Reduction in thrombosis and bacterial adhesion with 7 day implantation of S-nitroso-N-acetylpenicillamine (SNAP)-doped Elast-eon E2As catheters in sheep

Article abstract of DOI:10.1039/c4tb01839g

Thrombosis and infection are two common problems associated with blood-contacting medical devices such as catheters. Nitric oxide (NO) is known to be a potent antimicrobial agent as well as an inhibitor of platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels exhibit a NO flux of 0.5-4 × 10^-10 mol cm^-2 min^-1 that helps prevent thrombosis. Materials with a NO flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study, NO-releasing catheters were fabricated by incorporating S-nitroso-N-acetylpenicillamine (SNAP) in the Elast-eon E2As polymer. The SNAP/E2As catheters release physiological levels of NO for up to 20 days, as measured by chemiluminescence. Furthermore, SNAP is stable in the E2As polymer, retaining 89% of the initial SNAP after ethylene oxide (EO) sterilization. The SNAP/E2As and E2As control catheters were implanted in sheep veins for 7 days to examine the effect on thrombosis and bacterial adhesion. The SNAP/E2As catheters reduced the thrombus area when compared to the control (1.56 ± 0.76 and 5.06 ± 1.44 cm², respectively). A 90% reduction in bacterial adhesion was also observed for the SNAP/E2As catheters as compared to the controls. The results suggest that the SNAP/E2As polymer has the potential to improve the hemocompatibility and bactericidal activity of intravascular catheters, as well as other blood-contacting medical devices (e.g., vascular grafts, extracorporeal circuits). This journal is

Full text of DOI:10.1039/c4tb01839g

Journal of
Materials Chemistry B
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PAPER
Reduction in thrombosis and bacterial adhesion with
day implantation of S-nitroso-N-acetylpenicillamine
(SNAP)-doped Elast-eon E2As catheters in sheep
7
Cite this: J. Mater. Chem. B, 2015, 3,
639
1
a
a
a
a
Elizabeth J. Brisbois, Ryan P. Davis, Anna M. Jones, Terry C. Major,
a
b
c
Robert H. Bartlett, Mark E. Meyerhoff* and Hitesh Handa*
Thrombosis and infection are two common problems associated with blood-contacting medical devices
such as catheters. Nitric oxide (NO) is known to be a potent antimicrobial agent as well as an inhibitor of
platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels
ꢁ
10
ꢁ2 ꢁ1
exhibit a NO flux of 0.5–4 ꢀ 10
mol cm min that helps prevent thrombosis. Materials with a NO
flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study,
NO-releasing catheters were fabricated by incorporating S-nitroso-N-acetylpenicillamine (SNAP) in the
Elast-eon E2As polymer. The SNAP/E2As catheters release physiological levels of NO for up to 20 days,
as measured by chemiluminescence. Furthermore, SNAP is stable in the E2As polymer, retaining 89% of
the initial SNAP after ethylene oxide (EO) sterilization. The SNAP/E2As and E2As control catheters were
implanted in sheep veins for 7 days to examine the effect on thrombosis and bacterial adhesion. The
SNAP/E2As catheters reduced the thrombus area when compared to the control (1.56 ꢂ 0.76 and 5.06 ꢂ
Received 7th November 2014
Accepted 12th January 2015
2
1
.44 cm , respectively). A 90% reduction in bacterial adhesion was also observed for the SNAP/E2As
catheters as compared to the controls. The results suggest that the SNAP/E2As polymer has the
potential to improve the hemocompatibility and bactericidal activity of intravascular catheters, as well as
other blood-contacting medical devices (e.g., vascular grafts, extracorporeal circuits).
DOI: 10.1039/c4tb01839g
www.rsc.org/MaterialsB
Associated Infections that result in 99 000 deaths per year in the
1
. Introduction
4
United States alone. In addition, up to 40% of all indwelling
5
Blood-material interaction is critical to the success of many catheters become infected. An estimated 80 000 catheter-related
medical devices such as intravascular catheters, vascular gras, bloodstream infections occur in patients within ICUs in the
1
,2
6
stents, and extracorporeal life support circuits. Two common United States, resulting in as many as 28 000 deaths per year.
factors that can cause complications with blood-contacting
Over the last 50 years, much has been learned about surface-
devices are thrombosis and infection. As soon as blood comes induced thrombosis and many different strategies to create
1,7–9
in contact with foreign surfaces, platelets adhere and become hemocompatible materials have been reported.
