Biodegradable hydrogels for time-controlled release of tethered peptides or proteins

Article abstract of DOI:10.1021/bm901235g

Tethering drug substances to a gel network is an effective way of controlling the release kinetics of hydrogelbased drug delivery systems. Here, we report on in situ forming, biodegradable hydrogels that allow for the covalent attachment of peptides or proteins. Hydrogels were prepared by step-growth polymerization of branched poly(ethylene glycol). The gel strength ranged from 1075 to 2435 Pa; the degradation time varied between 24 and 120 h. Fluorescence recovery after photobleaching showed that fluorescently labeled bovine serum albumin (FITC-BSA) was successfully bound to the gel network during gel formation. Within 168 h, the mobility of the tethered molecules gradually increased due to polymer degradation. Using FITC-BSA and lysozyme as model proteins, we showed the potential of the developed hydrogels for time-controlled release. The obtained release profiles had a sigmoidal shape and matched the degradation profile very well; protein release was complete after 96 h.

Full text of DOI:10.1021/bm901235g

4
96
Biomacromolecules 2010, 11, 496–504
Biodegradable Hydrogels for Time-Controlled Release of
Tethered Peptides or Proteins
Ferdinand Brandl, Nadine Hammer, Torsten Blunk, Joerg Tessmar, and Achim Goepferich*
Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany
Received October 30, 2009; Revised Manuscript Received December 17, 2009
Tethering drug substances to a gel network is an effective way of controlling the release kinetics of hydrogel-
based drug delivery systems. Here, we report on in situ forming, biodegradable hydrogels that allow for the
covalent attachment of peptides or proteins. Hydrogels were prepared by step-growth polymerization of branched
poly(ethylene glycol). The gel strength ranged from 1075 to 2435 Pa; the degradation time varied between 24
and 120 h. Fluorescence recovery after photobleaching showed that fluorescently labeled bovine serum albumin
(FITC-BSA) was successfully bound to the gel network during gel formation. Within 168 h, the mobility of the
tethered molecules gradually increased due to polymer degradation. Using FITC-BSA and lysozyme as model
proteins, we showed the potential of the developed hydrogels for time-controlled release. The obtained release
profiles had a sigmoidal shape and matched the degradation profile very well; protein release was complete
after 96 h.
Introduction
Over the past decades, hydrogels have been extensively used
as matrices for controlled drug delivery and tissue engineering
decrease bioactivity, especially when peptides or proteins are
the target therapeutics. In addition, the anchor groups have to
2
be carefully designed in order to avoid unwanted side-effects
2
1
–6
during degradation in vivo.
applications. The popularity of these materials is based on
their enormous chemical versatility, which allows for the design
In this paper, we report the preparation and characterization
of in situ cross-linkable, biodegradable hydrogels for time-
controlled release of peptides or proteins. For this purpose,
branched poly(ethylene glycol) (PEG) was functionalized with
aromatic succinimidyl carbonate groups that readily react with
amino groups of other polymers, peptides, or proteins under
formation of biodegradable carbamate bonds. Aromatic succin-
imidyl carbonates have been originally described as linking
1
–4
of a broad range of hydrogels with varying properties.
Furthermore, hydrogel-based materials generally show excellent
biocompatibility because of their physicochemical similarity to
1
–4
the native extracellular matrix.
Finally, compared to other
commonly used biomaterials, such as poly(lactic acid) or
poly(lactide-co-glycolide), hydrophilic polymers are well suited
for the entrapment of biomolecules due to their lack of
hydrophobic interactions, which can affect the activity of these
1
2,13
groups for the reversible PEGylation of proteins.
While
2
,3
fragile species.
extensively applied for the preparation of soluble protein
prodrugs, this chemistry has never been used for the formation
of hydrogels. For the preparation of covalently cross-linked
hydrogels, PEG-succinimidyl carbonates were reacted with
Despite this multitude of advantageous characteristics, hy-
drogels also have several limitations. As a result of their high
water content, most hydrogels usually restrict the mobility of
encapsulated peptides or proteins only moderately. Conse-
quently, a large amount of the incorporated molecules will be
released within a few hours, which illustrates the necessity for
14,15
branched PEG-amines (Figure 1A). Based on previous studies,
the reactivity of primary amines toward succinimidyl carbonate
groups was expected to be influenced by their chemical
environment, resulting in different gel formation kinetics for
PEG chains terminated with different amino groups (e.g., amino
groups of alanine, 6-aminohexanoic acid, and lysine). These
amino acid moieties were chosen to represent the different amino
groups available in proteins (amino terminus and ε-amino groups
of lysine residues). Moreover, the chemical environment within
1
,2,5
more sophisticated strategies to prolong drug release.
Adjusting the cross-linking density is a common approach to
reduce gel permeability and slow down the release kinetics.
However, this approach is only effective in controlling the
release of relatively large molecules, such as proteins with
molecular weights of several thousand Daltons. Furthermore,
increasing the cross-linking density often results in decreased
the N-substituent may also influence the degradation rate of
2
hydrophilicity and hence decreased biocompatibility. Tethering
14,15
carbamate bonds.
During gelation, dissolved peptides or
drug substances to the gel network via degradable anchor groups
would be a further strategy to effectively control drug release.
In these systems, the release kinetics is ideally controlled by
the degradation of the anchor group while drug diffusivity only
proteins are expected to be immobilized within the gel network
via pendant PEG chains (Figure 1). This will improve handling
and flexibility of the drug delivery system, since any peptide
or protein could be incorporated without requiring chemical
modifications of these molecules. Similar to soluble prodrugs,
the anchoring PEG chains are cleaved during gel degradation,
1,2,5,7,8
plays a secondary role.
However, the drug molecules often
need to be chemically modified in order to allow for their
covalent attachment, which makes these drug delivery systems
and the native peptide or protein will be released into solution
9
–11
highly complex.
Furthermore, drug conjugation may also
12,13,16,17
(
Figure 1B).
Nondegradable gels (prepared from ali-
phatic PEG-succinimidyl carbonates and PEG-amines) served
as a control group. The prepared hydrogels were characterized
with respect to their mechanical properties, swelling capacities,
*
To whom correspondence should be addressed. Tel.: +49 941 943-
843. Fax: +49 941 943-4807. E-mail: achim.goepferich@chemie.uni-
regensburg.de.
