Synthesis and characterization of a fluorinated S-nitrosothiol as the nitric oxide donor for fluoropolymer-based biomedical device applications

Article abstract of DOI:10.1039/C8TB01814F

Fluorinated polymers are widely used as biomaterials in various biomedical implant and device applications. However, thrombogenicity, surface-induced inflammation, and the risk of microbial infection remain key issues that can limit their use. In this work, we describe the first nitric oxide (NO) releasing fluorinated polymer, in which a new fluorinated NO donor, S-nitroso-N-pentafluoropropionyl-penicillamine (C₂F₅-SNAP), is incorporated within a polyvinylidene fluoride (PVDF) tubing. The synthesis, decomposition kinetics, and NO-release characteristics of the C₂F₅-SNAP species are described in detail. Then, using a simple solvent swelling method, we demonstrate that C₂F₅-SNAP can readily be doped into PVDF tubing. The resulting tubing can release NO for 11 days under physiological conditions, with an NO flux >0.5 × 10^?10 mol cm^?2 min^?1 over the first 7 days. Due to fluorous-fluorous interactions, the leaching of the fluorinated NO donor and its decomposed products is shown to be very low (less than 5 nmol mg^?1, total). Furthermore, the new NO-releasing PVDF tubing exhibits significant antimicrobial activity (compared to undoped PVDF tubing) against both Gram positive and negative S. aureus and P. aeruginosa bacterial strains over a 7 d test period. This new NO-releasing fluorinated polymer is likely to have the potential to improve the biocompatibility and antimicrobial activity of various biomedical devices.

Full text of DOI:10.1039/C8TB01814F

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Zhou , Q.
Zhang, J. Wu, C. Xi and M. E. Meyerhoff, J. Mater. Chem. B, 2018, DOI: 10.1039/C8TB01814F.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Please do not adjust margins
Journal of Materials Chemistry B
Page 1 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
Synthesis and Characterization of a Fluorinated S-Nitrosothiol as
the Nitric Oxide Donor for Fluoropolymer-Based Biomedical
Device Applications
Received 00th January 20xx,
Accepted 00th January 20xx
Yang Zhou,a Qi Zhang,a Jianfeng Wu,b Chuanwu Xib and Mark E. Meyerhoff*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Fluorinated polymers are widely used as biomaterials in various biomedical implant and device applications. However,
thrombogenicity, surface-induced inflammation, and risk of microbial infection remain key issues that can limit their use.
In this work, we describe the first nitric oxide (NO) releasing fluorinated polymer, in which a new fluorinated NO donor, S-
nitroso-N-pentafluoropropionylpenicillamine (C2F5-SNAP), is incorporated within the polyvinylidene fluoride (PVDF) tubing.
The synthesis, decomposition kinetics, and NO-release characteristics of the C2F5-SNAP species are described in detail.
Then, using a simple solvent swelling method, we demonstrate that C2F5-SNAP can readily be doped into PVDF tubing. The
resulting tubing can release NO for 11 days under physiological conditions, with an NO flux > 0.5 × 10-10 mol/cm2·min over
the first 7 days. Due to fluorous-fluorous interactions, the leaching of the fluorinated NO donor and its decomposed
products is shown to be very low (less than 5 nmol/mg, total). Further, the new NO-releasing PVDF tubing exhibits
significant antimicrobial activity (compared to undoped PVDF tubing) against both gram positive and negative S. aureus
and P. aeruginosa bacterial strains over a 7 d test period. This new NO-releasing fluorinated polymer is likely to have the
potential to improve the biocompatibility and antimicrobial activity of various biomedical devices.
polymers are commonly employed in suture materials and surgical
meshes.1 In addition, a fluorosiloxane hydrogel (Lotrafilcon A) is
commercially available for use in the contact lens market.7
Introduction
Fluorinated polymers have attracted significant attention for
It is known that fluorinated polymers have much better
biocompatibility compared to corresponding non-fluorinated
polymers.5 In particular, fluorinated polymers have been reported
to offer both lower thrombogenicity and decreased inflammatory
response.5 Multiple studies provide direct evidence that fluorinated
polymers can reduce platelet adhesion and activation.5 For
instance, copolymers of PVDF and hexafluoropropylene (HFP)
exhibited low platelet reactivity and better blood compatibility than
more conventional non-fluorinated polymers.8 The biocompatibility
of non-fluorinated polymers can also be greatly increased through
the blending or surface fluorination with fluorinated polymers.9-20
Additionally, the biomedical application of both fluorinated
polymers and fluorinated surface-modifying polymers can decrease
inflammatory response, resulting from their highly hydrophobic
surface property.21 So far, many studies have reported this
antimicrobial activity through surface coating and/or doping of
fluorinated polymers. 22-25
use
in various biomedical implants/devices since
polytetrafluoroethylene (PTFE) was first discovered in 1983.1-5 This
class of polymers can be divided into two general categories based
on their corresponding components; perfluorinated and partially
fluorinated polymers.6 With fluorinated groups in the polymer
backbone, fluorinated polymers have distinct characteristics,
including high thermal stability, excellent chemical resistance, low
coefficient of friction, high tensile strength, enhanced
biocompatibility and interesting electrical properties.6 The use of
fluorinated polymers in blood contacting biomedical applications
have a long history because of these distinct properties.2 Currently,
the perfluorinated polymer expanded PTFE, and the partially
fluorinated polymer polyvinylidene fluoride (PVDF), are the two
commercial fluorinated polymers most widely used to prepare
biomedical implants and devices. For example, a variety of medical
materials/devices, such as vascular grafts, cardiovascular patches,
sutures and stents, are made of expanded PTFE material.2 PVDF
Although fluorinated polymers offer great potential for both
suppression of blood clots and reduction of bacterial adhesion and
proliferation,
surface-induced
thrombus
formation
and
a.
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. E-
mail: mmeyerho@umich.edu
Department of Environmental Health Sciences, University of Michigan School of
Public Health, Ann Arbor, MI 48109, USA
Electronic Supplementary Information (ESI) available: [details of any
inflammatory responses can still be triggered by such materials, and
further research is needed to address those issues.5 For example, in
two separate studies, visible adhered blood clotting was observed
b.
†
on a PTFE surface over a relatively short time.20, Fluorinated
26
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 2 of 12
View Article Online
DOI: 10.1039/C8TB01814F
ARTICLE
Journal Name
polyethylene was also found to stimulate thrombus formation.27 donors.51-61 In addition, the Handa group also recently proposed the
PVDF-HFP copolymers were also reported to elicit acute thrombotic potential biomaterial applications of NO releasing polymers
responses.28 In the practical application of PTFE catheters as insulin including silicone rubber,62, 63 CarboSil,64-67 and Elast-eon E2As.68, 69
infusion cannulas, the lifetime of these PTFE cannulas is about ~ 3 d Reynolds and coworkers reported NO-releasing Tygon and
due to the inflammatory response and also increased risk of polyphosphazene-based coating materials for biomedical
microbial colonization at the infusion site.29-31 To solve the applications.70-72 The Schoenfisch group investigated polyurethane
74
thrombus formation or inflammatory response from fluorinated fibers and silicone elastomers NO-releasing silica materials.73,
polymers, only a limited number of approaches were proposed Frost et al. also proposed poly(L-lactic acid) film within a cyclam-
using surface modifications with bioactive molecules. For instance, based SNAP, releasing physiological levels of NO for up to 3
expanded PTFE with surface coating of heparin provided a more months.75 Moreover, diazeniumdiolate coatings tethered on
thromboresistant blood contacting surface, which can greatly poly(ethylene terephthalate) and silicone elastomers showed
improve the vascular graft performance.32 A gendine-coated PTFE antibacterial efficacy toward Pseudomonas aeruginosa (P.
tubing was proposed as an insulin infusion cannula, to show aeruginosa) bacteria.76 Diazeniumdiolate-based acrylonitrile-co-1-
enhanced antimicrobial efficiency towards several pathogens.29 The vinylimidazole copolymers were reported to be used in sutures.77
A
surface modifications of PVDF film and membrane using a peptide- diazeniumdiolate-based poly(diol citrate) elastomer was also
click-poly(glycidylmethacrylate)
polymer
and
a
proposed by Ameer group as a coating film for expanded PTFE
polymethacrylamide-ammoniumbetaine copolymer also was found vascular grafts,78 in which NO release can only last for 2 days. These
to exhibit greater biocompatibility than the non-treated PVDF reports demonstrate that NO-releasing non-fluoropolymers have
34
polymer.33,
However, these surface modification methods are been extensively studied for potential applications in biomaterials.