Some of the
activated, forming thrombus within hours. Clinical use of cath- surface modications to improve hemocompatibility include
eters range from acute catheters placed in operating rooms, hydrophilic or hydrophobic surfaces, zwitterionic polymers,
emergency rooms, and intensive care units (ICUs) (typically for and immobilized heparin. However, in a clinical setting many
up to 7 days), to more permanent catheters for long-term nutri- devices still require the use of systemic or localized anti-
tion, dialysis access, etc. (months to years). Complications due coagulation (e.g., heparin lock solutions) to avoid device failure
10
to thrombosis and infection can result in extended hospital due to thrombosis. Long-term use of systemic anticoagulation
stays, increased healthcare costs, and even patient death. is not desirable because it can have adverse side effects such as
11
Thrombus formation on catheters decreases their patency and hemorrhage, thrombocytopenia, and thrombosis. Catheters
functional lifetime. Venous thrombosis has been detected by with antimicrobial coatings (e.g., containing silver compounds
3
Doppler imaging in 33% of intensive care unit patients. Infec- or antibiotics) are available, but are not completely effective at
tion is another signicant problem, with 1.7 million Healthcare preventing infection and biolm formation and also do not
5,12
address complications associated with activation of platelets.
A recent study reported that catheters coated with a silver alloy
had a similar infection rate as control catheters. Antibiotic
a
b
Department of Surgery, University of Michigan, Ann Arbor, MI, USA
13
Department of Chemistry, University of Michigan, 930 N. University Ave, Ann Arbor,
resistant bacterial strains are becoming more common in the
MI 48109, USA. E-mail: mmeyerho@umich.edu; Tel: +1-734-763-5916
14,15
c
hospital setting.
Further, bacteria have the ability to form
Department of Biochemical Engineering, University of Georgia, 220 Riverbend Road,
biolms (communities of bacterial encased in a self-synthesized
Athens, GA 30605, USA. E-mail: hhanda@uga.edu; Tel: +1-706-542-8839
This journal is © The Royal Society of Chemistry 2015
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30
extracellular matrix) that protect the bacteria from antiseptics, make clinical applications feasible. In this new study, intra-
16–19
antibiotics, and the host's defense system.
Biolm forma- vascular catheters were fabricated with the SNAP/E2As polymer
tion decreases the effectiveness of many antibiotics because using a dip coating method. The NO release from these cathe-
they cannot penetrate the biolm matrix, making the infections ters was monitored over a 20 day period under physiological
20
ꢃ
difficult to eradicate.
conditions (37 C, dark). Another key hurdle for potential clin-
The use of nitric oxide (NO)-releasing polymers is one ical applications is the ability of the NO-releasing materials to
approach that has great potential to improve the hemocompati- withstand sterilization. Therefore, the effect of ethylene oxide
bility and antimicrobial activity of blood-contacting devices such (EO) sterilization on the SNAP/E2As catheters was also evalu-
as catheters. Radomski et al. rst described NO as a potent ated. Finally, the SNAP/E2As catheters were assessed for their
vasodilator secreted by the normal endothelium that has the ability to reduce thrombosis and bacterial adhesion aer 7 days
ability to inhibit platelet adhesion and aggregation to the blood intravascular implantation in sheep.
21,22
vessel wall.
Nitric oxide is a natural inhibitor of platelet acti-
vation that is released from healthy endothelial cells at a ux into
2
. Materials and methods
ꢁ
10
ꢁ2
ꢁ1 23
the blood stream of 0.5–4.0 ꢀ 10
addition, NO that is released within the sinus cavities and by
neutrophils/macrophages functions as a potent natural antimi- N-Acetyl-DL-penicillamine (NAP), sodium chloride, potassium
mol cm min
.
In
2.1 Materials
24,25
crobial agent.
