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(1999) Biotechnol. Bioeng. 62, 269277 28 McLaren, A. D. and Packer, L. (1970) Adv. Enzymol. 33, 245308 29 Goldstein, L., Levin, Y. and Katchalski, E. (1964) Biochemistry 3, 19131919 30 Trevan, M. D. (1980) in Immobilized Enzymes: An Introduction and Applications in Biotechnology (Trevan, M. D., ed.), pp. 1155, John Wiley & Sons 31 Ruckenstein, E. and Sasidhar, V. (1984) Chem. Eng. Sci. 39, 11851200 32 Kasche, V. and Bergwall, M. (1977) in Insolubilized Enzymes (Salmona, C., Saranio, M. and Garattini, S., eds), pp. 7786, Raven Press 33 Rolinson, G. N. (1988) J. Antimicrob. Chemother. 22, 514 34 Liou, J. K. and Rousseau, I. (1986) Biotechnol. Bioeng. 28, 15821589 35 Bailey, J. E. and Chow, M. T. C. (1974) Biotechnol. Bioeng. 16, 13451357 36 Ruckenstein, E. and Rajora, P. (1985) Biotechnol. Bioeng. 27, 807817 37 Dickinson, M. and Fletcher, P. D. I. (1988) Enzyme Microb. Technol. 11, 5556 38 39 40 41 42 43 Klibanov, A. M. (1997) Trends Biotechnol. 15, 97101 Carrea, G., Ottolina, G. and Riva, S. (1995) Trends Biotechnol. 13, 6370 Tsai, S-W. and Dordick, J. S. (1996) Biotechnol. Bioeng. 52, 296300 Zacharis, E., Moore, B. and Halling, P. J. (1997) J. Am. Chem. Soc. 119, 1239612397 Bedell, B. A., Mozhaev, V. V., Clark, D. S. and Dordick, J. S. (1998) Biotechnol. Bioeng. 58, 654657 Khalaf, N., Govardhan, C. P., Lalonde, J. J., Persichetti, R. A., Wang, Y-F. and Margolin, A. L. (1996) J. Am. Chem. Soc. 118, 54945495 Lilly, M. D. and Woodley, J. M. (1985) in Biocatalysts in Organic Synthesis (Tramper, J., van der Plas, H. C. and Linko, P., eds), pp. 179191, Elsevier Scheper, T. (1990) Adv. Drug Delivery Rev. 4, 209231 Chang, H. N. and Furusaki, S. (1997) Adv. Biochem. Eng. 44, 2764 Nagayasu, T., Miyanaga, M., Tanaka, T., Sakiyama, T. and Nakanishi, K. (1997) Biotechnol. Bioeng. 43, 11181123 Tyn, M. T. and Gysek, T. W. (1990) Biotechnol. Bioeng. 35, 327338 Spieb, A., Schlothauer, R., Hinrichs, J., Scheidat, B. and Kasche, V. (1999) Biotechnol. Bioeng. 62, 267277

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Smart polymers and what they could do in biotechnology and medicine

Igor Y. Galaev and Bo Mattiasson
Stimulus-responsive or smart polymers undergo strong conformational changes when only small changes in the environment (e.g. pH, temperature, ionic strength) occur. These changes result in phase separation from aqueous solution or order-ofmagnitude changes in hydrogel size. Smart polymers are used in bioseparation and drug delivery, for the development of new biocatalysts, as biomimetic actuators, and as surfaces with switchable hydrophobichydrophilic properties.

ife is polymeric in its essence: the most important components of living cell (proteins, carbohydrates and nucleic acids) are all polymers. Nature uses polymers both for construction and as part of the complicated cell machinery. The salient feature of functional biopolymers is their all-or-nothing, or at least highly nonlinear, response to external stimuli small changes happen in response to a varying parameter until a critical point is reached, when a large change occurs over a narrow range of the varying parameter; after the transition is completed, there is no significant further response of the system. These nonlinear responses by biopolymers are caused by highly cooperative interactions. Despite the weakness of each particular interaction taking place in a separate monomer unit, when summed over hundreds and thousands of monomer units, these interactions can provide significant driving forces for the processes occurring in the whole system.

