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Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016-Kanpur, India
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund, Sweden
Received 22 February 2007; received in revised form 22 May 2007; accepted 22 May 2007
Available online 2 June 2007
Abstract
Smart polymers (SP) have become one important class of polymers and their applications have been increasing
signicantly. Last two to three decades have witnessed explosive growth in the subject. SP which are also known as stimuliresponsive solubleinsoluble polymers or environmentally sensitive polymers have been used in the area of biotechnology,
medicine and engineering. The present review is aimed to highlight the applications of SP when these polymers are
presented in three common physical forms (i) linear free chains in solution where polymer undergoes a reversible collapse
after an external stimulus is applied, (ii) covalently cross-linked reversible gels where swelling or shrinking of the gels can be
triggered by environmental change and (iii) chain adsorbed or surface-grafted form, where the polymer reversibly swells or
collapses on surface, once an external parameter is changed. Though there are number of reviews coming up in this area in
recent times, the present review mainly addresses the developments of SP in the last decade with specic application areas
of bioseparations, protein folding, microuidics and actuators, sensors, smart surfaces and membranes.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Smart polymer; Stimuli-responsive polymer; Bioseparation; Protein folding; Smart surfaces and membranes; Microuidics and
actuators
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206
Polymers as linear free chains in solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1. Bioseparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.1. Aqueous two-phase polymer system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.1.2. Afnity precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
Abbreviations: AA, acrylic acid; AML, afnity macroligand; ATPS, aqueous two-phase system; ATRP, atom transfer radical
polymerizations; CP, critical point; ConA, concanavalin A; EDTA, ethylenediaminetetraacetic acid; EOPO, ethylene oxide propylene
oxide; ELP, elastin like polymer; IPN, interpenetrating network; LCST, lower critical solution temperature; MAA, methacrylic acid;
NiPAAm, N-isopropylacrylamide; PEG, poly(ethylene glycol); poly(AA), poly(acrylic acid); poly(DMAAM), poly(N, N0 -dimethylacrylamide); PMAA, poly(methacrylic acid); PNiPAAm, poly(N-isopropylacrylamide); PVCL, poly(vinylcaprolactam); poly(VDF),
poly(vinylidene uoride); SP, smart polymers; SPP, 3-[N-(3-methacrylamidopropyl)-N, N-dimethyl] ammonio-propane sulfonate
Also to be corresponded to. Tel.: +91 512 2594010; fax: +91 512 2594051.
Corresponding author. Tel.: +46 46 2228264; fax: +46 46 2224713.
E-mail addresses: ashokkum@iitk.ac.in (A. Kumar), Bo.Mattiasson@biotek.lu.se (B. Mattiasson).
0079-6700/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2007.05.003
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3.
4.
1. Introduction
Polymers such as proteins, polysaccharides and
nucleic acids are present as basic components in
living organic systems. Synthetic polymers, which
are designed to mimic these biopolymers, have been
developed into variety of functional forms to meet
the industrial and scientic applications. The
synthetic polymers can be classied into different
categories based on their chemical properties. Out
of these, some special types of polymers have
emerged as a very useful class of polymers and
have their own special chemical properties and
applications in various areas. These polymers are
coined with different names, based on their physical
or chemical properties like, stimuli-responsive
polymers [1] or smart polymers (SP) [2,3] or
intelligent polymers [4] or environmental-sensitive polymers [5]. We shall use further on the name
smart polymers for such polymer systems in this
review. The characteristic feature that actually
makes them smart is their ability to respond to
very slight changes in the surrounding environment.
The uniqueness of these materials lies not only in
the fast macroscopic changes occurring in their
structure but also these transitions being reversible.
The responses are manifested as changes in one or
more of the followingshape, surface characteristics, solubility, formation of an intricate molecular
assembly, a sol-to-gel transition and others. The
environmental trigger behind these transitions can
be either change in temperature [6] or pH shift [7],
increase in ionic strength [7], presence of certain
metabolic chemicals [8], addition of an oppositely
charged polymer [9] and polycationpolyanion
complex formation [10]. More recently, changes in
electric [11] and magnetic eld [12], light or
radiation forces [13] have also been reported as
stimuli for these polymers. The physical stimuli,
such as temperature, electric or magnetic elds, and
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Fig. 2. Type-specic separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive
polymers, PNiPAAm (poly(NIPAM)). Adopted from [53] with permission.
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partitioning, the protein-like structure of the polymer is expected to enhance proteinpolymer interactions [67].
