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WO1999012975A1 - Entites bioactive couplees a des polymeres intelligents et utilisations de ces entites - Google Patents

Entites bioactive couplees a des polymeres intelligents et utilisations de ces entites Download PDF

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Publication number
WO1999012975A1
WO1999012975A1 PCT/US1998/018633 US9818633W WO9912975A1 WO 1999012975 A1 WO1999012975 A1 WO 1999012975A1 US 9818633 W US9818633 W US 9818633W WO 9912975 A1 WO9912975 A1 WO 9912975A1
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Prior art keywords
polymer
whiskers
solvent
composite article
solution
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English (en)
Inventor
David S. Soane
Michael R. Houston
Stephen E. Barry
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FLEXIMER LLC
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FLEXIMER LLC
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Priority to AU92246/98A priority Critical patent/AU9224698A/en
Publication of WO1999012975A1 publication Critical patent/WO1999012975A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0093—Purging against cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • 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

Definitions

  • This invention relates to hybrid composite articles comprising expandable and contractible polymer whiskers attached to bioactive entities and to their use in various medical, biological, pharmaceutical and industrial applications.
  • Catalysts are substances that increase the rate of a chemical reaction.
  • enzyme catalysts are used in many types of reactions, including redox, substitution, addition, elimination, cyclization, molecular rearrangements, and polymerization reactions.
  • catalyst usage problems include dispersion, control, collection and recovery of the catalysts from the reaction system, providing "dormant" catalysts for long-term storage of products, halting runaway exothermic reactions and catalyst poisoning and enzyme inhibition.
  • Cancer continues to be a major health care problem today. While great strides have been made in the treatment for and cure of cancer, the therapies now available remain less than ideal, commonly giving rise to harmful side effects and often failing to cure the disease.
  • the most widely used treatment modality is surgery. Surgical excision of a tumor is both quick and effective.
  • removal of a tumor does not guarantee that small extensions of the cancer, invisible to the surgeon, will be removed as well; in trying to compensate for this, large areas of normal tissue are often excised with the tumor, which may severely damage the appearance of the patient or the patient's ability to function.
  • surgery is impossible. Surgery cannot treat a cancer that has spread, or metastasized, throughout the body. And the surgical procedure itself, with the major risks attendant thereto, is traumatic to the patient.
  • Radiation therapy is also widely used to treat cancer.
  • radiation cannot treat widespread metastases, nor will it eradicate any cancer cells adjacent to the tumor but outside the x-ray or gamma ray beams. Additionally, radiation does not discriminate between healthy and tumorous cells and will damage and kill both indiscriminately.
  • Cancer-specific antigens have been discovered and identified, largely with the aid of monoclonal antibodies which selectively seek out cancerous cells and bind to their surfaces. By conjugating a toxic substance or other immunological marker to the monoclonal antibody, it was hoped that cancer cells could be selectively targeted and tagged, leading to destruction of the cancer cells by the body's immune system.
  • attempts to trigger an immunological response to kill the labeled cancer cells have met with only limited success, partly due to the immunological response to the monoclonal antibodies themselves which often destroys the monoclonal antibody-conjugate before it reaches the cancerous cells to be eradicated.
  • Stem cells are immature cells that later differentiate into various cells constituting the human blood. They are found in bone marrow and, to a lesser concentration, in peripheral blood. In the treatment of cancer using chemotherapy, the therapy often causes severe damage to bone marrow and the subsequent inability of the cancer patient to produce stem cells. Cancer patients undergoing chemotherapy can elect either to have some of their bone marrow removed and stored, or to have their stem cells harvested, cleansed, and stored, for re-introduction into the body after the toxic effects of chemotherapy lapse.
  • Bone marrow retrieval is a costly and painful operation, while peripheral blood collection is relatively straightforward.
  • stem cells are only present in small concentrations in peripheral blood. This necessitates extensive separation processing steps to obtain a sufficient number of stem cells for blood reconstitution after chemotherapy treatment.
  • the first is flow cytometry. This technique entails the cell-specific labeling of stem cells with a fluorescent conjugate. Stem cells are then sorted one-by-one as the entire collection of blood cells pass serially through a detection beam. As a result, the process takes a great deal of time and is not cost effective.
  • the second separation technique currently available uses high gradient magnetic fields to selectively sort stem cells in a process known as magnetic cell sorting (MACS).
  • MCS magnetic cell sorting
  • the collected blood is first centrifuged by a process known as leukapheresis, whereby white cells and stem cells sediment by virtue of their higher density, leaving red cells dispersed in the plasma.
  • the precipitate is then separated from the supernatant.
  • the precipitate is re-suspended for followed- on treatment.
  • the key of the process is the use of stem cell-specific antibody linked to tiny magnetic particles.
  • the antibody-laden magnetic beads are mixed with the re- suspended blood sediment. Stem cells are attracted to the magnetic beads, due to the antigen-antibody pairing.
  • the specific geometry of the cell-versus-bead combination dictates the number of beads thus bound to the stem cells.
  • a strong magnetic field is then applied to retain the beads, thus the bead-stem cell conjugates.
  • biotinylated- antibody can be first adsorbed onto the cancer cells.
  • the collected stem cells (with the cancer cells) can be eluted from a packed column, where the packing has avidin- treated surfaces.
  • the strong biotin-avidin pairing tendency pulls the cancer cells from the effluent, producing the desired product of cancer-free stem cells ready for storage and re-introduction after chemotherapy.
  • This second process involves the use of very expensive reagents.
  • the fabrication of colloidal magnetic particles, the surface treatment, and the tagging of antibody on the particles are labor-intensive.
  • the former may be a collection of peptides or oligonucleotides.
  • the latter generally have compounds derived from a pharmacophore (a kernel) upon which many variations of side-group substituents are attached.
  • the synthesis may be a solid-phase synthesis, examples of which are synthesis done on the surface of a chip, where each addressable pixel holds one compound, or on the surface of particles, where each particle is additionally tagged for later identification of the particular test compound in subsequent screening studies. Alternatively, the synthesis may be done in solution. Both solid-phase and solution-phase combinatorial syntheses have advantages and disadvantages.
  • Solid- phase synthesis makes it easier to conduct multi-step reactions and to drive reactions to completion because excess reagents can be added and then easily washed away after each reaction step. But with solid-phase procedures, the reactive sites are close together and the growing polymer compound may interfere with the synthesis process, for example blocking access of the reagents to the active site due to steric hindrances or the like. With solution-phase synthesis, a much wider range of reactions is available, with more effective contact between the reactants, and products can be more easily identified and characterized. However, the character and makeup of the large mixture of compounds thus synthesized cannot be easily controlled or directed, and it is often difficult to remove excess reactants and byproducts prior to the next reaction step. It would be desirable to find a process that combines the positive aspects of each of the solid-phase and the solution-phase methods, while avoiding the disadvantages.
  • the present invention substantially reduces or overcomes all of the above problems with the prior art.
  • This invention is directed to novel polymer hybrid composite articles having reversible activity and self-dispersion stabilities.
  • These polymer hybrid composite articles comprise reversibly expandable and contractible linear or branched polymer "whiskers” attached to bioactive entities, such as proteins that function as enzymes, antibodies, and soluble/bound receptors.
  • bioactive entities such as proteins that function as enzymes, antibodies, and soluble/bound receptors.
  • the smart polymer hybrid composite articles of this invention provide novel identification, recovery and/or purification methods.
  • specific uses include, but are not limited to: i) the use of smart polymer-coupled antibodies to extract and recover stem cells from peripheral blood, with the subsequent elimination of cancerous cells from the extract; ii) the use of smart polymer hybrid composite articles as platforms to synthesize directed combinatorial libraries of compounds such as polymers, oligonucleotides, small molecules and the like; iii) a technique that rapidly isolates specific compounds from a complex mixture of pharmaceutical drug candidates, which may be synthesized as a large library in a combinatorial chemistry-guided drug discovery effort; iv) the use of smart polymer- coupled enzymes in various laboratory and industrial enzyme-catalyzed reactions; and v) a treatment for selectively inactivating or killing cancer cells or other deleterious cells.
