WO1999012975A1

WO1999012975A1 - Smart polymer-coupled bioactive entities and uses thereof

Info

Publication number: WO1999012975A1
Application number: PCT/US1998/018633
Authority: WO
Inventors: David S. Soane; Michael R. Houston; Stephen E. Barry
Original assignee: Fleximer, Llc
Priority date: 1997-09-08
Filing date: 1998-09-08
Publication date: 1999-03-18
Also published as: AU9224698A

Abstract

Polymer hybrid composite articles having reversible activity and dispersion stabilities having polymer whiskers either chemical tethered to or physically adsorbed on the surfaces of medically or industrially active and important bioactive entities. The polymer whiskers are selected from those that exhibit distinct phase transitions that are induced by changing certain controllable thermodynamic parameters, such as temperature, pH, light, pressure, electric field strength, ionic strength, and solvent composition. Upon transition, the polymer whiskers, attached to the bioactive entity surfaces, undergo expansion or collapse, causing the polymer hybrid composite articles to disperse or coalesce. The bioactive entities chosen are from those that serve a wide range of functions, for example cells, proteins (e.g., enzymes, antibodies and receptors), nucleic acids, or small molecule functional groups. In the expanded polymer-whisker state, the bioactive entities of the composite articles perform their intended functions. When the polymer whiskers are switched into the collapsed-coil state, the bioactive entities are not active and the composite articles flocculate, allowing their facile elimination, collection or recovery. The present invention also provides a means to reversibly switch on and off the activity of an enzymatic catalyst. The present invention further provides a means for selecting and recovering target ligands, such as stem cells. Composite articles comprised of polymer whiskers attached to receptors having an affinity for the target ligand will, when the whiskers are expanded, expose the receptor to and allow attachment of the receptor with the target ligand. When the polymer whiskers are then collapsed, the target ligand is taken with the composite article as it flocculates, and can be collected and recovered. This method may be used in combinatorial chemistry, either in the synthesis of libraries of compounds or in the selection of targeted new molecular entities, based on structure-activity relationships.

Description

SMART POLYMER-COUPLED BIOACTIVE ENTITIES AND USES THEREOF

Field of the Invention:

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. Background Art:

Catalysts are substances that increase the rate of a chemical reaction. In industry, enzyme catalysts are used in many types of reactions, including redox, substitution, addition, elimination, cyclization, molecular rearrangements, and polymerization reactions.

Problems with catalyst usage 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.

Collection and recovery of catalysts is important both to purify a product and to re-use expensive catalysts, such as enzymes. Dispersion stability and particle settling are also problems faced when employing industrially active compounds. In view of the above, there exists a need in the art for stable active compositions for enzyme-catalyzed reactions that are easily dispersed in solvent and are easily collected and recovered from the solvent. Additionally, there is a need for catalyst compounds providing the ability to switch on and off the activity under specific reaction conditions. Also needed are catalytic compounds that can be stored indefinitely in a dormant state without triggering the catalytic reaction and can be switched on at will.

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. However, the shortcomings of surgery are many: 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. When cancer attacks certain vital structures, 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. However, while often effective on localized tumors, 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.

Anticancer drugs applied systemically to patients, the treatment known as chemotherapy, has been used to address some of the failings of surgery and radiation therapy. While used successfully in the treatment of some cancers, other cancers, including the most common types, are not presently curable with chemotherapy alone. Additionally, the chemotherapeutic drugs currently available will kill healthy cells as well as cancerous ones, causing serious side effects and limiting the doses that can be administered. Other side effects such as diarrhea, nausea, and hair loss, while not necessarily life-threatening, are often extremely unpleasant for the patient. There thus remains a need for a cancer treatment that selectively and effectively eradicates all cancer cells in the body while at the same time having little or no deleterious effect on healthy cells of the patient.

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. However, such 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. There thus remains a need for monoclonal antibody conjugates which are able to evade recognition by the body's immune system prior to attachment to the surface of cancerous cells, but which can be activated at a later time to initiate the destruction of the cells once attachment has occurred. 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. Unfortunately, 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. There thus exists a great need for an efficient procedure capable of economic extraction, separation and purification of cancer-free stem cells. Currently, two competing processes exist. 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). In this process, 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. Next, 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.

