Download Structure−Property Relationships in Poly(ethylene glycol)−Protein

Biomacromolecules 2005, 6, 1635-1641
1635
Structure-Property Relationships in Poly(ethylene
glycol)-Protein Hydrogel Systems Made from Various Proteins
Kirill I. Shingel* and Marie-Pierre Faure
Bioartificial Gel Technologies Inc., 400 Maisonneuve West, Suite 1156,
Montreal, H3A 1L4 Quebec, Canada
Received November 29, 2004; Revised Manuscript Received February 15, 2005
A series of poly(ethylene glycol)-protein hydrogels were synthesized with different proteins, and the resultant
structures were characterized in terms of swelling behavior and mechanical, optical, and drug release
properties. Irrespectively of the protein involved in polymerization with poly(ethylene glycol), all studied
systems were found to be loosely cross-linked networks, where both polymer and protein are completely
solvated, enabling as high as 96% water content. Changes in the apparent transparency of the hydrogels
synthesized with different proteins were attributed to the ability of the protein component to self-associate
via hydrophobic interactions. The polyelectrolyte nature of the protein component governs the pH
responsiveness of the network, which manifested itself in a pH-dependent mechanism of swelling and drug
release. It was demonstrated that there is great opportunity to modulate the final characteristics of the hydrogel
system to fit the need of specific biomedical application.
1. Introduction
On the basis of original research on poly(ethylene glycol)
(PEG)-bovine serum albumin conjugates by G. Fortier’s
group,1,2 we created a concept of protein-based biocompatible hydrogels for use in various cosmetic and pharmaceutical applications.3 This hydrogel represents a network
of the protein cross-linked with PEG macromolecules. Due
to the hydrophilic nature of both components, this material
may contain as much as 96% water in their structure, thus
forming an excellent liquid compartment for sustained drug
release. Although PEG is widely used in hydrogel chemistry
and readily available in numerous activated forms,4
PEG-protein drug delivery systems are less known. T. J.
Sanborn et al. has recently synthesized peptide-PEG hydrogels5 which, however, are not homologous to our material
since only a short peptide chain derived from fibrin was taken
in that study, as opposed to relatively high molecular weight
proteins used in this work for gel preparation.
High water content combined with the presence of a
natural polymer makes protein-containing hydrogels inherently biocompatible, which is an obvious advantage of such
hydrogels over widely used HEMA or polyacrylamide materials. It should be noted that the influence of the molecular
weight of PEG and its content on the swelling behavior and
mechanical and drug-releasing properties of the gel was
investigated in detail,6,7 whereas effects of the protein
component attracted less attention.
Therefore, to better understand how the molecular properties of the protein influence the physical appearance and
structural peculiarities of the materials, we synthesized a
series of hydrogels from soy protein, hydrolyzed soy protein,
* To whom correspondence should be addressed. Fax: (514) 280-7805.
E-mail: [email protected].
pea albumin, and casein. All these proteins became popular
and accepted in recent years as additives for cosmetic
formulation. Pea albumin, for example, is used to improve
the elasticity of the skin by decreasing its surface tension.8
A direct administration of bioactive proteins, however, may
be associated with allergic reactions and skin irritation. A
pegylation approach used in preparing protein-based hydrogels serves to eliminate these undesirable effects.
It should be mentioned that the protein hydrogel is expected to possess complex patterns of physicochemical characteristics because of the great diversity in the physicochemical properties of proteins which are taken or potentially
available for synthesis. This gives an opportunity to modulate
the final characteristics of the hydrogel, which, in turn, may
accelerate development of the optimal design of this drug
delivery system. Important factors that may influence hydrogel structure and properties are the polyelectrolyte nature
of the biopolymers and availability of hydrophobic regions
for intermolecular interactions.9 The idea that all these
properties of the proteins would inevitably govern the properties of the resultant hydrogel prompted us to perform this
study.