  • Smart polymers as defined and used herein are those polymers that can be induced to undergo a distinct thermodynamic transition by the adjustment of any of a number of environmental parameters (e.g., pH, temperature, ionic strength, co- solvent composition, pressure, electric field, etc.) without denaturing the modified bioactive entities to which the polymers are attached, and, if desired, without affecting the biological function of the bioactive entities.
  • the polymer whiskers allow dispersion of the bioactive entities in a solvent.
  • the polymer whiskers also allow control of the activity of or recovery of the bioactive entities.
  • the recovered bioactive entities may be conjugated or otherwise attached to targeted cells, such as stem cells, cancer cells, or fetal cells (to be used for prenatal genetic screening) by means of, for example, antibody-antigen or target- receptor interactions, resulting in the separation and recovery of these target cells from the solvent as well.
  • the system-based approach entails both novel polymer-linked bioactive entity hybrid composite articles and affiliated processes.
  • This invention thus embodies both hybrid composite articles and process innovation.
  • the polymer hybrid composite articles are suspended in solution.
  • the polymer whiskers undergo expansion and contraction in response to minor variations in one of several externally controlled thermodynamic parameters such as temperature, pH, light, pressure, electric field strength, ionic strength, and solvent composition.
  • the polymer hybrid composite articles When the polymer whiskers are in an expanded state, the polymer hybrid composite articles are dispersed in the solvent.
  • solvent as used herein is interchangeable with the terms “solution”, “media”, “reactant media”, “biological fluid”, and “biological media”.
  • the critical environmental stimulus of the solvent containing the polymer hybrid composite article such as temperature
  • the expanded polymer whiskers of the composite article contract.
  • the polymer whiskers coalesce in the solvent, activity is temporarily halted and the composite articles are easily removed from the solution.
  • the polymer hybrid composite article can be formulated to undergo transitions that switch the composite article between a dispersed state and a contracted or flocculated state in the solution.
  • the polymer hybrid composite articles having reversible activity and dispersion stabilities comprise a bioactive entity, and at least one polymer whisker attached to the bioactive entity, said one or more polymer whiskers being controllably expandable and contractible.
  • the polymer whiskers and the solvent interact to cause the polymer whiskers to reversibly expand and contract in response to one or more changes in the environmental conditions, such that at least some of the polymer whiskers are solvated when the polymer whiskers expand, and the absorbed solvent is expelled from the polymer whiskers when the polymer whiskers contract.
  • each composite article is dispersed in the solvent away from each of the other composite articles, allowing each bioactive entity to perform its function, whereas when the polymer whiskers are contracted, the plurality of composite articles are reversibly coalesced and the function of the bioactive entity is halted.
  • the dispersion of the plurality of the composite articles changes, depending on whether the polymer whiskers are in an expanded or contracted state.
  • the polymer whiskers are either UCST (Upper Critical Solution Temperature) or LCST (Lower Critical Solution Temperature) polymers.
  • the bioactive entity may be chosen from any entity exhibiting biological activity either alone or in combination or interaction with another entity.
  • the bioactive entity may be a cell; a protein, such as enzymes, antibodies and receptors; nucleic acid; a small molecule functional group; and the like, by way of example, in a preferred embodiment, the bioactive entities are cells and proteins, more preferably proteins. It is an advantage of the present invention that biological or bioactive entities in a solvent can be stably dispersed, recovered or collected.
  • a further advantage of the polymer hybrid composite articles is that biological environments or biological entities can be purified.
  • a still further advantage of the polymer hybrid composite articles of the invention is that catalytic compounds can be reversibly switched on and off.
  • Yet another advantage of the polymer hybrid composite articles is that deleterious biological entities can be inactivated or killed.
  • a further advantage of the polymer hybrid composite articles is the increased shelf life of reaction systems to retain their activity during periods of nonuse.
  • FIG. 1 schematically illustrates the transition between expanded and retracted or contracted polymer whiskers.
  • the polymer coil is shown in its expanded form on the left and in its contracted form on the right.
  • the transition is reversible, depending on a change in thermodynamic parameters.
  • FIG. 2 is an illustration of one type of thermodynamic phase diagram showing the transition temperature versus the polymer-solvent concentration of the polymer whiskers.
  • T means Temperature
  • LCST means Lower Critical Solution Temperature
  • UST Upper Critical Solution Temperature.
  • FIG.s 3A and 3B schematically illustrate the transition between dispersed expanded polymer hybrid composite articles and coalesced retracted polymer hybrid composite articles.
  • FIG. 3A shows the polymer composite articles in expanded form and dispersed throughout the solution.
  • FIG. 3B shows the polymer composite articles in their retracted form and coalesced at the bottom of the container.
  • the polymer coils reversibly collapse or extend depending on the environmental conditions.
  • FIG. 4 shows how the expansion and contraction process of the polymer whiskers reversibly switches on and off catalytic activity by covering the active sites on the surface of a catalytic enzyme.
  • the polymer-enzyme composite article is shown on the left in its expanded operational state; the extended polymer whiskers stabilize colloids and the enzyme is optimized for process speed and flexibility.
  • FIG. 5 shows the use of polymer hybrid composite articles to clean up a diverse mixture of synthesized chemicals (combinatorial library), of cells or of other targeted ligands by selection and separation of only those of interest.
  • 8 is the hybrid composite article in its expanded, operational state.
  • 11 is a set or library of chemicals or a mixture of cells, each different type of molecule or cell indicated by a different shape.
  • This invention is directed to smart polymer hybrid composite articles encompassing bioactive entities having linear or branched polymer "whiskers” attached.
  • the polymer hybrid composite articles are designed for specific applications in medical, biological, pharmaceutical and industrial areas.
  • the polymer whiskers are considered to be "switchable”; that is, the polymer whiskers provide dispersion and stabilization of the polymer hybrid composite articles in a solvent by expanding into the solution. This is opposed to traditional stabilization methods of compounds in solution, which rely on electrostatic or steric principles, which are difficult to switch on and off.
  • the polymer whiskers also provide control and recovery of the polymer hybrid composite articles by contracting or "coiling,” which causes the polymer hybrid composite articles to coalesce or flocculate.
  • the polymer hybrid composite articles are able to be switched back and forth between the dispersed and flocculated state. Alternatively, the polymer hybrid composite articles may be collected from the solution while in the coalesced or flocculated state.
  • smart polymers are chemically attached to or physically adsorbed on a biological macromolecule, such as an oligopeptide, polypeptide, protein, or protein- polysaccharide conjugate, unique performance characteristics can be achieved.
  • a biological macromolecule such as an oligopeptide, polypeptide, protein, or protein- polysaccharide conjugate
  • solvent When a plurality of the polymer hybrid composite articles are in a solvent, the polymer whiskers and the solvent interact to cause the polymer whiskers to expand or contract in response to one or more changes in environmental conditions.
  • solvent as used herein is interchangeable with the terms “solution”, “medium” or “media”, “reactant media”, “biological fluids” and “biological media”. At least some of the polymer whiskers are solvated when the polymer whiskers expand, and the absorbed solvent is expelled from the polymer whiskers when the polymer whiskers contract.
  • each of the polymer hybrid composite articles is dispersed in the solvent away from other polymer hybrid composite articles.
  • the attached polymer whiskers keep each bioactive entity at a distance from other likewise-modified bioactive entities.
  • the active sites of the bioactive entities are exposed, allowing the bioactive entity to perform its function.
  • the polymer whiskers are in the extended state, the polymer hybrid composite articles are stable and remain in the dispersed state.
  • the polymer hybrid composite articles coalesce (flocculate). At this time, the polymer hybrid composite articles may be either collected or left in the solvent for re- suspension.
  • a target molecule or entity such as a drug candidate or a cell
  • the target entity will be taken along with the composite article as it coalesces and can then be collected.
  • the collapsed polymer whiskers fold back onto the surface of the bioactive entities or onto the surface of the cell to which the bioactive entities are attached, effectively blocking the active sites of these molecules or cells.
