Photo-Cross-Linked Hydrogels from Thermoresponsive

PEGMEMA-PPGMA-EGDMA Copolymers Containing Multiple

Methacrylate Groups: Mechanical Property, Swelling, Protein

Release, and Cytotoxicity

Hongyun Tai,*

,†,‡,§

Daniel Howard,

|

Seiji Takae,

‡

Wenxin Wang,*

,

⊥

Tina Vermonden,

#

Wim E. Hennink,

#

Patrick S. Stayton,

‡

Allan S. Hoffman,

‡

Andreas Endruweit,

∇

Cameron Alexander,

|

Steven M. Howdle,

§

and Kevin M. Shakesheff

|

School of Chemistry, Bangor University, Bangor, LL57 2UW, United Kingdom, Department of

Bioengineering, University of Washington, P.O. Box 355061, Seattle, Washington 98195, School of

Chemistry, School of Pharmacy, and School of Mechanical, Materials and Manufacturing Engineering,

The University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom, Network of

Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland, and Department of
Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

Received June 23, 2009; Revised Manuscript Received August 25, 2009

Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials
via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolym-
erization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-cross-
linked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over
non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of
the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple
methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization
(ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M

n

) 475), poly(propylene glycol)

methacrylate (PPGMA, M

n

) 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent

cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence
on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a
model protein demonstrated a sustained release profile that varied dependent on the copolymer composition,
cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the
copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as
assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These
studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ
photopolymerizable materials for tissue engineering and drug delivery applications through a combination of
facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible
functionality for further structure modifications.

Introduction

Hydrogels are 3D networks of physically or chemically cross-

linked hydrophilic polymers and have been extensively used in
various biomedical applications.

1,2

The soft and hydrophilic

nature of hydrogels makes them particularly suitable as protein
delivery system or as a cell-entrapping scaffold in tissue
engineering.

3

In recent years, in situ curing gels, also called

injectable scaffolds, have attracted much attention because they
offer the possibility of homogeneously distributing cells and
molecular signals throughout the scaffolds and can be injected
directly into cavities with irregular shapes and sizes.

3-14

Finding

suitable materials that can solidify in situ with desired mechan-
ical and biological properties remains a challenge.

The in situ gelation can be achieved through either physical

or chemical cross-linking. Smart materials, which respond to
external stimuli such as temperature and pH can form hydrogels
via physical cross-linking.

15,16

In general, gels based on these

physical interactions are mechanically weak. In contrast, gelation
from macromonomers via chemical cross-linking can exhibit
much better mechanical performance. However, chemical cross-
linking can be a harsh procedure with respect to encapsulated
biological components. Photopolymerization as a cross-linking
method is a relatively mild procedure and provides many
benefits, including rapid polymerization, while maintaining
physiological conditions and good spatial and temporal control
over gelation.

8,17

Therefore, photo-cross-linking has been used

as an effective approach for the development of injectable
systems for a number of biomedical applications including
prevention of thrombosis, postoperative adhesion formation,
drug delivery, coatings for biosensors, and cell transplantation.

18-20

It has been shown that photo-cross-linkable materials have the

* To whom correspondence should be addressed. E-mail: h.tai@

bangor.ac.uk (H.T.); [e-mail] (W.W.).

†

Bangor University.

‡

University of Washington.

§

School of Chemistry, The University of Nottingham.

|

School of Pharmacy, The University of Nottingham.

⊥

National University of Ireland.

#

Utrecht University.

∇

School of Mechanical, Materials and Manufacturing Engineering, The

University of Nottingham.

Biomacromolecules 2009, 10, 2895–2903

2895

10.1021/bm900712j CCC: $40.75

 2009 American Chemical Society

Published on Web 09/11/2009

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

potential to be used for in vitro as well as in vivo applications
via minimally invasive procedures, such as laparascopic devices,
catheters, or subcutaneous injection with transdermal illumina-
tion. So far, a series of polyethylene glycol (PEG) based and
poly(lactic acid) (PLA) based linear multivinyl macro-
mers,

8,17,21-25

branched and star polymerizable polymers,

26-29

and dendrimers with acrylate end groups

12,14,30-32

have been

investigated as in situ gelling materials via photopolymerization
for regenerative medicine applications.

It is envisioned that polymers combining both thermorespon-

sive and photo-cross-linkable properties should exhibit superior
performance over solely thermoresponsive or photo-cross-
linkable materials for tissue engineering injectable scaffolds.

33,34

These polymer solutions can be rapidly confined within targeted
sites after immediate administration due to in situ thermal
gelation and, subsequently, form gels with the desired mechan-
ical properties by photo-cross-linking. Recently, we have
reported the successful synthesis of PEGMEMA-PPGMA-
EGDMA copolymers with both thermoresponsive and photo-
cross-linkable properties via one-step deactivation enhanced
atom transfer radical polymerization (ATRP).

35

In this paper,

the mechanical properties, swelling behavior, drug release, and
cytocompatibility of the photo-cross-linked gels derived from
these new smart copolymers were further investigated. The
results demonstrate that thermoresponsive PEGMEMA-PPGMA-
EGDMA copolymers offer potential as in situ photopolymer-
izable materials for tissue engineering and drug delivery
applications through a combination of facile synthesis, enhanced
mechanical properties, tunable cross-linking density, low cy-
totoxicity, and accessible functionality for further structure
modifications.

Experimental Section

Preparation of Thermoresponsive Copolymers Containing Multiple

Methacrylate Groups. The PEGMEMA-PPGMA-EGDMA copolymers
were synthesized according to the previously published method.