2. Materials and Methods
2.1. Materials. Soy protein isolate (SPI) with protein
content 96% was used “as received” from Archer Daniels
Midland Co. (Decatur, IL). Pea albumin was purchased from
Coletica (France). Casein of cosmetic grade was from
American Casein Co. (Burlington, NJ). Poly(ethylene glycol)
bis(p-nitrophenyl carbonate) or “activated PEG” was synthesized by the reaction of PEG-8000 with p-nitrophenyl
chloroformate in dichloromethane (CH2Cl2) as described
elsewhere,10 and was used as a macromolecular cross-linking
agent of proteins.
10.1021/bm0492475 CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/19/2005
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Biomacromolecules, Vol. 6, No. 3, 2005
All other chemicals purchased from Sigma or J.T. Baker
were of analytical, ACS reagent, or equivalent grade.
2.2. Protein Solubilization. It is known that the formation
of the solid hydrogel material upon cross-linking with
activated PEG is possible only at a certain optimal concentration of the protein6 dependent on the molar ratio of
activated carbonate groups of PEG and accessible amino
groups of the protein. Preliminary experiments revealed that
the optimal concentrations for pea albumin, SPI, casein, and
hydrolyzed soy protein are 80, 100, 125, and 120 mg/mL,
respectively, when the concentration of activated PEG is kept
constant at 220 mg/mL.
For the hydrogel synthesis, protein solutions in which the
protein concentration was 100 mg/mL for SPI, 125 mg/mL
for casein, and 120 mg/mL for hydrolyzed soy protein were
prepared in 0.16 M sodium hydroxide. The suspension
obtained upon dissolution of crude pea albumin in 0.16 M
sodium hydroxide was centrifuged, and only the supernatant
fraction diluted to the protein concentration of 80 mg/mL
was used for gel synthesis.
For analytical assays, soluble protein fractions from both
hydrolyzed soy protein (HSP) and SPI was extracted by 0.1
M Na2HPO4 at pH 9.3. The suspensions were incubated at
45 °C to facilitate protein extraction. After 3 h of incubation,
the suspensions were separated by centrifugation. Supernatants containing protein were filtered through a 0.22 µm
membrane filter and used for analysis.
2.3. Gel Synthesis. An aqueous solution of activated PEG
was mixed with an equal volume of the prepared protein
solutions, and the resultant mixture was placed between two
glasses to obtain a gel with a thickness of about 1.8 or 1.0
mm. The hydrogel matrix reticulation is known to appear
due to formation of the urethane links between free amino
groups of protein and PEG-carbonate moieties.6 After
the polymerization was completed, the gel was placed in
PBS buffer to wash out p-nitrophenol that was formed as
a byproduct of the cross-linking reaction. Here, the hydrogels are coded as SOYGEL, PEAGEL, CASGEL, and
HSOYGEL for the samples prepared with soy protein,
pea albumin, casein, and hydrolyzed soy protein, respectively.
2.4. Gel Characterization. Preweighed disks of the gels
(about 250 mg) were hydrolyzed in 3 mL of 1.0 M sodium
hydroxide at 45 °C. The solution thus obtained was subjected
to protein and PEG content determination by means of
bicinchoninic acid and iodine colorimetric assays, respectively,6 using a “Benchmark” microplate spectrophotometer
from BioRad. All the measurements were performed in
triplicate, and the data were expressed as an average.
The equilibrium water content (EWC) was estimated from
gravimetric measurements of the dry and completely swollen
hydrogels as follows: EWC (%) ) 100 (Ws - Wd)/Wd, where
Ws and Wd stand for the weights of the swollen and dry gels,
respectively. Transparency of the gel was characterized by
indexes of absorbance of 2.25 mm thick pastilles at 450 nm.
Additional measurements were performed for 1.18 mm thick
hydrogel samples.
The swelling behavior of the SOYGEL and PEAGEL
samples at different pHs was studied to determine the
Shingel and Faure
correlation between the solubility of the protein and the
ability of the corresponding gels to absorb water. In these
experiments, the solubility of the proteins in the range of
pH 3.5-9.0 were determined as the percentage of the soluble
fraction of the protein after reconstitution in a solvent,
incubation, and centrifugation of the corresponding initial
protein solutions.