4
10.1021/bm901235g
 2010 American Chemical Society
Published on Web 01/22/2010

Release of Tethered Peptides or Proteins
Biomacromolecules, Vol. 11, No. 2, 2010 497
Figure 1. Schematic illustration of biodegradable hydrogels for time-controlled release of tethered peptides or proteins. For gel formation, branched
PEG-succinimidyl carbonates are reacted with branched PEG-amines (A). The same chemistry allows for the immobilization of dissolved peptides
or proteins via pendant PEG chains. During gel degradation, these anchoring PEG-chains are cleaved, and the unmodified peptide or protein
will be released into solution (B).
1
8
and degradation rates. To prove their suitability as potential drug
delivery systems, the gels were loaded with fluorescein isothio-
cyanate (FITC) labeled bovine serum albumin (BSA) and
lysozyme as model proteins. The successful immobilization and
subsequent liberation of FITC-BSA was investigated by fluo-
rescence recovery after photobleaching (FRAP) experiments.
And finally, the in vitro release kinetics of FITC-BSA and
lysozyme were determined.
scribed. First, phthalimido-PEGs were obtained by reaction of
4armPEG10k-OH, phthalimide, triphenylphosphine, and diisopropyl
azodicarboxylate. The phthalimido groups were then converted into
primary amines by hydrazinolysis. Methoxy PEG-amines (2 kDa
molecular weight, mPEG2k-NH
armPEG10k-NH
Synthesis of Aliphatic PEG-Succinimidyl Carbonates (4armPEG10k-
SC). A total of 2.0 g of 4armPEG10k-OH (0.2 mmol) and 0.82 g of
DSC (3.2 mmol) were dissolved in 20 mL of dried acetonitrile. Then,
2
) were synthesized as described for
4
2
.
5
20 µL of pyridine (6.4 mmol) were added, and the reaction mixture
Experimental Section
was stirred overnight at room temperature. The next day, the solvent
was evaporated under reduced pressure, and 20 mL of methylene
chloride (DCM) were added to the residue. The insoluble residue was
filtered off, and the solution was concentrated under reduced pressure.
The product was crystallized at 0 °C under vigorous stirring by dropwise
addition of diethyl ether. For further purification, the crystallization
step was repeated. The precipitate was collected by filtration, washed
with cold diethyl ether, and dried under vacuum to yield 1.9 g (90%)
Materials. Hexane, 3-(4-hydroxyphenyl)propionic acic, methylene
chloride (DCM), and phthalimide were purchased from Acros Organics
(
Geel, Belgium). Boc-protected alanine (Boc-Ala-OH), Boc-6-amino-
hexanoic acid, and di-Boc protected lysine dicyclohexylammonium salt
Boc-Lys(Boc)-OH ·DCHA) were obtained from Bachem GmbH (Weil
am Rhein, Germany). Deuterated chloroform (CDCl ) and deuterated
dimethyl sulfoxide (DMSO-d ) were obtained from Deutero GmbH
Kastellaun, Germany). Phosphate-buffered saline (PBS) was purchased
(
3
6
1
of 4armPEG10k-SC (Figure 2A). H NMR (4armPEG10k-SC, DMSO-
(
d
(
-
6
, 600 MHz): δ 2.80 ppm (s, 16H, -C(O)CH
s, 8H, R CCH O-), 3.50 ppm (s, -OCH CH
OCH CH OC(O)O-), 4.39 ppm (t, 8H, -OCH
SynthesisofAromaticPEG-SuccinimidylCarbonates(4armPEG10k-
2
CH
O-), 3.69 ppm (t, 8H,
CH OC(O)O-).
2
C(O)-), 3.30 ppm
from Invitrogen GmbH (Karlsruhe, Germany). Four-armed poly(eth-
ylene glycol) (10 kDa molecular weight, 4armPEG10k-OH) was
purchased from Nektar Therapeutics (Huntsville, AL). All SDS-PAGE
reagents were obtained from Serva Electrophoresis GmbH (Heidelberg,
Germany). Acetonitrile, Coomassie brilliant blue G-250, N,N′-dicy-
clohexylcarbodiimide (DCC), fluorescein isothiocyanate (FITC)-labeled
bovine serum albumin (BSA), FITC-dextran (150 kDa molecular
weight), N-hydroxysuccinimide (HOSu), diisopropyl azodicarboxylate,
N,N′-diisopropylethylamine (DIPEA), lysozyme (from chicken egg
white), methoxy poly(ethylene glycol) (2 kDa molecular weight,
mPEG2k-OH) Sigmacote, and N,N′-discuccinimidyl carbonate (DSC)
were obtained from Sigma-Aldrich (Taufkirchen, Germany). Ethanol,
ethyl acetate, and diethyl ether were of technical grade and used without
further purification. All other chemicals were of analytical grade and
purchased from Merck KGaA (Darmstadt, Germany). Deionized water
was obtained using a Milli-Q water purification system from Millipore
3
2
2
2
2
2
2
2
dTyr-SC). In the first step, 0.67 g of 3-(4-hydroxyphenyl)propionic
acid (4.0 mmol), 0.46 g of HOSu (4.0 mmol), and 0.83 g of DCC (4.0
mmol) were dissolved in 20 mL of dried 1,4-dioxane and stirred
overnight at room temperature. The next day, the reaction mixture was
passed through a glass filter funnel to remove precipitated N,N′-
dicyclohexylurea (DCU), combined with a solution of 4armPEG10k-
2 3
NH (5.0 g, 0.5 mmol) and NaHCO (0.19 g, 2.2 mmol) in water (20
mL), and stirred overnight at 50 °C. Afterward, the solvent was
evaporated and the residue was dissolved in water. The raw product
was then extracted with DCM. The combined organic phases were dried
2 4
over anhydrous Na SO , and the solution was concentrated under
reduced pressure. The intermediate was crystallized at 0 °C under
vigorous stirring by dropwise addition of diethyl ether. The precipitate
was collected by filtration, washed with cold diethyl ether, and dried
under vacuum to yield 5.1 g (96%) of 4armPEG10k-dTyr. Thin layer
(
Schwalbach, Germany).
Synthesis of PEG-Amines. Branched PEG-amines (10 kDa molec-
ular weight, 4armPEG10k-NH ) were synthesized as previously de-
2

4
98 Biomacromolecules, Vol. 11, No. 2, 2010
Brandl et al.