35

Briefly, the reactions were conducted in 2-butanone (99.5%, HPLC
grade, Aldrich) at a volume ratio of total monomers and solvent of 1:1
with a Schlenk line system. Argon was bubbled through the solutions
to eliminate oxygen and liquids were transferred under argon by means
of septa and syringes or stainless steel capillaries. A round-bottom flask
fitted with a three-way stopcock was charged with copper(I) chloride
(CuCl, 95%, Acros), copper(II) chloride (CuCl

2

, 99%, Lancaster), and

2,2

′-bipyridine (bpy, Aldrich) and then connected to the Schlenk line.

Oxygen was removed by repeated vacuum-argon cycles. The degassed
PEGMEMA (M

n

) 475, Sigma-Aldrich), PPGMA (M

n

) 375, Sigma-

Aldrich), EGDMA (Sigma-Aldrich), and butanone were transferred into
the flask. Under magnetic stirring at 500 rpm, the initiator stock solution
methyl 2-chloropropionate (Aldrich) in 2-butanone was added, and the
polymerization was conducted at 60

°C in an oil bath for a desired

reaction time. After the polymerization, the solution was diluted with
acetone and passed through a silica column to remove copper catalyst.
The subsequent solutions were precipitated into a large excess of hexane
to remove PPGMA and EGDMA monomers. The precipitated mixture
of the polymer and PEGMEMA was dissolved in deionized water and
purified by dialysis (Spectrum dialysis membrane, molecular weight
cut off 3500) for 72 h in a dark environment at 4

°C to remove

PEGMEMA against fresh deionized water, while the water was changed
regularly. The pure polymer samples were obtained after freeze-drying,
then characterized by gel permeation chromatography (GPC) and

1

H

NMR. Number average molecular weight (M

n

), weight average mo-

lecular weight (M

w

), and polydispersity (M

w

/M

n

) were obtained by GPC

(PL-120, Polymer Laboratories) with an RI detector and multiangle
laser light scattering (MALLS) detector (mini-Dawn) supplied by Wyatt
Technology. The columns (30 cm PLgel Mixed-C, two in series) were

eluted by THF and calibrated with polystyrene standards. All calibra-
tions and analyses were performed at 40

°C and a flow rate of 1 mL/

min.

1

H NMR was carried out on a 300 MHz Bruker NMR with

MestRec processing software. The chemical shifts were referenced to
the lock CDCl

3

. Differential scanning calorimetry (DSC, TA 2920) was

used to measure the glass transition temperatures (T

g

) of the dried

copolymers.

Thermoresponsive Behavior of the Copolymers. The lower critical

solution temperatures (LCST) of the copolymers at 0.03% w/v solutions
in deionized water, phosphate buffered saline (PBS, pH 7.4), and cell
culture media (Dulbecco’s modified Eagle’s medium (DMEM)),
supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and
2.5 mg/mL amphotericin B (antibiotic/ antimycotic solution)) were
quantified by measuring their absorbance of 530 nm at temperatures
from 12 to 60

°C (heating rate ) 0.5 °C/sec) with a Beckman DU-640

spectrophotometer. The data were collected every 2 s. Moreover,
dynamic light scattering (DLS) was used to analyze sizes and
distributions of the copolymers in water solutions on a Malvern Nano
Zetasizer. Polymer solutions (0.01% w/v) were prepared in deionized
water and filtered prior to measurements using a 0.45 µm disposable
filter into a 12.5

× 12.5 mm polystyrene disposable cuvette.

Rheological, Mechanical, and Morphological Studies of Photo-

Cross-Linked Gels. Real-time photo-cross-linking rheological studies
were performed on an AR1000-N (TA Instruments) using parallel-plate
geometry (20 mm diameter) equipped with a UV light source (BluePoint
lamp 4, 350-450 nm, Honle UV technology, light intensity of 50 mW/
cm

2

), as described elsewhere.

35

The oscillatory measurements were

performed at 37 and 20

°C, respectively, for 5 min with a frequency

of 10 Hz, a strain of 0.5%, and a gap of 0.5 mm. The samples were
exposed to UV light for one minute after the first minute of data
collection. The elastic moduli of the photopolymerized gels were
obtained using a dynamic mechanical analyzer (DMA 2980, TA-
Instruments) in the controlled force mode, where a force ramp from
0.001 to 1.0 N at a rate of 0.1 N/min was applied at 25

°C. Photo-

cross-linked gels with a cylindrical shape and diameter of 8 mm were
prepared for DMA studies by curing PEGMEMA-PPGMA-EGDMA
copolymer solutions (400 µL) with 0.1% w/v Irgacure 2959 for 5 min
at 37

°C using a BluePoint lamp 4 (350-450 nm, Honle UV technology,

light intensity of 450 mW/cm

2

). Scanning electron microscopy (SEM)

was used to characterize the morphology of freeze-dried gels. The
samples were mounted on an aluminum stub using an adhesive carbon
tab and sputter coated with gold before images were obtained using a
JEOL JSM-6060LV SEM machine.

Preparation of Photo-Cross-Linked Gels for Swelling and

Release Studies. Copolymer solutions (15% w/v) were prepared using
0.1% w/v 2-hydroxy-4

′-(2-hydroxy-ethoxy)-2-methyl-propiophenone

(Ciba Irgacure 2959, Sigma-Aldrich) PBS stock solution. The solutions
(200 µL for swelling samples, 100 µL for release samples) were added
into 1 mL flat bottom vials, preheated in an oven at 37

°C, then photo-

cross-linked using Blak-Ray long-wave (365 nm) 100 W ultraviolet
lamp with spot bulb (model B100 AP) at an intensity of 8.9 mW/cm

2

for 10 min.