To cleanse the collected stem cells of cancerous cells, the same strategy can be employed, albeit with cancer-specific antibodies. Alternatively, 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 most important limitation is that the recovery and purification steps fall into the category of what is known as "single-stage" processes, whereas both the efficiency and purity levels increase with multi-staging. Therefore, there is a great need for a rapid, efficient, economic, and preferably multi-stage-able process for the extraction and purification of cancer-free stem cells.

In this disclosure, we present a revolutionary method for the recovery and purification of cancer-free stem cells as an illustration of the power of a new class of materials: conjugates of smart polymers and antibodies. Another example entails the coupling of smart polymers with proteinaceous entities, such as enzymes and soluble or membrane-bound receptors that form the majority of targets (gateways) where pharmaceutical drugs are designed to bind (interact). Before we discuss the full potential of our novel approach, we review the state-of-the-art of the drug discovery industry. Drug discovery has witnessed significant advances in recent years with the development of "combinatorial chemistry." Here, a large number of compounds are made with the hope of finding a few "hits" that bind with the target protein receptor or enzyme. This large array of compounds is termed a library. Both polymer-based and small molecule-based libraries are available. 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.

After synthesis, the library compounds must be individually screened for bioactivity. Micro-fluidics and microfabrication are needed to conserve the quantities of the targets and reagents. There is thus a major drive towards the development of "analysis on a chip", i.e., functionalized chips designed to test for bioactivity of the numerous compounds of the many libraries. Screening must be done for the numerous targets relevant for the different diseases. Although parallel processing of multiple samples (compounds) can be envisioned using multiple channels on a miniaturized device, bioactivity screening remains a daunting and time-consuming task. The need remains for a selective, high-throughput screening process to reduce a huge compound library to a handful of useful compounds, thus drastically streamlining the necessary work needed to identify the most promising drug candidates. Summary of the Invention:

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. Many unique performance characteristics can be envisioned by this novel combination of synthetic and biologically derived materials.

Generally, the smart polymer hybrid composite articles of this invention provide novel identification, recovery and/or purification methods. Examples of 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.

In typical operation, 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.

When the polymer whiskers are in an expanded state, the polymer hybrid composite articles are dispersed in the solvent. The term "solvent" as used herein is interchangeable with the terms "solution", "media", "reactant media", "biological fluid", and "biological media". When the polymer whiskers are expanded, the bioactive entity (such as an enzyme catalyst) is distanced from all other likewise-modified bioactive entities and each bioactive entity is then free to perform its function.

If the critical environmental stimulus of the solvent containing the polymer hybrid composite article, such as temperature, changes and attains or surpasses a critical point, the expanded polymer whiskers of the composite article contract. When the polymer whiskers are in a contracted state, the polymer hybrid composite articles coalesce in the solvent, activity is temporarily halted and the composite articles are easily removed from the solution.

Under controllable conditions, expansion and contraction of the polymer whiskers leads to reversible switching on and off of the activity of the bioactive entity.

No matter what type of bioactive entity is used, 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, in accordance with this invention, 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.

When the polymer hybrid composite article is in a solvent, 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. As a result, when a plurality of the polymer hybrid composite articles are in the expanded state in the solvent, 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.

In a preferred embodiment, 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.

These and other advantages will be clear to those skilled in the art in view of the following discussions.

As used herein and in the appended claims, "a" and "an" mean one or more. Brief Description of the Drawings: For a further understanding of the objects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying drawings, in which like parts are given like reference numerals and wherein: 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, and "UCST" means 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.

Three polymer-enzyme composite articles are shown on the right in their collapsed, inactive state, the polymer whiskers covering the active sites of the enzymes. Collapse of the polymer whiskers also induces flocculation, enabling enzyme or cell removal. The expansion and contraction is caused by a reversible thermodynamic transition.

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. Detailed Description of Modes for Carrying out the Invention:

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. When 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. Below are presented several applications to illustrate the power and utility of this synergistic combination of synthetic polymer and biologically derived material.

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. The term "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.

When the polymer whiskers are expanded, 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. When the polymer whiskers are in the extended state, the polymer hybrid composite articles are stable and remain in the dispersed state.

When the polymer whiskers collapse, two phenomena occur. First, 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. When a target molecule or entity, such as a drug candidate or a cell, is conjugated to the bioactive entity of the composite article, the target entity will be taken along with the composite article as it coalesces and can then be collected. Second, 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. Hence, when the active sites of the bioactive entity are blocked, 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. When a cell is enclosed by the collapsed polymer whiskers, 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. For example, when death of a target cell, such as a cancer cell, is desired, 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. Alternatively, when it is desirous to extract and purify a viable target cell, 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.