  • the functions of the polymer hybrid composite articles are temporarily halted until the polymer whiskers are re-expanded away from the bioactive entity surface and the polymer hybrid compound is again dispersed in the solution.
  • the cell is inactivated and cannot perform its function. If the polymer whiskers are not re-expanded, the cell may die.
  • the particular activity of the polymer hybrid composite articles may be controlled not only by the specific bioactive entity selected but also by the number and character of the polymer whiskers.
  • the number of polymer whiskers will be selected so that the cell is substantially enclosed by the whiskers, resulting in loss of the cell's ability to function.
  • the number of whiskers will be selected such that the cell is immobilized and coalesces together with the composite article to which it is conjugated, but the cell is not damaged or killed in the process.
  • a second bioactive entity or toxicological substance may be attached to the free end of the polymer whisker.
  • the two ends Upon coiling of the polymer chain, the two ends will be brought into close proximity. This will have the effect of enhancing the reaction rate and selectivity of chemical reactions which require a two- step enzyme-catalyzed reaction to occur.
  • a toxicological substance is attached to the free end of the polymer whisker, the other end of which is attached to a biological entity such as a cancer cell, the close proximity of the chain ends brought about by polymer coiling may hasten cell death.
  • FIG. 1 illustrates switchable polymer whiskers in solution undergoing abrupt thermodynamic transitions.
  • segment-segment interactions within the polymer whiskers are more favored over those between segment and solvent.
  • This changed state causes the polymer whiskers to collapse. This reversible transition is thus accompanied by drastic changes in the effective volume occupied by the polymer whiskers.
  • the above critical behavior can be further analyzed by plotting one of the environmental stimuli, such as transition temperature, versus the polymer- solvent concentration, as illustrated in FIG. 2.
  • one of the environmental stimuli such as transition temperature
  • lowering the temperature leads to polymer whisker collapse; i.e., phase separation from a homogeneous solution to an opaque two-phase mixture.
  • raising the temperature has the same effect.
  • the former systems are referred to as UCST (Upper Critical Solution Temperature) systems, while the latter are known collectively as LCST (Lower Critical Solution Temperature) systems.
  • LCST systems are those that exhibit abrupt polymer whisker expansion when cooled past the transition temperature, while UCST systems behave in an opposite fashion.
  • the temperature at which the LCST or UCST transition takes place depends on the composition of the system. With proper design of the polymer whiskers, similar solution transitions can be brought about at a fixed temperature by varying other thermodynamic parameters, such as by adjusting the local pH, medium ionic strength, light, pressure, electric field strength, or by titrating good solvents or non-solvents into the media.
  • thermodynamic parameters such as by adjusting the local pH, medium ionic strength, light, pressure, electric field strength, or by titrating good solvents or non-solvents into the media.
  • UCST systems are common, and their transition may occur frequently within conveniently observable thermodynamic parameter ranges.
  • LCST systems are less common, and appear predominantly in aqueous solutions. These systems are discussed in detail in the above-cited "Responsive Gels".
  • the more common type is associated with significantly different compressibilities of the polymer whiskers and the solvent, and is generally observed at temperatures and pressures near the critical point of the solvent.
  • the other type is thought to be caused by specific interactions between the polymer segments of the polymer whiskers and the solvents. Such interactions are generally believed to be dipolar or hydrogen-bonding in nature.
  • the polymer whiskers may be selected from the group N-isopropyl acrylamide and acrylamide; polyethylene glycol, di-acrylate and hydroxyethylmethacrylate; octyl/decyl acrylate; acrylated aromatic and urethane oligomers; vinylsilicones and silicone acrylate; polypropylene glycols, polyvinylmethyl ether; polyvinylethyl ether; polyvinyl alcohol; polyvinyl acetate; polyvinyl pyrrolidone; polyhydroxypropyl acrylate; ethylene, acrylate and methylmethacrylate; nonyl phenol; cellulose; methyl cellulose; hydroxyethyl cellulose; hydroxypropyl methyl cellulose; hydroxypropyl cellulose; ethyl hydroxyethyl cellulose; hydrophobically-modified cellulose; dextran; hydrophobically- modified dextran; agarose; low-gelling-temperature agarose
  • the whiskers may be employed.
  • multifunctional compounds such as bis-acrylamide and ethoxylated trimethylol propane triacrylate and sulfonated styrene may be employed.
  • the polymer whiskers comprise polyacrylamides, substituted polyacrylamides, polyvinylmethyl ethers, and modified celluloses.
  • the polymer whiskers are chosen based on their ability to interact with the solvent employed in a particular reaction, such as water, methanol, ethanol, isopropanol, butanol and higher alcohols, acetone, ethylene glycol, toluene, methyl ethyl ketone, tetrahydrofuran, polyethylene glycol, glycerol, aromatic silicone fluids, aliphatic silicone fluids and silicone copolymers and mixtures thereof.
  • the solvent employed in a particular reaction such as water, methanol, ethanol, isopropanol, butanol and higher alcohols, acetone, ethylene glycol, toluene, methyl ethyl ketone, tetrahydrofuran, polyethylene glycol, glycerol, aromatic silicone fluids, aliphatic silicone fluids and silicone copolymers and mixtures thereof.
  • the smart polymer portion In order to retain the viability of the biological entity portion of the hybrid conjugate, the smart polymer portion must undergo transitions within physiologically reasonable ranges of environmental conditions. For example, if temperature is relied upon to trigger either UCST or LCST, the transition point should preferably fall within the range of room temperature to a temperature below which the biological entity is not irreversibly altered from its physiologically-functional form (at most a few degrees over). If pH is used to cause transition, strongly acidic or basic conditions should be safely avoided. Similarly, mixed aqueous media can only be used up to a point where the target protein (or antibody) stays active.
  • NIPA poly(N-isopropylacrylamide), also known as NIPA, and its derivatives are ideally suited.
  • NIPA has a transition temperature between 32° to 33°C. It can be derivatized by co-polymerization or chemical modification to produce mixed acrylamides. Examples include isopropyl- and other alkyl-substituted acrylamide copolymers. Copolymers of NIPA and alkoxy acrylates can also be used to achieve different transition points. Transitions can be induced by pH changes, if NIPA is copolymerized (or polymerized, then hydrolyzed) to form NIPA and acrylic acid or NIPA and acrylamide copolymers.
  • Acrylic acid imparts a transition in the mildly acidic pH range, whereas the acrylamide functionality reaches charge neutrality in alkaline conditions.
  • the above suggested modifications of the well- studied NIPA systems are not intended to be exhaustive. Those skilled in the art of polymer thermodynamics can design other useful combinations.
  • block copolymer series (di-block, tri-block, and multi-block) of ethylene glycol and propylene glycol.
  • Combination can be made at any ratio from one smart polymer coil or whisker per protein to multiple smart polymer coils per bioactive entity.
  • the whiskers can even be attached via an inert spacer so as not to affect the function of the bioactive entity upon transition.
  • Attachment of Polymer Whiskers to a Bioactive Entity Anchoring of the polymer whiskers to a bioactive entity is accomplished by either physical adsorption of a constituent block of a block copolymer, or by chemically tethering the polymer whiskers directly to the bioactive entity. If the polymer whisker is physically adsorbed to the bioactive entity, then the whisker's free end will contain a constituent block.
  • the whisker will simply have a free-floating tail. In either case, the free block or the free tail of the attached polymer whisker can undergo the above-described UCST or LCST transitions.
  • the protein bioactive entity can be coupled with one to several polymer whiskers, depending on the relative size of the natural and synthetic macromolecules and the extent of bioactive entity surface coverage desired.
  • a strategically located polymer whisker can in principle reversibly block access to the active site on an enzyme, whereas multiple short polymer whiskers may be needed to effectuate reversible dispersion and flocculation of the protein bioactive entity of the polymer hybrid composite article.
  • the optimal number and length of the polymer whisker grafts will depend on the specific bioactive entity (i.e., protein-substrate-medium combination) being modified and can be determined without undue experimentation.