Synthesis of Methoxy PEG-Succinimidyl Carbonates (mPEG2k-
dTyr-SC). mPEG2k-dTyr-SC was synthesized in a similar manner to
1
that of 4armPEG10k-dTyr-SC with a 85% yield. H NMR (mPEG2k-
dTyr-SC, CDCl
3
, 600 MHz): δ 2.48 ppm (t, 2H, -C(O)CH
CH C(O)-), 2.98 ppm (t, 2H,
-), 3.38 ppm (s, 3H, H CO-), 3.64 ppm (s,
O-), 6.43 ppm (t, 1H, -NHC(O)-), 7.18 ppm (d, 2H,
OSu), 7.26 ppm (d, 2H, -C OSu).
Synthesis of Alanine-Modified PEG-Amines (4armPEG10k-
2 2
CH -),
2
.88 ppm (s, 4H, -C(O)CH
2
2
-
-
-
C(O)CH
2
CH
2
3
OCH CH
2
2
C
6
H
4
6 4
H
Ala). In the coupling step, 1.51 g of Boc-Ala-OH (8.0 mmol) and 0.83 g
of DCC (4.0 mmol) were dissolved in 20 mL of dried DCM. After
stirring for 30 min at room temperature, the solution was passed through
a glass filter funnel to remove precipitated DCU and combined with a
2
solution of 4armPEG10k-NH (5.0 g, 0.5 mmol) in dried DCM (50
mL). Then, 700 µL of DIPEA (4.0 mmol) were added, and the mixture
was stirred for 1.5 h at room temperature. The solution was concentrated
under reduced pressure and placed into an ice bath. The intermediate
was crystallized at 0 °C under vigorous stirring by dropwise addition
of diethyl ether. The precipitate was collected by filtration, washed
with cold diethyl ether, and dried under vacuum to yield 5.1 g (97%)
1
of 4armPEG10k-Ala-Boc. H NMR (4armPEG10k-Ala-Boc, CDCl
3
,
3
-
00 MHz): δ 1.36 ppm (d, 12H, -CH(NHBoc)CH
OtBu), 3.41 ppm (s, 8H, R CCH O-), 3.64 ppm (s, -OCH
), 5.20 ppm (4H, -CH(NHBoc)CH
3
), 1.44 ppm (s, 36H,
CH O-),
),
3
2
2
2
4
6
.15 ppm (4H, -CH(NHBoc)CH
.66 ppm (4H, -NHC(O)-).
3
3
To deprotect the amino acid moiety, 5.1 g of 4armPEG10k-Ala-
Boc (0.5 mmol) were dissolved in 55 mL of methanolic HCl (prepared
by dropping 5 mL of acetyl chloride into 50 mL of ice-cooled
methanol). After stirring for 30 min at room temperature, the solvent
was evaporated, and the residue was dissolved in 25 mL of water. The
raw product was extracted with DCM. The combined organic phases
2 4
were dried over anhydrous Na SO , and the solution was concentrated
under reduced pressure. 4armPEG10k-Ala was crystallized at 0 °C under
vigorous stirring by dropwise addition of diethyl ether. The precipitate
was collected, washed with cold diethyl ether, and dried under vacuum
1
to yield 4.7 g (96%) of the product (Figure 2B). H NMR (4armPEG10k-
Ala, CDCl
ppm (s, 8H, R
H, -NHC(O)-).
Synthesis of 6-Aminohexanoic Acid-Modified PEG-Amines
4armPEG10k-dLys). 4armPEG10k-dLys (Figure 2B) was synthesized
from 4armPEG10k-NH and Boc-6-aminohexanoic acid with a 95%
3
, 600 MHz): δ 1.34 ppm (d, 12H, -CH(NH
2 3
)CH ), 3.41
Figure 2. Polymers derived from branched poly(ethylene glycol).
Amine-reactive PEG-succinimidyl carbonates (A) and amino acid-
modified PEG-amines (B).
3
CCH O-), 3.64 ppm (s, -OCH CH O-), 7.55 ppm (t,
2
2
2
4
(
chromatography (TLC) indicated that the product was free of
2
1
1
4
armPEG10k-NH
NMR (4armPEG10k-dTyr, CDCl
C(O)CH CH -), 2.87 ppm (t, 8H, -C(O)CH
H, R CCH O-), 3.64 ppm (s, -OCH CH O-), 6.13 ppm (t, 4H,
NHC(O)-), 6.76 ppm (d, 8H, -C OH), 7.03 ppm (d, 8H,
OH).
The phenolic hydroxyl groups were then converted into amine-
2
, 3-(4-hydroxyphenyl)propionic acid, and HOSu. H
yield as described for 4armPEG10k-Ala. H NMR (4armPEG10k-dLys,
3
, 600 MHz): δ 2.43 ppm (t, 8H,
CH -), 3.41 ppm (s,
CDCl , 600 MHz): δ 1.36 ppm (m, 8H, -CH CH CH CH CH NH ),
3
2
2
2
2
2
2
-
8
-
-
2
2
2
2
1.47 ppm (m, 8H, -CH CH CH CH CH NH ), 1.66 ppm (m, 8H,
2
2
2
2
2
2
3
2
2
2
-CH CH CH CH CH NH ), 2.19 ppm (t, 8H, -CH CH CH
2
2
2
2
2
2
2
2
2
6
H
4
CH CH NH ), 2.70 ppm (t, 8H, -CH CH CH CH CH NH ), 3.41 ppm
2
2
2
2
2
2
2
2
2
6 4
C H
(
s, 8H, R
3
CCH
2
O-), 3.64 ppm (s, -OCH
2
CH O-), 6.23 ppm (t, 4H,
2
-
NHC(O)-).