Nitric oxide is known to have broad-spectrum chloride, sodium phosphate dibasic, potassium phosphate
antibacterial properties, where both Gram-positive and Gram- monobasic, ethylenediaminetetraacetic acid (EDTA), tetrahy-
26
negative bacteria can be killed. Therefore, use of NO-releasing drofuran (THF), sulfuric acid and N,N-dimethylacetamide
polymers has the advantage of enabling the creation of dual- (DMAc) were purchased from Sigma-Aldrich (St. Louis, MO).
functioning catheters that would possess both antithrombotic Methanol, hydrochloric acid and sulfuric acid were obtained
and antiseptic properties.
from Fisher Scientic (Pittsburgh, PA). Elast-eon™ E2As was
A wide variety of NO-releasing polymers have been reported obtained from AorTech International, plc (Scoresby, Victoria,
and investigated for their potential biomedical applica- Australia). All aqueous solutions were prepared with 18.2 MU
24,27–29
tions.
NO-releasing polymers have been shown to preserve deionized water using a Milli-Q lter (Millipore Corp., Billerica,
platelet count and reduced thrombus formation when tested in MA). Phosphate buffered saline (PBS), pH 7.4, containing
a 4 h rabbit model of extracorporeal circulation (ECC) throm- 138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate, 100 mM
30–34
bogenicity.
S-nitrosothiol-modied xerogels were shown to EDTA was used for all in vitro experiments.
signicantly reduce adhesion of platelets and bacteria (Pseu-
35,36
domonas aeruginosa) when tested in vitro.
Diazeniomdiolate-
2.2 SNAP synthesis protocol
doped poly(lactic-co-glycolic acid)-based lms exhibited anti-
biolm properties against both Gram-positive (Staphylococcus
aureus) and Gram-negative (Escherichia coli) bacteria when
SNAP was synthesized using a modied version of a previously
reported method. Briey an equimolar ratio of NAP and
sodium nitrite was added to a 1 : 1 mixture of water and
2 4
methanol containing 2 M HCl and 2 M H SO . Aer 30 min of
stirring, the reaction vessel was cooled in an ice bath to
precipitate the green SNAP crystals. The crystals were collected
by ltration, washed with water, and allowed to air dry. The
reaction mixture and resulting crystals were protected from
light at all times.
41
37
tested using a drip-ow bioreactor over a 7 days period.
Despite these promising literature reports, many of these NO-
releasing polymers have not been clinically applied due to
instability of the NO donor species within the polymers during
storage or sterilization, signicant leaching of unbound NO
donors, or the requirement of complex chemistry to covalently
30,38
bind the NO donor to the polymer.
For example, S-nitro-
sothiol-modied polyesters were effective at reducing platelet
adhesion/activation and had antimicrobial effect against
2.3 Preparation of intravascular catheters
Staphylococcus aureus and the multi-drug resistant Pseudomonas Catheters were prepared by dip coating polymer solutions on
aeruginosa strains, but the NO release capability was signi- 18 cm long stainless steel mandrels of 2.0 mm diameter
3
9,40
cantly inuenced by ethylene oxide sterilization.
In addition, (purchased from McMaster Carr). The E2As control catheter
ꢁ
1
most of the NO-releasing materials reported to date have been solution consisted of E2As dissolved in THF (150 mg mL ).
tested in vitro or in short-term animal models for their hemo- Thirty-ve coats of the E2As solution was applied on the
compatibility properties, so testing these materials in longer- mandrel by dip coating at 2 min intervals between each coat.
term animal models would be benecial to evaluate more The SNAP-doped E2As catheters had a trilayer conguration,
clinically relevant situations.
E2As top/base coats and a SNAP-containing middle active layer.
Herein we report one of the rst longer-term (7 days) in vivo The top/base coat solution consisted of E2As dissolved in THF
ꢁ
1
evaluation studies of novel NO-releasing catheters. As we have (150 mg mL ). The active solution was formulated to contain
previously reported, a S-nitroso-N-acetylpenicillamine (SNAP)- 10 wt% SNAP and 90 wt% E2As dissolved in THF with an overall
ꢁ1
doped Elast-eon E2As polymer formulation has very attractive total concentration of 150 mg mL
of solids. The trilayer
properties, in terms of its long-term NO release, enhanced shelf- catheters were prepared by dip coating 5 base coats of E2As
life stability, and its ability to preserve platelets and reduce solution, 25 coats of active solution, and 5 top-coats of E2As
thrombus during the 4 h extracorporeal circulation, that could solution. All catheters were allowed to dry overnight under
1640 | J. Mater. Chem. B, 2015, 3, 1639–1645
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ambient conditions, protected from light. Cured catheters were the NIH. This study was approved by the University of Michigan
removed from the mandrels and dried under vacuum for 48 h. Committee on Use and Care of Animals. Five adult sheep (Valley
Catheters had an i.d. of 2.08 ꢂ 0.06 mm and o.d. of 3.30 ꢂ View Farms, Dexter, MI) were utilized for the large animal
0
.12 mm, as measured with a Mitutoyo digital micrometer.