relatively complex, requiring multiple handling steps. In addition, However, fluoropolymers with NO-release properties have not yet
surface-grafted molecules carry the risk of potential toxicity. been reported. Therefore, it would be of great interest to explore
Nitric oxide (NO), an endogenously gaseous signaling molecule the application of NO-releasing fluoropolymers for preparation of
produced by oxidation of L-arginine by a family of NO synthase medical devices.
enzymes,35 has been well studied and understood for its broad and
In fact, based on the basic solubility principle of like dissolves
specific biofunctionalities,35-37 including blood vasodilation, anti- like, traditional hydrocarbon type NO donors are difficult to be
platelet activity, inhibition of bacterial growth, immune modulation, incorporated into fluorinated polymers because of the higher
anticancer activity, and biological process regulation. The NO flux polarity of hydrocarbons in comparison to fluorocarbons.
released by an endothelial layer of the inner wall of blood vessels is Therefore, we hypothesize that it should be possible to impregnate
estimated to be between 0.5 and 4.0 × 10-10 mol·cm-2·min-1.38 a synthetically fluorinated NO donor into fluoropolymers using the
However, molecular NO is highly reactive under physiological simple solvent-swelling method. In addition, fluorous-fluorous
conditions, and has a reported intravascular half-life of ~ 2 s.39 In interactions are widely present in the fluorinated materials, which
chemical and biochemical studies, various precursor molecules of have already been used in new affinity chromatography and
NO (NO donors) have been utilized for NO release in situ,40 such as enzyme immobilization technologies.79-82 Due to the specific
S-nitrosothiols,
N-diazeniumdiolates, metal
nitrosyls, N- fluorous-fluorous interaction between pentafluoropropionyl group
nitrosamines, organic nitrates and nitrites. Similar NO donors have (C2F5) and the fluoro-backbone in PVDF, we also expect a decrease
also been widely used as promising therapeutic agents.41-44 In in leaching of chemicals from any fluorinated NO donor possessing
particular, due to the potent antimicrobial and antithrombotic the C2F5 group when doped into fluoropolymers. Therefore, in this
properties of NO, such NO-releasing platforms have served as a work, we report the first NO-releasing fluorinated polymer via the
promising approach to improve the biocompatibility of medical incorporation of a novel fluorinated SNAP derivative (S-nitroso-N-
devices.38, 45-49 Such NO donors can be chemically synthesized as pentafluoropropionylpenicillamine, C2F5-SNAP, see Fig. 1) into a
part of the polymers (covalently attached) or doped into medical- PVDF polymer. The method used to synthesize C2F5-SNAP is
grade polymers. NO is then released from the surface of these described, and its stability is investigated by measuring its
parent polymers when the polymer is in contact with water/blood decomposition kinetics. In addition, the decomposition products
or other physiological fluid, to mimic the NO generated from derived from C2F5-SNAP are characterized. The synthetic C2F5-SNAP
physiological endothelial layers.
NO donor is then successfully impregnated into PVDF tubing via a
Due to the potential antimicrobial and antithrombotic solvent swelling approach using tetrahydrofuran (THF) as the
functionalities of NO, the development of novel NO-releasing swelling solvent. The NO-release profiles are evaluated from C2F5-
polymers has received considerable attention over the last SNAP-doped PVDF tubing under physiological conditions. Finally,
decade.38, 46, 50 Recent work from our laboratory has demonstrated the new NO-releasing PVDF tubing is also investigated for its
the increased biocompatibility and antimicrobial activity of many antimicrobial activity during 7 d of incubation with two bacterial
medical-grade polymers including poly(lactic-co-glycolic acid), Elast- strains, S. aureus and P. aeruginosa.
eon E2As, CarboSil polymer, silicone rubber, and polyurethane,51-61
via the incorporation of a nitrosothiol (e.g. S-nitroso-N-acetyl-
penicillamine, SNAP, in Figure 1) or N-diazeniumdiolates as the NO
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 3 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
F
fluorinated functional group. Target C2F5-SNAP was obtained as a
greenish oil, which had the characteristic color of SNAP.
F
F
O
CH3
O
F
F
O
O
NH
S
NH
S
HO
HO
H3C
H3C
O
O
N
N
CH3
CH3
C2F5-SNAP
SNAP
Fig. 1 The chemical structures of SNAP and C2F5-SNAP NO donors.
Results and discussion
Synthesis and stability test of C2F5-SNAP
F
F
F
F
F
O
F
F
O
F
F
O
O
NH2
SH
O
O
F
HCl/H2SO4
NaNO2
C2F5
OCH2CH3
NH
S
NH
SH
HO
HO
HO
H3C
Et3N, MeOH
H3C
H3
C
CH3
O
N
CH3
CH3
(a)
D-Penicillamine
C2F5-SNAP
C2F5-NAP
Scheme 1. Synthesis of C2F5-SNAP NO donor.
In our previous studies regarding the synthesis of non-
fluorinated SNAP derivatives as NO donors, we found that the
modification of the free carboxylic acid group of SNAP with amine
functional groups (to form amides) decreased both the thermal and
the photochemical stability of the desired NO donor.83 In addition,
tertiary S-nitrosothiols are known to be relatively thermally stable,
but very photochemically unstable.83 Furthermore, either the
substitution or the incorporation of fluorine functional groups into
the gem-dimethyl groups in the SNAP structure would likely cause
further instability of the resulting NO donors due to the strong
electron-withdrawing effect from these fluorine functional groups.
Therefore, our goal was to retain the free carboxylic acid group, the
tertiary structure of the nitrosothiol, and the gem-dimethyl groups
in our design of any fluorinated derivative of SNAP. The acetyl
group was finally selected as the site of interest for incorporation of
the fluorine atoms. The novel fluorinated SNAP derivative (C2F5-
SNAP) was therefore selected as the target species (Figure 1). The
synthesis of C2F5-SNAP was completed via two sequential reaction
(b)
1
Fig. 2 (a) H NMR spectra for the thermal stability of C2F5-SNAP oil
(CH: ~ 5.39 ppm; CH3: 2.12 and 2.01 ppm) at - 20 oC under argon
and dark conditions after (1) day 0, (2) day 4, (3) day 11, (4) day 18,
(5) day 25, and (6) day 39; (b) C2F5-SNAP remained percentage vs.
storage time at - 20 oC under argon and dark conditions.
S
-Nitrosothiols have been reported to be quite unstable under
both photochemical and thermal conditions.86, 87 With the presence
of a perfluoroalkyl chain (CF3CF2) in C2F5-SNAP, it is very important
to evaluate its stability due to the fluorinated functional group
steps from commercial
gram-scale synthesis proceeded without incident with ~ 84% overall
yield. In the first step, the nucleophile, -penicillamine, is readily
D-penicillamine in Scheme 1. This two-step
D
having
a strong electron withdrawing ability. Under vacuum
conditions at room temperature (23 oC) for 18 h, C2F5-SNAP
decomposed ~ 5%. The stability of C2F5-SNAP stored in a freezer at -
20 oC was examined by 1H NMR spectroscopy. The fluorinated SNAP
derivative was found to be slightly decomposed at -20 oC under
argon and dark conditions (see Fig. 2a) based on 1H NMR
spectroscopy data. After being stored in the freezer for 39 d, ~ 83%
of C2F5-SNAP remained (Fig. 2b). In addition, C2F5-SNAP
attacked at the carbonyl group at the corresponding fluorinated
ester,84 which is followed by a subsequent elimination of ethanol.