Swelling Behavior of Photo-Cross-Linked Gels. After the exact

weight of the gels was measured, 0.5 mL of PBS buffer (pH 7.4) was
added to allow the gels to swell at 37

°C. At regular intervals the

incubation buffer was removed and the weight of the gels was measured.
Swelling ratios were calculated as below:

where W

t

represents the weight of the hydrogels at a certain time point

and W

0

represents the original weight of the hydrogels. After the

swelling ratio was determined, 0.5 mL of fresh buffer was added again
and the samples were further incubated at 37

°C. The swelling

experiments were performed in triplicate.

Drug Release from Photo-Cross-Linked Gels. The gels were

saturated in carmoisine E122 (red food coloring) solution at room

swelling ratio ) W

t

/W

0

2896

Biomacromolecules, Vol. 10, No. 10, 2009

Tai et al.

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

temperature for a week. The dye-laden gels were washed gently with
deionized water to remove the dye at the surface before being transferred
into deionized water at 25 and 37

°C, respectively. Digital images were

taken at different time points to qualitatively demonstrate the effect of
temperature on the release behavior. For a quantitative release study,
lysozyme (from chicken egg white, M

w

) 14307 Da, Sigma-Aldrich)

was loaded within the copolymer solutions at a concentration of 1 mg/
mL before being photo-cross-linked to form gels according to proce-
dures described in the previous section. Then, 0.5 mL of PBS was added
to each photo-cross-linked gel sample to allow protein release. At each
time point, 0.35 mL of the supernatant was removed after gentle shaking
and the same volume of fresh PBS was added. The concentration of
lysozyme in the release samples was determined with the Micro BCA
(bicinchoninic acid) assay kit (Thermo Scientific). The percentage of
cumulative amounts of released lysozyme was calculated from standard
calibration curve, which was generated from standard protein solutions
at concentration range of 0.5-200 µg/mL. Release samples (150 µL)
were pipetted into a 96-microwell plate and 150 µL of working reagent
(BCA reagents A/B/C)50:48:2 v/v) was added. The plates were
incubated at 37

°C for 2 h. Subsequently, the absorbance was measured

at 562 nm with a TECAN Microplate Reader. Lysozyme activity was
measured on Micrococcus lysodeikticus cell walls using EnzChek
Lysozyme Assay Kit (Molecular Probes) following the experimental
protocol recommended by the supplier. The assay is based on the
hydrolysis of the outer cell membrane of Micrococcus lysodeikticus,
resulting in solubilization of the affected bacteria and consequent
decrease of light scattering.

36

A total of 50 µL of Micrococcus

lysodeikticus suspension (50 µg/mL in the buffer containing 0.1 M
sodium phosphate, 0.1 M NaCl, pH 7.5 and 2 mM sodium azide) was
added to each microplate well containing 50 µL of either release
samples or the standard curve samples, then the loaded plate was
incubated at 37

°C for 30 min while protected from light. The

fluorescence intensity of reactions was measured using a TECAN
fluorescence microplate reader. Digestion products have absorption
maxima at 494 nm and fluorescence emission maxima at 518 nm. The
release experiments were performed in triplicate.

Cytotoxicity Assessments. Mouse C2C12 myoblast cells were

cultured in Dulbecco’s modified Eagle’s medium (DMEM), supple-
mented with 10% FBS, 1% glutamine, and 2.5 mg/mL amphotericin
B (antibiotic/ antimycotic solution) in a humidified incubator at 37

°C

and 5% CO

2

. Prior to use, cells were trypsinised using 0.25% trypsin/

0.02% EDTA in PBS, centrifuged, and resuspended in DMEM.
Approximately 80000 cells and 2 mL media were added into each well
of a 24-well culture plates to allow cells to adhere and incubated at 37
°C for 4 h. A total of 10 µL of 15% w/v polymer solution was then
added into each of the wells to make the final polymer concentration
in the culture media and cultured at 37

°C for 5 days. Cell viability

was assessed using the Alamar Blue assay (Biosource Europe) to
measure metabolic activity of the cultured cells on days 1, 3, and 5.
The experiments were performed in triplicate. At day 5, the Live/Dead
viability/cytotoxicity assay (Molecular Probes, L-3224) was conducted
to measure the membrane integrity of cells. Fluorescence images were
taken using a Leica DMRB microscope, while viable cells fluoresce
green through the reaction of calcein AM with intracellular esterase,
and nonviable cells fluoresce red due to the diffusion of ethidium
homodimer across damaged cell membranes and binding with nucleic
acids. Light phase control microscope images were taken by Leica
DMRB inverted microscope. The cytotoxicity of the copolymers was
also assessed using lactate dehydrogenase (LDH) cytotoxicity detection
kit (Invitrogen), which measures cell damage/death in response to
chemical compounds or environmental factors using a coupled two-
step reaction. Cells were seeded in 96-well plates at a density of 12000
cells/cm

2

and allowed to adhere overnight. Polymer solutions at

concentrations of 10-1000 µg/mL in Phenol red media were added to
wells in triplicate. After cells had been incubated for 24 h with the
polymer solution, 100 µL of supernatant from each well of the cultured
cells was transferred to corresponding wells on a new plate. A total of
100 µL of LDH reaction solution was then added to each of the wells
using a repeating pipettor. The plate was then incubated on an orbital
shaker for 30 min at room temperature. The absorbance at 490 nm
was measured using a microplate reader.

