Gels and hydrogels

IntroductionStructure and properties of hydrogels

Types of hydrogelsApplications

Drug delivery systems

Reference: Gels, pages 490-, Encyclopedia of Smart Materials

What are hydrogels?

• Three-dimensional water-swollen structures

• Composed mainly by hydrophilic homopolymers or copolymers

• Insolubility is granted by chemical or physical cross-links that provide also the shape

• The swelling depends on the external environment

Classifications

• By structure:

• amorphous

• semicrystalline

• hydrogen bonded structures

• supermolecular structures

• hydro-colloidal aggregates

• By functional groups:

• neutral hydrogels

• ionic hydrogels

• By morphology:

• macroporous

• microporous

• nonporous

Sviluppo e applicazioni principali

• Developed from the early 1960. Poly(2-hydroxiethyl methacrylate).

• Principal applications in the biomedical and pharmaceutical fields

• Initially developed as a substitute of natural living tissue due to their rubber nature and high water content

• Properties C: swelling behaviour and network permeability

• Principal applications:

• contact lenses, biosensoris, sutures and dental materials

• controlled drug delivery

Drug delivery systems

• Permeability and swelling depend strongly on:

• chemical nature of the polymer

• structure

• morphology

• Drug delivery systems classification:

• swelling controlled

• chemically controlled

• environmentally responsive

• Smart and responsive hydrogels are ideally suited as intelligent drug delivery systems

Structure and properties of hydrogels

• From morphology the structure is defined by:

• Pores size: ξ

• Molecular weight between cross-links:

• Volume fraction of the polymer in the swollen gel:

�

υ 2,s = Volume polimeroVolume gel rigonfiato

=Vp

Vgel

= 1Q

�

Mc

Cross-links

�

X = M0

2Mc

Degree of cross-linking:Volume of polymer

Volume of swollen gel

Equilibrium swollen theory

• Free energy upon swelling:

• Neutral:

�

ΔG = ΔGelastico + ΔGmiscela

Elastic forces

Interaction energy polymer-solvent

�

1Mc

= 2Mn

−

υV1

⎛

⎝ ⎜

⎞

⎠ ⎟ ln 1−υ 2,s( ) + υ2,s + χ1υ 2,s[ ]

υ 2,rυ 2,s

υ2,r

3 − υ 2,s

υ 2,r

⎡

⎣ ⎢

⎤

⎦ ⎥

Mn : peso molecolare catene lineari senza cross - linkingυ 2,r : frazione volumetrica del polimero nello stato rilassatoυ : volume specifico del polimeroχ1 : parametro di interazione polimero solventeV1 : volume molare del solvente

It can be used to calculate the

molecular weight

: molecular weight of linear chains without cross-linking: volumetric fraction of polymer in the relaxed statespecific volume of the polymerpolymer-solvent interaction parametermolar weight of the solvent

Ionic

Swelling and equilibrium

• Free energy upon swelling:

• Ionic:

�

ΔG = ΔGelastico + ΔGmiscela + ΔGioni Ionic interactions

�

Δµ( )ionRT

= ln 1−υ 2,s( ) + υ 2,s + χ1υ2,s[ ] + V1

υMc

⎛ ⎝ ⎜

⎞ ⎠ ⎟ 1−

2Mc

Mn

⎛

⎝ ⎜

⎞

⎠ ⎟ υ 2,r

υ 2,s

υ2,r

3 − υ 2,s

υ 2,r

⎡

⎣ ⎢

⎤

⎦ ⎥

Δµ( )ion : contributo ionico al potenziale chimico

�

Δµ( )anionicoRT

= RTV14I

υ 2,s2

υ⎛

⎝ ⎜

⎞

⎠ ⎟

Ka

10−pH + Ka

⎛

⎝ ⎜

⎞

⎠ ⎟

2

Δµ( )cationicoRT

= RTV14I

υ 2,s2

υ⎛

⎝ ⎜

⎞

⎠ ⎟

Kb

10pH−14 + Ka

⎛

⎝ ⎜

⎞

⎠ ⎟

2

I : forza ionicaKa,Kb : costanti di dissociazione acido e base

Ionic contribution to the chemical potential

Ionic strengthDissociation constant for acid and base

Rubber elasticity theory

• The constitutive equations that defines the stress-strain behavior can be defined as:

