Posters

^aInstitute of Physical Chemistry, Faculty of Chemistry, Technical University of Brno, Purkyňova 118, CZ–612 00 Brno, Czech Republic

^bInstitute of Physics and Material Engineering, Faculty of Technology in Zlín, Technical University of Brno, nám. TGM 275, CZ–762 72 Zlín, Czech Republic

This research was conducted with the aim to prepare and characterize bio–compatible poly(propylene) (PP) based foils. The oxidative plasma surface activation method followed by concentrated ammonia etching was used for surface activation. It was found that the radical species generated are of enough reactivity and durability to continue with the consecutive chemical reaction with chemically treated atelocollagen. In such a way prepared bio–compatible layers the latter were characterized for adhesion strength both in a dry as well as in the swollen conditions by peel tests. Plasma–chemically modified surfaces were characterized by means of EPR, FTIR spectroscopy, contact angle measurements and SEM microscopy. It was found that with prolonged oxidative treatment PP surfaces were more hydrophobic, while after the concentrated ammonia etching more hydrophilic. As an addition primary amine groups were detected at the latter samples by ATR FTIR measurements.

The authors would like to express their gratitude for financing of this research by Ministry of Education, Youth and Sports of the Czech Republic (Grants Nos.: CZT90015, CZ690019).

Ľ. Lapčík, Jr., P. Peterková, B. Lapčíková, P. Horváth: Proceedings ICCE/7, University of New Orleans, Denver.

UTILIZATION OF MULTIFUNCTIONALIZED POLYISOBUTYLENE AS BUILDING BLOCKS FOR THE SYNTHESIS OF POLYMER NETWORKS FOR BIOMEDICAL APPLICATIONS

L. TOMAN^a, P. VLČEK^a, J. MICHÁLEK^a, J. VACÍK^a, K. SMETANA^a,b, B. DVOŘÁNKOVÁ^c, J. SPĚVÁČEK^a, P. HOLLER^a, I. KELNAR^a

^aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, CZ-162 06 Praha 6, Czech Republic

^bAnatomical Institute of the 1st Medical Faculty of the Charles University in Prague, U nemocnice 5, Praha 2, Czech Republic

^cThe 3rd Medical Faculty of the Charles University in Prague, Šrobárova 50, Praha 10, Czech Republic

Recently, a novel method of the direct synthesis of multifunctionalized reactive polyisobutylenes with isocyanate groups along the chains (PIB(NCO)_n) was discovered¹. In a mixture of isobutylene (IB) and an isocyanate agent RNCO (where RNCO is 3-isopropenyl-α,α-dimethylbenzyl isocyanate), cationic initiator reacts with the agent forming carbenium ion ⁺RNCO which then behaves as a comonomer with IB. Thus, multifunctionalized product is formed by cationic polymerization which can have isocyanate groups at the ends of the chains and, also along them. The isocyanate groups can be in situ transformed¹ into urethanes by reaction with, for example, 2-hydroxyethyl methacrylate (HEMA) and the product is subsequently copolymerized with HEMA in the presence of radical initiator (R*).

The direct synthesis of PIB(NCO)_n (1st step), reaction with HEMA and synthesis of amphiphilic polymer networks (2nd step) can be described by the following scheme:

MICELLES OF POLY[N-(2-HYDROXYPROPYL)METHACRYLAMIDE -block-BUTYL ACRYLATE] IN WATER / DIMETHYLFORMAMIDE MIXED SOLVENTS

Č . KOŇÁK^a, B. GANTCHEV^b, S.M. TEODORESCU^c, K. MATYJASZEWSKI^c, P. KOPEČKOVÁ ^d, J. KOPEČEK^d

^aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

^cDepartment of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.

^dDeparment of Bioengineering and of Pharmaceutics and Pharmaceutical Chemistry/ CCCD, University of Utah, Salt Lake City, Utah 84112, U.S.A.

Diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] (poly(HPMA)) and poly(butyl acrylate) (poly(BA)) were prepared by controlled living radical polymerization. All the copolymers were found to be molecularly soluble in dimethylformamide (DMF) and insoluble or less soluble in water. In water and mixed H₂O/DMF solvents, the copolymers were dispersed in micellar form by controlled addition of water to DMF solutions of the copolymers under continuous intensive stirring. The micellar solutions in water were prepared by dialysis of solutions in H₂O/DMF (5 vol. % of DMF) against water. Solution properties of poly(HPMA-b-BA) diblock copolymers were studied using static and dynamic laser light scattering to characterize the behavior of the copolymers on supramolecular level. The effects of preparation speed, organic solvent (DMF) and chemical composition of the copolymers on the formation of micelles were studied. While a slower mixing procedure was optimum for copolymers with short poly(HPMA) blocks, a faster mixing was better for copolymers having longer poly(HPMA) blocks. Finally, the particle parameters of micelles in water were evaluated. The most compact micelles were prepared from copolymers having short hydrophilic poly(HPMA) blocks. On the other hand, the copolymer with the longest poly(HPMA) block formed micelles of the smallest sizes and the lowest mean weight fraction of copolymer in the micelle (copolymer density) .

The support of the Grant Agency of the Czech Republic by grant No. 307/96/K226 and 203/00/1317 is gratefully acknowledged.

Hydrogels for encapsulation of cells: characterization and application in encapsulation of dopamin-producing cells

J. Lukáš^a, V. Palečková^a, J. Mokrý^b, J. Karbanová^b, B. Dvořánková^c

^aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

^cPrague Burn Centre, 3rd Faculty of Medicine, Charles University, Šrobárova 5,

The immunoprotection cell therapy is based on the encapsulation of cells producing biological active substances into polymer microcapsules and their subsequent transplantation into the patient body with effort to heal diseases caused by loss of secretory cell function. This concept is based on the separation of transplanted cells from the immune system of the host. Polymers used for cell encapsulation are porous hydrogels having besides the protection of cells also the function of semipermeable membrane allowing the passage of oxygen and low-molecular-weight nutrients into the capsule as well as diffusion of drugs from the capsule to the patient body. The choice of an appropriate polymer for encapsulation is restricted by a number of requirements, the most important are good biocompatibility with the recipient's tissue, mechanical stability, resistance to degradation and hydrolysis, and tolerance by entrapped cells.

The aim of our research is to develop new types of synthetic copolymers for cell encapsulation focusing on two main aspects - obtaining hydrogels with high diffusivity for low-molecular-weight substances and improving interactions between cells and capsule walls. In this way, a better transport of nutrients into the capsules, as well as prolongation of the viability and improvement of the secretory activity of encapsulated cells could be achieved.

New types of synthetic hydrogels designed for encapsulation of dopaminergic cells are based on copolymers of esters or amides of methacrylic acid prepared by solution radical polymerization at different ratios of monomers. Prior to cell encapsulation, we characterized physical and chemical properties of the copolymers, proved their encapsulation processability and performed number of biological tests to assess interaction of hydrogels with living cells. The results of in vitro and in vivo tests performed on 3T3 fibroblasts, PC-12 cells and rat fetal neural cells will be discussed.

The support of this work by Grant Agency of the Czech Republic, Grant No. 304/98/0267, is gratefully acknowledged.

Poly(N-isopropylacrylamide) with hydrolyzable lactic acid ester side groups: a new type of thermosensitive polymer suitable for drug delivery purposes

D. Neradovic¹, W.L.J. Hinrichs¹, J.J. Kettenes-van den Bosch², C.F. van Nostrum¹, W.E. Hennink¹

¹Department of Pharmaceutics and ² Department of Pharmaceutical Analysis, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands

Radical copolymerization of N-isopropylacrylamide (NIPAAm ) and 2-hydroxyethyl methacrylate (HEMA) monolactate was used to prepare poly(NIPAAm-co-HEMA-monolactate) with different compositions. A decrease in the lower critical solution temperature (LCST) of the copolymers is observed with increasing mol fraction of HEMA-monolactate, which indicates that incorporation of this monomer in poly(NIPAAm) increases the hydrophobicity.

To hydrolyze the lactate ester side group, the copolymers were incubated at pH 10.5 for 3 days. Hydrolysis of the lactate ester side group of the copolymers was accompanied by an increase in LCST, which demonstrates that a more hydrophilic copolymer is obtained after hydrolysis. Poly(NIPAAm-co-HEMA) was separately prepared from NIPAAm and HEMA for comparison with the hydrolyzed product. The LCST of the copolymers after hydrolysis was not significantly different from the LCST values of the NIPAAm-co-HEMA polymers, which demonstrates that, in agreement with HPLC and NMR data, the hydrolysis of the side groups was complete.

Thus, a new type of thermosensitive NIPAAm-based polymer with hydrolytically sensitive lactate ester side groups has been synthesized. Our current efforts are directed on the design of polymers, which exhibit an LCST above 37 ^oC (body temperature) after hydrolysis of the side groups. Such polymers have attractive properties, which can be exploited in drug delivery systems such as hydrogels and polymeric micelles.