reactive succinimidyl carbonate groups. For a typical synthesis, 5.3 g
of 4armPEG10k-dTyr (0.5 mmol) and 2.05 g of DSC (8.0 mmol) were
dissolved in 50 mL of dried acetonitrile. Then, 1300 µL of pyridine
Synthesis of Lysine-Modified PEG-Amines (4armPEG10k-
Lys). A total of 5.0 g of Boc-Lys(Boc)-OH·DCHA (9.5 mmol) was
suspended in 50 mL of ethyl acetate. A solution of 10% citric acid in
water was added until a clear mixture was obtained. After stirring for
(16.0 mmol) were added, and the reaction mixture was stirred overnight
at room temperature. The next day, the solvent was evaporated under
reduced pressure, and 50 mL of DCM were added to the residue. The
insoluble residue was filtered off, and the solution was concentrated
under reduced pressure. The product was crystallized at 0 °C under
vigorous stirring by dropwise addition of diethyl ether. For further
purification, the crystallization step was repeated. The precipitate was
collected by filtration, washed with cold diethyl ether, and dried under
3
0 min at 0 °C, the aqueous phase was separated and extracted with
ethyl acetate. The combined organic phases were washed with water
until neutral and dried over anhydrous Na SO . After evaporation of
the solvent, Boc-Lys(Boc)-OH was dried under vacuum to yield 3.2 g
97%). 4armPEG10k-Lys was prepared from 4armPEG10k-NH and
Boc-Lys(Boc)-OH in a similar manner to that of 4armPEG10k-Ala with
2
4
(
2
1
a 95% yield (Figure 2B). H NMR (4armPEG10k-Lys, CDCl
MHz): δ 1.32 - 1.54 ppm (m, 20H, -CH CH CH CH NH ), 1.76-1.85
ppm (m, 4H, -CH CH CH CH NH ), 2.69 ppm (t, 8H, -CH CH
), 3.32 ppm (4H, -NHC(O)CH(NH )-), 3.39 ppm (s, 8H,
O-), 3.61 ppm (s, -OCH CH O-), 7.54 ppm (4H,
NHC(O)-).
3
, 600
vacuum to yield 5.2 g (93%) of 4armPEG10k-dTyr-SC (Figure 2A).
1
H NMR (4armPEG10k-dTyr-SC, CDCl
H, -C(O)CH CH -), 2.88 ppm (s, 16H, -C(O)CH
ppm (t, 8H, -C(O)CH CH -), 3.41 ppm (s, 8H, R
ppm (s, -OCH CH O-), 6.36 ppm (t, 4H, -NHC(O)-), 7.17 ppm
d, 8H, -C OSu), 7.26 ppm (d, 8H, -C OSu).
3
, 600 MHz): δ 2.48 ppm (t,
CH C(O)-), 2.98
CCH O-), 3.64
2
2
2
2
2
8
2
2
2
2
2
2
2
2
2
2
2
-
CH
CCH
-OCH
2
CH
2
NH
2
2
2
2
3
2
R
3
2
2
2
2
2
(
6
H
4
6
H
4
2 2
CH

Release of Tethered Peptides or Proteins
Biomacromolecules, Vol. 11, No. 2, 2010 499
a
Table 1. Composition of the Prepared Hydrogels
polymer
concentration
PEG-succinimidyl
carbonate
group
pH
amine component
4armPEG10k-dLys (25 mg)
PEG-dLys
PEG-NH
PEG-Ala
PEG-Lys
control
5%
5%
5%
5%
5%
8.0
7.4
6.8
6.4
7.0
4armPEG10k-dTyr-SC (25 mg)
4armPEG10k-dTyr-SC (25 mg)
4armPEG10k-dTyr-SC (25 mg)
4armPEG10k-dTyr-SC (33 mg)
4armPEG10k-SC (25 mg)
2
4armPEG10k-NH (25 mg)
2
4armPEG10k-Ala (25 mg)
4armPEG10k-Lys (17 mg)
4armPEG10k-NH (25 mg)
2
a
The amine component was dissolved in 1000 µl of 25 mM phosphate buffer and added to the PEG-succinimidyl carbonate. The stoichiometric ratio
between succinimidyl carbonate and amino groups was balanced.
j
j
c
) m
Preparation and Rheological Characterization of Hydro-
gels. Gelation kinetics and gel strength were studied by performing
oscillatory shear experiments on a TA Instruments AR 2000 rheometer
weight between cross-links, M
c
, was calculated by M
p
/V
e
, where
m
p
is the total mass of PEG in the hydrogel. With this parameter, the
average network mesh size (ꢁ) was estimated by
2
2
(
TA Instruments, Eschborn, Germany) with parallel plate geometry.
For the preparation of hydrogels, accurately weighed amounts of the
amine component were dissolved in 1000 µL of 25 mM phosphate
buffer (Table 1) and cooled to 5 °C. Immediately before starting the
experiment, this polymer solution was added to 4armPEG10k-dTyr-
SC (degradable gels) or 4armPEG10k-SC (nondegradable gels, Table
2 Mj c 1/2
-
1/3
2s
1/2
Cn
ꢁ
) υ
l
(2)
(
)
Mr
1
). The stoichiometric ratio between succinimidyl carbonate and amino
groups was balanced, and the overall polymer concentration was 5%
w/v) for all hydrogels. After vortexing, the precursor solution was
where l is the bond length along the polymer backbone (taken as 0.146
-1
nm), M
and C
r
is the molecular mass of the PEG repeating unit (44 g ·mol ),
23
(
n
is the Flory characteristic ratio (here taken as 4 for PEG). All
poured onto the bottom plate of the rheometer, which was also cooled
to 5 °C. The upper plate (20 mm in diameter) was then lowered to a
gap size of 1000 µm, the temperature was raised to 25 °C, and the
measurement was started. The evolution of storage (G′) and loss moduli
experiments were carried out in triplicate and the results are expressed
as means ( standard deviations.
Degradation of Hydrogels. The gel samples were prepared as
described above. After gelation, the initial weight of each hydrogel
was determined. To study the degradation of the gel networks, the
samples were immersed in 10 mL of PBS (containing 0.025% sodium
azide) and incubated at 37 °C in a shaking water bath (approximately
(
G′′) was recorded as a function of time at 1 Hz oscillatory frequency
and a constant strain of 0.05. A solvent trap was used to prevent the
evaporation of water. The crossover of G′ and G′′ was regarded as the
gel point. After 90 min, the absolute value of the complex shear modulus
5
0 rpm). Every day, the samples were poured out over 24 mm Netwell
(
G* ) G′ + i·G′′) was determined as a measure for the gel strength.
Inserts (Corning GmbH, Kaiserslautern, Germany) with a 500 µm mesh
size polyester membrane and weighed. Degradation was assumed to
be complete when the sample passed through the Netwell Insert and
no remaining material could be detected.
All experiments were carried out in triplicate and the results are
expressed as means ( standard deviations.