The presence of triethylamine plays a critical role in neutralizing the
free carboxylic acid in
D-penicillamine. In the absence of
triethylamine, the reaction does not occur. The presence of free
carboxylic acid might potentially prevent the elimination step
following the completion of the nucleophilic addition reaction. The
resulting free thiol, C2F5-NAP, was used directly in the next step. As
expected, the nitrosylation of C2F5-NAP can be easily completed
using the classical SNAP synthetic method with nitrous acid.85
Interestingly, the desired product was able to be extracted by
dichloromethane during the workup, indicating that the polarity of
C2F5-SNAP is greatly decreased due to the presence of the
o
decomposed ~ 15% in a refrigerator set at 1 C over 7 d under dark
conditions (data not shown). These results suggest that C2F5-SNAP is
thermally unstable in its oil phase due to the presence of the CF3CF2
o
group. In contrast, SNAP is very stable at -20 C in the freezer, with
no decomposition observed over several years. The results above
suggest that the presence of the C2F5 group causes a decrease in
the observed thermal stability of the new C2F5-SNAP species.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 4 of 12
View Article Online
DOI: 10.1039/C8TB01814F
ARTICLE
Journal Name
Decomposition kinetics of C2F5-SNAP
decomposition studies of SNAP in a mixture of PBS and DMSO
To further evaluate the stability of C2F5-SNAP as an NO donor, (50:50, v/v). The corresponding kinetic data are summarized in Fig.
kinetic studies of its photochemical and thermal decomposition 1S and Fig. 2S (see Supporting Information). Photolysis of SNAP
were carried out using UV/Vis spectroscopy. Steady-state photolysis (250.0 µM) was observed to be a first-order reaction with rate
o
of C2F5-SNAP (~ 206.0 µM) at 23 C in a mixture of PBS and DMSO constant kobs = (1.50 ± 0.03) × 10-2 s-1 (t1/2 ~ 46 s). Therefore, C2F5-
(50:50, v/v) was carried out using as described in the experimental SNAP and SNAP exhibit similar lifetimes upon irradiation. Under the
section (see above). UV/Vis spectra of the photodecomposition of same thermal conditions at 37 oC, SNAP (206.0 µM) decomposes via
C2F5-SNAP are shown in Figure 3. The absorbance at 341 nm is the a zero-order reaction (kobs = (3.45 ± 0.02) × 10-5 µM Min-1).
characteristic UV-Vis band of the nO
ꢀ
π* transition, which was However, the observed rate constant (kobs = (3.45 ± 0.02) × 10-5 µM
observed to continuously decrease and finally flatten. The Min-1) of SNAP is lower than that of C2F5-SNAP (kobs = (4.39 ± 0.04) ×
corresponding plot of absorbance at 341 nm vs. time is shown in 10-5 µM min-1), indicating that C2F5-SNAP decomposes more rapidly
the Fig. 3 insert. The data were fitted to a first-order rate equation than SNAP. This result also agrees with the conclusion obtained in
(Fig. 3 insert, line), giving an observed rate constant kobs = (1.60 ± the stability studies reported above. That is, the thermal stability of
0.10) × 10-2 s-1 and a half-life of ~ 43 s.
C2F5-SNAP is decreased compared to SNAP due to the presence of
the fluorinated group.
Decomposition product analysis
The NO-generation mechanism from
S-nitrosothiols is well
characterized under thermal, light irradiation, and Cu+ mediated
reduction.86, 88 NO generation from these species is associated with
the homolysis of the S-N bond,88 to generate NO and thiyl radicals.
However, thiyl radicals are not generally observed as intermediates
during the reaction, as these radicals rapidly dimerize into a
disulfide final product.88 For the new C2F5-SNAP species, it is of
significance to quantify both the rate of NO release and the
corresponding decomposition products. To determine the
stoichiometry of NO release, a quantitative photolysis of C2F5-SNAP
Fig. 3 UV/Vis spectra for the photolysis of C2F5-SNAP (~ 206.0 µM)
at 23 oC in a mixture of PBS and DMSO (50:50, v/v). Inset: Plot of
absorbance at 341 nm vs. time for this reaction and solid line is fit
to first order rate equation line.
was conducted to quantitate the yield of NO gas generation.
A
solution of C2F5-SNAP (1.23 × 10-7 mol) in a mixture of PBS and
DMSO (50:50, v/v) was irradiated by 350 nm lamps in a NOA
reaction cell. The corresponding NO generation was analyzed by the
NOA instrumentation (see Fig. 5). The total NO release from three
independent photolysis experiments was determined to be 93% ±
2%.
Fig. 4 Plot of absorbance at 341 nm vs. time for the thermal
o
decomposition of C2F5-SNAP (~ 206.0 µM) at 37 C in a mixture of
PBS and DMSO (50:50, v/v).
o
The thermal decomposition of C2F5-SNAP (~ 206.0 µM) at 37 C
Fig. 5 NO determinations via NOA during photolysis of C2F5-SNAP
(1.23 × 10-7 mol) in a mixture of PBS and DMSO (50:50, v/v) at room
temperature.
was also carried out in a mixture of PBS and DMSO (50:50, v/v). The
kinetic study of this decomposition was also monitored at 341 nm
(see Fig. 4). The absorbance data vs. time was fitted with a zero-
order rate equation giving an observed rate constant kobs of (4.39 ±
0.04) × 10-5 µM min-1. The half-life for this thermal decomposition of
C2F5-SNAP at 37 oC is ~ 8.08 h.
In addition to NO, the corresponding disulfide was expected to
form as a side product of the photolytic decomposition of C2F5-
SNAP. To confirm the formation of C2F5-NAP disulfide, a C2F5-SNAP
sample in CDCl3 with 1% of tetramethylsilane (TMS) was analyzed
by 1H NMR spectroscopy before and after decomposition (see Fig.
To further compare with the NO release profiles between C2F5-
SNAP and SNAP, we also carried out the photolytic and thermal
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 5 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
o
6). Before decomposition, methide and gem-dimethyl groups within 11 d when the sample tubing is stored in PBS at 37 C. The NO flux
pure C2F5-SNAP exhibited chemical shifts at ~ 5.39, 2.12 and 2.01 number can reach up to 10 × 10-10 mol/cm2·min on the first day, but
ppm. After overnight thermal decomposition in the dark at room then drops to ~ 5.0 x 10-10 mol/cm2·min on the second day. The NO
temperature, the product observed had the methide (~ 4.74 ppm) release decreased on each succeeding day. The NO flux number
and gem-dimethyl groups (~ 1.52 and 1.43 ppm) shifted, which observed was > 0.5 × 10-10 mol/cm2·min for up to 7 d. In addition, a
corresponds to the disulfide dimer of C2F5-NAP. Full decomposition second swelling of PVDF tubing (1 inch in length, 1/16” ID × 1/8”
of C2F5-SNAP was observed after 4 days. The identity of C2F5-NAP OD) with a lower concentration of C2F5-SNAP (400.5 mg/mL in THF)
disulfide was further confirmed by HRMS results (m/z (ESI): demonstrated a total NO-release period of 8 d (NO-release > 0.5 ×
calculated for [M+H]+ 589.0519; found 589.0522). The conversion 10-10 mol/cm2·min for 5 d, see Fig. 3S). Hence, by swelling the PVDF
percentage to the C2F5-NAP disulfide was calculated to be ~ 95%, tubing with a higher concentration of C2F5-SNAP, NO-release time
based on the presence of the internal standard TMS. This can be extended, as expected. In fact, it is not practical to quantify
decomposed product fully agrees with the product generated the total amount C2F5-SNAP loaded into the PVDF tubing using this
during the stability testing (see Fig. 2a). These data confirm that solvent impregnation method, since C2F5-SNAP is unstable in
C2F5-SNAP can be used as a clean NO donor, and that the disulfide organic phase solutions. According to the NO release determined
dimer of C2F5-NAP is the only decomposition byproduct as each day by chemiluminescences measurements, the total loading
summarized in Scheme 2.
of C2F5-SNAP in PVDF tubing was estimated to be ~ 42.3 nmol/mg.
The corresponding total loading of C2F5-SNAP in PVDF tubing in Fig.
3S was estimated to be ~ 32.3 nmol/mg. In addition, C2F5-SNAP
doped PVDF tubes still showed the characteristic green color after
being stored < -20 oC for three months, indicating that NO-releasing
tubing is stable under long-term storage in a freezer.
Fig.
6
1H NMR spectra for C2F5-SNAP before and after
decomposition in CDCl3 at room temperature (1) before
decomposition, (2) overnight thermal decomposition, (3) completed
decomposition, (C2F5-SNAP (CH: ~ 5.39 ppm; CH3 : ~ 2.12 and 2.01
ppm); C2F5-NAP disulfide (CH: ~ 4.74 ppm; CH3: ~ 1.52 and 1.43
ppm), CH2Cl2: ~ 5.30 ppm).
F
F
F
F
F
Fig. 7 NO flux for the PDVF tubing incorporated with C2F5-SNAP
under physiological conditions (PBS with 100 µM EDTA at 37 oC in
the dark).
