Biomolecule_sensitive Glucose Hydrogels

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Advanced Drug Delivery Reviews 54 (2002) 7998www.elsevier.com/ locate/ drugdeliv

Biomolecule-sensitive hydrogelsa , a b*Takashi Miyata , Tadashi Uragami , Katsuhiko Nakamae

aUnit of Chemistry, Faculty of Engineering and High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan

bDepartment of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Rokko, Nada, Kobe 657-8501, Japan

Received 1 August 2001; accepted 17 August 2001

Abstract

Stimuli-sensitive hydrogels have attracted considerable attention as intelligent materials in the biochemical and biomedical

fields, since they can sense environmental changes and induce structural changes by themselves. In particular, biomolecule-

sensitive hydrogels that undergo swelling changes in response to specific biomolecules have become increasingly important

because of their potential applications in the development of biomaterials and drug delivery systems. This article provides an

overview of the important and historical research regarding the synthesis and applications of glucose-sensitive hydrogels

which exhibit swelling changes in response to glucose concentration. Enzymatically degradable hydrogels and antigen-

sensitive hydrogels are also described in detail as protein-sensitive hydrogels that can respond to larger biomolecules. The

synthetic strategies of other biomolecule-sensitive hydrogels are summarized on the basis of molecular imprinting and

specific interaction. The biomolecule-sensitive hydrogels reviewed in this paper are expected to contribute significantly to the

exploration and development of newer generations of intelligent biomaterials and self-regulated drug delivery systems.

2002 Elsevier Science B.V. All rights reserved.

Keywords: Stimuli-sensitive hydrogel; Biomolecule-sensitive hydrogel; Glucose-sensitive hydrogel; Antigen-sensitive hydrogel; Enzymati-

cally degradable hydrogel; Biomolecular interaction

Contents

1. Introduction ............................................................................................................................................................................ 80

2. Glucose-sensitive hydrogels ..................................................................................................................................................... 80

2.1. Glucose oxidase-loaded hydrogels.... .................... .................... ................... .................... .................... ................... ........... 81

2.2. Lectin-loaded hydrogels .................. .................... .................... ................... .................... .................... ................... ........... 82

2.3. Hydrogels with phenylboronic acid moieties ................... ................... .................... .................... .................... ................... . 85

3. Protein-sensitive hydrogels .................. .................... ................... .................... .................... ................... .................... .............. 87

3.1. Enzyme-sensitive hydrogels.......... .................... ................... .................... .................... ................... .................... .............. 873.2. Antigen-sensitive hydrogels.............................................................................................................................................. 89

4. Other molecule-sensitive hydrogels ................... .................... ................... .................... .................... .................... ................... . 93

4.1. Molecular imprinting of hydrogels .................... ................... .................... .................... ................... .................... .............. 93

4.2. Other biomolecule-sensitive hydrogels.................. .................... ................... .................... .................... ................... ........... 93

5. Conclusions ............................................................................................................................................................................ 95

Acknowledgements...................................................................................................................................................................... 95

References .................................................................................................................................................................................. 95

*Corresponding author. Tel.: 1 81-6-6368-0949; fax: 1 81-6-6330-3770.

E-mail address: [email protected] (T. Miyata).

0169-409X/ 02/ $ see front matter 2002 Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 9 - 4 09 X ( 0 1 ) 0 0 2 4 1 - 1


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80 T. Miyata et al. / Advanced Drug Delivery Reviews 54 (2002) 7998

1. Introduction [1722] and temperature- [2327] sensitive hydro-

gels have been prepared from polyelectrolyte and

The most important biosystems required to main- poly(N-isopropylacrylamide) (PNIPAAm), respec-

tain life are closely associated with natural feedback tively, for application in self-regulated drug delivery,

system functions such as homeostasis. For example, as temperature and pH are the most widely utilizedhormone release from secretory cells is regulated by triggering signals for modulated intelligent systems.

physiological cycles or specific input signals. Such Organs must respond to the presence of specific

natural feedback systems perceive specific ions or molecules, as well as physicochemical changes, such

biological molecules, such as hormones (sensor as pH and temperature, to maintain life. The next

function), and induce conformational changes or generation of biomaterials and drug delivery systems

rearrange their constitutional biomolecules to elicit will require biomolecule-sensitive hydrogels that

biological functions (effector function). Therefore, by recognize specific biomolecules and respond to them.

combining their functions in polymeric materials, For example, glucose-sensitive hydrogels that under-

natural feedback systems can be mimicked, thus go swelling in response to glucose can provide the

enabling us to fabricate intelligent systems that can tools for constructing self-regulated insulin delivery

be applied in the biomedical and biochemical fields. systems, in which a necessary amount of insulin canPolymeric materials, having both sensor and effector be administered in response to the blood glucose

functions, will contribute significantly to the con- concentration. Even though most biomolecule-sensi-

struction of the next generation of biomaterials and tive hydrogels still require further research before

drug delivery systems. practical application, they are likely to become quite

Hydrogels that exhibit both liquid-like and solid- important materials in the near future. This article

like behavior have a variety of functional properties, provides an overview of current research in the fields

such as swelling, mechanical, permeation, surface of synthesis and application of biomolecule-sensitive

and optical properties. Such properties have provided hydrogels which undergo swelling in response to

many potential applications of hydrogels in fields specific biomolecules, such as glucose, enzymes,

such as medicine, agriculture, biotechnology, etc. antigens, etc.