• then for an hydrogel cross-linked in the presence of the solvent we get:�

τ = ρRTMc

1− 2Mc

Mn

⎛

⎝ ⎜

⎞

⎠ ⎟ υ 2,r α − 1

α 2

⎡ ⎣ ⎢

⎤ ⎦ ⎥

τ : sforzoρ : densità polimeroα : allungamento (rapporto lunghezza finale / lunghezza iniziale)

�

τ = ρRTMc

1− 2Mc

Mn

⎛

⎝ ⎜

⎞

⎠ ⎟ υ 2,r α − 1

α 2

⎡ ⎣ ⎢

⎤ ⎦ ⎥

υ 2,r

υ 2,s

3

Stress

Polymer densityElongation ratio (ratio between the elongated and the initial length)

Pore dimension calculation

• ξ is the principal parameter for the rate of a drug delivery system

• It can be computed theoretically or measured directly using different techniques ranging from microscopy to laser-light scattering or indirectly by mercury porosimetry, rubber elasticity measurements or equilibrium swelling experiments:

�

ξ = 2Cn Mc

M 0

lυ 2,s

3

Cn : rapporto caratteristico di Floryl : lunghezza legame lungo le catene polimeriche

Flory characteristic ratio

length of the bonds along the backbone chains

Reversible mechanism

• The hydrogels may be responsive to pH changes or temperature changes

Cyclic response

• In the case of drug delivery systems the delivery can by cyclic

• It may be a regular cyclic delivery or on the need

• Example: physiological pH changes on the stomach

• For diabetic the delivery can be on the need

Classification

• pH-sensitive hydrogels

• Temperature-sensitive hydrogels

• Complexing hydrogels

• Materials sensitive to chemical or enzymatic reaction

• Magnetically responsive systems

Magnetic delivery

(a) No magnetic field: the drug is not delivered

(b) Application of a pulsating magnetic field: the drug can be delivered

(c) The hydrogel change swelling state from compression to extension stimulating the drug delivery

(d) As soon as the magnetic field is interrupted the drug delivery ends

P(MAA-g-EG)

• “grafted” polymers hydrogels

Applications

Contact lenses

• New high permeability lenses made of silicone based hydrogels

• Wear resistance has been improved thanks to specific surface treatments

• The hydrophobic surface of the silicone is converted into hydrophilic to avoid eye adesion and protein accumulation

• These systems have a higher permeability to oxygen

• Can be used up to 30 days without removing them

• They are covering now more than 2/3 of the market

Variable focus lenses

• The hydrogel swelling increases or decreases the pressure on the polymeric film deforming it

• The water-oil interface is the lens that may change the focal length with the pressure

• Micrometric dimensions

Hydrogels Biosensors

APRIL 2007 | VOLUME 10 | NUMBER 4 43

drug release; however, the extent of cleavage is limited by the degree

of crosslinker required to form a suitable gel (Table 1, entry 7).

Kumashiro et al.47 have proposed a delivery mechanism based upon

both a temperature range and enzyme activity. The group synthesized

temperature-responsive hydrogels that only allow enzyme-triggered

polymer degradation above a lower critical solution temperature and

below a higher critical solution temperature. They anticipate that this

technique will allow the release of drug molecules depending on both

enzyme selectivity and changes in body temperature (Table 1, entry 8).

We have developed a nondissolving, enzyme-responsive hydrogel

with physically entrapped guest molecules. Macromolecule release is

determined by charge-induced hydrogel swelling, which is controlled

enzymatically (Fig. 3, Table 1, entry 9) . A cleavable peptide chain is

modified to respond to a particular protease. Our studies detail the

release of 40 kDa dextran and avidin from Asp-Ala-Ala-Arg modified

gel particles following hydrolysis by thermolysin, a bacterial protease

(Fig. 3b)48. Previously, we demonstrated that the accessibility of

poly(ethyleneglycol acrylamide) particles can be controlled by varying

the extent of charge present. Enzymatic hydrolysis to remove positive

charge from the hydrogel results in structural collapse, reducting

molecular accessibility49. This approach may have applications in the

selective removal of (toxic) macromolecules in biological contexts.

Future work in this area is likely to focus on further optimization

of the biocompatibility of polymer systems, their delivery (including

intracellular delivery), and systems with multiple response modes.

