Dual-Action Biomaterial Surfaces with Quorum Sensing Inhibitor and

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Dual-action biomaterial surfaces with quorum sensing inhibitor and nitric oxide to reduce bacterial colonization Aditi Taunk, Renxun Chen, George Iskander, Kitty Ka Kit Ho, David StClair Black, Mark Willcox, and Naresh Kumar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00816 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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ACS Biomaterials Science & Engineering

Dual-action biomaterial surfaces with quorum sensing inhibitor and nitric oxide to reduce bacterial colonization Aditi Taunk,1 Renxun Chen,1 George Iskander,1 Kitty K. K. Ho,1 David StClair Black,1 Mark D. P. Willcox,2 Naresh Kumar1*

1

School of Chemistry, UNSW Sydney, Sydney, NSW 2052, Australia

2

School of Optometry and Vision Science, UNSW Sydney, Sydney, NSW 2052,

Australia

Author Address School of Chemistry University of New South Wales High Street, Sydney, NSW 2052 Australia Tel: 61 2 9385 4698; Fax: 61 2 9385 6141 E-mail: [email protected]

Keywords Biofilm, biomaterials, antibacterial, quorum sensing, dihydropyrrolone, nitric oxide

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Abstract Bacterial biofilms on implanted medical devices are a serious problem in longterm. At present, no effective strategies are available and the emergence of multi-drug resistance has highlighted the need to develop novel antibacterial coatings to combat device-related infections. One approach is to interfere with the bacterial communication pathway or quorum sensing (QS), which is responsible for biofilm formation and virulence factors, by incorporating QS inhibitors (QSIs) such as dihydropyrrolones (DHPs) on biomaterial surfaces. The endogenous biological signalling molecule nitric oxide (NO) is also a potential candidate for prevention of biomedical infections due to its antibiofilm activity. In this study, we have developed dual-action surface coatings based on DHPs and NO. X-ray photoelectron spectroscopy (XPS) and contact angle measurements confirmed successful immobilization of DHPs and NO, and the Griess assay revealed NO release from the coatings at 24 h. Bacterial colonization on the surfaces was assessed by confocal laser scanning microscopy (CLSM), where the DHP+NO surfaces demonstrated significantly higher efficacy in reducing colonization of Staphylococcus aureus and Pseudomonas aeruginosa via a nonbactericidal mechanism than the DHP or NO-releasing coatings alone. The excellent antibacterial activity of the novel coatings suggests the combination of DHP and NO has great potential to combat device-related bacterial infections.


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Introduction

In recent years, biomedical devices such as cardiovascular devices, orthopaedic implants, catheters, contact lenses and many other such devices have become an indispensable part of modern medical care.1–4 These life-saving devices are responsible for significantly improving the quality of life and also increasing the life expectancy of patients.5 However, the insertion or implantation of medical devices has been associated with a risk of bacterial infections. Despite the use of aseptic surgical techniques in hospitals, microorganisms are still found at the site of about 90 % of implants.6 The economic cost for the treatment of implant-associated infections in the United States alone was estimated to be almost $27 billion in one year.7 As we proceed further into the 21st century, the drastic increase in antibiotic resistance coupled with the lack of new antibacterial drugs has created a demand for alternate therapies for combating bacterial infections, particularly in the context of protecting biomedical devices. The ability of bacteria to adhere to materials and form biofilms plays an important role in generating and sustaining the pathogenicity of bacteria in device-related

infections.

The

production

of

virulence

factors

and

the

development of mature biofilms are regulated by an intercellular communication system in bacteria termed quorum sensing (QS).8,9 Therefore, blocking the QS system is a potential strategy to control virulence and biofilm formation. Importantly, antimicrobial agents that target QS are less prone to induce bacterial resistance due to their non-growth inhibitory mechanism that does not exert survival pressure on bacteria.10 Dihydropyrrolones (DHPs), structural analogues of natural QS inhibitory furanones isolated from the marine alga,