  • Physical anchoring of the smart polymer chain to a bioactive entity has the advantage that physical interactions (as opposed to stronger chemical interactions such as covalent bonding) are easily reversible and less permanent. Physical anchoring can be achieved by use of avidin:biotin binding. Physical anchoring can also be effected by using block or graft copolymers comprising blocks or grafts of different degrees of hydrophobicity and hydrophilicity (preferably having opposite properties).
  • di-block copolymers made of a polar or polyelectrolyte block (such as polyacrylic acid, polyacrylamide, and poly-4-vinylpyridine) and a nonpolar block (such as polystyrene, polyvinyl acetate, and polylauryl methacrylate) can be selectively adsorbed on the surface of proteins having a range of surface chemistry.
  • Hydrophilic entities for example, polar amino acid residues such as serine or cysteine
  • hydrophobic entities tend to absorb the hydrophobic block.
  • the partner block of the copolymer extends into the surrounding media.
  • the relative affinity of the blocks toward the surface and the surrounding media determines the particular spatial configuration of the block copolymer in the vicinity of the bioactive entity.
  • the above schemes can be utilized by those skilled in the art of polymer solution thermodynamics and surface phenomena to maximally differentiate the adsorption tendencies of the blocks (or grafts).
  • the dangling ends of the polymer whiskers exert the steric influence of dispersion or coagulation, depending on the prevailing thermodynamic state of the system in question relative to the UCST or LCST transition points. Protocols for performing block copolymerization reactions can be found in "Polymer Blends and Composites," J.A. Manson and L.H. Sperling, Plenum Press, N.Y., 1976, Chapters 5 and 7.
  • a bi-functional coupling agent may first be attached covalently to one or more sites on a bioactive entity by reacting with amino acid side chains (residues) on the surface of the protein of interest, for example, lodoacetate attachments to thiol side chains of serine, cysteine, and tyrosine residues, or nucleophilic substitution at exposed amino groups on lysine and arginine residues are well-known examples.
  • This procedure attaches one end of the bi-functional coupling agent to the biomolecule of interest, leaving the other end free to initiate the synthetic polymerization reaction.
  • One common coupling agent is diaminohexane. Equivalent ⁇ , acrylate and other functional groups (epoxy, amine, carboxylate, etc.) can be created on the surface by using the appropriate coupling agents.
  • the activated bioactive entities are mixed with reactive monomers that are subsequently polymerized in-situ.
  • N-isopropylacrylamide (NIPA) is one such example monomer.
  • NIPA N-isopropylacrylamide
  • the resulting polymer exhibits LCST behavior in aqueous solution.
  • the polymer whisker is in the expanded state, the polymer whisker is water-soluble, causing dispersion of the polymer hybrid composite article.
  • the polymer whiskers collapse and the polymer hybrid composite articles aggregate.
  • the hybrid composite articles precipitate from solution rapidly.
  • the flocculated compounds float in the solution and are easily separated from it.
  • substituted acrylamides can be used either as a homopolymer or copolymer to give transition points that can be engineered into the polymer whiskers by controlling its composition.
  • Substituted acrylamides can be copolymerized with alkyloxyacrylates or methacrylates to form the whiskers.
  • charged monomers such as acrylic or methacrylic acid salts, can be copolymerized with substituted acrylamides to fine- tune the transition behavior of the whiskers.
  • These charged polymers are susceptible to changes in pH, light, pressure, electric field strength or ionic strength.
  • Another example synthesis route involves grafting linear or branched polymer whiskers directly onto bioactive entity surfaces. Chemical- or radiation-induced grafting techniques abound in the literature.
  • Polymer whiskers with residual reactive functional groups for grafting can be either custom made or purchased from commercial sources, such as Shearwater Polymers, Polymer Sciences, and Chevron. These macromolecular whisker chains can be linked to the protein surface, depending on the nature of the available functional groups on the polymer whisker and on the protein surface or other bioactive entity.
  • the bioactive entities of the invention may be polymeric in nature, such as polymeric gel particles in which enzymatic reagents have been immobilized.
  • linking "smart" polymer whiskers 1 whiskers that undergo UCST or LCST transitions
  • industrially important polymer particles 2 as a special class of bioactive entities
  • FIG. 3A the polymer whiskers of the polymer hybrid composite articles are extended and the articles are dispersed throughout the solution.
  • the whiskers collapse and the composite articles flocculate together (3), as illustrated in FIG. 3B.
  • Industrially active polymer particles have routinely served as carriers or supports in reagent, catalyst, and substrate applications. These industrially important polymer carriers or supports can be synthesized by two known routes: attachment of functional groups to polymers and polymerization of functional monomers (protocols are found in "Principles of Polymerization", G. Odian, Wiley and Sons, 1981). The polymer hybrid composite articles are synthesized as described herein.
  • bioactive entities In general, polymeric reagents, catalysts, and substrates have previously had distinct advantages over their small molecule analogs. Foremost is the ease of separation of the insoluble polymer after use. However, in order to fully exploit the ease of separation, the prior art required that the bioactive entities must be larger than colloidal in dimension, giving rise to substantial mass transfer resistance and slowing down the relevant kinetics. Also in the prior art, the similar densities of the bioactive entities and the surrounding fluids hinder the typically deployed means of separation. Modification of bioactive entities by attaching smart polymer whiskers as disclosed in this invention allows very small (colloidal) bioactive entities, such as polymeric reagents, catalysts and substrates, to be used, accelerating mass transfer and the overall kinetics (see FIG. 3A), and at the same time facilitates separation of the polymer hybrid composite articles from the surrounding fluids after use (see FIG. 3B). Bioactive entities:
  • Bioactive entities useful in the present invention are selected for their special performance characteristics, which in turn will depend on the particular activity they are to perform. Exemplary activities and the biological entities useful therein are presented herein. Other examples of bioactive entities suitable for this invention will become apparent as the invention is further described.
  • FIG. 4 illustrates the reversible surface coverage and exposure of an enzyme catalyst bioactive entity 4, after polymer whiskers 5 capable of LCST or UCST transitions have been attached to the enzyme 6 (with an active site 7). Regardless of transition direction, when the polymer whiskers are expanded (as shown on the left side of FIG. 4), the catalytic activity resumes; whereas, when the polymer whiskers are retracted, the reaction stops or slows down because access to the active sites of the catalysts has been blocked (as shown on the right side of FIG.
  • the polymer whiskers are designed to contract at a temperature that is deemed to be a danger point of becoming a runaway reaction.
  • the polymer whiskers contract, thus blocking the active sites of the catalyst.
  • the reaction in this local area is stopped in a pervasive, microscopic and instantaneous manner.
  • the polymer whiskers will again expand and the reaction will continue. This control of the local reaction conditions prevents run-away reactions and possible damage to heat-sensitive enzymatic catalysts, and increases the quality of the product.
  • the attached polymer whiskers In addition to serving as a reversible blocker of the active sites, the attached polymer whiskers also cause flocculation of the polymer-enzyme catalyst hybrid composite articles after the desired catalytic reaction is complete. This conversion to a flocculated state of polymer-enzyme catalyst hybrid composite articles allows the ready elimination of the catalysts from the reaction mixture via collection and recovery of the sedimented mass (aided by simple filtration or centrifugation, for example, if necessary).
  • the polymer-catalyst hybrid composite articles of the invention are also useful in the long-term storage of enzymatic catalysts. For example, many enzymes undergo aging processes from the time they are first manufactured and concentrated until the time at which they can be used for their intended purpose. Enzyme shelf life may be usefully extended by attaching one or more polymeric whiskers described herein to an enzyme or other bioactive entity, followed by storage of the enzyme under conditions that produce polymer chain collapse. Because polymer coiling blocks access to much of the enzyme surface, including the active site, degradation of the enzyme during storage will be mediated until such a time as the enzyme is reactivated and used for its intended purpose.
