Lawrence Livermore Natl Lab
Univ of California-Berkeley
Berkeley, CA 94720
510-486-6581 Division of Biological Materials
Ward Bldg 10-116
Chicago, IL 60611-3008
Inst of Biomedical Engineering
Tokyo Women's Medical Univ
Tokyo, 162-8666 JAPAN
Ctr for Bioengineering
Univ of Washington
Seattle, WA 98195
* Invited paper
SESSION EE1: HYDROGEL SYSTEMS
8:30 AM *EE1.1
DESIGN OF POLYMERS TO INCREASE THE EFFICIENCY OF ENDOSOMAL
RELEASE OF DRUGS. Allan Hoffman , Chantal Lackey, Niren Murthy, Patrick
Stayton, Dept of Bioengineering, Oliver Press, Dept of Medicine; David
Tirrell, Dept of Chemical Engineering, University of Washington, Seattle,
Chair: Patrick S. Stayton
Monday Morning, April 5, 1999
Salon 15 (M)
The dose-limiting step of many therapeutic agents is delivery
to the appropriate cellular compartment. For example, in gene therapy and
immunotoxin therapy, there is a need to deliver DNA and toxins to cellular
targets such as the nucleus or the ribosomes, and to avoid their premature
degradation by lysosomal enzymes. An interesting class of polymers, called
``smart'' polymers, or stimuli-responsive polymers, may be incorporated
into polymeric drug delivery systems for enhancing the efficacy of intracellular
delivery of drugs such as DNA and immunotoxins. Since the endosome is at
a reduced pH, pH-sensitive polymers that become hydrophobic at the lower
pHs may disrupt the endosomal membrane, releasing the drug into the cytosol
before it is degraded by lysosomal enzymes. We are synthesizing and investigating
the ability of a variety of pH-sensitive homopolymers and copolymers to
disrupt lipid bilayer membranes, by measuring their abilities to hemolyse
red blood cells (RBC). We have also prepared graft copolymers of pH-sensitive
fusogenic peptides grafted to synthetic polymer backbones and showed that
they were much more effective in lysing RBCs than a physical mixture of
the two components. Results will also be presented on the hemolytic efficiency
of our polymers when the polymer is conjugated to a protein, which is relevant
to immunotoxin therapy.
9:00 AM EE1.2
HYDROGELS OF ASSOCIATING POLYMERS IN THE PHASE SEPARATING
REGIME. Giyoong Tae , Julie A. Kornfield, Caltech, Pasadena, CA; Jeffrey
A. Hubbell, Swiss Federal Institute of Technology (ETH), Zurich, SWITZERLAND.
Model hydrogels can be made from a long, water soluble
midblock (polyethyleneglycol, PEG) terminated at both ends by a hydrophobic
block (here a fluoroalkyl segment). Relative to more extensively studied
alkyl terminated PEG, the fluoroalkyl capped chains are of interest because
of the stronger hydrophobicity and consequently the stronger association
of - compared to -.
This difference accentuates the tendancy to form two phase (sol-gel) systems.
Two phase systems are of interest for biomedical applications because they
have the potential to provide slow, tunable erosion kinetics. First, we
present the phase behavior of these associating polymers, in particular
the effect of PEG length and hydrophobe length on the equilibrium composition
of coexisting sol and gel, using PEGs of 6K,10K, and 20K g/mol with -
of n = 8 or 10. Then we examine the linear viscoelasticity of their gel
phases as a function of molecular weight, n, and concentration. At the
concentration of the equilibrium gel, these systems show single relaxation
behavior, like hydrocarbon terminated PEG. More concentrated gels show
the appearance of a much slower relaxation process, which may reflect an
ordering transition of the micelle cores.
9:15 AM EE1.3
NUCLEOTIDES-RESPONSIVE HYDROGELS PREPARED FROM A FOOT-PRINTING
TECHNIQUE IN POLYION COMPLEXES. Yasumasa Kanekiyo , Seiji Shinkai, CHEMOTRANSFIGURATION
Project, Japan Science and Technology Corporation, Fukuoka, JAPAN.
The formation of the polyion complex was applied to the
preparation of the nucleotides-responsive hydrogels. Polyanion containing
boronic acid units can bind AMP by a boronate-diol ester formation. When
this polyanion forms a complex with a polycation in the presence of AMP,
the phosphate anionic charge of AMP affects the polycation/polyanion composition
of the polyion complex. Thus, after the removal of AMP from the polyion
complex a hydrogel which has excess cationic charges is created. When this
swollen hydrogel is immersed in aqueous AMP solution, deswlling occurs.
In contrast, AMP-analogues(adenosine, 2-deoxyAMP, and 2-deoxyadenosine)
do not induce the deswelling. Adenosine has cis-diol group but does not
have phosphate group, 2-deoxyAMP has no cis-diol group, and 2-deoxyadenosine
has neither phosphate group nor cis-diol group. These results indicate
that both the electrostatic interaction between anionic phosphate group
and polycation and the boronate-diol ester formation are indispensable
for the shrinkage.
9:30 AM EE1.4 PHOTOPOLYMERIZABLE POLY (VINYL ALCOHOL)
GELS. Penny Martens , Deanna Shook, Kristi S. Anseth, University of Colorado,
Department of Chemical Engineering, Boulder, CO.
Crosslinked poly (vinyl alcohol) (PVA) hydrogels are being
investigated as a replacement for current wound closure techniques. PVA
was chosen for this application for many reasons, including its history
in medical applications, its natural adhesive behavior, and the pendant
hydroxyl groups that can be easily functionalized. The design of a photopolymerizable
tissue adhesive has the potential of providing an alternative technique
that is easier, faster, and less painful than currents methods, while still
providing the same mechanical and cosmetic results. The photocrosslinkable
PVA hydrogels were prepared by acrylating 98-99
hydrolyzed PVA, adding UV sensitive initiators, and photo-curing the acrylated
PVA macromer solution. There are several variables that can be controlled
in our system, including the molecular weight of the PVA, crosslinking
density, PVA in the solution,
and the addition of degradable units. By rationally changing these variables,
we can alter the properties of our system, including reaction time, mechanical
properties, and adhesion. We have shown that high conversion can be reached
in under 10 minutes using a low intensity UV light source (4 mW/cm2).
