U.S. patent application number 15/116165 was filed with the patent office on 2017-02-02 for thromboresistant/bactericidal s-nitroso-n-acetylpenicillamine (snap)-doped nitric oxide release polymers with enhanced stability.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Elizabeth J. Brisbois, Hitesh Handa, Mark E. Meyerhoff.
Application Number | 20170028106 15/116165 |
Document ID | / |
Family ID | 51300259 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170028106 |
Kind Code |
A1 |
Brisbois; Elizabeth J. ; et
al. |
February 2, 2017 |
THROMBORESISTANT/BACTERICIDAL S-NITROSO-N-ACETYLPENICILLAMINE
(SNAP)-DOPED NITRIC OXIDE RELEASE POLYMERS WITH ENHANCED
STABILITY
Abstract
In an example of a method for making an NO-releasing polymeric
composition, a discrete RSNO adduct is dissolved in a solvent to
form a discrete RSNO adduct solution. A polymer material is soaked
in the discrete RSNO adduct solution for a predetermined time to
swell the polymer material and impregnate the polymer material with
the discrete RSNO adduct.
Inventors: |
Brisbois; Elizabeth J.; (Ann
Arbor, MI) ; Handa; Hitesh; (Ann Arbor, MI) ;
Meyerhoff; Mark E.; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN |
Ann Arbor |
MI |
US |
|
|
Family ID: |
51300259 |
Appl. No.: |
15/116165 |
Filed: |
November 5, 2014 |
PCT Filed: |
November 5, 2014 |
PCT NO: |
PCT/US2014/064145 |
371 Date: |
August 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US14/15086 |
Feb 6, 2014 |
|
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15116165 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/7007 20130101;
C08J 7/02 20130101; A61L 33/04 20130101; A61L 33/0088 20130101;
C08J 5/18 20130101; C08J 2383/04 20130101; A61L 2300/114 20130101;
A61L 29/085 20130101; C08J 7/065 20130101; C08J 2375/04 20130101;
A61K 31/198 20130101; A61P 7/00 20180101; A61K 31/197 20130101;
A61L 29/16 20130101; A61L 2300/404 20130101; A61L 33/0082 20130101;
A61K 33/00 20130101; A61L 29/085 20130101; C08L 75/04 20130101;
A61L 29/085 20130101; C08L 83/04 20130101 |
International
Class: |
A61L 29/16 20060101
A61L029/16; C08J 7/02 20060101 C08J007/02; A61L 29/08 20060101
A61L029/08; C08J 7/06 20060101 C08J007/06; A61K 31/198 20060101
A61K031/198; A61K 33/00 20060101 A61K033/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
EB-004527, EB-000783 K25HL111213 and NIH-R42-DK100161-02 awarded by
the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A method for making an NO-releasing polymeric composition,
comprising the steps of: dissolving a discrete RSNO adduct in a
solvent to form a discrete RSNO adduct solution; and soaking a
polymer material in the discrete RSNO adduct solution for a
predetermined time to swell the polymer material and impregnate the
polymer material with the discrete RSNO adduct.
2. The method as defined in claim 1 wherein the polymer matrix is
selected from the group consisting of a siloxane-based polyurethane
elastomer, silicone rubber, and a thermoplastic
silicone-polycarbonate-urethane, and wherein the discrete RSNO
adduct is S-nitroso-N-acetylpenicillamine (SNAP).
3. The method as defined in claim 1 wherein the dissolving and the
soaking are accomplished at room temperature ranging from about
17.degree. C. to about 24.degree. C.
4. The method as defined in claim 1 wherein dissolving is
accomplished by: adding the S-nitroso-N-acetylpenicillamine (SNAP)
to the solvent, wherein the solvent is selected from the group
consisting of tetrahydrofuran, chloroform, methylene chloride,
cyclohexanone, and combinations thereof; and stirring the solution
for a predetermined time.
5. The method as defined in claim 1, further comprising completely
immersing the polymer material in the discrete RSNO adduct solution
prior to the soaking.
6. The method as defined in claim 1 wherein the predetermined time
is about 24 hours, and wherein the soaking is accomplished in
darkness.
7. The method as defined in claim 1 wherein after the soaking for
the predetermined time, the method further comprises drying the
impregnated polymer material in darkness.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application Serial No. PCT/US2014/015086, filed Feb. 6, 2014, which
itself claims the benefit of U.S. Provisional Application Ser. No.
61/762,013, filed Feb. 7, 2013, both of which are incorporated by
reference herein in their entirety.
BACKGROUND
[0003] Nitric oxide (NO), a gas molecule endogenously produced by
various NO synthase (NOS) enzymes, has been shown to have several
important physiological functions, including its unique
vasodilating properties, cancer-fighting potency, antibacterial
and/or anti-biofilm properties (e.g., inhibiting proliferation of
bacteria, fungi and viruses), and anti-platelet activity. Although
NO is a stable radical, it may be highly reactive with hemoglobin
and oxygen, thus making delivery of NO to the target site
challenging. Stable hydrophilic NO donors, as well as hydrophobic
NO donors may be best to take advantage of the potency of NO for a
wide range of biomedical applications. These applications include
NO-releasing pharmaceuticals and the preparation of
thromboresistive hydrophobic polymeric coatings for medical
devices, such as intravascular catheters and extracorporeal
circuits (based on NO's antiplatelet activity). However, despite
the benefits of NO, the use of NO donors in polymeric systems has
been relatively limited for various reasons, including storage
stability, costly synthesis, and/or limited NO release
lifetimes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] Features and advantages of examples of the present
disclosure will become apparent by reference to the following
detailed description and drawings, in which like reference numerals
correspond to similar, though perhaps not identical, components.
For the sake of brevity, reference numerals or features having a
previously described function may or may not be described in
connection with other drawings in which they appear.
[0005] FIG. 1A is a schematic cut-away view showing thrombus
formation on a siloxane-based polyurethane elastomer (e.g.,
ELAST-EON.TM. E2As) control coated extracorporeal circulation (ECC)
circuit;
[0006] FIG. 1B is a schematic cut-away view showing a
S-nitroso-N-acetylpenicillamine (SNAP)/E2As coated ECC circuit,
which releases nitric NO and reduces thrombus formation;
[0007] FIG. 2A shows the structure of
S-nitroso-N-acetyl-D,L-penicillamine (SNAP);
[0008] FIG. 2B shows a scheme of S-nitrosothiol (RSNO)
decomposition, which can be catalyzed by metal ions (e.g.,
Cu.sup.+), light, and heat, yielding the disulfide (RSSR) product
and nitric oxide (NO);
[0009] FIGS. 3A and 3B show diffusion of SNAP from various polymer
films containing 10 wt % SNAP, monitored at 340 nm, films were
immersed in 4 mL phosphate buffered saline (PBS) in the dark at
room temperature, 22.degree. C. (FIG. 3A) or 37.degree. C. (FIG.
3B) (the data is the mean.+-.SEM (standard error of the mean)
(n=3));
[0010] FIG. 4A shows NO release behavior of 10 wt % SNAP/E2As film
at 37.degree. C. in the dark, ambient light, and 100 W floodlight
(the data is the mean.+-.SEM (n=3));
[0011] FIG. 4B shows NO release from 10 wt % SNAP in silicone
rubber (SR), CARBOSIL.RTM., and ELAST-EON.TM. E2As films at
37.degree. C. and continuously irradiated with the 100 W floodlight
(the data is the mean.+-.SEM (n=3));
[0012] FIG. 4C shows NO release from 5 wt % and 10 wt % SNAP in
Elast-Eon.TM. E2As films at 37.degree. C. continuously under
ambient light (amb) or the 100 W floodlight (the data is the
mean.+-.SEM (n=3));
[0013] FIG. 5A shows UV-vis spectra of a 10 wt % SNAP/E2As film,
1.0 mM SNAP, and E2As dissolved in N,N-dimethylacetamide (DMAc)
(the data is the mean.+-.SEM (n=3));
[0014] FIG. 5B shows cumulative NO release from 10 wt % SNAP/E2As
films incubated in PBS under various conditions: room temperature
(22.degree. C.) with ambient light, 37.degree. C. in the dark,
37.degree. C. under ambient light, and 37.degree. C. under the 100
W floodlight (the data is the mean.+-.SEM (n=3));
[0015] FIG. 6A shows diffusion of SNAP from 10 wt % SNAP-doped E2As
films soaking in 1 mL PBS in the dark, as monitored at 340 nm, at
room temperature (RT, 22.degree. C.) or 37.degree. C.;
[0016] FIG. 6B shows a comparison of the cumulative SNAP leaching
and cumulative NO release (from NOA-based or SNAP-based NO release
data) from the 10 wt % SNAP-doped E2As films soaking in PBS at
37.degree. C. in the dark. Nitric oxide release from SNAP-doped
E2As films can occur from thermal and/or photochemical
decomposition of SNAP within the polymer phase, or from SNAP that
leached into the aqueous phase. For the SNAP-doped E2As films,
approximately 26% of the total NO release is attributed to the SNAP
leaching;
[0017] FIG. 7 is a graph showing diffusion of SNAP from 10 wt %
SNAP in E2As films with 0, 2, or 4 top coats of E2As as monitored
at 340 nm by UV-vis, the films were soaked in 10 mM PBS containing
100 .mu.M EDTA, which was replaced daily after the UV-vis
measurement, at 37.degree. C. in the dark (the data is the
mean.+-.SEM (n=3));
[0018] FIG. 8 is a graph showing stability of 10 wt % SNAP in E2As
films stored dry with desiccant under various temperature and light
conditions, the films were dissolved in DMAc to determine the
amount of SNAP remaining at various times (compared to the initial
level) as monitored at 340 nm by UV-vis (the data is the
mean.+-.SEM (n=3));
[0019] FIG. 9 is a graph showing NO release behavior from 10 wt %
SNAP/E2As dry film at 37.degree. C. in the dark;
[0020] FIG. 10 is a schematic diagram of the extracorporeal circuit
(ECC) tubing coated with 5 wt % SNAP/E2As followed by a top coat of
E2As;
[0021] FIGS. 11A and 11B are graphs showing representative NO
surface flux profiles from a section of ECC tubing coated with 5 wt
% SNAP in E2As before (FIG. 11A) and after (FIG. 11B) blood
exposure, NO release was measured via chemiluminescence at
37.degree. C. under ambient light;
[0022] FIGS. 12A and 12B are graphs showing time-dependent effects
of the 5 wt % SNAP/E2As coating on platelet count (e.g.,
consumption) (FIG. 12A) and plasma fibrinogen levels (FIG. 12B)
during the 4 hour blood exposure in the rabbit thrombogenicity
model (the data is the mean.+-.SEM (n=4));
[0023] FIG. 13 is a graph showing results of in vitro fibrinogen
adsorption assays on the 5 wt % SNAP/E2As and E2As control coatings
(fluorescence assay in a 96-well plate that used goat anti-human
fibrinogen-FITC conjugated antibody to measure the level of
adsorbed human fibrinogen (3 mg/mL) on the coatings, the data is
the mean.+-.SEM (n=24));
[0024] FIG. 14 is a two-dimensional representation of thrombus
formation on the SNAP/E2As and control ECCs after 4 hour blood
exposure in the rabbit thrombogenicity model, as quantified using
ImageJ software from NIH (the data is the mean.+-.SEM (n=4));
[0025] FIG. 15 is a graph showing an NO release profile of
polyurethane tubing (a micro-polyurethane tubing available from
Scientific Commodities, Inc.) impregnated with SNAP, the tubing
having been soaked in a SNAP/acetone solution for either 1 or 2
days;
[0026] FIG. 16A is a schematic illustration of a catheter tubing
coated with an active layer of 5 wt % or 10 wt % SNAP/E2As followed
by a top coat of E2As;
[0027] FIG. 16B is a cross-sectional view of the catheter tubing of
FIG. 16A, taken along line 16B-16B of FIG. 16A;
[0028] FIG. 17 shows NO release profiles of 5 wt % and 10 wt %
SNAP/E2As catheters at 37.degree. C. in the dark (n=5);
[0029] FIG. 18 is a graph showing quantitation of thrombus area on
SNAP/E2As catheters and E2As control catheters after 7 day
implantation in sheep, as calculated with NIH ImageJ software using
a 2D representation of thrombus (the data are means.+-.SEM
(n=5)),*=p<0.05;
[0030] FIG. 19 is a comparison of bacterial adhesion (CFU/cm.sup.2)
on 1 cm piece of explanted SNAP/E2As catheters and E2As control
catheters (the data are means.+-.SEM (n=5)),*=p<0.05;
[0031] FIG. 20A is a graph illustrating the release of NO from SNAP
impregnated silicone Foley catheter tubings over a 30 day period
(data are the mean.+-.SEM (n=3));
[0032] FIG. 20B is a graph illustrating the NO release profile of
one SNAP impregnated catheter segment after 3 days of soaking in
phosphate buffered saline (PBS);
[0033] FIG. 21 is a graph illustrating the percentage of total
loaded SNAP that leaches from the surface of the silicone tubing
during the first 7 days of soaking in PBS at 37.degree. C. (data
are the mean.+-.SEM (n=3));
[0034] FIGS. 22A and 22B are, respectively, plate count results and
fluorescence images for S. epidermidis biofilms developed on Foley
catheters segments in a CDC biofilm reactor for 3, 7 and 14 days,
where FIG. 22A illustrates the plate count of the number of viable
bacteria adhered to the SNAP impregnated and control catheter
surfaces, and where FIG. 22B includes the black and white
representations of representative fluorescence images of the SNAP
impregnated and control catheter surfaces taken with an oil
immersion 60.times. objective lens;
[0035] FIGS. 23A and 23B are, respectively, plate count results and
fluorescence images for P. mirabilis biofilms developed on Foley
catheters segments in a CDC biofilm reactor for 3, 7 and 14 days,
where FIG. 23A illustrates the plate count of the number of viable
bacteria adhered to the SNAP impregnated and control catheter
surfaces, and where FIG. 23B are the black and white
representations of representative fluorescence images of the SNAP
impregnated and control catheter surfaces taken with an oil
immersion 60.times. objective lens or with a 10.times. objective
lens (the latter of which was used for the 14 day control catheter
only);
[0036] FIGS. 24A and 24B are, respectively, plate count results and
fluorescence images for P. mirabilis formation on SNAP impregnated
and control (no SNAP) Foley catheter segments after 3 days in CDC
reactor for catheter segments that were first soaked in PBS buffer
for 24 hours before the start of the biofilm formation experiments,
to reduce any initial higher flux of NO from the outer surface of
the SNAP-impregnated catheter segments, where FIG. 24A illustrates
the plate count of the number of viable bacteria adhered to the
SNAP impregnated and control catheter surfaces, and where FIG. 24B
includes the black and white representations of representative
fluorescence images of the SNAP impregnated and control catheter
surfaces taken with an oil immersion 60.times. objective lens;
and
[0037] FIG. 25 is a graph illustrating the nitric oxide flux of a
SNAP impregnated catheter segment after 3 days of P. mirabilis
biofilm growth in a CDC biofilm reactor (n=3), where the catheter
pieces had been pre-soaked in PBS buffer for 24 hours prior to the
start of the experiment, where the 96 hour and 120 hour time-points
correspond to the day of cell viability assessment and the
following day.