  • Catalysts employable in this invention are chosen from a wide variety of enzymes, as long as they are amenable to some form of smart-polymer attachment. Disclosed herein are some classes of catalysts that are employable as catalytic bioactive entities in the polymer hybrid composite articles of the invention. Cell Selection, Concentration, Isolation, Recovery. Depletion, or Viability Control:
  • Certain of the polymer hybrid composite articles of the present invention are useful for the separation and concentration of cells from blood or other solvent. This may include the separation of stem cells from peripheral blood for reinfusion after chemotherapy treatment, fetal cells from maternal blood for genetic testing, cancerous cells for blood purification purposes, particular targeted cells for research or manufacturing purposes, or cells for other similar purposes not mentioned herein.
  • Cell separation may proceed by the attachment of one or more polymer whiskers to certain bioactive entities or "receptors" that have an affinity for a particular ligand on the stem cells or other targeted cells. Examples of such receptors are antibodies that bind selectively to the surfaces of the desired cells.
  • polymer whiskers may be attached to one element of a receptor:ligand pair (e.g., avidin:biotin), where the other element has an affinity for, and has thus become attached to, the cell of interest.
  • a polymer hybrid composite article may be formed consisting of one or more polymer chains, a recepto ⁇ ligand pair, and a cell of interest.
  • contraction of the polymer chains or whiskers may be induced, thereby allowing the ready elimination of the cells from the supernatant mixture via collection and recovery of the flocculated mass.
  • stem, fetal, cancerous or other targeted cells may be effectively and economically separated from solutions containing large numbers of undesirable cells (i.e., positive selection). Furthermore, upon redispersing the collected cells into clean solvent, the steps may be repeated in a multi-staging process for further purification of the desired cells.
  • stem cells are selected and recovered from peripheral blood or other solvent by contacting the blood or solvent containing stem cells with one or more smart polymer whiskers coupled to an antibody specific to the stem cells.
  • the environmental conditions of the solvent is then adjusted to cause the polymer whiskers to expand, allowing the antibody to be exposed to the solvent and allowing the stem cells to be attracted to and attached to the antibody due to the antigen-antibody pairing.
  • the environmental conditions of the solvent are adjusted to cause the polymer whiskers to contract and coalesce. The coalesced composite article-stem cell conjugates are then collected.
  • the environment is adjusted to cause the whiskers to expand, and the antibody-stem cell conjugates are exposed to conditions where the stem cells are released from the antibody, which conditions are well-known to those skilled in the art.
  • the stem cells are then separated from the polymer-antibody composite articles, as for example by the polymer whiskers being caused to contract and the composite articles then coalescing and being removed from the solvent containing the free cells.
  • Cell membranes are portals where important nutrients and wastes pass through. Protein channels that can open and close reversibly control such cross- membrane transport. In order for a cell to survive, such transport mechanisms must remain intact.
  • a composite article comprising i) a receptor having an affinity for the targeted deleterious cells and ii) smart polymer whiskers attached to the receptor is placed in contact with the targeted cells.
  • the environmental conditions are adjusted to cause the polymer whiskers to expand and the cell to come into contact with and attach to the receptor, after which the environmental conditions are changed to cause the polymer whiskers to contract.
  • the whiskers enclose the deleterious cell, resulting in loss of the cell's ability to function.
  • the deleterious cell-composite article conjugate may then be separated out of the solvent, or the conditions causing contraction may be continued for a sufficient period of time until the cell is killed.
  • an anti-cancer antibody is linked to one or more smart polymers, and then this hybrid is injected into the human body.
  • polymer hybrid composite articles of the present invention are useful in combinatorial chemistry, either in the synthesis of libraries of compounds, or in the selection of targeted candidates for new drugs and pharmaceuticals or other desirable new molecular entities, based on structure-activity relationships.
  • Pharmaceutical drug discovery is one type of research that relies on the study of structure-activity relationships. In most cases, contemporary pharmaceutical research can be described as the process of discovering novel ligands with desirable patterns of specificity for biologically important receptors. Another example is research to discover new compounds for use in agriculture, such as pesticides and herbicides. Combinatorial chemistry has been developed as a way of creating a very large number of compounds or "libraries” en masse and identifying the most promising compounds for a particular use through screening of the libraries.
  • a "ligand” is a molecule or entity that is recognized by a particular receptor.
  • ligands examples include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oiigosaccharides, proteins, and monoclonal antibodies.
  • hormones e.g., opiates, steroids, etc.
  • hormone receptors e.g., opiates, steroids, etc.
  • hormone receptors e.g., opiates, steroids, etc.
  • peptides e.g., enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oiigosaccharides, proteins, and monoclonal antibodies.
  • a "receptor” is a molecule that has an affinity for a given ligand. Receptors may be naturally-occurring, biologically-derived, or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species.
  • Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance.
  • receptors that can be employed by this invention include, but are not limited to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles.
  • Receptors are sometimes referred to in the art as anti- ligands. As the term receptors is used herein, no difference in meaning is intended.
  • receptors that may be useful as the bioactive entity include, but are not limited to: a) Microorganism receptors: Determination of ligands that bind to receptors, such as specific transport proteins or enzymes essential to survival of microorganisms, is useful in a new class of antibiotics. Of particular value would be antibiotics against opportunistic fungi, protozoa, and those bacteria resistant to the antibiotics in current use. b) Enzymes: For instance, the binding site of enzymes such as the enzymes responsible for cleaving neurotransmitters.
  • Determination of ligands which bind to certain receptors to modulate the action of the enzymes that cleave the different neurotransmitters is useful in the development of drugs that can be used in the treatment of disorders of neurotransmission.
  • Hormone receptors For instance, the receptors for insulin and growth hormone. Determination of the ligands that bind with high affinity to a receptor is useful in the development of, for example, in the first case, an oral replacement of the daily injections that diabetics must take to relieve the symptoms of diabetes, and in the second case, a replacement for the scarce human growth hormone that currently can only be obtained from cadavers or by recombinant DNA technology.
  • a receptor 9 for the desired activity is modified by attachment of polymer whiskers 10 capable of LCST or UCST transitions to give a smart polymer-antibody (or other receptor) composite article 8, as illustrated in FIG. 5.
  • the composite articles are put into contact with a mixture of candidate compounds 11 in solution.
  • the solution environment is such that the polymer whiskers are in an extended configuration, making the receptors available to contact any ligands (compounds) in the mixture to which they have an affinity (see 12 of FIG. 5).
  • the solution environment is changed such that the polymer whiskers collapse.
  • the polymer-receptor composite articles, now conjugated to a target ligand will coalesce or flocculate (see 13 of FIG. 5) and can be collected, via filtration or any other suitable extraction method.
  • the collected conjugates are then placed into a second solvent conducive to expansion of the polymer whiskers, exposing the receptor-ligand (14, 15, and 16).
  • the ligand is then released from the antibody by any one of several methods known in the art to give the free ligands.
  • the solvent environment is then modified such that the polymer whiskers on the polymer-antibody composite articles are again collapsed, resulting in the articles coalescing. At this point, the composite articles are separated out, leaving the targeted ligands, which can then be further tested and developed as potential pharmaceutical drugs.
  • Combinatorial techniques generally assemble a selected set of building blocks to create a library of compounds. Libraries of DNA, peptide, carbohydrate, and glycoprotein sequences, as well as structurally related small molecules, can be synthesized through combinatorial chemistry techniques. See, for example, "Combinatorial Synthesis of Small Organic Molecules," F. Balkenhohl, et.al., Angewandte Chemie International Edition, English, 1996, Vol. 35, pp.
  • bioactive building blocks that may be employed include all those known in the art to be amenable to combinatorial synthesis, such as, but not limited to, one of the DNA bases (e.g., adenine, guanine, thymine, or cytosine), amino acids, nucleotides, and sugars, and derivatives of these compounds.
  • DNA bases e.g., adenine, guanine, thymine, or cytosine
  • amino acids e.g., amino acids, nucleotides, and sugars, and derivatives of these compounds.
  • Coupling expandable and retractable polymer whiskers with an industrial enzyme requires two steps. First, a block copolymer of polyethylene glycol and N- substituted polyacrylamide, such as N-isopropyl acrylamide block, is prepared.
  • Mono-acryloxy polyethylene glycol (of a given chain length, including, but not limited to, monomer, oligomer, short and long chains of ethylene glycol repeat units) is provided.