Delisea pulchra, have shown high inhibitory activity against QS-controlled processes

in

various

pathogens

while

having


low

cytotoxicity

towards


Page 4 of 38

mammalian cells.11 Significantly, DHP compounds retained their potent broadspectrum antimicrobial activity both in vitro and in vivo when covalently immobilized onto surfaces.12–14 Another promising strategy for the design of effective antibacterial biomaterials is to use the biologically ubiquitous nitric oxide (NO) to control biofilm formation. NO plays a critical role in the human body’s immune response against infections and as a signalling molecule in various bacteria.15,16 NO has shown to possess excellent biofilm inhibitory activity against both Gram-positive and

Gram-negative

Pseudomonas

bacteria,

aeruginosa

including

and

the

common

methicillin-resistant

human

pathogens

Staphylococcus

aureus

(MRSA).17–19 Further, exogenous sublethal doses of NO have been reported to increase the sensitivity of biofilms to antimicrobial treatments by transforming bacteria

in

resistant

biofilms

into

a

more

sensitive

planktonic

form.18

Additionally, research has shown that NO and QS are interconnected, with NO playing a very important role in controlling QS-dependent activities at molecular level such as biofilm formation, motility and virulence of different bacterial species.20,21

Therefore,

NO

can

be

used

as

an

effective

antimicrobial

therapeutic agent to regulate pathways in bacteria and control biofilm formation and pathogenicity. Given the potent biofilm inhibitory activity of NO, novel dual-action hybrid molecules have been synthesized by combining NO-releasing molecules such as N-diazeniumdiolate (NONOate) with potent QS inhibiting compounds. In conjunction with NO release, the hybrid compounds were more successful at eradicating biofilms of P. aeruginosa than the QS inhibitor alone.22,23 Therefore, co-administration of QS inhibitory agent and NO represents an attractive strategy for the control and prevention of bacterial infections.


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In this study, we have developed novel dual-action biomaterial surfaces by incorporating both DHP and NO donor onto fluorinated ethylene propylene (FEP) surface. Two DHP molecules with excellent antimicrobial properties in solution were selected and modified with an acrylate group (Fig. 1).11,24 The DHP-acrylates were covalently linked to the surface by Michael addition reaction followed by addition of NO releasing moieties on the surface. The resulting coatings were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements, their NO release profiles were determined by the Griess

assay,

and

bacterial

adhesion

analysis

was

performed

using

fluorescence microscopy against S. aureus and P. aeruginosa. This is the first study to develop dual-action surfaces by simultaneously grafting DHPs and NO donors onto a surface, and then investigating their effect on reducing bacterial adhesion. R

O

F

O

N H

O

N

N O

O

Active DHP R = H, ortho-F

DHP-1

DHP-2

Fig. 1. Functionalization of 4-aryl substituted DHPs with an acrylate group.

Materials and Methods Synthesis of acrylate-functionalized DHP derivatives The

acrylate-functionalized

DHPs

were

synthesized

previously developed by Kumar and Iskander.

11


according

to

method


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5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one (DHP-1) To a solution of 5-hydroxy-5-methyl-4-phenyl-dihydropyrrol-2-one (0.23 g, 1.22 mmol) in 10:1 dry dichloromethane/THF (11 ml) was added triethylamine (1.5 ml, 10.76 mmol) along with a few crystals of hydroquinone while stirring in an ice bath. A solution of acryloyl chloride in dry dichloromethane (3 ml) was added dropwise over a period of 10 min, and the mixture was stirred further for 3 h. The solvent was removed by evaporation under reduced pressure and the residue was purified by flash chromatography using dichloromethane as eluent to yield the diacrylate intermediate as a pale yellow solid (0.21 g, 75 %). A solution of the diacrylate intermediate (0.44 g, 1.48 mmol) in dichloromethane (9 ml) was treated with trifluoroacetic acid (1 ml) and stirred at room temperature for 2 h. The resultant mixture was neutralized with saturated sodium bicarbonate and water and then extracted into dichloromethane. The organic layer was dried over sodium sulphate and chromatographed on a silica column using dichloromethane as eluent to obtain the title product as a white solid (0.18 g, 51 %). M.p. 134-135 °C; 1H NMR (300 MHz, CDCl3): δ 5.42 (d,