Results and Discussion

Thermoresponsive Copolymers with Multiple Methacry-

late Groups Prepared by One-Step ATRP Copolymerization.
Water-soluble PEGMEMA-PPGMA-EGDMA copolymers with
multiple methacrylate groups were synthesized by copolymer-
ization of PEGMEMA (M

n

) 475), PPGMA (M

n

) 375), and

EGDMA via one-step deactivation enhanced ATRP approach
(Scheme 1).

35

This approach was first reported for the successful

Scheme 1. Dendritic PEGMEMA-PPGMA-EGDMA Copolymer from One-Step ATRP Copolymerization of Poly(ethylene glycol) Methyl
Ether Methacrylate (PEGMEMA), Poly(propylene glycol) Methacrylate (PPGMA), and Ethylene Glycol Dimethacrylate (EGDMA)

Photo-Cross-Linked Hydrogels

Biomacromolecules, Vol. 10, No. 10, 2009

2897

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

homopolymerization of divinyl benzene (DVB) and ethylene
glycol dimethacrylate (EGDMA) to yield soluble dendritic
homopolymers with multiple vinyl functional groups rather than
cross-linked macrogelation.

37

It was envisioned that this facile

approach could be further adopted for the design and synthesis
of soluble multifunctional dendritic copolymers using a high
level of bifunctional vinyl monomers to achieve tunable highly
branched structures. In this study, methyl 2-chloropropionate
and copper(I) and (II) chloride were used as the initiator and
catalyst during the reaction. The addition of Cu(II) species was
used to enhance the deactivation reaction, thus, the growth rate
of polymer chains was suppressed, leading to the delayed cross-
linking.

38

A yield of up to 75% of soluble dendritic copolymers

was obtained (Table 1). The GPC with a MALLS detector was
used to yield absolute molecular weight, which is found much
higher than the relative molecular weight obtained by RI detector
(see Supporting Information for GPC curves). The slope value
(ca. 0.25) of the conformation plot for the copolymers is much
lower than the typical range 0.5-0.6 for linear polymers,
indicating the branched structures. Moreover, the dendritic
structures of the copolymer products were also confirmed by

1

H NMR (the characteristic peaks at chemical shifts of 6.1 and

5.6 ppm are attributed to the vinyl functional groups). The
composition, double bond content, and branching degree of the
copolymer were calculated from the integral data of

1

H NMR

as described elsewhere.

35

The double bond content represents

the mol percentage of EGDMA with free vinyl functional groups
in the copolymer and the branching degree represents the mol
percentage of EGDMA as branching units (i.e., without vinyl
groups) in the copolymer. The copolymers are amorphous
viscous or tacky solids and their glass transition temperature
(T

g

) measured by DSC is about -46

°C.

Thermoresponsive Properties of PEGMEMA-PPGMA-

EGDMA Copolymers. The resultant dendritic PEGMEMA-
PPGMA-EGDMA copolymers were dissolved in deionized
water, phosphate buffered saline (PBS, pH 7.4), and cell culture
media. The solutions reversibly became cloudy when the
temperature was increased above 32

°C but were transparent

below this temperature. Figure 1a shows the temperature scans
of the copolymer solutions recorded by a UV spectrophotometer.
The LCSTs of the copolymers were measured as about 32

°C

in deionized water, suggesting this copolymer is soluble at
temperatures below LCST with a random coil conformation,
while above the LCST the polymer chains undergo a confor-
mational change, collapse, and aggregate. The LCSTs of this
copolymer in PBS (pH 7.4) and in cell culture media decreased
slightly (LCST

PBS

) 29 °C; LCST

media

) 28 °C) compared to

the LCST in pure water. This decrease agreed with prior data
on “salting out” of thermoresponsive polymers due to the
interactions and the hydrophilic/hydrophobic balance within the
polymer molecules in the presence of salt additives and protein
molecules.

39,40

Dynamic light scattering (DLS) was also used

to monitor temperature dependent changes in the conformation
of the macromolecules. The results on the phase transition

temperature LCST agreed well with the UV measurements.
Figure 1b shows a typical size distribution of the copolymers
in deionized water at 20 and 37

°C. As apparent from the peak

diameters increasing dramatically from 13.5 to 459 nm, the
thermoresponsive property of the polymer led to aggregations
at temperatures above LCST. The copolymers (100-300 mg)
were dissolved in deionized water (1.00 mL) at 4

°C and then

placed in 37

°C incubator to observe their gelation behavior.

Gel concentration was determined as no flow within 10 s by

Table 1. PEGMEMA-PPGMA-EGDMA Copolymers

GPC RI

GPC MALLS

entry

f

a

RT

b

(h)

conv

c

(%)

M

w

d

(kg/mol)

PDI

e

M

w

d

(kg/mol)

PDI

e

F

f

DB

g

(%)

BD

h

(%)

LCST

i

(

°C)

1

25/65/10

65

75

216

2.78

570

1.95

22:37:38

10

28

32 ( 0.5

2

30/40/30

42

72

207

2.90

521

1.98

21:26:53

18

35

33 ( 0.5

a

Monomer feed mole ratio, f

[PEGMEMA]/[PPGMA]/[EGDMA].

b

Reaction time.

c

Monomer conversion, estimated using peak areas for monomers and copolymers

in GPC traces.

d

Weight average molecular weight.

e

Polydispersity, M

w

/M

n

.

f

Polymer composition, F

[PEGMEMA]/[PPGMA]/[EGDMA].

g

Double bond content.

h

Branching

degree.

i

Lower critical solution temperature. Polymerization conditions: 60

°C in butanone; total monomers/butanone (v/v) ) 1:1; [I]/[total monomers]

(mol ratio) ) 1:100, [I]/[Cu

+

/Cu

2+

]/bpy ) 1:[0.375:0.125]:1. The initiator (I)/catalysts/ligand: methyl 2-chloropropionate/CuCl/CuCl

2

/bpy.