  • the chosen polyethylene glycol (PEG) is end-capped with an acrylate or methacrylate functional group. This material is then allowed to react with N- isopropylacrylamide by a standard free radical polymerization process. (S. L. Rosen, Fundamental Principles of Polymeric Materials, Wiley, 1993).
  • the next step is the activation of the terminal hydroxyl functionality of the polyethylene glycol block, in preparation for its attachment to an enzyme bioactive entity.
  • Several methods are cited here. (W. J. Fung, J. E. Porter and P. Bailon, Strategies for the Preparation and Characterization of Polyethylene Glycol (PEG) Conjugated Pharmaceutical Proteins, Polymer Preprints, Vol. 38, pp. 565-566, 1997, incorporated herein by reference).
  • N- hydroxysuccinimidyl (NHS) ester derivative which reacts with the free amines of the N-terminus or the lysine residues to form stable amide bonds
  • carbonyl imidazole derivative of polyethylene glycol (CI-PEG) which reacts with the enzyme bioactive entity resulting in an urethane linkage
  • CI-PEG polyethylene glycol
  • Teresyl-PEG tresyl derivative of PEG
  • Resyl-PEG which reacts with the primary amino group of the enzyme bioactive entity by forming a second amine group, thus maintaining the same charge of the native protein
  • aldehyde derivative of PEG AdPEG
  • VS-PEG vinyl sulfone derivative of PEG (VS-PEG), which selectively reacts with the protein sulfhydryl groups of the enzyme active compound under mild alkaline conditions to form a sulfo-linkage.
  • the enzyme thus modified has one to a few polymer whiskers extending from the surface of the enzyme globule at temperatures below the LCST of the poly-N- isopropylacrylamide (NIPA) moiety.
  • NIPA poly-N- isopropylacrylamide
  • Example 2 Formation and Separation of Polymer Coagulant from Saline Solution
  • the solution is shaken for several hours until complete dissolution of the PVME has occurred, producing a clear and homogenous mixture.
  • the glass vial containing the solution is placed into a temperature-controlled water bath, initially at room temperature.
  • the PVME solution turns cloudy.
  • a precipitate begins to form in the solution, which either settles to the bottom of the vial or sticks to the sides of the vial near the top at the liquid meniscus.
  • the polymer Upon further heating to 37 °C, the polymer continues to precipitate and aggregates to form larger agglomerations of polymer, termed coagulants.
  • the coagulated polymer lump(s) may be removed from the solution with tweezers and placed into a clean vial, or the supernatant can be decanted from the vial, leaving the solidified polymer behind.
  • the polymer coagulant can be resuspended in solution by adding room temperature PBS solution and agitating, after which the solution again becomes clear and homogeneous.
  • Example 3 Alternative Method for Polymer Coagulant Formation
  • a 1 wt% solution is formed as described in Example 2.
  • the vial is intermittently shaken by gently tipping the vial upside down and back again, and then replacing it into the heating bath. The shaking continues until the water bath reaches 35 °C, at which point much polymer coagulant has formed and settled to the bottom or stuck to the sides of the vial.
  • the coagulant which settles to the bottom of the vial generally forms a single lump which does not break apart with further shaking.
  • This technique will also be characterized by a supernatant that is more clear (less cloudy) than that seen in example 2.
  • the polymer coagulant can be enhanced in both size and strength as the solution is heated further to 37 °C.
  • Example 4 Alternative Method for Polymer Coagulant Formation and Separation A 1 wt% solution is formed as described in Example 2.
  • the vial is vigorously shaken so that small bubbles are formed throughout the solution and so that a foam of bubbles remains during the periods in the water bath. The shaking continues intermittently, approximately every 30 seconds, while heating continues.
  • the solution reaches 35 °C, a large amount of polymer coagulant has formed, generally consisting of a single or several large lumps, which may or may not be dispersed during shaking.
  • These polymer coagulants contain entrained air bubbles, and typically float to the top of the vial after each shake.
  • Example 5 Alternative Polymers for Polymer Coagulant Formation and Separation
  • a 1 wt% solution is formed as described in Example 2, except that any polymer from Table 1 may be used in place of PVME.
  • a 0J wt% solution is formed following the procedures described in Example 2, except that any polymer from Table 2 may be used in place of PVME.
  • DI cloud point temperatures listed in Table 1 or Table 2
  • the polymer solutions will form a precipitate. This precipitate may be settled, filtered, or otherwise collected.
  • the precipitate formed for each polymer may vary in size, strength, and density, but the solution will be characterized by cloudiness in all cases.
  • NIPA Polymerized NIPA was formed according to the following procedure: 1.13 grams of 9.1 wt % NIPA (Aldrich Chemical Co., Catalog No. 41,532-4, St. Louis, Missouri) solution in DI water was added to 0.54 grams of a 1.96 wt% solution of KPS (potassium persulfate, Aldrich Chemical Co., Catalog No. 21 ,622-4, St. Louis, Missouri) in DI water, in 8.3 grams of DI water. This solution was well-mixed, and then 0.03 grams of TEMED (tetramethylene ethyldiamine, ICN Pharmaceuticals, Inc., Catalog No. 195516, Costa Mesa, California) was added and the mixture was stirred and heated to 70 °C, and held for 1.5 hours. When the solution temperature reached 55-60 °C, the solution turned cloudy, indicating the polymerization of NIPA and precipitation of polyNIPA. TABLE 2 Cloud point temperatures of OJ wt% polymer solutions
  • Example 6 Alternative Polymer Mixtures for Polymer Coagulant Formation and Separation
  • a 1 wt% solution is formed as described in Example 2, except that any mixture of polymers from Table 3 may be used in place of a pure polymer.
  • both polymers will total 1 wt%, except in the case of EHEC + HPC mixture, in which case the polymer concentration should be 0.2 wt%.
  • the ratio of the polymers used by weight is listed in Table 3.
  • PBS buffered saline
  • the polymer coagulant may be settled, filtered, or otherwise collected. TABLE 3 Coagulation temperatures for polymer mixtures in PBS
  • a 1 wt% solution is formed as described in Example 2.
  • the solution is poured over a wire mesh screen (stainless steel, 635 mesh (20 micron), McMaster-Carr Supply Company, Catalog No. 34735K999, Los Angeles, California).
  • the mesh screen is preheated to at least the cloud point temperature by pouring heated saline over the screen just prior to adding the polymer solution.
  • the polymer coagulant is trapped on the screen, while the rest of the solution contents pass through.
  • the polymer coagulant may be washed with pure solution that has been heated to at least the cloud point temperature.
  • the polymer coagulant is collected with a spatula.
  • the polymer coagulant is redissolved by washing the screen with water or saline solution that has not been heated to the cloud point temperature. After several washings, or by letting the screen sit submersed in non-heated solution, the polymer will redissolve into solution.
  • Example 8 Alternative Method for Polymer Separation
  • a 2 wt% solution is formed as described in Example 2.
  • a glass chromatography column (Bio-Rad Laboratories, Catalog No. 737-1021 , Hercules, California) is packed with silica gel (Sigma, Catalog No. S-4883, St. Louis, Missouri) to a height of 7.5 cm.
  • the packing is wetted with saline (approximately 7 mL), then the column is heated to 37 °C with a heating tape on the outside. Once the temperature is equilibrated, 5 mL of the polymer solution is flowed through the column, followed by pure saline solution.
  • the solution collected at the bottom of the column will not contain an appreciable amount of polymer, as evidenced by the absence of a polymer coagulant upon heating the collected solution.
  • the flow through the column is stopped and the heating tape is turned off.
  • Example 9 Polymer Separation and Recovery from Glass Bead Mixture A. SOLUTION PREPARATION
  • deionized water Renionized water
  • the solution is heated while gently agitating every 30 seconds to one minute.
  • the polymer solution reaches its cloud point of 31 °C, white precipitate forms, which will sink to the bottom of the vial (as long as the agitation is not strong enough to produce bubbles).
  • the vial is opened and the liquid supernatant is decanted away, leaving the solid polymer coagulant behind.