J = 0.9 Hz, 1H, =CH2), 5.93 (dd, J = 1.8, 10.5 Hz, 1H, =CH2), 6.16 (d, J = 0.9 Hz, 1H, =CH2), 6.58 (dd, J = 17.1 and 1.8 Hz, 1H, =CH2), 6.70 (t, J = 1.2 Hz, 1H, -CH=), 7.45-7.53 (m, 6H, ArH and CH);

13C

NMR (75 MHz, CDCl3): δ

108.5 (CH2), 119.5 (CH), 128.7 (4 x ArCH), 130.1 (CH2), 130.4 (CH), 130.9 (CH), 141.8 (C), 155.5 (C), 163.4 (C), 165.8 (C=O), 168.6 (C=O); HRMS (ESI)

m/z calcd for C14H11NO2Na 248.0682 [M+Na]+, found 248.0684. 5-Methylene-1-(prop-2-enoyl)-4-(2-fluorophenyl)-dihydropyrrol-2-one (DHP-2) The title compound was synthesized following the same method used for the synthesis of DHP-1 to yield a pale yellow solid (0.23 g, 72 %). M.p. 121-122 °C;

1H

NMR (CDCl3): δ 5.25 (d, J = 0.6 Hz, 1H, =CH2), 5.93 (dd, J = 10.2

and 1.8 Hz, 1H, =CH2), 6.24 (d, J = 0.9 Hz, 1H, =CH2), 6.58 (dd, J = 16.5


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and 1.8 Hz, 1H, =CH2), 6.66 (t, J = 1.2 Hz, 1H, -CH=), 7.21-7.28 (m, 2H, ArH), 7.33-7.39 (m, 1H, ArH), 7.47-7.55 (m, 2H, ArH and CH);

13C

NMR (75

MHz, CDCl3): δ 108.2 (CH2), 116.3 (CH), 122.2 (CH), 124.3 (CH), 130.4 (CH), 130.8 (CH), 131.1 (CH2), 131.9 (CH), 141.7 (C), 149.2 (C), 158.1 (C), 161.4 (CF), 165.8 (C=O), 168.6 (C=O); HRMS (ESI) m/z calcd for C14H10FNO2Na 266.0593 [M+Na]+, found 266.0590. The spectral data of all compounds was consistent with that previously reported in the literature.11,25 Functionalization of FEP with carboxylic acid groups Before use, FEP sheets were cleaned with absolute ethanol then rapidly dried with a jet of nitrogen (FEP). The FEP samples were plasma-activated using acrylic acid in a custom-built plasma reactor according to a previously established procedure.26 Briefly, the reactor used was comprised of a cylindrical glass chamber (height = 350 mm, diameter = 170 mm). The reactor contained a horizontal disc electrode of diameter 150 mm on the bottom and a 6 mm rod electrode on the top, separated by 150 mm. The substrates were placed on the lower rectangular electrode of the plasma reactor. The acrylic acid monomer (Sigma-Aldrich, 98%) was degassed 5 times prior to deposition. The plasma deposition was carried out twice for 25 s with an initial pressure of 0.2 mbar (200 kHz, 20 W). The activated FEP samples with carboxylic acid groups (FEPAcid) were cut into roughly 6 × 6 mm pieces, stored in a sterile container and used within a week. The surfaces were washed with absolute ethanol prior to any surface modification. Attachment of spermine linker onto FEP surface The carboxylic acid functionalized FEP surfaces were immersed in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 30 mg/ml) in



Page 8 of 38

0.1 M sodium acetate buffer solution (pH 5.0) for 30 min with gentle shaking. The surfaces were then treated with a solution of spermine (3 mg/ml) in MilliQ water and left to react overnight at room temperature. The resultant sperminefunctionalized surfaces (FEP-Acid-Spermine) were washed with MilliQ water three times and air-dried before use. Attachment of DHP onto spermine-FEP The DHPs were immobilized on the amine-terminated FEP-Acid-Spermine according to a previously developed method.25 Briefly, a solution of DHP in absolute ethanol was prepared (6 mg/ml) and each spermine-functionalized surface was immersed in 500 µl of the DHP solution. The surfaces were left to react

overnight

with

agitation

at

room

temperature.

