Title: Polymer
1Nitric Oxide Releasing/Generating Polymers
Preparation, Characterization and Biomedical
Applications Mark E. Meyerhoff Department of
Chemistry University of Michigan
Polymer
2Patient Monitoring with Intra-Arterial Sensors
electrochemical or optical sensors
3Typical Accuracy Pattern Observed for
Intra-Arterial Measurements
P
O
125
2
mm
100
X
X
X
X
X
X
X
X
X
X
X
Hg
75
50
P
CO
mm
40
2
Hg
30
X
X
X
X
X
X
X
X
X
X
X
7.5
pH
X
X
X
7.4
X
X
X
X
X
X
X
X
7.3
4
2
6
8
Time (h)
X in vitro blood gas measurement
4Biological Response to Implanted Materials in
Blood
Fibrin and thrombus formation
Platelet adhesion
Protein adsorption
Platelet
Material surface
Fibrin
5NO Production Endothelial Cells
Vascular Endothelial Cell
Smooth Muscle Cell
Blood Vessel Interior
Red Blood Cell
Platelet
NO
surface flux 1 x 10-10 mol/cm2min
Nitric Oxide Synthase
6Solution Reactions of NO
2 NO O2 -------gt 2 NO2 2 NO2 lt-----------gt
N2O4 ----------gt NO2- NO3- NO NO2
lt---------gt N2O3 ---------gt 2 NO2- NO
Hb(II)-O2 ----------gt Hb(III) NO3-
H2O
H2O
7Concept of NO Release Polymeric Films for
Improved Biocompatibility of Chemical Sensors
sensor
8Hypothesized Mechanism of Diazeniumdiolate
Formation and Dissociation
Modified from the one proposed by R. S. Drago.
(Drago, R. S. Ragsdale, R. O. Eyman, D. P.
J. Am. Chem. Soc. 1961, 83, 4337.)
9Dimethylhexanediamine- diazeniumdiolate
(DMHD/N2O2)
Trilayer doped polymer configuration
10Chemiluminescence Detection of NO
11Tubular Amperometric Oxygen Sensor for Arterial
Implantation
Teflon coated Ag cathode
NO-adduct doped film
silicone rubber tubing
Ag/AgCl anode
0.15M KCl internal solution
cathode reaction O2 2H 4e-
2OH-
12NO Release Curve for Sensor Sleeves Coated with
DMHD/N2O2 Dispersed within a SR Matrix
flux from normal EC
NO-doped sensors were dip-coated in a solution of
90 silicone rubber, 8 plasticizer and 2
DMHD/N2O2 (w/w) in 5 ml of THF. NO release was
measured via chemiluminescence by soaking the
sensor sleeve in phosphate buffer saline
solution, pH7.4.
13Amperometric O2 Sensor Performance with and
without DMHD/N2O2
360 mmHg
control
NO-coated
225
Response times Control 53.3 21.0 sec
NO 60.0 19.2 sec
151 mmHg
150
current (nA)
72 mmHg
75
36 mmHg
0 mmHg
0
0
1500
3000
4500
6000
time (sec)
14Schematic of In Vivo Oxygen Sensor Experiment
15In Vivo Sensor Performance
125
100
75
PO2 (mmHg)
50
bench-top measurement
25
implanted control sensor
implanted NO sensor
0
0
3
6
9
12
time (h)
16Scanning Electron Micrographs of Sensors after 18
h Implantation in Canine Carotid Arteries
Control sensor surface
DMHD/N2O2 sensor surface
NO-doped sensors were dip-coated in a solution of
90 silicone rubber, 8 plasticizer and 2
DMHD/N2O2 (w/w) in 5 ml of xylene.
17In Vivo Oxygen Sensor Performance
Deviation of gt10 from in vitro blood-gas values
6 of 6 control sensors
1 of 6 NO-modified sensors
Severe Failure (gt50 deviation)
3 of 6 control sensors
0 of 6 NO-modified sensors
Failure Mechanism blood vessel
constriction and clotting
concomitant blood flow decrease
platelet adhesion
18Nitric Oxide Release Polymer Coatings for
Extracorporeal Circuits
Applications
- hemodialysis
- liver support systems
- extracorporeal oxygenation
- cardiopulmonary bypass
19Extracorporeal Rabbit Model
NO coating
NO
NO
NO
NO
NO
Pump
20Platelet Consumption During Extracorporeal
Circulation with and without NO Release
100
75
blood platelet concentration ( of initial)
50
25
0
0
1
2
3
4
time (h)
21Scanning Electron Micrographs of Extracorporeal
Circuits after 4 h without Heparinization
Control circuit
DMHD/N2O2 circuit
Both circuit segments are from the entry point
into the peristaltic pump.