Equilibrium Swelling of Hydrogels and Determination of
Network Parameters. For the swelling studies, accurately weighed
amounts of the amine component were dissolved in 1000 µL of 25
mM phosphate buffer and added to the PEG-succinimidyl carbonate
Mobility of Incorporated Macromolecules Determined by
Fluorescence Recovery after Photobleaching (FRAP). To investigate
the mobility of incorporated macromolecules with and without amino
groups, the hydrogels were loaded with FITC-BSA (66 kDa molecular
weight) and FITC-dextran (150 kDa molecular weight). The samples
were prepared by dissolving 12.5 mg of 4armPEG10k-NH in 450 µL
2
of 25 mM phosphate buffer, pH 7.4. Subsequently, 50 µL of the FITC-
BSA or FITC-dextran stock solution (prepared in the same buffer, c )
0 mg/mL) were added. The mixture was then added to 12.5 mg of
armPEG10k-dTyr-SC (degradable gels) or 12.5 mg of 4armPEG10k-
SC (nondegradable control gels), vigorously stirred, and cast into Lab-
Tek II Chambered Coverglasses (Thermo Fisher Scientific, Langensel-
bold, Germany). After 2 h of gelation, the samples were positioned on
the microscope stage.
The FRAP experiments were performed on a Zeiss Axiovert 200 M
microscope coupled to a LSM 510 META scanning device (Carl Zeiss
MicroImaging GmbH, Jena, Germany). A Plan-Neofluar 20× objective
lens with a numerical aperture of 0.50 was used. All bleaching
experiments were performed using the 488 nm-line of a 30 mW Ar-
ion laser operating at 50% output power. After the region of interest
has been brought into focus, a time series of digital images with a
resolution of 512 × 128 pixel was recorded using a highly attenuated
laser beam (0.5% transmission). The interval between two consecutive
images was 2 s. After acquisition of five prebleach images, a uniform
disk with a diameter of 36 µm was bleached at maximum laser intensity
(100% transmission). Immediately after bleaching, the laser intensity
was switched back to the previous value (0.5% transmission), and a
series of 75 images was acquired to measure the recovery of
fluorescence. To extract the experimental recovery curve from the image
stack, the mean fluorescence intensities inside the bleached region,
(Table 1). The stoichiometric ratio between succinimidyl carbonate and
amino groups was balanced, and the overall polymer concentration was
again 5% (w/v) for all gels. After vortexing, 250 µL of the precursor
solution were cast into cylindrical glass molds (7 mm inner diameter)
and allowed to gel for 2 h. The samples were then weighed in air and
hexane before and after swelling for 24 h in 10 mL of PBS using a
density determination kit (Mettler-Toledo, Giessen, Germany). To
minimize hydrogel degradation, the samples were incubated at 5 °C.
Based on Archimedes’ principle, the gel volumes after cross-linking
1
4
(
V
gc) and after swelling (Vgs) were determined. The volume of the dry
polymer (V ) was calculated from the mass of the freeze-dried hydrogel
and the density of the polymer in the dry state (taken as the density of
p
-
3
PEG, 1.12 g ·cm ). With these parameters, the polymer fraction of
the gel after cross-linking, υ2c ) V /Vgc, and in the swollen state, υ2s
/Vgs, was calculated. The reciprocal of υ2s is usually termed as the
volumetric swelling ratio (Q).
The number of moles of elastically active chains in the hydrogel
network, ν , was calculated using a modified version of the classical
Flory-Rehner equation
p
)
V
p
e
1
9–21
2
2s
Vp [ln(1 - υ ) + υ + ꢀ υ ]
2s
2s
1
νe ) -
·
(1)
V1υ2c
υ2s 1/3
υ2s
υ2c
2
f
-
[
(
)
(
)
]
υ2c
Here, ꢀ
1
is the Flory-Huggins interaction parameter for PEG in water
3
-1
(
taken as 0.43), V
1
is the molar volume of the solvent (18 cm ·mol ),
and f is the functionality of the cross-links (four in the case of four-
Ifrap(t), and inside a reference region were calculated for each time point
armed PEG). Assuming a defect-free network, the average molecular
using the NIH software ImageJ. In the next step, Ifrap(t) was normalized

5
00 Biomacromolecules, Vol. 11, No. 2, 2010
Brandl et al.
to the prebleach intensity, corrected for any bleaching effects that might
have occurred during image acquisition and normalized to the full scale.
To follow changes in the macromolecular mobility during gel
degradation, the samples were covered with 500 µL of PBS (containing
0
.025% sodium azide) and incubated at 37 °C. At predetermined time
points (t ) 48, 96, and 168 h) the FRAP experiments were repeated as
described above.
Reversible PEGylation of Lysozyme and Characterization by
Gel Electrophoresis. A total of 250 µL of a lysozyme stock solution
(prepared in 25 mM phosphate buffer, pH 7.4 at 50 mg/mL) was added
to 56 mg of mPEG2k-dTyr-SC and incubated for 2 h at room
temperature. The molar ratio of succinimidyl carbonate to protein amino
groups was 4:1. To demonstrate the elimination of attached PEG chains,
1
00 µL of this solution were diluted with 900 µL of 50 mM borate
buffer, pH 9.0, and incubated for 24 h at 50 °C. Nondenaturating SDS-
polyacrylamide gel electrophoresis (PAGE) was used to distinguish
PEG-lysozyme conjugates with varying degrees of substitution. The
electrophoresis was conducted using a polyacrylamide gel of 14% cross-
linking; gels were stained by a colloidal Coomassie brilliant blue G-250
dispersion.
Figure 3. Typical rheograms of degradable (9/0) and nondegradable
hydrogels (b/O). Both gels were prepared from 4armPEG10k-NH
2
at pH 7.4 (degradable gel) and pH 7.0 (nondegradable gel). The
closed symbols (9/b) represent G′, and the open symbols (0/O)
represent G′′.
Release of FITC-BSA and Lysozyme. The hydrogels were loaded
with FITC-BSA (66 kDa molecular weight) and lysozyme (14 kDa
molecular weight) to evaluate their suitability as potential drug delivery
systems. These two proteins were chosen because of their different
mass and different number of amino groups and serve as model
compounds for therapeutic peptides or proteins. To prepare the gel
Table 2. Gel Strength (|G*|), Volumetric Swelling Ratio (Q),
j
Molecular Weight between Cross-Links (M
Mesh Size (ꢁ) of the Prepared Hydrogels
c
), and Average Network
a
j
M
(g·mol-1
group
|G*| (Pa)
Q
c
)
ꢁ (nm)
PEG-dLys 1075 ( 68
36.8 ( 0.5 10477 ( 202 21.2 ( 0.3
2
samples, 25 mg of 4armPEG10k-NH were dissolved in 900 µL of 25
PEG-NH
PEG-Ala
2
1486 ( 132 34.7 ( 0.1
2137 ( 55 b 32.2 ( 0.3b
2435 ( 105 30.9 ( 0.3
9300 ( 82
19.6 ( 0.1
mM phosphate buffer, pH 7.4, and mixed with 100 µL of the respective
protein stock solution (prepared in the same buffer, c ) 10 mg/mL).