In osmotic deswelling experiments preweighed gel samples
were covered with a cellulose membrane for dialysis (molecular weight cutoff limit 10000). The samples were
incubated in the solutions of PEG 20000 with polymer
concentration ranging from 0.25 to 25.0 g/dL. After the
equilibrium between gel pastilles and the surrounding solutions was reached, the samples were weighed again. The
values of the polymer volume fraction were calculated from
gravimetric measurements, and the equilibrium swelling
pressure was then determined, as described by Stubbe et al.11
Similar experiments were performed with hydrogel samples
that were loaded with a 1% (w/v) solution of caffeine.
Mechanical properties of the hydrogel samples prepared
with different proteins were tested by using the Mach-1
compression system (Biosensing Inc., Laval, Quebec, Canada)
equipped with the 150 g load cell, which measured the load
(g) as a function of displacement (µm) during compression
and had a working surface of 0.503 cm2. The hydrogel
pastilles of known thickness (measured by a micrometric
magnifier) were subjected to 10-17 subsequent compression
ramps with an amplitude of the load cell displacement of
100 µm (about 5% of the initial pastille thickness) and a
compression velocity of 50.0 µm/s. The relaxation time after
compression was found to be 30 s, and this value was used
for all studied samples. The values of the load for each
compression-relaxation step was recorded, presented as a
function of the load cell displacement, and used for calculation of the elasticity modulus E:
E ) (∆N/∆L)h0
(1)
where N is the load at a displacement L and h0 is the initial
thickness of the hydrogel sample.
2.5. Drug Release. Samples were incubated in a 1%
(w/v) solution of caffeine in PBS buffer for 24 h to integrate
the drug into the hydrogel. The volume ratio of the gel
samples to caffeine solution was ∼1:100. After 6 h of
incubation the medium was changed to a new aliquot of a
fresh solution of the drug. The concentration of caffeine in
the gel samples after incubation was determined to be 0.98%
( 0.04%, indicating that the partition coefficient of caffeine
between the gel and bulk solution at equilibrium was close
to 1.0. The gel pastilles (about 250 mL) with integrated 1%
(w/v) caffeine were then immersed in PBS solution, pH 7.4,
for caffeine release. Aliquots taken at certain intervals of
time were subjected to chromatographic analysis for caffeine
quantification. The amount of caffeine released from the
hydrogels (Mt) was expressed as a percentage of the total
amount of the drug incorporated into the samples (Mt/M0).
These data were analyzed on the basis of Fick’s law of the
solute diffusion from the slab of thickness l: Mt/M0 )
4(Dt/πl2)1/2. Diffusion coefficients Dcaff (cm2/s) for caffeine
were derived from the slope of the linear part of the plot
PEG-Protein Hydrogels
Mt/M0 versus t0.5. Similar experiments were performed in
determining the rate constants of p-nitrophenol release,
kpNP (min-1).
The permeability of the hydrogels was studied using
streptomycin, caffeine, and methylparaben as model solutes.