D. Neradovic, W.L.J. Hinrichs, J.J. Kettenes-van den Bosch, W.E. Hennink, Macromol. Rapid Commun. 20, 577-581 (1999).

Hyaluronate as a tool for modification of the viscoelastic properties in connective tissue MATRIX

^aFaculty of Chemistry, Technical University of Brno, Purkyňova 118, CZ-612 00 Brno, Czech Republic

^bDepartment of Physics & Mat. Eng., Fac. Technology of Zlín, Technical University of Brno, nám. TGM 275, CZ-761 72 Zlín, Czech Republic

Hyaluronic acid, non-modified or modified by various functional groups, is a unique material, which is biocompatible to human body.

In solution, the hyaluronan polymer chain forms an extended random coil. Its structure allows building three-dimensional polymeric network with remarkable biophysical properties. In hyaluronan solutions random coil molecules are created and chain-chain interactions occur rarely. The chains are able to penetrate in solutions at higher concentration and to form highly entangled networks. Due to this fact hyaluronic acid in solution behaves as a molecular sponge capable to retain solvent water. This makes hyaluronan useful for many medical applications.

Structural characteristics of dilute solutions of hyaluronan were performed on HAAKE RS100 rheometer. Flow curves of HA solutions were measured at several temperatures and at different molecular weight. Investigation showed that hyaluronan solutions behaved as a pseudo-plastic material.

If the interaction forces between polymer and solvent molecules can be neglected (the so-called Q -solution) the polymer molecule is in unperturbed conformational state. In this situation, the molecular dimensions and limiting viscosity number can be predicted rather accurately. The critical coil overlapping concentration of polymer chain is typical for this state, too. Rudin equations were used to evaluate parameters as swelling factor or hydrodynamic swelling volume.

POLYMER CONJUGATES COMPRISING THE ANTIPROLIFERATIVE FERROCENE SYSTEM CARRIER-BOUND VIA ESTER LINKS

^a Department of Chemistry, University of the Witwatersrand, WITS 2050, South Africa

^b School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

The cytotoxic properties of certain water-soluble ferrocene derivatives and ferricenium salts are well on record^{1, 2}. Synthetic activities in this laboratory have been focused for several years now on conjugates in which the ferrocene group is carrier-anchored through carboxamide links susceptible to biological cleavage for release of the bioactive agent³. In the present communication we deal with analogous conjugates comprising ester (in place of amide) links as constituents of the anchoring tether. Such links are expected to undergo fission in the biological environment at different (possibly higher) rates and thus may alter the agents pharmacokinetic pathway. In this study water-soluble polyaspartamides bearing hydroxyl functions as side chain terminals serve as the carrier structures, to which the ferrocenylation agent, 4-ferrocenylbutanoic acid, is covalently anchored through O-acylation, mediated by a carbodiimide and catalyzed by 4-(dimethylamino)-pyridine. The conjugates, fractionated and purified by dialysis, are water-soluble and have iron contents of 3-7%. Cell culture tests probing the antiproliferative behavior of selected conjugates against the LNCaP human metastatic prostate adenocarcinoma cell line have revealed an activity pattern very similar to that of the polymers with amide-bound ferrocene, with IC₅₀ values typically in the range of 5-30 μg Fe/m. In this in vitro project the differences in release behavior between carboxamide and ester links as biocleavage sites are thus not significant. Further elucidation of this question must await in vivo studies.

1. P. Köpf-Maier, H. Köpf, and E.W. Neuse, J. Cancer Res. Clin. Oncol. 108, 336 (1984).

2. V.N. Babin, P.M. Raevskii, K.G. Shchitkov, L.V. Snegur, and Hu S. Nekrasov, Mendeleev Chem. J. 39, 17 (1995), a review.

3. M.G. Meirim, E.W. Neuse, and G. Caldwell, J. Inorg. Organomet. Polym. 8, 225 (1998).

P11

TRANSPORT OF POLY AMIDOAMINE (PAMAM) DENDRIMERS ACROSS MADIN-DARBY CANINE KIDNEY (MDCK) CELL LINES

^bDept. of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, U.S.A.

Introduction. Poly amidoamine (PAMAM) dendrimers are a family of branched synthetic polymers containing surface amine groups that have shown potential in controlled drug delivery¹. An important barrier to the oral bioavailability of macromolecular carriers is transport across epithelial cells of the intestine. The present study describes the transport of a series of PAMAM generations (G₀, G₁, G₂, G₃, G₄)² across Madin-Darby canine kidney (MDCK) cells as a tool for rapid membrane permeability screening³. Methods. Dendrimers with incremental increase in size and molecular weight were labeled by fluorescein isothiocyanate (FITC) and the least polydisperse fractions of the conjugates were collected by size exclusion chromatography techniques by procedure described previously². MDCK cells were grown on transwell filters for 4 days. The integrity of the cells was monitored by [¹⁴C]mannitol permeability. Detection methodology of the conjugates was developed by High Performance Liquid Chromatography (HPLC) connected to a fluorescence detector at l _em= 518 nm and l _ex= 494 nm. Reversed-phase separation was achieved by using C18 column. The mobile phase consisted of 88% phosphate buffer saline with pH adjusted to 7.25, 12% acetonitrile and flow rate at 1.0 ml/min. The permeability of the dendrimers was measured at two time intervals within 80 minutes on the MDCK cell lines. Results and discussion. A new methodology was successfully established for the detection of fluorescent labeled PAMAM dendrimers. The permeability of the PAMAM dendrimers was in the order of G₄>>G₁_»G₀>G₃>G₂. The permeability of mannitol in the presence of G₄ increased 9 fold, suggesting that PAMAM G₄ modulates the cell barrier. This result coupled with the order of the permeability of the polymers suggests that the PAMAM dendrimers’ permeability involves: i) polymer size, and ii) interaction of the cationic polymers with the cell lines. These observations are consistent with previous reports describing the generation-dependent cytotoxicity of cationic PAMAM dendrimers¹.

2) El-Sayed M et al, Proc. of the 27^th Int. Symp. on Cont. Rel. of Bioactive Materials, Paris, July (2000).

P12

WATER-SOLUBLE CONJUGATES FEATURING PLATINUM COMPLEXES CARRIER-BOUND THROUGH cis-DIHYDROXYLATOMETAL CHELATION

^a Department of Chemistry, University of the Witwatersrand, WITS 2050, South Africa

^b School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

Whereas compounds comprising cis-dicarboxylato-chelated platinum have come to occupy an important niche in cancer research and management¹, analogous complexes in which the metal is coordinated by a cis-dihydroxylato ligand system have been side-stepped in drug research because of the comparative lability of the Pt-O bond in such structures. Premature hydrolytic fission of this bond will create aquated species unable to reach endocytic space, while exerting unwanted toxicity. The reversible binding (anchoring) of a medicinal agent to a suitably designed macromolecular carrier may provide temporary protection against enzymatic attack², and this approach has been chosen in the present project. The polyamide resulting from polycondensation of diethyl L-tartrate with 4,7,10-trioxa-1,13-tridecanediamine, a bisamine-terminated oligo(ethylene oxide)³, lends itself as a water-soluble and biodegradable carrier for conjugation via cis-dihydroxylato chelation, and conjugates of this type are generated by treatment of the carrier with diaqua(cyclohexane-1,2-diamine)platinum(II) cation in aqueous solution. The conjugates are purified by aqueous dialysis and isolated by freeze-drying as water-soluble solids. The dialysis step causes some loss of the originally bound platinum. As a consequence, the platinum contents of the conjugates presently obtained do not exceed 5%. Preliminary cell culture tests against the LNCaP human metastatic prostate adenocarcinoma show these conjugates to possess activities equal to, or higher than, those displayed by a variety of polymer-platinum conjugates in which the metal is carrier-anchored through amine ligands.

3. M.G.Meirim, E.W. Neuse, and G. Caldwell, J. Appl. Polym. Sci. 73, 2143 (1999).

P13

Improved pharmacokinetics of poly-L-Lysine/DNA complexes stabilised with intracellularly cleavable surface crosslinking

Low levels of gene expression resulting from inefficient delivery of DNA to target cells in vivo is one of the main limitations of currently available synthetic polycation/DNA vectors. We propose that low delivery efficiency of complexes even after surface modification with PEG is a consequence of their low stability against polyelectrolyte exchange reactions in contact with cell surfaces, leading to partial decomplexation or loss of the protecting PEG layer.

A strategy that might result in significant complex stabilisation and thus increase the efficiency of delivery in vivo is to covalently crosslink primary amines on the surface of these complexes.

We have used reducible dimethyl-3,3’-dithiobispropionimidate to crosslink the surface of poly-L-lysine/DNA (pLL/DNA) complexes formed at N/P ratio of 2. Crosslinked complexes showed increased stability against poly-L-aspartic acid-mediated displacement of DNA. Following crosslinking, neither size nor positive surface charge of complexes was significantly altered. Surface modification of crosslinked complexes with PEG resulted in steric stabilisation against salt induced aggregation and in elimination of their positive surface charge.

Transfection efficiency of the crosslinked complexes in vitro decreased with increasing degree of crosslinking in a mouse melanoma cell line B16F10. This reduction was at least partly due to reduced cell association and uptake of the crosslinked complexes, compared to non-crosslinked pLL/DNA. Interestingly, gene expression of the crosslinked complexes microinjected into the cytoplasm of Xenopus oocytes or Rat-1 cells did not change compared to non-crosslinked pLL/DNA.

