Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
  • G01N33/555—Red blood cell
  • G01N33/556—Fixed or stabilised red blood cell

Definitions

• the present invention relates to novel conductive electroactive biomatenals, and processes for making such biomaterials.
• biomaterials In the search for a new generation of biomaterials, it has been sought to mimic the cell membrane in designing new polymeric materials that not only have similar chemistry to the cell membrane but also are reactive. To date, the majority of biomaterials have been relatively inert materials whose properties are determined and fixed at the time of their synthesis. Furthermore, man-made biomaterials or at least their bio-interfaces tend to be homogeneous in composition. This contrasts with the structure of most interfaces within the body where the emphasis is not only on a heterogeneous blend of different molecules but the whole structure is dynamically active.
• This dynamic activity is maintained at the level of the cell membrane that has a resting potential that can be altered and that has a broadly hydrophobic lipid component in which a mosaic of protein and carbohydrate structures are dispersed.
• the composition can be changed in time and some components, mucopolysaccharides and proteins, can be secreted from it in response to specific stimuli and as such current biomaterials are far from suitable.
• Conductive electroactive polymers such as polypyrrole, which have been previously described have a range of properties that would appear to be useful in the design of new biomaterials. They are dynamically active, have a resting potential and can be synthesised to contain significant amounts of protein which remains bioactive.
• biological macromolecule refers in the context of the present invention to any molecule of biological origin which has a high molecular weight and may be, but not necessarily, of polymeric construction. Examples of such macromolecules are proteins, lipids, carbohydrates, nucleic acids and the like.
• macromolecules are proteins, lipids, carbohydrates, nucleic acids and the like.
• macromolecule complex refers to larger macromolecular structures of increased complexity and includes viruses, bacteria, fungi and plant and animal cells.
• hydrophilic counterion refers in the context of the present invention to a counterion which when incorporated into the composite polymer of the invention causes the composite polymer to have a high water content.
• polyelectrolyte is meant a counterion which is a multi-charged species.
• cytopolymer refers to a composite polymer which incorporates biological materials such as viruses, bacteria, fungi or plant and animal cells. Throughout this specification by “macroscopically uneven” is meant that the distribution of polymer was such that there were bare patches of gold not covered by polymer. By “microscopically uneven” is meant that red blood cells (RBC) were distributed so that there were microscopic fields at 40x magnification that did not contain red blood cells.
• the invention consists in a conductive electroactive composite polymer comprising a hydrophilic counterion in combination with a biological macromolecule and/or a biological macromolecular complex.
• the invention consists in a process for making a conductive electroactive composite polymer having a hydrophilic counterion, comprising the step of oxidising a monomer in the presence of a hydrophilic counterion and a biological macromolecule and/or a biological macromolecular complex.
• the monomer of choice for the preparation of the composite polymers of the invention is pyrrole.
• other monomers or mixtures of monomers may be used, provided that they are compatible with the biological macromolecule or the biological macromolecular complex.
• aniline could also be a suitable monomer when the process used for its polymerisation satisfies the compatibility criterion.
• thiophene may also be used in the preparation of the polymer if a suitably water-soluble derivative of thiophene is used.
• the polymerisation process is carried out by electro-oxidation but suitable biochemical or chemical means of oxidation or reduction could also be used.
• the hydrophilic counterion is a polyelectrolyte selected from the group consisting of polyvinyl sulphonate (PVS), dextran sulphate, chondroitin sulphate, polyglutamic acid, polyacrylic acid, heparin sulphate, hyaluronic acid and mucopolysaccharides.
• PVS polyvinyl sulphonate
• dextran sulphate dextran sulphate
• chondroitin sulphate polyglutamic acid
• polyacrylic acid heparin sulphate
• hyaluronic acid mucopolysaccharides.
• the concentration of the counterion is about 250 to 500 mg/100 mL.
• any suitable counterion can be used in the present invention.
• the incorporated biological macromolecule is a protein, a glycoprotein or a lipoprotein, such as an enzyme, a hormone, a growth factor, a cytokine or an integral cell membrane component such as a receptor or a specific cell surface.