Alternatively, a second bioactive entity or toxicological substance may be attached to the free end of the polymer whisker. 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. When 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.

Critical Solution Transitions of Switchable Polymer Whiskers:

FIG. 1 illustrates switchable polymer whiskers in solution undergoing abrupt thermodynamic transitions. These systems are well known in the art and are explained in detail in "Responsive Gels: Volume Transitions I and II", K. Dusek, ed., Advances in Polymer Science, Vols. 109 and 110, Springer- Verlag, 1993, herein incorporated by reference. The polymer whiskers extend or collapse significantly upon minor changes in certain thermodynamic parameters, such as temperature, pH, light, pressure, electric field strength, ionic strength, or solvent composition. In a given thermodynamic state, the polymer segments along a backbone of a polymer whisker prefer to be surrounded by solvent molecules, leading to polymer whisker extension. The terms "extend" and "expand" are used interchangeably herein to mean "to open or stretch out," "to spread out" and "to increase in bulk by absorbing solvent".

Under a different condition, 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.

As an example, 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. For certain polymer-solvent combinations, lowering the temperature leads to polymer whisker collapse; i.e., phase separation from a homogeneous solution to an opaque two-phase mixture. For certain other systems, 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. In general, 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". A limited number of non-aqueous LCST systems exist. There are two types of LCST transitions. 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; and copolymers thereof. If crosslinking is desired between the whiskers, multifunctional compounds such as bis-acrylamide and ethoxylated trimethylol propane triacrylate and sulfonated styrene may be employed. In presently preferred embodiments, the polymer whiskers comprise polyacrylamides, substituted polyacrylamides, polyvinylmethyl ethers, and modified celluloses. In a preferred embodiment, 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.

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.

In this regard, 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.

Another main polymer system that exhibits useful transitions is the block copolymer series (di-block, tri-block, and multi-block) of ethylene glycol and propylene glycol.

Literature abounds in reports of water-soluble polymers that undergo coil- globule transitions. Certain polypeptide sequences are known to undergo such transitions. The above examples are known LCST systems. UCST systems are more numerous. Most if not all can be adapted for the intended purpose expressed here. We will term such useful polymers for coupling with the bioactive entities "smart polymers". "Coupling", "linking", and "tagging" will be used analogously in this disclosure to mean the combination of the bioactive entity and the smart polymer(s) by chemical attachment or physical adsorption. Both methods of combination must withstand the transition induced in downstream usage of the conjugate. 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. If the polymer whisker is chemically tethered, then 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. ("Polymeric Stabilization of Colloidal Dispersion", D.H. Napper, Academic Press, 1983).

Coupling a synthetic polymer with a protein is known in the art. The April 1997 issue of Polymer Preprints, Vol. 31 , No. 1 , a compilation of publications outlining the lectures delivered at the 1997 American Chemical Society, Division of Polymer Chemistry annual meeting, contains several articles reporting the attachment of polyethylene glycol or other water-soluble polymers to proteins and enzymes. Protocols are found throughout this issue, for example, pages 563-572. These articles are herein incorporated by reference; the objectives are to enhance protein stability in mixed aqueous environments, for industrial reactions.

Techniques of protein or enzyme coupling, traditionally to either an immobilization matrix or support, or a stabilizing encapsulating sheath of synthetic polymer whiskers, can be accomplished in a number of ways. Excellent references and protocols are found in the body of literature known as enzyme immobilization. (See, for example, "Immobilized Enzyme Principles", L.B. Wingard, E. Katchalski- Katzir, and L. Goldstein, eds., Academic Press, 1976, herein incorporated by reference). For example, substituted polyacrylamide and their copolymers may be employed as smart polymer whiskers. Numerous coupling methods have been documented, including activation with glutaraldehyde, conversion into acyl hydrazide derivative, activation by diazotization, activation of carboxyls with water-soluble carbodiimide, cyanogen bromide activation, etc. As the field of enzyme immobilization advances, new methodologies will be developed to couple proteins with synthetic polymers including the polymer whiskers of this invention. These advances can be exploited for carrying out the present invention.

Finally, 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). For example, 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) tend to adsorb the hydrophilic block. Conversely, 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.

The chemical attachment approach for making a hybrid relies on well- documented synthesis routes. 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.