I. Y. Galaev (igor.galaev@biotek.lu.se) and B. Mattiasson are at the Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, Lund, SE-221 00, Sweden.
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Not surprisingly, an understanding of the mechanism of cooperative interactions in biopolymers has opened the floodgates to attempts to mimic this cooperative behavior in synthetic systems. Recent decades have witnessed the appearance of synthetic functional polymers that respond in some desired way to a change in temperature, pH, electric or magnetic field, or some other parameter. These polymers were originally called stimulus responsive but the name smart polymers was coined based on their similarity to biopolymers1. Smart polymers and hydrogels undergo fast, reversible changes in microstructure from a hydrophilic to a hydrophobic state. These changes are triggered by small changes in the environment but are apparent at the macroscopic level as precipitate formation from a solution or order-of-magnitude changes in the size and water content of hydrogels. These macroscopic changes are also reversible, the system returning to its initial state when the trigger is removed2. The driving force behind these transitions varies, with common stimuli including the neutralization of charged groups by either a pH shift3 or the addition of an oppositely charged polymer4, changes in the efficiency of hydrogen bonding with an increase in temperature or ionic strength5, and

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Immobilized viable cells

Thermally controlled biocatalyst

Reversibly soluble biocatalyst

Immobilized biocatalyst Biomimetic actuators

Drug delivery

Smart polymer

Chemical valve

Bioseparation

Thermoresponsive surfaces
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Aqueous two-phase polymer systems Affinity precipitation

Thermoresponsive chromatography

Cell detachment

Figure 1 Uses of smart polymers in biotechnology and medicine.

collapse of hydrogels and interpenetrating polymer networks6. An appropriate balance of hydrophobicity and hydrophilicity in the molecular structure of the polymer is believed to be required for the phase transition to occur. Such highly nonlinear responses by smart polymer systems have mainly been observed in water and, occasionally, in organic solvents7 or polymer blends8. As the stimulus-responsive behavior occurs in aqueous solutions, these polymers and hydrogels are becoming increasingly attractive for biotechnology and medicine (Fig. 1). There have been numerous reviews of this field3,57,914 and so we will focus on recent developments. Biomimetic actuators There have been numerous attempts to mimic the efficient conversion of chemical energy into mechanical energy in living organisms15. Ionic hydrogels show a discontinuous volumetric change (a first-order phase transition) above a certain threshold of an external stimulus such as pH, temperature, ionic strength or concentration of organic solvent. At the phase-transition point, the swollen phases can be several hundred times the volume of the collapsed state and the transition can exert a significant force. An electric field is easily controlled and manipulated, and is an attractive example of a stimulus that induces a volumetric change in a polymer gel. A crosslinked gel of poly(vinyl alcohol) chains entangled with polyacrylic-acid chains has good mechanical properties and shows rapid electric-field-associated bending deformation: a gel rod of 1 mm diameter bends semicircularly within 1 sec on the application of