Intravenous administration of the crosslinked complexes into Balb/c mice resulted in a 10-fold increase of the amount of complexes remaining in the blood circulation 30 min post-injection, compared to non-crosslinked pLL/DNA.

The increased systemic exposure together with intracellularly triggered activation mechanism makes the described crosslinked complexes a promising system for targeted gene delivery in vivo.

P14

The establishment of gene therapy may be achieved by designing sophisticated gene carriers. Higher transfection efficiency than presently available carriers are wanted. Additionally, if location and timing, and/or duration period of the target gene expression can be specified, it must be very beneficial for the gene therapy. From this perspective, we are studying a thermo-responsive gene carrier system whose formation/dissociation of complexes with DNA can be controlled by temperature.

As illustrated in Fig. 1, strong complex formation with DNA is prerequisite to allow DNA uptake by cells and for resistance against endosomal digestion. On the other hand, DNA must be completely dissociated from the DNA-carrier complex in the transcription process. We have designed a novel polymeric gene carrier system which can modulate complex formation/dissociation behavior by external temperature.

Poly(N-isopropylacrylamide) is well known to exhibit a lower critical solution temperature (LCST). Below this LCST, this polymer is water-soluble and hydrophilic. Above the LCST, PIPAAm changes to a hydrophobic aggregate. We synthesized random copolymers composed of N-isopropylacrylamide, (dimethylamino)ethyl methacrylate, and butylmethacrylate. Copolymers were complexed with a -gal gene-containing plasmid, and applied to COS1 cells. One hour after the incubation with these complexes, the cells were washed with fresh medium, followed by 2 day incubation. During this 2 day incubation, incubation temperature was lowered to 20ºC for 3h. Results are shown in Fig. 2. For a copolymer, gene expression was enhanced by lowering temperature as almost 10 times as that incubated at 37ºC. This is a clear contrast to ordinal gene carrier polymers including poly((dimethylamino)ethyl methacrylate) homopolymer which reduce their gene expression by lowering temperature. This is the first gene expression control by external temperature with gene carriers. This system is feasible for site and time-specific gene expression, and this concept can be utilized to enhance transfection efficiency of non-viral gene carriers.

P16

INFLUENCE OF POLYMERIC STRUCTURAL FEATURES ON THE TRANSPORT OF POLY (AMIDOAMINE) DENDRIMERS ACROSS BIOLOGICAL BARRIERS.

^aDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N. Pine St., Baltimore, MD 21201-1180, U.S.A.

^bSchool of Biomedical Engineering, University of Tennessee at Memphis, 899 Madison Ave., Memphis, TN 38163, U.S.A.

Purpose. To study the influence of a controlled incremental increase in size and molecular weight of model polymeric drug carriers, i.e., a series of poly (amidoamine) (PAMAM) dendrimers, on their extravasation across the microvascular endothelium. Methods. PAMAM dendrimers, generations (G) 0-4 and linear amino-PEG (M_W 6,000 Da) were fluorescent-labeled using FITC. Purification and fractionation of the fluorescent-labeled polymers was done using size exclusion chromatography. The hamster cremaster muscle preparation was used as an in vivo model to study the extravasation of the fluorescent-labeled probes. The extravasation process was visualized and recorded using intravital microscopy techniques. Analysis of the recorded experiments was done using Metamorph Imaging System. Extravasation of the fluorescent-labeled probes is reported in terms of their extravasation time (t ), i.e., the time needed for the fluorescence intensity in the interstitial tissue to reach 90% of the fluorescence intensity in the neighboring microvessels. Results. Extravasation of PAMAM dendrimers across the endothelial barrier showed size and molecular weight dependence. The order of extravasation time (t ) of PAMAM dendrimers was G0 < G1 < G2 < G3 < G4. Extravasation time (t ) of linear PEG (M_W 6,000 Da) was longer than any of the PAMAM dendrimers. This difference in the extravasation time (t ) between PAMAM dendrimers and PEG may be due to the inherited difference in the molecular geometry and charge properties of the studied probes. Conclusions. Extravasation of PAMAM dendrimers across the microvascular endothelium showed size and molecular weight dependence. Results suggest that other physicochemical properties of polymeric drug carriers such as molecular geometry and charge may influence their microvascular extravasation. Such systematic studies will aid in the design of novel biomaterials with better extravasation properties.

P17

TARGETABLE HPMA COPOLYMER CONJUGATES: BIORECOGNITION, INTERNALIZATION AND CYTOTOXICITY IN COLON-ADENOCARCINOMA AND HEPATOCARCINOMA CELLS

^a Department of Pharmaceutics, The Hebrew University of Jerusalem, Jerusalem, Israel

^b Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, USA

The rationale for using carbohydrates as recognition factors to target cancer cells is based on the differences in lectin expression on normal versus cancer cells and the lectin-mediated endocytotic uptake of carbohydrate-containing drug carrier systems. However, one of the main difficulties associated with using carbohydrates to target lectins is the low binding affinity. To increase binding affinities, multiple carbohydrate moieties are often required. This research was designed to evaluate the biorecognition of polymer conjugates containing pendant saccharide moieties as targeting units. Three types of N-[(2-hydroxypropyl)methacrylamide] (HPMA) copolymers containing different pendant saccharide moieties [(a) galactose (b) lactose (c) tri-valent galactoside] were synthesized. The cell surface biorecognition and the internalization of polymer conjugates by three colon cancer cell lines (Colo-205, SW-480 and SW-620) and human hepatocarcinoma (HepG₂) cells were evaluated in vitro to identify those saccharide moieties with optimal binding affinity. The presence of Galectin-3 in the three human colon-adenocarcinoma cell lines was examined. The cytotoxicity of targetable HPMA copolymer-bound Doxorubicin, with a lysosomally degradable spacer, towards human colon adenocarcinoma and hepatocarcinoma cells was also assessed.

Biorecognition of the saccharide-containing HPMA copolymers by colon carcinoma and hepatocarcinoma cells was dependent on the type and content of the pendent carbohydrate moiety in the copolymers and on the type of cell line examined. The binding of the tri-valent galactoside and the galactoside-containing copolymers was found to be greater than the lactoside-containing copolymers in the examined colon adenocarcinoma cells. In the case of HepG₂cells, the binding of the tri-valent galactoside and the lactoside-containing copolymers was superior. The ability of lactose to drastically reduce (in colon cancer cells) or completely abolish (in HepG₂ cells) the cell binding of the copolymers, indicates the sugar specificity of the process involved. These results suggest that receptor mediated endocytosis is the process responsible for increased internalization of the saccharide-containing HPMA copolymers in various colon carcinoma and hepatocarcinoma cells tested. The presence of Galectin-3 and Galectin-3 specific RNA, as detected by Western blot and RT-PCR analysis, is consistent with Galectin-3 playing a role in the biorecognition of glycoconjugates on colon cancer cells. The cytotoxicity of the targeted HPMA copolymer-drug conjugates correlated well with the biorecognition of the saccharide moieties by target cells.

The study was supported by research grants from the BSF Grant no. 98-00449 and from the Israel Cancer Association grant no. 20000026-B.

P18

^aEscola Federal de Engenharia de Itajubá (EFEI - Itajubá, Minas Gerais, Brasil), e-mail: alencar@cpd.efei.br; ^bFaculdade de Ciencias Farmacêuticas (USP – São Paulo, Brasil); ^cInstituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA - Mar del Plata, Argentina), e-mail: icta308@ictp.csic.es; ^dInstituto de Ciencia y Tecnología de Polímeros (CSIC - Madrid, España).

In recent years, hydrogels have received considerable attention for use as specific sorbents in wound dressing materials. Thus, a number of polymers with superabsorbent properties have been developed for clinical applications such as liquefaction and removal of eschar, treatment of leg ulcers, pressure sores and prevention of tissue deterioration in patients with restricted mobility. In this work we report the sorption properties of wound dressings based on poly(vinyl alcohol) (PVA)/chitosan lactate (ChL) blends. Membranes of PVA/ChL were prepared by the phase inversion technique after blending solutions of poly(vinyl alcohol) (PVA, 99-100% hydrolyzed, M_v=8.6x10⁴), chitosan lactate (ChL, M_v= 4,3x10⁴) and nitrofurazone as a local anti-infective drug. The swelling capacities of the obtained membranes were measured in physiological solution (NaCl, 0.1 M) at different temperatures. The rate of nitrofurazone release was studied in a Hanson Research dissolution test station model SR6 in according to USP methodology. A positive swelling changes with temperature were observed with the increase of ChL in the blends. The results shows that absorption into the PVA/ChL membranes slows down, governed by the rate at which the dressing interacts with the physiological fluid. The non-linear shape of the absorption curve, when plotted over time, agrees with well-developed kinetic principles. The rate of release of nitrofurazone seems to depend on the ChL percentage on the blend as well as the pH of the solution. The results obtained in this work demonstrate that PVA/ChL may serve as a new type of potential wound dressing material.