In a second step, the activated bioactive entities are mixed with reactive monomers that are subsequently polymerized in-situ. N-isopropylacrylamide (NIPA) is one such example monomer. The resulting polymer exhibits LCST behavior in aqueous solution. When the polymer whisker is in the expanded state, the polymer whisker is water-soluble, causing dispersion of the polymer hybrid composite article. As soon as the temperature is heated above the LCST point, the polymer whiskers collapse and the polymer hybrid composite articles aggregate. In some cases, the hybrid composite articles precipitate from solution rapidly. In other cases, the flocculated compounds float in the solution and are easily separated from it. Other 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. Further, 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. For examples, see Vincent, B., Chem. Engineering Science, V. 48, pp. 429-436, 1993; and Albaglia, D., Bazan, G.C., Schrock, R.R., and Wrighton, M.S., Journal of the American Chemical Society V. 115, pp. 7328-7334, 1993. 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. As indicated in FIG. 3, linking "smart" polymer whiskers 1 (whiskers that undergo UCST or LCST transitions) to industrially important polymer particles 2 (as a special class of bioactive entities) endows the latter with reversible dispersion and flocculation properties. In FIG. 3A, the polymer whiskers of the polymer hybrid composite articles are extended and the articles are dispersed throughout the solution. As a result of a thermodynamic change, 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.

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.

The polymer hybrid composite articles also allow strong agitation and prolonged service life. Although not wishing to be bound by theory, we believe that these additional benefits arise due to attachment of the polymer whiskers, making the dispersed bioactive entities more robust and less fragile than the unmodified bioactive entities of the prior art. Catalysts: Certain of the polymer hybrid composite articles of the present invention are useful as enzyme catalysts. 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.

4).

For example, when the polymer-catalyst hybrid composite article of this invention is placed in an exothermic reaction, the polymer whiskers are designed to contract at a temperature that is deemed to be a danger point of becoming a runaway reaction. When local temperatures reach the "danger temperature", 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. When the local temperatures cool, 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.

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. Alternatively, 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. In this manner, a polymer hybrid composite article may be formed consisting of one or more polymer chains, a receptoπligand pair, and a cell of interest. At this point, 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. By choosing a bioactive entity selective to the appropriate cell type, 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.

As an example, in one embodiment of the invention 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. After a period of time sufficient for conjugation of the stem cells to the antibody, 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. To then collect the stem cells, 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. To selectively inactivate or kill deleterious cells, 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. When they are contracted, 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. As one example, in a treatment for cancer, first an anti-cancer antibody is linked to one or more smart polymers, and then this hybrid is injected into the human body.

Cancer cells will then be covered with this antibody-whisker hybrid entity. High fever is then induced in a patient, causing collapse of the whiskers, which then choke off the transport of materials into and out of the targeted cancer cells by blocking the transport portals. Ref: "Building Doors into Cells" by H. Bayley, Scientific American, September 1997, pp. 62-7. Combinatorial Chemistry:

Certain of the 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. Examples of ligands that can be investigated by this invention 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.

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. Examples of 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. Other examples of 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. c) 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. Other examples are the vasoconstrictive hormone receptors; determination of those ligands that bind to such a receptor may lead to the development of drugs to control blood pressure. d) Opiate receptors: Determination of ligands that bind to the opiate receptors in the brain is useful in the development of less-addictive replacements for morphine and related drugs. To screen for biological activity, a receptor 9 for the desired activity, such as an antibody, 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). After a period of time, 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.

An alternative application within the field of combinatorial chemistry for the polymer hybrid composite articles described herein is their use as platforms to synthesize directed combinatorial libraries of compounds such as polymers, oligonucleotides, small molecules and the like. 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. 2288-2337; "Combinatorial Chemistry and New Drugs," M.J. Plunkett and J.A. Eilman, Scientific American, April 1997; The Combinatorial Chemistry Catalog, Calbiochem-Novabiochem Corp., March 1998; Combinatorial Chemistry : Synthesis and Application, edited by Stephen R. Wilson, Anthony W. Czarnik, New York : Wiley, c1997; and, Combinatorial Chemistry, by Nicholas K. Terrett, Oxfordm New York : Oxford University Press, 1998; Series title: "Oxford Chemistry Masters 2".