Once the gels were photopolymerized, several mechanical tests were performed
to measure tensile strength, strain at break, the tensile modulus, and
work of adhesion and cohesion. The strength (.01-.1 MPa) and cohesiveness
(1-2 x 10-6 J/cm2) of these gels was determined as
a function of macromer content and crosslinking density. Progress will
be shown in studies of adhesion, degradation, and use as a tissue adhesive
in a rat model.
SESSION EE2: TISSUE ENGINEERING APPLICATIONS
10:15 AM *EE2.1
SELF ASSEMBLING BIOMATERIALS FOR TISSUE REPAIR. Samuel
I. Stupp , Univ of Illinois at Urbana-Champaign, Depts of Materials Science
and Engineering and Chemistry, Beckman Institute for Advanced Science and
Technology, Materials Research Lab.
Chair: Phillip B. Messersmith
Monday Morning, April 5, 1999
Salon 15 (M)
Our laboratory is interested in the design of self assembling
biomaterials for human tissue repair. Self assembly offers key opportunities
in this area including in situ organization of molecules into functional
materials during clinical procedures, and also the functionalization of
the large surface area of tissue engineering scaffolds. Self assembly could
also offer a road to interactive biomaterials that release nanostructures
as molecular implants that can control certain aspects of cell behavior.
This lecture will describe our efforts on synthesis of self assembling
lamellar solids containing cholesterol moieties as a universal ligand for
cell services. Fibroblasts cultured on these self assembling solids have
been found to attach on the surfaces of these lamellar nanostructures and
interact with charged and hydrophobic surfaces. Preliminary evidence has
been obtained for their strong interaction with cell membranes which may
be useful in the design of novel therapeutic biomaterials.
10:45 AM EE2.2
FUNDAMENTAL STUDIES OF BIODEGRADABLE HYDROGELS AS CARTILAGE
REPLACEMENT MATERIALS. Andrew T. Metters , Kristi S. Anseth, Christopher
Bowman, University of Colorado, Department of Chemical Engineering, Boulder,
Through intelligent control of monomer chemistry and gelling
techniques, biodegradable hydrogels with a range of mechanical strengths
and degradation timescales have been constructed. Biodegradable hydrogel
scaffolds have been focused upon because of their ability to provide structural
and vascular support for cell growth while eliminating complications due
to long-term cell-polymer interactions. A diacrylated poly(ethylene glycol)-poly(lactic
acid) copolymer chemistry developed by Hubbell et al. (Macromolecules 1993,
26, pg. 581-587) has been elaborated upon to produce synthetic networks
with equilibrium water contents (EWC) above 70% and initial compressive
moduli values exceeding 1 MPa, demonstrating its viability as a cartilage
replacement material. The hydrolytically-labile lactide linkages allow
biodegradability while the acrylated end-groups provide the means to form
a crosslinked network. Experiments have shown that the mechanical strengths,
EWCs, and useful lifetimes of these water-swellable networks are coupled
to their tailored copolymer chemistry as well as their processing conditions.
A systematic study utilizing photopolymerized gels has been undertaken
in order to elucidate the controlling factors behind the bulk-degradation
process, as well as monitor changes in network structure with degradation.
Factors that minimize the so-called burst effect commonly seen with bulk-degrading
systems during the final stage of mass loss will specifically be investigated.
A kinetic model will be used in conjunction with the experimental data
to explain the exponential modulus decay and primarily linear mass loss
observed versus degradation time for these hydrogels
11:00 AM EE2.3
INJECTABLE THERMO-RESPONSIVE HYDROGELS AS SCAFFOLDS FOR
TISSUE ENGINEERING APPLICATIONS. Ranee A. Stile , Kevin E. Healy, Northwestern
University, Division of Biological Materials and Department of Biomedical
Engineering, Chicago, IL; Wesley R. Burghardt, Department of Chemical Engineering,
Model injectable hydrogels that support tissue formation
in vitro were synthesized using the thermo-responsive polymer, poly(N
isopropylacrylamicle) [P(NIPAAm)]. NIPAAm, acrylic acid (AAc) (2.6
w/w total monomer), and BIS (0.16-0.20,
w/w total monomer) were polymerized in phosphate-buffered saline (PBS)
at room temperature (RT; 22C)
for 19 hours. At RT, the loosely crosslinked hydrogels were readily injectable
through a standard syringe (i.e., without a needle attached) and did not
demonstrate discernible macroscopic fracture following injection. When
heated to 37C, the hydrogels exhibited
a phase transition, as evidenced by an increase in opacity and a decrease
in ductility. Hydrogel characterization studies included: 1) verification
of the hydrogel chemistry with solid-state 1H-magic angle spinning
(MAS)-NMR spectroscopy; 2) determination of the lower critical solution
temperature (LCST) using a UV-Vis spectrophotometer; 3) estimation of the
water content at RT and 37C as
a function of swelling in PBS via a freeze-drying technique; 4) calculation
of the change in volume between RT and 37C
as a function of swelling in PBS by a water displacement method; and 5)
evaluation of the rheological properties using an oscillating rheometer.
In addition, bovine articular chondrocytes were seeded into the hydrogels
and the cell-loaded constructs were cultured in vitro . In situ
fluorescent viability studies and histological analyses were performed.
Overall, the 1H-NMR spectroscopic analyses were consistent with
the chemical structure of the hydrogels. Prior to swelling in PBS, the
P(NIPAAm-co-AAc) hydrogel contained 93.35
water at 37C and demonstrated
volume change between RT and 37C.
Additionally, the rheological properties changed considerably with temperature.