Bioresponsive hydrogels for sensingBiosensors are an obvious and increasingly important application of

bioresponsive hydrogels. In these systems, a biological recognition

event is coupled to a macroscopically observable change in hydrogel

properties. Specifically in biosensing applications, it is convenient

that many hydrogels can be readily micro- or nanopatterned to allow

the development of lab-on-a-chip devices. Hydrogel-based biosensor

surfaces are frequently based on PEG, which prevents nonspecific

adsorption of biomolecules. In cell-responsive sensors, this approach

ensures that the response is governed by the surface chemistry rather

than an adsorbed protein layer.

Holtz and Asher50 have developed a hydrogel-based photonic

crystal that acts as a glucose sensor for patients with diabetes mellitus.

Glucose oxidase is attached to arrays of polystyrene nanospheres,

which are then polymerized within a hydrogel matrix. The resulting

material reversibly swells in the presence of glucose (Table 2, entries

1, 2) , similar to the glucose-responsive systems described earlier.

The swelling event increases the mean separation between the

immobilized nanospheres, shifting the Bragg peak of diffracted light to

longer wavelengths and producing a red-shift in the optical properties

(i.e. a readily observed color change) of the polymer (Fig. 4) . This

system can be implanted as contact lenses or ocular inserts to detect

small changes in blood glucose levels indirectly via tear fluid. In this

modified system, boronic acid derivatives are attached to the array and

polymerized within a network of polyacrylamide-PEG. Glucose binds

to the derivatives, producing cross-links that shrink the hydrogel and

cause a blue-shift. The patient is then able to determine their blood

glucose levels via a color chart (Table 2, entry 1)51-53.

Another sensor with an optical output signal uses microlenses made

of poly(N-isopropylacrylamide-co-acrylic acid), or pNIPAm-co-AAc54.

The pNIPAm-co-AAc microlenses are functionalized with biotin to

detect avidin and antibiotin antibodies. Binding of these multivalent

proteins to surface-bound biotin causes additional cross-links to form

in the gel and increases the local refractive index of the hydrogel. The

change in optical properties of the gels can be measured qualitatively:

first by the appearance of ‘dark rings’ in the lenses and second by using

the lenses to focus a square image; the higher the concentration of

avidin or antibiotin, the larger the increase in refractive index and the

more focused the image (Fig. 5; Table 2, entry 2) .

Kim et al.55 recently reported an example of a whole-cell sensing

system using interactions between lymphocytes of the immune system.

Fig. 3 (a) Schematic of enzyme-controlled hydrogel swelling (top). Creation of a positively charged gel results in increased bead swelling. The hydrogel is loaded by altering the pH and, after neutralization, payload release occurs following a specific enzyme reaction (bottom). (b) Charged (avidin) and neutral (dextran) macromolecule release profiles in the presence of a cleaving and a noncleaving control enzyme. (Adapted and reprinted with permission from48. © 2007 Wiley-VCH.)

Entry Stimulus Hydrogel Application Output signal

1 Glucose50-53 PA-PEG Glucose biosensor

Optical, color

2 Protein54 pNIPAm-coAAc

Avidin, antibiotin biosensor

Optical, focusing

3 Peptide55 PEG Live cell biosensor

Biochemical, fluorescence

4 Enzyme58 Aromatic hydrogelator

β-lactamase Gel-formation

Table 2 Recent developments in hydrogel-based biosensors.

Bioresponsive hydrogels REVIEW

(b)(a)

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Contact lenses for diabetics

APRIL 2007 | VOLUME 10 | NUMBER 444

PEG hydrogel microwells are functionalized with antibodies that allow

the specific immobilization of T-cells in a regular pattern at the surface.

Antigen-capturing B-cells are overlaid on top, and act as receptors

for target molecules. Upon addition of a solution containing a model

peptide analyte, B-cells capture and process the peptide molecules,

presenting them to neighboring T-cells. Receptors on the T-cells

recognize the presented antigen, causing a biochemical pathway to

be triggered. Such activation of T-cells can be detected by fluorescent

monitoring of intracellular Ca levels (Fig. 6; Table 2, entry 3) .

The use of small-molecule hydrogels for enzyme (inhibitor) sensing

has been demonstrated by Yang et al.10,56,57. Hydrogel formation is

exploited in the biological sensing of β-lactamases, bacterial enzymes

that cause antibiotic inactivation in resistant bacterial strains (Table 2,

entry 4). In this work, treating a nongelling, β-lactam-containing

conjugate (β-lactam is a substrate for β-lactamases) with β-lactamase

cleaves the scissile β-lactam amide bond, thereby releasing a potent

hydrogelator. Gel formation is readily observed by the naked eye.