P19

Uptake of Poly[N-(2-hydroxypropyl)methacrylamide] in Organs and its cellular detection in tumor bearing rats

Introduction: Poly(N-2-(hydroxy-propyl)-methacrylamide) (pHPMA) is known to sequester in solid tumors, mainly due to its prolonged plasma circulation time and enhanced permeation and retention effect (EPR). To further exploit the in vivo disposition characteristics we evaluated the late biodistribution in organs with two molecular weight-pHPMAs in a rat prostate tumor model. Cellular distribution in different organs was demonstrated with biotinylated pHPMA in the same model.

Methods: Pieces of tumor of the R3327-AT1 Dunning prostate tumor were transplanted s.c. into the right thigh of male adult Copenhagen rats. 440 nMol/animal of 131I-pHPMA (MW 27.3 kD, 60 kD) were injected in rats carrying tumors of standardized size via the lateral tail vein. The dynamic accumulation of radioactivity in different regions (e.g. heart, liver tumor, whole animal) was monitored over 7 days using a g -camera. At day 7 animals were sacrificed and radioactivity content (% of injected dose/g tissue) were determined in various body compartments. Cellular distribution of pHPMA was performed with two biotinylated pHPMAs, applicated i.v in above concentration and MW in tumor bearing rats as described. Detection in paraffinated and rehydrated slides was done with a alkaline phosphatase-streptavidine conjugate, followed by staining with Fast Red.

Results: Uptake in tumors in the low molecular weight group was completed after 24 hrs, contrary to the high molecular weight group, who showed a continuing pHPMA uptake in tumors over 7 days. Tumor and organ content of drug after day 7 was dependent on MW and was in average twice as high for the 60 kD polymer. Cellular distribution: biotinylated pHPMA is restricted in kindney to the distal tubuli cytoplasm. In liver pHPMA of both MW are accumulated in vacuole-like structures in the cytoplasm of parencyma cells. In spleen accumulation occurs in RES associated macrophages. In tumor a diffuse staining with no cellular localization is demonstrated.

P23

ENCAPSULATION OF PROTEINS AND DNA USING LAYER-BY-LAYER ADSORPTION: METHOD AND APPLICATIONS

Elena A. Markvicheva, Gleb B. Sukhorukov*, Svetlana V. Kuptsova, Alexander A. Vikhrov

Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117871 Moscow, Russia; e-mail: lemark@ibch.siobc.ras.ru

*MaxPlanckInstitute of Colloids and Interfaces, Potsdam/Golm, 14476, Germany

Novel approach for fabrication of polyelectrolyte nanocapsules based on wide class of polymers has been recently introduced [1,2]. This method is based on layer-by-layer (LbL) adsorption of oppositely charged macromolecules onto surface of colloid particles. Organic and inorganic particles, cells and drug nanocrystals can be used to template LbL assembling. Decomposition of the core leads to formation of hollow capsules [2,3]. The capsules can be loaded with bioactive materials, and the capsule wall can provide desired release properties. The nanocapsules can be used as nanocarriers for biological species in drug delivery systems.

Another promising application is encapsulation of plasmid DNA for gene delivery. Transfection of eukariotic cells is a powerful tool in cell biology. Until recently, a major focus in this field was improving the methods for introduction of DNA into appropriate cells. Although the transfection efficiency of the polymer nanospheres reported usually is lower than that of lipofectamine and calcium phosphate controls in cell culture, the gene expression often is higher and more sustained than that achieved by naked DNA and lipofectamine complexes. The gene delivery system based on nanospheres/nanocapsules has several attractive features: 1) ligands can be conjugated to the nanoparticle for targeting or stimulating receptor-mediated endocytosis; 2) lysosomalytic agents can be entrapped to reduce degradation of the DNA in the endosomal and lysosomal compartments; 3) other bioactive agents or multiple plasmids can be co-encapsulated; 4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; 5) the nanoparticles can be lyophilized for storage without loss of bioactivity.

The aim of the current study was to develop a simple method for entrapment of macromolecule (proteins, DNA etc) in polymer nanocapsules (100 to 1000 nm). Plasmid DNA-loaded nanocapsules will be used for gene delivery. Our previous experiments demonstrated that DNA from calf thymus can be effectively entrapped in nanocapsules [2]. In this research, to study endocytosis in model systems we prepared a series of nanocapsule samples with FITC-labeled polymer molecules, such as gelatin, poly(allylamine hydrochloride), bovine serum albumin, chitosan.

Various biocompatible polysaccharides such as alginate, sulfate dextran, chitosan were used to compose the nanocapsule wall. To prepare nanocapsules these polyelectrolytes were adsorbed layer-by-layer from solution, alternating positively and negatively charged polymers on colloid core which was dissolved after assembling 7-10 polymer layers on its surface. The prepared FITC-labeled nanocapsules were added to CHO cells, and endocytosis was investigated by flow cytometry. Flow cytometry analysis was performed in 3, 5 and 24h after adding the capsules to the cell culture, and revealed 67.9, 81.1 and 89.7% of cells containing FITC-labeled capsules, respectively. Best results were obtained for the nanocapsules with sulfate chitosan layer on the surface. The cells were also characterized by fluorescent confocal microscopy. Thus, the proposed approach appears to be promising to prepare chitosan-based microcapsules with entrapped plasmid DNA for transfection of eukariotic cells.

References: 1. Sukhorukov, G.B. et al, Colloids Surfaces A 137, 253-266 (1998). 2. Sukhorukov, G.B. et al, Polymers for Advanced Technologies, 9, 759-767 (1998). 3. Donath, E. et al,. Angewandte Chemie, Intern. Edition, 37 (16) 2201-2205 (1998).

P25

SPECIFIC ANTITUMOR ACTIVITY OF POLY[N-(2-HYDROXYPROPYL) METHACRYLAMIDE] CONJUGATED BOVINE SEMINAL RIBONUCLEASE

J. SOUČEK^a, P. POUČKOVÁ^b, D. HLOUŠKOVÁ^b, M. ZADINOVÁ^b, Z. HRKAL^a, D.PLOCOVÁ^c, J. STROHALM^c, K. ULBRICH^c

^bInstitute of Biophysics, Medical Faculty, Charles University, 120 00, Praha 2,

^cInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity against various human tumor cell lines in vitro or when administered intratumorally (i.t) to the nude mice bearing human tumors. Ineffectivity of intravenous (i.v.) or intraperitoneal (i.p.) administration of BS-RNase led us to synthesis of several polymeric conjugates to ensure longer persitance in bloodstream and to prevent BS-RNase from its degradation. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS RNase modification and two PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated at the end of polymer side chains while “star-like” conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of wild BS RNase administered i.v. at a daily dose 10mg/kg, application of P-BS and S-BS conjugates at fivefold and twenty fold lower doses caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results we investigated the effects of i.v. administered S-BS on the metastatic process in C₅₇Bl₆inbred mice bearing Bl₆melanoma and on their survival. 60% of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated group was 75.5 days compared to 39.2 days in control group. Our experiments with [¹²⁵I]-labeled BS-RNase and S-BS have proved that S-BS can persist in blood for more than 24 hours at very high level, whereas wild BS-RNase disappears very quickly after its application from the bloodstream. PHPMA-BS-RNase conjugate was found to be a targetable potent anticancer and antimetastatic preparation what increases the possibility of its clinical use.

This work was supported by the Grant Agency of the Czech Republic grant No. 307/96/k226.

P27

Production of Fab fragments of Monoclonal Antibodies with the use of synthetic polyelectrolytes.

Application of monoclonal antibodies both for practical purposes and in fundamental studies is sometimes restricted by their divalent nature. Monovalent Fab fragments of antibodies containing only one binding site are used instead, e.g. for study of protein folding, in electron microscopy, medical diagnostics and in therapy. Production of Fab fragments with the use of different proteolytic enzymes was developed by Porter forty years ago. Since then the procedure remained essentially the same. The only exception is the use of gene technology for Fab production [King, 1992], but this method has not yet found a wide application.

The efficient protocol for Fab production should include the design of the conditions for proteolytic degradation of antibodies so that the splitting of antibodies into Fab and Fc fragments is nearly complete, while the binding center at the Fab fragment is not damaged by the proteolysis.

In the present paper we used polyanion and conjugate of polycation with attached antigen. It allowed solving of three main problems of the Fab fragment production, namely,

The conjugate was prepared by covalent attachment of the antigen to polycation, poly(N-ethyl-4-vinylpyridinium) bromide, containing 7% hydroxyethyl groups. Proteolysis of monoclonal antibodies bound to the conjugate by papain followed by i) precipitation of the conjugate induced by polyelectrolyte complex formation with added polyanion, poly(methacrylic) acid and pH-shift from 7.3 to 6.5 and ii) elution at pH 3.0 resulted in obtaining of 90 % immunologically competent Fab fragments. The required concentration of papain was 10 times lesser than in the case of free antibodies in solution. Rather small damage of the antibodies and high yield of the Fab fragments strongly suggest that the antigen-polycation conjugate not only protects binding sites of bounded monoclonal antibodies from proteolytic damage but also facilitates the proteolysis probably by exposing the antibody molecules in a manner convenient for proteolytic attack by the enzyme.