The injectable hydrogels sustained chondrocyte viability for at least 28
days of in vitro culture, during which the cells maintained a round
morphology. Histologically, the tissue formed in the hydrogels resembled
native articular cartilage and was composed of individual cells surrounded
by an extracellular matrix containing sulfated glycosaminoglycans.
11:15 AM EE2.4
EFFECT OF SCAFFOLD PORE SIZE ON CELL GROWTH AND EXTRACELLULAR
MATRIX DEPOSITION. Holly G. Alexander , Lee K. Landeen, Jonathan Mansbridge,
Anthony Ratcliffe, Advanced Tissue Sciences, Inc., La Jolla, CA.
Tissue engineering research often uses the approach of
three-dimensional culture of cells on biocompatible scaffolds to create
tissue-based systems that closely mimic
structures. The environment to which the cells in culture are exposed can
potentially influence the deposition of extracellular matrix (ECM) molecules.
In this study, we evaluated the effects of scaffold macro-architecture
on tissue formation using polystyrene (PS) and poly(DL-lactide-co--caprolactone)
(P[L:CL]) scaffolds in both non-porous, two-dimensional (2-D) and porous,
three-dimensional (3-D) forms. Porous and non-porous scaffolds were prepared
using a solvent (methylene chloride) casting technique. Mixing dissolved
polymer with NaCl particles of the various pore sizes (106-150 m,
150-300 m or 300-500 m)
and leaching yielded porous (85 void
fraction) sponge scaffolds. Scaffolds were cut into 1cm diameter discs
with a die punch, sterilized (2.0 MRad electron beam irradiation), and
seeded with adult canine smooth muscle cells or neonatal human dermal fibroblasts
(1x106 cells/scaffold). Seeded scaffolds were grown statically
in ascorbate-containing growth medium, assayed over 4 weeks and compared
to unseeded scaffolds that were used as controls. Overall, cell metabolic
activity, collagen, and elastin deposition increased over culture time
in all scaffolds, with greater values observed in 3-D scaffolds and with
increasing pore size. Additionally, P(L:CL) supported greater tissue growth
than the PS equivalents. The results of this study demonstrate that scaffold
macro-architecture, such as pore size, can influence the growth of cells
and the amount of ECM deposited.
11:30 AM *EE2.5
SYNTHETIC ECM ANALOGS: NOVEL CELL GROWTH SCAFFOLDS. Jennifer
L. West , Brenda Mann, Annabel Tsai, Department of Bioengineering, Rice
University, Houston, TX.
Synthetic hydrogels that mimic many of the properties
of the natural extracellular matrix have been developed for use as cell
growth scaffolds for tissue engineering and wound healing. These materials
are based on polyethylene glycol for biocompatibility and water solubility,
modified with various bioactive moieties, and terminated with acrylate
groups to allow photopolymerization. We have developed materials that mediate
biospecific cell adhesion, are proteolytically degraded by the normal matrix
remodelling process, and can regulate cell proliferation, motility, and
gene expression. Cells can be suspended in an aqueous solution of the hydrogel
precursor, and then become entrapped within the synthetic matrix upon photopolymerization.
High cell viability is maintained after photopolymerization, and cell proliferation
and tissue synthesis occurs within the hydrogel matrix.
SESSION EE3: BIOPOLYMERS AND BIOLOGICAL PRODUCTION
1:30 PM *EE3.1
POLYESTERS FOR A SUSTAINABLE CHEMICAL INDUSTRY. Simon
F. Williams , Oliver P. Peoples, Metabolix, Inc., Cambridge, MA.
Chair: Ashutosh Chilkoti
Monday Afternoon, April 5, 1999
Salon 15 (M)
In nature, polyesters are made by microorganisms to regulate
their metabolism using a series of elegant, rapid, and highly efficient
chemical transformations. The polyesters, known as PHAs (or polyhydroxyalkanoates),
are fascinating and unique thermoplastic polymers, with properties spanning
the range from materials that are similar to polypropylene to others that
are elastomers. Until now, however, the technologies for producing these
polymers through either chemical synthesis or traditional fermentation
have been limited, and this has prevented the introduction of this useful
class of materials for all but niche applications. At Metabolix, researchers
are using biotechnology to capture these natural polyester pathways, and
are developing new transgenic production systems which promise to deliver
PHA polyesters at prices competitive with traditional oil-derived thermoplastics.
The approach also provides the polymer chemist with extensive design space
by allowing polymer properties to be tailored from hundreds of monomer
options. As thermoplastics, the PHAs can be processed using a wide range
of conventional techniques, and they are also available in latex form.
Potential PHA products range from relatively inexpensive materials for
single use applications like packaging, fast-food serviceware, trash bags,
medical supplies, and diapers, to performance products such as specialty
coatings for the electronic, food, and paint industries, as well as materials
catering to biodegradable or green end uses. There are also potential uses
for the PHAs in high end medical applications, including tissue engineering,
drug delivery systems, and orthopaedic surgery.
2:00 PM EE3.2
SYNTHESIS AND CHARACTERIZATION OF GENETICALLY- ENCODED
ELASTIN-LIKE POLYPEPTIDES. Dan Meyer and Ashutosh Chilkoti , Department
of Biomedical Engineering, Duke University, Durham, NC.
We report the synthesis and characterization of environmentally-responsive
protein biopolymers based upon the repeating amino acid sequence VPGVG
(V = Valine, P = Proline G = Glycine) found in elastin. Elastin-like polypeptides
(ELPs) undergo a reversible phase transition at a specific temperature
(Tt). We are currently investigating the structure-property
relationships of ELPs using genetically-encoded synthesis because it allows
exquisite control over the primary variables that affect the phase transition,
namely the identity of the fourth residue, its degree of substitution,
and polymer chain length. A synthetic gene was designed to encode a repeating
pentapeptide sequence, incorporating three different residues at the fourth
position, with a targeted Tt of 42C.