  • the polymer coagulant is resuspended in solution by adding room- temperature deionized water to bring the total solution weight back to 10 grams.
  • the solution is again heated with gentle agitation, and the "washing" process is repeated by coagulating the polymer, decanting the supernatant, and redissolving the polymer in fresh deionized water. This process may be repeated indefinitely to continue washing the polymer and to effect a separation between the polymer and the glass beads.
  • the concentration of glass beads in each successive solution may be observed by placing a drop of solution onto a glass microscope slide and placing a cover slide on top of the drop.
  • the original solution (before any separations have taken place) has a very high density of beads, estimated to be >20,000 beads per mm 2 of area.
  • a liquid drop of the polymer solution shows a greatly decreased concentration of glass beads, estimated to be approximately 500 beads per mm 2 .
  • the glass bead concentration is reduced further yet, estimated to be approximately 10 beads per mm 2 .
  • no beads can be found in the 1-mm 2 field of view of the microscope.
  • the glass beads have been separated from the polymer solution, to the point where the polymer solution contains no glass beads above the detectable limit.
  • the polymer recovery from each washing step has been tested and calculated to be approximately 90%.
  • the polymer-glass bead separation and the polymer recovery may be improved by more efficient decantation techniques, which remove more of the supernatant but none of the polymer coagulant.
  • the coagulated polymer-glass bead mixture can be poured over a wire mesh screen to retrieve the polymer coagulant (see Example 7).
  • Example 10 Polymer Separation and Recovery from Yeast Cell Mixture
  • Dried yeast cells having a nominal density of 1.05 g/cc and a mean particle size of 5 microns (Saccharomyces cerevisiae (Bakers Yeast) Type II, Sigma, Catalog No. YSC-2, St. Louis, Missouri)) are added to the polymer solution in the amount of 0.1 g per 10 mL of solution, and the solution is well stirred.
  • the polymer coagulant is resuspended in solution by adding room- temperature saline to the new vial to bring the total solution weight back to 10 grams.
  • the solution is again heated with periodic agitation, and the "washing" process is repeated by coagulating the polymer, separating it into a clean vial (entraining as little supernatant as possible), and redissolving the polymer in fresh saline. This process may be repeated indefinitely to continue washing the polymer and to effect a separation between the polymer and the yeast cells.
  • the concentration of yeast cells in each successive solution may be observed by placing a drop of solution onto a glass microscope slide and placing a cover slide on top of the drop.
  • the original solution (before any separations have taken place) has a very high density of cells, estimated to be >10,000 beads per mm 2 of area.
  • a liquid drop of the polymer solution shows a greatly decreased concentration of yeast cells, estimated to be approximately 100 beads per mm 2 .
  • the yeast cell concentration is reduced further yet, estimated to be less than 1 cell per mm 2 .
  • no cells can be found in the 1-mm 2 field of view of the microscope.
  • Example 11 Smart-Polymer Conjugation to Stem-Cell Antibodies A. BACKGROUND
  • QbendIO and Thy1 antibodies Two known antibodies that recognize and bind with CD34+ progenitor cells are the QbendIO and Thy1 antibodies. These monoclonal antibodies (mAbs) discriminately bind to CD34+ progenitor cells by forming an antibody-antigen pair with the corresponding epitopes on the CD34 surface antigen.
  • mAbs monoclonal antibodies discriminately bind to CD34+ progenitor cells by forming an antibody-antigen pair with the corresponding epitopes on the CD34 surface antigen.
  • the QbendI O and Thy1 mAbs are first conjugated to smart-polymers to form a smart-polymer-coupled bioactive entity, as is described in this example.
  • Smart-polymer conjugation to mAbs is accomplished by reacting a crosslinking molecule between a smart-polymer chain and the mAb.
  • This cross-linker serves as a bridge molecule connecting the smart-polymer whisker and the antibody.
  • the cross-linker also acts as a molecular spacer, providing up to 10's of A between the smart polymer and antibody.
  • a particularly suitable cross-linker for conjugating smart-polymer chains and mAbs is 4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide (M 2 C 2 H, Pierce, Catalog No. 22303, Rockford, IL).
  • This reagent is a heterobifunctional cross-linking agent that possesses a carbonyl-reactive hydrazide group on one end and a sulfhydryl-reactive maleimide group on the other.
  • This cross-linker is especially useful because of the stability of the maleimide group, which allows conjugation reactions to be carried out in aqueous-phase solutions.
  • the conjugation reaction commences by reacting the maleimide group on the cross-linker with sulfhydryl groups present on the mAbs.
  • sulfhydryl groups present on the mAbs.
  • the presence of sulfhydryl groups in proteins and other molecules is typically low compared to other groups such as amines or carboxylates.
  • the use of sulfhydryl-reactive chemical reactions thus can restrict modification to only a limited number of sites within a target molecule, which greatly increases the chances of retaining activity after conjugation. When too few or no sulfhydryl groups are present, they must be generated from the reduction of indigenous disulfide groups.
  • An excellent reagent for this modification is N-succinimidyl S-acetylthioacetate (SATA, Pierce, Catalog No.
  • SATA is often used to form antibody-enzyme conjugates utilizing maleimide- containing heterobifunctional cross-linking agents. Most antibody molecules may be modified to contain up to about six SATA molecules per immunoglobin with minimal effect on antigen binding activity. This is because modification occurs predominantly on the crystallizable (Fc) portion of the mAb molecule, away from the antigen binding sites.
  • Fc crystallizable
  • the following protocol represents a generalized method for protein thiolation using SATA: (1) dissolve the protein to be thioiated (mAbs in this example) at a concentration of 1-5 mg/ml in 50 mM sodium phosphate, pH 7.5, containing 1-10 mM EDTA; (2) dissolve the SATA reagent in DMSO at a concentration of 65 mM; (3) add 10 ⁇ L (microliters) of the SATA solution to each milliiiter of protein solution; (4) mix and react for 30 minutes at room temperature; (5) separate modified protein from unreacted SATA and reaction by-products by dialysis against 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA.
  • the protocol is: (1) deprotect the acetylated -SH groups by adding 100 ⁇ L (microliters) of 0.5 M hydroxylamine hydrochloride in 50 mM sodium phosphate, 25 mM EDTA, pH 7.5, to each milliiiter of the SATA-modified protein solution; (2) mix and react for 2 hours at room temperature; (3) purify the sulfhydryl-modified protein by dialysis against 50 mM sodium phosphate, 1 mM
  • the deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation.
  • the sulfhydryl-activated mAb is mixed with an excess of M 2 C 2 H in physiologic buffer solution. The mixture is allowed to react overnight at room temperature, then is purified by dialysis. This reaction forms a permanent thioether bond of good stability between the cross-linker and mAbs.
  • the carbonyl-reactive hydrazide group at the other end of the heterobifunctional cross-linking agent will not react with the mAbs because in their native state, proteins, peptides, nucleic acids, and oligonucleotides contain no naturally occurring aldehyde residues. Thus, the hydrazide groups will be preserved for conjugation to the smart-polymer chains.
  • C. SMART-POLYMER CONJUGATION To complete the formation of the smart polymer-mAb entity, the mAb-cross- linker conjugate from above is reacted with the smart-polymer chains.
  • This reaction occurs between the hydrazide group on the M 2 C 2 H cross-linker and an aldehyde group on the smart-polymer chain.
  • Some smart polymers can be purchased with an aldehyde end-group (for example, Shearwater Polymers, Catalog No. M-ALD-20000, Huntsville, AL), while others must be mildly oxidized to produce aldehyde groups by treatment with sodium periodate.
  • Another route to formaldehyde formation is the oxidation of primary alcohols in the presence of pyridinium chlorochromate.
  • aldehyde functionalities may be formed according to common organic synthesis techniques as described in texts such as Organic Chemistry, 5th Edition, Robert Morrison and Robert Boyd, Allyn and Bacon, Inc., 1987.
  • the primary alcohol terminal group on PVME is oxidized to an aldehyde functionality in the presence of pyridinium chlorochromate.