P28

General Principles of creating of DNA-containing polyelectrolyte complexes with controllable stability

Complexes of DNA with synthetic oligo- and polycations have been successfully used for increasing the efficiency of transformation of cells by the plasmides and for protection of DNA from splitting by cell nucleases. The prospects for addressing DNA packed in PEC species to the target cell has motivated an extension of studies of DNA-containing PECs in order to give precise control over their stability.

Recently (Izumrudov et al, 1995) we developed the monitoring of the PEC destruction by following the ignition of fluorescence of ethidium bromide as a result of intercalation of the dye in free sites of DNA. This approach was shown to be applicable for PECs formed by DNA and different positively charged species, in particular linear and branched synthetic polyamines, basic polypeptides, histones, cationic dendrimers, cationic micelles etc.

In the present work we used this approach and ascertained the influence of different factors on stability of DNA-containing PECs in aqueous solutions, namely:

The data obtained appear to be the guideline in design of DNA-containing PECs with given and controllable stability. The design may be accomplished not only by the choice of proper polyamine, but by using of tailor-made polycations with revealed molecular characteristics as well. Thus, the destruction of PEC formed by DNA and random copolymer of 4-vinylpyridine and N-ethyl-4-vinylpyridinium bromide proved to be pH-sensitive and could be performed under pH and ionic strength closed to the physiological conditions. This result can be considered as a major step on a line of attack on the problem of creating of self-adjustment DNA-containing systems. It appears particularly promising for addressing DNA packed in PEC species to the target cell as well.

P29

effects of aging under dynamic conditions in physiological-like environments on the mechanical behavior of medical grade polyurethanes

Dept. Bioengineering, Politecnico di Milano, P.zza L. da Vinci, 32, 20133 Milan, Italy

A new methodology has been set up to evaluate the aging of polymers used for cardiovascular prostheses, under dynamic, physiological-like conditions. The purpose was the comparative evaluation of the stability of four biomedical segmented polyurethanes (a polyether-urethane, PEU, two polycarbonate-urethane, PCU and a polycarbonate-urethane-urea, PCUU).

Pellethane 2363-80A (PEU, Dow Chemical), Corethane 55D and Corethane 80A (PCUs, Corvita) and Coremer (PCUU, Corvita) were selected. Eight mm i.d. tubes were prepared by solvent casting from a DMAC solution. The aging media were: buffered saline solution (PBS, pH 7.4), lipid suspension in PBS (0.1% cholesterol, 0.25% phosphatidylcoline) and bovine serum. NaN₃ (0.02%) was added to each media in order to inhibit the bacterial growth. A testing apparatus, able to keep the materials in contact with the aging solutions at 37°C, under cyclically variable pressure (80-180mmHg, 0.5Hz), was designed and assembled. Tests were performed at three different times: 10, 20 and 30 days. Tensile tests were performed according to ASTM D1414 practice (standard method for testing rubber o-rings) on cylindrical samples cut from the tested tubes with a 5inch/min crosshead speed. Secant moduli at 100 and 300% elongation (E₁₀₀, E₃₀₀), ultimate tensile stress (_r) and strain (%_r)were recorded. Statistical analyses were performed to verify significant differences among the treatments.

Pellethane 2363-80A underwent the highest changes of mechanical properties, and was the only one that exhibited surface degradation, with evidence of surface cracking. Whereas after PBS aging no significant modifications were observed for all the materials, after lipids and serum treatments significant changes in mechanical properties were detected. For Pellethane and Coremer, a decrease in stiffness was detected after lipids aging, and an increase in _r and %_r values was shown after serum. Corethane 80A and Corethane 55D showed an opposite trend. Moreover, tensile tests also demonstrated modifications in the secant moduli after lipids and serum treatments. Mechanical properties modifications showed a trend from 10 days to 30 days with significant differences. All the examined materials proved to be rather stable under test conditions. Coremer, Corethane 55D and Corethane 80A demonstrated a higher stability than Pellethane.

The authors thank G.Laroche and D. Mantovani (IBQ, Quebec, Canada) for their contribution to this work.

P30

Designing POLYURETHANES FOR BIOMEDICAL DEVICES, TISSUE ENGINEERING AND DRUG RELEASE

Dipartimento di Bioingegneria, Politecnico di Milano, P.zza L. da Vinci, 32, 20133 Milan, e-mail: petrini@biomed.polimi.it

Biocompatibility, advantageous physical properties associated to their versatility have been making the polyurethanes primary candidates for many biomedical application.

By intelligent use of principles governing the structure/properties relationship of these polymers, a wide range of systems can be obtained, ranging from engineering materials to polymers with properties similar to those of living tissues.

This work describes different polyurethane structures, both linear and cross-linked, developed in our laboratory and in principle suitable for use in drug-release or tissue engineering applications.

Linear polyurethanes are block copolymers generally obtained from three types of base reagents: macrodiols, diisocyanates and chain extenders (i.e. low-molecular weight diols or diamines). Macrodiols will constitute the “soft”, flexible, segments, whereas hard, rigid, segments derive from the reaction of the other two reagents. The resulting phase-separated structure shows elastomeric properties. By the appropriate selection of the base reagents, the following materials were developed:

Furthermore, cross-linked poly-urethane foams have been developed with controlled porosity and density [4]. These foams were loaded with hydroxyapatites and/or calcium phosphates for bone ingrowth and reconstruction, or coated with drugs or biomolecules for soft tissue healing.

Full chemico-physical characterisation of all these materials will be provided, as well as the results of biological tests, with particular reference to cytocompatibility.

[1] P. Petrini, S. De Ponti, S. Farè, M.C. Tanzi, J. of Mat. Sci., Materials in Medicine 10, 635-640 (1999)

[2] P. Petrini, M.C. Tanzi, L. Visai, F. Casolini, P. Speziale, J. of Biomat. Sci., Polymer Edition, 1-13 2000, in press.

[3] P. Petrini, M.C. Tanzi, C.R. Moran, N.B. Graham, J. of Mat. Sci., Materials in Medicine, 10, 711-714 (1999)

[4] S. Farè, P. Petrini, S. Benvenuti, E. Piscitelli, M. L. Brandi, M.C. Tanzi, accepted for Aloha session, VI World Biomaterials Congress, Hawaii, 15-22 May 2000.

P32

¹Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

²CRC Institute for Cancer Studies, University of Birmingham B15 2AT, United Kingdom

The transport of genes to specific cells or tissues is a progressive method proposed for treatment of various human diseases. The DNA complexes with synthetic polycations are one of the vectors in nonviral gene delivery, which are intensively studied at present. We have studied systems based on polymers of the methacrylate and methacrylamide type with primary, tertiary and quaternary amino groups and also statistical copolymers of these types. The sizes and behaviour of the complexes in solution were characterised by light scattering methods. It was shown that stabilities of the complexes in salt solutions and in dilute buffers of various pH and their in vitro transfection activities depend on the detailed structure of the polycation and its molecular weight [1]. It was shown that in in vitro experiments the most active are polycations with primary amines. However, the polycation/DNA complexes are unstable in solutions of salt, where the surface charge is screened and particles start to precipitate. In the blood stream, these complexes interact with plasma protein albumin and are rapidly removed by RES. Coating of polycation/DNA complexes with nonionic hydrophilic polymers reduces the surface charge of the complexes and protects them from interaction with plasma proteins [2].

This work was supported by the Grant Agency of the Czech Republic (grant No. 307/96/K226), the European Union Biotechnology Program (IC20CT97005) and the Grant MECR TSR-006-99.

P34

ANTIPROLIFERATIVE MONOAMINE-COORDINATED PLATINUM COMPLEXES CONJUGATED TO WATER-SOLUBLE CARRIER POLYMERS

^a Department of Chemistry, University of the Witwatersrand, WITS 2050, South Africa

^b School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

The therapeutic effectiveness of cisplatin and related anticancer-active complexes containing the cis-diamineplatinum moiety in addition to other, leaving-group-type ligands has been well established and clinically proven¹. Analogous complexes comprising but a single amine ligand, although not invariably inactive^{2, 3}, have not, on the other hand, established themselves as clinically useful agents, and the reason for this neglect may be traced to the fact that monoamineplatinum compounds generally represent charged species and, thus, cannot readily enter the affected cell by the mechanism of passive diffusion common to most neutral agents. This realization prompted synthetic efforts in this laboratory to tie (anchor) the square-planar platinum complex system to polymeric carriers via a monoamine ligand in order to achieve improved bioavailability through altered pharmacokinetics. In this poster we highlight the synthesis of such monoamine-coordinated platinum conjugates, which proceeds through aqueous-phase platination of polyaspartamide carriers functionalized with primary amino groups as side chain terminals, the tetrachloroplatinate(II) anion serving as the platination agent. Under the aqueous work-up conditions, the primarily generated aminetrichloroplatinum complex tends to shed a chloro ligand, transforming into the amineaquadichlorometal complex structure postulated for the ultimate products. Representative conjugates of this study, tested in vitro against the LNCaP human metastatic prostate adenocarcinoma cell line, show high antiproliferative activity, with cytotoxic effects well on a par with those of analogous conjugates containing the metal in cis-diamine-chelated form. More extended screens in forthcoming work will serve to corroborate these findings.