• the biological macromolecule is releasable from the composite polymer.
• the invention consists in a composite conductive, electroactive polymer in communication with a virus or a bioactive cell which can be of bacterial, fungal, plant or animal origin.
• a virus or a bioactive cell which can be of bacterial, fungal, plant or animal origin.
• This type of composite polymer will be referred to as a "cytopolymer”.
• the polymer of the invention comprises a polymeric composite of polypyrrole:PVS in communication with a red blood cell.
• the invention consists in a method of delivering a biological macromolecule contained within a conductive electroactive composite polymer into an environment surrounding the polymer, said polymer comprising a hydrophilic counterion in combination with a biological macromolecule and/or a biological macromolecular complex, said method comprising the step of: applying a stimulus to said composite polymer to release said macromolecule into said surrounding environment from said composite polymer.
• the invention consists in a method of detecting a ligand comprising the steps of: a) introducing a conductive electroactive composite polymer comprising a hydrophilic counterion in combination with a biological macromolecule and/or a biological macromolecular complex into an environment containing a ligand which specifically binds to said macromolecule and/or macromolecular complex contained within the composite polymer; b) allowing the ligand to bind to said macromolecule and/or macromolecular complex; c) measuring the change in electrical properties of said composite polymer and thereby determining the presence and/or concentration of the ligand.
• the invention consists in a method of preventing or treating a disorder caused by or associated with a deficiency or absence of a biological macromolecule and/or a biological macromolecular complex comprising the step of administering to a host requiring such treatment a conductive electroactive polymer described above.
• the polymer made in accordance with the present invention has been found to have interesting hydrogel like properties of high water content and biocompatibility as well as the capacity for controlled release of bioactive protein and maintenance of cell integrity and viability.
• the biocompatible polymer is also electroconductive and electroactive. The electroactivity provides a mechanism for the control of several key properties for example the release of molecules such as drugs, proteins and other macromolecules and, if whole cells are incorporated, enables controllable modification of the cell membrane and cell activity.
• biocompatible polymers of the present invention over hydrogels and the facile control of their properties by application of small electrical potentials make them interesting candidates for the design and synthesis of a new generation of smarter biomaterials. Furthermore, the release of proteins from polymers is of interest for applications as both biomaterials and controlled drug delivery devices.
• biocompatible polymers enable the incorporation of very complex macromolecular structures such as viruses and even whole cells of bacterial, plant or animal origin.
• the viable cells can be incorporated directly during the polymerisation process with minimal lysis of the cells, thus producing a composite polymer comprising intact cells which remain biologically active.
• Such cytopolymers may be useful eg. as cell carriers or as diagnostic tools for the detection of antibodies or analytes via ligand-cell binding or interaction.
• the analyte- cytopolymer interaction may also be translated into a range of electrical signals since the composite polymer is electroactive and conductive, thus rendering the cytopolymers potentially useful as novel biosensors.
• In vitro study or characterisation of cell-ligand interaction may also be conducted using such structures, as well as modulation of cell function and/or structure.
• the cytopolymer may be used as a biosensor for determining eg. blood type or group as described below.
• the sensor may contain an appropriate type of cell.
• Figure 1 represents a view of the electrochemical cell used for polymer growth.
• Figure 7 represents a EQCM trace of polypyrrole:chondroitin sulphate during dehydration and rehydration.
• Figure 9 represents a cell culture of PC 12 cells on polypyrrole: chondroitin sulphate in the presence of soluble nerve growth factor (NGF).
• NGF nerve growth factor
• Figure 10 represents a high resolution light micrograph of red blood cells incorporated into a polymer.
• Figure 11 represents a scanning electron micrograph of a polymer incorporating red blood cells.
• Figure 12 represents microscopic appearance of red blood cells in (A) monomer mix containing 0.2M pyrrole and lg/L PVS in isotonic sucrose, and (B) isotonic sucrose solution alone.