DETAILED DESCRIPTION
[0038] Examples according to the present disclosure include a novel
RSNO-doped polymer formulation useful for making biomedical
devices. In some examples, the novel polymer formulations form
homogeneous films and exhibit RSNO stability even at 37.degree. C.
for 4 months (with only about a 10%-15% loss of NO).
[0039] In some examples, the novel polymer formulations may be
formulated and used as coatings to prevent thrombus (i.e., blood
clot) formation in, e.g., extracorporeal circulation (ECC)
circuits. FIG. 1A is a schematic cut-away view showing a
siloxane-based polyurethane elastomer (e.g., Elast-Eon.TM. E2As)
control coated ECC circuit 12 that exhibits thrombus formation. As
illustrated, the red blood cells 14 and platelets 16 clot together.
In contrast, FIG. 1B is a schematic cut-away view showing an
example 10 according to the present disclosure of a
S-nitroso-N-acetylpenicillamine (SNAP)-doped siloxane-based
polyurethane elastomer (e.g., SNAP/E2As) coated ECC circuit that
does not exhibit thrombus formation. As depicted in FIG. 1B, NO is
generated, which contributes to the red blood cells 14 and
platelets 16 not clotting together.
[0040] In some other examples, the novel polymer formulations may
be formulated by dissolving the SNAP (or other suitable discrete
RSNO adduct) in a suitable solvent to form a solution, and then
soaking a polymer (e.g., an existing low water uptake biomedical
polymer) in the solution. Soaking is allowed to occur for a
predetermined time (e.g., at least 24 hours) to swell the polymer
material and impregnate the polymer material with the discrete RSNO
adduct. Examples of the polymer include silicone rubber,
siloxane-based polyurethane elastomers, thermoplastic
silicone-polycarbonate-urethane, etc., and examples of the solvent
include tetrahydrofuran, chloroform, methylene chloride,
cyclohexanone, and combinations thereof. The swelling/impregnation
method disclosed herein is particularly suitable for introducing
SNAP into Foley catheters (i.e., indwelling urinary catheter formed
of a flexible tube and having two separated lumens). In an example,
the swelling/impregnation method is conducted at room temperature
(e.g., from about 17.degree. C. to about 24.degree. C.) with
commercially available Foley catheters, and therefore avoids
exposing the SNAP to elevated temperatures that are often
associated with catheter extrusion processes. As such, in the
swelling/impregnation method disclosed herein, the SNAP is not
exposed to elevated temperatures that would render it unstable and
cause it to prematurely decompose. Several advantages have been
observed with SNAP-impregnated Foley catheter tubing, such as a
steady release of NO for one month at physiological and
antimicrobial levels, and a decrease in the degree of microbial
biofilm formation after 14 days of exposure to flowing media
inoculated with P. mirabilis or Staphylococcus (S.) epidermidis,
two bacterial strains that most often induce catheter-associated
urinary tract infections (CAUTIs).
[0041] Blood/material interaction is important to the success of
implantable medical devices, ranging from simple catheters, stents
and grafts, to complex extracorporeal artificial organs that are
used in thousands of patients every day. Thrombosis is one of the
primary problems associated with clinical application of blood
contacting materials. Despite a thorough understanding of the
mechanisms of blood-surface interactions and decades of
bioengineering research effort, the ideal non-thrombogenic
prosthetic surface remains an unsolved problem. Over the last 50
years, much has been learned about surface-induced thrombosis and
attempts to prevent it with systemic anticoagulation and surface
modifications. Surface modifications have included using pure, very
smooth silicone rubber or polyurethane, pre-exposure of the
surfaces to albumin and other coating proteins, and surface binding
of heparin in an ionic as well as a covalent fashion. Despite
extensive research to develop a non-thrombogenic surface that
mimics the endothelium, none of these modifications have been
successful.
[0042] Nitric oxide (NO) has been found to be one of two potent
vasodilators secreted by normal endothelium that has the ability to
inhibit platelet adhesion/activation and aggregation to the blood
vessel wall. The NO-flux from a normal and stimulated endothelium
has been estimated to be in the range of 0.5.times.10.sup.-10 mol
cm.sup.-2 min.sup.-1 to 4.times.10.sup.-10 mol cm.sup.-2
min.sup.-1. Nitric oxide has been extensively studied for its
inhibitory effects on circulating platelet and monocyte activation
that leads to aggregation and ultimately initiation of thrombosis.
A wide range of NO donors such as S-nitrosothiols (RSNOs),
N-Hydroxy-N-nitrosoamines, N-diazeniumdiolates and nitrosyl metal
complexes have been studied at least over the past decade.
[0043] Nitric oxide (NO) can be released from an NO adduct/donor
species appended to polymers within a polymer coating. "Nitric
oxide adducts" (NO adducts) and "NO-donors" refer to compounds and
functional groups which, under physiological conditions, can donate
and/or release NO such that biological activity of the NO is
expressed at the intended site of action/target site.
[0044] Some examples according to the present disclosure include
the NO donor/adduct within a polymer coating. The NO donor/adduct
may be integrated into the polymer coating in any suitable manner,
examples of which include doping and swelling/impregnating.
Suitable NO adducts (examples of which include discrete adducts)
are generally those exhibiting capability of embedding (either by
covalent attachment and/or dispersion) into the polymer matrix and
exhibiting process preparation stability.
[0045] "Discrete NO adducts" as referred to herein are those NO
adducts (examples of which are RSNOs) which, when placed into a
polymer matrix, release therapeutically relevant fluxes of NO,
ranging from about 0.2.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 to
about 20.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 of NO from the
polymer phase. Those compounds that have their NO-releasing moiety
covalently attached to a polymer backbone are generally referred to
as "polymeric NO adducts." Examples of suitable polymeric NO
adducts include, but are not limited to, S-nitrosothiolated
polyurethanes, S-nitrosothiolated silicone rubbers, and/or mixtures
thereof. Some examples of the discrete NO adducts exhibit some
lipophilicity, but may be made more lipophilic by derivatization
with one or more alkyl groups.
[0046] As such, examples of the present disclosure are novel nitric
oxide (NO) releasing formulations formed from polymers doped or
impregnated with S-nitroso-N-acetylpenicillamine (SNAP) to prevent
thrombus formation in, e.g., extracorporeal circulation (ECC)
circuits and catheter tubing.
[0047] Various hydrophobic polymer materials may be employed in
examples of the material, method, and device as disclosed herein.
These include, but are not limited to materials such as
polyurethanes (PU), silicone rubbers (SR), copolymers of
polyurethane and silicone rubber (e.g., E2A), poly(vinyl chloride)
(PVC), polymethacrylates, polyacrylates, polycaprolactones, and/or
mixtures thereof. In other examples, the polymer material may
include both hydrophobic and hydrophilic domains. The polymer of
choice will be one capable of releasing NO from, for example,
covalently attached and/or dispersed S-nitrosothiol (RSNO) type
NO-adducts within the polymer. The polymer of choice may also
depend upon the application in which polymer coating/film will be
used and the desired NO release rate for that application. As
examples, a polymer having higher water uptake may be suitable in
applications where quick NO release is desirable, while a polymer
having lower water uptake may be suitable in applications were slow
NO release is desirable. In instances where prolonged NO release is
desirable, poly(lactic-co-glycolic acid) (PLGA) additives may also
be included in the polymer coating/film to create an acidic
environment to further stabilize the RSNO species.
[0048] Further, a system is contemplated as being within the
purview of the present disclosure that includes discrete RSNOs
doped into a polymer, with the polymer also having RSNO appended
thereto (e.g., by covalent attachment). For example, previously
prepared polyurethane polymers with appended RSNO functional groups
can be mixed with discrete RSNOs or similar species to create the
long-term NO release polymers enabled by the present
disclosure.
[0049] In some examples, the NO adduct of choice is one capable of
spontaneous release of NO when the polymer is exposed to solutions
and/or blood under physiological conditions. In other examples, the
NO adduct of choice is one capable of spontaneous release of gas
phase NO when the polymer is exposed to certain light conditions.
Some examples of NO adducts include discrete S-nitrosothiols
(RSNOs).
[0050] It is believed that examples of the present disclosure
including SNAP doped into siloxane-based polyurethane elastomers
(one example of which is E2As) may help stabilize the RSNO adduct,
thus advantageously allowing longer NO release from the RSNO
species and enhanced storage stability, even at higher temperatures
(e.g., 37.degree. C.).
[0051] Spontaneous release of NO from the polymer may be governed
by at least one process occurring between the NO adduct and the
surrounding environment. For RSNO species, these include, but are
not limited to temperature, moisture, and the presence of certain
wavelengths of light. For example, photolysis of the S--N bond in
the RSNO species liberates NO gas. Photolysis can occur with light
in either the 300 nm to 400 nm wavelength range or the 500 nm to
600 nm wavelength range. In this example, the efficiency of NO
release is generally greater in the higher wavelength range.
[0052] It is to be understood that discrete nitric oxide adducts
may be either covalently attached to the polymer matrix or may be
dispersed therein, or both. Some examples of discrete RSNOs
include, but are not limited to S-nitrosoglutathione (GSNO),
S-nitroso-N-acetylpenicillamine (SNAP, shown in FIG. 2A),
S-nitrosocysteine (CysNO), etc., and derivatized discrete RSNOs.
Derivatized RSNOs may be modified with alkyl group(s). As examples,
a derivative may have an alkyl group attached to the free carboxyl
group of SNAP and/or may have a longer alkyl (i.e., longer than
acetyl) attached to the amine group of S-nitrosopenicillamine. As
an example, an ester linkage may be formed between the desired
alkyl group and the free carboxyl group of SNAP. As another
example, a long chain alkyl (including from 4 to 10 carbon atoms)
may replace the acetyl group of SNAP so that the long chain alkyl
is attached to the amine nitrogen. As other examples, a sugar may
be attached to the carboxyl group of SNAP (e.g., glucose-SNAP,
mannose-SNAP, fructose-SNAP, etc.).
[0053] The SNAP-doped NO release siloxane-based polyurethane
elastomer coatings according to examples of the present disclosure
were evaluated in vitro and within a short-term in vivo rabbit
model of thrombogenicity. The novel coatings according to examples
of the present disclosure continuously released from 0.5 to
1.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 NO for 20 days in the
dark, soaking at 37.degree. C. in PBS. Additionally, the novel
coatings retained about 78% of the SNAP after 4 months at
37.degree. C. in the dark (i.e., not exposed to wavelengths that
could photolyze RSNO bonds) and in dry conditions (i.e., in the
presence of a desiccant). As discussed further herein, examples of
the novel coating materials were employed as inner wall coatings of
extracorporeal circuits used for 4 hours of extracorporeal
circulation (ECC) in a rabbit model of thrombogenicity to examine
the effect of the coatings on platelet function, clotting and
fibrinogen adsorption. The SNAP-doped NO release coatings were also
used to fabricate catheters, which were implanted in sheep veins
for 7 days to evaluate the effects on thrombus and bacterial
adhesion.
[0054] The SNAP impregnated catheter tubings were also evaluated in
vitro. These tubings exhibited significant anti-biofilm properties
using two bacteria strains (S. epidermidis and P. mirabilis) that
are responsible for high rates of nosocomial urinary catheter
associated infections.