Eight ELPs ranging in molecular weight from 4.8 to 71.0 kDa were expressed
from a gene library, which was synthesized by head-to-tail oligomerization
of a chemically-synthesized monomeric gene. Two ELPs were also expressed
as an N or C-terminal fusion to two recombinant proteins, thioredoxin and
tendamistat. To our knowledge, these artificial polypeptides are the first
examples of genetically-engineered elastin-like copolymers with precisely-specified
heterologous guest residues and defined chain length. Characterization
of the phase transition of free ELP and ELP fusion proteins revealed a
strong dependence of the Tt on both MW and ionic strength.
Interestingly, no significant difference in Tt was observed
between free ELP and ELP fusion proteins. The latter finding has led to
a novel application of these ELPs for purification of ELP-tagged fusion
proteins using thermally- or salt-induced reversible aggregation and resolubilization,
a process we term `inverse transition cycling'. Other biomedical and biotechnology
applications for ELP constructs are currently under investigation.
2:15 PM EE3.3
BIOSYNTHESIS OF A SELF-ASSEMBLING POLYPEPTIDE BLOCK COPOLYMER.
Yun Qu , Vincent P. Conticello, Dept of Chemistry, Emory University, Atlanta,
We report the design, biosynthesis, and characterization
of a block co-polypeptide based on the segmented structure of spider dragline
silk proteins. The sequence of spider dragline silk was chosen as our model
based on three key considerations: (1) the alternating sequence of structurally
distinct oligopeptide blocks, (2) the self-organization of individual blocks
into separate domains within the dragline silk fiber, and (3) the unique
and technologically significant combination of high tensile and compressive
strength in the natural material. The target polypeptide block copolymer
comprises alternating repeats of a 16-mer oligopeptide crystalline domain
and a 30-mer oligopeptide amorphous domain. The sequence of the crystalline
block is based on a self complementary, amphiphilic oligopeptide (AlaGluAlaGluAlaLysAla
Lys)2, which self assembles under ambient conditions into an
extremely stable sheet
structure. The sequence of the amorphous domain, which consists of six
(GlyProGlyGlnGln) repeats, is based on the flexible,
glycine-rich region of
fibroin 3. DNA cassettes encoding the individual domains were synthesized
independently and joined enzymatically to create a 153 base pair DNA monomer.
Concatameric genes encoding dimer, hexamer, octamer, and decamer repeats
of the target co polypeptide were isolated from enzymatic ligation
of the DNA monomer. The decamer gene was expressed in
and purified to homogeneity using immobilized metal affinity chromatography.
An optically-transparent membrane forms upon addition of water to a concentrated
formic acid solution of the polypeptide. This material is refractory toward
solubilization in most common solvents and denaturants. FT-IR spectroscopy
of the dried membrane exhibits absorptions consistent with formation of
a -sheet structure (Amide I
vibration at 1632 cm-1 and Amide II vibration at 1528 cm-1).
HRSEM of the membrane suggests a morphology in which -sheet
crystallites are embedded in an matrix of amorphous polypeptide.
2:30 PM EE3.4
SUPERMOLECULAR STRUCTURE OF COLLAGEN-LIKE PEPTIDE AMPHIPHILES.
Havazelet Bianco-Peled , Tushar Gore, Matthew Tirrell, Univ of Minnesota,
Dept of Chemical Engineering and Materials Science, Minneapolis, MN; Yeshayahu
Talmon, Technion-Israel Inst of Technology, Dept of Chemical Engineering,
The self-assembly of collagen-like peptide amphiphiles
was studied using small-angle neutron scattering and cryo-transmission
electron microscopy. Peptide-amphiphiles are synthetic molecules, in which
a peptide head group is covalently coupled with a hydrophobic tail. The
peptide sequence that was used contains a 15 amino acid sequence from the
triple helical domain of type IV collagen, known as peptide IVH1. A (GPP*)4
(P* = hydroxyproline) amino acid sequence, that was added to either one
end or to both ends of the IVH1 peptide, facilitate the folding of three
peptide strands into a triple helix conformation. The specific interactions
between the peptides and the amphiphilic nature of the molecules result
in a complex microstructure, characterized by at least two levels of organizations
within a single aggregate. The first level of organization, the basic unit,
is an aggregate composed of three molecules packed close together, having
their peptide portions folded into a triple helix conformation. The basic
units, which have a large head group compared to other common surfactants,
further aggregate and form various supermolecular structures, ranging from
disk-like objects to spherical micelles.
2:45 PM EE3.5
BIOSYNTHESIS AND CHARACTERIZATION OF ELASTOMERIC PROTEIN
POLYMERS. R. Andrew McMillan, Terrence A.T. Lee, Yingting Zhou, Vincent
P. Conticello , Department of Chemistry, Emory University, Atlanta, GA.
In order to elucidate the effects of molecular architecture
on gel properties, polypeptides based on elastin-mimetic repeat sequence
[(Val-Pro-Gly-Val-Gly)4(Val-Pro-Gly-Lys-Gly)] have been synthesized
using genetic engineering and microbial protein expression. This sequence
contains a chemically-reactive lysine residue at regular twenty-five amino
acid intervals. The amino groups of the lysine residues on adjacent chains
may be chemically cross-linked after synthesis and purification of the
nascent polypeptide. A synthetic gene of 3000 base pairs, which encodes
a repetitive polypeptide based on the aforementioned sequence, was isolated
and expressed in . A
new protein with a molecular mass of 90 kD accumulates to high levels and
may be purified to homogeneity in high yield. This elastin analogue has
been crosslinked into an extended network under ambient conditions
in either water or dimethylsulfoxide solutions using the activated diesters,
disuccinimidylsuberate (DMSO) or bis(sulfosuccinimidyl)suberate (water)
as reagents. These two cross-linking reagents should generate compositionally
identical networks, as they differ only in the identity of the leaving
groups. Both networks undergo reversible, temperature-dependent swelling
and collapse in aqueous solvents, in analogy to native elastin and chemosynthetic
elastin analogues. We report the results of structural studies on these
elastin-mimetic protein gels and related elastomeric protein polymers.
