ANTIBODY-TARGETED POLYMER CARRIERS OF DRUGS
K. ULBRICHa, V. ŠUBRa, M. PECHARa, D. PLOCOVÁa, J. STROHALMa, T. ETRYCHa, M. JELÍNKOVÁb, M. KOVÁŘb, B. ŘÍHOVÁb
a Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského n. 2, 162 06 Prague 6, Czech Republic
b Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic
Our previous results showed that water-soluble synthetic polymers conjugated with antibodies or their fragments provide a potential targetable drug carrier system facilitating specific delivery of anti-cancer drugs to model tumors or tumor cells inoculated into mice. We have also shown that biological activity of the antibody-targeted poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) conjugate with doxorubicin (DOX) significantly depends on its detailed structure. A prerequisite for the biological activity of the conjugate is attachment of the drug to the polymer carrier via biodegradable spacer enabling drug release at its target.
The design of an efficient polymer drug conjugate has to be based on careful selection of the proper polymer, potent drug and the antibody exhibiting specific and strong affinity to the receptors expressed on the surface of the target cells. However, no less important is also the choice of the proper structure of the conjugate and an appropiate conjugation reaction of the antibody and drug with the polymer.
We have synthesized conjugates in which both DOX and antibody were attached to the PHPMA chain by enzymatically degradable oligopeptide spacers situated as side chains along the polymer backbone or similar conjugates in which DOX was attached to the PHPMA-antibody carrier via hydrolytically sensitive hydrazon bonds. Physico-chemical and biological properties of such conjugates were compared with those of the conjugate in which a number of semitelechelic PHPMA chains, bearing the anti-cancer drug doxorubicin (Dox) attached with a biodegradable oligopeptide spacer, were linked to the central antibody molecule via their terminal carboxylic acid groups. Detailed structure of the PHPMA-DOX-antibody conjugate significantly influences in vitro cytotoxicity, biodistribution and the resulting efficiency of the treatment of various tumor models in mice.
Acknowledgements. The authors thank the Grant Agency of the Czech Republic for supporting the project by grant No. 307/96/K226.
NOVEL FUNCTIONAL POLYMERS FOR GENE DELIVERY
SUNG WAN KIM
Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City, UT 84112, USA
In this laboratory, we designed various polymeric gene carriers which contain specific targeting moiety or specific functional group for systemic delivery of therapeutic genes.
We designed a novel non-viral vector, a terplex system for receptor specific gene transfer with high gene transfection efficiency in a murine smooth muscle cell line, compared to DNA/Lipofectin(tm) and DNA/stearyl-PLL, respectively. The Terplex system is based on a balanced hydrophobicity and electrostatic interaction between stearyl-poly-L-lysine (stearyl-PLL), lower density lipoprotein (LDL) and DNA. The Terplex system showed negligible cytotoxicity both in the in vitro and in vivo studies. The DNA in the Terplex system is much more stable than naked DNA in serum. The pharmacokinetics and biodistribution studies showed that Terplex[32P]DNA complexed had much higher steady-state volume of distribution (Vd55, 2.61 L/kg) than naked DNA (0.95 L/kg) and after intravenous injection. The higher steady-state volume allows the Terplex gene carriers to have enough time to expose themselves to the vascular wall. The artery wall binding epitopes on LDL allow Terplex gene carriers to specifically target the vascular cells. Using bovine aortic primary culture cells, aortic endothelial cells and aortic smooth muscle cells, as in vitro gene transfer models, we demonstrated that gene transfer mediated by Terplex showed higher efficiency than that mediated by control gene carriers (Lipofectin(tm), and PLL-stearyl without LDL), with less cytotoxicity compared to control gene carriers. Terplex combines nonexistent pathogenicity with high efficiency, high vascular wall cell specificity of transfection and non-immunological reaction. Terplex gene carrier systems are new useful tools for vascular wall gene delivery.
We recently synthesized a biodegradable cationic polymer, poly[(-(4-aminobutyl)-L-glycolic acid] (PAGA). This polymer condenses DNA efficiently and is less cytotoxic than PLL. This carrier was used for the delivery of mouse IL-10 (mIL-10) gene which prevents insulitis in NOD mice. The efficiency of PAGA mediated transfection to 293T cells was significantly higher than that of naked DNA. Three week old NOD mice were injected with one shot of 100 (g of mIL-10 DNA complexed with PAGA (charge ration PAGA:DNA=2:1) in tail vein. IL-10 serum level was measured by ELISA, and serum IL-10 levels injected with PAGA mouse IL-10 plasmid DNA complex were significantly elevated. IL-10 level peaked at 5 days and was detected up to 7 weeks. The insulitis progress was reduced significantly by the injection of DNA/polymer complex (16%) compared to the naked DNA injection (35%). Non treated controls induced insulitis over 90%. We can conclude that the delivery of mIL-10 plasmid DNA with PAGA complex using intravenous injections reduces the severity of insulitis in NOD mice. This study presents that intravenous injections of mIL-10 plasmid DNA/PAGA complex have potential for the application to the prevention of autoimmune diabetes mellitus.