[0055] As mentioned above, nitric oxide (NO) is an endogenous gas
molecule that plays several key physiological roles, including
prevention of platelet adhesion and activation, inhibiting
bacterial adhesion and proliferation, enhancing vasodilation,
promoting angiogenesis, and aiding in wound healing. The effects of
NO are highly dependent on the location of the NO and its
concentration in the physiological system. For example, endothelial
cells that line the inner walls of healthy blood vessels produce an
estimated NO surface flux ranging from 0.5 mol cm.sup.-2 min.sup.-1
to 4.0.times.10.sup.-10 mol cm.sup.-2 min.sup.-1. The function of
many blood-contacting devices, including vascular grafts, stents,
intravascular sensors, intravascular catheters, and extracorporeal
life support circuits, can be impaired due to platelet activation
and thrombus formation. One approach to improve the
hemocompatibility of such devices is the use of coating materials
that mimic the endothelial cells with respect to NO release.
Indeed, in recent years there has been considerable interest in
developing NO-release and NO-generating materials that can be used
to improve the biocompatibility of such devices.
[0056] Nitric oxide also exhibits antimicrobial activity, including
killing bacteria and preventing biofilm formation. Bacterial
infections and biofilm formation are problems that can cause
complications with biomedical devices. Bacteria also possess the
ability to form biofilms on surfaces when the organism secretes a
polysaccharide matrix in which the bacteria will live. This matrix
provides both nutrients and protection against the host defense and
antibiotics. Biofilms can act as a source of chronic infection,
thereby prolonging the recovery time. Among its many biological
roles, nitric oxide functions as an antimicrobial agent and as an
accelerant to the wound healing process. Nitric oxide has
broad-spectrum antibacterial properties, killing both gram-positive
and gram-negative bacteria. Low levels of nitric oxide are also
reported to efficiently disperse biofilms that have formed on the
surface of indwelling medical devices.
[0057] Nitric oxide is highly reactive under physiological
conditions, and thus a wide range of NO donor molecules with
functional groups that can store and release NO have been studied
for potential biomedical applications. Such molecules include
organic nitrates, metal-NO complexes, N-diazeniumdiolates, and
S-nitrosothiols (RSNOs). Physiological RSNOs, such as
S-nitrosohemoglobin and S-nitrosoglutathione (GSNO), are considered
an endogenous reservoir of NO in vivo. Other synthetic RSNOs, such
as S-nitroso-N-acetyl-L-cysteine (SNAC) and
S-nitroso-N-acetylpenicillamine (SNAP, FIG. 2A) have been shown to
exhibit significant antimicrobial and antithrombotic effects. It
has also been demonstrated that RSNOs are both vasodilators and
potent inhibitors of platelet aggregation. RSNOs undergo thermal
decomposition, releasing NO and producing a corresponding disulfide
species (RSSR), as shown in FIG. 2B. The NO release from RSNOs can
be catalyzed by metal ions (e.g., Cu.sup.+) and by light, through
the irradiation at energies that correspond to the S-nitroso
absorption bands at 340 nm and/or 590 nm. It has been suggested
that the more potent activity of RSNOs vs. NO as antiplatelet
agents arises from the enhanced stability of RSNOs vs. NO, and
generation of NO from RSNOs locally at the surface of platelets by
membrane proteins that contain catalytic sites to convert RSNO to
NO.
[0058] Incorporation of RSNOs into polymers can extend the utility
of these NO donors to be applicable as coatings in biomedical
devices, providing localized NO release at the blood/device
interface. Several NO-release polymers consisting of small-molecule
RSNOs dispersed in various polymer matrices, including polyethylene
glycol (PEG), poly(vinyl alcohol), poly(vinyl pyrrolidone), and
PLURONIC.RTM. F127 hydrogel, have been suggested. These materials
have potential applications for topical NO delivery on wounds via
the diffusion of the hydrophilic RSNOs from the polymer to the
tissue. In fact, daily application of a GSNO-containing hydrogel
has been shown to accelerate the wound healing process. However,
the rapid leaching of the RSNOs from such polymers can
significantly shorten the NO/RSNO release lifetime, lasting only
several hours. An alternate approach has been to synthesize
RSNO-modified materials, where the RSNO functionality is covalently
bound to the matrix. Fumed silica particles, dendrimers,
polyurethanes, polyesters, polydimethylsiloxane (PDMS), xerogels,
self-assembled monolayers, and poly(vinyl methyl ether-co-maleic
anhydride) (PVMMA) have all been modified with RSNO
functionalities. RSNO-modified xerogels were found to release NO
for up to 14 days and exhibit reduced platelet and bacterial
adhesion. However, such RSNO-modified xerogels suffer from
synthesis complications leading to cracking and non-uniform films,
low RSNO conversion efficiency (maximum of 40% for the tertiary
RSNO-modified xerogels), and thermal instability at room
temperature that would limit their shelf-life. Many of the other
RSNO modified materials reported to date exhibit both thermal and
photoinitiated NO release, but many of these materials have not
proven clinically useful due to their limited NO release lifetimes
or lack of the RSNO functionality stability during storage, or low
conversion to RSNO during synthesis.
[0059] Another approach reported to achieve localized NO delivery
at a polymer/blood interface is to use NO-generating coatings, in
which immobilized catalysts (Cu(I/II) or organoselenium species)
can generate NO from endogenous RSNOs. For example, a NO generating
coating containing Cu.sup.0 nanoparticles was evaluated recently
using a rabbit model for extracorporeal circulation (ECC). However,
to achieve good efficacy in reducing thrombus formation, continuous
infusion of SNAP was required to supplement the endogenous RSNO
levels.
[0060] In order to avoid the continuous infusion of RSNO species,
the present disclosure includes several biomedical polymers that
are capable of storing RSNO species. The RSNO-doped coatings and
RSNO-impregnated polymers according to the present disclosure can
advantageously release NO, as well as potentially supplement the
endogenous RSNO levels, if NO generating catalysts are also
employed.
[0061] To further illustrate the present disclosure, examples are
given herein. It is to be understood that these examples are
provided for illustrative purposes and are not to be construed as
limiting the scope of the present disclosure.
Example 1
[0062] In this example of the present disclosure, five biomedical
grade polymers doped with S-nitroso-N-acetylpenicillamine (SNAP)
were investigated for their potential to control the release of NO
from the SNAP within the polymers, and further control the release
of SNAP itself. As discussed further herein, SNAP is quite stable
in the ELAST-EON.TM. E2As polymer, creating a homogeneous coating
that can locally deliver NO (via thermal and photochemical
reactions) as well as slowly release SNAP. E2As is an example of
suitable siloxane-based polyurethane elastomers contemplated as
being within the purview of the present disclosure. E2As is a
solution grade of E2A (see Table 1 below). The E2As polymer
containing SNAP was coated on the walls of extracorporeal circuits
(ECC) and exposed to 4 hour blood flow in a rabbit model of
extracorporeal circulation to examine the effects on platelet
count, platelet function, clot area, and fibrinogen adsorption.
After 4 hours, platelet count was preserved at 100.+-.7% of
baseline for the SNAP/E2As coated loops, compared to 60.+-.6% for
E2As control circuits (n=4). The SNAP/E2As coating also reduced the
thrombus area when compared to the control (2.3.+-.0.6 and
3.4.+-.1.1 cm.sup.2, respectively). As will be discussed further
herein, the SNAP/E2As catheters were also able to significantly
reduce the thrombus area and bacterial adhesion after 7 day
implantation in sheep veins. All of the results suggest that the
new SNAP/E2As coatings have potential to improve the
thromboresistance of intravascular catheters, grafts, and other
blood contacting medical devices.
[0063] The present inventors also examined the five biomedical
polymers (silicone rubber (SR), ELAST-EON.TM. E2As (a siloxane-base
polyurethane elastomer commercially available from Aortech
Biomaterials, Scoresby Victoria, Australia), CARBOSIL.RTM. (a
thermoplastic silicone-polycarbonate-urethane commercially
available from DSM Biomedical Inc., Berkeley, Calif.), TECOFLEX.TM.
SG80A and TECOPHILLIC.TM. SP-60D-60 (both polyurethanes
commercially available from The Lubrizol Corporation, Wickliffe,
Ohio)) for their potential to act as a storage reservoir for SNAP.
The ELAST-EON.TM. polymer has excellent intrinsic biocompatibility
and biostability properties, and exhibits low levels of blood
protein adsorption. Each of the SNAP-doped polymers is
characterized for its in vitro NO/SNAP release. The present
inventors have found that SNAP itself is stable for at least 4
months in the ELAST-EON.TM. E2As polymer, creating a coating that
releases NO thermally (at physiological temperature) and can also
serve as a reservoir to supplement endogenous RSNO levels (by SNAP
diffusion into blood from the polymer). The new SNAP/E2As polymer
was tested for potential biomedical applications via, e.g., an ECC
rabbit model of thrombogenicity to assess preservation of platelet
count and function, and thrombus area after 4 hours of ECC.
[0064] It is to be understood that other siloxane-based
polyurethane elastomers (aside from E2As) are also contemplated as
being suitable for use in the present disclosure. Further, it is to
be understood that other grades of ELAST-EON.TM. siloxane-based
polyurethane elastomers (commercially available from Aortech
Biomaterials, Scoresby Victoria, Australia) are contemplated as
being suitable for use in the present disclosure. Table 1 below is
a table of properties of various grades of ELAST-EON.TM. polymers.
In addition to those examples shown in Table 1, it is believed that
other suitable polymers include CARBOSIL.RTM., PURISIL.TM., or
silicone rubber.
TABLE-US-00001 TABLE 1 Elast-Eon .TM. Properties TEST E5-130
E5-325* E2A* E2-945 E2-852 E2-860 E2-862 E4 Durometer Hardness 77A
82A 90A 50D 55D 65D 68D 80D Tensile Strength, MPa 21 23 26 28 30 34
34 60 Elongation at Break, % >700 >500 >450 >400
>300 >200 >200 25 Tensile Stress, 100% E, MPa 4 5 8 12 15
23 -- -- Tensile Stress, 200% E, MPa 5 7 10 15 18 -- -- -- Tensile
Stress, 300% E, MPa 6 9 13 18 23 -- -- -- Modulus of Elasticity,
MPa 11 15 35 115 360 650 650 -- Tear Strength, kN/m 45 60 80 97 129
-- -- -- Physical Form Pellets Pellets** Pellets** Pellets Pellets
Pellets Pellets Pellets Melt Temperature .degree. C. 175-185
180-185 195-200 220 210-215 210-215 210-215 185 *Can also be
supplied in solution grade and is soluble in both THF and DMAc.
**Also supplied in dispersion form.
Preliminary In Vitro Characterization of Various SNAP-Doped Polymer
Films for Example 1
[0065] SNAP doped into all of the five biomedical polymers produced
homogeneous and transparent films of green color, without any
observable phase separation. The 10 wt % SNAP films stored
approximately 0.42 .mu.mol of SNAP per mg polymer film (or 6
.mu.mol/cm.sup.2), while the 5 wt % SNAP films stored approximately
0.21 .mu.mol of SNAP per mg polymer film (or 3
.mu.mol/cm.sup.2).
[0066] The 5 wt % and 10 wt % SNAP/polymer films were immersed in 4
mL PBS in the dark at room temperature (i.e., 22.degree. C.) or at
37.degree. C. The diffusion of SNAP into the PBS from the various
polymer films containing 5 wt % and 10 wt % SNAP was monitored
using UV-Vis absorption. As shown in FIGS. 3A and 3B, all of the
SNAP diffused out of the SG80A and SP-60D-60 polymer films during
the first day of soaking in PBS at room temperature (see FIG. 3A)
and at 37.degree. C. (see FIG. 3B). The SP-60D-60 polymer is
hydrophilic with a water uptake of about 60 wt %, while the SG80A
is more hydrophobic, having a water uptake of about 6 wt % (see
Table 2 below).
[0067] Table 2 illustrates the water uptake of the 5 biomedical
polymers used in the present disclosure. Polymer films (200 mg
polymer) were cast in Teflon.RTM. ring (d=2.5 cm) on Teflon.RTM.
plates. Small disks (d=0.7 cm) were cut from the parent films,
weighed, and immersed in PBS for 48 hours at 37.degree. C. The wet
films were wiped dry and weighed again. The water uptake of the
polymer films is reported in Table 2 in weight percent as follows:
water uptake (wt %)=(W.sub.wet-W.sub.dry)/W.sub.dry.times.100,
where W.sub.wet and W.sub.dry are the weights of the wet and dry
films, respectively.
TABLE-US-00002 TABLE 2 Polymer Water uptake [wt %] Silicone Rubber
1.2 .+-. 0.3 CarboSil 1.5 .+-. 0.3 Elast-Eon .TM. E2As 1.2 .+-. 0.1
Tecoflex SG80A 6.2 .+-. 0.7 Tecophilic SP-60D-60 64.5 .+-. 0.1
[0068] As shown in FIGS. 3A and 3B, all of the SNAP leaves the more
hydrophilic SP-60D-60 polymer during the initial 2 hours of
soaking, while the more hydrophobic SG80A leaches all of the SNAP
after 24 hours. Due to the rapid loss of the SNAP from the
SP-60D-60 and SG80A polymers, a very large initial burst of NO was
observed via chemiluminescence (with a Nitric Oxide Analyzer (NOA))
during the first day of soaking (Day 0), and the films exhibited no
SNAP/NO release after one day (data not shown). Therefore, these
two polymers provide a quick burst of NO/SNAP and were found not to
be suitable for longer-term release of NO/SNAP.
