bioactive hydrogels for implantable biosensors · pdf filearea where hydrogel existed a muscle...
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Bioactive Hydrogels for Implantable Biosensors
Gusphyl Justin, Abdur Rub Abdur Rahman, Anthony Guiseppi-Elie
Center for Bioelectronics, Biosensors and Biochips (C3B)Department of Chemical and Biomolecular Engineering, and
Department of Bioengineering, College of Engineering and Science, Clemson University
Clemson, South Carolina [email protected] url=http://www.biochips.org
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AcknowledgmentsClemson C3B ConsortiumClemson C3B ConsortiumDoDDoD USAMRMC PRMRP PRMRP
Undergraduates:Stephen FinleyAmber SmithLaura BrewerRyan GeorgianaDavid CochranRyan TrullBrad GordonNyan WinElizabeth HorahanMahammed AshrafCarolina FunkeyChris Nixon
Graduates:Ali BoztasAshwin RaoSheena Abraham Vandana GuptaJerome EdmonsonG. Scott TaylorBrad MagrumLouise LingerfeltGopakumar SethuramanMichael ZavatskyRachel Kahn
Post Docs:Gusphyl Justin, Ph.D.Abdur Rub Abdur Rahman, Ph.D.Marta Plonska, Ph.D.Walter Torres, Ph.D.Shufeng Liu, Ph.D.Sean I. Brahim, Ph.D.Marin Gheorghe, Ph.D.Chenghong Lei, Ph.D. High School Students
Lauren KochMatthew Sebastian
Special thanks:Dr. Kenneth ChristensenDepartment of ChemistryClemson University
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Implantable biosensors for glucose and lactate monitoring in trauma victims
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PSMBioChip Biotransducer Prototype
1
2
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50x
Counterelectrode
Referenceelectrode
Workingelectrode
50 µm
Sense Region 1(Lactate)
Sense Region 2(Glucose)
4 m
m
2 mm
(a) (b)
MDEA 5037 comprises 50 micron microdiscs, which number 37
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Proteins do not foul the surface of cells.Chapman, et al. (1984) suggested this was due the phosphorylcholine (PC) head group.Mimic the structural composition of a cell membrane by incorporating PC-containing moieties into polyHEMA-based hydrogels – “Biomimicry”
Molecular Engineering Based on Biomimicry to Achieve Implant “Stealth”
C
CH 3
OCO
CH 2
CH 2
O P
O -
O CH 2 CH 2 N +
O CH 3
CH 3
CH 3
CH 2
2-Methacryloyloxyethyl Phosphorylcholine (MPC)
Hayward J. A., Chapman D. “Biomembrane surfaces as models for polymer design: the potential for haemocompatibility”Biomaterials (1984) 4:135-142. Durrani A. A., Hayward J. A., Chapman D. “Biomembranes as models for polymer surfaces” Biomaterials (1986) 7:121-125
Membrane bi-layer
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Structure of the final bioactive composite
membrane containing covalently attached
enzyme
Guiseppi-Elie et al. J. Macromol. Sci. -Pure and App.Chem.(2001) A38(12), 1575.
