“The name 'smart ' was coined due to the similarity of the stimuli-responsive polymers to the . There is a strong belief that nature has always been striving for smart solutions in creating life. The goal for the scientists is not only to mimic biological processes, and therefore understand them better, but also to create novel species and invent new processes.” Smart polymeric Biomaterials: where Chemistry & Biology can merge By Ashok Kumar

Smart polymeric materials respond by large constructive elements and parts of changes due to small changes in environment. complicated machinery. The salient The polymers that form these smart materials feature of functional biopolymers is their all- are referred as “smart” polymers or “stimuli- or-none or at least highly nonlinear response responsive” polymers or “environmentally” to external stimuli. Small changes happens in sensitive polymers. The smart polymers response to varying parameter until the undergo fast and reversible changes in the critical point is reached, then the transition microstructure from a hydrophilic to a occurs in the narrow range of the parameter hydrophobic state that are triggered by small varied and after the transition is completed, stimuli in the environment. The changes are there is no significant further response of the apparent at the macroscopic level as precipitate system. Such nonlinear response of formation from a solution accompanied by biopolymers is warranted by highly phase separation from or cooperative interactions. Despite the order of magnitude changes in the hydrogel weakness of each particular interaction size. This whole phenomenon is reversible, the taking place in a separate monomer unit, system returning to its initial state when the these interactions when summed through trigger is removed. The driving force behind hundreds and thousands of monomer units these transitions varies, with common stimuli could provide significant driving forces for including neutralization of charged groups by the processes occurring in such systems. either a pH shift or the addition of an Understanding of the mechanism of oppositely charged , changes in the cooperative interactions in biopolymers has efficiency of the hydrogen bonding with an opened floodgates for attempts to mimic increase in or ionic strength, and cooperative behavior of the biopolymers in collapse of hydrogels and interpenetrating synthetic systems. Last two decades polymer networks. Even the latest among these witnessed the appearance of synthetic have been the electric, magnetic, light or functional polymers, which respond in some radiation induced reversible phase transitions. desired way to a change in temperature, pH, Such property change of smart polymers has electric or magnetic fields or some other shown various applications in biological parameters. These polymers were nicknamed systems. stimuli-responsive. The name 'smart polymers' was coined due to the similarity of Life is polymeric the stimuli-responsive polymers to the biopolymers. There is a strong belief that The most important components of living nature has always been striving for smart cells; , carbohydrates, nucleic are solutions in creating life. The goal for the polymers. The functions of living cells are scientists is not only to mimic biological regulated by these biopolymers that form the processes, and therefore understand them basis around which all major natural processes better, but also to create novel species and are controlled. Nature use polymers both as invent new processes. Recent developments

9 Fig. 1: pH-induced soluble-insoluble behaviour of synthetic (Eudragit S-100) and natural (chitosan) polymers have shown an explosive growth in the subject methacrylic (hydrophilic at high pH when where smart polymeric materials are being carboxy groups are deprotonated but more tailor made for application in hydrophobic when carboxy groups are and medicine. Of special interest are two most protonated) precipitate from aqueous common smart polymer systems with sharp solutions on acidification to pH around 5 while response to external stimuli and thus promising of methyl methacrylate enormous potential in biotechnology, (hydrophobic part) with dimethylaminethyl bioengineering and medicine. These systems methacrylate (hydrophilic at low pH when are developed from pH- and temperature- amino groups are protonated but more sensitive smart polymers. For applications, hydrophobic when amino groups are these polymers are utilized in many forms, as deprotonated) are soluble at low pH but can be dissolved in aqueous solution, adsorbed precipitate at slightly alkaline conditions. or grafted on aqueous-solid interfaces, or Hydrophobically modified cellulose cross-linked in the form of hydrogels. deri vatives with pending carboxy groups, e.g. hydroxypropyl methyl cellulose acetate succinate are also soluble at basic conditions but precipitate in slightly acidic media. The pH- pH-sensitive smart polymers induced precipitation of smart polymers is very sharp and requires usually the change in pH not This group of smart polymers consists of the more than 0.5 units (Fig. 1). polymers for which transition between soluble The copolymerization of some specific and insoluble state is created by decreasing net monomers results in the synthesis of a pH- charge of the polymer molecule. The net sensitive polymer with reversible transition in charge can be decreased by changing pH to the physiological range of pH 7.0-7.5, thus neutralize the charges on the macromolecule making them more suitable for biological and hence to reduce the hydrophilicity systems. The charges on the macromolecule (increase the hydrophobicity) of the can also be neutralized by addition of an m a c r o m o l e c u l e . C o p o l y m e r s o f efficient counterion, e.g., low molecular weight methylmethacrylate (hydrophobic part) and counter ion or a polymer molecule with the