This approach provides a low cost and easy to use method that could

be used to screen for inhibitors of this class of enzymes, which holds

promise for identification of next generation antibiotics58.

Bioresponsive hydrogels in tissue engineeringTissue engineering aims to regenerate damaged or diseased tissues

and organs59. The development of biomaterials that facilitate the

mechanical and cellular regeneration of tissue is crucial to its success.

Current strategies involve the production of porous scaffolds for

cells to colonize. Ideal scaffolds are those that mimic the ECM that

surrounds cells in their natural context. The current emphasis is on

creating materials that are highly hydrated, nanofibrous, directional,

of appropriate mechanical strength, and contain bioactive signals to

direct cell behavior23,25,34. Here, we cover recent research on materials

that respond to (cell-secreted) enzymes or are modified by enzymatic

action, thereby mimicking the adaptive properties of natural ECMs.

ECM-mimicking hydrogel scaffolds that permit cell migration have

been studied by Hubbell and coworkers22,60. The researchers use

oligopeptides as cross-linkers in PEG-based hydrogels. The peptide

sequences are cleavable by matrix metalloproteinases (MMPs) to form

a gel into which cells can infiltrate. MMPs are a family of enzymes

that have many roles including the breakdown of ECM molecules

during tissue remodeling and disease. Therefore, the integration of

MMP-cleavable sites is a logical approach toward ECM mimics. Human

Fig. 4 (a) Polystyrene particles arrayed within a three-dimensional hydrogel matrix. (b) Upon exposure to an analyte, the array changes in volume, causing a change in separation between the particles and a shift in the observed wavelength. (c) Diffraction shifts of up to ~170 nm are observed in response to changes in glucose concentration. (Reprinted with permission from52. © 2004 American Association for Clinical Chemistry.)

Fig. 5 (a) Synthesis of pNIPAm-co-AAc hydrogel microparticles by precipitation polymerization, and (b) their biotinylation. Binding of (c) avidin or (d) antibiotin causes additional cross-links to be formed in the hydrogel. (e) Formation of dark rings (left column) in different concentrations of avidin and the focusing of a square image (right column) in response to avidin binding. (Reprinted with permission from54. © 2005 American Chemical Society.)

REVIEW Bioresponsive hydrogels

(b)(a) (c)

(b)(a)(c)

(d)

(e)

p40_48.indd 44p40_48.indd 44 09/03/2007 11:39:2909/03/2007 11:39:29

APRIL 2007 | VOLUME 10 | NUMBER 444

PEG hydrogel microwells are functionalized with antibodies that allow

the specific immobilization of T-cells in a regular pattern at the surface.

Antigen-capturing B-cells are overlaid on top, and act as receptors

for target molecules. Upon addition of a solution containing a model

peptide analyte, B-cells capture and process the peptide molecules,

presenting them to neighboring T-cells. Receptors on the T-cells

recognize the presented antigen, causing a biochemical pathway to

be triggered. Such activation of T-cells can be detected by fluorescent

monitoring of intracellular Ca levels (Fig. 6; Table 2, entry 3) .

The use of small-molecule hydrogels for enzyme (inhibitor) sensing

has been demonstrated by Yang et al.10,56,57. Hydrogel formation is

exploited in the biological sensing of β-lactamases, bacterial enzymes

that cause antibiotic inactivation in resistant bacterial strains (Table 2,

entry 4). In this work, treating a nongelling, β-lactam-containing

conjugate (β-lactam is a substrate for β-lactamases) with β-lactamase

cleaves the scissile β-lactam amide bond, thereby releasing a potent

hydrogelator. Gel formation is readily observed by the naked eye.

This approach provides a low cost and easy to use method that could

be used to screen for inhibitors of this class of enzymes, which holds

promise for identification of next generation antibiotics58.

Bioresponsive hydrogels in tissue engineeringTissue engineering aims to regenerate damaged or diseased tissues

and organs59. The development of biomaterials that facilitate the

mechanical and cellular regeneration of tissue is crucial to its success.