[0069] In contrast, the silicone rubber, CARBOSIL.RTM., and E2As
polymers exhibit significantly lower amounts of SNAP diffusing into
the soaking buffer after one day (see FIGS. 3A and 3B). For all
three of these polymers, an initial burst of SNAP leaching was
observed during the first day of soaking, corresponding to rapid
water uptake by the polymer. This initial burst was about 10% of
the total SNAP molecules incorporated into the films. Small amounts
of SNAP continued to leach from these polymers during the
subsequent days of soaking. Silicone rubber, CARBOSIL.RTM. (a
thermoplastic silicone-polycarbonate-urethane), and E2As (a
siloxane-base polyurethane elastomer) all are hydrophobic polymers
due to their high PDMS content and also have the lowest water
uptake (see Table 1 above). SNAP is slightly hydrophobic.
Therefore, SNAP should have a preference for remaining in these
more hydrophobic polymer films. In addition, it is believed that
the hydrophobic property of these polymers also plays a significant
role in limiting the diffusion of SNAP into the buffer, due, at
least in part, to the minimal water uptake of these polymers.
[0070] The thermal and photoinitiated NO release from the three
SNAP-doped polymers (i.e., silicone rubber, CARBOSIL.RTM., and E2As
polymers) was also studied by NOA measurements. Nitric oxide
release can be turned on/off using the 100 W floodlight for all 3
film types. FIG. 4A illustrates the NO release behavior of the 10
wt % SNAP/E2As film at 37.degree. C. in the dark, in the ambient
light, and in the 100 W floodlight. As shown in FIG. 4A, there is
little difference in the NO release from the 10 wt % SNAP/E2As film
in the dark or under the ambient lab lights, since ambient
fluorescent lighting does not emit the wavelengths responsible for
decomposing RSNOs (340 nm or 590 nm). While the data for the 10 wt
% SNAP/E2As film is shown, it is to be understood that for all
three polymers, the total NO release detected by the NOA for films
continuously irradiated with the 100 W floodlight was about 100% of
the SNAP doped into the films. The photoinitiated NO release from
these three films was examined by continuously irradiating with a
100 W floodlight at 37.degree. C. and monitoring the NO released
with the NOA (see FIG. 4B). The SNAP-doped E2As and CARBOSIL.RTM.
films exhibited a gradual decrease in the photo-induced NO flux
over a 3 day period, while the SR-based films released NO for only
2 days under the same conditions. All three types of films
incubated at 37.degree. C. under ambient light yielded an initial
burst of NO on the first day of soaking, corresponding to release
of SNAP into the solution, and on subsequent days, the NO flux
ranged from 1.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 to
2.times.10.sup.-10 mol cm.sup.-2 min.sup.-1. This NO flux is still
potentially useful to inhibit platelet function and kill
bacteria.
[0071] It appears that the NO release may be more promising from
the film composed of 10 wt % SNAP in E2As under the 100 W
floodlight. Therefore, the wt % of SNAP in E2As was varied to 5 wt
% and examined in more detail (see FIG. 4C). The NO release and
SNAP leaching pattern is similar for the 5 wt % SNAP/E2As film, but
the NO release takes place over a shorter time period. The
biostability and biocompatibility of the Elast-Eon.TM. polymers in
combination with the NO release from SNAP makes this formulation
attractive for further in vitro and possible biomedical
applications.
Long-Term NO Release of SNAP/E2As Formulation for Example 1
[0072] In vitro studies were conducted with the SNAP/E2As films to
examine the long-term NO release and SNAP leaching from these
films. The NO release from the SNAP/E2As films over time was
determined based on the amount of SNAP decomposed within the
polymer phase (i.e., by measuring the SNAP remaining after
dissolving the films at given time points). The initial
concentration of SNAP in the 10 wt % films was 420 nmol SNAP/mg
film. FIG. 5A shows the UV-Vis spectra of 1.0 mM SNAP solution, a
10 wt % SNAP in E2As film redissolved in N,N-dimethylacetamide
(DMAc), and E2As dissolved in DMAc.
[0073] Due to thermal and/or photochemical decomposition of SNAP, a
decrease in the 340 nm absorbance band was observed as the 10 wt %
SNAP/E2As films were soaked in PBS under various conditions, and
the cumulative NO release based on that absorbance decrease is
shown in FIG. 5B. The 10 wt % SNAP/E2As films displayed an initial
burst of NO during the first day of soaking (see FIGS. 3A and 3B),
which corresponds to the thermal decomposition as well as diffusion
of SNAP out of the film. Films soaked at room temperature had the
lowest flux of NO release. However, films incubated at 37.degree.
C. in the dark or under ambient light exhibited a higher NO release
than the films at room temperature. This is due to the increased
thermal decomposition of SNAP. The films that were exposed to
ambient light yield essentially the same NO release profiles as the
films that were soaked in the dark. Nitric oxide release from the
SNAP/E2As films that are continuously irradiated with the 100 W
floodlight at 37.degree. C. only release NO for 3 days due to their
higher NO fluxes that rapidly deplete the SNAP reservoir. These
films provide NO release via both a thermal and photoinitiated
decomposition of SNAP.
[0074] Nitric oxide release from the SNAP-doped E2As can occur from
thermal and/or photochemical decomposition of SNAP either within
the polymer phase, or after SNAP enters the aqueous phase by
diffusion out of the polymer. In order to better understand the NO
release mechanism of the SNAP/E2As coating, the SNAP diffusion into
PBS was monitored over a 20 day period. As shown in FIG. 6A, the
films containing 10 wt % SNAP at 37.degree. C. exhibit an initial
burst of SNAP leaching on the first day. After this initial burst,
the SNAP continues to slowly diffuse from the E2As until the SNAP
reservoir is nearly depleted (with still measurable amounts of SNAP
leaching on day 20). The total moles of SNAP that leach from the
film accounts for about 27% of the total NO released (as detected
by NOA measurements) during the 20 day period (see FIG. 6B).
[0075] Additionally, the effect of the number of polymer top coats
on loss of SNAP was also evaluated. SNAP-doped E2As films without
any top coat exhibit higher levels of SNAP diffusion into the
buffer than films with at least 2 topcoats (see FIG. 7). The
thickness of the top coat allows control of the diffusion rate of
SNAP from the polymer reservoir. As such, in the examples disclosed
herein the SNAP-doped films may be coated with a polymer top coat.
Examples of suitable polymers for the top coat include the
siloxane-based polyurethane elastomers, poly(vinyl chloride),
crosslinked polyurethanes, crosslinked silicone rubber,
polytetrafluoroethylene, etc. (without the NO donor therein). In
some examples, the polymer used for the top coat is the same
polymer used for the underlying film.
Stability Study of the SNAP/E2As Films for Example 1
[0076] The stability of SNAP doped in the E2As polymer during dry
storage was also evaluated. SNAP incorporated in E2As can
potentially undergo thermal or photochemical decomposition during
storage, thus limiting the available NO release capacity at the
time of use. Therefore, 10 wt % SNAP/E2As films were stored with
desiccant in the freezer in the dark, dry in the dark or in ambient
light with desiccant at room temperature, and dry in the dark with
desiccant at 37.degree. C. and 50.degree. C. These stability
studies were conducted in a similar manner as the cumulative NO
release experiments, where films were dissolved in DMAc to
determine the amount of SNAP remaining in the polymer at various
time points (as described herein). The results indicate that SNAP
is stable within the E2As polymer matrix after 4 months. After 2
months, for example, the 10 wt % SNAP films stored in the freezer
(-20.degree. C.) in the dark maintain about 96% of the initial SNAP
species, compared to 89% for films stored at room temperature and
82% for films stored at 37.degree. C. (see FIG. 8). The results
shown in FIG. 8 illustrate the enhanced stability of SNAP during
storage. Tertiary RSNOs, such as SNAP, have greater stability than
primary RSNOs due to steric hindrance surrounding the sulfur atom.
The increased thermal stability of SNAP in combination with the
stabilization effect of the E2As polymer provides excellent storage
stability of the SNAP/E2As material.
[0077] Stability of RSNOs has been studied for viscous polymer
matrices containing such NO donors, including poly(ethylene
glycol), PLURONIC.RTM. F127 hydrogel, poly(vinyl alcohol) and
poly(vinyl pyrroloidone). RSNOs decompose according to the
following sequence of reactions:
RSNO.fwdarw.RS.+NO. (1)
RS.+RSNO.fwdarw.RSSR+NO. (2)
With the overall reaction:2RSNO.fwdarw.RSSR+2NO. (3).
[0078] The viscosity of the polymer matrix provides a cage effect
on the bond cleavage and radical pair recombination. In addition, a
viscous polymer matrix also limits the diffusion of the radical
species, favoring geminate recombination to reform RSNO. Thus, the
E2As polymer not only limits the diffusion of SNAP into the PBS,
but it also appears to provide an additional stabilization effect
to reduce the rate of SNAP decomposition.
[0079] An experiment was performed to test the storage stability of
SNAP in the E2A matrix. FIG. 9 illustrates the results. In
particular, FIG. 9 shows NO release behavior from 10 wt % SNAP in
E2As dry film at 37.degree. C. in the dark (n=1). The film was
dried at 37.degree. C. and then stored at 37.degree. C.
Approximately 5% of the total NO in the film was released during
the first hours of storage, followed by very low levels of NO
release. This corresponds with other storage/stability data
disclosed herein (see FIG. 8), which shows that the SNAP loses its
NO slowly during the 37.degree. C., dry storage (losing only 10-15%
of the SNAP after two months of storage in a dry state).
SNAP/E2As Coated ECC Loops and Effects on Rabbit Hemodynamics for
Example 1
[0080] The active ECC loops coated with 5 wt % SNAP in E2As and a
top coat of E2As (a schematic cross-section of which is shown in
FIG. 10) and control loops coated with E2As only were prepared. 5
wt % SNAP was used in these tests due, in part, to the short
duration of the ECC experiment. As described above, the SNAP/E2As
coating has an initial burst of SNAP diffusing into solution during
the first day of soaking. To reduce the effects of this burst
during the short-term ECC experiments, all loops were first soaked
overnight in saline, and the soaking solution was discarded prior
to the ECC experiments. Nitric oxide released from samples of the
coated ECC loops were measured with the NOA for NO release before
blood exposure (after overnight soaking in saline). The NO release
of the SNAP/E2As coated loops maintains an average flux of about
2.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 for 4 hours (at
37.degree. C. with ambient light) (see FIG. 11A). After 4 hours of
exposure to flowing blood, the ECC loops still exhibit a NO flux of
at least 1.5.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 for at an
additional 1 hour period (see FIG. 11B).
[0081] The hemodynamic effects of the SNAP/E2As coated ECC circuits
were also monitored over the 4 hours of blood exposure in the
rabbit ECC model. The mean arterial pressure (MAP) dropped
significantly for both SNAP/E2As and control loops within the first
hour, dropping to 35.+-.2 mmHg and 46.+-.2 mmHg, respectively. The
MAP was maintained at these levels for the 4 hours by continuous IV
fluid maintenance. The ECC blood flow dropped and remained at
64.+-.5 mL/min for SNAP/E2As ECC, but maintained at baseline levels
over the 4 hours (76.+-.6 mL/min) for controls. The MAP drop and
slower blood flow for the SNAP/E2As circuits is likely due to the
vasodilatory effects of SNAP diffusing from the coating into the
blood. The heart rate was maintained over the 4 hours and no
significant difference was noted between the SNAP/E2As and control
ECC loops, averaging 205.+-.2 beats/min. The activated clotting
time increased over the 4 hour period for both SNAP/E2As and
control circuits, likely due to the increase in intravascular
fluids (the hemodilution effect). Similar effects on MAP and flow
rate have been observed with SNAP infusion.
Effects of SNAP/E2As Coatings on Rabbit Platelet Function and
Thrombus Formation for Example 1
[0082] Platelet activation and function throughout the 4 hour ECC
was assessed by recording the platelet count (e.g., consumption,
see FIG. 12A) and plasma fibrinogen levels (see FIG. 12B), which
were both corrected for hemodilution due to the added IV fluids, as
well as % platelet aggregation. The baseline platelet counts
(.times.10.sup.8 platelets/mL) were 3.5.+-.0.6 and 4.8.+-.0.5 for
the 5 wt % SNAP/E2As and E2As control circuits, respectively. For
the SNAP/E2As circuits, the platelet count initially rose slightly
and was maintained at 100.+-.7% of baseline levels at the end of 4
hours on ECC. The platelet count for control circuits exhibited a
time-dependent loss in platelets, dropping to 60.+-.6% of baseline
after 4 hours. The percent of platelet functional aggregation was
determined by ex vivo collagen stimulation of PRP and measured by
optical turbidity. The platelets from blood taken from circulation
through the SNAP/E2As and control circuits showed similar response
to collagen-stimulated platelet aggregation during the 4 hour blood
exposure, both maintaining 56.+-.12% (with baseline values at
68.+-.6%).
[0083] As shown in FIG. 12B, the plasma fibrinogen levels were
maintained at baseline levels for the control circuits. For the 5
wt % SNAP/E2As circuits, the plasma fibrinogen levels during the
first hour of ECC dropped to 83% of baseline levels and remained at
that level for the 4 hour ECC. This decrease in plasma fibrinogen
levels can be attributed to fibrinogen binding to the surfaces, as
shown by the in vitro fibrinogen assay results (see FIG. 13).
Surprisingly, even with the enhanced adsorption of fibrinogen on
the SNAP/E2As coatings, these materials still exhibited
significantly less platelet loss than controls, suggesting that the
levels of NO produced overcome the enhanced fibrinogen adsorption
that would normally enhance activation of platelets.