• the polymers were cycled in 0.15M NaNO 3 , in the absence (A and C) or presence (B and D) of 40 ⁇ l of anti-Rh(D) monoclonal antibody.
• Scan rate 50mV/sec.
• a positively charged homopolymeric backbone which is counterbalanced by polyanionic complex carbohydrates such as dextran sulphate or chondroitin sulphate can be synthesised according to: Oxidise
• A" is a hydrophilic polyelectrolyte counterion incorporated during polymerisation.
• the presence of the counterion provides a facile route for the incorporation of chemical and biochemical functionalities eg. biologically active proteins, enzymes, cells.
• Composite supramolecular assemblages were synthesised by electrooxidation of pyrrole monomer in the presence of proteins and polysaccharides in their anionic forms. Intrusion of other anions was limited by extensive dialysis of the macroanions against water (purified to 18 mega ⁇ ) or use of salt free reagents, for example isotonic sucrose in the case of cells. To facilitate future cell culturing experiments composites were synthesised on transparent foils of mylar coated with gold (Cortaulds). Composites were grown by applying a constant electrical current (galvanostatic growth) at a density of 0.5m-A/cm for 0.5 to 4 min.
• the electrochemical cell was designed to provide a uniform parallel electrical field over the entire electrode and to eliminate or minimise edge effects.
• the electrochemical cell comprises a cell body 1 which contains the first electrode 2 made of reticulated vitreous carbon, the second electrode 3 coated with gold foil and a third electrode 4 which is a reference electrode (Ag/AgCl) comprising a salt bridge 5 (0.15M NaCl).
• the reference electrode is included to measure the potential.
• the solution in the electrochemical cell contained pyrrole distilled and purified over alumina by standard techniques immediately before use.
• pyrrole as supplied from Merck was distilled in standard equipment and the fraction distilling between 130-131°C was collected and stored in the dark under nitrogen in a deep freeze (approximately -10°C).
• a deep freeze approximately -10°C
• the concentration of pyrrole may be varied, preferrably between 0.1M and 0.3M. The optimal concentration could be determined by those skilled in the art with the aid of the present disclosure.
• polyvinyl sulphonate PVS, M r 900-1,000 Aldrich
• Dextran sulphate M r 50,000 Sigma or 1,000,000 Fluka
• chondroitin sulphate Sigma
• Heparin sulphate Sigma, Grade 1-A from porcine intestinal mucosa
• the prepared polymer films may be stored for periods of up to 9 to 12 months at room temperature, without deleterious effects on the properties of the polymer.
• Cyclic voltammetry, cyclic resistometry and electrochemical quartz crystal microbalance (EQCM) studies were performed as previously described (see John R, Wallace, G G, J. Electroanal. Chem. 1993, 354, 154-160; John R, Talaie A, Fletcher S and Wallace G G, J. Electroanal. Chem. 1991, 319, 365-371, and Mirmocheni A, Price W E and Wallace G G, J Membrane Science, 1995, 100, 239). Cyclic voltammetry was performed using a BAS CV27 potentiostat, a three-electrode cell in an electrolyte of 0.15M or 1.0M NaCl. Scan rates of 25 - 50 mV/sec were employed.
• EQCM crystals were cleaned in 0.4M H 2 SO 4 for 10 min. and then in 20% potassium ferrocyanide for 10 min. Polymers were grown galvanostatically as described above then washed in 0.15M NaCl or 0.15M NaNO 3 . For resistometry gold electrodes of 1 cm were used. Synthesis and cyclic voltammetry were performed as described in an electrolyte of 0.15M or 1.0M NaCl. Water content was estimated by gravimetric analysis after either drying over phosphorus pentoxide or on a vacuum pump overnight. Dynamic changes in water content were determined by using a quartz crystal microbalance.
• HRP horseradish peroxidase
• Rat PC 12 phaeochromocytoma cells were cultured in Dulbecco's Modification of Eagles Medium (DMEM) supplemented with foetal bovine serum (10% v/v) and with heat inactivated horse serum (5% v/v). No antibiotics were used and the cultures were maintained at 36.5°C in an atmosphere of CO 2 (5% v/v).