Current strategies involve the production of porous scaffolds for

cells to colonize. Ideal scaffolds are those that mimic the ECM that

surrounds cells in their natural context. The current emphasis is on

creating materials that are highly hydrated, nanofibrous, directional,

of appropriate mechanical strength, and contain bioactive signals to

direct cell behavior23,25,34. Here, we cover recent research on materials

that respond to (cell-secreted) enzymes or are modified by enzymatic

action, thereby mimicking the adaptive properties of natural ECMs.

ECM-mimicking hydrogel scaffolds that permit cell migration have

been studied by Hubbell and coworkers22,60. The researchers use

oligopeptides as cross-linkers in PEG-based hydrogels. The peptide

sequences are cleavable by matrix metalloproteinases (MMPs) to form

a gel into which cells can infiltrate. MMPs are a family of enzymes

that have many roles including the breakdown of ECM molecules

during tissue remodeling and disease. Therefore, the integration of

MMP-cleavable sites is a logical approach toward ECM mimics. Human

Fig. 4 (a) Polystyrene particles arrayed within a three-dimensional hydrogel matrix. (b) Upon exposure to an analyte, the array changes in volume, causing a change in separation between the particles and a shift in the observed wavelength. (c) Diffraction shifts of up to ~170 nm are observed in response to changes in glucose concentration. (Reprinted with permission from52. © 2004 American Association for Clinical Chemistry.)

Fig. 5 (a) Synthesis of pNIPAm-co-AAc hydrogel microparticles by precipitation polymerization, and (b) their biotinylation. Binding of (c) avidin or (d) antibiotin causes additional cross-links to be formed in the hydrogel. (e) Formation of dark rings (left column) in different concentrations of avidin and the focusing of a square image (right column) in response to avidin binding. (Reprinted with permission from54. © 2005 American Chemical Society.)

REVIEW Bioresponsive hydrogels

(b)(a) (c)

(b)(a)(c)

(d)

(e)

p40_48.indd 44p40_48.indd 44 09/03/2007 11:39:2909/03/2007 11:39:29

APRIL 2007 | VOLUME 10 | NUMBER 444

PEG hydrogel microwells are functionalized with antibodies that allow

the specific immobilization of T-cells in a regular pattern at the surface.

Antigen-capturing B-cells are overlaid on top, and act as receptors

for target molecules. Upon addition of a solution containing a model

peptide analyte, B-cells capture and process the peptide molecules,

presenting them to neighboring T-cells. Receptors on the T-cells

recognize the presented antigen, causing a biochemical pathway to

be triggered. Such activation of T-cells can be detected by fluorescent

monitoring of intracellular Ca levels (Fig. 6; Table 2, entry 3) .

The use of small-molecule hydrogels for enzyme (inhibitor) sensing

has been demonstrated by Yang et al.10,56,57. Hydrogel formation is

exploited in the biological sensing of β-lactamases, bacterial enzymes

that cause antibiotic inactivation in resistant bacterial strains (Table 2,

entry 4). In this work, treating a nongelling, β-lactam-containing

conjugate (β-lactam is a substrate for β-lactamases) with β-lactamase

cleaves the scissile β-lactam amide bond, thereby releasing a potent

hydrogelator. Gel formation is readily observed by the naked eye.

This approach provides a low cost and easy to use method that could

be used to screen for inhibitors of this class of enzymes, which holds

promise for identification of next generation antibiotics58.

Bioresponsive hydrogels in tissue engineeringTissue engineering aims to regenerate damaged or diseased tissues

and organs59. The development of biomaterials that facilitate the

mechanical and cellular regeneration of tissue is crucial to its success.

Current strategies involve the production of porous scaffolds for

cells to colonize. Ideal scaffolds are those that mimic the ECM that

surrounds cells in their natural context. The current emphasis is on

creating materials that are highly hydrated, nanofibrous, directional,

of appropriate mechanical strength, and contain bioactive signals to

direct cell behavior23,25,34. Here, we cover recent research on materials

that respond to (cell-secreted) enzymes or are modified by enzymatic

action, thereby mimicking the adaptive properties of natural ECMs.