model. All experiments were performed under sterile condi-
tions. Implantation of the catheters was performed under
general anesthetic. Propofol (APP Pharmaceuticals LLC,
Schaumburg, IL) (1 mg kg ) was used for induction followed by
isourane (Peramal Health Car Limited, Andhra Pradesh, India)
2.4 NO release measurements
ꢁ1
Nitric oxide release from the catheters was measured using
a Sievers chemiluminescence Nitric Oxide Analyzer (NOA),
model 280 (Boulder, CO). A sample catheter segment (1 cm)
(0.1–4%) anesthetic for maintenance. A small 2–3 cm incision
ꢃ
was created overlying the jugular vein. The right and le jugular
veins were then isolated and either control (E2As) or experi-
mental (SNAP/E2As) cannulas were placed under direct visual-
ization. Small 1–2 cm vertical and transverse incisions were
created over the right and le jugular veins. Catheters were then
placed using a modied Seldinger technique with one catheter,
either control (E2As) or experimental (SNAP/E2As), placed in
either the right or le jugular vein, and the other catheter in the
other vein. The skin was then re-approximated using skin
staples.
was placed in 4 mL PBS buffer at 37 C. Nitric oxide liberated
from the sample was continuously swept from the headspace of
the sample cell and purged from the buffer with a nitrogen
sweep gas and bubbler into the chemiluminescence detection
chamber. The ow rate was set to 200 mL min with a chamber
pressure of 5.4 Torr and an oxygen pressure of 6.0 psi. Catheter
segments were incubated in 4 mL of PBS buffer, pH 7.4, at 37 C
and tested for NO release at various time points. The NO release
from catheters is normalized by the surface area using the ux
ꢁ
1
ꢃ
ꢁ10
ꢁ2
ꢁ1
unit (ꢀ10
mol cm min ). The chemiluminescence nitric
Post-operative recovery protocol. Sheep were recovered from
anesthesia aer the catheter placements and returned to
animal housing. The sheep remained in animal housing
throughout the remainder of the experiment. Necropsy was
performed on day 7. Sheep were anesthetized using the same
anesthetic protocol described above. The right and le jugular
veins were dissected along their length and isolated. Sheep were
oxide analyzer (NOA) measures the NO release from samples in
ppb units within the gas phase of nitrogen that purges the
solution in which the catheters are placed. The NO ux is
calculated based on the surface area of the catheter sample and
using this raw data (NO level in ppb) and NOA constant (with
ꢁ1
ꢁ1
units of mol ppb
s ) of the instrument. The buffer was
replaced every day. Aer the 7 day chronic animal studies, 1 cm
sections of the implanted NO release catheters were tested for
NO release post-blood exposure. For dry vs. humidity experi-
ments, a catheter sample was placed in a dry NOA sample cell
ꢁ1
heparinized using approximately 100–150 IU kg bolus dose
and activated clotting time of >200 s was conrmed. The jugular
veins were then ligated and opened longitudinally. Catheters
were removed and placed in sterile saline for further analysis.
Catheter evaluation. Aer explanting, the catheters were
rinsed in sterile saline solution. Pictures were taken of the
exterior of the whole catheter using a Nikon L24 digital camera.
Catheter sections (1 cm) were cut for NO release testing and
bacterial adhesion measurements. To quantitate the viable
bacteria on the surfaces of the catheters, a 1 cm piece was cut
longitudinally and was homogenized in 1 mL sterile PBS buffer.
The resulting homogenate was serially diluted in sterile PBS.