SESSION EE4: POLYMERS AT SURFACES
3:30 PM EE4.1
MODIFIED POLYSACCHARIDE GRAFTED ON AN ATOMIC FORCE MICROSCOPE
TIP FOR MOLECULAR RECONGNITION INVESTIGATIONS. Michel Grandbois , Wolfgang
Dettmann, Martin Benoit and Hermann Gaub, Lehrstuhl fur angewandte physik,
Ludwig-Maximilians-University, Munich, GERMANY.
Chair: Kevin E. Healy
Monday Afternoon, April 5, 1999
Salon 15 (M)
The atomic force microscope (AFM) has become a versatile
tool in measuring force in the nanoscopic land scale. Recent developments
in piconewton instrumentation allow the manipulation of single molecules
and measurements of intermolecular as well as intramolecular forces. Here
we have used AFM tips on which polysaccharide modified with differents
biological receptors were grafted in order to investigate molecular recongnition
mechanisms. We have mesured the unbinding force for the receptor-ligand
couple streptavidin-biotin, lectin-sugar and antiboby-DNP. With these modified
AFM tips we also directly measured the interaction of such modified polysaccharide
with a living cell surface. Moreover, we mesured the interaction force
between bone cell or platelet immobilize on a AFM cantilever and modified
polysaccharide grafted on a solid surface.
3:45 PM EE4.2
BIOMIMETIC-INSPIRED ELECTROMAGNETIC FILTERS VIA SNAKE
INFRARED PIT SURFACE STRUCTURE. Morley Stone , Angela Campbell, Timothy
Bunning, AFRL/MLPJ, Hardened Materials Branch, Materials and Manufacturing
Directorate, Wright-Patterson AFB, OH.
Our biomimetic research is designed to apply the unique
infrared (IR) pit organ morphology to the improvement of current artificial
infrared technology. The same protein material, keratin, covers the entire
animal; therefore, the EM spectral differences we have observed must be
the result of varying surface architecture. Snakes from two families, Boidae
and Crotalidae, have the ability to sense IR radiation with special pit
organs. In contrast to the other body scales, the infrared pit scales possess
a unique surface morphology. We have characterized this morphology by scanning
electron microscopy (SEM) and atomic force microscopy (AFM). We have also
conducted a series of spectroscopy experiments which indicate that scales
from the infrared pit are less transparent to visible light and more transparent
to infrared light compared to body and eye scales. This experimental system
provides a stunning example of how nature has arranged a polymeric surface
to enhance a sensory function.
4:00 PM EE4.3
DESIGN AND DEVELOPMENT OF LINKERS TO TETHER BIOMOLECULES
TO GOLD SURFACES. Brenda Spangler , Dept of Chemistry and Biochemistry,
Bonnie Tyler, Dept of Chemical Engineering, Montana State Univ, Bozeman,
Biosensors based on surface plasmon resonance, surface
acoustic wave and quartz crystal microbalance technology are used for many
types of analyses, from kinetics of ligand binding and DNA hybridization
to electronic noses that provide taste and smell profiles for the food
industry. They are, in addition, a rapid, sensitive, selective method for
monitoring contaminants and pathogens in food, water and air. These sensors
have a metallic sensing surface, often gold, which requires that specific
ligands for analyte capture be attached to the gold in a stable manner.
A biosensor made by direct adsorption of ganglioside receptors on the gold
electrode of a quartz crystal microbalance set in a flow cell has been
used to monitor and detect several types of bacterial toxins, including
cholera toxin, Escherichia coli heat-labile enterotoxin, and the Shiga-like
toxins associated Escherichia coli O157:H7. As a refinement of this technology,
we are exploring novel bifunctional linkers and coatings designed to couple
biomolecular recognition molecules to gold for use in biosensors and surface
patterning. We will describe their assembly on gold, and their use in a
biosensor ensemble to preferentially orient antibodies without loss of
4:15 PM EE4.4
POLYSTYRENE AND POLY(4-HYDROXYSTYRENE): SURFACE MODIFICATION
AND QUARTZ CRYSTAL MICROBALANCE STUDY OF CELL RESPONSE. Qiu-Hong Hu , Julie
Gold, Claes Fredriksson1, Sofia Kihlman and Bengt Kasemo, Dept
of Applied Physics, Chalmers University of Technology, Guteborg University,
1Q-sense AB, Gothenburg, SWEDEN.
Several factors are important in studies of cell-surface
interactions, namely surface chemistry, surface topography, protein adsorption,
and mechanical forces. Recent advances in surface modification and characterization
techniques enable a surface to possess certain chemical and topographical
features to which the response of cells is examined. In addition, quartz
crystal microbalance (QCM) may provide a potential means to address mechanical
aspects of cell-surface interaction. Thus by identifying the contribution
of the surface chemistry, topographical features, and the mechanical forces,
understanding of cell-surface interaction can be improved. Potential applications
of the surfaces in biotechnology can be explored. To explore potential
biomedical applications of poly(4-hydroxystyrene) (PHS), chemical modification
of polystyrene and PHS was performed by oxygen plasma and UV-ozone treatments.
Topographical modification of the PHS surfaces was carried out by e-beam
and photolithography. The interaction of Chinese Hamster Ovary (CHO) cells
to the surfaces was studied by QCM. The treatments changed the chemical
composition of the surfaces and improved the wetting behavior, as monitored
by x-ray photoelectron spectroscopy and water contact angle measurement.
Cell attachment was observed with almost no cell spreading on the untreated
polystyrene surface. On UV-ozone treated polystyrene surfaces, both cell
attachment and spreading were observed. The cell response to the flat untreated
PHS surface showed no difference to the UV-ozone treated polystyrene surface.
On the PHS surfaces with grooves of depths 30 to 40 nm and width of 5000
nm, the orientation of the grooves with respect to the direction of oscillation
of the quartz crystal seems to influence the measured QCM parameters. This
might be linked to differences in cell response with respect to oscillation
direction. The possibility for topographical modification and its active
cell response without the need for chemical modification indicate that
PHS could be a suitable tissue culture material and a potential replacement
of tissue culture polystyrene.