The

resultant

DHP-

immobilized surfaces were rinsed with absolute ethanol three times, air-dried and stored in sterile containers. Attachment of N-diazeniumdiolate (NONOate) FEP, FEP-Acid-Spermine and DHP-immobilized FEP surfaces were placed in a Parr apparatus and clamped. The apparatus was then purged and evacuated with nitrogen gas three times and pressurized with NO gas to 5 atm at 25 °C for 48 h (Fig. 2). The surfaces were purged with nitrogen gas and then left under vacuum for 24 h to remove unreacted NO. The NO-treated surfaces were stored at 4 °C until required for further analysis.


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F E P

COOH COOH

H 2N

N H

COOH

EDC

Plasma activated acid surface O N H

N H

2

NH2

DHP acrylate

O N H

Spermine surface

NO gas

NH2

2

N H

NH

DHP

2

DHP surface

5 atm 48 h

NO gas

O

5 atm 48 h

O N H

N 2

NH2

Spermine+NO surface

N H

N

N

DHP+NO surface

DHP

2

=

O O N N

Fig. 2. Schematic representation of the immobilization of spermine and NO and the preparation of dual-action hybrid surface of DHP and NO. The hydrogen of the secondary amine group forms an ammonium (positively charged) group with the unreacted spermine chain after reaction with NO gas (Not shown). X-ray photoelectron spectroscopy (XPS) The surfaces were characterized using X-ray photoelectron spectroscopy (XPS; ESCALAB220-iXL, VG Scientific, West Sussex, England). The X-ray source was monochromated Al Kα and the photoenergy was 1486.6 eV with a source power of 120 W. The vacuum pressure was ≤ 10-8 mbar. All energies are reported as binding energies in eV taken from three samples of each type. Contact angle measurements Contact angles were measured using a contact angle goniometer (Rame-Hart, Inc. NRL USA, Model no. 100-00). Multiple drops of deionized water were



Page 10 of 38

placed on each surface using a micro-syringe. The angle between the droplet and the surface was measured using a 50 mm Cosmicar Television Lens (Japan). Rame-Hart Imaging software was used to calculate the contact angle. A minimum of fifteen measurements were made on five samples of each type. Determination of nitric oxide release by Griess assay NO released from the surface was determined using a standard Griess reagent kit (Molecular Probes® Life Technologies™). The surfaces to be tested were placed in PBS (1 ml) for 24 h. Then, a 150 µl aliquot of the PBS solution was added to 20 µl of freshly prepared Griess reagent in a 96-well plate. The mixture was topped up with 130 µl of PBS to make up a total volume of 300 µl, and incubated at room temperature for 30 min with agitation at 500 rpm. After that, the absorbance of the wells was measured at 580 nm using a FLUOstar® Omega Microplate Reader. All measurements were performed in triplicate and the results are mean values of three independent experiments. Solutions of 0 to 100 µM sodium nitrite were used to prepare a standard curve of

nitrite

absorbance

versus

concentration

under

the

same

experimental

conditions. Extrapolation from the standard curve gave the concentration of nitrite (µM) generated from different surfaces. Bacterial adhesion analysis The bacterial strains used for this study, Staphylococcus aureus SA38 and

Pseudomonas aeruginosa PA01, were streaked onto Luria-Bertani (LB) agar and incubated overnight at 37 °C. A single colony of bacteria from the plate was cultured overnight at 37 °C in 15 ml tryptone soya broth (TSB; Oxoid, UK). The bacteria were washed twice with fresh TSB by centrifugation. The optical density (OD) of the culture was adjusted to OD660 = 0.1 which corresponds to 108 CFU/ml. In a 12-well plate, the surfaces to be tested were