CCH3
OC
OH
O
CH2
CH2
CH2
CH2 CCH3
OC
OHCH2
CH2
O
CCH3
OCO
CH2
CH2
CH2
CH2
OC O
CH2
CH2
N
CH2
CCH
OCH2 C
CH3
OC
OH
O
CH2
CH2
CH2 COC
O
CH2
CH2
OC O
CH2 CCH3
OCO
CH2
CH2
O PO-
O CH2 CH2 N+
CH3
CH3
CH3
CH2 CH2
CCH3
OC
OH
O
CH2
CH2
CH2
CH2 CH2
O
C
H
C
OC
O
CH2
CH2
CH3
CH3
CH2
CH2
CH2
H
NH
CH2 CCH3
OC
OH
O
CH2
CH2
CH2
C OO
OCH2
CH2
C O
CCH3
OCO
CH2
CH2
O PO-
O CH2 CH2 N+
O CH3
CH3
CH3
CH2COCCH2 CH2C
CH3
CH2
CH2
CH2
NH
C OO
OCH2
CH2
C O
OCCH
O
CH2
CH2
O
H
CH2CH2
O
H
N H
NH
Ox
200
4
110PEGMA MPCHEMA
TEGDA
MPB
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Muscle
Area of Moderate Fibrosis and inflammation with foamy histocytesaround the site where hydrogel was placed
Residual area of Hemorrhage
Area where hydrogel existed
A
Muscle
Rim of new connective tissueforming a capsule and some residual inflammation around hydrogel
Residual area of Hemorrhage
Hydrogel
B
In-vivo Implantation in Sprague DawleyHemorrhage Model
After implantation for 2 weeks in the trapezius muscle
1 mol % MPC
un-modified p(HEMA)
Significant encapsulation and accumulation of foreign body material
Thin band of encapsulation and much reduced residual inflammation
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HMF viability and infiltration as a function of PEG and PC content of 3% crosslinked p(HEMA) hydrogel
Retention of human muscle fibroblasts as a function of hydrogel composition
Viability of human muscle fibroblasts as a function of hydrogel composition
Human Muscle Fibroblasts (HMF)ATCC Designation: SJCRH30 [RC 13, RMS 13, SJRH30]; CRL-2061
Growth Properties: AdherentMorphology: fibroblastOrganism: Homo sapiens (human)Tissue: Muscle, metastatic site
Central Composite Design
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Hydration percentage as a function of hydrogel composition
Hydration as a function of PEG and PC content of 3% crosslinked p(HEMA) hydrogel
100 x W
W-W hydration of Degreedry
drywet =
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02468
101214161820
0 0.2 0.4 0.6 0.8 1
Relative hydration w.r.t. p(HEMA)
Rel
ativ
e ce
ll re
tent
ion
w.r.
tp(
HE
MA
)0 mol% PEGMA0.25 mol% PEGMA0.5 mol% PEGMA
Correlation of cell retention and hydration -- relative to pure 3% crosslinked p(HEMA) polymer.
Cell (HMF) retention as
a function of hydration
d1
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Slide 11
d1 The hysteresis is used to plot the correlation between hydration and dynamic contact angle because 1) there are only 6 points (6 formulations were used)2) in the advancing and receding data there are is one point that does not follow the trend and hence throws the graph off the plot.glad, 5/8/2005
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STEP 1: Surface derivatization
Glass
O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O
Acryloyl-PEG(n=110)-NHS ester
CH
CO
O CH2 O N
O
O
H2C CH2 CO
O110
OSi
NHCH
CO
O CH2 OH2C CH2 CO
CH2
CH2
CH2
OSiCH2
CH2
CH2
O
NH2
O O
N
O
O
OH
+110STEP 2:
Surface functionalization
CC H3
OC
O H
O
CH2
C H2
CH2
CH2 C
OC
O
C H2
C H2
CH3
C H2 COCC H2
OH
O
CH2
CH2
200
C
H
OCO
CH2
CH2
CH2
O
OC
O
Si
NH
CH2
CH2
C H2
O
Si
CH2
CH2
C H2
O
NH2
CCH3
OC
O H
O
CH2
CH2
O O
O
P O CH2 C H2 N+
O CH3
C H3
C H3O-