Figure 1. Thermoresponsive properties of PEGMEMA-PPGMA-
EGDMA copolymer (2 in Table 1). (a) LCST behavior of the copolymer
in 0.03% w/v deionized water, PBS buffer, and the tissue culture
media, demonstrated by UV-vis spectroscopy; (b) Size distributions
measured by DLS (0.01% w/v aqueous solution); (c) Polymer solution
(15% w/v) became thermal gel when the temperature was raised
above its LCST from 20 to 37

°C. Published with permission from ref

35. Copyright 2009 American Chemical Society.

2898

Biomacromolecules, Vol. 10, No. 10, 2009

Tai et al.

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

visual observation (Figure 1c). It was found that gel points of
these copolymers at 37

°C were 15% w/v.

Rheological, Mechanical Properties, and the Morphology

of Photo-Cross-Linked Gels. The thermoresponsive PEG-
MEMA-PPGMA-EGDMA copolymers produced by the one-
step deactivation enhanced ATRP approach carry multiple
methacrylate groups, which can be used to form covalent cross-
linking via photopolymerization. The cross-linking of these
copolymer aqueous solutions by thermally induced gelation and
photopolymerization were studied by real-time rheological
measurements. The copolymer solutions were found to undergo
fast photo-cross-linking gelation, resulting in the crossover of
loss modulus (G

′) and storage modulus (G′′) within seconds of

UV exposure to form elastic gels (Figure 2a) with dramatically
increased moduli, that is, three orders of magnitude greater than
the physically thermal gels before photo-cross-linking. The
enhancement in mechanical strength was observed for gels at a
high copolymer concentration, photopolymerization at a tem-
perature above LCST, and using the copolymer with high double
bond content.

35

The storage modulus of 15% w/v copolymer 2

(f

[PEGMEMA]/[PPGMA]/[EGDMA

) 30/40/30, Table 1) gels produced

by photo-cross-linking at 37

°C is about 10 kPa, compared to

about 1 kPa for photo-cross-linked gels from copolymer 1
(f

[PEGMEMA]/[PPGMA]/[EGDMA

) 25/65/10, Table 1). Moreover, it was

demonstrated from DMA measurements (Figure 2b) that the
photo-cross-linked gels were elastic and displayed an increasing
elastic modulus (E) with increasing amounts of methacrylate
functional groups from 11 kPa for 15% w/v copolymer 1 gels
to 22 kPa for 15% w/v copolymer 2 gels. The morphology of
gels is an important factor in terms of the gel performance in
tissue engineering and drug delivery applications. In this study,
the freeze-dried gel samples were observed for their network
structure by taking SEM images. The freeze-dried gels prepared

from copolymer 1 formed a smooth monolith (Figure 3a), in
contrast, the freeze-dried gels prepared from copolymer 2
demonstrated a porous structure with an average pore diameter
about 1.63 µm (Figure 3b). The significant difference in the
morphology of the photo-cross-linked gels could be the com-
bined effect of the composition and cross-linking density on
phase separation, molecular assembly and the compactness of
molecules.

28

The impact of copolymer composition on the

morphology of photo-cross-linked gels will be further studied
by preparing a series of copolymers with various compositions.
It should be pointed out that the morphology of freeze-dried
gels is not the same as that for the gels after swelling, especially
for the gels yielded from thermoresponsive materials. A higher
swelling ratio was observed for copolymer 1 gels than copoly-
mer 2 gels. Therefore, the difference in pore size could be
reduced slightly after swelling. Nevertheless, the pore sizes and
porosity of the gels can be tailored by adjusting the copolymer
composition and concentration, thus, to tune the cross-linking
density so that to meet the specific needs for cells and/or guest
molecules encapsulation and transportation.

Figure 2. Photo-cross-linked gels (arrow pointed) from PEGMEMA-PPGMA-EGDMA copolymer (2 in Table 1). (a) Gel sample (diameter 20
mm, thickness 0.5 mm) after realtime rheological measurements; (b) Gel sample (diameter 8 mm, thickness 8 mm) for compression test.

Figure 3. SEM images for the freeze-dried photo-cross-linked gel samples. (a) Monolith obtained from copolymer 1 (Table 1); (b) Porous structure
obtained from copolymer 2 (Table 1).

Figure 4. Swelling of photo-cross-linked gels in PBS buffer (pH 7.4)
at 37

°C. The copolymer 1 with a lower cross-linking density

demonstrated a higher swelling ratio than the copolymer 2.

Photo-Cross-Linked Hydrogels

Biomacromolecules, Vol. 10, No. 10, 2009

2899

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

Swelling and Release Studies of Photo-Cross-Linked

Gels. The photo-cross-linked gels were soaked in PBS for
swelling at 37

°C until equilibrium to test the maximum water

uptake. It was found that the weight and volume of the gels
increased and the swelling reached equilibrium within 24 h
(Figure 4). The copolymer 1 with a lower cross-linking density
demonstrated a higher swelling ratio (about 1.2) than copolymer
2 (about 1.1). This indicates that the cross-linking density has
an impact on the swelling property of the photo-cross-linked
gels, thus influencing the mechanical strength of the gels and
mass transport within the gels.