4:30 PM EE4.5
4:45 PM EE4.6
EFFECT OF SULFOOCTYL SOFT SEGMENT SIDE CHAINS ON THE
PHYSICAL PROPERTIES AND PROTEIN ADSORPTION OF SEGMENTED POLYETHERURETHANES.
Ji-Ping Yang , Chuan-Qiu Luo, Hui-Ying Chen, Institute of Polymer Science,
College of Chemistry, Peking University, Beijing, P.R.CHINA.
In order to investigate the factors affecting the interaction
of polyetherurethanes and blood compatibility, a series of segmented polyetherurethanes
(SPEU) containing different sulfooctyl group content were synthesized using
sodium sulfooctyl dipolytetramethylene glycol (SSODPM) as the soft segments,
in which the sulfooctyl pendant groups were exactly located at the soft
segments. Bulk and surface characterization of the polymers was performed
using FTIR, ESCA, DCA, elemental analysis, DSC, et al. The microphase separation
and bulk physical properties were found to vary with sulfooctyl group content.
Using radioiodine labeling methods, the adsorption kinetics and adsorption
isotherms of bovine serum albumin (BSA) onto the SPEU surfaces was studied.
The amount of BSA adsorbed to SPEU was found to increase strongly when
sulfooctyl group was introduced into the soft segment of SPEU and increase
as the sulfooctyl content increased, which is consistent with the anticoagulant
properties based on the recalcification times.
SESSION EE5: DRUG DELIVERY APPLICATIONS
8:30 AM *EE5.1
DEVELOPMENT OF BIODEGRADABLE POLYMERIC MICROSPHERE DELIVERY
SYSTEMS FOR PROTEINS. Mark A. Tracy , Kevin L. Ward, Laleh Firouzabadian,
Maria Figueiredo, Nan Dong, Rulin Qian, Alkermes, Inc., Cambridge, MA.
Chair: Allan S. Hoffman
Tuesday Morning, April 6, 1999
Salon 15 (M)
Due to their short serum half-lives and negligible oral
bioavailability, therapy for protein drugs currently requires administration
by frequent injections (3-7 times per week in most cases). There has been
considerable interest in developing a biodegradable, injectable polymeric
controlled release system that provides more stable blood levels of these
drugs over time to minimize the number of required injections and thereby
improve patient compliance and convenience. Key obstacles in developing
polymeric protein delivery systems for clinical testing include maintaining
the drug integrity and achieving desirable release kinetics. This paper
presents approaches for stabilizing proteins in a polymeric delivery system
using examples, such as recombinant human growth hormone (rhGH), evaluated
in the clinic. Also, properties of the polymer play an important role in
the rate of microsphere degradation and therefore in achieving desirable
drug release kinetics. Poly(lactide-co-glycolide) (PLG) was used to encapsulate
the protein because of its biocompatibility and history of clinical use.
Data will be presented showing the effects of PLG properties such as the
type of end group and molecular weight on microsphere degradation rates
in-vivo and in-vitro. The degradation effects of encapsulating zinc carbonate,
a sparingly soluble basic salt, and rhGH in PLG microspheres will also
9:00 AM EE5.2
CONTROLLED RELEASE OF SOME PHARMACEUTICALS FROM TRANS-ESTERIFICATED
POLY-3-HYDROXYBUTYRATE MICROPARTICLES. Teresa Eligio , Rubén Sánchez,
Jacques Rieumont, Jaqueline Siquiera, Polymer Section, Advanced Material
Laboratory, North Fluminense State University, Campos, R.J., BRAZIL.
Polymeric devices can be used as a mean for microencapsulation
of drugs with the purpose of controlled release or to protect them from
the environment avoiding the loss of bioactivity. In this context poly-3-hydroxybutyrate
(PHB) is a biocompatible polymer suitable for biomedical purposes but its
crystallinity and low swelling do not contribute for a good releasing performance.
Thus, its modification by trans-esterification could improve its properties
as a polymeric matrix. In fact this modification procedure lead to a molecular
weight decrease from 527,100 to 307,600 by GPC. Furthermore, crystallinity
decreased about 10 according to its
x-rays diffractogram. Thionicotinamide is a drug useful against the Chagas
disease, a common healthy problem in South America and was encapsulated
with the aim to be used on the gastro-intestinal tract looking for a good
performance if with success attains the intestine. On the other side progesterone
was encapsulated to be used to regulate the menstrual cycle of cattle.
Emulsion encapsulation and sprays drying were used in order to obtain different
microparticles and as was shown by SEM they differ in morphology, size
or shape. Thionicotinamide is a water-soluble drug that requires a double
emulsion procedure (water-oil-water). However, progesterone is not soluble
in water so only a simple emulsion procedure (oil-water) was necessary.
In all cases microparticle diameters were ranging between 3-6 m.
Shape, size and morphology of the microparticles were studied by SEM. Spheroidal
particles were obtained and surfaces were not smooth in any case but irregular
with cavities. However, the modified material was not so wrinkled-folded
as in the case of PHB. Trans-esterificated PHB resulted a better matrix
for releasing that the original PHB material. Releasing rates were higher
but a two-step releasing profile was observed plus a burst. It seems that
a portion of the drug is distributed on the surface, near the surface and
into the core of the microparticles. A thoroughly kinetic in-vitro study
was performed using the different devices obtained.
9:15 AM EE5.3
SYTHESIS OF BIODEGRADABLE POLY(LACTIC-CO-GLYCOLIC ACID)
FILMS BY PULSED LASER DEPOSITION. J. Talton , G. Hochhaus, Dept. of Pharmaceutics,
Coll. of Pharmacy, J. Fitz-Gerald, R. Singh, Dept. of Materials Science
and Engineering, Coll. of Engineering, Univ of Florida, Gainesville, FL.
