Biofilm inhibition via delivery of novel methylthioadenosine

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Controlled Release and Delivery Systems

Biofilm inhibition via delivery of novel methylthioadenosine nucleosidase inhibitors from PVA-tyramine hydrogels while supporting mesenchymal stromal cell viability Isha Mutreja, Suzanne L. Warring, Khoon S. Lim, Tara Swadi, Keith Clinch, Jennifer M. Mason, Campbell Sheen, Dion Thompson, Rodrigo G. Ducati, Stephen Chambers, Gary B. Evans, Monica Gerth, Antonia Miller, and Tim B. F. Woodfield ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01141 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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ACS Biomaterials Science & Engineering

Biofilm inhibition via delivery of novel methylthioadenosine nucleosidase inhibitors from PVA-tyramine hydrogels while supporting mesenchymal stromal cell viability

Isha Mutreja,1,2 Suzanne L. Warring,3 Khoon S. Lim,1,2,4 Tara Swadi,5 Keith Clinch,6 Jennifer M. Mason,6 Campbell R. Sheen, Dion R. Thompson, Gary B. Evans,

4,6

7

Rodrigo G Ducati,

8,9

7

Stephen T. Chambers,5

Monica L. Gerth,3,4,10 Antonia G. Miller,

7,11

Tim B. F. Woodfield,1,2,4* 1

Christchurch Regenerative Medicine and Tissue Engineering (CReaTE) Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Centre for Bioengineering & Nanomedicine, University of Otago Christchurch, New Zealand. 2

Medical Zealand.

Technologies

Centre

of

Research

Excellence,

New

3

Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand. 4

Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand.

5

Department of Pathology, University of Otago Christchurch, New Zealand. 6

Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand. 7

Protein Science and Engineering, Callaghan Innovation, c/School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand. 8

Department of Biochemistry, Medicine, Bronx, NY 10461, USA

Albert

Einstein

College

of

9

Present Address – Centro de Ciências Médicas, Universidade do Vale do Taquari - Univates, Lajeado, RS 95914-014, Brazil 10

Present Address – School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand.

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11

Present Address – Plant & Food Research, Private Bag 4704, Christchurch Mail Centre, Christchurch 8140, New Zealand

* Corresponding author: [email protected]


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ABSTRACT

The

rise

of

antibiotic

resistance,

coupled

with

increased

expectations for mobility in later life, is creating a need for biofilm

inhibitors

surgical

implant

and

delivery

infection.

A

systems

that

limitation

of

will

some

reduce

of

these

existing delivery approaches is toxicity exhibited towards host cells. Here, we report the application of a novel inhibitor of the

enzyme,

enzyme

in

methylthioadenosine bacterial

metabolic

nucleosidase pathways,

(MTAN),

which

a

include

key S-

adenosylmethionine catabolism and purine nucleotide recycling, in combination with a poly(vinyl alcohol)-tyramine-based (PVATyr) hydrogel delivery system. We demonstrate that a lead MTAN inhibitor, selected from a screened library of 34 candidates, (2S)-2-(4-amino-5H-pyrrolo3,2-dpyrimidin-7ylmethyl)aminoundecan-1-ol

(31),

showed

a

minimum

biofilm

inhibitory concentration of 2.2 ± 0.4 µM against a clinical staphylococcal species isolated from an infected implant. We observed that extracellular DNA, a key constituent of biofilms, is significantly reduced when treated with 10 µM compound 31, along with a decrease in biofilm thickness. Compound 31 was incorporated into a hydrolytically degradable photocrosslinked PVA-Tyr hydrogel and the release profile was evaluated by HPLC studies. Compound 31 released from the PVA-hydrogel system significantly reduced biofilm formation (77.2 ± 8.4% biofilm inhibition). Finally, compound 31 released from PVA-Tyr showed no negative impact on human bone marrow stromal cell (MSC) viability, proliferation or morphology. The results demonstrate the potential utility of MTAN inhibitors in



treating infections caused by Gram-positive bacteria, and the development of a non-toxic release system that has potential for tuneability for timescale of delivery.

Keywords: biofilm inhibition, methylthioadenosine nucleosidase, PVA-Tyr, hydrogel, Staphylococcus, infection.


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Introduction Almost 500,000 total hip replacements were performed in the USA in 2012,1 with numbers projected to rise further over the next 15 years due to increases in life span and the expectation of the population that they will remain mobile longer.2,

3

Indeed,

numbers of hip and knee surgeries are expected to increase by >670% in the US by 2030.2 Acute or deep infections are some of the most devastating complications associated with the use of implants and similar medical device technologies. They occur in approximately 1–2% of these procedures and cost over 1 billion USD

per

annum

important

treat.3-9

to

strategy

for

Antimicrobial

reducing

the

prophylaxis

rates

of

is

an

post-surgical

infection and antimicrobial therapy may be effective for some implant infections, but their effectiveness is at risk from the spread of resistant organisms. The pathogenesis of implant infection involves the formation of a biofilm.10,

11

Biofilms consist of one or more microbial

species, which can be in different metabolic states, encased in a

self-produced

biopolymer

polysaccharides and DNA.4,

11,

matrix 12

composed

of

proteins,

A small subset of bacterial

species make up the majority of implant-associated pathogens, most prominently staphylococcal species, which account for close to

70%

of

organization

hip of

replacement the

biofilm

infections.13 provides

a

The

structure

barrier

to

and

immune

surveillance, limits diffusion of antibiotic therapy to the organisms, and supports metabolically inactive organisms that



Page 6 of 49

are poor targets for current therapies.12 The robustness of biofilms often requires surgical intervention to eradicate the infection, which is associated with high morbidity and cost. Efforts have been made to reduce biofilm formation to avoid surgical interventions. Different classes of materials have been tested

as

potential

inhibitors,14

bacterial

including

antimicrobial peptides,15 cationic polymers applied as coatings16 and antimicrobial nanoparticles like those of silver.17-19 The different modes of antimicrobial action of these compounds have made

them

attractive

candidates

for

reducing

bacterial

infection; however, their effective use is limited by their inherent

cytotoxicity.

nanoparticles applications catheters,

have such

they

For

attracted as

have

wound been

example, great

although

attention

dressings

associated

or with

for

as

silver clinical

coatings

cytotoxicity

genotoxicity in both in vitro and in vivo models.20,

21

on and

Similarly,

antimicrobial peptides have broad-range antimicrobial activity in vitro, but are limited by host cell-associated cytotoxicity and hemolytic activity.22,

23

These factors have driven the demand

for development of new agents or strategies that not only prevent bacterial colonization and biofilm formation to minimize implant related

infections,

interaction.10,

24

but

also

allow

implant/host

cell

Critically, it has been estimated that there

would be a 40–50% rate of post-operative infection following total hip replacement, with a death rate of ~30% of those


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infected,

should

pan-antimicrobial

resistance

become

widespread.25 Methylthioadenosine

nucleosidase

(MTAN)

catalyzes

the

hydrolytic deadenylation of methylthioadenosine (MTA) and Sadenosylhomocysteine in both Gram-negative and Gram-positive bacteria, including those involved in implant infection, such as S. aureus.26 In some Gram-negative bacteria, MTANs are also involved in the biosynthesis of autoinducers synthesized from S-adenosylmethionine. Autoinducers are key molecules involved in quorum sensing (QS), a mechanism of bacterial communication that can contribute to biofilm formation. In addition to these aspects, the absence of a mammalian equivalent that could be affected

by

antibiotic

inhibitor

resistance

candidates, and

the

view

the

continuing

that

current

rise

of

antibiotic

discovery is stalled,27 all make MTAN an attractive candidate for inhibitor design. Enzyme transition state theory and the methodology that allows the analysis of the structure of a transition state on-enzyme have

afforded

blueprints

for

a

variety

of

drugs

including

Mundesine, which has received regulatory approval in Japan for the treatment of peripheral T-cell lymphoma.28-31 This drug design process has been applied to MTANs from a variety of bacteria, and

has

afforded

some

of

the

tightest

binding

of

enzyme

inhibitors ever reported.32-39 MTAN inhibitors disrupt quorum sensing

and

reduce

biofilm

formation

in

the

Gram-negative

bacteria Escherichia coli and Vibrio cholerae.36 Furthermore,



Page 8 of 49

MTAN inhibitors kill Helicobacter pylori at concentrations lower than five of the antibiotics most commonly used to treat H. pylori infections.33 These findings highlight the importance of continued

efforts

inhibitors

in

to

investigate

bacteria.

In

the

MTANs

and

context

their of

candidate

Gram-positive

bacteria, which are known to infect implants, deletion of the gene encoding MTAN (pfs) in Staphylococcus aureus decreased biofilm

formation

under

both

static

and

dynamic

flow

conditions.40 Therefore, in this study we sought to screen a library

of

MTAN

clinically

inhibitors

relevant

against

biofilm

coagulase-negative

formation

in

a

staphylococcus,41

isolated from an infected implant. An efficient system is required to deliver these novel biofilm inhibitors at effective concentrations adjacent to the medical devices. Different delivery platforms have been designed to enable

sustained

delivery

of

anti-microbial

agents.

