree of swelling in a range of 20 to 170. These networks are a very interesting as fast swelling, antimicrobial superabsorber. To further explore this potential the water uptake of a slice of PBIN and also of the granulated material prepared by cutting the dried network was investigated on the example of crosslinked PBI5700. As seen in Figure 2a, a water drop 30 times the weight of the PBIN (molar ratio of NH2/bromine end groups of 1.50) is fully taken up within only one min at room temperature. The granulate of the PBIN (molar ratio of NH2/bromine end groups of 1.74) takes up the 48-fold of its weight within the same period of time forming a hydrogel (Figure 2b). This gel can still take up the same amount of water within two more minutes. The fast swelling of PBIN in water might be explained by the strong hydrophilicity of the highly charged PBI, which leads to ideal polymer-water interactions.58 Additionally, the polymer network prevents growth of the nosocomial bacterium Staphylococcus aureus (S. aureus). This was shown by treating a PBIN with a S.aureus suspension for 10 min and incubating this hydrogel in bacterial growth medium at 37 °C overnight. After this time no bacterial growth could be observed (Figure 2c). This shows the potential of the cross-linked PBI as antimicrobial superabsorber, which might be useful in wound dressings, given the non-hemolytic behavior of PBI in specific52 and the skin compatibility of hydrophilic, antimicrobial polycations in general59.



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Figure 2. a+b) Photographs of the rapid swelling of a PBIN film (molar ratio of NH2/bromine end groups of 1.50) (a) and granulated PBIN (molar ratio of NH2/bromine end groups of 1.74) (b) in water. c) Photographs of 15 mL falcon tubes filled with 4 mL growth medium: left) inoculated with 2.5*104 S. aureus cells/mL and incubated at 37 °C overnight, middle) incubated at 37 °C overnight without addition of bacterial cells, and right) 6 mg PBIN (NH2/bromine ratio 1.50, washed five times with water) treated with 10 µl of S. aureus suspension in growth medium (107 cells/mL) for 10 min, added to growth medium, incubated at 37 °C overnight. The photographs were taken after 16 h incubation at 37 °C and staining with TTC. While being useful as additive, the PBI network is too brittle in its dried state and too weak in its swollen state to be processed as a material. The hydrogel films tend to break during swelling and drying cycles. In order to create a hydrogel potentially usable as biomaterial, e.g., as wound dressing, the PBIN were altered into an interpenetrating polymer hydrogel using cross-linked poly(2-hydroxyethyl acrylate) (PHEA) as second network. Since the PBIN is not swellable in the monomer 2-hydroxyethyl acrylate (HEA), water was used as common solvent. 16 ACS Paragon Plus Environment

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The PBI5700 networks prepared using a molar ratio of 1.74 (PBIN1.74) and 1.50 (PBIN1.50), respectively, were chosen for all following experiments. The syntheses of the interpenetrating hydrogels (IPH) were performed by swelling the respective PBIN in a mixture of HEA (49 wt.%), cross-linker(0.5 wt.%), photoinitiators (0.45 wt.%) and water overnight, taking it out and radiating it with UV light. The degree of swelling (S) of each PBI network was terminated before radiating with UV light. Due to the complexity of the mixture, the degree of swelling showed a larger variation than that in water. The swelling of all prepared PBIN1.74(1) (S= 57-100) and PBIN1.50(1) (S= 25-36) was determined. Based on the individual degree of swelling, the PBI content in the final dried network was calculated. The resultant interpenetrating hydrogels were named IPH1.50(1) and IPH1.74(1). The PBI content of IPH1.74(1) was calculated to be in a range of 2.0 - 3.5 wt.% and that of IPH1.50(1) was in a range of 5.5 7.8 wt.%. The products are clear, elastic, and water-swellable materials. This suggests that both networks (PHEA and IPH) do not phase separate on the micrometer level. In order to remove all byproducts, the IPHs, were washed for 7 days in distilled water prior to further analysis, because the PBI starting networks could not be washed without being partially broken. The gel content was found to be 86-88 % indicating effective cross-linking. The structure of the resulting materials was investigated by scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX), transmission electron microscopy (TEM), and atomic force microscopy (AFM) on the example of IPH1.50(1), which should contain 7.8 wt.% PBI calculated from the starting material (Figure 3). As seen in Figure 3a, the bromine associated with PBI as counter ion of the quaternary ammonium function is well distributed within the matrix. The TEM and the AFM of cross-sections of the IPH show a distinguished nanostructure typical for this kind of networks.60-61 The dark phase in the AFM was attributed to PBI. The latter phase is isolated and embedded in the percolated PHEA phase, typical for



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this composition (8/92 wt/wt). The size of the PBI phase is approximately 3.6 nm. No larger phase separations could be observed with both methods.