The hydrogels prepared from thermo-responsive PEGMEMA-

PPGMA-EGDMA copolymers after photo-cross-linking were
tested for releasing model drug with changes in temperature.
The release of the red dye from the gels in the warm water

started immediately after transferring. However, in cold water
the dye was released from the samples at a much slower rate
(Figure 5a). Similar temperature dependent release profile was
also observed from lysozyme release studies at temperatures
above and below LCST of the copolymer, that is, 25 and 37

°C

(Figure 5b). The higher diffusion coefficient of encapsulated
compounds at a higher temperature could contribute to this
apparent fast release; in addition, the gel shrinking induced by
the changes in polymer chain conformation from hydrophilic
to hydrophobic might squeeze the model drug dye out of the
gel.

Lysozyme release studies were continued up to one week

using gels from copolymers 1 and 2. At 3 days, copolymer 1
exhibited

∼55% lysozyme release; while copolymer 2 showed

∼80% release (Figure 6). It is interesting that copolymer 2
showed a faster lysozyme release despite the higher cross-linking
density and lower swelling ratio. The porous structure (Figure
3) might be the key factor. A crossover point was also found
for the release profiles at the temperatures above and below
LCST, that is, gels initially released protein faster at a
temperature above LCST, then released slower at this temper-
ature after about one day (the crossover points in Figure 6).
However, owing to the experimental error bars, the differences
observed for the overall release profiles at 25 and 37

°C are

not considered to be significant. Statistical analysis performed
using Student’s t-test with a confidence level of 0.05 (p value)
on the release data shows that the difference for the release
profiles of copolymers 1 and 2 is statistically significant,
however, the difference for the release profiles of one copolymer
at two temperatures (25 and 37

°C) is not statistically significant.

It is reckoned that the release kinetics of PEGMEMA-PPGMA-
EGDMA photo-cross-linked gels are both diffusion and swelling
controlled, and influenced by the cross-linking density, gel
morphology, polymer chain conformation (from hydrophilic to
hydrophobic), and the interaction of the functional groups in
the copolymers with protein molecules.

41-43

A higher PPGMA

content in copolymer 1 provides a higher amount of OH groups

Figure 5. Release from photo-cross-linked gels prepared using PEGMEMA-PPGMA-EGDMA copolymer (2 in Table 1). (a) Carmoisine E122
(red food coloring) as the model molecule. The gels were releasing the carmoisine red dye at a faster rate in warm water, compared to a slow
release in cold water. Images were taken immediately (left images) and after 5 min (right images) of the gels being transferred into water. (b)
Cumulative release of the encapsulated lysozyme from photo-cross-linked hydrogels at a temperature above and below the LCST in 10 min.

Figure 6. Cumulative lysozyme release from photo-cross-linked gels
prepared using PEGMEMA-PPGMA-EGDMA copolymers (1 and 2
in Table 1) at temperatures below and above LCST.

2900

Biomacromolecules, Vol. 10, No. 10, 2009

Tai et al.

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

in the molecular structure than in copolymer 2. This could lead
to a stronger interaction for copolymer 1 with protein than
copolymer 2, thus contributing to a slower and incomplete
release even after 7 days (Figure 6). Moreover, the lysozyme
in the release studies demonstrated more than 85% remaining
activity in all cases determined by EnzChek Lysozyme Assay
and normalized with released lysozyme, indicating that experi-
mental conditions employed, including UV treatment, are not
harmful to lysozyme.

Cytotoxicity Assessments. Alamar Blue cell viability assays

of the copolymer solution at the concentration of 750 µg/mL at
days 1, 3, and 5 were performed and the results indicated that
the copolymers had a slight reduction in cell activity compared
to the control using cells alone (Figure 7a). Cells cultured in
the copolymer/culture media solutions (750 µg/mL) showed a
stellate morphology (Figure 8a), which was similar to those
cultured in the media only (Figure 8b). After 5 days culture,
the viable cells can be clearly seen to fluoresce green by Live/
Dead viability assay (Figure 8d). LDH cytotoxicity assay was
further used to measure the cell response to the addition of the
copolymers at the concentration of 10-1000 µg/mL. The results
indicated that the cells showed a sign of damage at a concentra-
tion of 1 mg/mL (Figure 7b), which was lower than the
concentration used as a gel in this study (15% w/v). Toxicity
at higher concentration suggests interactions between copoly-
mers and cells might exist even though these copolymers consist
of solely biocompatible PEG and PPG building blocks. It would
be necessary to perform further cellular interaction studies.

Possible cell interactions and penetrations of non-photo-polym-
erized polymers may be reduced by photopolymerization,
leading to a decrease in toxicity of photopolymerized gels.

34

Moreover, it was observed that with the increase in polymer
concentration, there were more precipitated copolymers covering
the surface of the cultured plates and cells, which led to less
contact surface areas for diffusion and at higher concentration
affecting the ability of cells to move and dividing cells to attach
and grow. Cell seeding experiments on the PEGMEMA-
PPGMA-EGDMA polymer films (Figure 8c) indicated they did
not adhere and spread on these materials. It is reported that
structure modifications by introducing cell adhesion peptide
moieties (i.e., RGD peptide) can improve cell adhesion
enormously.

23,44,45

Therefore, we currently conduct the ongoing

research to assess cell behavior by encapsulating cells within
the thermal gels and photo-cross-linked gels after modifying
polymer gel network by introducing cell adhesion peptides.

