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Spring 1999 logo1999 MRS Spring Meeting & Exhibit

April 5-9, 1999 | San Francisco
Meeting Chairs: Katayun Barmak, James S. Speck, Raymond T. Tung, Paul D. Calvert

Symposium EE—Polymers in Biotechnology


Mark Alper
Lawrence Livermore Natl Lab
Univ of California-Berkeley
MS 66
Berkeley, CA 94720
510-486-6581 Division of Biological Materials
Northwestern Univ
Ward Bldg 10-116
Chicago, IL 60611-3008

Teruo Okano
Inst of Biomedical Engineering
Tokyo Women's Medical Univ
Tokyo, 162-8666 JAPAN
81-3-3353-8111 x30233

Patrick Stayton
Ctr for Bioengineering
Univ of Washington
Seattle, WA 98195

Symposium Support

  • Army Research Office
* Invited paper
Chair: Patrick S. Stayton
Monday Morning, April 5, 1999
Salon 15 (M)
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, WA.

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 -$\rm C_nF_{2n+1}$ compared to -$\rm C_nH_{2n+1}$. 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 -$\rm C_nF_{2n+1}$ 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

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.

Chair: Phillip B. Messersmith
Monday Morning, April 5, 1999
Salon 15 (M)
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.

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, CO.

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, Evanston, IL.

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; $\approx$22$^{\circ}$C) for 19 hours. At RT, the loosely crosslinked hydrogels were readily injectable through a standard syringe (i.e., without a needle attached) and did not demonstrate discernible macroscopic fracture following injection. When heated to 37$^{\circ}$C, the hydrogels exhibited a phase transition, as evidenced by an increase in opacity and a decrease in ductility. Hydrogel characterization studies included: 1) verification of the hydrogel chemistry with solid-state 1H-magic angle spinning (MAS)-NMR spectroscopy; 2) determination of the lower critical solution temperature (LCST) using a UV-Vis spectrophotometer; 3) estimation of the water content at RT and 37$^{\circ}$C as a function of swelling in PBS via a freeze-drying technique; 4) calculation of the change in volume between RT and 37$^{\circ}$C as a function of swelling in PBS by a water displacement method; and 5) evaluation of the rheological properties using an oscillating rheometer. In addition, bovine articular chondrocytes were seeded into the hydrogels and the cell-loaded constructs were cultured in vitro . In situ fluorescent viability studies and histological analyses were performed. Overall, the 1H-NMR spectroscopic analyses were consistent with the chemical structure of the hydrogels. Prior to swelling in PBS, the P(NIPAAm-co-AAc) hydrogel contained 93.3$\pm$5$\%$ water at 37$^{\circ}$C and demonstrated a +5.3$\pm$6$\%$ volume change between RT and 37$^{\circ}$C. 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 ${\it in vivo}$ 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-$\epsilon$-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 $\mu$m, 150-300 $\mu$m or 300-500 $\mu$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.

Chair: Ashutosh Chilkoti
Monday Afternoon, April 5, 1999
Salon 15 (M)
1:30 PM *EE3.1
POLYESTERS FOR A SUSTAINABLE CHEMICAL INDUSTRY. Simon F. Williams , Oliver P. Peoples, Metabolix, Inc., Cambridge, MA.

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 42$^{\circ}$C. 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, GA.

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 $\beta$­sheet structure. The sequence of the amorphous domain, which consists of six (Gly­Pro­Gly­Gln­Gln) repeats, is based on the flexible, glycine-rich region of ${\it A.diadematus}$ 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 ${\it E.coli}$ 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 $\beta$-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 $\beta$-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, Haifa, ISRAEL.

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 ${\it E.coli}$. 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.

Chair: Kevin E. Healy
Monday Afternoon, April 5, 1999
Salon 15 (M)
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.

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, MT.

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 antigen-binding specificity.

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
Abstract Withdrawn.

4:45 PM EE4.6

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.

Chair: Allan S. Hoffman
Tuesday Morning, April 6, 1999
Salon 15 (M)
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.

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 be presented.

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 $\mu$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.

Chair: Mark Alper
Tuesday Morning, April 6, 1999
Salon 15 (M)
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.

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 37$^{\circ}$C and change to hydrophilic below 32$^{\circ}$C. 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 1000$^{\circ}$C 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 550$^{\circ}$C HTT, due to released hydrocarbons. GPC prepared at HTT 650$^{\circ}$C 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.