In this regard, smart polymers conjugated to a bioactive entity, which functions as a "building block", may serve as starting points for the chemical synthesis reactions typically employed in the field. 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. After each reaction step, polymer flocculation may be induced, allowing collection of the intermediate species for rinsing and purification. In this manner, excess reactants and byproducts can be easily separated from the reaction media, followed by re-dispersion of the polymer hybrid composite articles into solution. This method would allow for the advantages of both solution-phase synthesis (wide range of reaction steps, highly accessible reaction sites, etc.) and solid-phase synthesis (easy separation of reactants and products, etc.) to be realized in the formulation of highly diverse chemical libraries.

Example 1: Industrial Enzyme with a Tethered LCST or UCST Synthetic Polymer

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). These example methods include: (1) N- hydroxysuccinimidyl (NHS) ester derivative, which reacts with the free amines of the N-terminus or the lysine residues to form stable amide bonds; (2) carbonyl imidazole derivative of polyethylene glycol (CI-PEG), which reacts with the enzyme bioactive entity resulting in an urethane linkage; (3) tresyl derivative of PEG (Tresyl-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; (4) aldehyde derivative of PEG (AdPEG), which forms a secondary amine via the reductive amination in the presence of sodium cyanoborohydride; and (5) 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. The polymer-enzyme hybrid composite article thus remains freely dispersed in the aqueous solution indefinitely. Upon heating above the LCST point (but below the enzyme deactivation temperature), the extended polymer whiskers collapse into coils, effectively blocking the enzyme surface and causing enzyme precipitation.

The above tethering of polymer whiskers can be applied to all proteins amenable to coupling treatment, including enzymes (e.g., subtilisin). Example 2: Formation and Separation of Polymer Coagulant from Saline Solution A 1 wt% solution of PVME (polyvinylmethyl ether) in saline solution is formed by adding 0.114 grams of PVME (35 wt% in water, Mw = 200 k, Scientific Polymer Products, Inc., Catalog No. 899, Ontario, New York) to 3.89 grams of Dulbecco's PBS solution (Phosphate-Buffered Saline, JRH Biosciences, Catalog No. 59321 -78P, Lenexa, KS). The solution is shaken for several hours until complete dissolution of the PVME has occurred, producing a clear and homogenous mixture. Next, the glass vial containing the solution is placed into a temperature-controlled water bath, initially at room temperature. Upon heating the water bath to approximately 30 °C, the PVME solution turns cloudy. Upon further heating to 35 °C, 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. 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. When the polymer solution reaches its cloud point as it is being heated, 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. When the polymer solution reaches its cloud point as it is being heated, 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. When 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. There is typically no polymer precipitate at the bottom of the vial (unlike in Examples 2 and 3), and the supernatant is more clear (less cloudy) compared to Examples 2 and 3, particularly just after shaking has occurred. This lump at the top of the vial can be easily removed from the supernatant solution using tweezers or a spatula and can be placed into a separate clean vial for resuspension in solution.

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. Alternatively, 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. When the polymer solution is heated, it will turn cloudy at the cloud point temperatures listed in Table 1 or Table 2 ("DI" means "deionized"). Upon clouding and/or further heating, 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.

TABLE 1

Cloud point temperatures of 1 wt% polymer solutions.

¹ 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. For these solutions, 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. When these polymer mixtures are dissolved in buffered saline (PBS), they will form a visible polymer coagulant upon heating to the temperatures listed in Table 3. The polymer coagulant may be settled, filtered, or otherwise collected. TABLE 3 Coagulation temperatures for polymer mixtures in PBS

Example 7: Alternative Collection Method for Polymer Coagulant Separation

A 1 wt% solution is formed as described in Example 2. Upon formation of the polymer coagulant (see Examples 2, 3, or 4), 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. Alternatively, 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.

When the column cools to below the polymer cloud point, the column is eluted with four 5-mL increments of room-temperature saline. The polymer will elute with the washes, concentrated in the second and third washes, as evidenced by polymer coagulant formation upon heating. Example 9: Polymer Separation and Recovery from Glass Bead Mixture A. SOLUTION PREPARATION

A 2 wt% PVME solution is formed in a glass vial by adding 0.40 grams of polyvinylmethyl ether polymer (50 wt% in water, Mw = 90 k, Scientific Polymer Products, Inc., Catalog No. 025, Ontario, New York) to 9.6 grams of deionized water (Ricca Chemical Co., Catalog No. 9150-1 , Arlington, Texas). Hollow glass beads having a nominal density of 1.1 g/cc and a mean particle size of 11.7 microns (Potters Industries, Product No. 110P8, Valley Forge, Pennsylvania) are added to the polymer solution in the amount of 0.5 g per 10 mL of solution, and the solution is well stirred. B. POLYMER SEPARATION

To separate out the polymer from the beads, the solution is heated while gently agitating every 30 seconds to one minute. When 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). When the solution reaches 37 °C, the vial is opened and the liquid supernatant is decanted away, leaving the solid polymer coagulant behind.