Triplicate aliquots of each dilution (10 mL) of each dilution were
ꢃ
incubated at 55 C. Humidity was introduced by injecting PBS
buffer into the cell, with the catheter sample suspended in the
air above the buffer. The relative humidity in the sample cell
was measured using a Fieldpiece PRH2 psychrometer.
2.5 Effect of ethylene oxide (EO) sterilization
The SNAP-doped E2As catheters were sterilized by ethylene
oxide (EO) at the University of Michigan Hospital sterilization
facility. Briey, the EO sterilization procedure consists of the
ꢃ
following steps, all at 54 C: humidity and conditioning (1 h, 40–
plated on LB agar plates. The agar plates were incubated at
80% humidity); EO gas exposure (2–3 h, 40–80% humidity); and
ꢃ
3
7 C for 24 h followed by calculation of colony forming units
exhaust/aeration (14 h). Catheters were tested for the wt% SNAP
content before and aer EO sterilization by UV-Vis analysis.
Catheter pieces were weighed and dissolved in N,N-dimethyla-
cetamide (DMAc) for rapid determination of SNAP by UV-Vis
ꢁ2
per catheter surface area (CFU cm ).
2.7 Statistical analysis
(
absorbance at 340 nm). UV-Vis spectra were recorded in the Data are expressed as mean ꢂ standard deviation (SD). The
wavelength range of 200–700 nm using a UV-Vis spectropho- results between the control and SNAP catheters were analyzed
tometer (Lambda 35, Perkin-Elmer, MA) at room temperature. by a comparison of means using Student's t-test. Values of p <
The molar absorption coefficient for SNAP in DMAc at 340 nm 0.05 were considered statistically signicant for all tests.
ꢁ1
ꢁ1.
was determined to be: 3SNAP ¼ 1025 M cm
.6 Catheter implantation in sheep model
Sheep catheter implantation protocol. All animals received
3
. Results and discussion
2
3.1 In vitro NO release from catheters and effects of ethylene
oxide sterilization
care compliant with the “Principles of Laboratory Animal Care”
formulated by the National Society for Medical Research and As we have previously reported, the SNAP-doped E2As polymer
the “Guideline for the Care of Use of Laboratory Animals” has many properties that make it desirable for a variety
prepared by the National Academy of Sciences and published by of biomedical applications. The properties include long-term
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NO release, stability during long-term storage, and ability to
preserve platelets during short-term extracorporeal circula-
30
tion. Nitric oxide release from S-nitrosothiols (RSNOs), such as
+
SNAP, can be catalyzed by heat, moisture, metal ions (e.g. Cu ),
or light irradiation at the energies that correspond to the S-
42–45
nitroso absorption bands at 340 and/or 590 nm.
The spon-
taneous decomposition reaction of RSNOs (no redox reaction)
yields a disulde (RSSR) product and NO. In these catheters the
NO release from the SNAP-doped E2As polymer is initiated by
the heat and moisture when the devices are placed in the

owing blood. In this study, catheters were prepared with the
SNAP-doped E2As polymer using a dip coating method and the
trilayer conguration shown in Fig. 1. The top and base coats of
E2As were employed to reduce any initial burst of NO from any
leaching of SNAP from the surface of the catheters. There is
little concern regarding the low levels of SNAP leaching typically Fig. 2 NO release from SNAP/E2As catheters under physiological
ꢃ
observed from the SNAP/E2As polymer, since the parent thiol conditions (soaking in PBS buffer at 37 C in the dark). Data represents
46–49
the mean ꢂ SD (n ¼ 5).
(NAP) is clinically used to treat heavy metal poisoning.
Hence, the small amounts of SNAP, NAP, or NAP disulde that
could diffuse from the catheter into the blood are not likely to
create any toxicity issues. The NO release from SNAP/E2As
catheters was monitored using the NOA over a 20 day period
benecial to reduce intravascular catheter thrombosis and
bacterial adhesion.
In order for medical devices, such as the SNAP/E2As cathe-
ters, to be used clinically they must be able to withstand ster-
ilization and still retain their NO-releasing properties. Ethylene
oxide (EO) sterilization is one common method employed to
sterilize medical devices. This method is recommended for
heat-sensitive devices over other sterilization techniques (e.g.,
autoclave). Therefore, the SNAP/E2As catheters were also eval-
uated for their ability to withstand the EO sterilization process.