[1,2]. In addition, hydrogels have the unique proper-

ty of undergoing abrupt volume changes from theircollapsed and swollen states in response to environ-

mental changes [310]. The stimuli-sensitive hydro- 2. Glucose-sensitive hydrogels

gels that undergo volume changes in response to

environmental stimuli are intelligent materials, hav- Diabetes is caused by the inability of the pancreas

ing both sensor and effector functions. They can to control the blood glucose concentration. During

sense a stimulus as a signal and induce structural the treatment of diabetes, a necessary amount of

changes by themselves. With these points in view, insulin, a hormone which is secreted from the

various stimuli-sensitive hydrogels that respond to Langerhans islets of the pancreas and controls glu-

pH [7], temperature [6,8,9,11,12], electric field cose metabolism, must be administered while con-

[13,14], and other stimuli [15,16] have been studied stantly monitoring the blood glucose concentration.

experimentally and theoretically. Due to the fascinat- Many researchers have tried to develop self-reg-ing properties of the stimuli-sensitive hydrogels it is ulated insulin delivery systems in which insulin can

certain that they will have many future applications be released in response to the blood glucose con-

as suitable materials for the design of intelligent centration. Glucose-sensitive hydrogels are very

biomaterials and self-regulated drug delivery sys- useful for the development of self-regulated insulin

tems. Therefore, a variety of stimuli-sensitive hydro- delivery systems and enable us to construct an

gels have been developed for use in switches, artificial pancreas that can administer the necessary

sensors, mechanochemical actuators, drug delivery amount of insulin in response to the blood glucose

devices, specialized separation systems, bioreactors, concentration. The following subsections focus on

and artificial muscles. For example, a variety of pH- three types of glucose-sensitive hydrogels.


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T. Miyata et al. / Advanced Drug Delivery Reviews 54 (2002) 7998 81

2.1. Glucose oxidase-loaded hydrogels

Combining glucose oxidase with pH-sensitive

hydrogels to sense glucose and regulate insulin

release is the method that many researchers haveused to develop glucose-sensitive insulin delivery

systems. Within the pH-sensitive hydrogels contain-

ing glucose oxidase, glucose is converted to gluconic

acid by glucose oxidase, thus lowering the pH in the

hydrogels. Insulin can be released by the pH-sensi-

tive swelling of the hydrogels. Thus, the pH-sensitive

hydrogels containing glucose oxidase can control

insulin release in response to the glucose concen-

tration.

Ishihara et al. [28] combined a copolymer mem-

brane ofN

,N

-diethylaminoethyl methacrylate (DEA)and 2-hydroxypropyl methacrylate (HPMA) with a

cross-linked poly(acrylamide) membrane, in which

glucose oxidase was immobilized. The presence of

glucose enhanced insulin permeability through the

membrane containing glucose oxidase (Fig. 1). The

glucose-sensitive insulin permeation was achieved

based upon the combination of an enzymatic reaction

with a pH-sensitive swelling (Fig. 2). In this system,

glucose diffuses into the membrane and is catalyzed Fig. 2. Schematic representation of the glucose-sensitive hydrogelby glucose oxidase, resulting in the conversion of membrane consisting of a poly(amine) and glucose-oxidase-loaded

membrane.glucose to gluconic acid. The microenvironmental

pH in the membrane becomes low, due to the

production of gluconic acid. As the membrane

swells, resulting from ionization of the amine groups

by the lower pH, insulin permeability through the

membrane is enhanced. Thus, insulin permeation

through the membrane is strongly dependent upon

the glucose concentration. Further, Ishihara et al.

[29] investigated insulin release from polymer cap-

sules containing insulin and glucose oxidase, which

were prepared by a conventional interfacial precipi-

tation method. Insulin release was inhibited in theabsence of glucose, but was strongly enhanced in the

presence of glucose.

Horbett et al. [3032] entrapped glucose oxidase

within hydroxyethyl methacrylate-N,N-di-Fig. 1. Permeation profile of insulin through a glucose-sensitive methylaminoethyl methacrylate copolymer hydrogelpolymer membrane consisting of a poly(amine) and glucose membranes to construct a glucose-sensitive insulinoxidase-immobilized membrane. Glucose concentration: (m) 0 M;

delivery system. To obtain a high insulin permeabili-(d) 0. 1 M; (s) 0. 2 M; (n) 0.2 M without glucose oxidase.

ty, the hydrogel membranes were made porous via(Reprinted, with permission, from Ref. [28]. Copyright 1984 TheSociety of Polymer Science, Japan.) preparation under conditions that induced a phase


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separation during polymerization. The addition of Thus, the poly(MAAc-g-EG) hydrogels containing

glucose resulted in swelling of the membranes and glucose oxidase showed a glucose-sensitivity re-

an enhanced permeation of insulin from a reservoir sulting from the combination of the catalytic reaction

via diffusion through the swollen hydrogel mem- of glucose oxidase and the pH-sensitive complex

branes. Furthermore, to examine pH changes in the formation between carboxyl and etheric groups.glucose-sensitive hydrogel membranes, pH indicator Consequently, glucose-sensitive insulin release can

dyes that convert a pH change to a color change be achieved by using pH-sensitive hydrogels con-

were introduced into the membranes. Based on the taining insulin and glucose oxidase.

results of the experiments, a mathematical model

was developed to describe the steady-state behavior 2.2. Lectin-loaded hydrogels

of the glucose-sensitive hydrogel membranes. Theo-

retical and experimental studies demonstrated that Lectins, which are carbohydrate-binding proteins,

glucose-sensitive hydrogel membranes containing interact with glycoproteins and glycolipids on the

glucose oxidase can achieve a maximum response at cell surface and induce various effects, such as cell

sub-physiological glucose concentrations and not agglutination, cell adhesion to surfaces, and hor-

respond to higher glucose concentrations. The model mone-like action. The unique carbohydrate-bindingrevealed that the glucose-sensitive hydrogel mem- properties of lectins are very useful for the fabrica-

branes with sufficiently low glucose oxidase loading tion of glucose-sensitive systems. Therefore, some

show a progressive response to glucose concen- researchers have focused on the glucose-binding

trations in the physiological range. properties of concanavalin A ( Con A), a lectin

Peppas et al. [33,34] copolymerized methacrylic possessing four binding sites.

acid (MAAc) with poly(ethylene glycol) mono- Brownlee et al. [35] and Kim et al. [36,37] were

methacrylate in the presence of activated glucose pioneers in the development of glucose-sensitive

oxidase in order to prepare glucose-sensitive poly- insulin release systems using Con A. Their strategy

(methacrylic acid-g-ethylene glycol) (poly(MAAc-g- was to synthesize a stable, biologically active glyco-