Next, the polymer coagulant is resuspended in solution by adding room- temperature deionized water to bring the total solution weight back to 10 grams. When all of the polymer has redissolved into solution, 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. C. RESULTS

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 mm² of area. After the first washing step, a liquid drop of the polymer solution (after redissolution into clean deionized water) shows a greatly decreased concentration of glass beads, estimated to be approximately 500 beads per mm². After a second washing step, the glass bead concentration is reduced further yet, estimated to be approximately 10 beads per mm². After a third washing step, no beads can be found in the 1-mm² field of view of the microscope.

Thus, with three subsequent washing steps, 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. Also, 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. Alternatively, instead of using decantation, 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

A. SOLUTION PREPARATION

A 2 wt% PVME solution is formed in a glass vial by adding 2.0 grams of PVME (Mw = 200 k) from a 10 wt% stock solution in saline to 8.0 grams of buffered saline solution. 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.

B. POLYMER SEPARATION To separate out the polymer from the cells, the solution is heated while agitating every 30 seconds to one minute. The agitation is vigorous enough to produce small bubbles in the solution each time, and usually lasts no longer than 5 seconds. When the polymer solution reaches its cloud point of 31 °C, white precipitate forms, which will float to the top of the vial. When the solution reaches 37 °C, the vial is opened and the solid polymer coagulant is removed from the vial and placed into a clean vial using a spatula, leaving the liquid supernatant behind. Small portions of the polymer coagulant sometimes have to be scraped from the sides of the vial where it sticks; recapping the vial and shaking periodically will help free the coagulant from the vial sides.

Next, 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. When all of the polymer has redissolved into solution, 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. C. RESULTS

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 mm² of area. After the first washing step, a liquid drop of the polymer solution (after redissolution into clean saline) shows a greatly decreased concentration of yeast cells, estimated to be approximately 100 beads per mm². After a second washing step, the yeast cell concentration is reduced further yet, estimated to be less than 1 cell per mm². After a third washing step, no cells can be found in the 1-mm² field of view of the microscope.

Thus, with three subsequent washing steps, the yeast cells have been separated from the polymer solution, to the point where the polymer solution contains no yeast cells above the detectable limit. Example 11 : Smart-Polymer Conjugation to Stem-Cell Antibodies A. BACKGROUND

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. To selectively modify, coagulate, collect, and purify CD34+ progenitor cells from a diverse mixture of cells in accordance with this invention, 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.

B. CROSS-LINKER CONJUGATION TO MONOCLONAL ANTIBODIES

A particularly suitable cross-linker for conjugating smart-polymer chains and mAbs is 4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide (M₂C₂H, 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. 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. 26102, Rockford, IL), which reacts with terminal amines to create a protected sulfhydryl group through a stable amide linkage. This modified, protected sulfhydryl group may be stored without degradation, and can be subsequently deprotected as needed with an excess of hydroxylamine.

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. For example, SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity, and it has also been used in the formation of therapeutically useful toxin conjugates with recombinant CD4.

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 μL (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. To deprotect the sulfhydryl groups for subsequent reaction with maleimide- containing cross-linking agents, the protocol is: (1) deprotect the acetylated -SH groups by adding 100 μL (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

EDTA, pH 7.5. The deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation.

The above modification protocols are given in the text Bioconjugate Techniques by Greg T. Harrison, Academic Press, 1996, herein incorporated by reference, and many other protocols and schemes are available for the controllable creation of sulfhydryl groups on proteins.

To conjugate the heterobifunctional cross-linking agent to the mAbs of interest (i.e., QbendIO or Thy1), the sulfhydryl-activated mAb is mixed with an excess of M₂C₂H 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.

Note, the carbonyl-reactive hydrazide group at the other end of the heterobifunctional cross-linking agent (M₂C₂H) 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 M₂C₂H 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. Alternatively, 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.

