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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
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
Implantable biosensors for glucose and lactate monitoring in trauma victims
PSMBioChip Biotransducer Prototype
1
2
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
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
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
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
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
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 =
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
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
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.
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)
-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
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…
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
-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
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.
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).
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
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