  • the final conjugation reaction will proceed by mixing the cross-linker-mAb conjugate with excess aldehyde-terminated PVME chains in physiological buffer and allowing the reaction to proceed overnight.
  • the aldehyde end-group functionality on the polymer will react with the hydrazide group on the M 2 C 2 H cross-linker to form hydrozone bonds, thus producing a fully coupled smart polymer-mAb conjugate, abbreviated by PVME-M 2 C 2 H-Thy1 or PVME-M 2 C 2 H-Qbend10.
  • Example 12 Stem Cell Separations Using Smart Polymer-mAbs Conjugates
  • the smart polymer-mAbs conjugates formed in Example 11 are used to selectively mark the stem cells, and to effectuate reversible flocculation and precipitation out of solution upon switching of the smart-polymer chains.
  • 1 mg of smart polymer- mAbs conjugate is added to give a thousand-fold excess number of antibody conjugates for binding to the stem cells. The mixture is stirred for 1 hour to allow for the formation of suitable antibody-antigen interactions.
  • the solution temperature is controlled to be slightly less than the cloud point temperature of the smart polymer being used; for the PVME polymer described in the previous examples, the solution may be at room temperature or at any temperature below 30 °C.
  • the cell mixture is typically suspended in 1 liter of PBS solution.
  • the polymer conjugate-cell mixture is heated and agitated as described in Examples 2, 3 or 4.
  • the polymer coagulant that results upon heating to above the cloud point temperature of the polymer is collected by means of filtration, centrifugation, decantation, or other suitable collection means.
  • the polymer coagulant is then resuspended in 1 liter of fresh PBS, and the procedure may be repeated until the desired purity of stem cells is reached (typically 2-4 washes).
  • the stem cells are collected and cleaned by simple centrifugation or filtration at a temperature below the cloud point of the polymer, removing the excess polymer conjugate in solution and leaving purified stem cells.

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Abstract

Cette invention se rapporte à des articles composites hybrides polymères qui possèdent une activité réversible et des stabilités de dispersion, ces articles étant constitués par des filaments polymères qui sont soit attachés chimiquement soit adsorbés physiquement sur les surfaces d'entités bioactives importantes et actives dans le domaine médical ou industriel. Les filaments polymères sont choisis parmi ceux qui présentent des transitions de phase distinctes qui sont induites par la variation de certains paramètres thermodynamiques régulables, tels que la température, le pH, l'émission de lumière, la pression, la force du champ électrique, la force ionique et la composition de solvant. Lors de leur transition, ces filaments polymères, attachés aux surfaces de l'entité bioactive, subissent une dilatation ou un repli, provoquant ainsi la dispersion ou la coalescence des articles composites hybrides à base de ces polymères. Les entités bioactives choisies sont parmi celles qui remplissent une large gamme de fonctions, telles que cellules, protéines (par exemple enzymes, anticorps et récepteurs), acides nucléiques ou groupes fonctionnels de petites molécules. Lorsque les filaments polymères sont à l'état dilaté, les entités bioactives de ces articles composites remplissent les fonctions voulues. Lorsque les filaments polymères passent à l'état replié et enroulé, les entités bioactives ne sont plus actives et les articles composites subissent une floculation, de sorte qu'on peut facilement les éliminer, les recueillir ou les récupérer. Cette invention présente également un moyen pour activer et désactiver de façon réversible un catalyseur enzymatique. Cette invention propose en outre un moyen permettant de sélectionner et de récupérer des ligands cible, tels que des cellules souches. Les articles composites constitués par ces filaments polymères attachés à des récepteurs ayant une affinité pour les ligands cibles vont, lorsque les filaments sont dilatés, exposer le récepteur au ligand cible et permettre audit récepteur de se fixer audit ligand. Lorsque les filaments polymères sont ensuite repliés, le ligand cible s'associe à l'article composite à mesure qu'il subit la floculation et il peut alors être recueilli et récupéré. Ce procédé peut être utilisé en chimie combinatoire, soit dans la synthèse de bibliothèques de composés soit dans la sélection de nouvelles entités moléculaires ciblées, se fondant sur les relations entre structure et activité.
PCT/US1998/018633 1997-09-08 1998-09-08 Entites bioactive couplees a des polymeres intelligents et utilisations de ces entites Ceased WO1999012975A1 (fr)

Priority Applications (1)

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AU92246/98A AU9224698A (en) 1997-09-08 1998-09-08 Smart polymer-coupled bioactive entities and uses thereof

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US5816397P 1997-09-08 1997-09-08
US60/058,163 1997-09-08

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016496A1 (fr) * 2000-08-23 2002-02-28 National Institute Of Advanced Industrial Science And Technology Complexe polymere / polymere sensible a la temperature
WO2007005792A3 (fr) * 2005-07-01 2007-06-21 Univ Pittsburgh Reseaux polymeres de cicatrisation
EP1580260A4 (fr) * 2002-12-25 2008-07-09 Nat Inst Of Advanced Ind Scien Appareil de separation et de collecte de cellules et procede de separation et de collecte de cellules
EP2498755A4 (fr) * 2009-11-13 2013-06-19 Icx Agentase Nanoparticules thermoréactives dynamiques utilisées pour stabiliser des enzymes à des températures élevées

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4975375A (en) * 1985-07-02 1990-12-04 Canon Kabushiki Kaisha Biocatalyst immobilization with a reversibly swelling and shrinking polymer
JPH07136505A (ja) * 1993-11-17 1995-05-30 Terumo Corp アフィニティー分離材料
WO1996002577A1 (fr) * 1994-07-18 1996-02-01 Gel Sciences, Inc. Perles de gel polymere sensibles
WO1996006134A1 (fr) * 1994-08-19 1996-02-29 Gel Sciences, Inc. Gels reactionnels eliminant une cible selectivement d'un environnement et procedes afferents
JPH08108069A (ja) * 1994-10-07 1996-04-30 Toyobo Co Ltd 幹細胞分離方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4975375A (en) * 1985-07-02 1990-12-04 Canon Kabushiki Kaisha Biocatalyst immobilization with a reversibly swelling and shrinking polymer
JPH07136505A (ja) * 1993-11-17 1995-05-30 Terumo Corp アフィニティー分離材料
WO1996002577A1 (fr) * 1994-07-18 1996-02-01 Gel Sciences, Inc. Perles de gel polymere sensibles
WO1996006134A1 (fr) * 1994-08-19 1996-02-29 Gel Sciences, Inc. Gels reactionnels eliminant une cible selectivement d'un environnement et procedes afferents
JPH08108069A (ja) * 1994-10-07 1996-04-30 Toyobo Co Ltd 幹細胞分離方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 9530, Derwent World Patents Index; Class A97, AN 95-227588, XP002089920 *
PATENT ABSTRACTS OF JAPAN vol. 096, no. 008 30 August 1996 (1996-08-30) *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016496A1 (fr) * 2000-08-23 2002-02-28 National Institute Of Advanced Industrial Science And Technology Complexe polymere / polymere sensible a la temperature
US6863437B2 (en) 2000-08-23 2005-03-08 National Institute Of Advanced Industrial Science And Technology Temperature-responsive polymer/polymer complex
JP4904511B2 (ja) * 2000-08-23 2012-03-28 独立行政法人産業技術総合研究所 温度応答性高分子間コンプレックス
EP1580260A4 (fr) * 2002-12-25 2008-07-09 Nat Inst Of Advanced Ind Scien Appareil de separation et de collecte de cellules et procede de separation et de collecte de cellules
WO2007005792A3 (fr) * 2005-07-01 2007-06-21 Univ Pittsburgh Reseaux polymeres de cicatrisation
US8029774B2 (en) 2005-07-01 2011-10-04 University Of Pittsburgh Wound healing polymeric networks
EP2498755A4 (fr) * 2009-11-13 2013-06-19 Icx Agentase Nanoparticules thermoréactives dynamiques utilisées pour stabiliser des enzymes à des températures élevées
US9121017B2 (en) 2009-11-13 2015-09-01 Flir Detection, Inc. Dynamic thermoresponsive nanoparticles for stabilization of enzymes at high temperatures

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