[0084] To determine the differential formation of thrombus in the
thrombogenicity chamber of the ECC circuit (i.e., the 3/8 inch ID
Tygon.RTM. tubing, 8 cm in length within the ECC loop),
2-dimensional (2D) image analysis was performed after 4 hours of
blood exposure. The thrombus area was analyzed by using Image J
software and represents the 2D area of thrombus formation
(pixels/cm.sup.2) in each thrombogenicity chamber. The thrombus
area was quantitated and data are shown in FIG. 14. The thrombus
area is significantly reduced for the SNAP/E2As circuits when
compared to controls, although the E2As controls also had
relatively low thrombus area, likely resulting from the enhanced
intrinsic biocompatibility of the E2As polymer.
[0085] One of the effects of the new SNAP/E2As coating is the
hypotension caused by the diffusion of SNAP into the blood stream.
The co-administration of intravenous fluids counteracts this, but
may in some instances pose difficulties in a clinical situation.
Applications of SNAP have been reported to cause hypotension,
hyperglycemia and impaired insulin secretion, and decreased cell
viability. However, when used as catheters for coatings for small
implantable devices, the surface area to volume (of blood) ratios
will be quite small, and thus the amounts of SNAP lost to the blood
will generally not be a significant issue. Furthermore, endogenous
thiols and superoxide dismutase will reduce many of the adverse
effects. The parent thiol, N-Acetyl-DL-penicillamine (NAP),
however, has been used clinically to treat mercury poisoning and
cystinuria with minimal side effects. Although the SNAP/E2As
coatings disclosed herein do exhibit a hypotension effect, the
daily levels of SNAP delivered by the coating are well below the
reported levels causing the other potential adverse side effects
described above.
Impregnation Method for Making SNAP-Doped Polymers for Example
1
[0086] The present disclosure further includes a method for loading
commercial SR or PU tubing with SNAP. Commercial SR and PU include
medical grade tubing, including those available from US plastics,
Cole Palmer, Professional Plastics, ICORally, and Thermedics, Inc.
FIG. 15 shows an NO release profile of polyurethane (PU) tubing
that was soaked in a SNAP/acetone solution for either 1 or 2 days.
The tubing was then rinsed with acetone and dried prior to the NOA
testing. The tubing was soaked in PBS at 37.degree. C. for the NOA
testing.
[0087] This impregnation approach enables the incorporation of the
SNAP species within the walls of existing commercial
catheters/tubings. This approach avoids problems that may arise
when attempting to extrude SNAP into a polymer tubing under normal
hot extrusion conditions due to the thermal instability of NO
donors (e.g., SNAP and related species) at high temperatures. While
acetone was used in the impregnation approach described herein, it
is believed that other solvents (or mixtures of solvents) that may
be used include ethyl acetate, cyclohexane, isopropanol, methanol,
butanone, tetrahydrofuran (THF), etc.
SNAP-Doped Polymers for Catheter Tubing Applications for Example
1
[0088] FIG. 16A is a schematic illustration of an E2As catheter
tubing prepared with 5 wt % or 10 wt % SNAP/E2As according to an
example of the present disclosure followed by the application of a
top coat of E2As. FIG. 16B is a cross-section of the catheter
tubing, illustrating the various layers. In general, the trilayer
catheters were prepared by dip-coating 5 base coats of an E2As
solution, 25 coats of the respective active solutions, and 5 top
coats of the E2As solution. The SNAP/E2As catheters were kept in
phosphate buffered saline (PBS) in the dark at 37.degree. C.
[0089] FIG. 17 shows 20 day NO release of the SNAP/E2As catheters
at 37.degree. C. in the dark in the phosphate buffered saline
(PBS). The results in FIG. 17 illustrate that the 10 wt % SNAP/E2As
catheters were able to release NO for up to 20 days and that the 5
wt % SNAP/E2As catheters were able to release NO for up to 7 days
at the specified conditions.
[0090] E2As control catheters and the 10 wt % SNAP/E2As catheters
were implanted in sheep veins for 7 days. After explanation, the
SNAP/E2As catheters were found to have significantly less thrombus
(FIG. 18) and a 90% reduction of bacterial adhesion (FIG. 19) than
the E2As control catheters. Together FIGS. 17 through 19
demonstrate the potential application of the SNAP-doped polymers in
the catheter configuration.
Materials for Example 1
[0091] N-Acetyl-D,L-penicillamine (NAP), sodium chloride, potassium
chloride, sodium phosphate dibasic, potassium phosphate monobasic,
ethylenediaminetetraacetic acid (EDTA), tetrahydrofuran (THF),
sulfuric acid and N,N-dimethylacetamide (DMAc) were purchased from
Sigma-Aldrich (St. Louis, Mo.). Methanol, hydrochloric acid and
sulfuric acid were obtained from Fisher Scientific (Pittsburgh,
Pa.). TECOPHILIC.TM. SP-60D-60 and TECOFLEX.TM. Sg-80A were
products of Lubrizol Advanced Materials Inc. (Cleveland, Ohio). Dow
Corning RTV 3140 Silicone Rubber (SR) was purchased from Ellsworth
Adhesives (Germantown, Wis.). CARBOSIL.RTM. 20 90A was from the
Polymer Technology Group (Berkeley, Calif.). ELAST-EON.TM. E2As was
obtained from AorTech International, PLC (Scoresby, Victoria,
Australia). Human plasma fibrinogen containing >90% clottable
proteins was a product of Calbiochem (La Jolla, Calif.), and
fluorescein-labeled goat IgG (polyclonal) against denatured human
fibrinogen was purchased from MP Biomedicals, LLC (Solon, Ohio).
Black, polypropylene 96-well microtiter plates used for
fluorescence measurements were obtained from Nalge Nunc
International (Rochester, N.Y.). All aqueous solutions were
prepared with 18.2 M.OMEGA. deionized water using a Milli-Q filter
(Millipore Corp., Billerica, Mass.). Phosphate buffered saline
(PBS), pH 7.4, containing 138 mM NaCl, 2.7 mM KCl, 10 mM sodium
phosphate, 100 .mu.M EDTA was used for all in vitro
experiments.
Synthesis of SNAP for Example 1
[0092] SNAP was synthesized using a modified version of a
previously reported method (I. Chipinda, R. H. Simoyi, Journal of
Physical Chemistry B 2006, 110, 5052). Briefly, an equimolar ratio
of NAP and sodium nitrite was added to a 1:1 mixture of water and
methanol containing 2 M HCl and 2 M H.sub.2SO.sub.4. After 30
minutes of stirring, the reaction vessel was cooled in an ice bath
to precipitate the green SNAP crystals. The crystals were collected
by filtration, washed with water, and allowed to air dry. The
reaction and crystals were protected from light at all times.
Preparation of SNAP-Doped Films for Example 1
[0093] The polymer films containing 5 wt % and 10 wt % SNAP were
prepared by solvent evaporation. For the 10 wt % SNAP films, the
casting solutions were prepared by dissolving 180 mg of the
respective polymer in THF. The polyurethanes (SP-60D-60, SG-80A,
CARBOSIL.RTM. and ELAST-EON.TM. E2As) were dissolved in 3 mL THF,
and SR was dissolved in 1 mL THF. SNAP (20 mg) was then added to
the polymer solution, and the mixture was stirred for 10 minutes.
The 5 wt % SNAP films were prepared similarly with SNAP (10 mg) and
polymer (190 mg). The film solution was cast in a TEFLON.RTM. ring
(d=2.5 cm) on a Teflon.RTM. plate and dried overnight under ambient
conditions. Small disks (d=0.7 cm) were cut from the parent films
and were dip coated 2 times with a topcoat solution (200 mg of the
respective polymer (no SNAP added) in 4 mL THF) and dried
overnight. As such, the topcoat for each sample was made of the
same polymer as the parent film. The weight of each small disk was
recorded prior to topcoating. All films and film solutions were
protected from light. The final films had a SNAP-doped layer that
was about 150 .mu.m thick and a top coat that was about 50 .mu.m
thick.
Preparation of SNAP/E2As Coated ECC Loops for Example 1
[0094] The ECC configuration employed in the in vivo rabbit study
consisted of 16-gauge and 14-gauge IV polyurethane angiocatheters
(Kendall Monoject Tyco Healthcare, Mansfield, Mass.), two 16 cm in
length 1/4 inch inner diameter (ID) Tygon.RTM. tubing, and an 8 cm
length of 3/8 inch ID Tygon.RTM. tubing that created a
thrombogenicity chamber where thrombus could form more easily due
to more turbulent blood flow.
[0095] As previously mentioned, due to the short duration of the
ECC experiments (4 hours), the NO release ECC loops were coated
with only 5 wt % SNAP in E2As. The SNAP/E2As solution was prepared
by dissolving SNAP (125 mg) and E2As (2375 mg) in THF (15 mL). The
E2As control solution consisted of E2As in (2500 mg in 15 mL).
SNAP/E2As loops were first coated with 2 layers of the SNAP/E2As
solution, followed by 1 coat of the E2As control solution. E2As
control loops were coated with 2 coats of the E2As control
solution. ECC loops were allowed to air dry for 1 hour in the dark
between each coat. The completely coated ECC was welded together
using THF, starting at the left carotid artery side, with the
16-gauge angiocatheter, one 15 cm length 1/4 inch ID tubing, the 8
cm length thrombogenicity chamber, the second 15 cm length 1/4 inch
ID tubing and finally the 14-gauge angiocatheter. The
angiocatheters were interfaced with tubing using two luer-lock PVC
connectors. The assembled ECC loops were dried under vacuum while
protected from light for at least 48 hours. Prior to the ECC
experiment, the loops were filled with saline solution for
overnight soaking, and this solution was discarded immediately
before the rabbit experiment.
In Vitro Characterization of SNAP-Doped Films for Example 1
UV-Vis Spectra
[0096] All UV-Vis spectra were recorded in the wavelength range of
200 nm-700 nm using a UV-Vis spectrophotometer (Lambda 35,
Perkin-Elmer, Mass.) at room temperature. The presence of the S--NO
group of SNAP provides characteristic absorbance maxima at 340 nm
and 590 nm, corresponding to the .pi..fwdarw..pi.* and
nN.fwdarw..pi.*electronic transitions.
[0097] Diffusion of SNAP from SNAP-Doped Polymer Films Immersed in
PBS
[0098] Top coated films were placed in individual vials soaked in
10 mM PBS, pH 7.4, containing 100 .mu.M EDTA to minimize any trace
metal ion catalyzed decomposition of SNAP. Films were incubated in
the dark at room temperature (22.degree. C.) or 37.degree. C. At
various time points, the UV-Vis spectra of a 1 mL aliquot of the
PBS was taken for rapid determination of the SNAP concentration.
The aliquots were protected from light and were immediately
returned to the sample vials for the duration of the experiment.
The films were placed in fresh PBS buffer daily. The molar
absorption coefficient for SNAP in PBS at 340 nm was determined to
be: .epsilon..sub.SNAP=1024 M.sup.-1 cm.sup.1.
[0099] Cumulative NO Release from SNAP/E2As Films
[0100] After the 10 wt % SNAP in E2As films were prepared, the
UV-Vis spectra were recorded of individual films dissolved in DMAc
to determine the initial concentration of SNAP within the films
(nmol SNAP/mg film). Equivalent films were then placed in
individual vials containing 3 mL PBS (pH 7.4) containing 100 .mu.M
EDTA. Films were incubated under various conditions: RT under
ambient light, 37.degree. C. under ambient light, 37.degree. C. in
dark, and 37.degree. C. under a 100 W floodlight. The fluorescent
lights in the laboratory are referred to as ambient light. Films
were placed in fresh PBS daily. At various time points, the films
were dissolved in DMAc for rapid determination of the SNAP present
in the film. The amount of NO released was determined indirectly
from the amount of SNAP decomposed at various time points. The
cumulative NO released over time ([NO].sub.t) was calculated by the
difference between the initial amount of SNAP in the film
([SNAP].sub.0) and the amount of SNAP at time t ([SNAP].sub.t):
[NO].sub.t=[SNAP].sub.0-[SNAP].sub.t (where concentrations are in
nmol/mg film). This calculation was based on the fact that the
decay of the 340 nm absorption band of SNAP is directly associated
with the hemolytic cleavage of the S--NO bond and concomitant NO
release. The molar absorption coefficient for SNAP in DMAc at 340
nm was determined to be: .epsilon..sub.SNAP=1025 M.sup.-1
cm.sup.-1.
[0101] NO Release Measurements
[0102] Nitric oxide released from the films was measured using a
Sievers chemiluminescence Nitric Oxide Analyzer (NOA) 280 (Boulder,
Colo.). Films were placed in the sample vessel immersed in PBS (pH
7.4) containing 100 .mu.M EDTA. Nitric oxide was continuously
purged from the buffer and swept from the headspace using an
N.sub.2 sweep gas and bubbler into the chemiluminescence detection
chamber. Clear glass sample vessels were used for the ambient light
and photoinitiated NO release experiments. A 100 W halogen
floodlight (GE model 17986) was placed about 60 cm from the sample
cell for the photolysis experiments. Films were incubated in the
PBS under the same conditions as the NOA measurements (ambient
light or 100 W floodlight irradiation at 37.degree. C.).
[0103] SNAP/E2As Stability Study
[0104] SNAP/E2As films (consisting of 10 wt % SNAP) were placed
under the following conditions in vials with desiccant: room
temperature with ambient light, room temperature in dark,
37.degree. C. in dark, and in the freezer (-20.degree. C.) in dark.
At various time points over a 4 month period, films were dissolved
in DMAc, and the UV-Vis spectra was recorded to determine the %
SNAP remaining in the film, as compared to the initial 10 wt %
SNAP.