10 Fig. 2: Temperature response for thermo-sensitive polymers. (A) soluble phase(below LCST); (B) insoluble phase (above LCST) opposite charges. The latter systems are separation takes place. An aqueous phase combined under the name of polycomplexes. containing practically no polymer and a The cooperative nature of interaction between polymer enriched phase are formed. Both two polymers with the opposite charges makes phases can be easily separated by decanting, polycomplexes very sensitive to the changes in centrifugation or filtration. The temperature of pH or ionic strength. Many polymer systems phase transitions depends on polymer have thus been designed that can show the concentration and molecular weight (MW) reversible solubility property in any desired pH (Fig. 2). The phase separation is completely range where one needs to utilize them. reversible and the smart polymer dissolves in when the temperature is reduced below the transition temperature. Thermo-sensitive smart polymers Two groups of thermo-sensitive smart polymers are most widely studied and used. The reversible solubility of thermosensitive · Poly(N-alkyl substituted acrylamides) and smart polymers is caused by changes in the most well-known of them, poly(N- hydrophobic-hydrophilic balance of isopropyl acrylamide) with transition uncharged polymer induced by increasing temperature of 32 °C. temperature or ionic strength. The uncharged polymers are soluble in water due to the · Poly(N-vinylalkylamides) like poly(N- hydrogen bonding with water molecules. The vinyliso-butyramide) with transition efficiency of hydrogen bonding reduces with temperature of 39°C or poly(N-vinyl increase in temperature. The phase separation caprolactam) with transition temperature 34- of polymer takes place when the efficiency of 36°C (depending on polymer molecular hydrogen bonding becomes insufficient for weight). solubility of macromolecule. On raising the Other polymers with different transition temperature of aqueous solutions of smart from 4-5°C for poly (N-vinyl polymers above a cer tain critical piperidine) to 100°C for poly(ethylene glycol) temperature(which is often referred as are available at present. Increase in the transition temperature, lower critical solution hydrophilicity of the polymer by incorporation temperature, LCST, or 'cloud point'), phase of hydrophilic co-monomers or coupling to

11 hydrophilic ligands, increases the transition Applications in Biotechnology and temperature while hydrophobic co-monomers Medicine and ligands have the opposite effect. Block copolymers with a thermosensitive Smart polymers may be physically mixed with “smart” part consisting of poly (NIPAAM) or chemically conjugated to biomolecules to form reversible on increase in temperature yield a large family of polymer-biomolecule while random copolymers separate from systems that can respond to biological as well as aqueous solutions by forming concentrated to physical and chemical stimuli. Biomolecules polymer phase. Thus, the properties of smart that can be polymer conjugated include polymers important for biotechnological and proteins and oligopeptides, sugars and medical applications could be controlled not polysaccharides, single and double-stranded only by the composition of co-monomers but oligonucleotides, DNA plasmids, simple lipids also by the polymer architecture. The phase and phospholipids and wide range of other transition at increased temperature of ligands and synthetic drug molecules. These thermosensitive polymers is the result of polymer-biomolecule complexes, are referred hydrophobic interactions between polymer as affinity smart biomaterials or intelligent molecules. As hydrophobic interactions are bioconjugates. Also such polymers have been promoted by high salt concentrations, the used in developing smart hydrogels and smart addition of salts shifts cloud point to lower surfaces that can respond to external stimuli. temperatures. On the other hand, addition of Such polymeric biomaterials have shown a organic solvents, detergents and chaotropic range of different applications in the fields of agents increases transition temperature as these biotechnology and medicine (Fig. 3).The compounds deteriorate hydrophobic researchers have used these polymers for interactions. biomedical applications to downstream

Immobilized Thermally controlled Reversible soluble viable cells biocatalyst biocatalyst

S upramolecular Immobilized cell design biocatalyst detachment

Smart Biomimetic polymer Thermoresponsive actuator s surfaces

D rug delivery Bioseparation Thermoresponsive chromatography

Aqueous two-phase Affinity polumer system precipitation

Fig. 3: Application of smart polymeric materials in biotechnology and bioengineering

12

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S uppramooleecularr Immoobiiliizeedd cell dessignn bioccaattaalysst detaachmeennt

Smarrt Biiomimeettic polyymerr Thermooresponssiivvee aacttuuattoo rss surffaces

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Aqueouus two-phhassee Afffinitty ppoollumerr system ppreecippiitatiion