COLLAGEN BASED BIOMATERIALS
R. ZEEMANa, L.D.D. OLDE DAMINKa, M. HENDRIKSb, P.T. CAHALANb, P.B.VAN WACHEMc, L.A.BROUWERc, M.J.A.VAN LUYNc, M.J.B. WISSINKa, G.H.M. ENGBERSa, W.G.VAN AKENa, A.A. POOTa, P.J.DIJKSTRAa, J. FEIJENa.
aDepartment of Chemical Technology and Institute for Biomedical Technology, P.O.Box 217, 7500 AE, Enschede, The Netherlands.
bMedtronic Bakken Research Center B.V., Endepolsdomein 5, 6229 GW, Maastricht, The Netherlands.
cFaculty for Medical Sciences, Cell Biology and Biomaterials, University of Groningen, Bloemsingel 10/B2, 9712 KZ, The Netherlands.
Collagen is an essential protein, which can be found in skin, connective tissue, blood vessels, bone and other parts of the body. Different types of collagen have different chemical structure and organization and are adjusted to function at a specific site of the body. Collagen and also denatured collagen (gelatine) are applied or are being developed as biomaterials or as matrices for the controlled delivery of drugs. For the application of collagen as a biomaterial, cross-linking is required to prevent early degradation and to suppress immune reactions. In this presentation the following subjects will be addressed:
1. Organization of collagen and cross-linking of collagen using glutaraldehyde, carbodiimide, epoxy-compounds and combined treatments. The cross-linked structures were characterized by chemical methods and shrinkage temperature and the mechanical properties, enzymatic degradation and the degradation in vivo after subcutaneous implantation in rats will be related to the cross-linking method. The cross-linking techniques have been successfully applied on porcine heart valves to reduce calcification.
2. Collagen fibres were used as a component of a hybrid blood vessel prosthesis, to be applied for the replacement of small diameter blood vessels. Collagen fibres were introduced in a porous matrix of a non-degradable prosthesis. After cross-linking of the collagen, heparin was covalently attached. Subsequently, growth factors for endothelial cells were physically bound to the heparinized collagen. In this application, collagen serves to close the pores of the wall, heparin to prevent initial clotting and to bind and stabilize the growth factor, and the growth factor to stimulate the outgrowth of seeded endothelial cells. The performance of the structures in terms of functional behaviour of heparin, release of growth factor and outgrowth of endothelial cells will be discussed.
TEMPERATURE RESPONSIVE INTELLIGENT POLYMERS FOR TARGETED DRUG DELIVERY
T.OKANO, J.E.CHUNG, M.YOKOYAMA
Institute of Biomedical Engineering, Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, JAPAN
We have proposed the utilization of temperature responsive properties of Poly(N-isopropylacrylamide) (PIPAAm) and its hydrogels as on-off switches for drug release and attachment/detachment of cells. PIPAAm in aqueous solution is well-known to exhibit a reversible temperature responsive phase transition at 32°C (Lower Critical Solution Temperature, LCST). PIPAAm is water-soluble and hydrophilic, showing an extended chain conformation below its LCST, and undergoes a phase transition to an insoluble and hydrophobic aggregate above the LCST.
AB-type block copolymers consisting of a PIPAAm segment and an hydrophobic segment can form core-shell micellar structures below the PIPAAm LCST. This polymeric micellar structure comprises a hydrophilic outer shell of hydrated PIPAAm segments and a hydrophobic inner core. The inner core can be loaded with hydrophobic drugs, while the PIPAAm outer shell plays the role of aqueous solubilization and temperature-responsiveness. Polymeric micelles with a hydrophilic PIPAAm outer shell and a favorable size (< 100 nm) may exhibit specific targeting of solid tumor sites by a passive targeting mechanism that inhibit non-selective scavenging by the reticuloendothelial system (RES) and can be utilized as tumor-targetable drug carriers based on the EPR (enhancement of permeability and retention) effect. Furthermore, the temperature responsiveness of these micelles can increase the targeting efficiency via a stimuli-responsive targeting process that utilizes local heating at solid tumor sites. Selective accumulation of micelles at malignant tissue sites could be increased by micellar adsorption to cells mediated by hydrophobic interactions between polymeric micelles and cells. Simultaneously, this strategy can also achieve temporal drug delivery control: drug is released and expresses its bioactivity only for a time period defined by local heating and cooling. ON/OFF control of drug delivery associated with temperature responsive micellar structural changes was regulated by physical and chemical control of the inner core, such as design of polymer flexibility, hydrophobicity and degradability.
Reversible and sensitive temperature responsive drug delivery from the polymeric hydrogels and micelles comprises a novel multifucntional drug delivery system achieving both spatial targeting and temporal dosing control in conjunction with external controlled system of local heating.
Engineering Molecular and Cellular Recognition in Artificial Proteins
David A. Tirrell
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 USA
Even strictly linear polymer chains are highly heterogeneous in terms of length, sequence and stereochemistry. A conventional polymer sample constitutes a ”combinatorial library” of enormous diversity, many times larger than the largest libraries now being explored by our colleagues in biological and medicinal chemistry. Although we cannot yet sort through this complexity in a productive fashion, it is a fact of life in polymer chemistry and physics, and in some very practical ways provides advantages in terms of materials performance.