• DMEM Dulbecco's Modification of Eagles Medium
• foetal bovine serum % v/v
• horse serum 5% v/v
• No antibiotics were used and the cultures were maintained at 36.5°C in an atmosphere of CO 2 (5% v/v).
• the polymer films tested were cut to a size that fitted into the bottom of rectangular multi-well culture dishes (Nunc cat. no. 176600). Cells were seeded at 10 4 cells/cm 2 and left for 2 to 4 days before the polymer was cycled through potentials -0.8V to +0.7V (vs Ag/AgCl) using Electro
• Nerve growth factor was added to the culture medium at a concentration of
• Cyclic resistometry showed that for all polymer systems the polymer resistance increased on reduction but the polymers remained relatively conductive when reduced (see Figure 6) compared to polypyrrole chloride that shows a resistance change of 100- 200 ⁇ when reduced. The polymers were also less conductive than polypyrrole chloride when in the fully oxidised state (60-70 ⁇ vs 40-50 ⁇ ).
• Enzyme assay showed that appreciable HRP activity could be incorporated into the polymer during synthesis of PVS:HRP:pyrrole composite. Longer synthesis times showed lower activity measured in the polymer.
• haemoglobin Mr 64,500, pi 6.8, a neutral protein
• Table 3 shows that as the pyrrole concentration is increased from 0.1M to 0.5M the extent of incorporation of protein is reduced. Furthermore, the amount of protein released from the polymer is significantly reduced (Table 4). This effect is probably caused by the denaturing effect of the hydrophobic solvent pyrrole.
• the HSA was retained within the polymer and did not diffuse from it when incubated in saline solution, 0.15M NaCl; after 7 hours 98 ( ⁇ 3)% remained and even after 3 days 95 ( ⁇ 10)% remained.
• the polymer was reduced by applying a cyclic voltage ramp from +0.5 V to -0.7, -0.8 or -0.9 V, appreciable protein was released into the saline electrolyte solution. The amount of protein released was greater for more negative potentials.
• the time course of release was investigated by applying a fixed negative potential for times ranging from 2 to 32 seconds. The protein was released rapidly from the polymer. Within 2 seconds 20 to 30 percent of the protein contained within the polymer had been released. After only 32 seconds most of the contents of the polymer had been released. When oxidising potentials (+0.5 V) were applied or potentials close to the rest potential of polypyrrole (+0.17 V) no protein was released.
• an anionic polyelectrolyte such as dextran sulphate
• release was induced by application of negative potentials (see Figure 8B). Applying -0.7V produced some release of NGF. However, release evoked at pH 7.0 was detected only 64 seconds after the application of potential. When more negative potentials were applied (-0.9V) greater and more rapid release was detected within 2 seconds of application of potential. The amount of protein released increases with the time of application of potential so that up to 45% of the NGF contained in the polymer was released within 1 minute. Soaking a polymer containing NGF in 0.15M NaCl at 4°C for 3 - 4 weeks caused no more growth factor to diffuse out of the polymer.
• the mode of release of NGF is such that no protein leaks out of the polymer by diffusion but within 2 seconds of application of the electrical potential significant protein is released.
• Such control of protein release from a polymer has not been achieved before.
• the amounts of NGF released are sufficient to cause differentiation of PC 12 cells.
• the PC 12 cells will begin to differentiate when NGF concentration in culture medium reaches about 16ng/mL.
• the maximum NGF released in the above study was 450ng in 1 minute. Release of this amount of NGF into a 4 mL culture volume would be more than sufficient to cause stimulation of PC 12 cells growing upon the polymer.
• Factors that affected the extent of protein incorporation were the time of synthesis and the concentration of pyrrole in the monomer solution.
• the mechanism of release of the protein is thought to be expulsion of anions from the polymer when it is reduced.
• the open, porous, hydrophilic matrix is responsible for the facile expulsion of the negatively charged protein.