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first sterilized with 70% w/v ethanol, then thoroughly washed with sterile phosphate buffered saline (PBS) three times and finally placed in 4 ml of the adjusted bacterial culture. The surfaces were incubated at 37 °C for 24 h. The media was then replaced by fresh TSB (4 ml) and further incubated for 24 h at 37 °C. Subsequently, the samples were washed twice with PBS before examination by fluorescence microscopy. Samples with adherent bacterial cells were stained with Live/Dead BacLight Bacterial Viability Kit (Molecular Probes, Inc, Eugene, OR, USA) according to the manufacturers’ procedure. Briefly, 2 µl of the two components were mixed thoroughly in 1 ml of PBS; 100 µl of the solution was then placed on each sample and allowed to incubate at room temperature in the dark for 15 min. The excess stain was gently washed by PBS (200 µl). Bacteria were fixed by adding 50 µl of 4 % formaldehyde to each sample for 5 min and rinsed with PBS. The samples were then placed on a glass microscopy slide for microscopic observation and image acquisition which were performed with an Olympus FV1200 Confocal Inverted Microscope. Bacterial cells that stained green were considered to be viable, while those that stained red or both green and red were considered to be dead. Images from 10 representative areas on each

of

triplicate

samples

for

each

surface

from

a

minimum

of

three

independent experiments were taken and analysed using ImageJ software. The results were reported as the average percentage coverage of live and dead cells of the fields of view. Further analysis of data was done by the one-way analysis of variance (ANOVA) using GraphPad Prism 7.03 software. Statistical differences were analyzed using post hoc Tukey correction. Results were considered significant at p < 0.05.



Results and discussion XPS characterization of coated surfaces The elemental composition of the surfaces before and after modification was assessed by XPS. The data for untreated FEP (FEP), FEP-Acid, FEP-AcidSpermine, DHP and NO coated surfaces are summarized in Table 1. The blank FEP surface was found to contain mainly 31.3 % carbon and 68.5 % fluorine, with a small amount of oxygen (0.2 %). Acrylic acid plasma deposition on FEP surface resulted in a polymer layer that was rich in carbon (73.1 %), oxygen (23.8 %) along with traces of nitrogen (0.7 %). The decrease in the fluorine content to 2.4 % after plasma deposition could either be due to defluorination of the polymer surface induced by secondary electrons created during XPS analysis27 or due to the presence of a thick coating of acrylic acid on the surface.28,29 The attachment of the aliphatic diamine linker spermine on the acid surface (FEP-Acid-Spermine) was confirmed by increase in carbon percentage to 75.0 % and nitrogen percentage to 2.1 %. The subsequent attachment of DHP-acrylates further increased the carbon and nitrogen compositions (77.7–78.8 % C and 3.1–3.2 % N). Both DHPs displayed similar attachment efficiency on the spermine surface, as indicated by the similar values of carbon, nitrogen and oxygen contents for DHP-1 and DHP-2. However, the fluorine content decreased from 1.6 to 1.2 % for DHP-1 whereas for DHP-2 it increased from 1.6 to 2.3 %, which could be attributed to the presence of the ortho-fluorine group in DHP-2. The incorporation of NOdonating moieties on the spermine and DHP-1/DHP-2 surfaces resulted in further increases in nitrogen and oxygen values.


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Table 1 XPS elemental composition of blank, acid, spermine, DHP and NOmodified FEP surfaces. Contact Surface

% C

% N

% O

% F

angle (°) (±1)