EG)) hydrogels. Carboxyl groups of MAAc formed a sylated insulin derivative able to form a complex

complex with etheric groups of EG at a low pH, but with Con A. The glycosylated insulin derivative

the complex dissociated at a high pH, due to could be released from its complex with Con A inionization of the carboxyl groups. Therefore, the the presence of free glucose, based on the competi-

poly(MAAc-g-EG) hydrogels collapsed at a low pH, tive and complementary binding properties of glyco-

due to complexation between carboxyl groups and sylated insulin and glucose to Con A. Furthermore,

etheric groups, but were swollen at a high pH. Thus, Kim et al. [38] bound the self-regulated insulin

poly(MAAc-g-EG) hydrogels showed pH-sensitivity delivery systems, enclosed in polymer membranes, to

caused by formation and dissociation of the complex soluble, bead immobilized or cross-linked Con A.

in response to pH changes. The glucose oxidase- Polymer membranes or microcapsules containing

loaded poly(MAAc-g-EG) hydrogels showed a Con A and succinyl-amidophenyl-glucopyranoside

slower swelling rate at high glucose concentrations insulin (SAPG-insulin) quickly controlled the release

as seen in hyperglycemic conditions (200500 mg/ of SAPG-insulin in response to changes in the

dl) than that at the lower glucose concentrations of glucose concentration (Fig. 3), based on the mecha-normal blood (80 mg / dl). As glucose oxidase in the nism of the competitive and complementary binding

poly(MAAc-g-EG) hydrogels catalyzed glucose oxi- properties of SAPG-insulin and glucose to Con A.

dation, the microenvironmental pH in the hydrogels Polymer membranes and microcapsules containing

decreased, due to the production of gluconic acid. Con A and SAPG-insulin were used as self-regulated

Lowering the pH made the poly(MAAc-g-EG) hy- insulin release devices in vitro and could be opti-

drogels collapse caused by a complexation between mized for use in in vivo studies. Their studies

carboxyl and etheric groups. It is postulated that the provided new concepts with regard to the competi-

poly(MAAc-g-EG) hydrogels squeeze out insulin tive and complementary binding properties of glu-

during their collapse in the presence of glucose. cose derivatives and glucose to Con A, as well as


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that Con A can recognize pendant glucose groups of

PGEMA and that the PGEMACon A complex is

sensitive to monosaccharides. Therefore, the

PGEMACon A complex is a promising develop-

ment for the fabrication of a novel glucose sensor ora glucose-sensitive insulin release system.

Novel glucose-sensitive hydrogels were prepared

using complex formation between Con A and the

pendant glucose groups of PGEMA to produce cross-

linked points in the hydrogels [41,42]. The Con

A-entrapped PGEMA hydrogels were obtained by

copolymerization of a monomer with a pendant

glucose (GEMA) and a divinylmonomer in the

presence of Con A. The density of cross-linkage of

the resultant Con A-entrapped PGEMA hydrogels

increased with increasing Con A, suggesting thatCon A acts as a cross-linking agent. The immersion

of Con A-entrapped PGEMA hydrogels in an aque-

ous glucose solution resulted in their swelling and

the swelling ratios were strongly dependent upon theFig. 3. Release of SAPG-insulin in response to stepwise changes

glucose concentration. Compressive modulus mea-in the glucose concentration. (Reprinted, with permission, from

surements revealed that the cross-linking density ofRef. [38]. Copyright 1990 Elsevier Science B.V.)Con A-entrapped PGEMA hydrogels decreased with

increasing glucose concentration. Therefore, the

their suitability for the fabrication of glucose-sensi- glucose-sensitive swelling of Con A-entrapped

tive hydrogels. PGEMA hydrogels was due to the presence of free

A variety of polymers containing saccharide res- glucose, which resulted in the dissociation of the

idues have been synthesized because of their high complex via competitive exchange (Fig. 4). Mannosepotential as promising biomaterials for biochemical caused the swelling of Con A-entrapped PGEMA

and biomedical applications [39]. Some synthetic hydrogels more effectively than glucose and the

polymers with well-defined saccharide residues have swelling ratio did not change in the presence of

been used for the investigation of saccharide-recog- galactose (Fig. 5). As mannose is a stronger inhibitor

nition processes in proteins. Nakamae et al. [40] of PGEMACon A complex formation than glucose,

investigated the complex formation between Con A the former can induce the dissociation of the com-

and a polymer with pendant glucose groups, poly(2- plex more effectively than the latter. A change in the

glucosyloxyethyl methacrylate) (PGEMA). Aqueous swelling ratio of Con A-entrapped PGEMA hydro-

PGEMA was flocculated by the addition of Con A, gels in an aqueous galactose solution was not

due to the complex formation between Con A and observed, because Con A was not able to form

the pendant glucose groups of PGEMA. The turbid complexes with galactose. Thus, the Con A-entrap-PGEMACon A complex solution became transpar- ped PGEMA hydrogels are able to recognize a

ent on the addition of free glucose or mannose, but specific monosaccharide and induce structural

not on the addition of free galactose. This is because changes. These results suggest that Con A-entrapped

free glucose and mannose act as inhibitors, thereby PGEMA hydrogels have many potential applications

inducing the dissociation of the PGEMACon A as a novel glucose sensor and as new closed-loop

complexes. The effect of these monosaccharides on insulin delivery systems.

the dissociation of the PGEMACon A complexes Park et al. [43,44] prepared a new type of hydro-

can be explained by the difference in affinity of Con gel capable of solgel phase-reversible transitions

A for the monosaccharides. These results suggest based upon changes in the glucose concentration. It


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Fig. 4. Schematic representation of glucose-sensitive swelling changes in a poly(GEMA)Con A hydrogel. ( Reprinted, with permission,

from Ref. [41]. Copyright 1996 WileyVCH.)

concentration of free glucose in the environment had

to be at least four times that of pendant glucose to

induce the phase transition from gel to sol. The

solgel phase transition in response to the free

glucose concentration was repeated more than 10

times without any problems. The hydrogel was able

to sense changes in the glucose concentration of the

environment and respond to them in a reversible

manner. Park et al. [45] also investigated the release

of lysozyme and insulin as model proteins throughglucose-sensitive hydrogel membranes using a diffu-

sion cell. Porous poly(hydroxyethyl methacrylate)

(PHEMA) membranes were used to sandwich

glucose-containing polymers and Con A between the

donor and receptor chambers of the cell. The release

rate of the proteins from the receptor chamber to the

donor chamber was strongly dependent upon the free

glucose concentration (Fig. 6). Their studies demon-Fig. 5. Swelling ratio changes of a PGEMACon A gel as a strated that the glucose-sensitive solgel phase tran-function of time when the gel was immersed in a buffer solution

sition can be used to regulate insulin release incontaining 1 wt% of monosaccharide: (s) glucose; (j) mannose;

response to the free glucose concentration in the(d) galactose. (Reprinted, with permission, from Ref. [41].environment.Copyright 1996 WileyVCH.)