Figure 3. a-c) Scanning electron microscopy (SEM, a) transmission electron microscopy (TEM, b), atomic force microscopy (AFM, c) images of an IPH1.50(1) after 7 days of washing at 37 °C in water.. a) The inserted box marks a typical area where counts for the standard element analysis by SEM-EDX are accumulated. d) The table shows bromine content of the IPH1.50(1) and IPH1.74(1), after 7 days of washing in water, determined by SEM-EDX and the respective PBI content in the IPH, calculated from these values (see Table S6 in the supplements). e) Photograph of a typical PHEA(1), IPH1.50(1), and IPH1.74/(1) network, respectively, in dried state.

These measurements confirm that the synthesized material is indeed a nanophase separated IPHs. The smaller structure found in the TEM image compared to the AFM is typical for this dimension of nanostructure, because the transmission image of the 50 to 100 nm thick slice shows an overlap of several layers of the structure seen in the AFM image62-65. The bromine content determined by EDX was used to calculate the composition of the networks. The IPH 18 ACS Paragon Plus Environment

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prepared from PBIN1.50 contains 5.6 wt.% PBI indicating that some 25% of the PBI was washed out (calculated starting value 7.8 wt.%). The IPH based on PBIN1.74 contains 1.4 wt.% PBI, which corresponds to a washout of 40% PBI (calculated starting value 2.5 wt.%). This higher washout might be due to the fact that the cross-linking effectiveness of PBIN1.74 is lower and thus less polymer chains might be linked. A series with different cross-linker amount in the PHEA phase was prepared to investigate the controllability of the water swelling of the networks. As seen in Figure 4, the swellability of the IPHs is expectedly controlled by the amount of cross-linker in the PHEA phase. IPH1.74 networks with the lower PBI content swell to the same degree as the respective cross-linked PHEA. A higher PBI content in the IPH1.50 results in higher swelling compared to the PHEA networks indicating that the PBI phase starts to influence the properties of the hydrogels at this content. However, the high swelling rate of the PBIN was not found for the IPHs, which need 24 h to reach equilibrium swelling. The swelling curve of IPH1.50(1) is given as a representative example in Figure S1 in the supplements. The mechanical properties of the IPHs and the PHEA networks were investigated regarding Young’s modulus, maximum tensile stress and maximal elongation. The Young’s moduli found are similar for all networks being in a range of 0.64 - 1.34 MPa. The maximal tensile stress was found in a narrow range of 0.28 to 0.42 MPa for all samples. Only the maximal elongation changed with GDMA content in the PHEA phase from some 450% for 1 wt.% GMDA to around 180% for 5 wt.% of the cross-linker. In all cases, the mechanical properties are not significantly influenced by the PBI content (see Table S5 in the supplements).66



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Figure 4. Degrees of swelling (S) in bidistilled water after 72 h of PHEA, IPH1.50 and IPH1.74 networks with 1 wt.% (grey), 3 wt.% (grey lined) or 5 wt.% (white) GDMA as crosslinker with respect to used amount of HEA. All measurements were performed in a triplicate, and the error bars are the standard deviation. (Compare to Table S4 in the supplements)

The antimicrobial properties of the hydrogels were tested by bringing bacterial cells in contact with the surface in PBS buffer for 3 h. Then the adhered cells were removed by ultrasound and the colony forming units (CFUs) were counted on agar plates. The found numbers of CFUs, which are equal to the number of surviving bacterial cells, were compared to the number of CFUs found on the respective PHEA hydrogel without PBI. The experiments were carried out with three different clinically relevant bacterial cell lines, the Gram-positive bacterial strain S. aureus, and the Gram-negative bacterial strains Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In order to see if the hydrogels are long-term active they were washed in water (24 mL/1 g sample) at 37 °C with daily change of water for 12 days in case of IPH1.74(1) and 27 days in case of IPH1.50(1).