F
F
F
O
F
F
O
OH
H3
C
CH3
NH
O
NH
S
O
S
S
O
O
thermal/hv
HO
HN
NO
+
1/2
H3
C
CH3
HO
H3
C
F
O
N
CH3
F
F
Nitric oxide is well-known to inhibit platelet activation and
prevent bacterial proliferation/adhesion. Therefore, NO-release
materials have been already well established/documented as a
promising platform for developing antithrombotic and antimicrobial
materials that can improve the biocompatibility of devices for
biomedical applications.38, 50 Once NO is released from biomaterials
via doping or grafting the appropriate NO donors (e.g., from
catheters), the improved biocompatibility of various polymeric
materials is always observed in vivo (in animal studies). This has
F
F
C2F5-SNAP
C2F5-NAP disulfide
Scheme 2. NO release from C2F5-SNAP NO donor.
NO release from C2F5-SNAP doped PVDF tubing
Three PDVF tubes (1 inch in length, 1/16” ID × 1/8” OD) were
swelled in a high concentration of C2F5-SNAP stock solution (~ 634.0
mg/mL in THF) for 24 h. After being dried under vacuum, these
tubes were washed with CH2Cl2 (10 times) to completely remove
the surface-attached chemicals. After drying under vacuum for
been reported in many previously published animal studies using
another 24 h, the resulting PVDF tubes impregnated with C2F5-SNAP various polymeric materials including Elast-eon E2As,52,
60, 68
67, 74
63
61
CarboSil,54,
silicone rubber,57,
and Tygon59,
and so on.
exhibited a dark green color characteristic of this NO donor. The
corresponding NO-release profile from these PDVF tubes was
recorded by an NOA instrument in PBS at 37 oC, and the results are previously in the literature for NO release polymers, we can
Based on the antithrombotic/antiplatelet properties reported
anticipate that our new NO-releasing PVDF tubing will demonstrate
summarized in Fig. 7. Interestingly, NO release can be observed for
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 6 of 12
View Article Online
DOI: 10.1039/C8TB01814F
ARTICLE
Journal Name
similar antithrombotic properties for at least 7 days, since the NO
flux released from the new C2F5-SNAP doped PVDF tubing over the
first days is > 0.5 x 10-10 mol/cm2min.
Cumulative leaching test
The leaching problem remains a challenge for the application of
NO donor-doped polymers since the NO donors employed are
typically not covalently bonded with the matrix polymers. Leaching
of NO donors from the polymer can greatly decrease the efficacy of
the NO-releasing flux number and longevity of NO release. Further,
the loss of NO donors to the bathing medium raises a concern
about toxicity, since an excess amount of NO donor could be rapidly
consumed by the local oxyhemoglobin (oxyHb) within blood via the
rapid reaction of NO with oxyHb. In addition, the leaching of
byproducts such as free thiols and disulfides derived from NO donor
decomposition can also create potentially toxic species. Therefore,
chemical leaching of the initial C2F5-SNAP species and potential
products (C2F5-NAP and its disulfide etc.) from the PDVF tubing was
examined. Throughout the measurement of the NO-release
process, the concentrations of C2F5-NAP, C2F5-NAP disulfide and
C2F5-SNAP were monitored by HPLC-MS daily. Calibration curves of
C2F5-NAP and C2F5-NAP disulfide with the HPLC-MS instrument were
established using standard solutions using the corresponding pure
chemicals (see Fig. 4S). The leaching of C2F5-NAP disulfide was
found to be ~ 0.6 nmol per mg of PVDF tubing on the first day (see
Fig. 8a). After 9 d of soaking, the total C2F5-NAP disulfide doubled (~
1.2 nmol/mg, 6% of loading of C2F5-SNAP in PVDF tubing). C2F5-NAP
was also detected in the soaking solution with an initial
concentration of 1.1 nmol per mg of PVDF tubing (see Fig. 8b). After
being stored in soaking solution for 9 d, the leaching of C2F5-NAP
increased to 1.39 nmol/mg (3% of the total loaded C2F5-SNAP in the
PVDF tubing). The detection of free thiols in the leaching
experiment from NO donor-doped polymer is quite common, and
(a)
(b)
Fig. 8 Cumulative leaching of C2F5-NAP disulfide (a) and C2F5-NAP (b)
into PBS soaking solution (2.0 mL) from the PDVF tubing
incorporated with C2F5-SNAP at 37 oC in the dark.
Antimicrobial and anti-biofilm studies
NO-releasing polymers are primarily applied as biomaterials for
46
medical devices.38,
The antimicrobial property derived from
surface released NO plays a significant role in the prevention of
surface-related infection. Biofilm formation on device surfaces can
greatly decrease antibiotic efficiency and opsonophagocytic activity,
further leading to chronic infections or inflammations.89 Therefore,
it is of interest to test the antimicrobial and anti-biofilm behavior of
the new C2F5-SNAP doped PDVF tubing. Since the doped amount
can be estimated according to the total NO gas released, using a
higher concentration (~ 634.0 mg/mL in THF) of C2F5-SNAP in the
swelling process will lead to a higher total loading of C2F5-SNAP in
the PVDF tubing (~ 42.3 nmol/mg), compared to ~ 32.3 nmol/mg
achieved when swelling in a lower concentration of C2F5-SNAP
(400.5 mg/mL in THF). Thus, a higher concentration of C2F5-SNAP
used in that process will also result in a longer NO release time and
possibly resulted from the reduction of disulfide or hydrolysis of S-
nitrosothiols.54 In addition, a small amount of C2F5-SNAP (~ 0.1
nmol/mg) was observed on the first day, which was < 1% of the
total loading of C2F5-SNAP in PVDF tubing. A trace amount of C2F5-
SNAP was detectable on the second day; however, the C2F5-SNAP
NO donor was not detected on third day of the HPLC-MS results. In
comparison with the chemicals leaching observed from SNAP doped
CarboSil, E5-325, and silicone rubber over the first 10 days,54 the
corresponding concentrations of chemical leaching from C2F5-SNAP
doped PVDF tubing is dramatically less. It is likely the fluorous-
fluorous interactions present in the fluorinated materials could be
responsible for these low leaching rates.79-82 More importantly, the
absence of C2F5-SNAP within the leaching test solution on the third
day and the very low leaching of C2F5-SNAP (< 1%) indicate that the
NO-release from C2F5-SNAP doped PVDF tubing mainly comes from
C2F5-SNAP species within the polymer phase, rather than any donor
species that leached out into the soaking solution. The total
leaching of chemicals (C2F5-SNAP, C2F5-NAP, and C2F5-NAP disulfide)
was < 10% of the initial amount of C2F5-SNAP originally present
after 11 d.
a
higher loading of C2F5-SNAP. In our previous studies, the
antimicrobial activity of NO toward P. aeruginosa was reported
even when the NO flux number from a polymer surface is < 0.5 x
10-10 mol/cm2min.90 In addition, with a higher NO flux rate,
enhanced antimicrobial properties are always observed.90
Therefore, we anticipate that the C2F5-SNAP-doped PVDF tubing (~
42.3 nmol/mg) will exhibit better antimicrobial properties than the
one with a total loading of ~ 32.3 nmol/mg, since the total
corresponding NO release amount is higher. Therefore, in the
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 7 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
following antimicrobial and antibiofilm studies, the C2F5-SNAP- significantly inhibit the growth of any bacteria on this surface. In
doped PVDF tubing with the higher impregnated amount of C2F5- addition, a significant difference is observed between Fig. 9e and 9f,
SNAP (~ 42.3 nmol/mg) were used.
illustrating the effect on inhibiting P. aeruginosa biofilm formation.
Tests were conducted with both gram positive and negative It should be noted that in comparison to our previous studies with
bacterial strains (S. aureus and P. aeruginosa, respectively). All other polymeric biomaterials,53-55 our control group of PVDF
antimicrobial and anti-biofilm studies were carried out in a CDC without NO-release also demonstrated bacterial inhibition
biofilm reactor as previously described.53, 54 Both control and NO- (compared to what was observed with non-fluorinated tubing)
releasing PVDF tubes (labeled as PVDF-NO) were suspended and which is consistent with the innate antimicrobial properties of
incubated in a flowing media with each bacterial strain for 7 d at 37 fluorinated polymers reported in literature.5 However, via the
oC. After 7 days of incubation, the greenish color was still observed addition of surface NO-release, the C2F5-SNAP doped PVDF tubing
in all NO-release tubes, indicating that a significant amount of C2F5- can more effectively reduce risk of bacterial infections.