The deposition of poly(lactic-co-glycolic acid) (PLGA)
films produces nano-thin coatings that reduce the release rate of drug
particles in vitro. Coatings are applied by an excimer laser (248 nm) that
enters the deposition chamber through a quartz window and interacts with
a PLGA target. The laser radiation is absorbed by the polymer target and
expands from the surface in a plume of monomer, polymer, and polymer clusters
that is then deposited onto the fluidized drug particle. Factors that affect
the PLGA molecular weight distribution, coating morphology, and thickness
include the background gas and pressure, laser energy, laser freuqency,
and particle size. Characterization using SEM and FEM, FTIR, 3H-NMR, and
SEC, as well as in vitro dissolution rate, was studied.
9:30 AM EE5.4
CHIRAL INFLUENCE ON THE SOLUTION STRUCTURE OF DOUBLE
CHAINED CATIONIC LIPIDS. Henrich H. Paradies , Markische Fachhochschule,
Biotechnology & Physical Chemistry, Iserlohn and University of Paderborn,
Chemistry & Chemical Engineering, Paderborn, GERMANY; Shaun F. Clancy,
Witco Corporation, Greenwich, CT.
A detailed physical analysis of the data obtained from
static and dynamic light scattering experiments with polymer-like C(18)-C(16)
dimethylammonium X micelles (CLPs) will be presented for dilute and semidilute
concentrations, and compared with those obtained in the presence of racemic
and enantiomeric anions, e.g. S-(+)-lactate or enantiomers of arylpropionic
acids. CLPs are important colloidal aggregates, which have a high potential
for gene ferries,drug delivery sytems,transfection models and thin film
technology. Not only differences in the CMCs are being noticed but also
in the concentration dependence of the entanglements as determind from
rheologic and light scattering measurements. An important understanding
of this system is the influence of the chiral counterions on the CLPs on
the decay time distribution of these aggregates which is almost independent
of the chain lengths, but strongly dependent on the chirality & nature
of the anions. The fast mode reflects the co-operative motions of the transient
network formed throughout the chain entanglements, which has a positive
exponent. The CLP-concentration dependence of the relaxation rates of the
slow component in the presence of chiral counterions scales with 1.70 -
1.79. In addition the slow hydrodynamic motion is independent of the scattering
vector (Q), so it cannot be diffusive, and therefore not correlated with
the particle size. One explanation can be offerred considering the different
giant micelles as living polymers, where the slow relaxation is connected
with the kinetics of breaking up the micelles, and reforming new CLPs in
the presence of chiral anions. This would also explain the values found
for racemic anions, which reveal a different temperature dependence vs.
the ones for enantiomeric anions. The rheological parameters obtained for
this systems can be explained in terms of the correlation length. The time
correlation function of the concentration fluctuations decays as a purely
exponential relaxation, which can be characterized through the gel-diffusion
coefficient including the gel modulus and the osmotic modulus.
SESSION EE6: STIMULI-RESPONSIVE AND BIOACTIVE SYSTEMS
10:15 AM *EE6.1
COMPARTMENTAL REACTION CONTROL USING LIPID VESICLES:A
STRATEGY FOR TRIGGERING BIOMATERIAL FORMATION. Eric Westhaus, William L.
Murphy, Phillip B. Messersmith , Northwestern University, Division of Biological
Materials and Department of Biomedical Engineering, Chicago, IL.
Chair: Mark Alper
Tuesday Morning, April 6, 1999
Salon 15 (M)
In nature, phospholipid assemblies (e.g. lipid bilayers)
act as physical barriers that partition the aqueous phase into distinct
cellular, subcellular, and extracellular compartments that are chemically
and functionally distinct. In our laboratory we are utilizing a similar
strategy to control chemical reactions that result in the rapid in-situ
formation of biomaterials intended to repair, replace, or augment living
tissues. Specifically, we are exploiting the barrier properties of phospholipid
vesicles to entrap and isolate reactive chemical species of an aqueous
suspension. We are designing the liposomes to release the entrapped species
in response to an applied stimulus, such as light, temperature, pH, etc.
Upon release, the entrapped species reacts with other components of the
solution to form a solid or semi-solid biomaterial. The use of this approach
will be illustrated by examples in which calcium phosphate minerals and
polymer hydrogels are rapidly formed from liposome-containing fluids upon
warming from ambient to body temperature. Potential clinical applications
include hard and soft tissue reconstruction, wound healing, and drug delivery.
10:45 AM *EE6.2
2D AND 3D MANIPULATIONS OF CELL SHEETS USING TEMPERATURE-RESPONSIVE
CULTURE SURFACES FOR RECONSTRUCTION OF TISSUE ARCHITECTURES. Masayuki Yamato
, Ai Kushida, Chie Konno, Akihiko Kikuchi, Yasuhisa Sakurai, Teruo Okano,
Tokyo Women's Medical Univ, Inst of Biomedical Engineering, Tokyo, JAPAN.
We have focused on novel cell manipulation techniques
as a key technology in tissue engineering. By grafting a temperature-responsive
polymer, poly(N-isopropylacrylamide), we have developed temperature responsive
culture surfaces that are hydrophobic at 37C
and change to hydrophilic below 32C.
Various cell lines adhere, spread, and proliferate on these grafted surfaces
similarly to those on ungrafted commercial tissue culture dishes. By reducing
culture temperature, cells are spontaneously liberated only from the grafted
surfaces without the need for typical enzymatic digestion. Highly trypsin-susceptible
cells such as hepatocytes and retinal pigmented epithelial cells retained
higher activities of specific cell functions after recovery by reducing
temperature. Confluent cells are recovered from grafted surfaces as a single
contiguous monolayer sheet with intact cell-cell and cell-extracellular
matrix junctions. Therefore, viable cell sheets can be transferred from
temperature-responsive surfaces to other surfaces of culture dishes or
devices (2D cell sheet manipulation). Furthermore, since several cell types
are co-organized into defined cell sheet layers in natural tissues, we
have used 3D cell sheet manipulation to reconstruct complex stratified
tissue architectures from cell sheets. Overlaying two monolayer sheets
of hepatocytes and endothelial cells obtained from polymer-grafted culture
surfaces provides a viable construct for in vitro fabricated liver lobule-like
tissue. Cultured lamellar cell sheets preserve each cell phenotype and
basic cell functions. We believe that these 2D and 3D cell manipulation
techniques will become new revolutionary tools for tissue engineering.