This mixture was then added to 25 mg of 4armPEG10k-dTyr-SC and
vigorously vortexed. In each case, 250 µL of these precursor solutions
were cast into cylindrical glass molds (7 mm inner diameter) and
allowed to gel for 2 h. The protein loading was 250 µg per gel.
Afterward, the gels were removed from the glass molds, immersed in
7905 ( 123 17.6 ( 0.2
PEG-Lys
control
b
b
2429 ( 73
30.6 ( 0.8
6946 ( 122 16.3 ( 0.2
a
Data are presented as means ( standard deviations (n ) 3). The
differences in the mean values are statistically significant (p < 0.05).
b
Statistically not significant (p > 0.05).
1
3
0 mL of PBS (containing 0.025% sodium azide), and maintained at
7 °C in a shaking water bath (approximately 50 rpm). All vials were
environment of the reacting amino groups, the polymers used
for gel formation were further modified with alanine (mimicking
the amino terminus of proteins), 6-aminohexanoic acid, and
lysine (Figure 2B). All syntheses were straightforward, and the
desired products were obtained with high yields. End-groups
treated with Sigmacote to prevent the adsorption of proteins. At
specified time intervals, 500 µL of the release medium were collected
and replaced with fresh buffer. Blank hydrogels without protein and
protein solutions (250 µg in 10 mL of PBS) served as control groups.
The released protein amounts were determined as described by
Bradford. In brief, 100 µL of sampled release buffer were pipetted
into a microtiter plate and incubated with 100 µL of Bradford reagent
for 10 min. The protein content was quantified by measuring the
absorption at 595 nm using a Shimadzu CS-9301PC 96-well plate reader
1
were almost fully converted, as indicated by H NMR spectra.
2
4
Physicochemical Characterization of Hydrogels. Hydrogels
were prepared by step-growth polymerization of branched,
amino acid-modified PEG-amines with branched PEG-succin-
imidyl carbonates. As it was expected, gelation kinetics and
gel strength were strongly dependent on the polymers used for
gel preparation. In preliminary tests, the buffer pH value was
therefore adjusted for each gel type (Table 1). When the buffer
pH value was too low, polymerization was slow and the gel
strength remained comparatively low. On the other hand, if the
pH value was too high, the gels solidified immediately and could
no longer be placed on the rheometer. Although the pH values
used for gel preparation (pH 6.4 to 8.0) differed in part from
the physiological value, the prepared hydrogels would still be
(
Shimadzu GmbH, Duisburg, Germany). Calibration curves were
obtained from known concentrations of FITC-BSA and lysozyme. All
experiments were carried out in triplicate and the results are expressed
as means ( standard deviations.
Statistical Analysis. The results from mechanical testing, swelling
studies, and FRAP experiments were compared using single-factor
analysis of variance (ANOVA) and Tukey’s multiple comparison test;
p < 0.05 was regarded as statistically significant. Statistical analysis
was performed using SigmaStat 3.0 software (Systat Software, San Jose,
CA).
26
applicable without causing tissue damage.
Nondegradable hydrogels were prepared from 4armPEG10k-
2
SC and 4armPEG10k-NH at pH 7.0. At the beginning of the
Results and Discussion
experiment, the sample behaved like a free-flowing liquid (G′′
> G′); gelation occurred after 9.2 ( 0.4 min. During the course
of the experiment, cross-linking further proceeded as indicated
by the steadily increasing value of G′ (Figure 3). After 90 min,
the value of G′ exceeded that of G′′ by several orders of
magnitude (G′ . G′′); the gel strength was 2429 ( 73 Pa (Table
2). These data agreed very well with those reported previously
for gels prepared from branched PEG-succinimidyl propionates
The aim of the present study was to prepare in situ forming
hydrogels that allow for the time-controlled release of incor-
porated proteins. For this purpose, branched PEG-succinimidyl
carbonates were synthesized (Figure 2A). These polymers react
with primary amino groups of other polymers, peptides, or
proteins under formation of carbamate bonds. In case of proteins,
succinimidyl carbonates typically react with N-terminal amino
2
5
18
acids and ε-amino groups of lysine residues. Because bond
and PEG-amines. For the preparation of biodegradable gels,
formation and cleavage may vary depending on the chemical
2
4armPEG10k-NH was polymerized with 4armPEG10k-dTyr-

Release of Tethered Peptides or Proteins
Biomacromolecules, Vol. 11, No. 2, 2010 501
SC at pH 7.4. These samples solidified in less than 1 min and
the gel strength was 1486 ( 132 Pa (Table 2). Compared to
4armPEG10k-SC, the reactivity of 4armPEG10k-dTyr-SC seemed
to be considerably increased (Figure 3). This could explain both
the accelerated polymerization as well as the lower strength of
these gels. With increasing reactivity of succinimidyl carbonate
groups, their susceptibility to hydrolysis will simultaneously
1
3
increase. This will result in lower cross-linking densities and,
hence, reduced gel strengths. In the case of 4armPEG10k-dLys,
the buffer pH value was increased to pH 8.0. This was required
because of the high pK
moiety (10.6 ( 0.1). The prepared hydrogels solidified after
.1 ( 0.2 min and the absolute value of G* was 1075 ( 68 Pa
Table 2). However, when 4armPEG10k-Ala and 4armPEG10k-
a
value of the 6-aminohexanoic acid
1
(
Lys were used for gel preparation, the buffer pH value was
decreased to pH 6.8 and pH 6.4, respectively. Otherwise,
gelation occurred within a few seconds and the samples could
not be placed on the rheometer. The high reactivity of
Figure 4. Degradation of hydrogels. Gels prepared from 4armPEG10k-
Lys (b) and 4armPEG10k-Ala (9) degraded within 48 h. Hydrogels
made from 4armPEG10k-NH
tegrated over 4 and 5 days, respectively. The nondegradable gels
O) remained stable over the observation period. The experiments
were carried out in triplicate and the results are presented as means
standard deviations.