These

platforms, aimed at releasing antimicrobial agents from the time of

implantation,

are

effective

in

preventing

bacterial

colonization.42 One of the prominent examples involves combining antibacterial

agents

within

polymethylmethacrylate

(PMMA)

cements used in orthopedic procedures, either as coatings or as beads. A PMMA-based antibiotic delivery platform has effectively eliminated surgery associated infection.43 However, due to its non-resorbable nature, it needs to be removed at a later stage.42 In contrast, degradable polylactide loaded with gentamicin has shown long-term sustained antibiotic release; however acidic


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degradation products and consequent local acidity accumulation result in toxicity.44 Bioactive molecules such as hyaluronic acid and

chitosan

effectively

inhibit

bacterial

adhesion

and

proliferation but very little is known about host cell response performance on these coatings.45 Because of these limitations, strategies

involving

the

use

of

biodegradable

hydrophilic

matrices, such as hydrogels, have drawn significant attention as localized drug delivery platforms.46 This is attributed to their biocompatibility, highly hydrated nature and relative ease of drug dispersion within the porous matrix. Amongst the range of synthetic and naturally derived hydrogels that

have

been

tested

for

drug

and

antibiotic

delivery,

synthetic PVA (polyvinyl alcohol)-based hydrogels have drawn great attention for varied biomedical applications for their biocompatibility, modification.47 demonstrated

hydrophilicity

Previous

that

by

reports

and

ease

published

functionalizing

the

of

by

PVA

Lim

chemical et

backbone

al. with

photo-crosslinkable tyramine residues (PVA-Tyr), hydrogels of controlled

spatial

and

temporal

characteristics

can

be

fabricated.48 Furthermore, hydrolytically degradable ester bonds can be introduced into the PVA-Tyr and the degradation profile can be tailored from days to several weeks by altering the formulations of the hydrogel,49 allowing for tuning of the in vivo degradation profile for a desired function.48 Another important aspect that requires consideration to ensure effective

utilization

of

an

antibacterial


coating

is

its


Page 10 of 49

interaction with surrounding host cells and tissues. Most of the systems previously published in literature focus on only one of the two key attributes pertaining to the success of an implant, which

is

preventing

implications

of

infection

such

systems

without

on

understanding

surrounding

the

cells.13

host

Understanding the importance of interactions between the host cells and microbes and the biofilm inhibiting coating, we sought to investigate not only the effect of MTAN inhibitor on biofilm inhibition of a clinical strain, but also its effect on human bone marrow-derived mesenchymal stromal cells (MSCs). Therefore, the key aims of this study were to systematically investigate and identify a lead MTAN inhibitor from a library of

molecules,

to

inhibit

biofilm

formation

in

a

clinical

Staphylococcus cohnii isolate. Furthermore, we investigated the release

of

the

lead

MTAN

inhibitor

compound

from

a

hydrolytically-degradable visible-light cross-linked hydrogel delivery system by tuning the macromer concentration and photocrosslinking successful

conditions.

release

of

Finally,

unmodified

we

demonstrate

MTAN

inhibitor

both at

the

target

concentrations and retention of its ability to inhibit biofilm formation

of

Staphylococcus

cohnii

in

vitro,

without

concomitantly interfering with MSC viability and proliferation capacity

compared

to

controls

cultured

released MTAN inhibitors.

Experimental Procedures


in

the

absence

of

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Preparation of MTAN transition-state analogue inhibitors A panel of 34 MTAN transition-state analog inhibitor compounds were

synthesized

(Compounds

1

to

34,

Figure

1).

Full

experimental details are provided in the Supporting Information for all new compounds. Experimental details for the previously reported sulfoxide 13 and sulfone 14 derivatives of 338 are also provided in the Supporting Information. Unless otherwise stated, inhibitors were prepared as stock concentrations ranging from 1–7.2 mM in sterile MilliQ H2O. Where further solubilization was required, the pH was reduced to pH 3–5 using HCl either with (compounds 4, 6–10, 12, 18, 24, 27– 28) or without sonication (compounds 5, 11, 29–32, 34). All stocks

were

passed

through

a

0.2-µm

filter

and

their

concentration was determined by spectrophotometry (ε = 8.5 mM−1 cm−1 at 275 nm)

38

and stored at −20 °C when not in use.

Staphylococcus species characterization and culture conditions A

coagulase-negative,

isolated

from

an

biofilm-producing

infected

titanium

Staphylococcus

implant

at

was

Christchurch

Hospital, Christchurch, New Zealand. The species of the isolate was identified as Staphylococcus cohnii by 16S ribosomal RNA gene sequencing as described in the Supporting Information. The organism was cultured in either Luria Bertani (LB) broth or tryptic soy broth supplemented with 1% w/v D-(+)-glucose (TSBglucose).



Page 12 of 49

Static biofilm compound screening assays and determination of minimum biofilm inhibitory concentrations For

initial

compound

screening

assays,

static

microplate

biofilm assays were conducted as previously described.50 Briefly, S. cohnii was cultured overnight at 37 °C then diluted 1/500 in fresh media. Diluted bacteria (180 µL) and MTAN inhibitor (20 µL) were sequentially added to each well of a 96-well U-bottomed plate and incubated at 37 °C for 24 h. Vehicle-only (sterile MilliQ H2O) controls, and media-only controls were also included. After incubation, the plate was gently washed three times with H2O, to remove planktonic bacteria, and 200 μL of 0.1% (w/v) crystal violet was added to each well and incubated for 10 min at room temperature. The plates were again washed with H2O, then 200 μL of 33% (v/v) acetic acid was added to each well and the plate was incubated for 10 min. Aliquots (125 μL) from each well were

transferred

to

a

flat-bottomed

96-well

plate

and

the

absorbance at 570 nm was measured in a SpectraMax plate reader (Molecular Devices). Minimal biofilm inhibitory concentrations (MBIC) were obtained using crystal violet staining of adherent biofilms as described above, with minor modifications. Cultures of S. cohnii were grown

in

overnight

at

37

°C

with

shaking

in

TSB-glucose.

Overnight cultures were diluted in fresh media to an OD600 ~0.1 and 135 µL was added to each well of a 96-well U-bottomed plate. Fifteen microliters of either MTAN inhibitor or sterile MilliQ H2O were added to each well and the plate was incubated at 37 °C


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under static conditions. Crystal violet staining was performed as described above, and the absorbance at 590 nm was measured using a CLARIOstar microplate reader (BMG Labtech).

Cloning and expression of S. cohnii MTAN A plasmid expressing an MTAN fusion protein with the structure of N-His6-glutathione S-transferase-3C protease cleavage siteMTAN was generated as follows. A codon-optimised DNA fragment for S. cohnii MTAN (Integrated DNA Technologies) was cloned into pOPINJ vector,51 linearised with KpnI and HindIII, using an Infusion

cloning

manufacturer’s

kit

(Takara

instructions.

Bio),

according

Infusion

to

reactions

the were

transformed into Stellar E. coli and grown on LB agar with 100 µg/ml carbenicillin. Plasmid DNA was prepared from overnight LB cultures

using

a

NucleoSpin

Plasmid

EasyPure

kit

(Macherey

Nagel) and plasmids were sequenced (Macrogen). E. coli Tuner pLacI cells (Novagen) were transformed with pOPINJ-MTAN vector and grown on Luria Broth agar (with 100 µg/ml carbenicillin and 25 µg/ml chloramphenicol) at 37°C. Colonies were picked and grown in 500 ml LB (with 100 µg/ml carbenicillin and 25 µg/ml chloramphenicol) until early log phase. Cultures were then transferred to 25°C and expression of S. cohnii MTAN was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside. Cells were harvested by centrifugation (6000 rcf; 15min) and the supernatant was removed. The pellet was re-suspended in Tris pH 7.8 with 0.1 mg/ml lysozyme and then frozen at −80°C. Cells were



Page 14 of 49

thawed and incubated at 37°C for 30 minutes, then sonicated on ice for 3 min using a Vibra-cell VC 750 (SONICS). Cell debris was pelleted by centrifugation (6,000 rcf; 15 min) and the supernatant was passed through a 0.22-µm filter. The supernatant was loaded onto a 5 mL Hi-trap GST column (GE Healthcare), equilibrated with 50mM Tris pH 8. MTAN was eluted using an increasing gradient to 10 mM GSH. Eluted protein was buffer exchanged back into 50mM Tris pH 8 and stored at -80°C until required. Protein concentration was determined by Bradford assay using ovalbumin as a standard. SDS-PAGE analysis was undertaken to confirm the MW of the protein. Activity of the final product was tested using the method of Dunn et al which monitors cleavage of 5′-deoxy-5′-(methylthio)adenosine (MTA) in a coupled assay where xanthine oxidase utilises the freed adenosine to reduce p‐iodonitrotetrazolium, measured as an increase in absorbance at 470 nm.52 Activity was performed in a 50mM HEPES pH7.4 buffer.