FEP

31.3

-

0.2

68.5

108

FEP-Acid

73.1

0.7

23.8

2.4

59

75.0

2.1

21.3

1.6

41

Spermine+NO

66.4

3.0

23.6

7.0

47

DHP-1

78.8

3.1

16.9

1.2

49

DHP-1+NO

72.7

5.5

19.4

2.4

54

DHP-2

77.7

3.2

16.8

2.3

52

DHP-2+NO

70.9

4.6

20.5

4.0

55

FEP-AcidSpermine



Page 14 of 38

The high-resolution curve-fitting results and proposed assignments for C 1s and N 1s are shown in Table 2. The FEP C 1s spectrum revealed the presence of C-C at 284.8 eV, CF2 at 290.5 eV, CF3 at 292.4 eV and O-CF2 at 294.5 eV. After acrylic acid plasma deposition, peaks for new species emerged at 286.3 eV, 287.7 eV and 289.1 eV. These peaks were attributed to C-O/C-N, C=O and O-C=O respectively, confirming the presence of carboxylic acid groups on the FEP-Acid surface. The next step involved spermine conjugation, which resulted in the appearance of a peak at 288.7 eV corresponding to the newly formed amide bond (N-C=O). The percentage of the surface acid groups (OC=O at 289.1 eV) also decreased from 2.2 to 1.2 % as expected after spermine attachment. These results indicated successful reaction of spermine with the carboxylic acid groups on the FEP substrate to form an aminefunctionalized surface (FEP-Acid-Spermine). The coupling reaction of DHP-1 and DHP-2 with the spermine linker showed further increase in the peak intensity for C-O/C-N, C=O and N-C=O signals. Additionally, complete attenuation for the acid peak (O-C=O) was observed, suggesting a thick coating of DHPs had formed on the surface. The addition of NONOate groups slightly changed the ratio of the carbon components for DHP+NO and spermine+NO. The narrow N 1s scan of spermine and DHP samples showed the presence of two common nitrogen species assigned to N-H (399.7 eV) and tertiary N/NH3+ (401.8 eV) (Table 2). The N 1s binding energy scans of DHP-1 before and after attachment of NO are shown in Fig. 3. Upon coupling of the N-H groups with NO to form NONOate, the intensity of the N-H peak was reduced and a new peak arose at 405.8 eV with a small shoulder at 407.6 eV. By reference to

published

respectively

values, from

the

these

values

NONOate

were donors

assigned attached


to on

NNO the

and

NNNO

surface.30,31

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Furthermore, there was also an increase in the intensity for the peak at 401.8 eV which was assigned to nitrolysis species, such as R-(NO)2 or NO dimers.32,33 The N 1s survey scan therefore indicated successful incorporation of NO on the DHP-1 coated surface. Similar changes in intensity of N 1s peaks were observed upon NO attachment for spermine+NO and DHP-2+NO modified surfaces.


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

Page 16 of 38

C 1s

Surface

N 1s

284.8

286.3

287.7

288.7

289.1

292.2

399.7

eV

eV

eV

eV

eV

eV

eV

C-C

C-O/C-

C=O

N-C=O

O-C=O

CF3

N-H

N

401.8 eV Tertiary N/ NH3+/R(NO)2

405.8 eV NNO

407.6 eV NNNO

-

-

-

-

-

91.1

8.9

-

-

2.7

6.8

47.6

17.5

28.5

6.3

3.9

-

-

88.7

11.3

-

-

9.1

5.6

-

-

58.5

14.5

23.0

3.9

17.8

8.1

4.0

-

-

75.8

24.2

-

-

19.1

8.3

5.5

-

-

54.7

14.9

25.9

4.5

FEP-Acid

76.0

15.1

6.7

-

2.2

-

FEP-Acid-

74.8

16.6

6.5

0.9

1.2

Spermine+NO

67.7

16.3

6.6

-

DHP-1

71.7

17.0

7.4

DHP-1+NO

66.2

19.1

DHP-2

70.1

DHP-2+NO

67.1

Spermine


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Table 2 XPS binding energies for C 1s and N1s and proposed assignments with percentage peak intensities.



(A) DHP-1 surface (before NO incorporation)

(B) DHP-1+NO surface (after NO incorporation)


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Fig. 3. XPS N 1s narrow scan spectra of DHP-1 surfaces (A) before treatment with NO and (B) after treatment with NO. Contact angle measurements Water contact angle measurements were performed to determine the changes in hydrophobicity of the surface after each modification step. The hydrophobicity of materials is a useful parameter that is correlated with cell-biomaterial interfacial interactions.34,35 The blank surface was found to be highly hydrophobic with high contact angle value