an electric field16. An artificial fish with a gel tail swam forward at a velocity of 2 cm s 1 as the gel oscillated under sinusoidally varied electric fields; a mechanical hand composed of four gel fingers could pick up a fragile quail egg (9 g) from a sodium-carbonate solution and hold it without breaking it, controlled by an electrical signal16. Polymer gels capable of mechanical response to electric field have also been developed using the cooperative binding of positively charged surfactant molecules to the polyanionic polymer poly(2acrylamido-2-methyl-1-propanesulfonic acid)17. The models mentioned above are driven in solution. A mechanical hand composed of two fingers capable of working in air was constructed from hybrid gels containing internal platinum wires as electrodes. The fingers could bend inwards simultaneously and catch a piece of paper18. Another solid-state artificial muscle based on poly(pyrrole)poly(epichlohydrin-co-ethylene oxide)LiClO4 (a solid polymeric electrolyte) has recently been reported19. The state of the gel (swollen or collapsed) depends on the balance of attractive and repulsive forces between the polymer chains (which are usually electrostatic). Copolymer gels consisting of N-isopropylacrylamide and acrylic acid would be useful for constructing biochemomechanical systems. A pH-induced change in the COOH ionization of acrylic acid alters the repulsive force; the attractive force is produced by hydrophobic interactions arising from the dehydration of the N-isopropylacrylamide moieties. Glucose dehydrogenase catalyses the conversion of neutral glucose into gluconic acid, accompanied by a decrease in pH. When this reaction occurs inside the gel, the repulsive forces are eliminated owing to the protonation of COO groups. As a result, the attractive forces dominate, followed by the collapse of the gel. Changes in the gels size (about 1.5 times) using immobilized glucose dehydrogenase are initiated by pulses of a 40 mM glucose solution. However, the process is relatively slow, taking 6 h for the changes to complete20,21. Biomimetic actuators composed of smart polymers form a biochemomechanical system capable of transforming chemical energy directly into mechanical work, a property also exhibited by living organisms. The biomimetic actuators could be used in future soft machines that are designed using more biological than mechanical principles. In contrast to biological systems, however, biomimetic actuators can withstand very hostile environments. Immobilized biocatalysts When an enzyme is covalently coupled to a smart polymer either in solution or in a hydrogel, drastic changes in the polymer conformation produced by environmental changes can significantly affect enzyme activity and substrate access to the enzyme molecule. The transition between the soluble and insoluble states of smart polymers has been used to develop reversibly soluble biocatalysts. These biocatalysts catalyse an enzymatic reaction in their soluble state and hence can be used in reactions with insoluble or poorly soluble substrates or products: as soon as the reaction is complete and the products are separated, the conditions are changed to cause the catalyst to precipitate so that it can be separated and used in the next cycle, after redissolution.
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A wide range of smart polymers has been used for the development of reversibly soluble biocatalysts whose solubility is controlled by pH2225 or temperature2632. For example, -glucosidase immobilized on a pH-responsive, reversibly soluble polymer (hydroxypropylmethylcellulose acetate succinate) was used for repeated hydrolysis of the water-insoluble compound phlorizidin, retaining about 75% of its initial activity after five cycles25. When Arthrobacter simplex cells were immobilized inside beads of a thermosensitive polymer gel as a biocatalyst, thermal cycling caused cyclical changes between the collapsed and swollen states of the hydrogel beads. This system thus worked as a hydraulic pump33, driving the mass transfer of the substrate (hydrocortisone) into the gel and the product (prednisolone) out. However, external heating of the gel beads caused a dehydrated, dense skin to form on the gel surface, which could hinder the mass transport. A biocatalyst preparation sensitive to magnetic fields has been produced by immobilizing invertase and Fe2O3 in a poly(N-isopropylacrylamide-co-acrylamide) gel. The heat generated by the exposure of -Fe2O3 to a magnetic field causes the gel to collapse, which is followed by a sharp decrease in the rate of sucrose hydrolysis34. As the heating in this case occurs inside the gel, no skin is formed and the -Fe2O3 beads could be used to develop a more-efficient magnetically driven hydraulic pump. Drug delivery When an enzyme is immobilized in smart hydrogels, the products of the enzymatic reaction could themselves trigger the gels phase transition. It would then be possible to translate the chemical signal (e.g. presence of the substrate) into the environmental signal (e.g. pH change) and then on into the mechanical signal, namely shrinking or swelling of the smart gel. We have discussed above the application of these systems as biomimetic actuators but this idea also lies behind the development of drug-delivery systems in which a drug is liberated in response to a chemical signal (e.g. insulin release in response to rising glucose concentration). The swelling or shrinking of smart hydrogel beads in response to small changes in pH or temperature can be used successfully to control drug release, because the diffusion of the drug out of the beads depends on the gel state35,36. When a smart polymer is integrated into a microcapsule wall37 or a liposomal lipid bilayer38, the conformational transition of the polymer affects the integrity of the microcapsule or liposome and allows the regulated release of the drugs loaded into the microcapsule or liposome. The development of a glucose-sensitive insulinreleasing system for diabetes therapy is a long-standing challenge for biomedical engineers. Unsurprisingly, therefore, it became a popular model for the systems using smart polymers. An insulin-loaded matrix has been produced by mechanical mixing and compressing a dry pH-responsive polymer, poly[(N,N-dimethylamino)ethyl methacrylate-co-ethylacrylamide], glucose oxidase, bovine serum albumin and insulin. Exposure to glucose resulted in the oxidation of glucose to gluconic acid and thus a decrease in the pH, protonation and swelling of the polymer, accompanied by insulin
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Insulin

Insulin H+ Glucose

Glucose H+ Glucose oxidase

Figure 2 Schematic presentation of a chemical valve. Glucose oxidase is immobilized on pH-responsive polyacrylic acid, which is grafted onto a porous polycarbonate membrane. (a) Poly(acrylic acid) is in an expanded conformation blocking insulin transport. (b) The oxidation of glucose is accompanied by a decrease in pH and the transition of poly(acrylic acid) into a compact conformation, resulting in the opening of the pores and the transport of insulin (redrawn from Ref. 44).