22Possible Mechanisms of Nitric Oxide Release from
DMHD/N2O2 Films
23Chromatograms of DMHD Standard and Polymer
Soaking Solutions after FMOC-Cl Derivatization
3.93
3.91
7.17
7.20
2.30
2.29
5.86
15.47
15.42
(B)
(A)
Film soaking solution
DMHD standard
Solute peaks at 2.3, 3.9 and 7.2 min are due to
excess FMOC.
24More Lipophilic Diamine Diazeniumdiolates Under
Investigation
25Extended NO Release of DBHD/N2O2 in Silicone
Rubber Coating on Oxygen Sensor
with anion sites (tetraphenylborate derivative)
10
Surface Flux (x10-10 mol/cm2/min)
5
w/o lipophilic anion sites
0
10 20 30 40
50
Time (h)
NO release was measured via chemiluminescence by
soaking sensor sleeves prepared by dip coating a
plasticized silicone rubber solution (79 wt SR,
8 wt DOS) containing 5 wt DBHD/N2O2 and 8 wt
KTpClPB (ratio 11) in phosphate buffer saline
solution (pH7.4, 100 mM phosphate, 137 mM NaCl,
2.7 mM KCl).
26Schematic of Using Lipophilic Anionic Species to
Buffer the Polymer Matrix and Prolong NO Release
KB-
H OH-
KB-
2 NO
KB-
KB-
H OH-
K B-
H
K
2 NO
B- tetrakis-4-chlorophenylborate
anion Raliphatic alkyl side chain
27Determination of pH Changes in Polymer Films
B.
A.
With KTpClPB
Without KTpClPB
Increasing time
Increasing time
Visible spectra of Chromoionophore II
incorporated into a 12 PVC/DOS matrix containing
DBHD/N2O2 soaked in PBS buffer (pH 7.4) as a
function of time with (A) and without (B)
KTpClPB. The protonated absorption peak of
chromoionphore II is at 650 nm and the
deprotonated band is at 514 nm.
28Oxygen Levels by Intravascular Sensors Coated
with DBHD-SR Compared to Control
9-3-02
29Oxygen Sensors Coated with DBHD/N2O2-SR After
16h in Femoral Porcine Arteries
30Average Deviation in Oxygen Level Determined by
NO-Releasing DBHD-SR and Control Sensors Compared
to Standard Blood Gas Analysis (In Vivo Porcine
Model)
N 9 controls and 9 NO-Release Sensors
31Application of Diazeniumdiolates Vascular
GraftsMacroscopic Images and Thrombus Free
Surface Area Results
NO Release
Control
Venous
Arterial
Venous
Arterial
DBHD/N2O2 in PVC coated on 21 cm Commercial
Vectra grafts implanted in sheep for 21 d.
plt0.001
32H2N(CH2)nNH(CH2)3Si(OCH3)3
C
H
C
H
C
H
3
3
3
Si
H
O
O
Si
O
Si
O
H
dibutyltin dilaurate
x
C
H
C
H
C
H
3
3
3
Rxn Scheme to Prepare NO Release Silicone Rubber
NO, 80psi
n 2 or 6
33Effect of SR Film Thickness on Amount of NO
Released
NO release profiles (average of three films) of
DACA-6/N2O2-SR (6X, diam. 1 cm2) films with
different thickness in PBS at 37 oC, measured
indirectly by determining the level of the
oxidized NO product (nitrite) using the Griess
assay.
34Chemiluminescence Determination of NO-Release
from DACA-6/N2O2 Coated Oxygen Sensor
coating thickness100 µm
35Oxygen Levels Determined by Intravascular Sensors
Coated with DACA-6 SR Compared to Control
5-21-02
36Oxygen Sensors After Removal From Porcine
Arteries (16-18 h)
37Percent Deviation ( s.d) in Oxygen Levels
Determined by Intravascular Sensors Compared to
Standard In Vitro Blood Gas Measurement
N 10 controls and 10 NO Release Sensors--in 5
Pigs
38Schematic of NO Release from Sil-N2O2 Embedded
Polymer Matrices
Nitric oxide gas is released as water molecules
diffuse into polymer/Sil-N2O2 matrices.
39Synthesis of Sil-N2O2 Particles
- R Sil-N
-
- H Sil-1N-H
- CH3 Sil-1N-C1
- (CH2)2NH2 Sil-2N2
- (CH2)6NH2 Sil-2N6
- M Na, K, Li
FS
Si
OH
O
FS
Si
O
Si(CH
)
NHR
2
3
O
O
FS
Si
O
Si(CH
)
NHR
2
3
O
N2O2-M
40Platelet-Count Drop for Sil-N2O2 Coated and
Control Circuits after 4 h ECC in Rabbits
Less platelet-count drop was observed for
NO-release circuits, indicating improved blood
compatibility via NO-release.