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Figure 5. a) Reduction of bacterial cells on the surface of the in water washed networks IPH1.74(1) (12 days at 37 °C) and IPH1.50(1) (27 days at 37 °C) after an incubation time of 3 h in PBS. The numbers of bacterial cells on the control PHEA were the following: E. coli in PBS (3.3 ± 2.0)*105 cells/cm2; S. aureus in PBS, (5.3 ± 0.9)*104 cells/cm2; P. aeruginosa in PBS, (7.5 ± 9.0)*106 cells/cm2; (Compare to Table S7 in the supplements). b) Image of S. aureus colonies formed after being sprayed on an agar plate with an embedded IPH1.50(1) network (washed for 7 days with water), incubated at 37 °C overnight and stained with TTC.



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The results show a log 5 reduction for S. aureus and E.coli for both IPHs (see Figure 5a). Here, even the low PBI concentration afforded practically full killing of all adhered bacterial cells. In case of P. aeruginosa, the IPH1.74(1) with some 1.4 wt.% PBI afforded a log reduction of 4, while the IPH1.50(1) reduced the bacterial cell number by 6-7 logs. This shows that the PBI content has a certain influence of the killing efficiency for the obviously less susceptible P. aeruginosa. However, in all cases the IPHs are highly antimicrobially active against adhering antimicrobial cells, even after being exposed for several weeks in water at 37 °C. There are only few examples of antimicrobial hydrogels in the literature that withstand such a challenge, e.g., the slow PHMB release hydrogel of Zhang et. al.67 and the amphotericin B release dextran hydrogel of Zumbuehl et. al.6. Also cellulose hydrogel superabsorbers with intrinsic antimicrobial properties have been recently published by the group of Chang.4, 37 To explore the influence of proteins on the killing efficiency of the IPHs, the experiment was repeated in an albumin containing growth medium containing S. aureus cells. While IPH1.50(1) showed a log 5 reduction, the IPH1.74(1) afforded a log reduction of 3. This shows that the antimicrobial activity of the IPHs is also dependent on the nature of respective environment.


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Figure 6. a) Calibration curve of PBI5700 in water determined according to the procedure in Scheme S1 in the supplements; b) Concentration of PBI in the daily changed washing water. Washing was performed by 24-fold excess volume of water at 37 °C. (Compare to Table S8 and S9 in supplements)

Having established the high antimicrobial efficiency of the IPHs, it was tested, if the killing is caused on contact or by releasing a biocidal compound. The only antimicrobial compound in the IPH should be PBI. In order to detect this compound quantitatively at low concentrations, an optical detecting test was developed to determine up to 49 mg/L PBI in water taking advantage of the selective complexation of PBI with fluorescein (see calibration curve given in Figure 6a). Due to concentrating the sample solution, a final concentration as low as 2.5 mg/L could be detected in the washing solution. The washing test for leaching PBI was performed in water at 37 °C with daily changing the water and determining the PBI content. As seen in Figure 6b the freshly prepared IPHs are showing a high initial release of PBI. After 7 days of washing, the released PBI/day becomes constant. The released amount is more than 7 times lower than MICS. aureus for both IPHs, indicating that the network might not kill bacterial cells in the surrounding, but only at its surface. In order to test this, S. aureus cells were sprayed on a washed sample embedded in growth agar and incubated overnight at 37 °C. As seen in Figure 6b the grown bacterial colonies reach the hydrogel surface, but do not grow on the hydrogel, i.e., there is no inhibition zone visible would indicate leaching of a biocide in toxic concentration. Additionally, the hydrogel IPH1.50(1) was incubated for 3 h in growth medium in the amount used for testing the antimicrobial activity (50 mg hydrogel/1 mL growth medium) at 37 °C and taken out. The medium was the inoculated with S. aureus cell (105 cells/mL) and incubated overnight. The bacterial cells were fully grown after this time showing that no biocide in toxic concentration was in the medium. These experiments 23 ACS Paragon Plus Environment


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show that the minor amount of released PBI is not sufficient to kill bacterial cells in the surroundings of the IPH and thus the killing indeed takes place right at the surface of the gel. The potential application of wound dressings requires hemocompatibility. This was tested by swelling the PBINs and the IPHs, respectively, in CPD buffer for 48h and bringing them in contact with porcine red blood cells for 3 h. In all cases, a maximal cell lysis of

Cross-Linking of a Hydrophilic, Antimicrobial Polycation toward a Fast

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