Determination

of

S.

cohnii

MTAN

kinetic

parameters

and

inhibition constants Steady-state kinetic parameters and inhibition constants were determined for the S. cohnii MTAN reaction following conditions previously described.53 The catalytic activity was monitored in a Cary 100 spectrophotometer (Varian) using an absorbance-based coupled enzyme assay.


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Steady-state kinetic parameters and the catalytic efficiency were determined in assays containing varying concentrations of substrate (1 to 100 µM), 1 U of xanthine oxidase, and were initiated with 50 nM S. cohnii MTAN. Reactions mixtures were monitored for 5 min, and the substrate saturation curve was fitted to a Michaelis-Menten hyperbolic function. The equilibrium inhibition constant (Ki*) for S. cohnii MTAN with compound 31 was determined in assays containing substrate at 1 mM, 1 U of xanthine oxidase, inhibitor at concentrations ranging from 3 nM to 1 mM, and were initiated by addition of 20 nM S. cohnii MTAN. Reactions mixtures were monitored for up to 2 h to permit development of slow-onset binding. The reaction rates obtained were fitted to the Morrison enzyme inhibition kinetics equation for slow-onset, tight-binding inhibitors.54

Hydrogel formulation and incorporation of compounds 3 and 31 Tyramine-functionalized

poly(vinyl

alcohol)

synthesized as described previously.48,

55

(PVA-Tyr)

was

Briefly, for grafting

tyramine moieties onto the polymer backbone, 1g PVA (13–23 kDa, 98% hydrolyzed, Sigma) was first carboxylated using succinic anhydride (0.045g) and triethylamine (0.061ml) to achieve 2% carboxylation. precipitated

in

The

carboxylated-PVA

ethanol,

dissolved

(PVA-COOH) in

water,

was

dialyzed

then and

freeze-dried. The lyophilized PVA-COOH (1g) was then dissolved in

dry

DMSO

(10ml)

dicyclohexylcarbodiimide

and

calculated

(0.271g)

and


amounts

of

1,3-

N-hydroxysuccinimide


Page 16 of 49

(0.151g) were used for activating carboxyl groups that were then conjugated

with

precipitation

in

tyramine

(0.121g)

acetone.

The

for

24

h,

precipitated

followed PVA-Tyr

by was

redissolved in water and filtered, then dialyzed against water using a 10-kDa cellulose membrane (Sigma), before being freezedried for further use. The percent conjugation of tyramine groups

onto

the

PVA

backbone

was

calculated

using

nuclear

magnetic resonance (1H NMR, 300 MHz Bruker Advance DPX-300 spectrometer)

by

comparing

the

area

of

the

proton

peaks

corresponding to the aromatic moieties (δ = 6.5-7.5ppm) to the area of the methylene protons in the PVA backbone (δ = 4.0ppm). The degree of tyramination was defined as the percentage of hydroxyl groups substituted with tyramine moieties, and was calculated to be 2%. Freeze-dried PVA-Tyr (20% (w/w)) was dissolved in phosphatebuffered saline (PBS) at 80 °C. Upon complete dissolution, the macromer solution was cooled to room temperature, after which the photo-initiators Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru; Sigma) and sodium persulfate (SPS; Sigma) were added at a 0.5/5 (mM/mM) ratio. The final concentration of PVATyr was adjusted to 10 wt% using PBS. The samples were irradiated under 30 mW/cm2 of 400–450 nm visible light (Rosco IR/UV filter combined with OmniCure S1500, Excelitas Technologies) for 3 min. Initial biofilm screening work identified compounds 3 and 31 as candidates for further investigation for the remainder of the study.

For

MTAN

inhibitor-incorporated


PVA-Tyr

hydrogels,

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calculated volumes of stock solution (1.53 mM compound 3 or 6.69 mM compound 31) were mixed with PVA-Tyr prior to the addition of photo-initiators. The final MTAN inhibitor concentration was adjusted to 100 µM. The initial dimensions of the fabricated hydrogels were approximately 5 mm in diameter and 1 mm in height. To study the effect of incorporating compound 31 on the photocrosslinking reaction, sol-gel analysis was executed on the prepared gels. Briefly, PVA-Tyr gels with or without 100 µM compound 31 were prepared under the conditions as previously mentioned. All gels were weighed immediately to determine the initial wet weight (minitial,

t=0),

after which three gels were

freeze dried to obtain their dry weight (mdry,

t=0).

To determine

the swelling ratio, the remaining gels were left in PBS for 24 h at 37 °C. After 24 h incubation, gels were removed from the incubator, blot-dried and weighed (mswollen) and the samples were freeze-dried

and

weighed

again

(mdry).

These

were

used

to

determine the swelling ratio of the cast gels using the equation as follows: 𝑞=

𝑚𝑠𝑤𝑜𝑙𝑙𝑒𝑛 𝑚𝑑𝑟𝑦

(eq. 1)

Some of the PVA-Tyr-only control and compound 31-containing gels were left in PBS in the incubator and were monitored every 24 h until complete degradation.

Quantitation of compounds 3 and 31 released from hydrogels and assessment on impact of biofilms



Page 18 of 49

The hydrogel discs (5 mm × 1 mm) containing either MTAN inhibitor 3 or 31 were incubated in 1 mL 1× PBS for 24 h at 37 °C. After 24 h, the extracts were collected and analyzed by HPLC to confirm and quantitate the release of 3 and 31 from the hydrogels. The concentration of either 3 or 31 released from the gels was determined from the standard curve constructed by determining the peak area for each compound prepared to known concentrations. Compound 3 was resolved on a Synergi Fusion-RP column

(2.5

μm,

triethylammonium

100

Å,

acetate

50 in

× H2O,

3 pH

mm,

Solvent

6.5;

A:

Solvent

10 B:

mM 100%

acetonitrile; Gradient 0–15% B over 9 min, 0.5 ml/min) with detection at a wavelength of 230 nm. Compound 31 was resolved on a Kinetix C-18 column (2.6 μm, 100 Å, 50 × 3 mm; Solvent A: 0.1% formic acid in H2O; Solvent B: 100% methanol. Gradient 5– 100% B over 10 min, 0.5 mL/min) with detection at a wavelength of 254 nm. PVA-Tyr hydrogels were loaded with different concentrations of compound 31 (25–100 µM), fabricated as previously described, and incubated in 1 mL of 1× PBS at 37 °C for 24 h. The extracts were then

filter-sterilized

and

used

for

biofilm

inhibition

and

bacterial growth assays as described. PVA-Tyr hydrogels without compound 31 (control PVA-Tyr) were prepared in parallel as controls to assess the effect of PVA-Tyr on biofilm formation. Fluorescent confocal laser scanning microscopy (CLSM) analysis Overnight cultures of S. cohnii in TSB at 37 °C were diluted to OD600 ~0.1 in TSB-glucose. A polycarbonate membrane was


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placed on a TSB-agar plate and inoculated with 100 μl of diluted culture. For biofilms treated with compound 31, the inhibitor was

added

directly

to

the

dilute

inoculum

at

a

final

concentration of 1 or 10 µM before being pipetted onto the polycarbonate membranes and dried. The membranes were dried in a sterile laminar flow hood for 1 h, then incubated at 37 °C for 24 h. The biofilms were stained with Syto-9 and propidium iodide for live/dead staining (Filmtracer LIVE/DEAD Biofilm Viability Kit, ThermoFisher) and Toto-3 for extracellular DNA staining (eDNA; ThermoFisher).

The

dyes

were

diluted

in

PBS

to

final

concentrations of 10 µM Syto-9, 60 µM propidium iodide and 2 µM Toto-3, as per manufacturer instructions, and 500 µL of the diluted dye mix was then immediately pipetted gently onto each S. cohnii biofilm. The biofilms were incubated in the dark at room temperature for 20–25 min, after which each biofilm was rinsed three times with sterile PBS and then imaged. Fluorescent confocal images were recorded using a Zeiss LSM 710 confocal laser-scanning microscope (Carl Zeiss Microscopy GmbH, Germany) with

a

Zeiss

AxioImager

Z2

microscope

frame.