Fig.4: Shielding effects of a conjugated smart polymer on the binding of a small ligand (biotin) and a large macromolecular ligand (a biotinylated ) to streptavidin. (Sour ce: Natur e (2001); 411, 59-62). processing and biocatalysis and several efficient conversion of chemical energy into breakthrough findings have emerged. In the mechanical energy in living organisms. A cross streak of these stimulatory research findings is linked of poly(vinyl alcohol)chains the latest thrilling breakthrough achieved by entangled with chains has good the group of Stayton and Hoffman, at the mechanical properties and shows rapid electric University of Washington, USA. The field associated bending deformation: a gel rod researchers developed a clever way to use smart of 1mm diameter bends semi circularly within polymers that provide size selective switches to 1 sec on the application of an electric field. An turn proteins on and off. If smart polymer artificial fish with a gel tail swam forward at a chain is attached to the protein molecule velocity of 2 cm/ sec as the gel oscillated under farther from the active site, the extended sinusoidally varied electric fields; a mechanical polymer chain would shield the site, blocking hand composed of four gel fingers could pick larger molecule from attaching. However, up a fragile quil egg (9g) from a sodium when coiled, the chain was far enough out of carbonate solution and hold it without way, so the big molecule could bind to the site. breaking, controlled by an electric signal. Such polymers act as a kind of shield or Polymer gels capable of mechanical response molecular gatekeeper that regulates, based on to electric field have also been developed using size, the kind of molecules that can bind to a the cooperative binding of the positively protein (Fig. 4). This could help bioengineers charged surfactant molecules to the precisely control the function of proteins and polyanionic polymer poly (2 acrylamido- 2 could enable a new class of powerful diagnostic methyl- 1- propane sulfonic acid). Copolymers and sensing devices. gels consisting of N-isopropylacrylamide and acrylic acid would be useful for constructing Biomimetic actuators biochemomechanical systems. A pH induced change in the -COOH ionization of acrylic There have been attempts to mimic the acid alters the repulsive force; the attractive

13 force is produced by hydrophobic interactions smart polymers have been used for the arising from the dehydration of the N- development of reversibly soluble biocatalysts isopropylacrylamide moieties. Glucose whose solubility is controlled by pH or dehydrogenase catalyses the conversion of temperature. For example, trypsin immobilized neutral glucose into gluconic acid, on a pH-responsive of accompanied by a decrease in the pH. When methylmethacrylate and methacrylic acid the reaction occurs inside the gel, the repulsive (Eudragit S100) is used for repeated hydrolysis forces are eliminated owing to the protonation of casein. Water-insoluble compound of the COO- groups. As a result, the attractive phlorizidin is hydrolyzed by a reversible forces dominate, followed by the collapse of biocatalyst of b -glucosidase immobilized on a the gel. Changes in the gel's size (about 1.5 pH responsive, reversibly soluble polymer times) using immobilized glucose (hydroypropylmethylcellulose acetate dehydrogenase are initiated by pulses of a 40 succinate) and was reused for five cycles. mm glucose solution. Similarly, when Arthrobacter simplex cells are The biomimetic actuators could be used in immobilized inside beads of a thermosensitive future 'soft' machines that are designed using polymer gel as a biocatalyst, thermal cycling more biological than mechanical principles. cause cyclical changes between the collapsed The gel hand consisting of smart polymer can and the swollen states of the hydrogel beads. be used as a very useful tool in picking up very The system thus work as a 'hydraulic pump', tiny little objects (so tiny we can't even see it) in driving the mass transfer of the substrate the aqueous solution. All we have to do is to (hydrocortisone) into gel and the product insert the gel hand and raise the temperature a (prednisolone) out. A biocatalyst sensitive to little bit. As the polymer layer shrinks, the magnetic field is produced by immobilizing g - “hands” will contract and grab on to the target invertase and Fe2O3 in a poly (N- object. This is totally reversible. When the isopropylacrylamide-co-acrylamide ) gel. The g - target compound within the gel hand is heat generated by exposure of Fe2O3 to a destined for release, just decrease the magnetic field causes the gel to collapse, which surrounding temperature. In contrast to is followed by a sharp decrease in the rate of biological systems, biomimetic actuators can sucrose hydrolysis. Polymer bound smart withstand very hostile environments. catalysts are useful in waste minimization, catalyst recovery , and catalyst reuse. Polymeric smart coatings have been developed that are Reversible biocatalysts capable of both detecting and removing hazardous nuclear contaminants. Such applications of smart materials involving catalysis chemistry, chemistry, and The ability of the smart polymers to form a chemistry relevant to decontamination separate phase in the aqueous solutions methodology are especially applicable to following a slight change in the external environmental problems. conditions can be used to create reversibly soluble biocatalysts, when the Thus the biocatalysts obtained by immobilizing molecule is bound covalently to the polymer. and cells with the aid of “smart” These biocatalyst catalyze an enzyme reaction polymers acquire potentially useful properties. in their soluble state and thus can be used in Such biocatalyst can combine the advantages reactions with insoluble or poorly soluble of homogeneous and heterogeneous catalyst, substrates or products. As the reaction is can serve as convenient 'chemical switches' complete, the conditions are changed to cause sensitive to slight changes in the external the catalyst to precipitate so that it can be conditions, and are distinguished by a higher separated from the products and used in the productivity as a result of the operation of the next cycle, after redissolution. Wide range of 'thermal pump'.