[0105] In Vitro Fibrinogen Adsorption Assay
[0106] The in vitro fibrinogen adsorption immunofluorescence assay
was performed in a 96-well format. The SNAP/E2As and E2As control
polymer solutions used to prepare the ECC circuits were also
employed to coat microwells of the 96-well microtiter plates and
were dried under the same conditions as the ECC loops. Briefly,
human fibrinogen was diluted to 3 mg/mL with Dulbecco's
phosphate-buffered saline (dPBS) without CaCl.sub.2 and MgCl.sub.2
(Gibco Invitrogen, Grand Island, N.Y.), equivalent to the human
plasma concentration, and then used for adsorption experiments. One
hundred .mu.L of this solution were added to each well and the
coated wells were incubated with this solution for 1.5 hours at
37.degree. C. This was followed by eight washing steps using wash
buffer (100 .mu.L) for each wash, which consisted of a 10-fold
dilution of the AbD Serotec Block ACE buffer (Raleigh, N.C.)
containing 0.05% Tween 20 (Calbiochem La Jolla, Calif.). To block
nonspecific antibody binding, coated wells were incubated with 100
.mu.L of blocking buffer (4-fold dilution of Serotec Block ACE
buffer) for 30 minutes at 37.degree. C. After rinsing 3 times with
wash buffer (100 .mu.L per well), a background fluorescence
measurement of the plates was performed at 485 nm (excitation) and
528 nm (emission) on a Synergy 2 fluorescence microplate reader
(Biotek Winooski, Vt.). To detect the adsorbed fibrinogen,
fluorescein-labeled goat anti-human fibrinogen antibody was diluted
(1:10) in a 10-fold dilution of the Serotec Block ACE buffer and
100 .mu.L of this final solution was added to each well. The
antibody was allowed to bind to the surface-adsorbed fibrinogen for
1.5 hours at 37.degree. C. Human fibrinogen adsorption to
non-coated polypropylene was used as an internal control to
normalize the fluorescence signals within different plates. All
measurements were conducted in triplicate.
Rabbit ECC Thrombogenicity Experiments for Example 1
[0107] All animal handling and surgical procedures employed were
approved by the University Committee on the Use and Care of Animals
in accordance with university and federal regulations. A total of 8
New Zealand white rabbits (Covance, Battle Creek, Mich.) were used
in this study. All rabbits (2.5 kg-3.5 kg) were initially
anesthetized with intramuscular injections of 5 mg/kg xylazine
injectable (AnaSed.RTM. Lloyd Laboratories Shenandoah, Iowa) and 30
mg/kg ketamine hydrochloride (Hospira, Inc., Lake Forest, Ill.).
Maintenance anesthesia was administered via isoflurane gas
inhalation at a rate of 1.5%-3% via mechanical ventilation which
was done via a tracheotomy and using an A.D.S. 2000 Ventilator
(Engler Engineering Corp. Hialeah, Fla.). Peek inspiratory pressure
was set to 15 cm of H.sub.2O, and the ventilator flow rate set to 8
L/min. In order to aid in maintenance of blood pressure stability,
IV fluids of Lactated Ringer's were given at a rate of 10 mL/kg/h.
For monitoring blood pressure and collecting blood samples, the
rabbits' right carotid artery were cannulated using a 16-gauge IV
angiocatheter (Jelco.RTM., Johnson & Johnson, Cincinnati,
Ohio). Blood pressure and derived heart rate were monitored with a
Series 7000 Monitor (Marquette Electronics Milwaukee, Wis.). Body
temperature was monitored with a rectal probe and maintained at
37.degree. C. using a water-jacketed heating blanket. Prior to
placement of the arteriovenous (AV) custom-built extracorporeal
circuit (ECC), the rabbit left carotid artery and right external
jugular vein were isolated and baseline hemodynamics as well as
arterial blood pH, pCO.sub.2, pO.sub.2, total hemoglobin and
methemoglobin were measured using an ABL 825 blood-gas analyzer and
an OSM3 Hemoximeter (Radiometer Copenhagen, DK). In addition,
baseline blood samples were collected for platelet and total white
blood cell (WBC) counts which were measured on a Coulter Counter Z1
(Coulter Electronics Hialeah, Fla.). Plasma fibrinogen levels were
determined using a Dade Behring BCS Coagulation Analyzer (Siemens,
Deerfield, Ill.), activated clotting times (ACT) were monitored
using a Hemochron Blood Coagulation System Model 801 (International
Technidyne Corp., Edison, N.J.), and platelet function was assessed
using a Chrono-Log optical aggregometer model 490 (Havertown,
Pa.).
[0108] After baseline blood measurements, the AV custom-built ECC
was placed into position by cannulating the left carotid artery for
ECC inflow and the right external jugular vein for ECC outflow. The
flow through the ECC was initiated by unclamping the arterial and
venous sides of ECC, and blood flow in circuit was monitored with
an ultrasonic flow probe and flow meter (Transonic HT207, Ithaca,
N.Y.). Animals were not systemically anticoagulated during the
experiments.
[0109] After 4 hours on ECC, the circuits were clamped, removed
from animal, rinsed with 60 mL of saline and drained. Any residual
thrombus in the larger tubing of ECC (i.e., thrombogenicity
chamber) was photographed, and the degree of thrombus was
quantitated using Image J imaging software from National Institutes
of Health (Bethesda, Md.). Prior to euthanasia, all animals were
given a dose of 400 U/kg sodium heparin to prevent necrotic
thrombosis. The animals were euthanized using a dose of Fatal Plus
(130 mg/kg sodium pentobarbital) (Vortech Pharmaceuticals,
Dearborn, Mich.). All animals underwent gross necropsy after being
euthanized, including examination of the lungs, heart, liver and
spleen for any signs of thromboembolic events.
Blood Sampling for Example 1
[0110] Rabbit whole blood samples were collected in
non-anticoagulated 1 cc syringes for ACT, and in 3.2% sodium
citrate vacutainers (Becton, Dickinson, Franklin Lakes, N.J.) with
3 cc volumes for cell counts and aggregometry, and 1 cc syringes
containing 40 U/mL of sodium heparin (APP Pharmaceuticals, LLC,
Schaumburg, Ill.) for blood-gas analysis. Following the initiation
of ECC blood flow, blood samples were collected every hour for 4
hours for these in vitro measurements. Samples were used within 2
hours of collection to avoid any activation of platelets, monocytes
or plasma fibrinogen.
Platelet Aggregometry for Example 1
[0111] Rabbit platelet aggregation was assayed based on the Born's
turbidimetric method using a Chrono-Log optical aggregometer.
Briefly, citrated blood (1:10 blood to 3.2% sodium citrate
solution) was collected (6 mL), and platelet-rich plasma (PRP) was
obtained by centrifugation at 110.times.g for 15 minutes.
Platelet-poor plasma (PPP) was obtained by another centrifugation
of the PRP-removed blood sample at 2730.times.g for 15 minutes and
was used as the blank for aggregation.
[0112] PRP was incubated for 10 minutes at 37.degree. C. and then
25 .mu.g/mL collagen (Chrono-PAR #385 Havertown, Pa.) was added.
The percentage of aggregation was determined 3 minutes after the
addition of collagen using Chrono-Log Aggrolink software.
Preparation of SNAP-Doped E2As and E2As Control Catheters for
Example 1
[0113] Catheters were prepared by dip-coating polymer solutions on
18 cm long stainless steel mandrels of 2 mm diameter (purchased
from McMaster Can). For the E2As control catheters, the polymer
solution consisted of E2As dissolved in THF (150 mg/mL).
Thirty-five coats of the E2As solution was applied on the mandrel
by dip-coating at an interval of 2 min between each coat. For the
SNAP/E2As catheters, two different solutions, namely a top/base
coat solution and an active solution, were prepared to make the
trilayer catheters (see FIG. 16). The top/base coat solution
consisted of E2As dissolved in THF (150 mg/mL). The active solution
was made up of 10 wt % SNAP and 90 wt % E2As was dissolved in THF
with overall concentration of 150 mg/mL. Trilayer catheters were
prepared by dip-coating 5 base coats of E2As solution, 25 coats of
active solution, and 5 top coats of E2As solution. Catheters used
for sheep studies were 15 cm long.
Long-Term (7 Day) Implantation of Catheters in Sheep for Example
1
Sheep Catheter Implantation
[0114] All animals received care compliant with the "Principles of
Laboratory Animal Care" formulated by the National Society for
Medical Research and the "Guideline for the Care of Use of
Laboratory Animals" prepared by the National Academy of Sciences
and published by the NIH. This study was approved by the University
of Michigan Committee on Use and Care of Animals.
[0115] Five adult sheep were utilized in the large animal model.
All experiments were performed under sterile conditions. The first
3 sheep procedures were performed using 1% Lidocaine subcutaneously
for local anesthetic. Small (1 cm to 2 cm) vertical and transverse
incisions were created over the right and left jugular vein.
Cannulas were then placed using a modified Seldinger technique with
one cannula either control (E2As) or experimental (SNAP/E2As)
placed in either the right or left jugular vein. The skin was then
re-approximated using skin staples.
[0116] The final 2 sheep experiments were performed under general
anesthetic. Propofol (1 mg/kg) was used for induction followed by
Isoflurane (0.1-4%) anesthetic for maintenance. A small 2-3 cm
incision was created overlying the jugular vein. The right and left
jugular veins were then isolated and either control (E2As) or
experimental (SNAP/E2As) cannulas were placed under direct
visualization. Sheep were then recovered and returned to animal
housing.
[0117] Animals remained in animal housing throughout the remainder
of the experiment. Catheters were tested on a daily basis for
patency. Cannulas were initially attempted to be aspirated and then
flushed with 15 mL of saline. If aspiration was initially
difficult, 15 mL of normal saline was attempted to be infused, and
the process and aspiration and flushing were again tested. Patency
data was recorded every 24 hour for 7 days.
[0118] Necropsy was performed on day 7. Sheep were anesthetized
using the same anesthetic protocol described above. The right and
left jugular veins were dissected along their length and isolated.
The sheep were heparinized using approximately 100-150 IU/kg bolus
dose and activated clotting time of >200 seconds was confirmed.
The jugular veins were then ligated and opened longitudinally.
Catheters were removed and placed in sterile saline for further
analysis.
[0119] Catheter Evaluation
[0120] After explanting, the catheters were rinsed in PBS. Pictures
were taken of the exterior of the whole catheter and the interior
of a 1 cm piece cut longitudinally using a Nikon L24 digital
camera. The degree of thrombus was quantitated using Image J
imaging software from NIH. To quantitate the viable bacteria, a 1
cm piece was cut longitudinally and was homogenized in 1 mL PBS
buffer. The optimal homogenizing speed was found using a separate
experiment where different homogenizing speeds and times were
compared. The resulting homogenate was serially diluted in sterile
PBS. Triplicate aliquots of each dilution (10 .mu.L) were plated on
agar plates. The agar plates were incubated at 37.degree. C. for 24
hours, followed by calculation of colony forming units per catheter
surface area (CFU/cm.sup.2).
Statistical Analysis for Example 1
[0121] Throughout this disclosure, data are expressed as
mean.+-.SEM (standard error of the mean). Comparison between the
various SNAP/E2As and E2As control polymer groups were analyzed by
a comparison of means using student's t-test. Values of p<0.05
were considered statistically significant for all tests.
Conclusions for Example 1
[0122] Examples of the present disclosure have shown that
hydrophobic polyurethanes, e.g., siloxane-based polyurethane
elastomers (one example of which is the ELAST-EON.TM. E2As polymer)
are excellent matrices to act as a reservoir for SNAP, and the
resulting films can be used for the controlled release of NO and
SNAP. SNAP slowly diffuses from the polymer film, and NO release
from the film/coating can be initiated by light and/or thermal
decomposition when blood flows through an ECC loop. A stability
study demonstrates that SNAP is quite stable within the E2As
matrix, even during storage for 4 months at 37.degree. C. While the
E2As polymer has excellent innate biocompatible properties on its
own, incorporating SNAP into the E2As polymer matrix provides
controlled delivery of NO/SNAP to further improve polymer
hemocompatibility. The SNAP/E2As coated ECC loops significantly
preserved platelet count and function during 4 hours of ECC blood
flow, while also reducing the clot area when compared to
corresponding E2As coated control loops. In addition, the NO
released from SNAP/E2As catheters was able to significantly reduce
thrombus and bacterial adhesion during 7 day implantation in sheep,
thereby improving catheter patency. Incorporating SNAP within
ELAST-EON.TM. E2As polymer films/coatings provides a simple way to
locally deliver NO/SNAP, and has potential for improving the
hemocompatibility of a wide variety of blood-contacting medical
devices.
[0123] In summary, examples as disclosed herein include novel
nitric oxide (NO) releasing coatings including siloxane-based
polyurethane elastomers doped with S-nitroso-N-acetylpenicillamine
(SNAP) to prevent thrombus formation in, e.g., extracorporeal
circulation (ECC) circuits and catheter tubing. In addition, the NO
release from these formulations is likely to serve as a very
effective bacterial agent.