Nature approaches macromolecular synthesis differently. The synthesis of proteins and nucleic acids is tightly controlled by the molecular genetic apparatus of the cell, and provides striking examples of the extent to which macromolecular structure and function can be regulated through the use of molecular templates to dictate chain length and sequence.
In recent years, our work has attempted to bridge the gap between natural and synthetic polymers, by combining the precision of biological polymerization with the versatility of synthetic chemistry. This lecture will address the use of protein engineering methods to prepare artificial macromolecules with predetermined molecular and cellular recognition behavior.
SYNTHETIC VECTORS FOR INTRACYTOPLASMIC DELIVERY
R DUNCAN1, S RICHARDSON1, N PATTRICK1, P FERRUTI2
1Centre for Polymer Therapeutics,
The School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX,
UK. and
2Dipartimento di Chimica Organica e Industriale,
Universita’ degli Studi di Milano, Via Venezian 21, 20133,
Milano, Italy.
Greater understanding of the genetic basis of diseases brings novel opportunities for therapy in the form of target-directed drug design and high through-put screening. However many of the emerging therapeutics have limited ability to pass across the plasma and intracellular membranes and gain access to the desired intracellular compartment where the target resides. At the cellular level, limited endosomal escape of macromolecular therapeutics is recognised as a major barrier limiting effective delivery and therapeutic efficacy. Although many polymeric systems have been under study as components of non-viral intracytoplasmic delivery systems, poor biocompatibility, tendency to localise in the liver and lung after i.v. administration and inability to promote efficient endosomal escape have limited their development from laboratory to clinical application.
Our research has taken a systematic approach to identify polymers that may have all the necessary attributes to make them useful in vivo. The aim of this study being the design of an endosomolytic vector that can be safely targeted to specific diseases (e.g. cancer) after i.v. administration to the patient. Of all the polymers we have studied (poly-L-lysine, polyethyleinimine, PAMAM dendrimers, chitosans and linear polyamidoamines (PAAs)), the PAAs are the most interesting with greatest potential to progress further. Whereas most polycations are relatively toxic to cells (IC50 10-1000 m g/ml), PAAs have been identified which are relatively non-toxic (IC50 >1mg/ml), which do not rapidly localise in liver and lung after i.v. administration and moreover target tumours by the EPR effect. In vitro PAAs display pH-dependant membrane lysis and they can promote both toxin and gene delivery in vitro. Using i.v. administration of a hepatotropic PAA, we have observed that endocytic capture is followed by reversible changes in intracellular vesicle density suggesting that this PAA can modulate early endosome/lysosome fusion events in vivo. The alteration in the pattern of intracellular enzyme distribution is PAA dose-dependant and is consistent with the ability of this PAA to cause pH-dependant membrane rupture in vivo. The biological properties of the polymeric vectors so far studied will be compared and discussed.
Acknowledgements: We would like to thank MRC and BBSRC and the Italian Ministry of University and Technological Development (MURST) for supporting this work.
DEGRADABLE AND RESORBABLE HYDROGELS FOR LOCAL DELIVERY AND MODULATION OF BIOLOGIAL RESPONSES
J. A. HUBBELL, Institute for Biomedical Engineering, ETH Zurich and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland (hubbell@biomed.mat.ethz.ch)
Biomimetic approaches are being increasingly employed in the design for materials for tissue engineering. There exist numerous motivations and applications in the engineering of tissue healing in which one would seek to manipulate cell adhesion in both two and three dimensions, such as to reduce adhesion, to target the adhesion of particular cell types and to induce specific cellular responses that may be related to adhesion. Of particular interest in biomimetic materials is to replace the key features of the extracellular matrix, e.g. in the development of materials for cell in-growth matrices in tissue regeneration and wound healing: the presentation of adhesion signals, the display of growth factor binding sites, and the ability to be degraded by enzymes that are used by cells as they migrate. Two approaches toward this end will be discussed.
(1) Fibrin represents an interesting platform material as a cell in-growth matrix, especially since it can be readily remodeled by cell-derived proteases. A deficiency of fibrin is that it does not possess many of the adhesion proteins that are used as developmental cues in morphogeneisis and that it does not display high affinity binding sites for many interesting morphogenetic growth factors. To address this, an enzymatic modification, using synthetic substrates for the transglutaminase factor XIIIa, has been developed by which to incorporate adhesion factors, heparin binding sites for growth factors, or growth factors directly, to thus incorporate within fibrin these important developmental cues.
(2) Synthetic mimics of fibrin have been developed, consisting of water-soluble polymer chains cross-linked by plasmin-sensitive (or collagenase-sensitive, in a different protein context) reactive peptides, by reaction with thiols on the peptide with conjugated unsaturations on the polymer. Adhesion peptides, as well as growth factor binding peptides, have been incorporated as dangling chains in this network. Such materials, although totally synthetic, can be degraded by cell-derived proteases and remodeled naturally in vivo.