of

108°

hydrophobicity

which of

the

is

in

surface

accordance decreased

with

reported

markedly

to

values.36,37

The

59°

acid

after

functionalization of the blank FEP surface (Table 1). This finding is consistent with previous reports where functionalization of untreated FEP resulted in a reduction in hydrophobicity.37,38 A further reduction in hydrophobicity was observed after the reaction with spermine (41°). The DHP-modified surfaces showed slight increase in contact angle value (49° for DHP-1 and 52° for DHP2) due to addition of hydrophobic components (phenyl ring and fluorine atom) from the DHPs. The subsequent reaction with NO for spermine and both the DHP surfaces resulted in further increase in contact angle of 3 to 6°. Nitric oxide release from coated surfaces The concentration of NO released from DHP and spermine surfaces was determined by the Griess assay, which is the most frequently used colorimetric technique to assess NO release.39 The blank FEP surface was also treated with NO and used as a control to confirm the attachment of NONOate on spermine and DHP substrates. The samples were placed in PBS for 24 h, and the NO released was converted to nitrite in oxygen-containing aqueous media



and quantified using the Griess reagent. The amount of NO released from the surfaces was measured over 24 h and the results are shown in Table 3. The efficacy of NO-releasing surfaces is greatly dependent on the concentration of NO produced under physiological conditions. Studies have shown that polyamine-conjugated NONOates undergo a first order decomposition reaction in solution, producing approximately 2 equiv. of NO per mole of NONOate.40 In this study, it was found that the NO coatings showed release of NO after 24 h in the presence of a buffered solution. A previous study has shown that the NO release for the first 24 h is sufficient to significantly reduce bacterial infections and enhance the longevity of the medical device.41 After 24 h incubation in PBS, spermine+NO, DHP-1+NO and DHP-2+NO exhibited a much higher NO release of 19.7 ± 2.5, 20.8 ± 1.9 and 22.9 ± 1.1 µM, which is equivalent to total NO release of 547.2, 577.7 and 636.1 nmol/mm2 of the surface respectively, compared to the blank surface (1.9 ± 1.5 µM; 52.7 nmol/mm2). This could be attributed to the fact that the blank does not possess any amine groups that could react with NO to form the NONOate functionality. Additionally, the similar amount of NO release exhibited by the spermine, DHP-1 and DHP-2 surfaces suggested that the attachment efficiency of NO gas to form NONOate releasing moieties is comparable for the three surfaces, and is consistent with the XPS data. The Griess assay results therefore indicated the successful formation of NONOate donors on spermine and DHP-modified substrates.

Table 3: Assessment of NO release from the surfaces after 24 h incubation in PBS as determined using the Griess assay.


Page 20 of 38

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Surface FEP+NO FEP-AcidSpermine+NO

NO release (µM) 1.9 ± 1.5 19.7 ± 2.5

DHP-1+NO

20.8 ± 1.9

DHP-2+NO

22.9 ± 1.1

Antibacterial activity In the current investigation, the combined effect of NO and DHPs on bacterial surface colonization was systematically examined against both Gram-positive S.

aureus and Gram-negative P. aeruginosa. In order to determine the adhesion and viability of bacteria on the surfaces, samples were incubated at 37 °C for 48 h in an adjusted bacterial culture (108 CFU/ml), followed by staining of the surfaces with BacLight Live/Dead reagent, which stains live bacteria green and dead bacteria red. Representative micrographs of adherent S. aureus and P.

aeruginosa on controls (acid and spermine-treated), and selected DHP and DHP+NO samples are shown in Fig. 4. The relative proportion of live (greenstained) and dead bacteria with damaged membranes (red-stained) on the modified surfaces was evaluated by image analysis and the results for S.

aureus and P. aeruginosa are shown in Fig. 5. Extensive bacterial colonization was observed on acid and spermine control surfaces by both S. aureus and P. aeruginosa, as indicated by the high coverage of the green-stained (live) bacteria (Fig. 4). In contrast to the control samples, the surfaces modified by DHP-1 showed considerably less bacterial



coverage. Further reduction in adherent bacteria was observed on surface coated with DHP-1+NO compared to the DHP-1 surface.