For PVME conjugation to occur via the above mechanism, 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 M₂C₂H cross-linker to form hydrozone bonds, thus producing a fully coupled smart polymer-mAb conjugate, abbreviated by PVME-M₂C₂H-Thy1 or PVME-M₂C₂H-Qbend10. D. ALTERNATIVE APPROACHES

It is possible, and perhaps even desirable, that more than one smart-polymer linkage will occur with each mAb, through multiple cross-linker attachments to the mAb. Those experienced in the art will be able to recognize and select cross-linking agents which attach to mAbs at multiple sites, or in which each cross-linking molecule possesses multiple attachment sites for smart-polymer linkages. With or without multiple smart-polymer linkages to the mAb, alternative chemical attachment schemes between the mAb and the cross-linking agent and/or between the cross- linker and the smart-polymer chain may also be envisioned. A detailed discussion of suitable cross-linkers and their conjugation chemistry can be found in Bioconjugate Techniques by Greg T. Harrison, Academic Press, 1996. A list of suitable cross- linkers may be chosen from Table 4, and using the appropriate coupling reaction may be used to form a smart polymer-mAbs conjugate.

TABLE 4 Suitable Cross-Linking Reagents for the Formation of Smart-Polymer-Monoclonal

Antibody Conjugates

^' Suifonated, water-soluble analog also available. Example 12: Stem Cell Separations Using Smart Polymer-mAbs Conjugates In order to separate stem cells from a diverse mixture of cells, 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. For a cell mixture containing approximately 10¹⁰ total cells and 10⁸ (100 million) stem cells, 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.

Approximately 22 grams of polymer are added and dissolved (below the polymer cloud point temperature) to create a 2 wt% polymer mixture.

Next, 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). Finally, 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.

Claims

WHAT IS CLAIMED IS:

1. A composite article comprising: a bioactive entity; and at least one polymer whisker attached to said bioactive entity, said polymer whiskers being controllably expandable and contractible.

2. A composite article according to claim 1 wherein said composite article is in a solvent or a reactant medium, said polymer whiskers and said solvent or medium interact to cause said polymer whiskers to reversibly expand and contract in response to one or more changes in environmental conditions, at least some of said polymer whiskers being solvated when said polymer whiskers expand, and said absorbed solvent being expelled from said polymer whiskers when said polymer whiskers contract.

3. A composite article according to claim 1 wherein said polymer whiskers are selected from the group consisting of N-isopropyl acrylamide and acrylamide; polyethylene glycol, di-acrylate and hydroxyethyimethacrylate; 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; and copolymers thereof.

4. A composite article according to any of claims 1 , 2 or 3 wherein said bioactive entity is selected from the group consisting of a cell, an enzyme, an antibody, a receptor, nucleic acid, and a small molecule functional group.

5. A composite article according to claim 4 wherein said bioactive entity is a receptor having an affinity for a ligand.

6. A composite article according to claim 4 wherein said bioactive entity is an antibody having an affinity for a stem cell.

7. A method for selecting and recovering stem cells from media, the method comprising: contacting media containing stem cells with a composite article comprising i) an antibody specific to said stem cells, and ii) at least one polymer whisker attached to said antibody, said polymer whiskers being controllably expandable and contractible; adjusting environmental conditions of said media to cause said polymer whiskers to expand, allowing said antibody to be exposed to said media and allowing said antibody to be attracted to and attached to said stem cells due to antigen- antibody pairing; after a period of time sufficient for conjugation of said stem cells to said antibody, adjusting environmental conditions of said media to cause said polymer whiskers to contract and coalesce; and collecting coalesced composite article-stem cell conjugates.

8. A method according to claim 7 wherein the steps are repeated one or more times.

9. A method according to claim 7 which comprises the further steps of. exposing said coalesced composite article-stem cell conjugates to conditions wherein said stem cells are released from said antibody; and separating said stem cells from said composite articles.

10. A method for selectively inactivating or killing deleterious cells, the method comprising: contacting a solvent containing deleterious cells with a composite article comprising i) a receptor having an affinity for said deleterious cells, and ii) at least one polymer whisker attached to said receptor, said polymer whiskers being controllably expandable and contractible; adjusting environmental conditions of said solvent to cause said polymer whiskers to expand, allowing said receptor to be exposed to said solvent and allowing said deleterious cells to be attracted to and attached to said receptor; and after a period of time sufficient for attachment of said deleterious cells to said receptor, adjusting environmental conditions of said solvent to cause said polymer whiskers to contract; wherein, the number of said polymer whiskers is selected so that said deleterious cell is substantially enclosed by said whiskers, resulting in loss of said cell's ability to function.