14 Smart systems years for the intelligent release of (Fig. 5 & 6). The application of the smart polymers for drug delivery shows great promise due to modulated or pulsatile drug release pattern to mimic biological demand. Stimuli occurring externally or inter nally include temperature, photoirradiation, electric current, pH, and metabolic chemicals. When an enzyme is immobilized in smart hydrogels, the products of enzymatic reaction could themselves trigger the gel's phase transition. It would then be possible to translate the chemical signal (e.g., presence of substrate) into the environmental signal (e.g. pH change) and then into the Fig. 5: Insulin delivery system mechanical signal, namely shrinking or swelling (Source: TIBTECH (1999), 17(8):335-40) of smart gel. The swelling or shrinking of smart hydrogel beads in response to small change in pH or temperature can be used successfully to control drug release, because diffusion of the drug out of the beads depends on the gel state. When a smart polymer integrated into microcapsule wall or a A B liposomal lipid bilayer, the conformational transition of the polymer affects the integrity of the microcapsule or liposome and allows the regulated release of the drug loaded into the microcapsule or liposome. In a temperature sensitive polymer, a dilute solution (1-3%) of the polymer is watery liquid, while on warming C D to body temperature the solution gels, becoming viscous and clinging to surface in a Fig. 6: Schematic of composite hydrogel

'' form. The hydrogel therefore A- Gel porosity is reduced by affinity crosslinks provides an effective way to administer drugs, that exclude large molecules, e.g. insulin (large blue dot). either topically or mucosae, over longer B- Glucose (small red dot) diffuses in and timescales than otherwise possible, by competitively displaces affinity crosslinks. dissolving them in a solution of the hydrogel, C- Insulin is able to diffuse into the more highly porous gel. which also contains hydrophobic regions. By D- Insulin diffuses through the gel providing the using such drug formulations incorporated concentration gradient and glucose into hydrogels pharmaceutical companies will concentration is maintained. be able to increase the efficiency, cost- effectiveness and range of applications for One latest model explains how specific release existing therapeutics. of insulin can be achieved in response to As an intelligent drug delivery scheme, the glucose in the form of 'chemical valve'. develo pmen:t of glucose-sensitive insuln- Glucose oxidase can be immobilized on a pH releasing system for diabetes therapy has responsive poly(acrylic acid) layer grafted onto become a popular model for the systems using a porous polycarbonate membrane. Under smart polymers. There have been several neutral conditions, polymer chains are densely schemes which researchers developed over the charged and have an extended conformation,