The

LSM

was

configured with a 405-diode laser, argon laser, green HeNe and red HeNe laser, phase objectives and mercury burner. Images were captured

using

Fiji/Image

J

Zeiss

software

ZEN and

2009

software

COMSTAT2.56

and

Details

processed of

analyses are available in the Supporting Information.


the

with image


Impact

of

compound

31

on

viability

and

Page 20 of 49

proliferation

of

cultured human bone marrow-derived mesenchymal stromal cells (MSCs) Human MSCs were isolated from bone marrow aspirates from patients undergoing spinal fusion surgery after informed consent (New Zealand Health and Disability Ethics Committee URA-08-08049) in accordance with a previously published protocol.57 Cells were cultured and expanded up to passage 2 in α-minimum essential medium supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 0.1 mg/mL streptomycin and 1 ng/mL basic fibroblast growth factor prior to use. P2 MSCs were seeded in a 48-well plate at a density of 5000 cells/cm2 in 500 µL of media and incubated for 24 h. After incubation, the media was replaced with fresh media supplemented with different concentrations (2.5 µM to 25 µM) of either compound 31 alone or extract collected after compound 31 was released from PVA-Tyr hydrogels. Cells were then incubated for an additional 48 h. Following incubation, cell metabolic activity was assessed using

Alamar

Blue

to

determine

the

percentage

cell

growth

inhibition. Different concentrations of ethanol (4%, 5% and 7.5%) were included as positive controls. This is in accordance with

ISO

10993-5

standards

for

assessing

the

biological

performance and cytotoxicity of medical devices.23 The percentage cell growth inhibition was determined using: Cell growth inhibition (%) = (1 − (Asample/Acontrol)) × 100 (eq. 2)


Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


where Asample is the metabolic activity measured for each sample and Acontrol being the metabolic activity for control untreated cells. MSC proliferation was determined using a fluorometric CyQUANT Cell Proliferation Assay Kit (ThermoFisher Scientific) following manufacturer’s instructions. Briefly, cells were treated with different concentrations of either 31 alone or 31 extracts collected from PVA-Tyr hydrogels (2.5–25 µM) for 48 h. The cells were then washed with PBS and lysed using lysis buffer provided in the kit. One-hundred microliters of cell lysate was then mixed with 100 µL of 2X-GR dye and incubated for 60 min in the dark, following which the fluorescence was measured at 520 nm emission, following excitation at 480 nm, in a plate reader (Thermo Scientific Varioskan Flash). The morphology of the MSCs after different treatments was determined through actin staining. Cells were cultured under different conditions, as described for the proliferation assay, for 48 h, after which the cells were washed with PBS and fixed in 4% neutral buffered formalin for 30 min. Post fixation, cells were washed with PBS and permeabilized with 0.25% (v/v) Triton X-100 for 5 min, after which they were incubated with FITCphalloidin (1:500 in PBS; Abcam, Australia) for 30 min and Hoechst 33342 (1:1000 in PBS; Invitrogen, USA) for 10 min. Samples were washed twice with PBS before they were imaged by fluorescence

microscopy

(Zeiss

Axio

imager

Microscopy GmbH, Germany).


Z1,

Carl

Zeiss


Page 22 of 49

Statistical analysis MBIC data was fitted with a non-linear regression using GraphPad Prism 6.0, using the variable-slope, four-parameter model. Unless otherwise stated, data were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test using GraphPad Prism 6.0.

Results and discussion MTAN inhibitors reduce static biofilm formation in S. cohnii NCBI BLAST analysis of 16S ribosomal RNA gene sequencing revealed that the coagulase-negative Staphylococcus isolated from the infected implant showed 100% sequence identity to S. cohnii. This organism is of clinical relevance, given that S. cohnii has been reported to be pathogenic in human diseases,5860

and is frequently reported during screens for coagulase-

negative staphylococci present in the clinical environment.61 Alarmingly, one study indicated S. cohnii was the predominant species isolated in an ICU pediatric ward, with 97% of the S. cohnii

isolates

resistant

to

methicillin.41

Another

report

described an S. cohnii isolate that was resistant to linezolid, an

antibiotic

that

is

used

to

combat

drug-resistant

Gram-

positive bacteria.62 A library of 27 MTAN inhibitors (Figure 1) was initially screened against the clinical isolate of S. cohnii to assess their impact on biofilm formation. Five of these compounds (3, 4, 5, 17 and 18) reduced biofilm formation by more than 70% in


Page 23 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a static biofilm assay (Figure 2). Of these, compound 3, also known as methylthio-DADMe-Immucillin-A (MTDIA), was chosen as the model compound for investigation in the hydrogel delivery system as it was one of the best inhibitors identified. Following

our

initial

hydrogel

release

assays,

where

the

elution of MTDIA from the hydrogel polymer was tracked, HPLC analysis showed two other peaks together with MTDIA (Figure 3A). It was hypothesized that MTDIA was being oxidized, resulting in the release of three potential oxidation products in addition to MTDIA. These oxidation products could be formed from MTDIA under the conditions used to polymerize the hydrogel and were proposed to be the diastereomeric mixture of sulfoxides 13, sulfone 14, and sulfone N-oxide 15. We synthesized and fully characterized 13, 14, and 15 and tested their ability to limit biofilm production and found that none of these compounds acted as inhibitors (Figure 2). Using these compounds as standards in our HPLC analysis of MTDIA elution from the hydrogel showed that the oxidation products were a mixture of diastereomers of 13 having the same retention times as the standard (Figure 3A). Given the generation of these oxidation side-products, and the need for release of an unmodified MTAN inhibitor, additional compounds were designed. As the side-products isolated from hydrogel polymerization involved oxidation of the sulfur atom, we focused on sulfur-free MTAN inhibitors from our initial library

of

compounds.

Compound

31

was

chosen

for

further

examination because our previous research has shown that the



Page 24 of 49

2'(S) stereochemistry was a key component of our most potent acyclic inhibitors when compared with the 2'(R),63, more

easily

synthesized

on

a

multi-gram

scale

64

and it was than

other

compounds like 17. We also elected to alter the length of the hydrophobic side-chain of compound 31 to determine whether there was an optimal number of carbons required for the best antibiofilm activity. We synthesized compounds 28, 29, 30, 32 and 33, which, together with compounds 19-27 from our original screen, allowed us to observe the trend between the ability of these compounds to reduce biofilm formation and their sidechain length. The optimal sidechain length consisted of the 9 carbon atoms

contained

in

compound

31.

The expanded

library

of

34

compounds yielded 10 that reduced static biofilm formation by >70% in assays, namely compounds 3, 4, 5, 17, 18, 29, 30, 31, 32 and 34 (Figure 2). To further confirm the impact of MTAN inhibitor stereochemistry on biofilm inhibition, we synthesized the enantiomer of compound 31, compound 34. As mentioned above, our previous work on acyclic MTAN inhibitors demonstrated that the enantiomer 34 would be expected to be a poorer inhibitor of biofilm formation in the functional assay when compared with compound 31. Minimal biofilm inhibitory

concentration

(MBIC)

values

were

assessed

for

compounds 31 and 34 using the crystal violet assay. The MBIC for compound 31 was 2.2 ± 0.4 µM, while the MBIC for enantiomer 34 was approximately 10fold higher at 19.1± 0.4 µM (Supplementary Figure S1).


Page 25 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 31 was taken forward as our model candidate for further

biofilm

inhibition

and

release

studies,

given

its

scalable synthetic route and low MBIC.

Inhibition of purified S. cohnii MTAN by compound 31 Purification of GST-tagged S. cohnii MTAN was carried out using affinity chromatography, yielding approximately 3 mg of purified protein. SDS-PAGE indicated a highly purified sample with a predominant band just above 50kDa, which was in-line with MW expected in calculations for a GST-tagged MTAN (Calculated MW = 52.5 kDa). S. cohnii MTAN exhibits a Km value of 0.70 ± 0.02 µM, a kcat value of 1.140 ± 0.005 s−1, and a catalytic efficiency (kcat/ Km) value of 1.63 × 106 M−1s−1 for 5′-methylthioadenosine (substrate). Compound 31 yielded Ki and Ki* values of 78 ± 7 pM and 45 ± 4 pM, respectively.

PVA-Tyr as delivery system for release of MTAN inhibitors After confirmation of biofilm inhibition, experiments were conducted to evaluate systems to deliver MTAN inhibitors. FDAapproved PVA-based synthetic hydrogel systems have been used in different biomedical applications and this is attributed to their

biocompatibility,

modification.65

In

this

low

toxicity

case,

a

and

visible

ease

of

light

chemical

crosslinked

tyramine-modified PVA (PVA-Tyr) hydrogel system was selected for immobilizing MTAN inhibitors. We have previously demonstrated that

efficient

utilization

of

light-mediated


cross-linking


enables

spatial

and

fabrication process.66,

temporal 67

control

Page 26 of 49

over

the

hydrogel

Moreover, hydrolytically labile ester

bonds are included within the PVA-Tyr polymer backbone, allowing the unique tailorable and predictable degradation profiles for both in vitro and in vivo applications.48,

55

By tuning the

macromer concentration and photo-crosslinking conditions, the release of the MTAN inhibitors could be tailored to ensure a release over an initial few days, which is critical for targeting post-operative bacterial adhesion as a key step in biofilm formation in the clinical setting following surgery.68 To ensure compound 31 release over the first few hours and complete degradation of the fabricated hydrogel up to 5 days, different

macromer

and

photo-initiator

concentrations

were

tested. Based on the swelling and degradation profiles, 10% (w/v) with 0.5/5 Ru/SPS were selected. As the visible light polymerization chemistry employed to fabricate PVA-Tyr hydrogels in

this

study

is

mediated

through

radicals,

the

biofilm-

inhibiting molecules might scavenge the radicals required to crosslink PVA-Tyr, negatively affecting the overall hydrogel physicochemical

properties.

Therefore,

the

physicochemical

properties of PVA-Tyr hydrogels incorporated with compound 31 were characterized. The swelling ratio remain unaltered (P>0.05) when

different

concentrations

of

the

compound

31

were

incorporated within the fabricated hydrogel. The swelling ratio for compound 31-containing PVA-Tyr was 23 ± 1 and the gels were completely degraded in 5 ± 1 days. This data confirmed that the


Page 27 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incorporation of biofilm inhibiting compounds, even at high concentrations (100 µM), did not interfere with the lightinitiated

crosslinking

reaction.