Kokufuta et al. [46] combined the carbohydrate-

binding properties of Con A with the temperature-

was shown that the mixture of a vinylpyrrolidinone- sensitive property of poly(N-ispropylacrylamide)

allylglucose copolymer with Con A led to the (PNIPAAm) to prepare saccharide-sensitive hydro-

immediate formation of hydrogels, due to complex gels. Con A was loaded on a cross-linked PNIPAAm

formation between pendant glucose groups and Con hydrogel that underwent a volume phase transition at

A. The addition of free glucose led to a phase 348C. The Con A-loaded PNIPAAm hydrogel was

transition of the hydrogel into the sol state. The shown to swell abruptly in the presence of the ionic


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Fig. 6. Release of lysozyme through a glucose-sensitive hydrogel at initial glucose concentrations of 1 mg/ ml (d) and 4 mg/ml (s).

(Reprinted, with permission, from Ref. [45]. Copyright 1997 Elsevier Science B.V.)

saccharide dextran sulphate at temperatures close to means that complex formation between phenylboron-

the volume phase transition point. The abrupt swell- ic acid and a polyol compound has many potential

ing of the Con A-loaded PNIPAAm hydrogel caused applications as a glucose-sensitive material.

by the ionic saccharide dextran sulphate was attribu- Kitano et al. [47,48] synthesized copolymers with

ted to the ionic osmotic pressure exerted by the phenylboronic acid moieties (poly(NVP-co-PBA))

ionized saccharide. The replacement of the ionic by copolymerizing N-vinyl-2-pyrrolidone (NVP) and

saccharide dextran sulfate with a non-ionic sac- 3-(acrylamido)phenylboronic acid (PBA). Due to the

charide led to the collapse of the hydrogel to its reversible complex formation between phenylboronicnative volume. The temperature-sensitive property of acid of poly(NVP-co-PBA) and poly(vinyl alcohol)

PNIPAAm in the hydrogel contributed to a dramatic (PVA), the competitive binding of phenylboronic

change in the swelling ratio, due to a shift in volume acid with glucose and PVA could be utilized to

phase transition by complex formation. construct a glucose-sensitive system. The formation

and dissociation of the poly(NVP-co-PBA)/PVA2.3. Hydrogels with phenylboronic acid moieties complex could be investigated by observing the

change in viscosity.Viscosity measurements revealed

All of the preceding studies utilized proteins, such that poly(NVP-co-PBA) formed a complex with PVA

as glucose oxidase or lectins, for the fabrication of in the absence of glucose, however the complex

glucose-sensitive hydrogels. This section focuses on dissociated in the presence of glucose. These results

the preparation of glucose-sensitive hydrogels with- led to the concept of a glucose-sensitive insulinout biological components such as proteins, but release system using poly(NVP-co-PBA) and PVA

instead complex formation between a phenylboronic (Fig. 7) [48]. An electrode coated with the poly-

acid group and glucose. (NVP-co-PBA)/PVA complex is an example of the

Phenylboronic acid and its derivatives form com- potential of this system for the development of

plexes with polyol compounds, such as glucose in glucose-sensitive devices [49]. The presence of free

aqueous solution. The complex between phenylbor- glucose resulted in swelling of the poly(NVP-co-

onic acid and a polyol compound can be dissociated PBA) / PVA complex hydrogel, due to complex dis-

in the presence of a competing polyol compound sociation. Thus, the electrode coated with poly(NVP-

which is able to form a stronger complex. This co-PBA)/PVA complex hydrogel could be utilized as


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complex as a glucose-sensitive insulin release sys-

tem, because of its intrinsic instability at a physio-

logical pH of 7.4. To stabilize complex formation

between phenylboronic acid and glucose at a physio-

logical pH of 7.4, amino groups were introducedeither into the polymer or in the vicinity of the

phenylboronic acid moiety [50]. Furthermore, a

glucose-sensitive hydrogel possessing both phenyl-

boronic acid and amino groups was prepared for the

development of a novel glucose-sensitive insulin

delivery system at physiological pH [51]. This

glucose-sensitive insulin delivery system was based

upon the complex formation between gluconated

insulin and the phenylboronic acid groups in the

hydrogel. The gluconated insulin was released from

the hydrogel in the presence of free glucose, whichinduced the dissociation of the complex. This system

was able to achieve an insulin release in response to

the glucose concentration at a physiological pH.

PBA exists in equilibrium between the uncharged

and the charged form (Fig. 8). Complex formation

between the uncharged form and glucose was shown

to be unstable because of its high susceptibility to

hydrolysis, while charged phenylboronic acid groups

were able to form a stable complex with glucose.

Complex formation between the charged phenyl-Fig. 7. Concept of a glucose-sensitive insulin release system using boronic acid groups and glucose caused a shift in the

PVA/poly(NVP-co-PBA) (polymer capsule type). equilibrium towards an increase of charged phenyl-boronic acid groups. Therefore, the total amount of

a glucose sensor, as the current changes were charged phenylboronic acid groups increased and

proportional to the glucose concentration. uncharged groups decreased when glucose was

It is difficult to use the poly(NVP-co-PBA)/ PVA added. A change in the ratio of the uncharged to

Fig. 8. Equilibria of (alkylamido)phenylboronic acid ( 1).