One centimeter sections of the SNAP/E2As catheter were EO
sterilized by the University of Michigan Hospital sterilization
facility. Precautions were taken to minimize any excessive
exposure to light, so that only the effects of the EO sterilization
process could be observed. The SNAP/E2As catheters (before
and aer EO) were dissolved in DMAc to quantitate the wt%
SNAP by UV-Vis. The catheters that had been EO sterilized had
(Fig. 2). The catheters exhibited a higher level of NO release
upon rst exposure to physiological conditions (soaked in PBS
ꢃ
at 37 C) on day 0. The initial water uptake of the polymer and
leaching of SNAP on the outer surface of the catheters
contributes to the higher level of NO release observed during
this initial test period. In blood, NO has a short half-life due to
50
its rapid scavenging by hemoglobin; therefore, initial NO
release levels that are somewhat higher than physiological
levels, such as what is observed on day 0, are not a signicant
concern. This is because the surface areas of the catheters
studied here are very small compared to the entire surface areas
of the inner walls of all blood vessels within the body that are
producing NO at physiological levels continuously. As shown,
the SNAP/E2As catheters continue to release physiological levels
ꢁ
10
ꢁ2
ꢁ1
of NO (>0.5 ꢀ 10
mol cm min ) for up to 20 days. The NO
8
8.8 ꢂ 1.7% of the original SNAP (see Fig. 3), and the NO release
release from the catheters at that time approaches the lower
end of the normal endothelium range, which may still prove
prole aer sterilization was not signicantly affected (data not
shown). During the EO sterilization process, the SNAP/E2As
catheters were not only exposed to the EO gas, but also were
ꢃ
exposed to elevated temperatures (54 C) and humidity (40–80%
RH). The NO release from catheter sections was observed with
ꢃ
the NOA under dry and humid conditions at 55 C. As shown in
Fig. 4, the NO release from the catheters is low under dry
conditions, but dramatically increases upon exposure to
humidity (ꢄ50% RH). This demonstrates that the NO loss
observed during EO sterilization may, in part, be caused by the
high humidity exposure. This loss of SNAP could be signi-
cantly reduced if the devices can be EO sterilized under dry
conditions.
3.2 Evaluation of thrombus formation and bacterial
adhesion on catheters in a 7 day sheep model
Thrombus formation and infection are the two main prob-
lems that cause complications with indwelling catheters. The
Fig. 1 Schematic of SNAP-doped E2As catheters with a trilayer
configuration.
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Surface thrombi on the explanted catheters were photo-
graphed and the degree of thrombus area was quantitated using
Image J imaging soware from National Institutes of Health
(Bethesda, MD). Fig. 5A, shows representative images of clot
formation on the exterior of SNAP/E2As and control catheters.
These thrombi area measurements were quantitated and, as
shown in Fig. 5B, the thrombus area of the SNAP/E2As catheters
was signicantly reduced compared to the control catheters,
2
1
.56 ꢂ 0.76 and 5.06 ꢂ 1.44 cm , respectively (n ¼ 5 each).
One centimeter catheter sections in 1 mL sterile PBS were
homogenized to detach the bacteria from the inner and outer
catheter surfaces and cultured as described in the Experi-
mental section above. The bacterial colonies were counted
ꢁ
2
the following day and are represented as CFU cm in Fig. 6. A
.0 log reduction (90% reduction) in living bacteria adhered to
the surfaces was observed for SNAP/E2As catheters vs. the
1
Fig. 3 The SNAP content (mg SNAP/mg catheter) of catheters before controls (n ¼ 5 each). Post-implanted catheters had a NO ux of
ꢁ
10
ꢁ2
ꢁ1
and after ethylene oxide (EO) sterilization. Data represents the mean ꢂ
SD (n ¼ 4).
0
.6 ꢂ 0.3 ꢀ 10
mol cm min on the day of explantation,
which is at the lower end of the normal range of NO from the
2
3
endothelium. This data is consistent with previously reported
animal data, where it was shown that NO release is not
31,33
compromised due to blood exposure.