More interesting application has been shown for
the separation of animal cells by coupling an
antibody (against a cell surface protein) to a temperature-sensitive SP such as PNiPAAm (Fig. 2). The
ATPS composed of PEG and dextran was developed where PNiPAAm was used as a ligand carrier
for specic separation of animal cells. Monoclonal
antibodies were conjugated with the carrier and
added to the polyethylene glycol 8000-dextran T500
aqueous two-phase system. About 80% of the
animal cells which specically bind to the antibody
polymer conjugate partitioned to the top phase of
ATPS. As a model system, CD34+-positive human
acute myeloid leukemia cells (KG-1) were specically separated from human T lymphoma cells
(Jurkat) by applying anti-CD34 conjugated with
PNiPAAm in the ATPS [53].
2.1.2. Affinity precipitation
The selective precipitation of a target molecule
from a mixture is a very attractive approach in
bioseparations. Precipitation can be highly selective
technique for protein purication or enrichment.
Traditionally precipitation of the target protein is
achieved by the addition of large amounts of salts,
like ammonium sulfate, organic solvents miscible
with water, like acetone or ethanol or by the
addition of polymers, like PEG [68]. It is not
expected to have high selectivity to be achieved by
traditional precipitation techniques, as the selectivity of precipitation is limited to the differences in
integral surface properties of protein molecules.
Thus, the introduction of high selectivity to the
precipitation techniques is of great importance.
Afnity precipitation of proteins using SP emerged
in the early 1980s. Since then it has evolved as a
technique capable of simple, fast, and efcient
purication of a variety of proteins [6971]. As a
general rule, there are ve basic steps in afnity
precipitation: (i) carrying out afnity interactions in
free solution, (ii) precipitation of the afnity
reagenttarget protein complex from the solution,
(iii) recovery of the precipitate, (iv) dissociation and
recovery of the target molecule from the complex,
and nally, (v) recovery of the afnity reagent.
Afnity precipitation methods have two main
approaches which have been described in the
literature [70], as precipitation with homo- and
hetero-bifunctional ligands. However, as the homo-
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Fig. 3. Scheme of metal chelate afnity precipitation of proteins. Reproduced from [79] with permission.
metal ions and the target protein binds the metalloaded co-polymer in solution via the interaction
between the histidine on the protein and the metal
ion. Many proteins both containing natural metalion binding residues and recombinant proteins
containing His-tag residues have been puried using
metal chelate afnity precipitation [78]. Therefore,
His-tagged protein or cells or bioparticles (with
surface accessible co-ordinating groups) can be
puried through the precipitation of target moleculemetal loaded polymer complex from the mixture.
The precipitated complex is solubilized by reversing
the precipitation conditions and the target molecule
is dissociated from the precipitated polymer by
using imidazole or EDTA as eluting agent. The
biomolecule is recovered from the co-polymer by
precipitating the latter at elevated temperature in
the presence of NaCl. In a recent study, purication
of extracellularly expressed six histidine-tagged
single chain Fv-antibody fragments (His6-scFv
fragments), from recombinant Escherichia coli cell
culture broth was performed. Quantitative precipitation of the His6-scFv fragments was tested at
different loads of the cell supernatant using Cu(II)
and Ni(II) loaded co-polymers of vinylimidazole
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[(VPGVG)2(VPGKG) (VPGVG)2]21 were synthesized and the free amino groups on the lysine
residues were modied by reacting with imidazole2-carboxyaldehyde to incorporate the metal-binding
ligands into the ELP biopolymers. Biopolymers
charged with Ni(II) were able to interact with a
His-tag on the target proteins. Purications of two
His-tagged enzymes, b-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the application of metal afnity precipitation
using this new type of afnity reagent. The bound
enzymes were easily released by the addition of
either EDTA or imidazole and over 85% recovery
was observed in both cases. The recovered ELPs
were reused with no observable decrease in the
purication performance. This has been the rst
report exploiting the features of ELPs for protein
purication based on metal-afnity purication.
The capability of modulating purication conditions by simple temperature triggers and their low
cost of preparation will probably make the ELPbased metal-afnity precipitation a useful method in
future, not only for protein purication but also for
diverse applications in bioseparation such as DNA
purication and environmental remediation [86].
Another interesting example has been a one-pot
afnity precipitation purication of carbohydratebinding protein reported by Sun et al [90]. By
designing thermally responsive glyco-polypeptide
polymers, which were synthesized by selective
coupling of pendant carbohydrate groups to a
recombinant triblock ELP, glyco-afnity precipitation purication of carbohydrate-binding protein
was demonstrated.
Other types of metal-chelating polymers for afnity
precipitation of proteins were reported by synthesizing
highly branched co-polymers of NiPAAm and 1,2propandiol-3-methacrylate (GMA: glycerol monomethacrylate), poly(NiPAAm-co-GMA) using the
technique of RAFT polymerization using a chain
transfer agent that allows the incorporation of
imidazole functionality in the polymer chain-ends.