release. The insulin release stopped within 10 min of glucose removal and could be restimulated by glucose addition39. A variety of designs for an insulin-delivery system that responds to glucose were also developed by Klumb and Hotter using glucose-sensitive membranes containing glucose oxidase40. The swelling of a gel in response to glucose can be designed in a different way, using reversible complex formation. Copolymers of allyl glucose and N-vinyl2-pyrrolidone form a hydrogel when mixed with concanavalin A, a lectin that can bind four glucose molecules. The gel can be loaded with insulin, which is released under exposure to glucose. The glucose in solution competes with glucose ligands coupled to the polymer and finally disintegrates the gel network by destroying the interactions with concanavalin A41,42. The efficiency of the system is increased when the smart-polymer device is covered by an outer membrane permeable to glucose and insulin but impermeable to the glucosecontaining polymer and concanavalin A43. The specific release of insulin in response to glucose could also be designed in the form of a chemical valve (Fig. 2). Glucose oxidase can be immobilized on a pHresponsive poly(acrylic acid) layer grafted onto a porous polycarbonate membrane. Under neutral conditions, polymer chains are densely charged and have an extended conformation, preventing insulin transport through the membrane by blocking the pores. Upon

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exposure to glucose, the pH drops and the polymer chains become protonated and adopt a more compact conformation. The blocking of pores is reduced and insulin is transported through the membrane44. Membrane permeability can be regulated via the conformational changes of the poly(methacrylic acid)45, poly(2-ethylacrylic acid)46, poly(benzyl glutamate)47 or poly(N-isopropylacrylamide) alone48,49, or in combination with poly(methacrylic acid)49 grafted inside the pores. This has been used in the construction of heatand pH-sensitive chemical valves. For hydrogel beads composed of crosslinked poly(N-isopropylacrylamideco-acrylamide)50 or porous polymer beads with incorporated poly(N-isopropylacrylamide)51, the pore-size change with changes in temperature was proved by determining the elution profiles of probe molecules with different molecular weights. As the temperature is raised, the substances are eluted earlier, indicating shrinking of the pores. A novel set of polymeric composite films formed from multilayers of amine-terminated dendrimers and poly(maleic anhydride-co-methylvinyl ether) grafted onto gold-coated silicon wafers have recently been reported52. These composite membranes exhibit fully reversible, pH-switchable permselectivity for both cationic and anionic redox-active probe molecules. Thermoresponsive surfaces Modifying the inner surfaces of a membranes pores by grafting smart polymers onto them allows the membranes permeability to be controlled. Grafting thermoresponsive polymers onto the surfaces endows the surface with considerable thermoresponsive properties hydrophilic below the critical temperature of the polymer transition and hydrophobic above it. The change in hydrophobicity of a surface with grafted poly(N-isopropylacrylamide) was demonstrated by contact-angle measurements53 and water absorbancy54. Surfaces with thermoresponsive hydrophobic hydrophilic properties have been used in the HPLC separation of steroids55,56 and drugs57. However, less impressive results were obtained for protein chromatography with poly(N-isopropylacrylamide-co-Nhydroxyethylacrylamide)-grafted porous glass58. Poly(N-vinyl caprolactam), a thermoresponsive polymer with a critical temperature of about 35 C, interacts efficiently with Cibacron Blue (a triazine dye), which is widely used as a ligand in the dye-affinity chromatography of various nucleotide-dependent enzymes (e.g. lactate dehydrogenase59). The polymer binds strongly by multiple interactions with the dye ligands. At high temperatures, the polymer molecules are in a compact globular conformation capable of binding to only a few ligands on the matrix. Lactate dehydrogenase from porcine muscle has good access to the ligands and binds to the column. On lowering the temperature, the polymer molecules undergo a transition to a more expanded coil conformation; the polymer molecules now interact with more ligands and begin to compete with the bound enzyme for the ligands, thereby displacing the enzyme. Lactate dehydrogenase has been purified using only the temperature change as the eluting factor60. This approach seems quite promising because it eliminates the step of separating the target protein from the eluent, which (for dye-affinity