SNAP was still present within the PVDF tubing. The results of
Interestingly, compared with the enhanced antimicrobial
adhered viable cells for S. aureus and P. aeruginosa are presented activity of non-fluorinated polymers that have been observed when
in Figures 9a and 9b. After one week, the NO-release tubing showed doping with SNAP or other NO release agents, for our tests against
significant inhibition for both bacterial strains. In the test against S. S. aureus and P. aeruginosa, somewhat lower antimicrobial effects
aureus, the NO-release tubing had > 1 order of magnitude less (in terms of total log decrease in bacterial counts) were observed in
bacterial counts than that of the control group. A greater effect for comparison with the control tubing segments. This is mostly due to
the prevention of P. aeruginosa was observed, with > two orders of the fact that the PVDF tubing itself has enhanced intrinsic
magnitude difference in comparison with the control tubing antimicrobial activity compared to nonfluorinated polymers.3 Since
segments.
the PVDF tubing used in this work already has less cell adhesion to
Our prior research has reported that a 5-log reduction of the start with, it is less meaningful to compare our antimicrobial results
total viable S. aureus cells was observed when the NO release was > with those of the other types of nonfluorinated polymers tested to
0.5 x 10-10 mol/cm2min flux during 7 days of release from SNAP date that have been doped with SNAP or other NO release agents.
impregnated polymers.54 In addition, > 2-log reduction of P.
aeruginosa was observed in only 3 h when the NO was 0.5 x 10-10
(a) S. aureus
mol/cm2min flux.90 These literature results indicate that NO release
demonstrates significant antimicrobial properties when the flux is ≥
0.5 x 10-10 mol/cm2min. The NO release data shown in Fig. 7
illustrates that the NO flux for the C2F5-SNAP doped PVDF tubing is
> 0.5 x 10-10 mol/cm2min during first 7 days of soaking. From our
cumulative leaching test studies, we found that leaching of the C2F5-
SNAP NO donor is very small, less than 1% of total originally present
in the polymer tubing. Further, the total leaching of C2F5-NAP
disulfide and C2F5-NAP species is only 1.2 nmol/mg (6%) and 1.39
nmol/mg (3%) after 9 d of soaking, respectively. Hence, due to the
very low leaching of all C2F5-SNAP species, the measured surface NO
release (~ 42.3 nmol/mg) represents > 99% of the C2F5-SNAP NO
donor originally doped within the PVDF tubing. Hence, it is very
likely that the antimicrobial effects must be coming from released
NO, not due to any antimicrobial effect of C2F5-NAP (without NO on
it) or the dimeric species.
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Control
PVDF-NO
(b) P. aeruginosa
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Furthermore, the antimicrobial findings were further confirmed
by representative fluorescence images of the biofilms. A clear
difference in the images are shown in Fig. 9c and 9d, indicating the
preventative role of NO release from tubing with regard to S.
aureus growth. In Fig. 9d, the surface observed is basically the
inherent texture of surface rather than any biofilm/bacteria itself.
To provide further evidence the less bacterial adhesion, the
corresponding image (to the one with normal exposure shown in
Fig. 9d) is provided (see Fig. 5S). In fact, there is only one small red
spot located above the image center in Fig. 5S, which is consistent
with that in Fig. 9d. Therefore, only very few dead bacteria are
adhered on the surface of the NO-releasing PVDF tubing. This result
also indicates that the NO-release from PVDF tubing can
Control
PVDF-NO
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 8 of 12
View Article Online
DOI: 10.1039/C8TB01814F
ARTICLE
Journal Name
aeruginosa bacterial strains after 7 d. These results confirmed the
potential for C2F5-SNAP doped PVDF tubing to further improve the
biocompatibility of fluorinated polymers. To achieve much longer-
term NO release purposes, currently, the synthesis of new,
relatively stable fluorinated NO donors is underway in our
laboratory, which can be further incorporated into fluorinated
polymers. Finally, we anticipate that the novel C2F5-SNAP doped
PVDF composite with NO release capability described here may
provide new opportunities to enhance the biocompatibility of
biomedical devices for a variety of potential applications.
Experimental section
General materials and methods
All chemicals were directly used without any further
purification. The organic reactions were carried out using ACS grade
solvent under aerobic conditions. PVDF tubing was purchased from
Cole-Parmer company (1/16’’ ID × 1/8’’ OD, LOT#WQ11443-0002).
The synthetic compounds were characterized by 1H and 19F NMR
spectroscopy, and high-resolution mass spectrometry (HRMS) with
an electrospray ionization source. 1H and 19F NMR spectra were
Fig. 9 Antimicrobial and anti-biofilm studies with S. aureus and P.
aeruginosa bacterial strains for 7 d. (a) Plate count number of viable
S. aureus on the tubing surfaces. (b) Plate count number of viable P.
aeruginosa on the tubing surfaces. (c) and (d) Representative
fluorescence images of the biofilms on the tubing surfaces with S.
aureus ((c): Control, (d): PVDF-NO). (e) and (f) Representative
fluorescence images of the biofilms on the tubing surface with P.
aeruginosa ((e): Control, (f): PVDF-NO).
recorded using
a Varian 400/500/700 MHz spectrometer.
Phosphate-buffered saline (PBS, 10.0 mM, pH 7.40, with 100 µM
EDTA) was prepared using NaCl, KCl, Na2HPO4 and KH2PO4, and the
pH of PBS was adjusted by diluted HCl pH solution (referred to a
“PBS” throughout).
Synthesis of C2F5-NAP and C2F5-SNAP
N
-Pentafluoropropionylpenicillamine
synthesized by the reaction between
pentafluoropropionate. To a stirred solution of
(3.0120 g, 20.186 mmol) and Et3N (2.90 mL, d = 0.726 g/mL, 20.8
mmol) in MeOH (250 mL) was added ethyl
(C2F5-NAP)
-penicillamine and ethyl
-penicillamine
was
D
Conclusions
D
In summary, the fluorinated NO donor C2F5-SNAP was
successfully synthesized, and NO release from a fluoropolymer
PVDF impregnated with this new NO donor agent was investigated.
C2F5-SNAP was found to be thermally unstable due to the presence
of its fluorinated functional group. Its corresponding photolytic and
thermal decompositions were further characterized in the solution
phase. The clean decomposition of C2F5-SNAP into NO and C2F5-NAP
pentaperfluoropropionate (3.90 mL, d = 1.299 g/mL, 26.4 mmol)
dropwise. After stirring for 25 h at room temperature, the solvent
was evaporated to dryness and
a colorless oil product was
obtained. This oil product was then dissolved in water (40 mL) and
further acidified with concentrated HCl (37%, 15 mL). After stirring
for 15 min, the aqueous solution was extracted with EtOAc (3 × 50
mL). All organic layers were washed with saturated brine (100 mL),
dried over Na2SO4, and concentrated in vacuo to afford a colorless
oil as the desired product (5.364 g, 90%). 1H NMR (500 MHz, CDCl3):
disulfide was demonstrated using
a
combination of NO
determination by NOA and C2F5-NAP disulfide analysis by 1H NMR
spectroscopy. The PDVF tubing that was swelled in C2F5-SNAP
solution (~ 634.0 mg/mL in THF) for 24 h showed a loading
efficiency of C2F5-SNAP ~ 42.3 nmol/mg, which allowed NO release
for 11 d under physiological conditions. In the first 7 d, the NO flux
number was observed to be greater than that of the physiological
level (0.5 flux number). This NO-releasing PVDF tubing
demonstrated very low leaching of chemicals including C2F5-SNAP,
C2F5-NAP, and C2F5-NAP disulfide, likely resulting from fluorous-
fluorous interactions between these fluorinated molecules and
PVDF polymer. The low leaching also confirmed that the NO release
from C2F5-SNAP doped PVDF tubing primarily comes from the
surface release of NO from C2F5-SNAP within the polymer. Finally,
antimicrobial and anti-biofilm studies of C2F5-SNAP doped PVDF
tubing demonstrated that NO-releasing PVDF can significantly
inhibit the growth of both positive and negative S. aureus and P.
δ 7.32 (d,
(s, 3 H), 1.41 (s, 3 H). 19F NMR (471 MHz, CDCl3): δ -83.26 (s), -
123.74 (d,
= 4.71 Hz). HRMS m/z (ESI): calculated for [M+Na]+
318.0194, found 318.0193.