Importantly,

compound

31

remained functionally unaltered during the photo-crosslinking process and after being released from the hydrogel (Figure 3C). HPLC analysis indicated that 50 ± 4% of compound 31 was detected at the conclusion of a 24-h PVA-Tyr release study. The loading concentrations within the hydrogel were therefore normalized to the release data to enable comparison with biofilm data obtained for compound 31 shown in Figure 2 and Supplemental Figure 1. Figure 4 indicates that static biofilm formation was reduced by more than 60% when compound 31 was loaded into the hydrogel up to a concentration of 5 µM, with 20% inhibition at 2.5 µM and no inhibition below 1 µM. PVA-Tyr controls (no compound 31) did not show any significant biofilm inhibition relative to the PBS control, indicating that biofilm inhibition observed from compound 31-loaded hydrogels were from compound 31

released

from

fabricated

hydrogels,

and

not

related

to

degradation products or interference from the PVA-Tyr hydrogel. Furthermore, the inhibition profile of compound 31 released from PVA-Tyr hydrogels was similar (P>0.05) to that observed from compound

31

added

directly

to

the

media

(Figure

2).

Specifically, 20 µM compound 31 loaded in PVA-Tyr resulted in 77.2 ± 8.4% biofilm inhibition (Figure 4) which is comparable to the 86.03 ± 2.7% inhibition of compound 31 alone at 10 µM. These results confirm that incorporation of compound 31 into the



PVA-Tyr

hydrogels

did

not

affect

Page 28 of 49

the

unique

tailorable

physicochemical (swelling and degradation profiles) capability of PVA-Tyr, but importantly also preserved the bioactivity of the

loaded

compound,

thereby

offering

an

ideal

candidate

hydrogel platform suitable for release of robust and functional biofilm inhibitor (compound 31) over a period of days. Compound 31 does not affect MSC viability and proliferation at concentrations where biofilms are inhibited A

key

consideration

in

therapeutic

strategies

involving

delivery of antimicrobial agents is the requirement that any adopted

material

should

not

affect

the

performance

of

the

surrounding host cells.69 MSCs play a key role in wound healing by homing to the wound site where they recruit other local cells and secrete growth factors to initiate the repair process.70,

71

Therefore, human bone marrow-derived MSCs were selected as a model cell type to assess the influence of compound 31, either alone

or

released

from

hydrogel,

on

cell

viability

and

proliferation. The same concentrations of compound 31 that were used for previously described biofilm inhibition studies were selected (0.5–20 µM). Increasing concentrations of compound 31 showed no negative effect (P>0.05) on metabolic activity of MSCs relative to the control, as assessed by Alamar Blue assay (Figure 5A),

thereby

indicating

no

cytotoxic

effect.

Different

concentrations of ethanol (4%, 5% and 7.5% (v/v)) were included as

positive

controls

for

cytotoxicity,

with

increasing

concentrations of ethanol significantly decreasing (P0.05) in the presence of compound 31, as represented in Figure 5B. Similarly, when comparing the effect of varying concentrations of compound 31 released from PVA-Tyr hydrogels, no impedance on either MSC metabolic activity or cell proliferation was observed (Figure 5A and 5B). This result suggests that the released compound 31 is not cytotoxic as per the ISO10993-5 standards. Across all tested concentrations of compound 31 released from PVA-Tyr hydrogels, cell metabolic activity was significantly higher

than

the

control

(P0.05) was observed in cell proliferation in the presence of biofilm-inhibiting compound released from PVA-Tyr relative to the control. Morphological evaluations of MSCs after treatment with varying concentrations of compound 31, either added directly to the media or after being released from the PVA-Tyr hydrogel, showed no difference to the untreated cells (Figure 6). Cells exhibited a multipolar spindle-shape with a well-organized cytoskeletal structure as well as several filopodia and lamellipodia, even after being exposed to 20 µM compound 31. Taking the cell viability and proliferation data together, the results suggest that

the

MTAN

inhibitors,

when

combined

with

the

hydrogel

platform, did not introduce any cytotoxic or growth inhibitory effect to MSCs. These experiments demonstrate the tolerability



Page 30 of 49

of the system by a host cell and support the further development of

a

PVA-Tyr-based

injectable

system

that

can

be

photo-

crosslinked in situ and could be used as biofilm-inhibiting delivery vehicle for medical devices.

Compound 31 reduces eDNA levels and biofilm formation Following demonstration of successful release of compound 31 from the hydrogel and measurement of its impact on biofilm formation and MSC performance, we next sought to understand the mechanistic role by which compound 31 affects biofilm formation. A previous report indicates that deletion of the S. aureus gene encoding MTAN (pfs), decreased the amount of eDNA released, resulting in a significant reduction in biofilm formation. In order to quantify the effect of compound 31 on S. cohnii eDNA levels and biofilm structure, scanning laser confocal microscopy on treated and untreated colony biofilms was performed (Figure 7 and Supplementary Figure S2). The eDNA content (expressed as the ratio of eDNA:live cells) was reduced in biofilms treated with 10 µM compound 31 (Figure 7A). Additionally, 10 µM of compound 31 reduced biofilm thickness from 39 ± 4 μm in the untreated control, to 20 ± 4 μm (Figure 7B). Interestingly, Bao et al. previously reported that reduction of

biofilm

formation,

and

the

resulting

decrease

in

eDNA

following pfs deletion, occurred independently of Autoinducer-2 (AI-2)

production.40

AI-2

is

a

key

quorum

sensing

molecule

involved in biofilm formation in Gram-negative bacteria. MTAN


Page 31 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


catalyzes the conversion of S-adenosyl homocysteine to S-ribosyl homocysteine, which can be catalyzed further by the LuxS enzyme to generate 4,5-dihydroxy-2,3-pentanedione and, ultimately, a series of furanones collectively referred to as AI-2. Even though AI-2 is produced in staphylococcal species, its role is unclear, as although luxS deletion results in reduction of virulence and biofilm formation in some staphylococci,57,

72

a

report observed that the effects of luxS deletion in S. aureus may

be

due

to

its

role

in

metabolism

rather

than

through

reduction of quorum-sensing molecules.73 Efforts have been made to unpick the relationship between AI-2 production and MTAN activity with respect to observed phenotypes in staphylococcal species; indeed, MTAN is critical for AI-2 production in S. aureus.26 However, when considering the impact of both AI-2/LuxS and MTAN/pfs on phenotype, pfs knockout reduced virulence of S. aureus in a mouse sepsis model. However, this effect was not observed in a luxS mutant, suggesting the virulence triggered by MTAN is independent of LuxS/AI-2.26 Taking these findings together, future work will explore the effect of our lead compound 31 on metabolism versus AI-2 production, along with further exploration of the impact of compound 31 on autolytic genes and proteins involved in eDNA production in our clinical isolate, along with other clinically important staphylococcal species.

Conclusions



Page 32 of 49

We report the successful development of a versatile, lightactivated biodegradable PVA-Tyr-based hydrogel delivery system that has enabled the targeted release of a lead MTAN inhibitor (compound 31). The PVA-Tyr system chosen here offers a burst release of the loaded MTANi of over ~50% being released in the first 24 h. This release profile is important as the first 24 h post-implantation

are

considered

crucial

and

has

been

acknowledged as a ‘decisive period’ whereby inhibiting bacterial adhesion offers long-term success of the implant. Furthermore, this inhibitor can significantly reduce biofilm formation in a clinical staphylococcal isolate, through reductions in eDNA levels, with minimal impact on MSC proliferation and metabolic activity.

We

recognize

that

whilst

the

biofilm

screening

experiment provided key information, more detailed studies are required to evaluate the direct impact of compound 31 on both bacterial growth and biofilm formation, with a view to further understanding the function of MTAN.

Future studies will also

focus on combining this technology with implant surfaces (e.g. titanium)

and

assessing

the

effect

of

the

identified

lead

compound on cell function, primarily focusing on osteogenic differentiation of MSCs following prolonged exposure to these MTAN molecules.


Page 33 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Figure 1: MTAN inhibitors screened against a coagulase-negative Staphylococcus



Page 34 of 49

Figure 2. Screening of MTAN inhibitors for disruption of biofilm formation. S. cohnii biofilms were grown in the presence of 10 μM each MTAN inhibitor. The percent biofilm formation relative to

an

untreated

(no

inhibitor)

control

was

measured

using

crystal violet staining of the bacterial biomass. Filled circles represent the mean of six technical replicates, bars represent the mean inhibition from at least two independent experiments (n=2–3), and lines represent the range.