The LCST of the co-polymers can be controlled by
the amount of hydrophobic and GMA co-monomers
incorporated during co-polymerization procedures.
The co-polymers demonstrated LCST below 18 1C
and were successfully used to purify a His-tagged
BRCA-1 protein fragment (a protein implicated in
breast cancer) by afnity precipitation [91,92]. An
interesting example of the use of poly(N-acryloylpiperidine) terminally modied with maltose for afnity precipitation of thermolabile a-glucosidase was
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Fig. 4. Schematic view of the mechanism of temperature-stimuli-articial chaperone assisted protein refolding in vitro.
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Fig. 5. Illustration of hydrogel volume changes as the gels temperature varies. The top (a) and bottom (b) rows correspond, respectively,
to temperatures above and below the critical temperature (TC). The left and right columns provide, respectively, top and side views.
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anionic poly (2-acrylamido-2-methylpropane sulfonic acid) (PAMPS) gel with cationic surfactant
N-alkylpyridinium chloride (C10PyCl) exhibits
greatest and fastest chemo-mechanical movement
under DC current thus exhibiting biomimetic
pulsation. The gel shrinkage was attributed to the
neutralization of negative sulfonate groups of the
gel due to complex formation with surfactant
cations through electrostatic interaction which leads
to decrease in the osmotic pressure difference
between the interior of the gel and the surrounding
solution. When the DC voltage was turned on, the
surfactant molecules move by electrophoresis and
bind with the gel, which cause an isotropic
contraction of the gel [155]. Novel polymeric
biomimetic gels with self-oscillating function have
been developed which generates periodic mechanical energy from the chemical energy of the
BelousovZhabotinsky reaction. The self-oscillating
gel may be useful in a number of application like
pulse generator or chemical pacemaker, self-walking
(auto-mobile) actuators or micropumps, etc [156].
When looking into the mechanical characterization
of an electric and pH-responsive gel consisting of a
co-polymer of PVApoly(acrylic acid), which expands and contracts in response to external stimuli,
the gel was found to be viscoelastic, mechanically
compressible over a relatively large deformation.
These properties could be useful in developing
them into biomimetic actuators or as scaffolds for
articial organs [157,158].
The triggered control of interfacial properties
provided by immobilized SP at the solid water
interface has applications in designing of microuidics bioanalytical devices [159]. SP-hydrogels
provide actuation pressure required for both valving
and dispensing functions in microdispensing device.
The microdispensing device uses an array of
responsive hydrogels to deform a exible membrane
above a uid reservoir chamber. When the microvalve is open, deformation of the membrane reduces
the volume of the reservoir chamber and pushes
uid through the microvalve. When the microvalve
is closed, expansion of hydrogel array generates a
storable pressure source that will result in uid
dispensing once the microvalve is opened [160].
A phenylboronic acid-based hydrogel was used to
construct a smart microvalve that responds to the
change in the glucose and pH concentrations [161].
Random co-polymers composed of poly(acrylic
acid) and poly(vinyl sulfonic acid) have been
analyzed for their swelling ratios to characterize
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with experiment [178]. The model explains reasonably well the experimental data for the temperaturedependent conformational changes of PNiPAAm
grafted on gold and on silicon oxide over a range of
surface densities and molecular weights obtained
using neutron reection in D2O. The largest
conformational changes were observed for intermediate grafting densities and high molecular
weights [179].
The grafting of SP to the surface could be
achieved via chemical bonding between reactive
groups on the gel surface and reactive terminal
groups of the preformed polymer (so called grafting
to) [180182]. The obvious advantage here is that
one can beforehand determine the properties
(molecular mass, MW distribution) of the to-begrafted polymer. The problem is that the surface
should have reactive groups suitable for grafting
and the grafted chain should carry the proper
functionality at the end. It is very difcult to achieve
high grafting densities using the grafting to methods
because of steric crowding of reactive sites at the gel
surface by already bound polymer molecules.
Moreover, the efciency of grafting to methods is
pretty low resulting in pronounced losses of the
terminally modied polymer. Hence, an alternative
approach also called grafting from has been mainly
used for the production of SP brushes. Attachment
of active precursors, such as initiator, transfer agent
or co-monomer onto the surface followed by radical
polymerization of NiPAAm allows for a ne control
of the density and thickness of polymer brushes
[183,184]. Alternatively, deposition of polymer at
the surface from monomer vapor under plasma
glow discharge was successfully used [185]. Atom
Fig. 7. Preparation of patterned dual thermoresponsive polymer-grafted culture dishes. Reproduced from [192] with permission.