-Nitroso- -pentafluoropropionylpenicillamine (C2F5-SNAP) was
J = 8.5 Hz, 1H), 4.69 (d, J = 9.0 Hz, 1 H), 1.99 (s, 1 H), 1.63
J
S
N
generated from the reaction between C2F5-NAP and NaNO2 under
acidic conditions. During the reaction process, light exposure was
minimized to prevent the decomposition of final target product. HCl
(10 mL, 1.0 M) solution was added to a stirred solution of C2F5-NAP
(1.0135 g, 3.4329 mmol) in MeOH (10 mL) in one portion. Then, a
concentrated H2SO4 (4.0 mL) solution was added into the reaction
mixture. After cooling to room temperature using an ice-water
bath, a stock solution of NaNO2 (766.0 mg, 11.10 mmol) dissolved in
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 9 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
water (2 mL) was slowly added dropwise. After stirring for 1 h, the spectroscopy. After further storage for 4 d in the dark at room
reaction mixture was extracted with CH2Cl2 (3 × 50 mL), dried over temperature, the final spectrum of the resulting sample was
NaSO4 and concentrated in vacuo to afford a greenish oil as target measured by 1H NMR spectroscopy again.
product (1.035 g, 93%). The product was characterized by UV-Vis
spectroscopy, 1H/19F NMR spectroscopy and HRMS. UV-Vis: 341 Preparation of C2F5-SNAP doped PVDF tubing
(nO→π*) and 595 nm (nN→π*). 1H NMR (700 MHz, DMSO-d6): δ
10.14 (d, = 9.1 Hz, 1H), 5.33 (d,
(s, 3 H). 19F NMR (377 MHz, DMSO-d6): δ -82.29 (s), -121.22 (d,
11.31 Hz). HRMS m/z (ESI): calculated for [M-H]- 323.0130, found prepared C2F5-SNAP (~ 634.0 mg) dissolved in anhydrous THF (1.0
C2F5-SNAP doped PVDF tubing was prepared using a simple
= 9.1 Hz, 1 H), 2.05 (s, 3 H), 1.98 solvent swelling method. Commercially available PVDF tubing (1
inch long , 1/16” ID × 1/8” OD) was swelled in a solution of freshly
J
J
J
=
323.0145.
mL) for 24 h. Then, the swollen PVDF tubing was dried under
vacuum for 24 h. This tubing was washed with CH2Cl2 (10 times) to
completely remove the surface attached chemicals, including any
Decomposition kinetic studies of C2F5-SNAP and SNAP
The thermal and photo decomposition of the C2F5-SNAP and C2F5-SNAP and its decomposition products. After being dried under
SNAP NO donors under aerobic conditions were monitored by vacuum for another 24 h, the resulting dark green tubing was
either a PerkinElmer Lambda 35 or a Shimadzu UV-1601 UV-Vis tested for NO release, chemical leaching, or antimicrobial studies
spectrophotometer. The photodecomposition of C2F5-SNAP and (see below).
SNAP was carried out using a Rayonet photochemical reactor (RMR-
600) equipped with eight 350 nm lamps (4 W). C2F5-SNAP NO donor Characterization of C2F5-SNAP doped PVDF tubing
(~ 206.0 µM) in a mixture of PBS and DMSO (50:50, v/v) was
The NO release profile of C2F5-SNAP doped PVDF tubing was
manually scanned by the UV-Vis spectrophotometer at room recorded by a Sievers 280i chemiluminescence Nitric Oxide Analyzer
o
temperature (23 C) after each irradiation time of 0, 15, 30, 45, 60, (NOA). A given piece of C2F5-SNAP doped PVDF tubing was loaded
90, 120, 180, 240, 360 and 480 s. The kinetic data was analyzed at onto NOA every day to determine the average NO flux in a solution
341 nm, by fitting to a first-order rate equation. In a separate of PBS (4 mL) at 37 oC. Between each measurement, the tubing was
o
o
thermal decomposition kinetic study at 37 C, C2F5-SNAP NO donor soaked in PBS (2 mL), stored in a 37 C oven under dark conditions
(~ 206.0 µM) was dissolved in a mixture of PBS and DMSO (50:50, (within a glass bottle). During the storage process, the bottle’s cap
v/v). The kinetic data was monitored at 341 nm with a time period had a small hole to release gas pressure built up in the bottle, which
of 10 min. The resulting kinetic data at 341 nm versus time was was also tightly covered by a parafilm tape to prevent the
fitted into a zero-order rate equation.
evaporation of water. The soaking buffer solution obtained on each
Using a similar kinetic method as employed for the C2F5-SNAP day was utilized to test for leaching of C2F5-SNAP and/or
NO donor, photolysis of SNAP (~ 250.0 µM) in a mixture of PBS and decomposition products. After each measurement of NO release,
DMSO (50:50, v/v) was manually scanned after each irradiation the tubing was re-soaked in a fresh PBS (2 mL). The leaching test
time of 0, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480 and 780 s. The was performed using an Agilent 6520 Accurate-Mass Quadrupole
kinetic data was then analyzed using the absorbance changes at 342 Time-of-Flight (Q-TOF) LC/MS in the electrospray positive-ion mode
nm with a first-order rate equation. In a thermal decomposition with a ZORBAX RRHD Eclipse Plus C18 reversed-phase column (2.1 x
kinetic study of SNAP at 37 oC, the SNAP NO donor (~ 206.0 µM) in a 50 mm, 1.8 µm). The soaking solution obtained daily from the
mixture of PBS and DMSO (50:50, v/v) was monitored at 342 nm tubing was used for the determination of C2F5-SNAP, C2F5-NAP, and
over a time period of 10 min. The resulting absorbance changes at C2F5-NAP dimer that leached from the C2F5-SNAP doped PVDF
342 nm versus time was fitted to a zero-order rate equation.
tubing. In the HPLC method, a gradient was carried out with eluent
A (water with 0.1% formic acid) and eluent B (acetonitrile with 0.1%
formic acid) from 95% A to 0% A over a 10 min period with a flow
Analysis of decomposition products derived from C2F5-SNAP
NO generated from C2F5-SNAP NO donor (1.23 × 10-7 mol) was rate of 0.4 mL/min. Corresponding calibration curves for pure C2F5-
measured using a Sievers 280i chemiluminescence Nitric Oxide NAP and C2F5-NAP dimer were obtained to determine their
Analyzer (NOA). For rapid NO release, the C2F5-SNAP NO donor in a concentrations.
mixture of PBS and DMSO (3 mL, 50:50, v/v) was irradiated under a
Rayonet photochemical reactor (RMR-600) equipped with eight 350 Antimicrobial and anti-biofilm studies
nm lamps (4 W) at room temperature (23 oC). The total moles of NO
Three pieces of C2F5-SNAP doped PVDF tubing (4 cm, 1/16” ID ×
released from the C2F5-SNAP NO donor (1.23 × 10-7 mol) were 1/8” OD) and three pieces of PVDF tubing without the NO donor (4
calculated via integration of NO moles detected over time. The cm, 1/16” ID × 1/8” OD) were sterilized by 70% ethanol before the
C2F5-NAP disulfide formed after NO release during thermal antimicrobial and anti-biofilm studies. These pieces of tubing were
decomposition (in the dark) was further characterized by 1H NMR mounted on the holders of a CDC biofilm reactor (Biosurface
spectroscopy. For this experiment, a C2F5-SNAP NO donor sample Technologies, Bozeman, MT) using rubber bands. The bioreactor
1
in CDCl3 was prepared and characterized by H NMR spectroscopy. was supplemented continuously with 10% LB broth medium at a
This NMR sample was covered with foil and stored in a dark drawer flow rate of 100 mL/h. The bacteria, S. aureus, and P. aeruginosa
overnight, and was then again characterized by 1H NMR (1% of overnight grown), were inoculated into the bioreactor,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 10 of 12
View Article Online
DOI: 10.1039/C8TB01814F
ARTICLE
Journal Name
respectively (in separate experiments). The bioreactor was 13. J.-C. Lin, S.-L. Tiong and C.-Y. Chen, J. Biomater. Sci. Polym.
incubated at 37 oC under dark conditions for 7 d. After the pieces of
Ed. , 2000, 11, 701-714.
14. L. Pedrini, M. Dondi, A. Magagnoli, F. Magnoni, E. Pisano, E.
Del Giudice and M. Santoro, Ann. Vasc. Surg. , 2001, 15, 679-
PVDF tubing were removed, and the parts below the rubber bands
were divided into two portions, which were used for surface-
683.
fluorescence imaging of biofilm formation and for the
15. A. R. Jahangir, W. G. McClung, R. M. Cornelius, C. B.
McCloskey, J. L. Brash and J. P. Santerre, J. Biomed. Mater.
determination of viable cell counts adhered on the surfaces by
using a plate count method. For plate counting, the tubing segment
was placed in a centrifuge tube with 2 mL of 10 mM sterilized PBS
(pH 7.4) and vortexed to homogenize the biofilm and form a single
cell suspension. Then, the solutions were 10-fold serially diluted
and plated on LB agar plates for further overnight incubation at 37
oC. To determine the degree of biofilm formation, surface-
fluorescence images were taken by fluorescence microscopy
(Olympus IX71, Center Valley, PA), after the tubing segments were
stained with Live/Death BacLight Bacterial Viability Kit (Life
technology, Grand Island, NY). The SYTO-9 green fluorescent dye
was analyzed with the excitation light source at 488 nm and
emission at 520 nm. Propidium iodide dye was visualized with an
excitation light source at 535 nm and emission at 617 nm.