2
(2) and 4armPEG10k-dLys (() disin-
4
armPEG10k-Ala was attributed to the comparatively low pK
value of the alanine moiety (8.2 ( 0.3). And in case of
armPEG10k-Lys, gelation was most likely facilitated by the
a
(
4
(
increased number of amino groups per macromer (8 vs 4). The
stiffness was 2137 ( 55 Pa for gels prepared from 4armPEG10k-
Ala and 2435 ( 105 Pa for those prepared from 4armPEG10k-
Lys (Table 2).
swelling capacity. After a critical amount of bonds had been
cleaved, the wet weight decreased and the gels dissolved slowly.
Interestingly, the two gels prepared from 4armPEG10k-Lys and
When compared to each other, the gels prepared from
4
armPEG10k-Ala disintegrated within the first 48 h. This was
4
armPEG10k-dLys had the lowest cross-linking density of all
not expected, as these hydrogels had the highest cross-linking
density of all biodegradable gels. In contrast to that, the
hydrogels containing 4armPEG10k-NH and 4armPEG10k-dLys
2
were stable over 72 and 96 h, respectively, and dissolved within
the following 24 h (Figure 4).
degradable hydrogels (estimated by G*), whereas those made
from 4armPEG10k-Lys showed the highest value. This cor-
responded well with the results of equilibrium swelling studies.
The volumetric swelling ratio Q was inversely related to gel
strengthandwashighestforhydrogelspreparedfrom4armPEG10k-
dLys and lowest for those prepared from 4armPEG10k-Lys
Obviously, the nature of the amino groups not only influenced
gelation kinetics and gel strength but also affected degradation
rate. It is known from the literature that the reactivity of phenyl
carbamates increases with increasing polar effects within the
N-substituent. Phenyl carbamates of amino acid amides or
dipeptides, for example, were hydrolyzed within a few hours.
Phenyl N-ethylcarbamates, however, showed a half-life of
(
Table 2). Using eqs 1 and 2, these data also allowed for the
calculation of the average network mesh size (ꢁ). The gels with
the lowest cross-linking density showed the highest value of ꢁ
(
21.2 ( 0.3 nm); hydrogels with higher stiffness and hence
higher cross-linking density exhibited lower values of ꢁ (Table
). For gels prepared from 4armPEG10k-Lys, the average
2
15
several days. The same effects could account for the degrada-
network mesh size could not be calculated, as these gels
contained macromers of two different functionalities and eq 1
can only be applied for gels containing one single type of
tion profiles of the different hydrogels. In case of 4armPEG10k-
Lys and 4armPEG10k-Ala, the amino group is in close
proximity to an amide linkage, which makes the resulting
carbamates more susceptible to hydrolysis. In contrast to that,
1
9–21
cross-links.
Degradation of Hydrogels. Samples were incubated in PBS
at 37 °C and weighed at predetermined time points to study
hydrogel degradation. As expected, the gels of the control group
did not degrade within the observation period. During the first
2
the chemical structures of 4armPEG10k-NH and 4armPEG10k-
dLys resemble those of phenyl N-ethylcarbamates, which can
explain the better hydrolytic resistance of the corresponding
hydrogels.
As a compromise between both, gel strength and degradation
2
time, hydrogels prepared from 4armPEG10k-NH were chosen
2
4 h, the wet weight of these samples first increased due to
swelling. However, after the nondegradable gels were swollen
to equilibrium, their wet weight remained constant (Figure 4).
In these networks, the individual macromers are linked together
by aliphatic carbamate bonds, which are stable under the
for all further experiments. In future studies it may be also
possible to prolong the degradation time by introducing strongly
electropositive carboxylic acid moieties into the N-substituent,
because these groups were reported to increase the hydrolytic
1
4
experimental conditions. In degradable gels, however, the
polymer chains were held together by aromatic carbamate
groups. These carbamates hydrolyze in neutral and basic
solutions by an E1cB elimination reaction involving the
15
resistance of phenyl carbamates.
Mobility of Incorporated Macromolecules. To show their
suitability as a drug delivery system, the developed hydrogels
were loaded with FITC-BSA (66 kDa molecular weight) and
FITC-dextrans (150 kDa molecular weight). The hydrodynamic
1
4,15
intermediate formation of an unstable isocyanate.
The
isocyanate readily reacts with water and disintegrates into a
primary amine and carbon dioxide (see Figure 1 for compari-
27
diameters of these two macromolecules were 7.2 and 16.6
1
4,15
son).
28
nm, respectively. When comparing these values with the
During the initial phase of the study, the wet weight of the
degradable samples first increased (Figure 4). The hydrolysis
of carbamate groups obviously enlarged the average network
mesh size of these hydrogels, which resulted in an increased
average network mesh sizes of nondegradable (16.3 nm) and
degradable gels (prepared from 4armPEG10k-NH , 19.6 nm),
2
the diffusivity of both macromolecules should be restricted only
to a minor extent. The diffusivity of the incorporated FITC-

5
02 Biomacromolecules, Vol. 11, No. 2, 2010
Brandl et al.
Figure 6. Elimination of PEG chains from PEGylated lysozyme. Lane
1, PEGylated lysozyme after hydrolysis for 24 h at pH 9.0 and 50 °C;
lane 2, modified lysozyme directly after PEGylation; lane 3, native
lysozyme; lane 4, molecular weight marker (6.5, 14.2, 20, 24, 29,
36, 45, and 66 kDa).
5
A). Because the hydrodynamic diameter of FITC-BSA was
well below the average network mesh size of the prepared gels,
it was concluded that the protein molecules were successfully
immobilized by covalent attachment to the hydrogel backbone.
This demonstrates the general potential for tethering therapeutic
peptides or proteins to the gel backbone by simply dissolving
them together with the gel-forming polymers. Hydrogel cross-
linking and drug loading can be performed simultaneously
without the need for additional synthetic steps.