ECM-mimicking hydrogel scaffolds that permit cell migration have

been studied by Hubbell and coworkers22,60. The researchers use

oligopeptides as cross-linkers in PEG-based hydrogels. The peptide

sequences are cleavable by matrix metalloproteinases (MMPs) to form

a gel into which cells can infiltrate. MMPs are a family of enzymes

that have many roles including the breakdown of ECM molecules

during tissue remodeling and disease. Therefore, the integration of

MMP-cleavable sites is a logical approach toward ECM mimics. Human

Fig. 4 (a) Polystyrene particles arrayed within a three-dimensional hydrogel matrix. (b) Upon exposure to an analyte, the array changes in volume, causing a change in separation between the particles and a shift in the observed wavelength. (c) Diffraction shifts of up to ~170 nm are observed in response to changes in glucose concentration. (Reprinted with permission from52. © 2004 American Association for Clinical Chemistry.)

Fig. 5 (a) Synthesis of pNIPAm-co-AAc hydrogel microparticles by precipitation polymerization, and (b) their biotinylation. Binding of (c) avidin or (d) antibiotin causes additional cross-links to be formed in the hydrogel. (e) Formation of dark rings (left column) in different concentrations of avidin and the focusing of a square image (right column) in response to avidin binding. (Reprinted with permission from54. © 2005 American Chemical Society.)

REVIEW Bioresponsive hydrogels

(b)(a) (c)

(b)(a)(c)

(d)

(e)

p40_48.indd 44p40_48.indd 44 09/03/2007 11:39:2909/03/2007 11:39:29

Avidin-antibiotin for focusing lenses

APRIL 2007 | VOLUME 10 | NUMBER 444

PEG hydrogel microwells are functionalized with antibodies that allow

the specific immobilization of T-cells in a regular pattern at the surface.

Antigen-capturing B-cells are overlaid on top, and act as receptors

for target molecules. Upon addition of a solution containing a model

peptide analyte, B-cells capture and process the peptide molecules,

presenting them to neighboring T-cells. Receptors on the T-cells

recognize the presented antigen, causing a biochemical pathway to

be triggered. Such activation of T-cells can be detected by fluorescent

monitoring of intracellular Ca levels (Fig. 6; Table 2, entry 3) .

The use of small-molecule hydrogels for enzyme (inhibitor) sensing

has been demonstrated by Yang et al.10,56,57. Hydrogel formation is

exploited in the biological sensing of β-lactamases, bacterial enzymes

that cause antibiotic inactivation in resistant bacterial strains (Table 2,

entry 4). In this work, treating a nongelling, β-lactam-containing

conjugate (β-lactam is a substrate for β-lactamases) with β-lactamase

cleaves the scissile β-lactam amide bond, thereby releasing a potent

hydrogelator. Gel formation is readily observed by the naked eye.

This approach provides a low cost and easy to use method that could

be used to screen for inhibitors of this class of enzymes, which holds

promise for identification of next generation antibiotics58.

Bioresponsive hydrogels in tissue engineeringTissue engineering aims to regenerate damaged or diseased tissues

and organs59. The development of biomaterials that facilitate the

mechanical and cellular regeneration of tissue is crucial to its success.

Current strategies involve the production of porous scaffolds for

cells to colonize. Ideal scaffolds are those that mimic the ECM that

surrounds cells in their natural context. The current emphasis is on

creating materials that are highly hydrated, nanofibrous, directional,

of appropriate mechanical strength, and contain bioactive signals to

direct cell behavior23,25,34. Here, we cover recent research on materials

that respond to (cell-secreted) enzymes or are modified by enzymatic

action, thereby mimicking the adaptive properties of natural ECMs.

ECM-mimicking hydrogel scaffolds that permit cell migration have

been studied by Hubbell and coworkers22,60. The researchers use

oligopeptides as cross-linkers in PEG-based hydrogels. The peptide

sequences are cleavable by matrix metalloproteinases (MMPs) to form

a gel into which cells can infiltrate. MMPs are a family of enzymes

that have many roles including the breakdown of ECM molecules

during tissue remodeling and disease. Therefore, the integration of

MMP-cleavable sites is a logical approach toward ECM mimics. Human

Fig. 4 (a) Polystyrene particles arrayed within a three-dimensional hydrogel matrix. (b) Upon exposure to an analyte, the array changes in volume, causing a change in separation between the particles and a shift in the observed wavelength. (c) Diffraction shifts of up to ~170 nm are observed in response to changes in glucose concentration. (Reprinted with permission from52. © 2004 American Association for Clinical Chemistry.)