Res., 2002, 60, 135-147.
16. T. M. Massa, W. G. McClung, M. L. Yang, J. Y. C. Ho, J. L.
Brash and J. P. Santerre, J. Biomed. Mater. Res. A, 2007, 81A
178-185.
,
17. T. Hasebe, S. Yohena, A. Kamijo, Y. Okazaki, A. Hotta, K.
,
Takahashi and T. Suzuki, J. Biomed. Mater. Res. A, 2007, 83A
1192-1199.
18. X.-W. Wen, S.-P. Pei, H. Li, F. Ai, H. Chen, K.-Y. Li, Q. Wang
and Y.-M. Zhang, J. Mater. Sci., 2010, 45, 2788-2797.
19. D. C. Leslie, A. Waterhouse, J. B. Berthet, T. M. Valentin, A.
L. Watters, A. Jain, P. Kim, B. D. Hatton, A. Nedder, K. Donovan,
E. H. Super, C. Howell, C. P. Johnson, T. L. Vu, D. E. Bolgen, S.
Rifai, A. R. Hansen, M. Aizenberg, M. Super, J. Aizenberg and D.
E. Ingber, Nature Biotechnol., 2014, 32, 1134.
20. K. Tokuda, T. Ogino, M. Kotera and T. Nishino, Polym. J.,
2014, 47, 66.
21. A. Muñoz-Bonilla and M. Fernández-García, Prog. Polym.
Sci., 2012, 37, 281-339.
Conflicts of interest
22. S. Krishnan, R. J. Ward, A. Hexemer, K. E. Sohn, K. L. Lee, E.
R. Angert, D. A. Fischer, E. J. Kramer and C. K. Ober, Langmuir,
2006, 22, 11255-11266.
The authors declare no competing financial interest.
23. E. Martínez-Campos, T. Elzein, A. Bejjani, M. J. García-
Granda, A. Santos-Coquillat, V. Ramos, A. Muñoz-Bonilla and J.
Acknowledgements
Rodríguez-Hernández, ACS Appl. Mater. Interfaces, 2016,
6344-6353.
8,
We gratefully acknowledge the Juvenile Diabetes Research
Foundation (JDRF), Grant # 2-SRA-2016-230-Q-R, for supporting
this research.
24. J. Lin, X. Chen, C. Chen, J. Hu, C. Zhou, X. Cai, W. Wang, C.
Zheng, P. Zhang, J. Cheng, Z. Guo and H. Liu, ACS Appl. Mater.
Interfaces, 2018, 10, 6124-6136.
25. H. Ni, T. Jiang, P. Hu, Z. Han, X. Lu and P. Ye, J. Biomater.
Sci. Polym. Ed. , 2014, 25, 1920-1945.
26. R. I. Leininger, R. D. Falb and G. A. Grode, Ann. N. Y. Acad.
Sci. , 1968, 146, 11-20.
Notes and references
1. M. F. Maitz, Biosurf. Biotribol., 2015, 1, 161-176.
2. S. Ebnesajjad, in Expanded PTFE Applications Handbook,
William Andrew Publishing, Oxford, 2017, ch. 9, pp. 193-211.
3. V. Cardoso, D. Correia, C. Ribeiro, M. Fernandes and S.
Lanceros-Méndez, Polymers, 2018, 10, 161.
4. A. J. T. Teo, A. Mishra, I. Park, Y.-J. Kim, W.-T. Park and Y.-J.
Yoon, ACS Biomater. Sci. Eng. , 2016, 2, 454-472.
5. H. Mori, A. Gupta, S. Torii, E. Harari, H. Jinnouchi, R. Virmani
and A. V. Finn, Expert Rev. Med. Devices, 2017, 14, 707-716.
6. H. Teng, Appl. Sci., 2012, 2, 496.
7. P. C. Nicolson and J. Vogt, Biomaterials, 2001, 22, 3273-
3283.
8. T. Koppara, K. Sakakura, E. Pacheco, Q. Cheng, X. Zhao, E.
Acampado, A. V. Finn, M. Barakat, L. Maillard, J. Ren, M.
Deshpande, F. D. Kolodgie, M. Joner and R. Virmani, Int. J.
Cardiol., 2016, 222, 217-225.
9. T.-Y. Liu, W.-C. Lin, L.-Y. Huang, S.-Y. Chen and M.-C. Yang,
Polym. Adv, Technol., 2005, 16, 413-419.
27. M. V. Sefton, A. Sawyer, M. Gorbet, J. P. Black, E. Cheng, C.
Gemmell and E. Pottinger-Cooper, J. Biomed. Mater. Res.,
2001, 55, 447-459.
28. S. L. Chin-Quee, S. H. Hsu, K. L. Nguyen-Ehrenreich, J. T. Tai,
G. M. Abraham, S. D. Pacetti, Y. F. Chan, G. Nakazawa, F. D.
Kolodgie, R. Virmani, N. N. Ding and L. A. Coleman,
Biomaterials, 2010, 31, 648-657.
29. M. A. Jamal, K. Garoge, J. S. Rosenblatt, R. Y. Hachem and I.
I. Raad, Antimicrob. Agents Chemother., 2015, 59, 4397-4402.
30. L. Heinemann, J. Walsh and R. Roberts, J. Diabetes Sci.
Technol. , 2014,
31. L. Heinemann and L. Krinelke, J. Diabetes Sci. Technol. ,
2012, , 954-964.
8, 199-202.
6
32. P. C. Begovac, R. C. Thomson, J. L. Fisher, A. Hughson and
A. Gällhagen, Eur. J. Vasc. Endovasc. Surg. , 2003, 25, 432-437.
33. F. He, B. Luo, S. Yuan, B. Liang, C. Choong and S. O.
Pehkonen, RSC Adv., 2014, 4, 105-117.
10. A. M. Garfinkle, A. S. Hoffman, B. D. Ratner, L. O. Reynolds
and S. R. Hanson, ASAIO J., 1984, 30, 432-439.
11. H. Dong Keun, J. Seo Young, K. Young Ha and M. Byoung
Goo, J. Biomater. Sci. Polym. Ed. , 1992, 3, 229-241.
12. A. Welle, M. Grunze and D. Tur, J. Colloid Interface Sci.,
1998, 197, 263-274.
34. X. Li, X. Hu and T. Cai, Langmuir, 2017, 33, 4477-4489.
35. D. S. Bredt, Free Radic. Res., 1999, 31, 577-596.
36. H.-T. Chung, H.-O. Pae, B.-M. Choi, T. R. Billiar and Y.-M.
Kim, Biochem. Biophys. Res. Commun., 2001, 282, 1075-1079.
37. N. Toda, K. Ayajiki and T. Okamura, Pharmacol. Rev., 2009,
61, 62-97.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 11 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Journal Name
ARTICLE
38. Y. Wo, E. J. Brisbois, R. H. Bartlett and M. E. Meyerhoff, 65. J. Pant, J. Gao, M. J. Goudie, S. P. Hopkins, J. Locklin and H.
Biomater. Sci., 2016, , 1161-1183. Handa, Acta Biomater., 2017, 58, 421-431.
4
39. X. Liu, M. J. S. Miller, M. S. Joshi, H. Sadowska-Krowicka, D. 66. Q. Liu, P. Singha, H. Handa and J. Locklin, Langmuir, 2017,
A. Clark and J. R. Lancaster, J. Biol. Chem., 1998, 273, 18709- 33, 13105-13113.
18713. 67. M. J. Goudie, B. M. Brainard, C. W. Schmiedt and H. Handa,
40. P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and A. J. J. Biomed. Mater. Res. A., 2017, 105, 539-546.
Janczuk, Chem. Rev., 2002, 102, 1091-1134. 68. E. J. Brisbois, T. C. Major, M. J. Goudie, M. E. Meyerhoff, R.
41. A. D. Ostrowski and P. C. Ford, Dalton Trans., 2009, 10660- H. Bartlett and H. Handa, Acta Biomater., 2016, 44, 304-312.