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charged forms by the addition of glucose influenced

the solubility of the polymer in water. The change in

solubility indicated that the shift in the equilibrium

between the uncharged and charged forms of the

phenylboronic acid groups can be used to developglucose-sensitive systems. Kataoka et al. [50] pre-

pared a copolymer of N,N-dimethylacrylamide and

PBA, which had a low critical solution temperature

(LCST) of about 278C, in a buffer solution of pH

7.4. The fact that a copolymer without the phenyl-

boronic acid groups had no LCST suggested that the

phenylboronic acid groups played an important role

in the appearance of LCST. LCST of the copolymer

with the phenylboronic acid groups increased con-

tinually with the addition of glucose. This change in

the glucose-sensitive LCST was attributed to anincrease in hydrophilic, charged phenylboronic acidFig. 9. Temperature dependence of swelling curves for PNIPAAm

groups caused by complex formation between thecopolymer gel with phenylboronic acid moieties at different

phenylboronic acid groups and glucose. glucose concentrations. ( Reprinted, with permission, from Ref.This fascinating property of phenylboronic acid [53]. Copyright 1998 American Chemical Society.)

was combined with the temperature-sensitive proper-

ty of PNIPAAm to improve the glucose-sensitive

LCST of copolymers containing phenylboronic acid 3. Protein-sensitive hydrogels

groups [52]. A ternary polymer was synthesized by

copolymerizing NIPAAm, PBA, and N-(2-di- 3.1. Enzyme-sensitive hydrogels

methylaminopropyl)acrylamide) (DMAPAA). LCST

of the ternary polymer with phenylboronic acid Biodegradable polymers have become increasingly

groups was influenced by the glucose concentration. important in biomedical fields because of their highThis indicates that the solubility of the polymer chain potential for tissue engineering, drug delivery sys-

is strongly dependent upon the glucose concentration tems, etc. [54,55]. Since some biodegradable poly-

at a constant temperature. Based on the glucose- mers can be digested by specific enzymes, enzyme-

sensitive solubility change, Kataoka et al. [53] sensitive hydrogels can be prepared from such

prepared totally synthetic hydrogels (from NIPAAm biodegradable polymers. Some enzymes are used as

and phenylboronic acid) showing glucose sensitivity. important signals for diagnosis to monitor several

These hydrogels underwent a sharp transition in the physiological changes, and specific enzymes in spe-

swelling ratio in response to the external glucose cific organs have become useful signals for site-

concentration (Fig. 9). No insulin was released from specific drug delivery. Therefore, the enzyme-sensi-

the hydrogel in a buffer solution containing less than tive hydrogels are promising candidates as enzyme

1 g / l glucose, but a remarkable release of insulin sensors and enzyme-sensitive drug delivery systems.took place with a glucose concentration of 3 g/ l. The microbial enzymes that are predominantly

Onoff regulation of insulin release from the hydro- present in the colon can be used as signals for

gel was successfully repeated in response to stepwise site-specific delivery of drugs to the colon. Hovgaard

changes in the glucose concentration (Fig. 10). et al. [56] focused on the fact that microbial enzymes

These results suggest that a glucose-sensitive insulin in the colon, such as dextranases, can degrade the

release system can be constructed by exploiting the polysaccharide dextran. They prepared dextran hy-

complex formation properties of phenylboronic acid drogels cross-linked with diisocyanate for colon-

groups and glucose, without the use of biological specific drug delivery. The dextran hydrogels were

components, such as proteins. degraded in vitro by a model dextranase, as well as


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Fig. 10. Repeated on off release of FITC-insulin from a glucose-sensitive hydrogel at 288C, pH 9.0, in response to the external glucose

concentration. (Reprinted, with permission, from Ref. [53]. Copyright 1998 American Chemical Society.)

in vivo in rats and in a human colonic fermentation in the stomach, due to their low swelling ratio at a

model. Release of a drug from the dextran hydrogels low pH. However, when the hydrogels pass through

can be controlled by the presence of dextranase. the gastrointestinal tract they swell due to ionization

Drug release from the dextran hydrogels in the of carboxylic acid groups. In the colon, azoreductase

absence of dextranase was observed to be based on becomes accessible to the cross-links in the swollen

simple diffusion processes, however in the presence hydrogels and can degrade the matrix to release the

of dextranase it was mainly governed by the degra- protein drugs. These studies are examples showing

dation of the dextran. Thus, it follows that dextran that the combination of enzyme sensitivity with pH

hydrogels are dextranase-sensitive and may hold sensitivity enables site-specific drug delivery.promise as intelligent systems for colon-specific drug Some potential applications of stimuli-sensitive

delivery. hydrogels in the medical field require the ability to

Azoreductase is also useful for colon-specific drug sense physiological changes from several diseases at

delivery as it is an enzyme produced by the micro- the same time. Yui et al. [63,64] prepared dual-

bial flora of the colon. To construct colon-specific stimuli-sensitive hydrogels that can be degraded in

drug delivery systems, a few researchers used the presence of two enzymes as biological stimuli.

azoaromatic bonds, which can be degraded by The dual-stimuli-sensitive hydrogels consisted of

azoreductase [5762]. Kopecek et al. [5862] used interpenetrating polymer networks (IPNs) of

azoaromatic bonds as cross-linking agents to prepare oligopeptide-terminated poly(ethylene glycol) (PEG)

azoreductase-sensitive hydrogels for colon-specific and dextran. Only the presence of both papain and

drug delivery. The hydrogels were pH-sensitive and dextranase could induce the degradation of the IPNbiodegradable as they contained both acidic hydrogels, while the presence of only one of the two

comonomers and azoaromatic cross-links. The hy- enzymes was ineffective. Such a dual-stimuli sen-

drogels were based on biocompatible copolymers of sitivity of the IPN hydrogels could act as a fail-safeN,N-dimethylacrylamide (DMAAm), as well as tert- mechanism for guaranteed drug delivery to a certain

butylacrylamide (BAAm) to improve mechanical diseased tissue (Fig. 11). Further, dual-stimuli-sensi-

properties, acrylic acid (AAc) to introduce pH sen- tive hydrogels were prepared by sequential cross-

sitivity, and cross-linking agents containing linking of gelatin and methacryloylated dextran

azoaromatic bonds. The hydrogels can protect pro- below the solgel transition temperature (T ) oftrans

tein drugs against digestion by proteolytic enzymes gelatin [65]. The gelatindextran IPN hydrogels