COCC H2
OCCH
O
CH2
CH2
O
H
CH2
4
CH3
CH2
110
CH2
PEGMA
MPC
HEMA
TEGDA
Partial hydrogel structure
C
CH3
OCO
CH2
CH2
O P
O-
O CH2 CH2 N+
O CH3
CH3
CH3
CH2CCH 3
OC
O H
O
CH 2
CH 2
CH 2
HEMA TEGDA PEGMA
STEP 3:UV irradiationλ= 366 nm,
10 min, Ar blanket
Methacrylate monomer cocktail
PIDMPA
CH2
O
C
CH
HC
OC
O
CH2
CH2
CH2
O
CCH3
OC
O
CH2
CH2
CH2
OH 200 MPC
STEP 1: Surface derivatization
Glass
O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O
STEP 1: Surface derivatization
Glass
O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O
O
Si
NH2
CH2
CH2
CH2
O
Si
CH2
CH2
CH2
O
NH2
O O
Acryloyl-PEG(n=110)-NHS ester
CH
CO
O CH2 O N
O
O
H2C CH2 CO
OCH
CO
O CH2 O N
O
O
H2C CH2 CO
O110
OSi
NHCH
CO
O CH2 OH2C CH2 CO
CH2
CH2
CH2
OSiCH2
CH2
CH2
O
NH2
O O
N
O
O
OH
+110
OSi
NHCH
CO
O CH2 OH2C CH2 CO
CH2
CH2
CH2
OSiCH2
CH2
CH2
O
NH2
OSi
NHCH
CO
O CH2 OH2C CH2 CO
CH2
CH2
CH2
OSiCH2
CH2
CH2
O
NH2
O OO OO O
N
O
O
OH
+110STEP 2:
Surface functionalization
CC H3
OC
O H
O
CH2
C H2
CH2
CH2 C
OC
O
C H2
C H2
CH3
C H2 COCC H2
OH
O
CH2
CH2
200
C
H
OCO
CH2
CH2
CH2
O
OC
O
Si
NH
CH2
CH2
C H2
O
Si
CH2
CH2
C H2
O
NH2
CCH3
OC
O H
O
CH2
CH2
O O
O
P O CH2 C H2 N+
O CH3
C H3
C H3O-
COCC H2
OCCH
O
CH2
CH2
O
H
CH2
4
CH3
CH2
110
CH2
PEGMA
MPC
HEMA
TEGDA
Partial hydrogel structure
C
CH3
OCO
CH2
CH2
O P
O-
O CH2 CH2 N+
O CH3
CH3
CH3
CH2CCH 3
OC
O H
O
CH 2
CH 2
CH 2
HEMA TEGDA PEGMA
STEP 3:UV irradiationλ= 366 nm,
10 min, Ar blanket
Methacrylate monomer cocktail
PIDMPA
CH2
O
C
CH
HC
OC
O
CH2
CH2
CH2
O
CCH3
OC
O
CH2
CH2
CH2
OH 200 MPC
Substrate surface modification, derivatization, monomer casting and hydrogel synthesis
Sheena Abraham, Sean Brahim, Kazuhiko Ishihara and Anthony Guiseppi-Elie “Molecularly engineered hydrogels for implant biocompatibility” Biomaterials (2005), 26(23), 4767-4778.
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0.30
0.35
0.40
0.45
0.50
0.55
0.60
48 50 52 54 56 58 60 62Potential (mV)
Nor
mal
ized
Cur
rent
(µA
)
hydrogel coateduncoated
0 .05 .0
1 0 .01 5 .02 0 .02 5 .03 0 .03 5 .0
4 5 5 0 5 5 6 0 6 5P o te n t ia l (m V ) v s . A g /A g C l
Cur
rent
(nA
)
∆I
ν2IC DL
∆=
Cyclic voltammetry in Tris/KCl (pH 7.2)
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-40
-20
0
20
40
60
0 200 400 600 800Potential (mV) vs. Ag/AgCl
Cur
rent
Den
sity
(µA
/cm
2 )
-400
-200
0
200
400
600
0 200 400 600 800Potential (mV) vs. Ag/AgCl
Cur
rent
Den
sity
(µA
/cm
2 )
-400
-200
0
200
400
600
0 200 400 600 800Potential (mV) vs Ag/AgCl
Cur
rent
den
sity
(µA
/cm
2 )
A B
C
Ferrocene-ferrocenium oxidation-reduction at microdisc electrode arrays (MDEAs)
MDEA 050
MDEA 050 + hydrogel
MDEA 5037
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Chemical structure, electrochemical and electrical properties of conducting electroactive polypyrrole films
E (V) vs. SCE-1.0 -0.5 0.0 0.5 1.0
Con
duct
ivity
(S/c
m)
1e-6
1e-5
1e-4
1e-3
1e-2
cath
odic
I
(mA
)
anod
ic-0.6
-0.4
-0.2
0.0
0.2
0.4
Cyclic Voltammogram Electrical Conductivity
Guiseppi-Elie et al. In Handbook of Conductive Polymers 2nd Edition (1997), Chapter 34, p 963.Guiseppi-Elie et al. In, Electrical, Optical, and Magnetic Properties of Organic Solid State Materials, Mat. Res. Soc. Symp. Proc. Vol. 413; 1996, p 439.