The hemocompati-
bility of these catheters likely could be further improved by
exploring methods to increase the NO ux from the SNAP/E2As
polymer, perhaps by adding polymeric additives that increase
the degree of water uptake by the E2As polymer.
Fig. 4 Representative NO release from SNAP/E2As catheters under
ꢃ
ꢃ
various conditions: dry and room temperature (25 C); dry and 55 C;
and humid (ꢄ50% RH) and 55 C.
ꢃ
catheters were implanted for 7 days in the jugular veins of sheep
(1 SNAP/E2As catheter and 1 E2As control catheter per animal).
This 7 day implantation period was selected to mimic potential
clinical applications (e.g., Intensive Care Units, etc.) where
51
thrombosis and infection causes complications. Following
catheter implantation, sheep were monitored closely for
changes in behavior, weight, appearance, and activity level. All
the sheep recovered rapidly from the surgical procedure and
returned to a normal activity level. At the time of catheter
explantation, precautions were taken to remove the catheter
from the vessel without disrupting the catheter surface. The
vessel was cut longitudinally to carefully remove the entire
catheter. The catheter was briey rinsed in sterile saline solu-
tion. Any residual thrombus on the catheter was photographed.
Fig. 5 Representative images of SNAP/E2As and E2As control cathe-
ters after 7 days of implantation in sheep veins (A). The catheters used
Explanted catheters were systematically cut into 1 cm sections in this study had an i.d. of 2.08 ꢂ 0.06 mm, and o.d. of 3.30 ꢂ 0.12 mm,
and were 15 cm long. Two-dimensional representation of thrombus
formation on the SNAP/E2As and E2As control catheters after 7 days of
starting at the distal tip for post-implantation NO release
measurements and bacterial adhesion testing.
implantation in sheep veins (B). Data represents the mean ꢂ SD (n ¼ 5).
*
¼ p < 0.05, SNAP/E2As vs. E2As control.
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Fig. 6 Bacterial adhesion on SNAP/E2As and E2As control catheters
after 7 days of implantation in sheep veins. Data represents the mean ꢂ
SD (n ¼ 5). * ¼ p < 0.05, SNAP/E2As vs. E2As control.
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. Conclusions
In this study, the SNAP-doped E2As polymer previously char- 14 M. A. Fischbach and C. T. Walsh, Science, 2009, 325, 1089–
30
acterized was used to fabricate intravascular catheters and
1093.
these devices were evaluated for their hemocompatibility and 15 G. Taubes, Science, 2008, 321, 356–361.
antimicrobial properties in a sheep model. The SNAP-doped 16 J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber
ꢁ
10
E2As catheters release physiological levels of NO (>0.5 ꢀ 10
and H. M. Lappin-Scott, Annu. Rev. Microbiol., 1995, 49, 711–
745.
ꢁ
2
ꢁ1
mol cm min ) for up to 20 days. The SNAP/E2As catheters
were found to withstand ethylene oxide sterilization, main- 17 L. Hall-Stoodley, J. W. Costerton and P. Stoodley, Nat. Rev.
taining ca. 89% of the original SNAP aer such treatment. It was
Microbiol., 2004, 2, 95–108.
further determined that the SNAP/E2As catheters signicantly 18 G. O'Toole, H. B. Kaplan and R. Kolter, Annu. Rev. Microbiol.,
reduce the amount of thrombus and bacterial adhesion, in
2000, 54, 49–79.
comparison to non-NO release control catheters, aer 7 days of 19 M. R. Parsek and P. K. Singh, Annu. Rev. Microbiol., 2003, 57,
intravascular implantation in sheep. This study demonstrates
677–701.
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to improve the hemocompatibility and antimicrobial properties
Microbiol., 2005, 13, 34–40.
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implantation periods.
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2
2
2
Acknowledgements
This work was supported by grants from the National Institutes
of Health (EB-000783, EB-004527 and K25HL111213). The
authors wish to thank AorTech International for the gi of
Elast-eon E2As polymer. The authors would also like to thank Dr
Chuanwu Xi and Dr Jianfeng Wu for their assistance in
providing access to the bacteria homogenizer.
2
2
27 A. W. Carpenter and M. H. Schoensch, Chem. Soc. Rev.,
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28 M. C. Frost, M. M. Reynolds and M. E. Meyerhoff,
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