P35

^* Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic

^** The Fraunhofer Institute of Physical Measurement Techniques, Freiburg, Germany

Preparation of assemblies consisting of multiple molecular layers of bovine serum albumin (BSA), monoclonal antibodies against horseradish peroxidase (anti-HRP), and monoclonal antibodies against methotrexate (anti-MTT) and interaction of the assemblies with human blood plasma were observed using a grating coupler (GC) and Young interferometer (YI).

The arrangement of molecular layers in the assemblies and total deposits irreversibly adsorbed from human blood plasma on GC chips (tantalum oxide waveguide) coated with the assemblies are shown in Table I. The deposits are given relative to that on the uncoated GC surface I:

Assembly	Deposit (rel.)	Assembly	Deposit (rel.)
uncoated GC surface (I)	1	BSA¾ anti-HRP (V)	0.040
anti-MTT (II)	0.16	BSA¾ BSA (VI)	0.030
anti-MTT/PDA/anti-MTT/PDA/anti-MTT (III)	0.072	anti-MTT¾ anti-MTT (VII)	0.022
anti-MTT/PDA/anti-MTT (IV)	0.047	anti-HRP¾ anti-HRP (VIII)	0.016

Monomolecular anti-MTT layer II was immobilized covalently on the GC surface and the surface passivated by BSA adsorption. Assemblies III and IV were prepared by successive electrostatic adsorption of poly(diallyldimethylammonium chloride) (PDA) polycations and anti-MTT. Assembly V was prepared by covalent binding anti-MTT to a crosslinked BSA monolayer. Covalently crosslinked protein double layers VI, VII, and VIII were prepared by successive adsorption ¹.

Composition of plasma deposits studied by the sensor response to the binding of antibodies specific for plasma proteins - immunoglobulins G and M (IgG and IgM), human serum albumin (HSA), and fibrinogen (Fb) is shown in Table II. The values of sensor response to the binding of specific antibodies to a surface are given relative to the response to anti-IgG binding to the same surface.

Assembly	anti-IgG	anti-IgM	anti-HSA	anti-Fb
I	1	1.44	3.15	5.62
V	1	0.6	0	1
VI	1	1.05	0.25	0.15
VII	1	1	0	0.42
VIII	1	1.07	0	0.71

An anti-HRP double layer was immobilized in a waveguiding branch of YI and a similar anti MTX double layer was immobilized in the other branch. The nonspecific responses to blood plasma in the two branches were compensated and sensor responded to addition of HRP or MTX to plasma.

P36

IMMUNOGENIC ECITY OF AMFIFILIC WATER SOLUBLE CONJUGATES OF SERUM PROTEINS WITH TEMPERATURE-SENSETIVE POLY(N-IZOPROPYLACRYLAMIDE)

Zeynep Mustafaeva^a, Myroslava Demchenko^a, Erhan Ergen, Aydan Salman Dilgimen^a, and Mamed Mustafaev^a*

^a Institute for Genetic Engineering and Biotechnology, TUBITAK Marmara Research Center, Gebze, Kocaeli, Turkey

We have conjugated Bovine Serum Albumin (BSA) to poly (N-isoproprylacrylamide-co-acrylic acid) (Poly(NIPAAm-AA)) by using water soluble carbodiimide, and the effects of the bulk mass ratio of protein to polymer (r) on the formation of polymer-protein conjugates have been studied. HPLC, electrophoresis, viscosimetry and fluorescence spectroscopy suggest that the mode of covelent binding of BSA to Poly (NIPAAm-AA) depends upon the weight concentration ratio ( r ) of BSA to Poly (NIPAAm-AA). At r ≤ 1, free Poly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches 1 the amount of free polymer is too small to be observed. It is shown that depending on the ratio r, two types of conjugate particles were formed : at r<1, the protein molecules in the structure of conjugate particles are densely covered as a shell by polymer chain and practically ”fenced off” from water environment ; at r>1 the forming conjugate particles possess more friable structures in which protein molecules are practically exposed to the solvent. The complex formation involving electrostatic interactions between BSA and carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transition of macromolecules in low- and thermally induced precipitation in more concentrated solutions of bioconjugates has observed.

The immunogenic properties of covalent conjugates of CP-BSA were investigated and the temperature modulated solubility – immunogenicity alterations was analyzed. A single immunization of mice with conjugates at the thermally precipitating concentration without adjuvant evoked increased specific immune response to BSA, which practically did not depend in the initial conjugation ratio of components. Such a modulated system is attractive for application as a novel immunogenic system in vaccine technology.

Institute for Genetic Engineering and Biotechnology, TUBITAK Marmara Research Center, Gebze-Kocaeli, TURKEY

Physico-chemical behaviors of Biopolymer Systems comprising haptens (estradiol, progesterone and betulin), protein antigens and synthetic polyelectrolytes were investigated. The effects of the polymer nature (temperature-negative and –positive solubility), methods of binding (multipoint metal induced complexation, covalent coupling, etc.) and self-assembly principle in such systems were analysed. It is shown that depending on the monomer composition, and ratio of components two types of polycomplex particles were formed: 1) the protein molecules in the structure of particles are densely covered as a shell by polymer chains and practically ”fenced off” from water environment; 2) the polycomplex particles possess more friable structures in which protein molecules are practically exposed to the solution.

These Biopolymer Systems possess simultaneously highly protein- and hormon-specific immunogenecity without traditional adjuvants and a single immunization with functions following the immunization gave rise the development of hormone-specific monoclonal antibodies.

Metal-induced formation of Biopolymer Systems possess simultaneously highly immunogenecity and radiostability. In these systems Cu²⁺ ions promoted: (1) the binding of anionic polyelectrolytes to similarly charged protein antigen, (2) intermolecular aggregation of polycomplex particles and (3) protection the polymer and protein components against radiation damage. The mechanism of observed immunogenic and protection effects are discussed.

P37

Design of microcapsule for encapsulation of living cells based on polyelectrolyte complexation: Role of the anti-gelling cation

^aPolymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 842 36 Bratislava, Slovak Republic

^bCenter for Microgravity Research and Applications, Box 1743 Station B, Vanderbilt University, Nashville, TN, 37235, U.S.A.

A few years ago the Vanderbilt University Cell encapsulation team announced a new capsule for encapsulation of living cells [1-3]. It is based on the polyelectrolyte complexation in a single-step reaction of five active components. The reaction proceeds between the mixture of two polyanions (high viscosity sodium alginate and cellulose sulfate) and solution of cations containing polycation poly (methylene-co-guanidine), gelling calcium chloride and anti-gelling sodium chloride [2]. This capsule was successfully used in the diabetic rodent animal models, including the NOD mice, to immunoisolate the transplanted islets of Langerhans, which reversed diabetes for a number of months up to one year.

A manifold role of sodium chloride has been recognized and utilized in adjusting the capsule properties. Generally, the presence of sodium chloride has been understood as a requirement to provide the osmotic pressure for the cells. However, since the membrane is formed by the mutual reaction of oppositely charged polyelectrolytes in the presence of the gelling calcium cation, one should anticipate an additional role of sodium chloride. We emphasized and quantified the anti-gelling effect of sodium cations in the competitive reaction of two other components of cation solution – calcium ions and poly (methylene-co-guanidine) – with the polyanion matrix. The membrane permeability can be simply adjusted by a minor modification of molar ratio between sodium and calcium cations [3]. Another effect is a control of the surface morphology, when the extent of surface roughness / smoothness measured by atomic force microscopy is also controlled by the ratio between sodium and calcium cations [4]. A smoother surface is obtained by slowing down the rate of gelling reaction. Based on this understanding, a continuous process providing both smooth surface and mechanically and chemically stable membrane was recently developed [5].

P43

V. ŠUBR^a, T. ETRYCH^a, K. ULBRICH^a, T. HIRANO^b, T. KONDO^c, T. TODOROKI^c

Superoxide dismutase [EC1.15.1.1] is an enzyme preventing tissue damage from cytotoxic oxygen radicals produced in pathological states such as inflammatory diseases, ulcerative colitis and ischemic/reperfusion injury during transplantation. The use of superoxide dismutase is limited by its short circulation time (about 5 min) and by development of immune response after prolonged use.

Modification of superoxide dismutase with a synthetic water soluble polymer is a possible way to increase the circulation time and decrease the protein immunogenicity. The polymers on the basis of poly[N-(2-hydroxypropyl)-methacrylamide] are good candidates for synthesis of these conjugates.

”Classic” and ”star-like” conjugates of superoxide dismutase with poly[N-(2-hydroxypropyl)methacrylamide] were prepared and their physicochemical and biological properties were studied. The conjugates showed better thermal stability and better stability to the oxidation with H₂O₂ in comparison with free superoxide dismutase.

Preliminary in vivo activity of superoxide dismutase conjugates was evaluated by intravital fluorescence microscopy on rat liver after induced ischemia/reperfusion injury. Both types of conjugates showed a higher protective effect on ischemia/reperfusion injury in comparison with free superoxide dismutase.