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Fig. 12. Lipid microsphere release from a gelatin/ dextran IPN

hydrogel in phosphate buffer at 378C. (s) 5 U / m l a-chymo-

trypsin1 0.5 U/ml dextranase; (n) 5 U/ml a-chymotrypsin; (h)

0.5 U /ml dextranase. ( Reprinted, with permission, from Ref. [65].Copyright 1998 Elsevier Science B.V.)

Fig. 11. Concept of dual-stimuli-sensitive drug release by IPN-

structured hydrogel.

structing a fail-safe system for guaranteed drug

prepared below T showed an enzymatic degra- delivery and medical micromachines.trans

dation in the presence of both a-chymotrypsin and

dextranase, however those prepared above T did 3.2. Antigen-sensitive hydrogelstrans

not. This result suggests that the degradation be-

havior of dual-stimuli-sensitive IPN hydrogels is An antibody has recognition sites to bind with a

strongly governed by the IPN structure, i.e. physical specific antigen through multiple noncovalent bonds,

entanglements between chemically different polymer such as electrostatic interactions, hydrogen bonds,

networks. Lipid microspheres, acting as drug mi- hydrophobic interactions, and van der Waals interac-croreservoirs, were released from the dual-stimuli- tions. Such unique features of antibodies are associ-

sensitive hydrogels in the presence of both a-chymo- ated with the immune responses to protect the

trypsin and dextranase, however their release was organism from infection. Antibodies have been em-

completely hindered in the presence of either enzyme ployed in a variety of immunological assays which

alone (Fig. 12). Another type of dual-stimuli-sensi- utilize their specificity and versatility in order to

tive hydrogel was also prepared by combining the detect biological substances [68]. Thus, the specific

temperature-sensitivity of PNIPAAm with enzymatic antigen-recognition function of an antibody can

degradation [66,67]. These hydrogels, consisting of provide the basis for constructing sensors with

NIPAAm, DMAAm, butyl methacrylate (BMA) and various uses for immunoassays and antigen sensing.

a novel biodegradable cross-link, exhibited tem- This section describes novel antigen-sensitive hydro-

perature-sensitive biodegradation. The hydrogels gels that undergo swelling changes in response to awere degraded by an enzyme at low temperatures, specific antigen.

but not at higher temperatures. At higher tempera- Antigen-sensitive hydrogels were prepared by

tures, the formation of an enzymesubstrate complex using antigenantibody bonds at cross-linking points

was sterically hindered by the increased cross-linking in the hydrogels [69,70]. For example, rabbit im-

density of the hydrogels. Therefore, temperature- munoglobulin G (IgG), the antigen, was chemically

sensitive changes in the cross-linking density enabled modified by coupling it with N-succinimidylacrylate

an onoff switch of enzymatic degradation of the (NSA) in phosphate buffer solution to introduce

hydrogels. Hydrogels that are dual-stimuli sensitive vinyl groups into the rabbit IgG. The resultant vinyl-

for enzyme and temperature can be used for con- rabbit IgG was mixed with the antibody, goat anti-


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rabbit IgG (GAR IgG), to form an antigenantibody BIAcore system using surface plasmon resonance

complex. The vinyl-rabbit IgG was then copoly- [71,72]. Measurements of the affinity constant re-

merized with acrylamide (AAm) as a comonomer vealed that the binding of the antibody with polymer-

and N,N9-methylenebisacrylamide (MBAA) as a ized antigen was much weaker than that with the

cross-linker in the presence of GAR IgG, resulting in native antigen. Therefore, the addition of free, nativea hydrogel containing antigenantibody bond sites antigen can induce the dissociation of the complex

(antigenantibody entrapment hydrogel). between antibody and antigen grafted to the hydrogel

After the swelling ratio attained equilibrium in a network. Furthermore, determination of the hydrogel

buffer solution without rabbit IgG, the antigenanti- modulus clarified that the cross-linking density of the

body entrapment hydrogel was immersed in a buffer antigenantibody entrapment hydrogel decreased

solution containing rabbit IgG as a free antigen [69]. gradually in proportion to the increasing free antigen

In the presence of a free rabbit IgG, the antigen concentration in a buffer solution. Consequently, the

antibody entrapment hydrogel showed signs of swell- antigen-sensitive swelling of the antigenantibody

ing. The equilibrium swelling ratio of the antigen entrapment hydrogel can be explained by the com-

antibody entrapment hydrogel was strongly depen- plex exchange mechanism as follows: in the antigen

dent upon the antigen concentration of the buffer antibody entrapment hydrogel in a buffer solutionsolution (Fig. 13). Furthermore, the presence of a containing a free antigen, the free antigen induces

free rabbit IgG resulted in a dramatic increase in the the dissociation of the antigenantibody bonds

swelling ratio of the antigenantibody entrapment grafted to the network, due to the stronger affinity of

hydrogel, but the presence of free goat IgG did not. the antibody for the free antigen than for the antigen

To determine the mechanism responsible for the grafted to the network. Therefore, the hydrogel

antigen-sensitive swelling, the affinity of the anti- underwent swelling in the presence of the free

body for modified antigen was investigated by the antigen because the dissociation of the antigen

antibody bonds resulted in a decrease in the cross-

linking density. Thus, the antigenantibody entrap-

ment hydrogel showed antigen-sensitive behavior on

the basis of the competitive binding properties of the

free antigen and network-grafted antigen to antibody.In general, most potential applications of stimuli-

sensitive hydrogels require a reversible behavior in

response to environmental stimuli changes. In the

case of the antigenantibody entrapment hydrogel,

however, the antibody entrapped in the network

leaked out of the hydrogel, while it underwent

swelling in response to a specific antigen. As a result

of the leak of the antibody, the antigenantibody

entrapment hydrogel did not show reversible swell-

ingshrinking behavior in response to stepwise

changes in the antigen concentration. Therefore, suchhydrogel structures must be designed to prepare

reversible antigen-sensitive hydrogels. To do this, the

antibody must be immobilized within the network so

that it can build a complex with the antigen graftedFig. 13. Effect of the antigen concentration in a phosphate buffer to the network in a buffer solution without a freesolution on the equilibrium swelling ratio of a PAAm hydrogel antigen. With this in view, a reversible, antigen-(s) and an antigenantibody entrapment hydrogel (d), after

sensitive hydrogel was prepared by the fabrication ofswelling equilibrium was attained in a phosphate buffer solution

a semi-interpenetrating polymer network (semi-IPN)containing Rabbit IgG. ( Reprinted, with permission, from Ref.[69]. Copyright 1999 American Chemical Society.) structure, consisting of a linear polymer containing