• Simple anions like chloride diffuse from the polymer by ion exchange. There was no such tendency for albumin to diffuse despite the high water content. Therefore, there must be other forces retaining the protein within the polymer matrix. This could include hydrophobic effects as well as ion pairing, hydrogen bonding and steric restriction.
• Glutamic acid is relatively hydrophobic when protonated. Thus it is thought that those groups strongly ion paired with positive charges on the backbone would contribute a hydrophobic nature to the composite compared to the hydrophilic carbohydrate units.
• Human red blood cells collected at the Red Cross Blood Bank from anonymous donors were fractionated from whole blood and were stored in Citrate, Adenine, Phosphate, Dextrose (CAPD) solution. Before use they were washed and suspended in isotonic media such as Adenine, Citrate, Dextrose (ACD), CAPD, 0.9% w/w saline or 92.4 g/L sucrose. A monomer solution was then made from the packed cells.
• Citrate, Adenine, Phosphate, Dextrose (CAPD) solution Before use they were washed and suspended in isotonic media such as Adenine, Citrate, Dextrose (ACD), CAPD, 0.9% w/w saline or 92.4 g/L sucrose.
• a monomer solution was then made from the packed cells.
• a typical monomer solution contained pyrrole at concentrations from 0.1 to 0.3M, an osmotic agent and or an energy source, such as sucrose at a concentration of 92.4 g/L, a polyelectrolyte selected from polyvinyl sulphonate (PVS), dextran sulphate, chondroitin sulphate, polyglutamic acid, polyacrylic acid, heparin sulphate, hyaluronic acid or mucopolysaccharides, for example polyvinyl sulphonate at a concentration of 1 g/L and red blood cells at 3 to 6 x 10 12 /L.
• PVS polyvinyl sulphonate
• dextran sulphate dextran sulphate
• chondroitin sulphate polyglutamic acid
• polyacrylic acid polyacrylic acid
• heparin sulphate hyaluronic acid
• mucopolysaccharides for example polyvinyl s
• Integrity of red cells in the monomer mix before and after synthesis of polymer was performed by determination of a full blood count on a Coulter S-Plus IV, electronic cell counter.
• the state of the haemoglobin in the monomer mix was determined by analysis on a Radiometer blood gas analyser.
• the integrity of AB and the D-antigen on the cells was determined by performing a ABO and Rhesus blood group typing using standard reagents. Morphology of red blood cells in the monomer mix was determined by
• Polymers were grown on plastic foil coated with gold in a standard electrochemical cell (see Figure 1) using galvanostatic growth at 0.5-1.5 mA/cm 2 for 0.5 to 1.5 minutes, temperature was maintained at less than 17°C. Higher or lower temperatures can be used but, as is well known, synthesis of any type of polypyrrole polymer is temperature sensitive; lower temperatures usually producing polymers with greater electrical-conductivity and better mechanical properties. Lower temperatures are also desirable because it enables the production of polymers that are macroscopically even and preserves the integrity of biological components.
• Red blood cells that were incorporated into the polymer appeared intact at high resolution light microscopy but not always in their usual biconcave form (Figure 10) This may be an artefact of fixation caused by the picric acid in the fixative used.
• Other fixatives can be used but picric acid present as a modified Zamboni fixative composed of 4% formaldehyde, 0.25% glutaraldehyde, 40% v/v saturated picric acid, in 0.1 M sodium phosphate buffer pH 7.4 gave superior results.
• Formaldehyde vapour fixation better preserves the biconcave form but is not suitable for large-scale use.
• the biconcave form or morphology of the red blood cell is not important in itself. It is the state of antigens/enzymes etc. in the membrane as a function of the use of the cytopolymer which is crucial. Scanning electron microscopy shows that the red blood cells are incorporated into as well as onto the polymer ( Figure 11).
• MALE Mean cell volume: 80.8 - 97.7fL
• More "even" polymers can be grown by reducing the concentration of red blood cells to l.5 to 3.0 x l0 1 /L.
• CAPD and ACD caused the polymer to be macroscopically and microscopically uneven. Cells washed in sucrose produced more even polymers.