Example 2
[0124] In this example, the impregnation method described in
Example 1 was further studied. SNAP-impregnated silicone Foley
catheters were generated via a solvent swelling method. As will be
discussed in more detail herein, the catheters of Example 2
generated NO surface-fluxes >0.7.times.10.sup.-10 mol min.sup.-1
cm.sup.-2 for over one month under physiological conditions, with
minimal SNAP leaching; and prevented biofilm formation over 3, 7,
and 14 day periods by microbial species commonly causing CAUTIs. In
Example 2, it is also shown that segments of SNAP impregnated Foley
catheter tubings reduced the colony-forming units (CFU) of
gram-positive S. epidermidis biofilms grown over 14 days by almost
4 log units. Further, the SNAP-impregnated silicone Foley catheters
of Example 2 abated cell viability of biofilms by 6 log units
formed over 14 days by gram-negative Proteus mirabilis, the
principle bacterium in causing urinary catheter encrustation
infections. Finally, the toxicity assessment data in Example 2
demonstrates that SNAP-impregnated silicone Foley catheters are
fully biocompatible (as extracts of the catheter tubings scored 0
on a 3-point grading scale using an accepted mouse fibroblast
cell-line toxicity model).
Materials for Example 2
[0125] N-Acetyl-D,L-penicillamine (NAP), sodium chloride, potassium
chloride, sodium phosphate dibasic, potassium phosphate monobasic,
ethylenediaminetetraacetic acid (EDTA), and tetrahydrofuran (THF)
were purchased from Sigma Aldrich (St. Louis, Mo.), and used as
received. Methanol (MeOH), hydrochloric acid (HCl) and sulfuric
acid (H.sub.2SO.sub.4) were obtained from Fisher Scientific
(Pittsburgh, Pa.). Luria Bertani (LB) broth and LB agar were also
obtained from Fisher Scientific Inc. Aqueous solutions were
prepared with deionized water using a Milli-Q filter (18M.OMEGA.
cm.sup.-1; Millipore Corp., Billerica, Mass.). Phosphate buffered
saline (PBS), pH 7.4, containing 10 mM sodium phosphate, 138 mM
NaCl, 2.7 mM KCl, and 100 .mu.M EDTA was used for all in vitro
experiments of Example 2.
[0126] Silicone 2-way Foley Balloon Catheters, size 18 Fr (outer
diameter (o.d.)=0.59 cm, inner diameter (i.d.)=0.30 cm), were
purchased from Fortune Medical Instrument Corp (New Taipei City,
TAIWAN). S. epidermidis ATCC 14990 and P. mirabilis ATCC 29906 were
obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.).
Synthesis of SNAP for Example 2
[0127] SNAP was synthesized using the method described in Example
1, except that an equimolar ratio of NAP and sodium nitrite was
added to a 3:1 mixture by volume of methanol and water containing 2
M HCl and 2 M H.sub.2SO.sub.4.
Preparation of SNAP Impregnated Silicone Foley Catheters for
Example 2
[0128] SNAP was first dissolved in THF at a concentration 125 mg/mL
of solvent, for 15 minutes. Existing FDA approved silicone Foley
catheter tubing was cut either into 1 cm long sections (for NO
release measurements, SNAP % loading and leaching assessments) or
into 2 cm long sections (for biofilm studies). All of the segments
were completely immersed into SNAP-containing THF solutions, within
glass vials, for 24 hours in the dark. After this
swelling/impregnation period, the catheter sections were dried in
dark for 72 hours within a fume hood to remove any residual
solvent. Control Foley catheter segments (designated for the
biofilm experiments) were swollen in a vial containing only THF for
24 hours, followed by a 72 hour drying process within the fume
hood.
NO Release Measurements for Example 2
[0129] Nitric oxide release from the surfaces of 1 cm SNAP
impregnated Foley catheter segments (total surface area=3.19
cm.sup.2) was measured using a Sievers chemiluminescence Nitric
Oxide Analyzer (NOA) 280i (Boulder, Colo.). The catheter segments
were placed in an amber glass vessel (placed in a water bath at
37.degree. C.) containing 4 mL of PBS (pH 7.4) with 100 .mu.M EDTA.
Nitric oxide released from the catheter segment was continuously
purged from the buffer and swept from the headspace using an
N.sub.2 sweep gas and bubbler into the chemiluminescence detection
chamber. When not being tested with the NOA, the SNAP impregnated
catheters were incubated in 10 mM PBS (pH 7.4) with 100 .mu.M EDTA
at 37.degree. C., avoiding exposure to light. All experiments were
conducted in triplicate.
SNAP Loading Efficiency for Example 2
[0130] Since the reaction of 2 RSNO.fwdarw.RSSR+2 NO is catalyzed
by light in the visible region of the spectrum, the total loading
of SNAP into the 1 cm catheter segments was determined by the total
amount of NO (in moles) released over time in the presence of a
high intensity white light source (until no additional NO could be
detected above baseline by the NOA). By integrating the signal from
the NOA over time, the total amount of SNAP impregnated into each
catheter segment was calculated. The wt % of SNAP within the
catheter material was quantitated using the initial weight of the
catheter segment.
[0131] NO release was measured via the NOA, by immersing the
catheter tubing sample in a clear glass vessel (kept inside a water
bath at 37.degree. C.) containing 50 mM CuCl.sub.2 and 10 mM
cysteine. The added cysteine catalyzed the reduction of Cu.sup.2+
into Cu.sup.+, which then promoted the SNAP decomposition and the
release of NO. A 100 W halogen floodlight (GE model 17986) was
placed 20 cm away from the sample, and was used to photo-initiate
the NO release from the SNAP impregnated catheter pieces. All
experiments were conducted in triplicate.
SNAP Leaching from Surface of SNAP Impregnated Foley Catheters for
Example 2
[0132] 1 cm long catheter segments were completely immersed into 1
mL of 10 mM PBS (with 100 .mu.M EDTA) inside individual amber glass
vials and stored at 37.degree. C. for the entire duration of the
experiment. At every time point, each of the soaking solutions,
where catheter segments had been stored, was tested by UV-Vis to
detect the SNAP characteristic UV-vis absorbance band maximum at
340 nm. Using the Beer-Lambert law (A=c.epsilon.I), the moles of
SNAP leached out at every time point was calculated. Since the wt %
of total SNAP loading was determined from total NO release (see
SNAP loading efficiency for Example 2), the % of leached SNAP can
be quantitated. All UV-Vis spectra were recorded in the wavelength
range of 250-650 nm using a UV-Vis spectrophotometer (Lambda 35,
PerkineElmer, Mass.). The presence of the S--NO group of SNAP
provides characteristic absorbance maxima at 340 nm and 590 nm,
corresponding to the .pi..sub..fwdarw..pi.* and
n.sub.N.sub..fwdarw..pi.*electronic transitions. The molar
absorption coefficient for SNAP in PBS at 340 nm was determined to
be .epsilon..sub.SNAP=1075M.sup.-1 cm.sup.-1. PBS was used as the
blank and all experiments were conducted in triplicate.
[0133] Biofilm Growth Conditions and Plate Counting
[0134] S. epidermidis Biofilm
[0135] S. epidermidis ATCC 14990 was tested as a bacterial strain
to develop 3, 7, and 14 day-old biofilms on the surfaces of both
control and SNAP impregnated silicone Foley catheter segments. A
CDC biofilm reactor (Biosurface Technologies, Bozeman, Mont.) was
used for the biofilm growth. The CDC biofilm reactor and its coupon
holders were autoclaved before use. Both control and SNAP
impregnated catheters segments were mounted on the coupon holders
and the reactor was supplemented with 10% LB medium by a
peristaltic pump with a continuous flow rate of 100 mL/h. Overnight
cultures of S. epidermidis (grown under shaking conditions at
37.degree. C.) were diluted by 1:100 and inoculated into the glass
vessel of the CDC reactor aseptically. The liquid growth medium was
circulated through the vessel and a shear force was generated by a
magnetic stir bar rotated by a magnetic stir plate. The CDC biofilm
reactor was placed inside an incubator and biofilms were grown at
37.degree. C. After 3, 7 or 14 days of growth, the catheters were
aseptically removed and each catheter was cut into two 1 cm
segments.
[0136] One of the 1 cm segments was utilized for fluorescent
imaging of live bacteria on the surface, while the other 1 cm
segment was used for plate count experiments. Each 1 cm segment to
be used for plate counting was placed into a centrifuge tube,
containing 2 mL of 10 mM sterilized PBS (pH 7.2). The 1 cm segment
was then homogenized for 30 seconds in order to disintegrate the
biofilm clumps and form a homogeneous single cell suspension.
Finally, to assess cell viability, samples were 10-fold serially
diluted and plated onto LB agar plates. All experiments were
conducted in triplicate.
[0137] P. mirabilis Biofilm
[0138] The bacterium P. mirabilis ATCC 29906 was also tested for
the development of 3, 7, and 14 day-old biofilms on the surfaces of
both control and SNAP impregnated silicone Foley catheter segments.
Growth conditions were the same as those used for S. epidermidis
biofilms. Procedures to assess cell viability were also performed
as described above for S. epidermidis. For the second studies with
P. mirabilis, the SNAP catheter segments were presoaked for 24
hours before being placed in the bioreactor. All experiments were
conducted in triplicate.
Biofilm Imaging for Example 2
[0139] Control and SNAP impregnated catheter segments designated
for fluorescent imaging were stained with LIVE/DEAD BacLight
Bacterial Viability kit (L7012, Invitrogen, Carlsbad, Calif.)
according to its instructions. Fluorescent images were acquired
with an inverted fluorescence microscope (Olympus 1X71, Center
Valley, Pa.) equipped with Fluorescence Illumination System (X-Cite
120, EXFO) and filters for SYTO-9 (excitation=488 nm/emission=520
nm) and Propidium Iodide (excitation=535 nm/emission=617 nm)
fluorescence. Images were obtained using an oil immersion 60.times.
objective lens. Only the fluorescent micrographs of control Foley
catheters surfaces upon which P. mirabilis had been grown for 14
days were taken with a 10.times. objective lens due to the high
biofilm biomass on these pieces of tubing. For these catheters, the
use of the 60.times. objective lens would have impeded obtaining a
clear focus of the biofilm layer on the catheter surfaces). All
experiments were conducted in triplicate, and different surface
areas of the catheter segments were randomly chosen for
imaging.
Statistical Analysis for Example 2
[0140] Data for all of the experiments are expressed as mean.+-.SEM
(standard error of the mean). A comparison of means was performed
using Student's t-test to analyze whether there was a statistical
difference between the data for SNAP impregnated versus control
silicone Foley catheter materials. Values of p<0.05 were
considered statistically significant and graphically illustrated
with a*.
[0141] NO Release from SNAP-Impregnated Silicone Foley
Catheters
[0142] The total SNAP amount loaded into Foley catheters segments
by the solvent swelling/impregnation method was found to be
5.43.+-.0.15 wt % (n=3) using photolysis to emit the entire payload
of NO in a reasonable time frame. Upon placing the dried tubing
segments loaded with SNAP into PBS buffer at 37.degree. C., SNAP
decomposition within the walls of the tubing commenced due to the
combination of both the presence of moisture and elevated
temperature. The NO release data in FIG. 20A clearly show that the
SNAP impregnated catheter tubing can release NO at a flux between
1.4 and 0.8.times.10.sup.-10 mol min.sup.-1 cm.sup.-2 for at least
30 days. For this experiment, both the outer and the inner wall
surfaces of the pieces of tubing were utilized to calculate flux
levels.
[0143] The NO release in the nitrogen-purged solution phase using
the NOA was analyzed. As shown in FIG. 20B, the steady-state NO
release profile, at any given time point for testing the silicone
tubing, was relatively stable after about 30 minutes.
[0144] From the data in FIG. 20A, it is clear that the catheter
tubing displayed an initial burst of NO during the first hours of
soaking on day 1. This was likely due to the rapid reaction and
leaching of SNAP present in the outermost surface layers of the
silicone tubing. The long-term NO release data confirms that the
swelling/impregnation method disclosed herein, conducted at room
temperature, allows for the successful incorporation of SNAP within
the polymer, and that NO release from the incorporated SNAP can
last for at least 1 month at near constant flux levels over this
period. This demonstrates the clear advantage of using the solvent
swelling/impregnation approach over extruding the polymer with the
NO donor present, since the latter would completely degrade the
SNAP (loss in NO) due to the elevated extrusion temperatures.
[0145] SNAP Leaching from the Catheter Surface
[0146] To ensure that SNAP leaching from the catheter polymeric
surface was primarily limited to the first day of soaking due to
rapid water uptake at the outermost surface of the silicone rubber,
leaching studies were conducted using UV-Vis absorbance
spectroscopy over the first 7 days of soaking. At various time
points, the UV-Vis spectra of aliquots of the PBS soaking solution
were assayed for SNAP concentration. Since the total SNAP amount
loaded into these 1 cm long catheter sections had previously been
quantified, it was determined that only 3.51.+-.0.04% of the total
loaded SNAP leached from the catheter segment on the first day of
soaking (see FIG. 21). Minimal amounts of SNAP continued to leach
from the Foley catheters during the subsequent days of soaking.
Indeed, as shown in the inset of FIG. 21, only 10.92.+-.0.05% of
the total SNAP was lost during the first week (168 hours).
[0147] N-acetyl-penicillamine (NAP) and/or NAP disulfide may also
leach from the catheter surface. NAP may be used to treat mercury
and other heavy metal poisoning, and thus its leaching has minimal
toxicity concerns.
[0148] SNAP Impregnated Foley Catheters Anti-Biofilm Properties
Against S. Epidermidis
[0149] Catheterization is a principle risk factor for bacterial
adhesion, colonization and subsequent infection. As such, the
antimicrobial properties of the SNAP impregnated silicone Foley
catheter segments with bacterial strains known to be associated
with CAUTIs were examined.