To follow the mobility of FITC-BSA over time, the gel
samples were covered with PBS. In nondegradable hydrogels,
protein mobility did not change substantially during the
observation period. After 168 h of incubation, approximately
20% of the incorporated protein molecules seemed to be mobile
(
Figure 5B). This increase can be attributed to hydrolysis of
aliphatic carbamate groups, slow degradation of the protein or
release of the fluorescence label. In degradable hydrogels,
however, FITC-BSA mobility gradually increased (Figure
5B,C). After 168 h, when the hydrogel was completely degraded,
all protein molecules were mobile. The lower degradation rate
most likely results from differences in the experimental setup
Figure 5. Mobile fractions of FITC-dextran and FITC-BSA in nonde-
gradable (0) and degradable (9) hydrogels directly after cross-linking
(
see Figure 4 for comparison). The FRAP experiments indicate
(
A). *Indicates statistical significance (p < 0.05). Mobile fractions of
that the developed hydrogels would allow for the time-controlled
release of therapeutic peptides or proteins. As a result of the
degradation mechanism, encapsulated peptides or proteins are
expected to be released in the unaltered state. This would be
FITC-BSA in nondegradable (O) and degradable (b) hydrogels over
time (B). Mobility of FITC-BSA in degradable hydrogels (C). The
recovery profiles were acquired 0 (b), 48 (9), 96 (2), and 168 h (()
after cross-linking.
8
an advantage over previous drug delivery systems, as it
preserves bioactivity and lowers the risk of unwanted immune
reactions.
2
9,30
dextran served as benchmark and allowed for the evaluation of
the successful immobilization of FITC-BSA.
Elimination of PEG Chains from PEGylated Lysozyme.
To demonstrate the complete reversibility of protein tethering,
lysozyme was PEGylated with a monofunctional derivative of
4armPEG10k-dTyr-SC (PEGylation using mPEG2k-dTyr-SC).
Directly after PEGylation, most protein molecules were carrying
3-5 PEG chains, as indicated by SDS-PAGE (Figure 6, lane
2). However, after incubation for 24 h at pH 9.0 and 50 °C,
native lysozyme as well as slightly PEGylated protein molecules
could be detected (Figure 6, lane 1). Together with the proposed
Directly after cross-linking, 73% of the FITC-dextran mol-
ecules incorporated into nondegradable hydrogels were mobile.
In the case of degradable gels, even 98% of the incorporated
FTIC-dextrans were mobile (Figure 5A). Due to the lack of
amino groups, FITC-dextrans cannot be bound to the gel
network by reaction with PEG-succinimidyl carbonates. The
obtained results further indicate that incorporated macromol-
ecules will not be immobilized within the gel network by
physical entrapment. In FITC-BSA-loaded hydrogels, however,
the situation was different. Directly after hydrogel preparation,
most of the incorporated protein molecules were immobilized
within the gel network. The mobile fractions were 4% in
nondegradable gels and 20% in degradable hydrogels (Figure
14,15
mechanism of carbamate degradation,
these findings clearly
demonstrate the complete reversibility of protein modifications
using aromatic PEG-succinimidyl carbonates. During gel deg-
radation, the tethered protein molecules will be detached from
their anchoring PEG chains in a similar manner; released

Release of Tethered Peptides or Proteins
Biomacromolecules, Vol. 11, No. 2, 2010 503
slow degrading macromers) could be combined to adjust the
resultant release profiles. In the end, this might allow for a
constant release of therapeutic peptides or proteins over a time
period of several days.
Conclusion
We successfully synthesized different derivatives of poly-
(ethylene glycol) that allow for the preparation of in situ forming
hydrogels. Gel strength and degradability could be tailored by
altering the polymer end-groups. Because cross-linking is
performed in situ, the developed hydrogels could be easily
delivered by injection. During the gelation process, dissolved
proteins were covalently bound to the polymer backbone, as
shown by FRAP experiments. In the same way, therapeutic
peptides or proteins could be tethered to the hydrogel network
without the need for chemical modifications of these molecules.
The nonradical cross-linking approach is, thereby, favorable to
the stability of these fragile molecules. During hydrogel
degradation, the incorporated proteins were released into solu-
tion. Release kinetics will depend on both the incorporated
proteins and the polymers used for gel formation. The chosen
linker group disintegrates without leaving any polymer residues
attached to the protein, which is an advantage over previous
drug delivery systems. Altogether, the developed hydrogels
proved to be suitable for the time-controlled release of incor-
porated molecules. Further modifications of the described
polymers might result in long-lasting hydrogels that would allow
for the sustained release of therapeutic peptides or proteins over
a time period of several days up to a few weeks.
Figure 7. Release of FITC-BSA (b) and lysozyme (O) from biode-
gradable hydrogels. Data are presented as means ( standard
deviations (n ) 3).
peptides or proteins will not carry substantial amounts of residual
polymer, which is an advantage over previously reported
8
approaches.
Release of FITC-BSA and Lysozyme. In the final experi-
ment, the release of FITC-BSA and lysozyme was quantified.
As expected from degradation studies and FRAP experiments,
almost no FITC-BSA was released during the first 24 h (Figure
7
). With the onset of gel degradation, however, more and more
protein was released into solution. The obtained release profile
had a sigmoidal shape and matched the degradation profile very
well. After 96 h, the release of FITC-BSA was completed. In
FRAP experiments, only 75% of the incorporated protein
molecules were mobile after the same time period. These
differences are most likely due to the different amounts of PBS
used for FRAP and release experiments (500 µL vs 10 mL of
PBS). Compared to FITC-dextrans, which were released from
Acknowledgment. The authors wish to acknowledge the
financial support by the German Research Foundation (“Deut-
sche Forschungsgemeinschaft”, DFG), Grant Number GO 565/
1
6-1. We thank Dr. Thomas Burgemeister and Fritz Kastner
1
8
similar gels almost completely within 24 h, the covalent
attachment considerably prolonged the release of incorporated
protein molecules. In addition to FITC-BSA, the hydrogels were
also loaded with lysozyme. In general, the resulting release
profile was similar to that of FITC-BSA (Figure 7). During the
initial phase, however, the release of lysozyme was significantly
higher. After 54 h, approximately 30% of the total amount of
lysozyme was released into solution. In the case of FITC-BSA,
however, only 15% of the incorporated protein molecules were
released at the same time point. Furthermore, the release profile
of lysozyme was almost linear during the first 54 h.
These variations are explained by the different characteristics
of the encapsulated proteins. In the case of lysozyme, one protein
molecule can be bound to the gel network by a maximum of
seven amino groups (amino terminus and ε-amino groups of
lysine residues). Bovine serum albumin, in contrast, is bearing
for recording NMR spectra.
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