11:15 AM EE6.3
ADSORPTION OF HYDROPHOBICALLY MODIFIED RESPONSIVE POLYMERS
ON A MODEL SURFACE. Edel Ruske , Tatiana Golubeva, Alexander Gorelov, Kenneth
Dawson, Dept. of Chemistry, University College Dublin, Dublin, IRELAND.
We studied the adsorption of the hydrophobically modified
responsive copolymer, N-isopropylacrylamide (NIPA), N,N-dimethylacrylamide
(DMA) and pyrenemethylacrylamide on the surface of a polystyrene latex.
Pyrene was also introduced to a second thermoresponsive copolymer of NIPA,
DMA and acrylamidoundecanoic acid (AAuA). Pyrene serves as a hydrophobic
group and as a reporter to follow the adsorption of the copolymer on the
polystyrene surface. Fluorescence spectroscopy and dynamic light scattering
were used to establish the structure of the polymer layer on the surface
at different stages of adsorption. Also the adsorption isotherm and structural
changes in polymer layer upon the increase of temperature were investigated.
The presented result can give a useful insight into surface modification
by thermoresponsive polymers with pendant photoreactive groups.
11:30 AM EE6.4
USE OF SILVER ZEOLITES IN THE DEVELOPMENT OF BIOACTIVE
ELASTOMERIC COMPOSITES. Regina S. Nascimento , Marcio B. Oliveira, Universidade
Federal do Rio de Janeiro, Instituto de Quimica, Rio de Janeiro, BRAZIL;
Maria H. Rocha-Leao, Universidade Federal do Rio de Janeiro, Escola de
Quimica, Rio de Janeiro, BRAZIL; Luciana M. Estevao, Universidade Federal
do Rio de Janeiro, Instituto de Quimica, Rio de Janeiro, BRAZIL.
A variety of polymer additives have been studied by our
group in order to develop different types of interactive polymers. Among
the novel technologies being sought, those playing an active role in microorganism
growth-control have been deserving special attention, since one of the
present day challenges is to produce materials which provide high and long-lasting
bactericidal effects. It is known that certain metal ions such Ag+
have high affinity with sulphydryl, amino, carboxyl and phophate groups,
inactivating bacteria enzymes. The aim of this project was to develop non-toxic
elastomeric materials containing anti-microbial agents supported on aluminum
silicate carriers, liable to be used for the manufacturing of shoe soles,
insoles, mats and rubber articles in general. Silver-exchanged NaY zeolites,
with nominal silver contents of 25 and
50 were processed with an SBR matrix
in a roll mill, at the filler levels of 0.25, 0.50, 1.00, 1.50, 2.00 phr.
The mixtures were then pressed and the obtained specimens submitted to
tensile strength and biocide testing. Saccharomyces cerevisiae and
Escherichia coli cells were submitted to growth in the complex rich
medium, of a) 2 glucose, 2
peptone and 1 yeast extract and b) 2
sucrose, 0.5 peptone, 0.3
meat extracts, respectively, under stress metabolic conditions i.e. AgY
zeolites at 0,04 mg/ml. As expected, the presence of zeolites in the SBR
formulations increased the rubberís tensile elastic modulus and lowered
both strength and elongation at break. AgY zeolites produced an 87
and 100 inhibition of the growth of
Saccharomyces cerevisiae and Escherichia coli respectively,
when compared with control systems.
11:45 AM EE6.5
IMPROVEMENT OF THE BIOCOMPATIBILITY OF GLASSY POLYMERIC
CARBON CARDIAC PROSTHESIS. Marcello G. Rodrigues , Robert L. Zimmerman,
FFCLRP-USP, Dept of Physics and Mathematics, RibeirãPreto, SP, BRAZIL;
Hélio P. de Magalhães, HP-Biopróteses, São
Paulo, SP, BRAZIL; Daryush Ila, Alabama A&M University, Center For
Irradiation of Materials, Normal, AL.
Glassy Polymeric Carbon (GPC) is made of carbon in layers
of graphitic planes, which are random ordered in space, forming an amorphous
organic polymeric material. Due to high inertness and chemical biocompatibility,
GPC has been used as biomaterial, mainly in the manufacturing of prosthetic
heart (mainly Biplus and Disc models) and hydrocefalic valves. The mechanical
cardiac valves are in general more durable, but they are less bio-compatible
than biological cardiac valves. GPC is produced by the pyrolysis of cross
linked resins like Resol or Furfural alcohol, in inert environment upon
1000C Heating Treatment Temperature
(HTT), at very low temperature rates to avoid changing the shape or disruption,
due to volatile products of the carbonization. Such rates must be chosen
according the thickness of the artifacts. The structure of the material
changes during the carbonization, making it progressively conducting after
550C HTT, due to released hydrocarbons.
GPC prepared at HTT 650C presents
maximum permeability due to presence of pores opened in its bulk; after
this temperature, the pores progressively get closed, decreasing the permeability
as the graphitic planes aggregate themselves to form the GPC structure.
Energetic ion bombardment (gold, carbon, silicon and oxygen, MeV) and plasma
etching have been used for increasing the permeability even at final stages
of carbonization where the GPC material is non-permeable. This points out
the possibility for using it in drug delivery. Ion bombardment also changes
the surface of the material making it more biocompatible. Our experience
has shown that independent of HTT, the roughness of GPC artifacts can be
increased and controlled with ion bombardment and plasma treatment. To
test the biocompatibility, tissue adherence and thromboresistance, two
kinds of GPC artifacts were manufactured to be used in cardiac cavities
in vivo animal experiments.