15 preventing insulin transport through the membrane by blocking the pores. Upon exposure to glucose the pH drops and the polymer chains become protonated and adopt Temperature Responsive a more compact conformation. The blocking Polymer of pores is reduced and insulin is transported 0 through the membranes. Membrane Cell culture at 3 7 0C Cell detachment at 20 C permeability can be regulated via the c o n f o r m a t i o n a l c h a n g e s o f t h e poly(methacrylic acid), poly(2-ethylacrylic acid) or poly(N-isopropylacrylamide) grafted inside the pores. Thermosensitive polymers as carriers for DNA delivery are recently looked at as an efficient mode of gene transfection and also these source http://www.cellseed.com/ Smart polymer grafted surfaces for cell detachment Polymer are engineered as carriers of therapeutic proteins. Drug release from This enables the collection of cultured cells as a thermoresponsive self assembled polymeric single "sheet". Cell-sheet is highly effective micelles composed of cholic acid and poly(N- when transplanted to patients due to tight isopropylacryalmide) were studied as model communication between cells and cells. This system to monitor the rate of drug release technolog y has recently now been based on temperature changes. commercialized. Similarly surfaces with thermoresponsive Stimuli-responsive surfaces hydrophobic-hydrophilic properties have been used in modifying chromatographic matrices. Temperature responsive size-exclusion The change in surface properties of the c h r o m a t o g r a p h y u s i n g p o l y ( N - t h e r m o r e s p o n s ive p o l y m e r s f r o m isopropylacrylamide) is used for high protein hydrophobic above the critical temperature to resolution with low non-specific interactions. hydrophilic below it has been used in tissue Affinity interactions are also controlled by culture applications. Mammalian cells are grafting responsive polymers on affinity cultivated on a hydrophobic solid culture matrices (Fig. 7). More interestingly, dishes and are usually detached from it by combinations of pH and temperature- protease treatment, which also cause damage to responsive polymers have been used in the cells. This is rather an inefficient way in that aqueous chromatography to separate ionic only some detached cells are able to adhere bioactive compounds. These provide the onto new dishes because the rest are damaged. 0 mutual influences of both electrostatic and At temperature of 37 C, a substrate surface hydrophobic interactions between the c o a t e d w i t h g r a f t e d p o l y ( N - separating compounds and the modified isopropylacryalmide) is hydrophobic because chromatographic matrix. this temperature is above the critical temperature of the polymer and the cells grow well. However, when the temperature is Bioseparations decreased to 200C, resulting the surface to become hydrophilic, the cells can be easily detached without any damage. The cells can be The use of smart polymers in bioseparation used for further culturing. The cells are has developed into a broad area of application detached maintaining the cell-cell junction. since it was begun in the late 1980 s. In affinity

16 Fig. 7: Modification of affinity chromato-graphic surfaces with temperature responsive polymers to influence the binding and recovery of biomolecules. precipitation of biomolecules, the co-ordination sites available for proteins (Fig. bioconjugate is synthesized by coupling a 8). ligand to a water soluble “smart” polymer. The ligand-polymer conjugate selectively binds the target protein from the crude extract and the protein-polymer complex is precipitated from the solution by the changes in the environment like pH, temperature, ionic strength or addition of some reagents. Finally, the desired protein is dissociated from the polymer and the later can be recovered and reused for another cycle. Various ligands like protease inhibitors, , nucleotides, metal chelates, carbohydrates, and trizine dyes have been used in affinity precipitation. New approach is recently developed for purifying His-tag Fig. 8: Smart polymer-metal conjugate proteins using thermoresponsive polymers by for protein purifications metal chelate affinity precipitation. The incorporation of relatively hydrophilic Other emerging strategy involves selective imidazole moieties into the poly(NIPAM) portioning of proteins between two phases in backbone hinder the hydrophobic interactions aqueous two-phase systems (ATPS). This is an and increase the precipitation temperature of efficient tool for purifying proteins, cells and the copolymer. However, when Cu(II) ions are some low molecular weight substances. be loaded on the copolymer, the precipitation However, the main problem is how to separate efficiency is promoted by adding NaCl and the target biomolecules from the phase increasing temperature. The flexible backbone forming polymer. Smart polymers provide a of the water soluble poly(NIPAM) allowed simple solution to this problem as simple proper co-ordination between the imidazole precipitation of the phase forming polymer groups and metal ions and also leaving some leaves the protein in supernatant. There have

17 b e e n s e v e r a l e x a m p l e s w h e r e This seems to be only beginning of the myriad uses and thermoresponsive polymers such as applications of smart polymers in biotechnology. The poly(ethylene oxide-co-propylene oxide) or application of these polymers is only limited by our poly(N-vinyl caprolactam-co-vinyl imidazole) imagination. Even though the class of these “smart” from two phase systems with dextran and have materials is still very much in the research and been used to purify proteins. Another development stage, it would not be long before we can interesting approach is a combination of really utilize their applications in our daily lives. On the affinity precipitation with the extraction in theoretical side, we expect to gain a better understanding ATPS. Type specific separation of animal cells of the mechanism of cooperative interactions in these using pH-sensitive polymer Eudragit S-100 and polymer and to increase our knowledge of structure- temperature sensitive polymer poly(NIPAM) property relationships to enable the rational synthesis of as ligand carrier in ATPS has been established. smart polymers with predefined properties.

About the author: Dr. Ashok kumar is an Associate professor in the Department of Biological Sciences & Bioengineering at IIT Kanpur. He obtained his Ph.D. jointly from IIT Roorkee and Institute of Genomics & Integrative Biology, Delhi. He has postdoctoral research experience from Sweden & Japan. He was a faculty at Lund University, Sweden. His areas of research interest include smart polymeric biomaterials for biotechnological applications, bioprocess technology and bioseparations, affinity interactions, stem cell separations, biosensors and ligand-receptor interaction analysis.

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