10669.
42. M. R. Miller and I. L. Megson, Br. J. Pharmacol., 2007, 151
305-321.
69. M. J. Goudie, E. J. Brisbois, J. Pant, A. Thompson, J. A.
Potkay and H. Handa, Int. J. Polym. Mater., 2016, 65, 769-778.
70. M. N. Mann, B. H. Neufeld, M. J. Hawker, A. Pegalajar-
,
43. T. Munzel and A. Daiber, Vasc. Pharmacol., 2018, 102, 1- Jurado, L. N. Paricio, M. M. Reynolds and E. R. Fisher,
10. Biointerphases, 2016, 11, 031005.
44. W. Fan, B. C. Yung and X. Chen, Angew. Chem. Int. Ed., 71. J. M. Joslin, S. M. Lantvit and M. M. Reynolds, ACS Appl.
2018, 57, 8383-8394. Mater. Interfaces, 2013, , 9285-9294.
45. M. C. Frost, M. M. Reynolds and M. E. Meyerhoff, 72. A. Lutzke, J. B. Tapia, M. J. Neufeld and M. M. Reynolds,
Biomaterials, 2005, 26, 1685-1693. ACS Appl. Mater. Interfaces, 2017, , 2104-2113.
46. Z. Sadrearhami, T.-K. Nguyen, R. Namivandi-Zangeneh, K. 73. A. Koh, A. W. Carpenter, D. L. Slomberg and M. H.
5
9
Jung, E. H. H. Wong and C. Boyer, J. Mater. Chem. B, 2018,
2945-2959.
6,
Schoenfisch, ACS Appl. Mater. Interfaces, 2013, 5, 7956-7964.
74. E. M. Hetrick, H. L. Prichard, B. Klitzman and M. H.
47. P. Singha, J. Locklin and H. Handa, Acta Biomater., 2017, Schoenfisch, Biomaterials, 2007, 28, 4571-4580.
50, 20-40. 75. C. W. McCarthy, J. Goldman and M. C. Frost, ACS Appl.
48. T. C. Major, H. Handa, G. M. Annich and R. H. Bartlett, J. Mater. Interfaces, 2016, , 5898-5905.
Biomater. Appl., 2014, 29, 479-501. 76. G. Fleming, J. Aveyard, J. Fothergill, F. McBride, R. Raval
49. E. M. Hetrick and M. H. Schoenfisch, Chem. Soc. Rev., 2006, and R. D’Sa, Polymers, 2017, , 601.
35, 780-789. 77. A. Lowe, W. Deng, D. W. Smith and K. J. Balkus,
50. A. W. Carpenter and M. H. Schoenfisch, Chem. Soc. Rev., Macromolecules, 2012, 45, 5894-5900.
8
9
2012, 41, 3742-3752. 78. H. C. Zhao, M. C. Serrano, D. A. Popowich, M. R. Kibbe and
51. W. Cai, J. Wu, C. Xi and M. E. Meyerhoff, Biomaterials, G. A. Ameer, J. Biomed. Mater. Res. A., 2010, 93A, 356-363.
2012, 33, 7933-7944.
52. E. J. Brisbois, H. Handa, T. C. Major, R. H. Bartlett and M. E. Biotechnol. , 1989,
Meyerhoff, Biomaterials, 2013, 34, 6957-6966. 80. D. Chopra and T. N. G. Row, CrystEngComm, 2011, 13
53. A. R. Ketchum, M. P. Kappler, J. Wu, C. Xi and M. E. 2175-2186.
Meyerhoff, J. Mater. Chem. B, 2016, , 422-430. 81. H. M. Rapp, S. Bacher, A. Ahrens, W. Rapp, B. Kammerer,
79. R. K. Kobos, J. W. Eveleigh and R. Arentzen, Trends
7, 101-105.
,
4
54. Y. Wo, Z. Li, E. J. Brisbois, A. Colletta, J. Wu, T. C. Major, C. G. U. Nienhaus and W. Bannwarth, ChemPlusChem, 2012, 77
Xi, R. H. Bartlett, A. J. Matzger and M. E. Meyerhoff, ACS Appl. 1066-1070.
,
Mater. Interfaces, 2015,
55. A. Colletta, J. Wu, Y. Wo, M. Kappler, H. Chen, C. Xi and M. Cryst. Growth Des., 2015, 15, 2835-2841.
E. Meyerhoff, ACS Biomater. Sci. Eng., 2015, , 416-424. 83. A. R. Ketchum, Ph. D. Thesis, The University of Michigan,
56. H. Ren, J. L. Bull and M. E. Meyerhoff, ACS Biomater. Sci. Michigan, USA, 2016.
Eng., 2016, , 1483-1492. 84. T. I. Gorbunova, D. N. Bazhin, A. Y. Zapevalov and V. I.
7
, 22218-22227.
82. H. Omorodion, B. Twamley, J. A. Platts and R. J. Baker,
1
2
57. Y. Wo, E. J. Brisbois, J. Wu, Z. Li, T. C. Major, A. Saloutin, Russ. J. Appl. Chem., 2013, 86, 992-996.
Mohammed, X. Wang, A. Colletta, J. L. Bull, A. J. Matzger, C. Xi, 85. L. Field, R. V. Dilts, R. Ravichandran, P. G. Lenhert and G. E.
R. H. Bartlett and M. E. Meyerhoff, ACS Biomater. Sci. Eng., Carnahan, J. Chem. Soc. Chem. Commun., 1978, 249-250.
2017,
58. Y. Wo, L.-C. Xu, Z. Li, A. J. Matzger, M. E. Meyerhoff and C. 87. D. L. H. Williams, Acc. Chem. Res., 1999, 32, 869-876.
A. Siedlecki, Biomater. Sci., 2017, , 1265-1278. 88. C. Zhang, T. D. Biggs, N. O. Devarie-Baez, S. Shuang, C.
3, 349-359.
86. D. L. H. Williams, Chem. Comm., 1996, 1085-1091.
5
59. E. J. Brisbois, M. Kim, X. Wang, A. Mohammed, T. C. Major, Dong and M. Xian, Chem. Comm., 2017, 53, 11266-11277.
J. Wu, J. Brownstein, C. Xi, H. Handa, R. H. Bartlett and M. E. 89. C. Cucarella, M. Á. Tormo, E. Knecht, B. Amorena, Í. Lasa, T.
Meyerhoff, ACS Appl. Mater. Interfaces, 2016, 8, 29270-29279. J. Foster and J. R. Penadés, Infect. Immun., 2002, 70, 3180-
60. E. J. Brisbois, R. P. Davis, A. M. Jones, T. C. Major, R. H. 3186.
Bartlett, M. E. Meyerhoff and H. Handa, J. Mater. Chem. B, 90. H. Ren, J. F. Wu, A. Colletta, M. E. Meyerhoff and C. W. Xi,
Front. Microbiol., 2016, 7, 1260.
2015,
3, 1639-1645.
61. T. C. Major, H. Handa, E. J. Brisbois, M. M. Reynolds, G. M.
Annich, M. E. Meyerhoff and R. H. Bartlett, Biomaterials, 2013,
34, 8086-8096.
62. M. J. Goudie, J. Pant and H. Handa, Sci. Rep., 2017, 7,
13623.
63. E. J. Brisbois, T. C. Major, M. J. Goudie, R. H. Bartlett, M. E.
Meyerhoff and H. Handa, Acta Biomater., 2016, 37, 111-119.
64. P. Singha, J. Pant, M. J. Goudie, C. D. Workman and H.
Handa, Biomater. Sci., 2017, 5, 1246-1255.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Journal of Materials Chemistry B
Page 12 of 12
View Article Online
DOI: 10.1039/C8TB01814F
Synthesis and Characterization of a Fluorinated S-Nitrosothiol as the Nitric
Oxide Donor for Fluoropolymer-Based Biomedical Device Applications
Yang Zhou,a Qi Zhang,a Jianfeng Wu,b Chuanwu Xib and Mark E. Meyerhoff*a
The first nitric oxide (NO) releasing fluorinated polymer was developed via incorporating a new
fluorinated NO donor into polyvinylidene fluoride tubing.
ORCID
Yang Zhou: 0000-0003-0377-4338
Qi Zhang: 0000-0003-2496-8844
Jianfeng Wu: 0000-0002-9770-5538
Chuanwu Xi: 0000-0002-7554-1915
Mark E. Meyerhoff: 0000-0002-7841-281X