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the antibodies and a network containing the antigens polymerized GAR IgG. The linear polymerized

[70]. The synthetic strategy and structure of the antibody interpenetrated the antigen-containing net-

antigenantibody semi-IPN hydrogel are shown in work, resulting in an antigenantibody semi-IPN

Fig. 14. As shown in Fig. 14b, both rabbit IgG hydrogel. The linear polymerized antibody did not

(antigen) and GAR IgG (antibody) were chemically leak out of the semi-IPN hydrogel because it wasmodified by coupling them to NSA to produce a entangled with the network.

vinyl-antigen and a vinyl-antibody. The resultant Similar to the antigenantibody entrapment hydro-

vinyl-GAR IgG was copolymerized with AAm to gel, the antigenantibody semi-IPN hydrogel was

create a polymerized GAR IgG that acts as the linear also able to swell immediately after the addition of

chain in a semi-IPN hydrogel. Then, the antigen free rabbit IgG to the buffer solution [70]. The

antibody semi-IPN hydrogel was prepared by the antigenantibody semi-IPN hydrogel possessed the

copolymerization of the vinyl-rabbit IgG, AAm, and antigen-sensing function, as its swelling ratio was

MBAA as a cross-linker in the presence of the strongly dependent upon the antigen concentration in

Fig. 14. Strategy for the preparation of an antigen-sensitive hydrogel with a semi-IPN structure. (Reprinted, with permission, from Ref.

[70]. Copyright 1999 Nature Publishing Group.)


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Fig. 15. Swelling ratio changes of the antigenantibody semi-IPN

hydrogel following the addition of goat IgG (s) and rabbit IgG

(d) after the swelling equilibrium had been attained in phosphate

buffer solution at 258C. The concentration of the antigen in the

phosphate buffer solution was 4 mg/ml. (Reprinted, with permis-

sion, from Ref. [70]. Copyright 1999 Nature Publishing Group.)

Fig. 16. Reversible swelling changes and antigen-sensitive per-the buffer solution. Furthermore, the antigenanti- meation profiles of hemoglobin through a PAAm semi-IPNbody semi-IPN hydrogel responded only to rabbit hydrogel (s) and an antigenantibody semi-IPN hydrogel (d) in

response to stepwise changes in the antigen concentration betweenIgG, but not other IgG (Fig. 15). These results imply0 and 4 mg/ ml. (Reprinted, with permission, from Ref. [70].that the antigenantibody semi-IPN hydrogel is alsoCopyright 1999 Nature Publishing Group.)

antigen-sensitive. Compressive modulus measure-

ments of the antigenantibody semi-IPN hydrogel

demonstrated that its cross-linking density decreased antigen, thus differing from the antigenantibody

with increasing rabbit IgG concentration in a buffer entrapment hydrogel. In addition, the cross-linking

solution. Therefore, the antigen-sensitive swelling of density of the antigenantibody semi-IPN hydrogel

the antigenantibody semi-IPN hydrogel was caused showed a reversible behavior in response to stepwise

by a decrease in its cross-linking density, due to the changes in the antigen concentration. Therefore, by

dissociation of the antigenantibody bonds in the introducing a semi-IPN structure the hydrogel was

presence of the free antigen. Consequently, the able to undergo reversible swelling changes in

antigenantibody semi-IPN hydrogel was able to response to the antigen concentration, most likelyrecognize a specific antigen and induce structural with the following mechanism (Fig. 14a). The an-

changes. tigenantibody semi-IPN hydrogel swelled in the

The reversibility of the antigen-sensitive swelling presence of a free antigen, due to the dissociation of

shrinking behavior of the antigenantibody semi-IPN the antigenantibody bonds, acting as cross-linking

hydrogel was investigated to reveal the effect of the points. In the swollen semi-IPN hydrogel, polymer-

semi-IPN structure [70]. As shown in Fig. 16, the ized antibody could not leak out of the hydrogel as it

antigenantibody semi-IPN hydrogel was able to was trapped in the network containing grafted an-

swell immediately in the presence of a free antigen, tigen. Therefore, the hydrogel was able to shrink

however it shrunk gradually in the absence of reversibly in the buffer solution without free antigen,


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because the complex between the polymerized anti- molecular cavity in order to memorize the print

body and grafted antigen was able to form anew. molecule. Recently, it was reported that temperature-

Consequently, the antigenantibody semi-IPN hy- sensitive hydrogels, prepared using a small amount

drogel exhibits a reversible antigen-sensitive be- of cross-linker, could memorize a print molecule in

havior. their collapsed state via molecular imprinting [78To investigate the possibility of an antigen-sensi- 80].

tive hydrogel as an intelligent system for novel drug Watanabe et al. [78] synthesized temperature-

delivery applications, the permeation of a model sensitive hydrogels by copolymerizing NIPAAm and

drug through an antigenantibody semi-IPN hydro- acrylic acid (AAc) with a cross-linker in the presence

gel membrane was investigated in the presence and of a print molecule and then removing the print

absence of rabbit IgG as a free antigen [70]. The molecule from the resultant hydrogel. The NIPAAm-

model drug permeated through the antigenantibody AAc hydrogel exhibited swelling at a low tempera-

semi-IPN hydrogel membrane in the presence of ture and collapsed at a high temperature. The swol-

rabbit IgG, but not in its absence. As shown in Fig. len NIPAAm-AAc hydrogel at a low temperature