Page 35 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3: HPLC traces depicting detection of compounds 3 and 31. A) 3 is released from PVA-Tyr hydrogels (Retention time, tR, 9.2 min, detection at 230 nM), sulfoxide diastereomers 13 are also observed (tR 5.1 and 5.5 min); B) Standard solution of 3; C) 31 released from PVA-Tyr (tR

5.85 min, detection at 254

nM). Ru(bpy)32+ is also observed at tR 2.9 min; D) Standard solution of 31



Page 36 of 49

Figure 4: Reduction in static biofilm formation using PVA-Tyr hydrogel supernatants containing 31. The concentrations relate to the doses loaded into the hydrogel. Filled circles represent the mean of six technical replicates, bars represent the mean inhibition

from

three

independent

experiments,

and

lines

represent the range. Asterisks denote significantly reduced biofilm formation as compared with the untreated control (n=3; *P ≤ 0.01, **P ≤ 0.001, as determined by ANOVA with Dunnett’s multiple comparisons test).


Page 37 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5: Compound 31 does not decrease viability or affect proliferation of bone marrow stromal cells (MSCs), as shown by (A)

Alamar

Blue

assay

and

(B)

cell

proliferation

assay,

respectively. e4, e5 and e7.5 represent 4%, 5% and 7.5% (v/v) ethanol used as positive controls. The concentrations relate to the doses loaded into the hydrogel. Dark grey bars represent compound 31 in solution (not encapsulated in PVA-Tyr); light grey

bars

represent

compound

31

following

release

from

hydrogels. Filled circles represent the mean of six technical



Page 38 of 49

replicates, the bars represent the mean inhibition from three independent experiments, and lines represent the range. (n=3; **P ≤ 0.001 against untreated control, as determined by ANOVA with Dunnett’s multiple comparisons test)

Figure

6:

Compound

31

does

not

affect

the

morphology

or

distribution of human bone marrow stromal cells as shown by Factin staining of cells after 48 h of treatment. Cells were stained with phalloidin (green) and counterstained with nuclear


Page 39 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stain Hoechst 33342 (blue). The concentrations relate to the doses loaded into the hydrogel. The left panels represent the group in which compound 31 was added directly to the media, whereas

the

right

panels

show

cells

treated

with

varying

concentrations of compound 31 following release from hydrogels. Images are representative of the average phenotype across 3–5 technical and 3 biological replicates.



Page 40 of 49

Figure 7. Quantification of the effect of compound 31 on biofilm eDNA content and biofilm thickness. A) Ratio of TOTO-3-stained eDNA to live cells in the colony biofilms. B) Colony biofilm thickness in the presence of 0, 1 or 10 µM compound 31. Bars represent the mean values from three independent experiments (n=3),

filled

circles

represent

the

mean

of

3–5

technical

replicates per experiment, and lines represent the range.


Page 41 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT Supporting Information The supporting information included in the manuscript provides a summary of: synthesis of different families of MTAN inhibitor; S.cohnii describing

culture

and

minimal

characterization

biofilm

inhibitory

protocol;

and

concentration

data (MBIC)

values from crystal violet assay (Figure S1) and processing of confocal laser scanning microscopic images for MTAN treated S. cohnii biofilms (Figure S2). See “Supporting Information.docx”

AUTHOR INFORMATION Corresponding author *

Tim

B.

F.

Woodfield;

E-mail:

[email protected];

Telephone: (+64) 3 3641086 Christchurch (CReaTE)

Regenerative

Group,

Musculoskeletal

Medicine

Department Medicine,

of

Centre

and

Tissue

Orthopaedic for

Engineering Surgery

&

Bioengineering

&

Nanomedicine, University of Otago Christchurch, PO Box 4345, Christchurch 8100, New Zealand.

Author contributions



Page 42 of 49

AGM, IM, SW, TS, JM, KC, CS, DT, RGD, KL designed and conducted experiments,

analyzed

data,

and

contributed

to

writing

the

manuscript. AGM, MLG, SC, GBE, KL, TBFW designed experiments, analyzed data and assisted with writing of the manuscript.

Notes No potential conflicts of interest relevant to this article were reported.

ACKNOWLEDGMENTS The authors wish to acknowledge the New Zealand Ministry of Business Innovation and Employment for financial support through a Smart Idea Grant (RTVU1504). KL is supported by a New Zealand Health

Research

Council

Emerging

Researcher

First

Grant

(15/483). TBFW is a Royal Society of New Zealand Rutherford Discovery Fellowship (RDF-UOO1204). IM, KL, TBFW are supported by funding from the Medical Technologies Centre of Research Excellence (MedTech CoRE). MLG, GE, TBFW, KL are supported by funding

from

the

Maurice

Wilkins

Centre

Biodiscovery.

ABBREVIATIONS


for

Molecular

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MTAN,

5′-methylthioadenosine

nucleosidase;

MTANi,

5′-

methylthioadenosine nucleosidase inhibitor; methylthio-DADMeImmucillin-A

(MTDIA);

PVA-Tyr,

tyramine-functionalized

poly(vinyl alcohol), PVA-COOH, carboxylated-PVA; Ru, (Tris(2,2′bipyridyl)dichlororuthenium(II)

hexahydrate;

SPS,

sodium

persulfate; CSLM, confocal laser scanning microscopy; MSCs, human bone marrow stromal cells.



References 1. Lehil, M. S.; Bozic, K. J., Trends in total hip arthroplasty implant utilization in the United States. J Arthroplasty 2014, 29 (10), 1915-8, doi: 10.1016/j.arth.2014.05.017. 2. Kurtz, S.; Ong, K.; Lau, E.; Mowat, F.; Halpern, M., Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030. J Bone Joint Surg Am 2007, 89 (4), 780-5, doi: 10.2106/JBJS.F.00222. 3. Campoccia, D.; Montanaro, L.; Moriarty, T. F.; Richards, R. G.; Ravaioli, S.; Arciola, C. R., The selection of appropriate bacterial strains in preclinical evaluation of infection-resistant biomaterials. Int J Artif Organs 2008, 31 (9), 841-7, doi: 10.1177/039139880803100913. 4. Tran, P. L.; Hamood, A. N.; Reid, T. W., Antimicrobial Coatings to Prevent Biofilm Formation on Medical Devices. In Antibiofilm Agents, Springer: 2014; pp 175-204, doi: 10.1007/978-3-642-538339_9. 5. Harding, J. L.; Reynolds, M. M., Combating medical device fouling. Trends Biotechnol 2014, 32 (3), 140-6, doi: 10.1016/j.tibtech.2013.12.004. 6. Connaughton, A.; Childs, A.; Dylewski, S.; Sabesan, V. J., Biofilm Disrupting Technology for Orthopedic Implants: What's on the Horizon? Front Med (Lausanne) 2014, 1, 22, doi: 10.3389/fmed.2014.00022. 7. Cochis, A.; Azzimonti, B.; Della Valle, C.; Chiesa, R.; Arciola, C. R.; Rimondini, L., Biofilm formation on titanium implants counteracted by grafting gallium and silver ions. J Biomed Mater Res A 2015, 103 (3), 1176-87, doi: 10.1002/jbm.a.35270. 8. Grigg, C.; Baggs, J.; Slayton, R., Association Between Surgical Antimicrobial Prophylaxis Regimen and Risk of Periprosthetic Joint Infection Following Total Hip and Knee Arthroplasty. Open Forum Infect. Dis. 2016, 3 (suppl_1), 1477-1477, doi: 10.1093/ofid/ofw172.1179. 9. Gao, A.; Hang, R.; Chu, P. K., Recent advances in anti-infection surfaces fabricated on biomedical implants by plasma-based technology. Surface and Coatings Technology 2017, 312, 2-6, doi: 10.1016/j.surfcoat.2016.04.020. 10. Mauffrey, C.; Herbert, B.; Young, H.; Wilson, M. L.; Hake, M.; Stahel, P. F., The role of biofilm on orthopaedic implants: the “Holy Grail” of post-traumatic infection management? Eur. J. Trauma Emerg. Surg. 2016, 42 (4), 411-416, doi: 10.1007/s00068-016-0694-1. 11. Manavathu, E. K.; Vazquez, J. A., Biofilms: Emerging Importance in Infectious Diseases. J. Multidiscip. Pathol. 2015, 2 (1), doi: 10.1146/annurev.micro.57.030502.090720. 12. Nana, A.; Nelson, S. B.; McLaren, A.; Chen, A. F., What's New in Musculoskeletal Infection: Update on Biofilms. J Bone Joint Surg Am 2016, 98 (14), 1226-34, doi: 10.2106/JBJS.16.00300. 13. Raphel, J.; Holodniy, M.; Goodman, S. B.; Heilshorn, S. C., Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants. Biomaterials 2016, 84, 301-14, doi: 10.1016/j.biomaterials.2016.01.016. 14. Ng, V. W.; Chan, J. M.; Sardon, H.; Ono, R. J.; Garcia, J. M.; Yang, Y. Y.; Hedrick, J. L., Antimicrobial hydrogels: a new weapon in the arsenal against multidrug-resistant infections. Adv Drug Deliv Rev 2014, 78, 46-62, doi: 10.1016/j.addr.2014.10.028. 15. D'Este, F.; Oro, D.; Boix-Lemonche, G.; Tossi, A.; Skerlavaj, B., Evaluation of free or anchored antimicrobial peptides as candidates for the prevention of orthopaedic device-related infections. J. Pept. Sci 2017, doi: 10.1002/psc.3026. 16. Mi, F. L.; Wu, Y. B.; Shyu, S. S.; Schoung, J. Y.; Huang, Y. B.; Tsai, Y. H.; Hao, J. Y., Control of wound infections using a bilayer chitosan wound dressing with sustainable antibiotic delivery. J Biomed Mater Res A 2002, 59 (3), 438-449, doi: 10.1002/jbm.1260. 17. Agnihotri, S.; Mukherji, S.; Mukherji, S., Antimicrobial chitosan–PVA hydrogel as a nanoreactor and immobilizing matrix for silver nanoparticles. Appl Nanosci 2012, 2 (3), 179-188, doi: 10.1007/s13204-012-0080-1. 18. Neethirajan, S.; Clond, M. A.; Vogt, A., Medical biofilms—nanotechnology approaches. J Biomed. Nanotechnol 2014, 10 (10), 2806-2827, doi: 10.1166/jbn.2014.1892.