Acknowledgement: This work was supported by the Grant Agency of the Czech Republic (grant No. 307/96/K226). The authors thank the Wyatt Technologies Corp. for kind loaning a multiangle light scattering detector DAWN DSP-F.

P44

*PAOLO FERRUTI, ^†RANIERO MENDICHI,^°MARIO CASOLARO,^††SIMON C. W. RICHARDSON, ^††NICOLA G. PATTRICK AND ^††RUTH DUNCAN

*Dipartimento di Chimica Organica e Industriale, Università di Milano, Via Venezian 21, 20133 Milano, Italy;^†Istituto di Chimica delle Macromolecole del CNR, Via A.Corti 12, 20145 Milano, Italy;^°Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi, Università di Siena, Piano dei Mantellini 44, 53100 Siena, Italy; ^††Centre for Polymer Therapeutics, The School of Pharmacy, University of London, 29-39 Brunswick Sq., London WC1N 1AX, UK.

Poly(amido-amine)s (PAAs) are a family of polymers with interesting properties that can be exploited in biomedical and other applications (reviewed in ^1,2). They are usually water-soluble and contain hydrolysable bonds in their main backbone allowing degradation in aqueous media³. The ability of PAAs to promote pH-dependant membrane lysis⁴ has been used to enhance intracellular delivery of DNA and toxins⁵.

Here, five poly(amido-amine)s (PAAs) carrying two ter-amino groups and one carboxyl group per repeating unit were prepared by hydrogen-transfer polyaddition of 2-methylpiperazine (ISA 23), 1,2-bis(N-methylamino)ethane (DMEDA-BAC), 1,2-bis(N-ethylamino)ethane (DEEDA-BAC), 1,3-bis(N-methylamino)propane (DMEPDA-BAC) or 1,6-bis(N-methylamino)hexane (DMEXA-BAC) in each case to 2,2-bis(acrylamido)acetic acid (BAC). The resultant PAAs had an Mn in the range 7,900 - 25,000 g/mole and an Mw in the range 11,500 - 42,500 g/mole. Considerable differences were observed in the basicity of the amino groups present (logK˚₁ = 7.5-9.5; logK˚₂ = 3.2-8.4), whereas the logK˚₃ value (2-3) of the carboxyl groups was consistent with a fairly strong acid.

To measure general cytotoxicity, B16F10 mouse melanoma cells were incubated with each PAA and cell viability was assessed. DEEDA-BAC, DMEDA-BAC and ISA 23 were non toxic (IC₅₀ > 5 mg/mL). Those PAAs with highest log˚K₂ values were more cytotoxic (IC50 = 3.55 mg/mL for DMEPDA-BAC, and IC₅₀ = 0.23 mg/mL for DMEXA-BAC. An in vitro haemolysis assay shown previously to correlate well with endosomal/lysosomal rupture in vivo was used to measure pH-dependent PAA-membrane interaction over time. The pH values chosen for these studies were representative of the systemic circulation (pH 7.4), the endosome (pH 6.5) and secondary lysosome (pH 5.5). All the PAAs displayed pH-dependent haemolysis (most lytic at pH 5.5) consistent with their proposed use as endosomolytic polymers.

(2) Ferruti, P.; Duncan, R.; Richardson, S. In: ”Targeting of Drugs 6: Strategies for Stealth Therapeutic Systems”; Gregoriadis, G., McCormack, B., Eds.; Plenum Press: New York., 1998; p207-224.

(3) Ranucci, E.; Spagnoli, G.; Ferruti, P.; Sgouras, D.; Duncan, R., Journal of Biomaterial Science, Polymer Edition 1991, 2, 303-315.

(4) Duncan, R.; Ferruti, P.; Sgouras, D.; Tuboku-Metzger, A.; Ranucci, E.; Bignotti, F., Journal of Drug Targeting, 1994, 2, 341-347.

(5) Richardson, S. C. W.; Pattrick, N. G.; Man, S.; Ferruti. P.; Duncan, R. Gene Therapy 2000, submitted.

P49

Immunological Features of Macromolecular prodrug for controlled release of antracycline antibiotic

^aA.V.Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninsky Prospect, 29, 117912, Moscow, Russia

^bP.A.Gertsen Research Institute of Oncology, 2^nd Botkinsky Pr. 3, 125284, Moscow, Russia

Soluble macromolecular prodrugs for controlled release of antracycline antibiotic daunomycin (DM) were synthesized; its structure and solution properties were studied. It was shown on the number of tumor models (P 388, B-16 melonoma, Lewis lung carcinoma and etc) that those macromolecular prodrugs possessed the more antitumor activity and decreased toxicity compared to free DM.

It is well known that immunological damages are one of the main side actions of most antitumor drugs, included DM, and often determinate the progress of the anticancer treatment. On this reason we studied the immunological features of macromolecular prodrug for controlled release of antracycline antibiotic DM. In order to light the action of those systems some model compounds - conjugates DIVEMA with various amount hydrophobic units (long-chain amines) - were studied also. The results indicated that effect of those systems on immune response determined by its hydrophobicity. Then, the immunomodulating action of DM-conjugates was studied. It was shown that macromolecular prodrugs unlike free DM caused stimulation of immune response. The conjugates of DIVEMA exhibited increasing number of nuclear cells (~200%) and antibody-forming cells (~180%) compared to the control.

The most interesting are data about interferoninducing activity of DIVEMA and conjugates. DIVEMA, that was administrated intraperitoneally, caused the interferon production in the serum of experimental animals as 160-320 units/ml, with maximum production through 24 h after it administration. In the case of conjugates the interferon level was 320 units/ml through the same time.

In conclusion – the correct choose of polymeric carrier allows to compensate of characteristic for DM immunodepresant effect.

P52

A NEW DISPERSION TECHNIQUE FOR MICRO- AND NANOPARTICLE FORMATION OF DRUG DELIVERY CARRIERS BASED ON POLY(ACRYLIC ACID)

Biomaterials and Drug Delivery Laboratories, School of Chemical Engineering, Purdue University, 1283 CHME Bldg., West Lafayette, IN 47907, U.S.A.

The availability of large molecular weight protein- and peptide-based drugs due to the recent advances in the field of molecular biology has given us new ways to treat a number of diseases. Oral drug delivery is the preferred route of administration. However, the instability of these drugs results in extremely low bioavailability and thus presents a considerable challenge in our field. One approach of increasing the bioavailability of these potent drugs is to use bioadhesive drug delivery systems and thus increase their residence time at the absorption site. The residence time can be further increased by utilizing particulate carriers in the lower micrometer or nanometer range which are able to penetrate the mucosal lining of the GI tract.

The pH-sensitive polymer poly(acrylic acid) (PAA) was selected as the primary component of the drug delivery systems since it has been shown to possess excellent mucoadhesive characteristics and to protect proteins and peptides from proteolytic degradation. Poly(ethylene glycol) (PEG) was incorporated into the particles using PEG-acrylate macromonomers. The hydrogel particles were prepared by free radical dispersion polymerization technique using water as the outer phase and their size evaluated by Photon Correlation Spectroscopy and Transmission Electron Microscopy. The effects of the cross-linking ratio, AA/EG feed ratio (molar ratio of AA units and EG units in the PEG macromonomer) and the amount of surfactant on particle size were examined.

The average particle size decreased with increasing amount of the sodium dodecyl sulfate (SDS) surfactant. It is important to notice that pure PAA particles of average particle size of 1100 nm were synthesized even in the absence of any surfactant.

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^a Department of Chemistry, University of the Witwatersrand, WITS 2050, South Africa

^b School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

^c Department of Immunology, University of Pretoria, Pretoria 0001, South Africa

The pharmacokinetic fate of a medicinal agent may be altered significantly through the expediency of tying (anchoring) the compound reversibly to a water-soluble polymeric carrier, thereby achieving critically important biomedical advantages unavailable to the free drug^{1, 2}. The polymer-drug conjugate may experience extended serum half-life, facilitated cell entry through pinocytosis, preferential accumulation in the tumor tissue, and, under suitable conditions, lessened propensity for eliciting drug resistance in the target cells. Exploiting this concept, we have anchored the antifolate-type anticancer drug methotrexate (MTX)³ to polyaspartamide carriers featuring hydroxyl terminals on short side chains. Conjugation is brought about through ester formation between these carrier-attached hydroxyl functions and a carboxylic acid group of the glutamyl moiety in MTX. This step is performed in aprotic medium in the presence of coupling agent and catalyst, and the water-soluble conjugates are purified by aqueous dialysis and are physically isolated by freeze-drying. The molar feed ratios are chosen so as to achieve drug loading at typical levels of 5-20% MTX by mass. Encouraging results are obtained in preliminary cell culture tests, giving IC₅₀ values below 1 μg MTX/m against the human adenocarcinoma cell line. This range coincides with that observed in activity screens against the HeLa human cervix epithelioid carcinoma line of analogous conjugates containing the drug in amide-linked form. More extended comparative screens will follow.