Fig. 5 (a) Synthesis of pNIPAm-co-AAc hydrogel microparticles by precipitation polymerization, and (b) their biotinylation. Binding of (c) avidin or (d) antibiotin causes additional cross-links to be formed in the hydrogel. (e) Formation of dark rings (left column) in different concentrations of avidin and the focusing of a square image (right column) in response to avidin binding. (Reprinted with permission from54. © 2005 American Chemical Society.)

REVIEW Bioresponsive hydrogels

(b)(a) (c)

(b)(a)(c)

(d)

(e)

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Tissue regeneration - scaffolds

APRIL 2007 | VOLUME 10 | NUMBER 4 45

fibroblasts are encouraged to invade the hydrogel through integrin-

binding domains (Arg-Gly-Asp-Ser-Pro) that are incorporated via PEG

linkers. The fibroblasts then cause a local breakdown of the hydrogel

cross-links via secreted MMPs (Table 3, entry 1) . The potential for

bone tissue engineering was tested by loading the gel with bone

morphogenetic protein-2 (BMP-2), which is known to be involved in

bone formation. An assessment of the degradation behavior of MMPs

and the cell invasion of provisional matrices revealed that the healing

response in vivo depends on the enzymatic sensitivity of the matrix.

Raeber et al.61 (Table 3, entry 2) subsequently tested the suitability

of two proteolytically degradable PEG hydrogels as ECM mimics

that allow regulated cell migration in three dimensions (Fig. 7) . In

one system, the PEG hydrogel is chemically cross-linked with an

MMP-sensitive sequence (M-PEG). In the second, a plasmin-sensitive

sequence (P-PEG) is used. Both sequences have been previously found

to allow proteolytic remodeling of bone defects. The three-dimensional

migration patterns and cell morphology of human fibroblasts in

M-PEG and P-PEG have noticeable differences, with cell migration

only observed in M-PEG. To regulate the MMP function of the PEG

hydrogels, two new hydrogels were made, one containing an MMP

inhibitor and the other an MMP stimulator linked to the PEG hydrogel.

The former results in complete suppression of cell migration, while the

latter results in a significant increase in cell migration. These results

indicate that migration in M-PEG gels is highly sensitive to MMP

modulation. The ability of a gel to respond to a single class of enzyme

represents an effective communication between cells and the matrices.

Kim et al.62 have created an injectable hydrogel of pNIPAm-co-AAc

to mimic the ECM (Table 3, entry 3). These hydrogels are prepared by

cross-linking an MMP-13/collagenase-3-degradable peptide sequence

and NIPAm in the presence of Arg-Gly-Asp-modified poly(AAc). The

proteolytic degradation and cell adhesion properties of this hydrogel

were studied using rat calvarial osteoblasts. Collagenase was found to

degrade the hydrogel, with the rate dependent on the concentration

of collagenase in relation to the poly(AAc) chain. Migration of

osteoblasts is observed in hydrogels both with and without the Arg-

Gly-Asp peptide. However, greater migration is seen in those hydrogels

Fig. 6 (a) B-cells capture pathogens, such as the different viral particles shown, and present them as peptide-major histocompatibility complex (p-MHC) assemblies. T-cell receptors (TCRs) on T-lymphocytes recognize p-MHCs, causing a signaling pathway response. (b) This interaction is employed in a sensing system. (c) The T-cell signaling pathway can be detected by fluorescence. (A) A fluorescence micrograph of microwells immediately after pathogen addition. (B) The microwells after 10 min exposure to the pathogen. (Reprinted with permission from55. © 2006 Wiley-VCH.)

Entry Material Stimulus Cell type

1 Oligopeptides

Ac-CGYGRGDSPG60

Metalloproteinase

(MMP)

Human fibroblasts

2 Polyethylene glycol (PEG)61 MMP Dermal fibroblasts

3 pNIPAm-Co-AAc62 MMP-13 Rat calvarial osteoblasts (RCOs)

4 Gelatin/gellan65 TGase Fibroblasts NIH T3 cells

5 Gelatin66 mTGase Retinal tissue

6 Three-dimensional fibrin hydrogels modified with αVβ3 receptor68 TGase Human umbilical cells

7 CH3(CH2)14CO-GTAGLIGQRGDS69 MMP-2 Dental pulp cells

8 Nap-FFGEY70 (i) kinase

(ii) phosphatase

HeLa cells

9 Polyurethane/polycaprolactone/PEG73 Elastase Endothelial cells

10 Phosphoester/PEG72 Alkaline phosphatase Mesenchymal stem cells

Table 3 Enzyme-responsive gels for tissue regeneration and culture.