Page 44 of 49

Page 45 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19. Gilabert-Porres, J.; Martí, S.; Calatayud, L.; Ramos, V.; Rosell, A.; Borrós, S., Design of a nanostructured active surface against Gram-positive and Gram-negative bacteria through plasma activation and in situ silver reduction. ACS Appl. Mater. Interfaces 2015, 8 (1), 64-73, doi: 10.1021/acsami.5b07115. 20. AshaRani, P.; Low Kah Mun, G.; Hande, M. P.; Valiyaveettil, S., Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 2008, 3 (2), 279-290, doi: 10.1021/nn800596w. 21. Burd, A.; Kwok, C. H.; Hung, S. C.; Chan, H. S.; Gu, H.; Lam, W. K.; Huang, L., A comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models. Wound Repair Regen 2007, 15 (1), 94-104, doi: 10.1111/j.1524-475X.2006.00190.x. 22. Pacor, S.; Giangaspero, A.; Bacac, M.; Sava, G.; Tossi, A., Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use. J. Antimicrob. Chemother 2002, 50 (3), 339-348, doi: 10.1093/jac/dkf141. 23. Bacalum, M.; Radu, M., Cationic antimicrobial peptides cytotoxicity on mammalian cells: an analysis using therapeutic index integrative concept. Int J Pept Res Ther 2015, 21 (1), 47-55, doi: 10.1007/s10989-014-9430-z. 24. Figueiredo, A. M. S.; Ferreira, F. A.; Beltrame, C. O.; Côrtes, M. F., The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus. Crit. Rev. Microbiol. 2017, 43 (5), 602-620, doi: 10.1080/1040841X.2017.1282941. 25. Smith, R.; Coast, J., The true cost of antimicrobial resistance. BMJ 2013, 346, f1493, doi: 10.1136/bmj.f1493. 26. Bao, Y.; Li, Y.; Jiang, Q.; Zhao, L.; Xue, T.; Hu, B.; Sun, B., Methylthioadenosine/Sadenosylhomocysteine nucleosidase (Pfs) of Staphylococcus aureus is essential for the virulence independent of LuxS/AI-2 system. Int J Med Microbiol 2013, 303 (4), 190-200, doi: 10.1016/j.ijmm.2013.03.004. 27. Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K.; Wertheim, H. F.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens, H.; Greko, C.; So, A. D.; Bigdeli, M.; Tomson, G.; Woodhouse, W.; Ombaka, E.; Peralta, A. Q.; Qamar, F. N.; Mir, F.; Kariuki, S.; Bhutta, Z. A.; Coates, A.; Bergstrom, R.; Wright, G. D.; Brown, E. D.; Cars, O., Antibiotic resistance-the need for global solutions. Lancet Infect Dis 2013, 13 (12), 1057-98, doi: 10.1016/S1473-3099(13)70318-9. 28. Izawa, K.; Aceña, J. L.; Wang, J.; Soloshonok, V. A.; Liu, H., Small-Molecule Therapeutics for Ebola Virus (EBOV) Disease Treatment. Eur. J. Org. Chem. 2016, 2016 (1), 8-16, doi: 10.1002/ejoc.201501158 29. Schramm, V. L., Freezing Time-Targeting the briefest moment in chemistry may lead to an exceptionally strong new class of drugs. Scientist 2012, 26 (5), 30, doi: 30. Schramm, V. L., Transition States and transition state analogue interactions with enzymes. Acc Chem Res 2015, 48 (4), 1032-9, doi: 10.1021/acs.accounts.5b00002. 31. Gebre, S. T.; Cameron, S. A.; Li, L.; Babu, Y. S.; Schramm, V. L., Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase. J Biol Chem 2017, doi: 10.1074/jbc.M117.801779. 32. Namanja-Magliano, H. A.; Stratton, C. F.; Schramm, V. L., Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis. ACS Chem Biol 2016, 11 (6), 1669-76, doi: 10.1021/acschembio.6b00144. 33. Wang, S.; Cameron, S. A.; Clinch, K.; Evans, G. B.; Wu, Z.; Schramm, V. L.; Tyler, P. C., New Antibiotic Candidates against Helicobacter pylori. J Am Chem Soc 2015, 137 (45), 14275-80, doi: 10.1021/jacs.5b06110. 34. Haapalainen, A. M.; Thomas, K.; Tyler, P. C.; Evans, G. B.; Almo, S. C.; Schramm, V. L., Salmonella enterica MTAN at 1.36 Å Resolution: A Structure-Based Design of Tailored Transition State Analogs. Structure 2013, 21 (6), 963-974, doi: 10.1016/j.str.2013.04.009.



35. Singh, V.; Schramm, V. L., Transition-state analysis of S. pneumoniae 5'-methylthioadenosine nucleosidase. J Am Chem Soc 2007, 129 (10), 2783-95, doi: 10.1021/ja065082r. 36. Gutierrez, J. A.; Crowder, T.; Rinaldo-Matthis, A.; Ho, M. C.; Almo, S. C.; Schramm, V. L., Transition state analogs of 5'-methylthioadenosine nucleosidase disrupt quorum sensing. Nat Chem Biol 2009, 5 (4), 251-7, doi: 10.1038/nchembio.153. 37. Singh, V.; Shi, W.; Almo, S. C.; Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Painter, G. F.; Lenz, D. H.; Mee, S.; Zheng, R.; Schramm, V. L., Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae. Biochemistry 2006, 45 (43), 12929-41, doi: 10.1021/bi061184i. 38. Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee, S.; Painter, G. F.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; Howell, P. L.; Schramm, V. L., Femtomolar transition state analogue inhibitors of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli. J Biol Chem 2005, 280 (18), 18265-73, doi: 10.1074/jbc.M414472200. 39. Gutierrez, J. A.; Luo, M.; Singh, V.; Li, L.; Brown, R. L.; Norris, G. E.; Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Painter, G. F.; Lenz, D. H.; Schramm, V. L., Picomolar inhibitors as transition-state probes of 5'-methylthioadenosine nucleosidases. ACS Chem Biol 2007, 2 (11), 725-34, doi: 10.1021/cb700166z. 40. Bao, Y.; Zhang, X.; Jiang, Q.; Xue, T.; Sun, B., Pfs promotes autolysis-dependent release of eDNA and biofilm formation in Staphylococcus aureus. Med Microbiol Immunol 2015, 204 (2), 215-26, doi: 10.1007/s00430-014-0357-y. 41. Szewczyk, E. M.; Piotrowski, A.; Rozalska, M., Predominant staphylococci in the intensive care unit of a paediatric hospital. J Hosp Infect 2000, 45 (2), 145-54, doi: 10.1053/jhin.1999.0754. 42. Montali, A., Antibacterial coating systems. Injury 2006, 37 (2), S81-S86, doi: 10.1016/j.injury.2006.04.013. 43. Giavaresi, G.; Bertazzoni Minelli, E.; Sartori, M.; Benini, A.; Della Bora, T.; Sambri, V.; Gaibani, P.; Borsari, V.; Salamanna, F.; Martini, L., Microbiological and pharmacological tests on new antibiotic-loaded PMMA-based composites for the treatment of osteomyelitis. J. Orthop. Res. 2012, 30 (3), 348-355, doi: 10.1002/jor.21531. 44. Taylor, M.; Daniels, A.; Andriano, K.; Heller, J., Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J Appl Biomater 1994, 5 (2), 151-157, doi: 10.1002/jab.770050208. 45. Goodman, S. B.; Yao, Z.; Keeney, M.; Yang, F., The future of biologic coatings for orthopaedic implants. Biomaterials 2013, 34 (13), 3174-3183, doi: 10.1016/j.biomaterials.2013.01.074. 46. Salomé Veiga, A.; Schneider, J. P., Antimicrobial hydrogels for the treatment of infection. J. Pept. Sci 2013, 100 (6), 637-644, doi: 10.1002/bip.22412. 47. Martens, P.; Blundo, J.; Nilasaroya, A.; Odell, R. A.; Cooper-White, J.; Poole-Warren, L. A., Effect of poly (vinyl alcohol) macromer chemistry and chain interactions on hydrogel mechanical properties. Chem. Mater. 2007, 19 (10), 2641-2648, doi: 10.1021/cm0626381. 48. Lim, K. S.; Alves, M. H.; Poole-Warren, L. A.; Martens, P. J., Covalent incorporation of nonchemically modified gelatin into degradable PVA-tyramine hydrogels. Biomaterials 2013, 34 (29), 7097-7105, doi: 49. Lim, K. S.; Roberts, J. J.; Alves, M. H.; Poole-Warren, L. A.; Martens, P. J., Understanding and tailoring the degradation of PVA-tyramine hydrogels. J Appl Polym Sci 2015, 132 (26), doi: 10.1002/app.42142. 50. Merritt, J. H.; Kadouri, D. E.; O'Toole, G. A., Growing and analyzing static biofilms. Curr Protoc Microbiol 2005, Chapter 1, Unit 1B 1, doi: 10.1002/9780471729259.mc01b01s00. 51. Berrow, N. S.; Alderton, D.; Sainsbury, S.; Nettleship, J.; Assenberg, R.; Rahman, N.; Stuart, D. I.; Owens, R. J., A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic acids research 2007, 35 (6), e45, doi: 10.1093/nar/gkm047. 52. Dunn, S. M.; Bryant, J. A.; Kerr, M. W., A simple spectrophotometric assay for plant 5′-deoxy-5′-methylthioadenosine nucleosidase using xanthine oxidase as a coupling enzyme. Phytochemical Analysis 1994, 5 (6), 286-290, doi: 10.1002/pca.2800050603