Conclusions

PEGMEMA-PPGMA-EGDMA copolymers with both thermo-

responsive and photo-cross-linkable properties were synthesized
via one-step deactivation enhanced atom transfer radical po-
lymerization (ATRP). The photo-cross-linked gels prepared from
these copolymers at a temperature above the LCST showed
excellent mechanical properties. The swelling ratio and the
lysozyme release rate of these gels could be controlled by simply
adjusting the monomer composition within the polymers. Also,

Figure 7. Cytotoxicity assessments of PEGMEMA-PPGMA-EGDMA copolymer (1 in Table 1). (a) Alamar Blue cell metabolism assay at the
copolymer concentration of 750 µg/mL. (b) LDH cell damage assay at the copolymer concentration of 10-1000 µg/mL.

Photo-Cross-Linked Hydrogels

Biomacromolecules, Vol. 10, No. 10, 2009

2901

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

these materials were found to have low toxicity for mouse
C2C12 myoblast cells as assessed with LDH, Alamar Blue, and
a Live/Dead assay at concentrations less than 1 mg/mL. Further
structure modifications by introducing cell-adhesion functionality
are needed to improve cytocompatibility of the photo-cross-
linked gels. These in situ photo-cross-linked hydrogels from
thermoresponsive copolymers with multiple methacrylate groups
have many advantages for the potential applications in tissue
engineering and drug delivery. First, thermal gelation due to
thermoresponsive properties upon injection allows easy handling
and holds the shape of the gels prior to the photopolymerization
during clinical practice. Second, in situ photo-cross-linked
hydrogels from thermoresponsive polymers have enhanced
mechanical strength and stability compared to non-photo-cross-
linked gels. Third, the multiple methacrylate groups within the
copolymer dendritic structure can provide a tunable high cross-
linking density to allow tailoring the mechanical properties,
swelling, and release profiles of the gels. Therefore, this novel
thermoresponsive copolymer synthesized by one-step deactiva-
tion enhanced ATRP approach has great potential as a smart
injectable system for regenerative medicine such as wound
healing and tissue repair. The biodegradability of the gels can
be further introduced by copolymerizing macromonomers with
biodegradable building blocks, for example, PLGA based
dimethacrylate, or using a biodegradable cross-linker.

Acknowledgment. H.T. is supported by EPSRC with a Life

Science Interface Fellowship (EP/E042619/1). The British
Council and Platform Be`ta Techniek are thanked for their
financial support through Partnership Programme in Science
(PPS RV19).

Supporting Information Available. GPC curves of dendritic

copolymers. This material is available free of charge via the
Internet at http://pubs.acs.org.

References and Notes

(1) Khademhosseini, A.; Langer, R. Biomaterials 2007, 28, 5087-

5092.

(2) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. AdV. Mater.

2006, 18, 1345–1360.

(3) Van Tomme, S. R.; Storm, G.; Hennink, W. E. Int. J. Pharm. 2008,

355, 1–18.

(4) Hou, Q. P.; De Bank, P. A.; Shakesheff, K. M. J. Mater. Chem. 2004,

14, 1915–1923.

(5) Declercq, H. A.; Cornelissen, M. J.; Gorskiy, T. L.; Schacht, E. H. J.

Mater. Sci. 2006, 17, 113–122.

(6) Yamada, Y.; Ueda, M.; Naiki, T.; Nagasaka, T. Clin. Oral Implants

Res. 2004, 15, 589–597.

(7) Elisseeff, J. Exp. Opin. Biol. Ther. 2004, 4, 1849–1859.
(8) Anseth, K. S.; Metters, A. T.; Bryant, S. J.; Martens, P. J.; Elisseeff,

J. H.; Bowman, C. N. J. Controlled Release 2002, 78, 199–209.

(9) Ibusuki, S.; Fujii, Y.; Iwamoto, Y.; Matsuda, T. Tissue Eng. 2003, 9,

371–384.

(10) Peretti, G. M.; Xu, J.-W.; Bonassar, L. J.; Kirchhoff, C. H.; Yaremchuk,

M. J.; Randolph, M. A. Tissue Eng. 2006, 12, 1151–1168.

(11) Hoemann, C. D.; Sun, J.; Legare, A.; McKee, M. D.; Buschmann,

M. D. Osteoarthr. Cartilage 2005, 13, 318–329.

(12) Grinstaff, M. W. Chem.sEur. J. 2002, 8, 2838–2846.
(13) Carnahan, M. A.; Grinstaff, M. W. J. Am. Chem. Soc. 2001, 123, 2905–

2906.

(14) Wathier, M.; Jung, P. J.; Camahan, M. A.; Kim, T.; Grinstaff, M. W.

J. Am. Chem. Soc. 2004, 126, 12744–12745.

(15) Yin, X.; Hoffman, A. S.; Stayton, P. S. Biomacromolecules 2006, 7,

1381–1385.

(16) Henry, S. M.; El-Sayed, M. E. H.; Pirie, C. M.; Hoffman, A. S.;

Stayton, P. S. Biomacromolecules 2006, 7, 2407–2414.

Figure 8. Light phase control microscope images for the cells cultured (a) in PEGMEMA-PPGMA-EGDMA copolymer (1 in Table 1) culture
media solutions (750 µg/mL); (b) in the culture media without polymers, (c) on the photo-cross-linked polymer films. (d) Live/Dead viability assay
for the cells cultured in the copolymer/culture media solutions after 5 days. The viable cells fluoresce green, whereas the nonviable cells fluoresce
red (pointed by the arrow).