Bioresponsive hydrogels REVIEW

(b)(a) (c)

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Wound-care dressing (skin regeneration)

• The hydrogel is self-supporting and stimulates the regeneration of the skin

• It is permeable to the exudate produced by the wound that can be espelled

• The hydrogel can be loaded with drugs that treat the wound

Drug delivery systems

• Drug-hydrogel composite that may release the pharmaceutical in a controlled way and timing

• The drug delivery may be done:

• as a constant release over a long period

• cyclic release over a long period

• on the need based on external stimuli

• Goals:

• maximize therapy effects

• minimize over- or under-doses

• Extremely important for drugs that cannot be delivered by oral or by injection

Controlled drug delivery

• Pro

• controlled delivery between minimum and maximum dose

• reduced external assistance

• optimal use of drug

• improved tolerance by the patient

• Cons:

• possible intolerance to the hydrogel or materials used

• formation of unwanted compounds by degeneration of the carrier

• possible surgical intervention to insert the system

• possible uneasiness of the patient

• cost

Carriers ideal characteristics

• The ideal drug delivery system should be:

• inert

• biocompatible

• mechanically resistant

• acceptable by the patient

• able to carry high doses of pharmaceuticals

• avoid accidental release

• easy to handle, insert, remove

• easy to produce, stock and sterilize

Traditional and controlled system for drug delivery

Traditional injection of the pharmaceutical at regular intervals

Controlled release (there are system to go from 1 day to 1 month up to 5 years)

Matrix release system

• The matrix progressively releases the pharmaceutical and the drug is homogeneously distributed in the matrix

• The release rate decreases with time as the drug has to do a longer path from the internal core and the area decreases with r2

• If the release rate decreases the drug delivery goes for longer time

Source (tank) releasing system

• More constant release rate over time

• the rate is determined by the thickness of the polymer/hydrogel and remains constant over time

• (a) oral or internal system

• (b) skin patch system

Possible biodegradable systems

• Biodegradable polymers

• Polymers containing the drug that with slower or faster degradation release the pharmaceutical

• More often are aggregation of polymer particles that can be dispersed in the environment

• Hydrogels responsive to the environment

• This kind of system are collapsed (dry) before entering the human body. Inside they capture water swelling and releasing the drug

Hydrogels for drug delivery

(a)Source system: the drug is contained in the tank, the hydrogel membrane swell opening the pores and releasing the drug

(b)Matrix system: the hydrogel contains the drug inside and releases it by swelling and opening of the porosity

Example: oral ingestion

• With hydrogels we can realize delivery using oral ingestion

• Anionic hydrogels are used

• From mouth to stomach the pH is low and the drug is not delivered

• In the intestine the pH increases and the drug is delivered

Diabetics and insulin

• Need to monitor the glucose level in the blood

• Glucose-oxidase immobilized over the system

• The reaction glucose/glucose-oxidase lower the pH of the system

• The low pH causes the pores opening and drug release

Membrane in copoliymer: poly(methacrylic acid-g-poly(ethylene glycol))

Drug delivery systems with hydrogels

Stimulus Hydrogel Mechanism

pH Acidic or basic hydrogel Change in pH — swelling — release of drug

Ionic strength Ionic hydrogel Change in ionic strength — change in concentration of ions inside gel-change in swelling — release of drug

Chemical species Hydrogel containing electron-accepting groups

Electron-donating compounds — formation of charge/transfer complex — change in swelling — release of drug

Enzyme-substrate Hydrogel containing immobilized enzymes

Substrate present — enzymatic conversion — product changes swelling of gel — release of drug

Magnetic Magnetic particles dispersed in alginate microshperes

Applied magnetic field — change in pores in gel — change in swelling — release of drug

ThermalThermoresponsive hrydrogel

poly(N-isopro-pylacrylamide)

Change in temperature — change in polymer-polymer and water-polymer interactions — change in swelling — release of drug

Electrical Polyelectrolytehydrogel

Applied electric field — membrane charging — electrophoresis of charged drug — change in swelling — release of drug

Ultrasound irradiation Ethylene-vinyl alcohol hydrogel Ultrasound irradiation — temperature increase — release of drug