41Applications of Diazeniumdiolates Glucose Sensor
Schematic of Amperometric Glucose Sensor
Calibration Curve
to recording device
DBHD/N2O2 doped PU/PDMS membrane
Ag/AgCl reference electrode coil
glucose oxidase membrane
nafion/cellulose acetate membrane
Dynamic Response
sensing membrane over Pt-Ir electrode
Pt/Ir electrode
PTFE insulation
0.25 mm
inserted into tissue
in collaboration with George Wilson
42Application of Diazeniumdiolates Glucose Sensors
NO Release
Control
Representative histology images of (A) a control
glucose sensor implant site and (B) an NO release
glucose sensor implant site. The sensors were
implanted subcutaneously using a rat model.
After 24 h, the sensors and tissues were excised
for histology studies. Pink indicates skeletal
muscle and subcutaneous tissue. The dark purple
regions are neutrophil infiltration indicating an
inflammatory response. NO Release exhibits
statistically less inflammatory response vs.
controls (N6 each)-plt0.001-- as quantitated by
scoring the degree of neutrophil infiltration
Courtesy of Raeann Gifford and Dr. George S.
Wilson, University of Kansas
43Nitric Oxide Cycle
L-Arginine
NO synthase
NO
NO Scavengers (Hb, thiols, etc.)
Nitrite reductase
NO2-
Reutov, V. P. Biochem. 1999, 64, 538.
R-S-NO
44Estimates of S-Nitrosothiols/Nitrite Levels
Present in Circulating Blood
1(a) arterial, (v) venous levels in blood, all
other values in plasma
Kelm, M Biochim. Biophys. Acta 1411 (1999)
273-289.
45S-Nitrosothiols as NO Donors
2RS-
2Cu2
RSSR
46Schematic Diagram of Catalytic NO Generation at
the Polymer/Solution Interface
B. K. Oh, M. E. Meyerhoff, J. Amer. Chem. Soc.
125, 9552 (2003)
47Decomposition of SNAP to NO by Cu(II)-Complex
Doped Film
Film formulation 65.3 wt PVC 32.6 wt NPOE
2.1 wt Cu(II)-complex 0.4 cm disk from parent
film
SNAP S-Nitroso-N-acetyl-DL-penicillamine
Background solution deoxygenated 10 mM PBS, pH
7.4 and 100 mM EDTA NO generation was measured
by NO analyzer (NO chemiluminescence)
48Decomposition of SNAP at the Surface of
Cu(II)-Complex Doped Film
49Sham Catheter Sensors Coated withPolymer Layer
Containing Cu(II)-DTTCT
A. Control
B. NO Generating
Preliminary In Vivo Results (in pigs) -45 of
control coatings show visible thrombus (N11) -0
of NO-generating coatings show visible clot (N11)
50- Amperometric Detection Scheme of RSNO Sensor
Pt wire
0.75 V
Tip of glass capillary
- 3e-
Platinized Pt surface 1
NO3-
Thin PTFE membrane
NO
Thin hydrophilic PU layer
containing Cu sources, e.g.,
Cu(II)-DTTCT,
Cu(II)-phosphate, or
Cu(0)-particles
Cu(II)
Ox.
RS- NO
Red.
RSNO
Cu(I)
Sample solution phase
- Example
( SNAP )
( Ascorbate )
Reducing equivalents
RSNO
1) Lee, Youngmi Oh, Bong Kyun Meyerhoff, Mark
E., Anal Chem. 76 (2004) 536-544
51Reversibility RSNO Sensor Signal
amperometric response toward SNAP
Experimental Conditions at room temperature,
with 10 µM EDTA and 10 µM sodium ascorbate in pH
7.4 PBS aqueous solution
52Sensor Sensitivity to RSNO species
k values of RSNOs (with no added RSH) RSNO
k CysNO 24500 500 SNAP
20 1 SNAC 0 GSNO 0
(decomposition rate k Cu2RSNO )
D. Williams, Acc. Chem. Res. 32 (1999) 869
Experimental Condition at room temperature,
under 10 µM EDTA and 10 µM sodium ascorbate in pH
7.4 PBS aqueous solution
53 Detection of NO Generating Ability of Fresh
Blood Using Electrochemical RSNO Sensor
54NO-Release Collaborators
Graduate Students/Undergraduates/Post-Docs
- Kelly Mowery Pawel Parzuchowski
Zhengrong Zhang - Mark Schoenfisch Huiping Zhang Sangyeul
Hwang - Jeffrey Politis Melissa Batchelor Sarah
Ingersoll - Bong Oh Megan Frost Yiduo Wu
- Qingshan Ye Cecilia Espadas-Torre
Vanessa Oklejas - Monica Rader Youngmi Lee Wansik Cha
Financial Support
Faculty/Other
- NIH-GM-56991 - NIH-GM-28882 - NIH-ECMO
Center - Michigan Critical Care
Consultants Inc. - Bolton Medical
- Larry Keefer (NIH)
- Steven Rudich (UM-Surgery)
- Charles Shanley (Beaumont Hospital)
- Robert Bartlett (UM-Surgery)
- Gail Annich (UM-Pediatrics)