chromatography) usually involves adding a competing nucleotide or increasing the salt concentration. The change in surface properties of the polymer from hydrophobic above the critical temperature to hydrophilic below it has been used successfully to detach mammalian cells from their substrate. Mammalian cells are cultivated on a hydrophobic solid substrate and are usually detached from it by protease treatment, which often damages them. At a temperature of 37 C, a substrate surface coated with grafted poly(N-isopropylacrylamide) is hydrophobic because this temperature is above the critical temperature of the polymer and the cells grow well. However, decreasing the temperature to 25 C results in the surface becoming hydrophilic, and the cells can then be easily detached without any damage61,62. Bioseparation All bioseparation processes include three stages: (1) preferential partitioning of the target substance and impurities between two phases (liquidliquid or liquid solid); (2) mechanical separation of the phases (e.g. separation of the stationary and mobile phase in a chromatographic column); and (3) recovery of the target substance from the enriched phase. The use of smart polymers in bioseparation has developed into a broad area of application since it was begun by Hoffman63 and Cussler64 in the late 1980s. Precipitation Affinity precipitation is based on using a conjugate of smart polymers with covalently coupled ligands specific for the target protein. The conjugate first forms a complex with the target protein and phase separation of the complex is triggered, transforming the polymer backbone into an insoluble state. The target protein is then either eluted from the insoluble-macroligand protein complex or the precipitate is dissolved, the protein dissociated from the macroligand and the ligandpolymer conjugate reprecipitated without the protein, which remains in the supernatant in a purified form. Various ligands, such as triazine dyes, sugars, protease inhibitors, antibodies and nucleotides, have been successfully used for affinity precipitation65. Triazine dyes, which are robust affinity ligands for many nucleotide-dependent enzymes, have been used successfully in conjugates with Eudragit S100 (a copolymer of methacrylic acid and methylmethacrylate that precipitates at a low pH) to purify dehydrogenases from various sources by affinity precipitation66. Sugar ligands are another attractive alternative for use in bioseparating lectins67. The incorporation of relatively hydrophilic imidazole moieties into the thermoresponsive polymer poly(N-isopropylacrylamide) hindered the hydrophobic interactions and resulted in a drastic increase in the precipitation temperature68. The effect could be counteracted by the addition of salt, which reduces the repulsive electrostatic interactions and promotes hydrophobic interactions, leading to polymer precipitation. The efficient precipitation of Cu(II)-loaded polymers by high salt concentrations at mild temperatures is very convenient for affinity precipitation with immobilized metal ions, because high salt concentrations do not interfere with proteinmetal-ion interactions and it reduces the nonspecific binding of foreign proteins to
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the polymer both in solution and when precipitated69. The flexibility of polymer chains in solution allows several imidazole ligands on a polymer molecule to move close enough to interact with the same Cu(II) ion and thus to provide strong polymerCu(II) interactions. Kunitz soybean trypsin inhibitor70 and -amylase inhibitors from wheat69 and ragi seeds71 have been successfully purified using this technique. A particularly interesting group of smart polymers is the nonstoichiometric polyelectrolyte complexes, which consist of two oppositely charged polymer molecules. The polyanion poly(methacrylic acid) and the polycation poly(N-ethyl-4-vinyl-pyridinium bromide) form a complex that undergoes reversible precipitation from aqueous solution at any pH value in the range 4.56.5, depending on the ionic strength and polycation:polyanion ratio in the complex. The precipitation is accompanied only by small nonspecific coprecipitation of proteins. Polyelectrolyte complexes have been successfully used for the purification of antibodies raised in rabbits against inactivated glyceraldehyde-3-phosphate dehydrogenase72. When the monoclonal antibodies specific for inactivated dimers were covalently coupled to the polyanion, polyelectrolyte complexes have been successfully used to separate inactivated from active glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle73. This system can be regarded as a simplified model of chaperone action in living cells assisting in the separation of active protein molecules from misfolded ones. Two-phase systems Two aqueous polymer solutions become mutually incompatible and form two separate phases with a clear interface when the critical concentration of polymers is exceeded. The selective partitioning of proteins between the two phases is an efficient tool for purifying proteins and some low molecular weight substances. However, the main problem is how to separate the target protein from the phase-forming polymer and this has not yet been completely solved. Smart polymers provide an elegant solution to this problem simple precipitation of the phase-forming polymer leaves the protein in the supernatant. Thermoresponsive polymers such as poly(ethylene oxideco-propylene oxide)74,75 or poly(N-vinyl caprolactam-covinyl imidazole)76 form two-phase systems with dextran and have been used to purify proteins. The target protein partitioned into the phase formed by the thermosensitive polymer and the two polymer phase were separated. The target-containing phase was heated so that it precipitated, leaving the purified target protein in aqueous supernatant (Fig. 3). One interesting approach is a combination of affinity precipitation with the extraction in aqueous two-phase systems77. Future developments In the future, we look forward to further developments and the commercial introduction of new smart polymers with transition temperatures and pHs in the range at which the target biomolecules are most stable (415 C and pH 58). On the theoretical side, we expect to gain a better understanding of the mechanism of cooperative interactions in these polymers
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Discarded Phase separation Addition of crude protein

Concentrated polymer phase reused after dissolution in cold butter 1. Heating 2. Phase separation Purified protein in buffer solution

Figure 3 Schematic diagram showing a scheme for protein purification using aqueous-polymer two-phase extraction, with the top phase-forming polymer being thermosensitive.

and to increase our knowledge of structureproperty relationships to enable the rational synthesis of smart polymers with predefined properties. Acknowledgment The support of the Swedish Competence Center for Bioseparation is gratefully acknowledged. References
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