[0150] S. epidermidis is a species associated with both
intravascular and urinary infections. S. epidermidis represents a
commensal inhabitant of healthy mucosal microflora, and it has the
capacity to form high-biomass biofilms and colonize biomaterials.
Biofilm-forming clinical strains of S. epidermidis, isolated from
urinary tract infections, are significantly more resistant to a
wide array of antibiotic treatments (e.g., ampicillin,
ciprofloxacin, gentamicin, levofloxacin) than non-biofilm producing
strains. The biofilm extracellular polysaccharide substance (EPS)
may also represents a physical and chemical barrier to antibiotics.
The polymeric matrices of biofilms may retard the penetration rate
of antibiotics enough to induce the expression of genes that
mediate antibiotic resistance. Low susceptibility to antibiotics
may also be attributed by the metabolic state of microorganisms in
a biofilm. As the cells located within the biofilm experience
nutrient limitation, this condition can result in a stationary
phase-like dormancy, thus influencing biofilm resistance to
antibiotics as compared to bacteria in a planktonic stage. For at
least these reasons, S. epidermidis was used in these
experiments.
[0151] To simulate the development of bacterial biofilms on the
surface of urinary Foley catheters, a CDC biofilm reactor was
utilized. This reactor enables growth of mature biofilms and
generates high shear force (flow-related turbulence) and is a
standardized model to replicate urinary tract conditions in vivo.
The total viable S. epidermidis adhered on the catheter tubing
surface was determined after growing biofilms for either 3, 7, or
14 days at 37.degree. C.
[0152] FIG. 22A shows the plate count data for 3 day old, 7 day old
and 14 day old S. epidermidis biofilms on the surface of the SNAP
impregnated catheter tubing and on the surface of the control
tubing. As depicted in FIG. 22A, the plate count data for 3 day old
S. epidermidis biofilms shows that the viable bacteria attached on
the surface of the SNAP impregnated catheter tubing was about 50%
less than on the controls (n=3, p value=0.014). A possible
explanation for observing only a moderate reduction in cell
viability may be that the bacteria had not yet developed into a
mature biofilm, but were still in a micro-colony growth stage. At 3
days, the bacterial counts on the surfaces of the control catheters
were relatively low, and thus a high percentage difference between
the SNAP-doped and control catheters was not observed. However, the
plate count data for 7 day S. epidermidis biofilms exhibited a
significant 2.5 logarithmic unit difference in viable bacteria
between control and NO releasing (i.e., SNAP impregnated) catheter
segments (n=3, p value=0.015). Similarly, when S. epidermidis
biofilms were grown for 14 days and reached a matured growth stage,
the reduction in adhered viable bacteria on SNAP impregnated
catheters compared to controls is even more pronounced, with a 3.7
logarithmic unit difference (n=3, p value=0.013). It is clear from
the data shown in FIG. 22A that that the SNAP impregnated catheter
segments can progressively reduce formation of S. Epidermidis
biofilms.
[0153] The plate count data was substantiated by fluorescence
imaging data. FIG. 22B illustrates black and white representations
of the original fluorescence images that were taken. As shown in
FIG. 22B, it is observed that while biofilm thickness and surface
coverage increases with incubation time on control catheters,
biofilm thickness and surface coverage remain relatively low over
time on the surfaces of the SNAP impregnated catheters.
[0154] The results shown in FIGS. 22A and 22B illustrate that the
SNAP impregnated silicone Foley catheters are capable of
significantly limiting the viability of a prototypic
biofilm-forming bacterium (S. epidermidis). As such, the SNAP
impregnated silicone Foley catheters disclosed herein may be used
to successfully prevent nosocomial urinary infections.
[0155] SNAP Impregnated Foley Catheters Anti-Biofilm Properties
Against P. mirabilis
[0156] The same experiments performed with S. epidermidis were
performed with P. mirabilis, which is the primary bacterium
associated with complicated urinary tract infection. This urease
positive bacterial strain is also well known for its ability to
establish mature crystalline biofilms. This bacterium elevates
urine pH by catalyzing the conversion of urea into ammonia,
inducing calcium/magnesium phosphates to precipitate from urine and
become incorporated within the biofilm. For at least these reasons,
P. mirabilis was used in these experiments.
[0157] FIG. 23A shows the plate count data for 3 day old, 7 day old
and 14 day old P. mirabilis biofilms on the surface of the SNAP
impregnated catheter tubing and on the surface of the control
tubing. As shown in FIG. 23A, the results for 3 day biofilms
already show a 3 logarithmic unit difference in cell viability
between the SNAP impregnated and control catheter segments (n=3, p
value=0.02). Although the growth period is rather short, these
biofilms had developed to a relatively mature stage.
[0158] Comparing FIGS. 22A and 23A, the plate count data (at 3
days) of the control catheter segments for the P. mirabilis
experiments were significantly higher than the S. epidermidis
biofilms. These results are also supported by the black and white
representation of the fluorescent micrographs, shown in FIG. 23B,
that show significantly greater biomass than the S. epidermidis
biofilms (see FIG. 22B). Without being bound to any theory, it is
believed that that P. mirabilis, by expressing
mannose-resistant/Proteus-like (MR/P) fimbriae and P. mirabilis
fimbriae (PMF), may be able to more rapidly and efficiently
adhere/colonize onto the abiotic surfaces. Similar observations and
comparisons between P. mirabilis and S. epidermidis can also be
made for 7 day and 14 day biofilms, and an even greater decrease in
biofilm formation is observed for P. mirabilis compared to S.
epidermidis for the SNAP impregnated catheter pieces (see FIGS. 22A
and 23A). In FIG. 23A, the cell counts for the SNAP impregnated
catheter tubing are 5 logarithmic units (at 7 days, n=3, p
value=0.02) and 6 logarithmic units (at 14 days, n=3, p
value=0.007) lower than those for controls. It FIG. 23A, it the
difference in viable P. mirabilis cell counts between SNAP
impregnated catheter pieces and controls increases with biofilm
growth time, indicating that the SNAP-doped catheters, by releasing
NO, can progressively reduce biofilm development. This finding is
further corroborated by the fluorescent imaging data (black and
white representations of which are shown in FIG. 23B). FIG. 23B
shows that bacterial surface coverage on the SNAP impregnated
catheters segments is noticeably less than the controls. These
results demonstrate that NO releasing SNAP impregnated Foley
catheters may be suitable for preventing catheter surface
encrustation by crystalline biofilm-forming bacterial species.
[0159] The data set forth herein with P. mirabilis illustrate that
a synthetic NO-donor, with extremely stable NO release at
low/non-toxic fluxes, is capable of preventing P. mirabilis mature
biofilm formation. Other systems and/or methods have been used in
attempts to prevent P. mirabilis mature biofilm formation. In some
instances, biofilm inhibition had been observed only against
non-mature biofilm communities. In other instances, the methods
have not been successful for several reasons, such as instability
and decay of the NO donor species, no protection against
gram-negative bacteria, and antibiotic tolerance exhibited by the
bacteria.
P. mirabilis Anti-Biofilm Study after Pre-Soaking SNAP Catheters
for 24 Hours for Example 2
[0160] Chemiluminescence data, via the NOA, shows that the SNAP
impregnated catheters display an initial burst of NO during the
first day of soaking at 37.degree. C., which is due to the thermal
decomposition and the diffusion of SNAP out of the polymeric
surface of the catheters. The bactericidal efficacies of NO
releasing materials improve with increasing initial NO flux. To
investigate non-toxic NO flux thresholds in urinary tract tissues,
this experiment aimed for SNAP impregnated catheters to generate NO
within ranges similar to those released by healthy endothelial
cells (0.5-4.times.10.sup.-10 mol min.sup.-1 cm.sup.-2). To ensure
that SNAP impregnated catheters' antibiofilm properties were not
due to the bactericidal effect of an initial high NO flux
(4.5.times.10.sup.-10 mol min.sup.-1 cm.sup.-2) during the initial
hours of anti-bacterial studies in the bioreactor, additional
experiments were conducted in which the SNAP catheters were first
pre-soaked in PBS at 37.degree. C. for 24 hours.
[0161] After this pre-soaking step, 3 day P. mirabilis
anti-biofilms studies were conducted exactly as described above.
FIG. 24A illustrates the plate count data of viable bacteria
adhered on the catheters' surfaces. This data clearly illustrates
that there is more than 3 logarithmic units difference between
controls and pre-soaked SNAP impregnated catheters (n=6, p
value=0.010). As such, in previous experiments, the initial release
of high NO levels during day 1 was not the major factor
contributing to the anti-biofilm properties of SNAP impregnated
catheters. Indeed, even when SNAP impregnated catheters are
pre-soaked for 24 hours before the start of the biofilm experiment,
a significant reduction in viable cell counts can be observed for
NO releasing catheters as compared to those for controls. These
results are also supported by fluorescent images showing that
bacterial surface coverage of pre-soaked SNAP impregnated catheters
is noticeably less than the controls (black and white
representations of the fluorescent images are shown in FIG.
24B).
[0162] SNAP Impregnated Foley Catheters NO Releasing Properties
Post P. mirabilis Anti-Biofilm Experiment
[0163] To ensure that the SNAP impregnated catheter pieces can
still release NO at a flux >0.5.times.10.sup.-10 mol min.sup.-1
cm.sup.-2 near the end of the antibiofilm experiments, the NO
releasing profile of three SNAP catheter segments, pre-soaked in
PBS prior to the beginning of a 3 day P. mirabilis biofilm
experiment, were tested via the NOA. As shown in FIG. 25, the SNAP
impregnated catheter segments can still release NO at a flux
>1.times.10.sup.-10 mol min.sup.-1 cm.sup.-2 at the end of the
biofilm experiment (96 hours post initial soaking step) (FIG. 25,
n=3).
[0164] Further, the NO releasing properties of the same catheter
segments were then also tested on the following day, and the NO
flux remained relatively constant (120 hours post initial soaking
step) (FIG. 25, n=3). These results provide further evidence that
the antibiofilm activity of SNAP impregnated catheters is due to
the steady NO release properties of these novel biomedical
devices.
Conclusions for Example 2
[0165] The data for Example 2 demonstrates that an FDA approved
silicone Foley catheter material can be impregnated with SNAP via a
solvent swelling method, and that the resulting catheters can
release a relatively steady NO flux at their surfaces for 30 days.
Since no extrusion process is required for the incorporation of the
NO donor/antimicrobial agent into the catheter walls with little or
no chemical degradation of the SNAP, the catheters should maintain
functionality with stable NO release capability.
[0166] The SNAP impregnated catheter tubings also exhibit
significant anti-biofilm properties using two bacteria strains (S.
epidermidis and P. mirabilis) that are responsible for high rates
of nosocomial urinary catheter associated infections.
[0167] Furthermore, the preliminary assessment of the toxicity of
SNAP impregnated catheter tubing was provided by Wuxi AppTec Inc.
(St. Paul, Minn.) using ISO-based GLP biocompatibility studies. The
catheter tubing received the safest scores possible (0) for in
vitro toxicity testing (0-1=safe, 3-4=toxic) on L-929 Mouse
Fibroblast Cells (extracts were taken from the catheter pieces
stored for 24 hours at 37.degree. C. in Eagle's Minimal Essential
Media), as well as for in vivo testing in mice (extracts were taken
from the catheter pieces stored for 72 hours at 37.degree. C. in
both saline and sesame oil). Hence, it is believed that the SNAP
impregnated Foley urinary catheters provide a cost effective and
suitable approach to dramatically reduce the frequency of
nosocomial CAUTIs.
[0168] It is to be understood that the ranges provided herein
include the stated range and any value or sub-range within the
stated range. For example, a range of about 0.2.times..sup.-10 mol
cm.sup.-2 min.sup.-1 to about 20.times..sup.-10 mol cm.sup.-2
min.sup.-1 should be interpreted to include not only the explicitly
recited limits of 0.2.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 to
about 20.times.10.sup.-10 mol cm.sup.-2 min.sup.-1, but also to
include individual values therebetween, such as 1.times.10.sup.-10
mol cm.sup.-2 min.sup.-1, 14.5.times.10.sup.-10 mol cm.sup.-2
min.sup.-1, etc., as well as sub-ranges therebetween, such as from
0.75.times..sup.-10 mol cm.sup.-2 min.sup.-1 to about
17.times.10.sup.-10 mol cm.sup.-2 min.sup.-1, from about
5.times.10.sup.-10 mol cm.sup.-2 min.sup.-1 to about
15.times.10.sup.-10 mol cm.sup.-2 min.sup.-1, etc. Furthermore,
when "about" or "approximately" or the like is/are utilized to
describe a value, this is meant to encompass minor variations (up
to +/-10%) from the stated value.
[0169] Reference throughout the specification to "one example",
"another example", "an example", and so forth, means that a
particular element (e.g., feature, structure, and/or
characteristic) described in connection with the example is
included in at least one example described herein, and may or may
not be present in other examples. In addition, it is to be
understood that the described elements for any example may be
combined in any suitable manner in the various examples unless the
context clearly dictates otherwise.
[0170] Furthermore, in describing and claiming the examples
disclosed herein, the singular forms "a", "an", and "the" include
plural referents unless the context clearly dictates otherwise.
[0171] While several examples have been described in detail, it
will be apparent to those skilled in the art that the disclosed
examples may be modified. Therefore, the foregoing description is
to be considered non-limiting.
* * * * *