Page 46 of 49

Page 47 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


53. Ducati, R. G.; Harijan, R. K.; Cameron, S. A.; Tyler, P. C.; Evans, G. B.; Schramm, V. L., Transition-state analogues of Campylobacter jejuni 5'-methylthioadenosine nucleosidase. ACS chemical biology 2018, 13 (11), 3173-3183, doi: 10.1021/acschembio.8b00781. 54. Morrison, J. F.; Walsh, C. T., The behavior and significance of slow-binding enzyme inhibitors. Advances in enzymology and related areas of molecular biology 1988, 61, 201-301, doi: 10.1002/9780470123072.ch5 55. Lim, K. S.; Ramaswamy, Y.; Roberts, J. J.; Alves, M. H.; Poole-Warren, L. A.; Martens, P. J., Promoting Cell Survival and Proliferation in Degradable Poly(vinyl alcohol)-Tyramine Hydrogels. Macromol Biosci 2015, 15 (10), 1423-1432, doi: 10.1002/mabi.201500121. 56. Heydorn, A.; Nielsen, A. T.; Hentzer, M.; Sternberg, C.; Givskov, M.; Ersboll, B. K.; Molin, S., Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 2000, 146 ( Pt 10), 2395-407, doi: 10.1099/00221287-146-10-2395. 57. Schrobback, K.; Klein, T. J.; Woodfield, T. B. F., The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models. Tissue Eng Pt A 2015, 21 (11-12), 1785-1794, doi: 10.1089/ten.tea.2014.0691. 58. Yamashita, S.; Yonemura, K.; Sugimoto, R.; Tokunaga, M.; Uchino, M., Staphylococcus cohnii as a cause of multiple brain abscesses in Weber-Christian disease. J Neurol Sci 2005, 238 (1-2), 97-100, doi: 10.1016/j.jns.2005.06.009. 59. Soldera, J.; Nedel, W. L.; Cardoso, P. R.; d'Azevedo, P. A., Bacteremia due to Staphylococcus cohnii ssp. urealyticus caused by infected pressure ulcer: case report and review of the literature. Sao Paulo Med J 2013, 131 (1), 59-61, doi: 10.1590/S1516-31802013000100010 60. Bhattacharya, G.; Dey, D.; Bhattacharya, A.; Das, S.; Banerjee, A., Bacteraemia by staphylococcus cohni isubsp. urealyticusin a diabetic patient: A case report. World J. Pharm. Pharm. Sci. 2016, 5, 1864-1869, doi: 10.1590/S1516-31802013000100010 61. Wojtyczka, R. D.; Orlewska, K.; Kepa, M.; Idzik, D.; Dziedzic, A.; Mularz, T.; Krawczyk, M.; Miklasinska, M.; Wasik, T. J., Biofilm formation and antimicrobial susceptibility of Staphylococcus epidermidis strains from a hospital environment. Int J Environ Res Public Health 2014, 11 (5), 4619-33, doi: 10.3390/ijerph110504619. 62. Chen, H.; Wu, W.; Ni, M.; Liu, Y.; Zhang, J.; Xia, F.; He, W.; Wang, Q.; Wang, Z.; Cao, B.; Wang, H., Linezolid-resistant clinical isolates of enterococci and Staphylococcus cohnii from a multicentre study in China: molecular epidemiology and resistance mechanisms. Int J Antimicrob Agents 2013, 42 (4), 317-21, doi: 10.1016/j.ijantimicag.2013.06.008. 63. Clinch, K.; Evans, G. B.; Frohlich, R.; Furneaux, R. H.; Kelly, P. M.; Mason, J. M.; Schramm, V. L.; Tyler, P. C.; Gulab, S. A. A., Acyclic amine inhibitors of 5-methytioadenosine phosphorylase and nucleosidase. US Patent 8,383,636: 2013, doi: 64. Clinch, K.; Evans, G. B.; Frohlich, R. F. G.; Gulab, S. A.; Gutierrez, J. A.; Mason, J. M.; Schramm, V. L.; Tyler, P. C.; Woolhouse, A. D., Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics. Bioorg. Med. Chem. 2012, 20 (17), 5181-5187, doi: 10.1016/j.bmc.2012.07.006. 65. Kumar, A.; Han, S. S., PVA-based hydrogels for tissue engineering: A review. International Journal of Polymeric Materials and Polymeric Biomaterials 2017, 66 (4), 159-182, doi: 10.1080/00914037.2016.1190930. 66. Lim, K. S.; Levato, R.; Costa, P. F.; Castilho, M. D.; Alcala-Orozco, C. R.; van Dorenmalen, K. M.; Melchels, F. P.; Gawlitta, D.; Hooper, G. J.; Malda, J. J. B., Bio-resin for high resolution lithographybased biofabrication of complex cell-laden constructs. 2018, 10 (3), 034101, doi: 10.1088/17585090/aac00c. 67. Bertlein, S.; Brown, G.; Lim, K. S.; Jungst, T.; Boeck, T.; Blunk, T.; Tessmar, J.; Hooper, G. J.; Woodfield, T. B.; Groll, J. J. A. M., Thiol–ene clickable gelatin: a platform bioink for multiple 3D biofabrication technologies. 2017, 29 (44), 1703404, doi: 10.1002/adma.201703404.



68. Hetrick, E. M.; Schoenfisch, M. H., Reducing implant-related infections: active release strategies. Chem Soc Rev 2006, 35 (9), 780-9, doi: 10.1039/b515219b. 69. Sun, D.; Xu, D.; Yang, C.; Shahzad, M. B.; Sun, Z.; Xia, J.; Zhao, J.; Gu, T.; Yang, K.; Wang, G., An investigation of the antibacterial ability and cytotoxicity of a novel cu-bearing 317L stainless steel. Sci. Rep. 2016, 6, 29244, doi: 10.1038/srep29244. 70. Maxson, S.; Lopez, E. A.; Yoo, D.; Danilkovitch-Miagkova, A.; LeRoux, M. A., Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl Med 2012, 1 (2), 142-149, doi: 10.5966/sctm.2011-0018. 71. Lin, W.; Xu, L.; Zwingenberger, S.; Gibon, E.; Goodman, S. B.; Li, G., Mesenchymal stem cells homing to improve bone healing. J Orthop Translat 2017, doi: 10.1016/j.jot.2017.03.002. 72. Wang, Y.; Zhang, W.; Wu, Z.; Zhu, X.; Lu, C., Functional analysis of luxS in Streptococcus suis reveals a key role in biofilm formation and virulence. Vet Microbiol 2011, 152 (1-2), 151-60, doi: 10.1016/j.vetmic.2011.04.029. 73. Doherty, N.; Holden, M. T.; Qazi, S. N.; Williams, P.; Winzer, K., Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not quorum sensing. J Bacteriol 2006, 188 (8), 2885-97, doi: 10.1128/JB.188.8.2885-2897.2006.


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For Table of contents use only Biofilm inhibition via delivery of novel methylthioadenosine nucleosidase

inhibitors

from

PVA-tyramine

hydrogels

while

supporting mesenchymal stromal cell viability. Isha Mutreja, Suzanne L. Warring, Khoon S. Lim, Tara Swadi, Keith Clinch, Jennifer M. Mason, Campbell R. Sheen,

Dion R.

Thompson, Rodrigo G Ducati, Stephen T. Chambers, Gary B. Evans,

Monica L. Gerth, Antonia G. Miller, Tim B. F. Woodfield,


Biofilm inhibition via delivery of novel methylthioadenosine

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