• the conditions for and the stability of the cytopolymers on storage is to a larger degree governed by the type and viability characteristics of the cells incorporated in the polymer and to a smaller extent by the chemical composition of the polymer.
• a polypyrrole:PVS:RBC:sucrose polymer was cycled in 0.15M NaNO 3 in the presence of anti-D antibody (the antibody was monoclonal, at a concentration of Img/mL, obtained from Gamma Biologicals, USA).
• a polypyrrole:PVS polymer which does not contain red blood cells served as a blank polymer, where no interaction involving red blood cells could take place.
• thin polymers i.e. grown at 0.5 mA/cm 2 for 1 min.
• thick ones grown at 5.0 mA/cm 2 for 1 min.
• anion exchange is predominant in thin polymers whereas both cation and anion exchange processes occur on thicker polymers.
• Cyclic voltammetry showed that a good electroactive polymer film was produced. There was no interference due to the iron present in the haemoglobin.
• the cyclic voltammogram was similar to polymer grown with only PVS as a counterion.
• the net mass of the polymer increased as would be expected when antibody was bound to the polymer. This was not artificial as shown by the absence of mass increase when the antibody was added to a blank polymer that did not contain red blood cells.
• An electroactive polymer shows a characteristic sigmoidal resistance change with hysteresis as the polymer is oxidised or reduced.
• Rh control Human, Bovine serum, Gamma Biologicals
• Blood Grouping Reagent Anti-D Blood Grouping Reagent Anti-D (Gamma Biologicals), a monoclonal ⁇ polyclonal blend.
• Red blood cell containing polymers show different changes upon addition of antibody (anti-D) and antibody control (see Figure 14). However, when anti-D is added the change in resistance is inverted ie. the resistance change between 0.0V and -0.8V, it becomes negative instead of positive.
• the conducting polymer By changing the nature of the conducting polymer, eg. using substituted pyrroles or thiophenes, it is possible to change the redox properties (eg the potential at which the transition from oxidised to reduced state takes place) and also chemical properties (eg solubility of polymer).
• redox properties eg the potential at which the transition from oxidised to reduced state takes place
• chemical properties eg solubility of polymer
• pharmacological or physiological responses could be elicited. Illustrated here are four test cases (1) horseradish peroxidase, (2) haemoglobin, (3) human serum albumin and (4) nerve growth factor. Release of the latter protein causes profound changes in the behaviour of cells growing on the composite polymer. It causes them to stop dividing and to differentiate into cells containing long axonal processes. This test case illustrates the feasibility of release of any protein growth factor in a controlled fashion to cause cellular changes. It is envisaged that similar materials could be constructed containing for example endothelial cell growth factor that would induce arterial grafts to become lined with a full covering of endothelial cells in vivo.
• cytopolymers wherein the cell remains bioactive may be useful as biosensors or diagnostic tools.
• Such tools may be useful in, for example, various biological reactions such as the reaction of an antigen incorporated in the polymer with an antibody specific for that antigen.
• Such structures have been found to have a high water content and to be hygroscopic.
• This environment is suited for cell integration and allows access of proteins (eg. analytes or antibodies) to the cell surface or receptors in subsequent sensor uses.
• proteins eg. analytes or antibodies
• Red blood cells are probably the most fragile of cells and thus provide a suitably rigorous test system.
• Other cells such as white blood cells (granulocytes, monocytes/macrophages, lymphocytes), for detection of anti-HIV or gp 140 , or for analysis of MHC antigens, stable cell lines or primary cultures of liver, kidney or other tissue cells, for assessment of cell surface markers, receptors and their respective tumour cell lines, whether primaiy or stable cell lines, may be used.
• Which cell is inco ⁇ orated depends upon the ultimate use required of the cytopolymer.
• Sucrose, dextrose, mannitol, trehalose and other agents known for their ability to aid in sustaining cell viability and integrity through providing an appropriate energy source or isotonicity may also be inco ⁇ orated during the synthetic process.

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