3. J.R. Piper, in Cancer Chemotherapeutic Agents, W.O. Foye, Ed., Am. Chem. Soc. Washington, DC, 1995, p.96.

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NOVEL DESIGN OF SMART HYDROGELS FROM SYNTHETIC AND GENETICALLY ENGINEERED POLYMERS

Departments of Bioengineering^a, and of Pharmaceutics and Pharmaceutical Chemistry^b, University of Utah, 30 S 2000 E Rm 201, Salt Lake City, Utah 84112-5820, U.S.A.

Smart hydrogels are polymeric networks capable of undergoing volume phase transition in response to external stimuli such as temperature. We are interested in utilizing the techniques of molecular biology to engineer bio-based polymers as new materials, and proposed the design of a new class of smart hydrogels consisting of water-soluble synthetic polymers and engineered protein domains.

The coiled-coil is a well-defined protein motif of multiple a -helices, and can be used as crosslink for water-soluble polymers such as poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Several recombinant proteins containing terminal histidine tags and coiled-coil domains derived from motor protein kinesin have been cloned, expressed in E. coli, and studied using circular dichroism (CD) and analytical ultracentrifugation (AUC). Hybrid hydrogels containing these genetically engineered coiled-coils will display interesting properties of swelling that are predetermined by the characteristic unfolding processes of these proteins. These hydrogels are potentially applicable in many biomedical fields such as drug delivery and tissue engineering.

Fig. 1. Stimuli-responsive conformational change of genetically engineered proteins (left half of the figure) determines the volume phase transition of hybrid hydrogels (right half of the figure).

NOVEL DESIGN OF SMART HYDROGELS FROM SYNTHETIC AND GENETICALLY ENGINEERED POLYMERS

Departments of Bioengineering^a, and of Pharmaceutics and Pharmaceutical Chemistry^b, University of Utah, 30 S 2000 E Rm 201, Salt Lake City, Utah 84112-5820, U.S.A.

Fig. 1. Stimuli-responsive conformational change of genetically engineered proteins (left half of the figure) determines the volume phase transition of hybrid hydrogels (right half of the figure).

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^a Krebs Institute for Biomolecular Science, Department of Chemistry, University of Sheffield, Brook Hill, Sheffield, S3 7HF, United Kingdom

^b Department of Medicine, Division of Clinical Sciences, University of Sheffield, Northern General Hospital, Herries Road Sheffield, S5 7AU, United Kingdom

Current research will rapidly demonstrate the synthesis, and characterise the morphology and mechanical properties of a range of materials based on polyether soft segments and polypeptide hard segments. The polymers will then be converted into integral skin, micro-cellular bandages for application in the treatment of burns and ulcers.

Block copolymers of peptides and polyethers could provide the ideal solution to the requirements of a synthetic skin substitute for use as a micro-cellular bandage. The local molecular structure of the materials controls its biological specificity/compatibility. Use will be made of block copolymers of peptides and polyethylene glycol. Low molecular weight polyethylene glycol is non-toxic and can be eliminated from the body by excretion. The sequence of the peptide component will be used to regulate the interaction of the synthetic material with its biological environment.

The macroscopic structure can be built into the material at the processing stage to make an asymmetric foam which will control permeability whereby the upper surface will be water permeable but will prevent bacteria from entering the wound, while the inner surface will have large pores to promote vascularisation and natural healing of the wound.

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DICARBOXYL-FUNCTIONALIZED CARRIER POLYMERS AS PRECURSORS OF MACROMOLECULAR PLATINUM DRUGS

Department of Chemistry, University of the Witwatersrand, WITS 2050, South Africa

Dicarboxylatoplatinum complexes in which the metal is chelate-bound by two carboxyl groups in addition to other functional ligands have found challenging applications as second-generation anticancer agents evolved from the parent platinum drug cisplatin¹, and several polymeric platinum complexes featuring this structural pattern are in various stages of development (recently surveyed²). As part of a program to conjugate biomedicinal agents to polymeric carriers for enhancement of therapeutic effectiveness³, we have synthesized a number of water-soluble macromolecular compounds featuring side chain-attached 1,1- or 1,2-dicarboxyl functions lending themselves to metal bonding through dicarboxylatometal chelate formation. The class of carriers comprising the 1,1- dicarboxylate system is derived from malonic acid substituted at C-2 by functional groups suitable for polymerization with the aid of polyaddition or polycondensation reactions. Analogously, carriers containing the 1,2-dicarboxylate system are accessible by copolymerization of aspartic acid or, more efficiently, of suitably N-substituted aspartic acids, with complementary comonomers. The water-soluble polymeric products are purified, fractionated, and isolated by standard methods and are structurally characterized by spectroscopic and microanalytical techniques. A model experiment involving platinum binding to a selected carrier with diaqua(cyclohexane-1,2-diamine)platinum(II) dication as the platination agent serves to demonstrate the feasibility of platinum chelation through the polymers side groups, and this opens up a promising inroad into the field of macromolecular, platinum-containing anticancer drugs.

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TRANSPORT OF POLY AMIDOAMINE (PAMAM) DENDRIMERS ACROSS MADIN-DARBY CANINE KIDNEY (MDCK) CELL LINES

^bDept. of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, U.S.A.

2) El-Sayed M et al, Proc. of the 27^th Int. Symp. on Cont. Rel. of Bioactive Materials, Paris, July (2000).

Supplement:

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ChipWare, ChemWare, InfoWare

Beyond the Mono-Parameter Paradigm--Looking at the Whole Elephant

Dept. of Bioengineering, University of Utah, 50 S. Campus Center Dr.,Salt Lake City, UT 84112-9202 USA

KIM’S ELEPHANT

In the early 80's, during an interview for a local newspaper, Sung Wan Kim told the reporter:

”A blind man trying to identify an elephant by examining only the trunk faces a nearly impossible task.”

TOO MANY TRUNKS –NO ELEPHANTS!

Today Science--and most NIH study sections--insist on single hypothesis, single parameter proposals. To look at the whole elephant is considered to be ”too ambitious”, ”lacking a specific hypothesis”, ”a fishing expedition”, or other negative descriptor. How can one understand the elephant by examining only its toe, or its ear, or its trunk? By the time we formulate enough single parameter hypotheses and experiments, the elephant--and surely the investigator--would likely be dead.

There are many examples where our dependence on single parameter hypotheses can get us into trouble. In fact, the general public is now skeptical of much scientific and medical research because of the contradictory output from overly simplistic, mono parameter studies.

Albert Einstein said: ”Science should be as simple as possible, but not simpler”.

(a set of newspaper clippings on medical and health single parameter studies). These are primarily chemical, biochemical, examples

A BIOCHEMICAL ELEPHANT – METABOLISM

The most well known and yet least understood biochemical elephant is metabolism. We have all seen and used biochemical maps and metabolic pathway charts. Such a map makes it crystal clear that biochemical reactions do not exist or operate in isolation. Every reaction is obviously dependent on many other reactions through the principles of reaction kinetics and equilibria. And yet we continue to look for ”magic” single chemical parameter disease correlations. We study them and present those results to the general public in the form of lifestyle recommendations., never warning them that this is such a tiny part of the biochemical elephant that it may well be irrelevant to the overall system.

MONOPARAMETER BRAINWASHING

We have indeed learned much from our simplistic single parameter approaches -- often called the reductionist method. That enormous knowledge base can now be used to understand the systems we have been reducing and dissecting for many decades.

The problem is that study sections, proposal reviewers, and most of the scientific system are now programmed, hard-wired, to only appreciate and understand reductionist science. We have all been doing single parameter reductionism for so long that it's all we know. The hard wired scientific establishment -- and perhaps most of us -- have great difficulty in appreciating that we now have the information, tools and skills to deal directly with multiparameter complex systems. Fortunately, the National Science Foundation has recognized this potential and this need with its new initiatives on biocomplexity.

THE 4M LAB – APPROACHING THE COMPLEXITY OF THE METABOLOME

We are beginning an initiative on biochemical complexity--to begin the modeling and simulation of major segments of metabolism, coupled with the means to directly measure many different metabolite concentrations simultaneously, thereby providing the multiparameter chemical data required for the development and utilization of models of complex biochemical networks. This work of course uses the fundamental principles of chemical reaction kinetics and reaction equilibria which Sung Wan Kim, and his famous Ph.D. supervisor, Henry Eyring, so extensively studied and developed.

Our new Laboratory for the Modeling, Measurement, and Management of the Metabolome (the 4M Lab) includes the following projects:

FROM COMPLEXITY TO SIMPLICITY—BACK TO COMPLEXITY

Although our work on complex systems is focused initially on metabolism, the mathematical modeling and multiparameter measurement and presentation approaches should have application to all areas of science. Modern scientific and analytical tools permit the monitoring and measurement of many different parameters simultaneously. Modern mathematical and data analysis tools permit us to deal with multiparameter data sets in a highly efficient and effective manner. It is no longer necessary to design and conduct experiments using only monoparameter hypotheses. Indeed, given today's tools and skills, it is incredibly inefficient and misleading to do so. Nevertheless, we are all simple hypothesis, monoparameter trained and therefore hard wired. It will be difficult to overcome those decades of monoparameter brainwashing. Let us try.

POSTERS

The three posters on ChipWare, ChemWare, and InfoWare present our current approach.