Polyelectrolyte
Redox switchable (ms)
High chemical stability
High hydrolytic stability
Demonstrated biocompatibility
polypyrrolepolypyrrole
A-
+
Developed in late 1970s as materials for polymer batteries…
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Electropolymerization of Polypyrrole (PPy) Within p(HEMA)-based bioactive hydrogels
Apparatus connected to a Princeton Applied Research (PAR) Potentiostat/Galvano-stat in conjunction with a Solartron frequency response analyzer (FRA) for electropolymeriza-tion (+0.85V for 100 sec) and impedance measurements (1Hz to 10kHz).
Hydrogel (UV crosslinked)
0.4M pyrrole in 0.1M PBS/0.1M KCl solution (pH = 6)
Large area Pt counter electrode
Ag|AgCl 3M Cl-
reference electrode
Au-coated slide (working electrode)
2cm
2cm
Cloning cylinder
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-90-80-70-60-50-40-30-20-10
01 10 100 1000 10000
Frequency (Hz)
Thet
a
1
10
100
1000
10000
100000
1 10 100 1000 10000Frequency (Hz)
|Z|
-2500.00
-2000.00
-1500.00
-1000.00
-500.00
0.00
0 20 40 60 80 100 120
Time (s)
Cur
rent
(µA
)
PPy-Hydrogel
Hydrogel
PPy-Hydrogel
Hydrogel
Electropolymerization kinetics
0.85V vs. Ag/AgCl
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Viability Study: Human muscle fibroblasts (RMS 13) and Rat pheochromocytoma cells
(PC12)
Neuronal progenitor cell line (PC12) and human muscle fibroblasts (RMS13) grown in F-12K and RPMI 1640, respectively.
Cells seeded onto hydrogel-polypyrrole coated gold surfaces.
Cell viability determined.
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Summary
Cell viability is enhanced by the incorporation of phosphorylcholine moieties into the pHEMA-based hydrogel..
Polypyrrole reduces the electrical impedance of hydrogel coated gold electrodes.
Hydrogel coated onto MDEAs increases impedance (decreased diffusion coefficient compared to aqueous solution).
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AcknowledgmentsClemson C3B ConsortiumClemson C3B ConsortiumDoDDoD USAMRMC PRMRP PRMRP
Undergraduates:Stephen FinleyAmber SmithLaura BrewerRyan GeorgianaDavid CochranRyan TrullBrad GordonNyan WinElizabeth HorahanMahammed AshrafCarolina FunkeyChris Nixon
Graduates:Ali BoztasAshwin RaoSheena Abraham Vandana GuptaJerome EdmonsonG. Scott TaylorBrad MagrumLouise LingerfeltGopakumar SethuramanMichael ZavatskyRachel Khan
Post Docs:Gusphyl Justin, Ph.D.Abdur Rub Abdur Rahman, Ph.D.Marta Plonska, Ph.D.Walter Torres, Ph.D.Shufeng Liu, Ph.D.Sean I. Brahim, Ph.D.Marin Gheorghe, Ph.D.Chenghong Lei, Ph.D. High School Students
Lauren KochMatthew Sebastian
Special thanks:Dr. Kenneth ChristensenDepartment of ChemistryClemson University