11. A method according to claim 10 wherein said polymer whiskers enclose said deleterious cell for a period of time sufficient to kill said cell.

12. A method according to claim 10 or 11 wherein said deleterious cell is a cancer cell.

13. A method of selecting and isolating a targeted ligand from a mixture of ligands, the method comprising: contacting a mixture of ligands with a composite article comprising i) a receptor having an affinity for a target ligand, and ii) at least one polymer whisker attached to said receptor, said polymer whiskers being controllably expandable and contractible; adjusting environmental conditions of said mixture to cause said polymer whiskers to expand, allowing said receptor to be exposed to said mixture and allowing said target ligand to be attracted to and attached to said receptor; after a period of time sufficient for conjugation of said target ligand to said receptor, adjusting environmental conditions of said mixture to cause said polymer whiskers to contract and coalesce; and collecting coalesced composite article-target ligand conjugates.

14. A method according to claim 13 which comprises the further steps of: exposing said coalesced composite article-target ligand conjugates to conditions wherein said target ligand are released from said receptor; and separating said target ligands from said composite articles.

15. A method for controlling the activity of a bioactive entity, the method comprising: adding to a solvent a composite article comprising i) a bioactive entity having active sites, and ii) at least one polymer whisker attached to said bioactive entity, said polymer whiskers being controllably expandable and contractible in response to changes in environmental conditions of the solvent; adjusting environmental conditions of the solvent to expand said polymer whiskers away from said bioactive entity, so that said active sites are exposed and said bioactive entity is active; and adjusting environmental conditions of the solvent to contract said polymer whiskers so that said whiskers coil around said bioactive entity to block said active sites, thereby rendering said bioactive entity essentially inactive.

16. A method according to claim 15 wherein the steps are repeated one or more times, depending on said changes in environmental conditions of the solvent.

17. A method according to claim 15 or 16 which further comprises an additional step of collecting and removing coalesced composite articles from the solvent, said composite articles being coalesced when said polymer whiskers are contracted.

18. A method according to claim 15, 16, or 17 wherein said bioactive entity is selected from the group consisting of a cell, an enzyme, an antibody, a receptor, nucleic acid, and a small molecule functional group.

19. A method according to claim 15, 16, or 17 wherein said bioactive entity is an enzyme catalyst and said solvent is a reactant medium.

20. A method for synthesizing a combinatorial library of compounds, the method comprising: adding to a solution of combinatorial building blocks a composite article comprising: i) a selected initial building block, and ii) at least one polymer whisker attached to said initial building block, said polymer whiskers being controllably expandable and contractible; adjusting environmental conditions of said solution to cause said polymer whiskers to expand, allowing said initial building block to be exposed to said nucleic acids in solution and allowing attachment of additional building blocks to said initial building block, either in a random or a controlled manner, to provide a sequence of building blocks; after a time sufficient for attachment of said additional building blocks to said initial building block, adjusting environmental conditions of said solution to cause said polymer whiskers to contract and coalesce; and collecting coalesced composite article-building block sequence.

21. A method according to claim 20 which comprises the further steps of: adding said coalesced composite article-building block sequence to a second solution of building blocks; adjusting environmental conditions of said second solution to cause said polymer whiskers to expand, allowing said building block sequence to be exposed to said building blocks in the second solution and allowing attachment of additional building blocks to said building block sequence; after a time sufficient for attachment of said additional building blocks to said building block sequence to give a longer building block sequence, adjusting environmental conditions of said second solution to cause said polymer whiskers to contract and coalesce; and collecting said coalesced composite article-longer building block sequence; wherein the steps are repeated one or more times to attach additional building blocks to said building block sequence.

22. A method according to claim 20 or 21 which comprises the further steps of: exposing said coalesced composite article-building block sequences to conditions wherein said building block sequence is released from said initial building block, and separating said building block sequences from said composite articles.

23. A method according to any of claims 7 to 22 wherein said polymer whiskers are selected from the group consisting of 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; and copolymers thereof.

24. A composite article according to any of claims 4 to 6 wherein said changes in environmental conditions that cause said polymer whiskers to reversibly expand and contract are selected from the group consisting of temperature, pH, light, pressure, electric field strength, ionic strength and solvent composition.

25. A method according to any of claims 7 to 23 wherein said changes in environmental conditions that cause said polymer whiskers to reversibly expand and contract are selected from the group consisting of temperature, pH, light, pressure, electric field strength, ionic strength and solvent composition.