Page 22 of 38

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S. aureus

P. aeruginosa

(A) FEP-Acid

(E) FEP-Acid

(B) FEP-Acid-Spermine

(F) FEP-Acid-Spermine

(C) DHP-1

(G) DHP-1

(D) DHP-1+NO


(H) DHP-1+NO


Fig. 4. Confocal microscopy images of S. aureus and P. aeruginosa adhered to FEP-Acid (A and E), FEP-Acid-Spermine (B and F), DHP-1 (C and G) and DHP-1+NO (D and H) modified surfaces. Live bacterial cells were stained green and bacteria with damaged membranes were stained red. Magnification 200×. Scale bar = 100 µm. To compare the amount of live and dead bacteria on the samples, quantitative image analysis was performed, which confirmed high coverage of S. aureus on the FEP (10.5 ± 0.5 %), FEP-Acid control (14.1 ± 1.1 %), and FEP-AcidSpermine control (15.2 ± 1.4 %) surfaces (Fig. 5A). In contrast, the DHP and DHP+NO coatings displayed over 3-fold lower bacterial coverage compared to the control surfaces. The DHP-1 and DHP-2 surfaces displayed reductions in bacterial adhesion by 70.9 ± 1.1 % and 60.3 ± 1.3 % respectively compared to the spermine control (p < 0.001). In the case of P. aeruginosa, the FEP (13.7 ± 1.5 %), FEP-Acid (13.1 ± 1.5 %) and FEP-Acid-Spermine (14.8 ± 1.1 %) surfaces exhibited high density of bacterial colonization (Fig. 5B). In contrast, the DHP-1 and DHP-2 coated surfaces exhibited significant reductions in adherent bacteria by 61.9 ± 0.4 % and 56.7± 0.3 % respectively when compared to the spermine control (p < 0.001). Amongst the two DHP compounds, the unsubstituted DHP compound (DHP-1) was more active against bacterial adhesion compared to the fluorine-substituted DHP (DHP-2). This correlates with previous findings where DHP-1 showed the best activity on glass


Page 24 of 38

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substrates, with reductions of up to 79.3 % and 65.8 % against S. aureus and

P. aeruginosa respectively, as well as on polymer beads with 5-log unit reduction of S. aureus bacterial count.13,25 As expected, the addition of NO donors on the DHP-modified samples resulted in further improvements in activity compared to the DHP coatings alone, with more pronounced reductions of S. aureus adhesion by 83.1 ± 0.5 % for DHP1+NO and 71.4 ± 0.5 % for DHP-2+NO compared to FEP-Acid-Spermine (p < 0.001). Importantly, both DHP-1+NO (p < 0.05) and DHP-2+NO (p < 0.01) were more active than their corresponding DHP parent coatings (42.8 ± 0.7 % and 28.0 ± 0.4 % greater reduction in coverage respectively), indicating the addition of NO provides additional inhibitory activity to the DHP surfaces. For P.

aeruginosa,

further

reduction

in

percentage

bacterial

coverage

was

also

observed for both the NO-treated DHP coatings when compared to FEP-AcidSpermine (77.1 ± 0.3 % for DHP-1+NO and 64.6 ± 0.3 % for DHP-2+NO). However, while DHP-1+NO showed significantly higher reduction than the DHP1 surface (40.0 ± 0.6 %; p < 0.05), the reduction in coverage for DHP-2+NO (18.2 ± 0.7 %) was found not significant compared to DHP-2 alone. Overall, the DHP-1+NO coating displayed the best broad-spectrum antibacterial activity amongst all the modified samples.



A

Bacterial coverage - S. aureus 18

Bacterial coverage (%)

16 14 12 10 8

#

6

*

4

*

×

*

^ *

2

Dead bacteria Live bacteria

0

B

Bacterial coverage - P. aeruginosa 18 16

Bacterial coverage (%)

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

Page 26 of 38

14 12

××

10 8 6 4

#

*

^ *

2 0


*

*

Dead Bacteria Live bacteria

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Fig. 5. Percentage bacterial coverage of live and dead bacteria for (A) S.

aureus and (B) P. aeruginosa (mean ± standard error of mean); *indicates p < 0.001 and

××indicates

p < 0.1 compared to spermine control; ^indicates p

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