16, the antigenantibody semi-IPN hydrogel mem- showed no change in swelling after the addition of

brane enabled the pulsatile permeation of a model an excess of print molecule, however the hydrogelsdrug in response to stepwise changes in the antigen in the collapsed state at high temperatures showed an

concentration. The antigen-sensitive hydrogel could increase in swelling ratio with increasing print

lead to a novel drug delivery system, in which a drug molecule concentration in water. This suggests that

can be released in the presence of a specific antigen the imprinted hydrogel in the collapsed state can

and the drug release can be stopped in its absence. memorize the print molecule but that in the swollen

Thus, the antigen-sensitive hydrogel is a promising state it cannot. For example, the NIPAAm-AAc

candidate for the fabrication of an intelligent device hydrogel prepared using norephedrine as a print

to modulate drug release in response to a specific molecule was able to swell with increasing nor-

antigen. ephedrine concentration in water, but exhibited no

change in swelling after increasing the adrenaline

concentration (Fig. 17). This implies that the

4. Other molecule-sensitive hydrogels NIPAAm-AAc hydrogel prepared using norephedrineas a print molecule is norephedrine-sensitive. It is

4.1. Molecular imprinting of hydrogels noticeable that the preparation conditions strongly

affected the memorization of the hydrogel prepared

Some proteins, such as enzymes and antibodies, by molecular imprinting. The NIPAAm-AAc hydro-

can recognize specific substrates, based upon the gel prepared in 1,4-dioxane in the presence of a print

correct fit of guest molecules in their molecular molecule underwent a specific volume change in

cavities via noncovalent interactions. Molecular im- response to the print molecule, but when prepared in

printing is an attractive technique to create water it did not. These results were supported by

biomimetic polymers possessing such molecular studies on a general approach for creating hydrogels

cavities for molecular recognition [7377]. In molec- that are able to recognize and capture a target

ular imprinting, some functionalized monomers are molecule by multiple-point interaction, as reportedprearranged around a print molecule via noncovalent by Tanaka et al. [79,80]. Consequently, molecular

interactions and then polymerized. Afterwards, the imprinting is a very useful method to synthesize

print molecule is removed from the resultant poly- molecule-sensitive hydrogels that undergo changes in

mer, resulting in a molecular cavity. The polymer swelling in response to a specific molecule.

with the molecular cavity can thus recognize the

print molecules by a combination of reversible 4.2. Other biomolecule-sensitive hydrogels

binding and shape complementarity. In most molecu-

lar imprinting approaches, a large amount of cross- Deoxyribonucleic acid (DNA) and ribonucleic

linker has been necessary to fix the structure of the acid (RNA) are composed of the nucleotides


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between uracil moieties at a lower temperature, but

became water-soluble above an upper critical solu-

tion temperature (UCST). In addition, the presence

of adenosine resulted in a shift of its UCST to a

lower temperature, because of complex formationbetween the uracil moiety and complementary

adenosine. The effect of the addition of adenosine on

the UCST of PAU was different from that of

guanosine, due to the different interaction of uracil

moieties with adenosine and guanosine. Therefore,

PAU is a nucleic acid base-sensitive polymer, as its

solubility in water changes in response to the species

of additive nucleic acid base and could be used for

the fabrication of a novel nucleic acid base-sensitive

hydrogel for drug delivery purposes.

In addition, Aoki et al. [82] synthesized a tem-perature-sensitive copolymer composed ofN-(S)-sec-

-butylacrylamide ((S)-sec-BAAm) and NIPAAm,

which exhibited hydration changes in response to

foreign, optically active compounds. The LCST

(23.18C) of the resultant poly((S)-sec-BAAm-co-

NIPAAm) with a (S)-sec-BAAm content of 50 mol%

was shifted to 28.7 and 34.58C in the presence of

D-tryptophan (D-Trp) and L-Trp, respectively. The

shift of LCST was strongly dependent upon theL-Trp concentration. The remarkable shift in LCST

of poly((S)-sec-BAAm-co-NIPAAm) in the presence

of L-Trp could be attributable to the stereospecificinteraction between the optically active (S)-sec-

BAAm in the copolymer and L-Trp. These results led

to the concept that a temperature-sensitive polymer

with optically active moieties can respond to enantio-

mers. Based upon this concept, optically active

poly(N-(L)-(1-hydroxymethyl)propylmethacrylamideFig. 17. Equilibrium swelling ratios at 508C as a function of the(L-PHMPMA) and DL-PHMPMA were synthesized,concentration of either norphedrine (d) or adrenaline (s) in

water for molecular recognition gels prepared in the presence of which had optically active moieties and a chemicalnorphedrine (A) and adrenaline (B). (Reprinted, with permission, structure similar to PNIPAAm [83]. An aqueousfrom Ref. [78]. Copyright 1998 American Chemical Society.)

PHMPMA solution exhibited quite a different be-

havior in the temperature-sensitive phase transitionbetween L- and DL-PHMPMA. This indicates that

L-PHMPMA has a unique temperature sensitivity,

adenine, cytosine, guanine, thymine and uracil, and due to the optically active moieties and might

form double or triple strands with their complemen- respond to enantiomers on the basis of this specific

tary base pairs via hydrogen bonding and stacking of interaction. Therefore, enantiomer-sensitive hydro-

their bases. Focusing on complementary hydrogen gels such as poly((S)-sec-BAAm-co-NIPAAm) hy-

bonding between nucleic acid bases, Aoki et al. [81] drogel and L-PHMPMA hydrogel can be prepared

synthesized poly(6-(acryloyloxymethyl)uracil) from such temperature-sensitive polymers with opti-

(PAU) containing uracil moieties as side chains. PAU cally active moieties [84]. The temperature-sensitive

was insoluble in water, due to the complex formation swelling behavior of these hydrogels was strongly


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influenced by the addition of L-Trp. Consequently, from the Ministry of Education, Science, Sports, and

they are enantiomer-sensitive hydrogels that can Culture, Japan.

recognize the difference between L- and D-Trp and

initiate structural changes in response. Such enantio-

mer-sensitive hydrogels are useful for constructing Referencesintelligent systems to sense enantiomers.

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