2902

Biomacromolecules, Vol. 10, No. 10, 2009

Tai et al.

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j

(17) Elisseeff, J.; McIntosh, W.; Anseth, K.; Riley, S.; Ragan, P.; Langer,

R. J. Biomed. Mater. Res. 2000, 51, 164–171.

(18) Nguyen, K. T.; West, J. L. Biomaterials 2002, 23, 4307–4314.
(19) Hubbell, J. A. J. Controlled Release 1996, 39, 305–313.
(20) Hillwest, J. L.; Chowdhury, S. M.; Slepian, M. J.; Hubbell, J. A. Proc.

Natl. Acad. Sci. U.S.A. 1994, 91, 5967–5971.

(21) Mann, B. K.; Gobin, A. S.; Tsai, A. T.; Schmedlen, R. H.; West, J. L.

Biomaterials 2001, 22, 3045–3051.

(22) Vernon, B.; Tirelli, N.; Bachi, T.; Haldimann, D.; Hubbell, J. A.

J. Biomed. Mater. Res. A 2003, 64A, 447–456.

(23) Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 39, 266–

276.

(24) Bryant, S. J.; Bender, R. J.; Durand, K. L.; Anseth, K. S. Biotechnol.

Bioeng. 2004, 86, 747–755.

(25) Jeon, O.; Bouhadir, K. H.; Mansour, J. M.; Alsberg, E. Biomaterials

2009, 30, 2724–2734.

(26) Carter, S.; Rimmer, S.; Rutkaite, R.; Swanson, L.; Fairclough,

J. P. A.; Sturdy, A.; Webb, M. Biomacromolecules 2006, 7, 1124–
1130.

(27) Rimmer, S.; Carter, S.; Rutkaite, R.; Haycock, J. W.; Swanson, L.

Soft Matter 2007, 3, 971–973.

(28) Hiemstra, C.; Zhou, W.; Zhong, Z. Y.; Wouters, M.; Feijen, J. J. Am.

Chem. Soc. 2007, 129, 9918–9926.

(29) Li, Y. T.; Tang, Y. Q.; Narain, R.; Lewis, A. L.; Armes, S. P. Langmuir

2005, 21, 9946–9954.

(30) Velazquez, A. J.; Carnahan, M. A.; Kristinsson, J.; Stinnett, S.;

Grinstaff, M. W.; Kim, T. Arch. Ophthalmol. 2004, 122, 867–870.

(31) Luman, N. R.; Kim, T.; Grinstaff, M. W. Pure Appl. Chem. 2004, 76,

1375–1385.

(32) Sontjens, S. H. M.; Nettles, D. L.; Carnahan, M. A.; Setton, L. A.;

Grinstaff, M. W. Biomacromolecules 2006, 7, 310–316.

(33) Vermonden, T.; Fedorovich, N. E.; van Geemen, D.; Alblas, J.; van

Nostrum, C. F.; Dhert, W. J. A.; Hennink, W. E. Biomacromolecules
2008, 9, 919–926.

(34) Fedorovich, N. E.; Swennen, I.; Girones, J.; Moroni, L.; van

Blitterswijk, C. A.; Schacht, E.; Alblas, J.; Dhert, W. J. A. Biomac-
romolecules 2009, 10, 1689–1696.

(35) Tai, H.; Wang, W.; Vermonden, T.; Heath, F.; Hennink, W. E.;

Alexander, C.; Shakesheff, K. M.; Howdle, S. M. Biomacromolecules
2009, 10, 822–828.

(36) Van Tomme, S. R.; De Geest, B. G.; Braeckmans, K.; De Smedt,

S. C.; Siepmann, F.; Siepmann, J.; van Nostrum, C. F.; Hennink, W. E.
J. Controlled Release 2005, 110, 67–78.

(37) Wang, W.; Zheng, Y.; Roberts, E.; Duxbury, C. J.; Ding, L. F.; Irvine,

D. J.; Howdle, S. M. Macromolecules 2007, 40, 7184–7194.

(38) Matyjaszewski, K.; Nanda, A. K.; Tang, W. Macromolecules 2005,

38, 2015–2018.

(39) Schmaljohann, D. AdV. Drug DeliVery ReV. 2006, 58, 1655–1670.
(40) Magnusson, J. P.; Khan, A.; Pasparakis, G.; Saeed, A. O.; Wang, W.;

Alexander, C. J. Am. Chem. Soc. 2008, 130, 10852–10853.

(41) de Jong, S. J.; van Eerdenbrugh, B.; van Nostrum, C. F.; Kettenes-

van de Bosch, J. J.; Hennink, W. E. J. Controlled Release 2001, 71,
261–275.

(42) Siepmann, J.; Peppas, N. A. AdV. Drug DeliVery ReV. 2001, 48, 139–

157.

(43) Peppas, N. A.; Huang, Y.; Torres-Lugo, M.; Ward, J. H.; Zhang, J.

Annu. ReV. Biomed. Eng. 2000, 2, 9–29.

(44) Burdick, J. A.; Anseth, K. S. Biomaterials 2002, 23, 4315–4323.
(45) Hubbell, J. A. Curr. Opin. Biotechnol. 1999, 10, 123–129.

BM900712J

Photo-Cross-Linked Hydrogels

Biomacromolecules, Vol. 10, No. 10, 2009

2903

Downloaded by UNIWERSYTET WARSZAWSKI on October 24, 2009 | http://pubs.acs.org

Publication Date (Web): September 11, 2009 | doi: 10.1021/bm900712j