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porous membranes. Grafted PEG chains are believed to accelerate the diffusion of water molecules
to PNiPAAm grafts, showing more rapid detachment of cell sheet compared to only PNiPAAmgrafted membranes [216].
Microbial cells, like Halomonas marina (ATCC
25374) capable of adhesion to hydrophobic surfaces
can be also bound to PNiPAAm-grafted surfaces
above the transition temperature and released with
about 90% efciency at reduced temperature [217].
The change in the pattern of attachment of common
oral bacteria Streptococcus mutans following cycling of PNiPAAm brushes above and below the
transition temperature was correlated with changes
in the surface properties as a result of the phase
transitions [218]. A similar correlation of short-term
attachment of gram negative and motile bacteria
(Salmonella typhimurium) and gram positive, nonmotile species (Bacillus cereus) with changes in
surface properties was observed for surfaces with
grafted co-polymers of NiPAAm with acrylamide or
N-tert-butylacrylamide [182].
Temperature-regulated detachment of mammalian cells requires cell metabolic activity requiring
ATP consumption, signal transduction and cytoskeleton reorganization. Precoating PNiPAAmgrafted surfaces with bronectin improves spreading of less adhesive cultured hepatocytes and
reduces the temperature at which cultured cells are
released from bronectin-adsorbed grafted surfaces.
Immunostaining with anti-bronectin antibodies
revealed that only bronectin located beneath the
cultured cells is removed from culture surfaces after
reducing temperature. Fibronectin adsorbed to
surface areas lacking direct cell attachment remained surface-bound after reducing temperature
[219]. Principal component analysis using time-ofight secondary ion mass spectrometry indicates
that molecular ion fragments of amino acids are
present on the surface after low-temperature liftoff
from PNiPAAm brushes. Seeding new cells on
surfaces from which an initial layer of cells was
removed indicates that liftoff dissociation leaves
behind surfaces that better promote cell adhesion as
compared to cell detachment by enzymatic detachment. It was concluded that the removal of bovine
aortic endothelial cells via low-temperature liftoff
from PNiPAAm brushes is less damaging to the
extracellular matrix proteins remaining at the surface as compared to the enzymatic methods [220].
At present, the low-temperature liftoff of cell
sheets from surfaces grafted with SP presents a
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Fig. 8. Chromatograms of steroids on a PNiPAAm terminally-modied column at 10 and 50 1C using pure water as a mobile phase.
Peaks: (1) hydrocortisone; (2) prednisolone; (3) dexamethasone; (4) hydrocortisone acetate; and (5) testosterone. HPLC conditions: owrate, 1.0 mL min1; monitoring, UV at 254 nm. Reproduced from [225] with permission.
multipoint interactions and the difculty in disrupting multivalent interactions is one of the main
problems in designing afnity techniques for cell
separation. The conformational changes of SPs
allow for a nely tuned modication of the surface
properties and hence give a good tool to control
cellsurface interactions. For example, adsorption
of anti-mouse CD80 monoclonal antibodies onto
PNiPAAm-grafted polypropylene membrane at
37 1C and their desorption at 4 1C was exploited
for the capture of mouse-CD80 transfected cells at
37 1C which facilitated detachment of captured cells
by washing at 4 1C. With this method, mouse CD80or mouse CD86-transfected cells were enriched from
a 1:1 cell suspension to 72% or 66%, respectively,
and with high yield [231].
Recently we have exploited the heat-induced
shrinkage of macroporous hydrogel prepared by
cross-linking polymerization of NiPAAm under
semi-frozen conditions (so called cryogel) for the
improved detachment of afnity bound cells. When
bearing Cu(II)-iminodiacetate ligands, PNiPAAm
cryogel monoliths bound E. coli cells. The bound
cells were eluted with only 65% efciency using
0.2 M imidazole buffer at 25 1C i.e. below the
transition temperature. However, when elution
was carried out with the same buffer at 40 1C, i.e.
above the transition temperature, the PNiPAAm
cryogel shrank almost instantaneously upon contact
with the warm buffer, resulting in the release of
85% of the bound cells [232]. Correspondingly,
whereas conventional elution of yeast cells captured
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Increase in
temperature
Fig. 9. Schematic presentation of the mechanism of detachment of captured cells by heat-induced shrinkage of macroporous PNiPAAm
cryogel.
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Fig. 10. Schematic representation of channels made of magnetic polystyrene latex covered with PNiPAAm) and built in the PVA gel
matrix: (a) off state below the collapse transition temperature; (b) on state above the collapse transition temperature. Arrows indicate
the diffusive mass transfer in the channels of PVA membrane. Reproduced from [258] with permission.
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