Hydrogel samples were soaked separately in 1% (w/v)
solutions of the drugs, and release of the molecule was
monitored in the experiments with static cells,12 as reported
previously.13 Quantification of the drugs in the release media
was carried out by means of HPLC, and the data were
analyzed in terms of the quantity of the drug that was loaded
(C0) and released from the gels at time t (Ct). Permeability
coefficients (P, cm2/s) were calculated from the plots of
(Vδ/2A) ln(1 - (2Ct/C0) versus t, where V designates the
volume ratio of the gel and receptor medium, A is the surface
area, and δ is the thickness of the gel slab.14
2.6. Chromatography. The molecular weight of the
proteins (native and hydrolyzed soy proteins, pea albumin,
and casein) was estimated by means of size-exclusion
chromatography (SEC). SEC was carried out by using a
Hewlett-Packard 1050 chromatography system (Agilent
Technologies, Inc., Delaware). The molecular weights of
native soy proteins are known to vary considerably, from
10000 to 100000; therefore, to achieve satisfactory separation
within this range, we used a TSK G4000PWXL (300 × 7.8
mm i.d., 5 µm particle size) column packed with a methacrylate-based hydrophilic stationary phase (TosoBiosep,
Stuttgart, Germany). To eliminate any undesirable effects
of protein sorption on the polymeric phase, a solution of 0.05
M potassium phosphate (pH 9.0) containing 20% (v/v)
methanol was used as the mobile phase. The molecular
weights of the proteins under investigation were derived from
the calibration curve constructed using the following proteins: aprotinin, Mr ) 6500; R-lactalbumin, Mr ) 14200;
carbonic anhydrase, Mr ) 36000; ovalbumin, Mr ) 45000;
bovine serum albumin, Mr ) 66000; fructose-6-phosphate
kinase, Mr ) 84000; β-galactosidase, Mr ) 116000; myosin,
Mr ) 205000. Denaturing SEC was performed with 0.05 M
potassium phosphate (pH 9.0), where urea was added up to
a final concentration of 0.5 M. In both SEC experiments the
flow rate was 1.0 mL/min.
The component composition of the proteins was also
studied by means of reversed-phase chromatography
(RP-HPLC). RP-HPLC was performed in the gradient mode
on a system consisting of a type 600E pump, a 776
autosampler, and a 996 photodiode array detector, all
obtained from Waters (Milford, MA). The column used
was an Ace C4 (150 × 3.9 mm i.d., particle size 5 µm)
silica-based column thermostabilized at 40 °C. The mobile
phase composed of 0.1% TFA in water (eluent system A)
and 0.075% TFA in 60% (v/v) acetonitrile (eluent system B) was pumped at a flow rate of 1.0 mL/min, and the
column effluent was measured within the wavelength range
210-300 nm at a 4.8 nm resolution. The elution was carried
out with a linear gradient of B from 0 to 90% for 30 min
followed by isocratic elution in 90% B for 5 min. Before
chromatography, the mobile phases were filtered through
membrane filters (pore diameter 0.22 µm) (Millipore) and
degassed for 20 min.
Biomacromolecules, Vol. 6, No. 3, 2005 1637
Figure 1. Size-exclusion chromatograms of soy protein (a), hydrolyzed soy proteins (b, c), and pea albumin (d) used for gel synthesis
in 0.05 M potassium phosphate (pH 9.0) with 20% (v/v) methanol
(TSK G4000PWXL column, flow rate 1.0 mL/min).
Analysis of caffeine, streptomycin, and methylparaben in
drug release experiments was carried out on a HewlettPackard 1050 chromatography system (Agilent Technologies)
in isocratic mode on an Ace C18 reversed-phase column,
using a 20% (v/v) methanol or 25% (v/v) acetonitrile solution
in 0.025 M phosphoric acid as the mobile phase. Detection
was performed at 272 nm (caffeine), 210 nm (streptomycin),
254 nm (methylparaben), or 317 nm (p-nitrophenol), followed by quantification of the drug, using a five-point
standard calibration curve.
3. Results and Discussion
3.1. Molecular Weight of Proteins. Size-exclusion analysis revealed that all proteins are not homogeneous with
respect to molecular weight (see Figure 1). Native soy protein
(SPI) was found to contain two major components eluted in
the volumes that corresponded to values of 92800 and 34600.
Similar results were obtained recently for β-conglycinin and
glycinin components of this protein from SDS-PAGE
experiments,15 although the values of 18000016 and
302000-37500017 are more commonly known for soybean
proteins in the aggregated state.
Acid hydrolysis of proteins leads to a decrease of the
molecular weight of the proteins, which was indeed found
for hydrolyzed soy protein (see Figure 1). The chromatographic profiles of hydrolyzed soy protein samples corresponded to a molecular weight of 38000-43000, although
some minor high molecular weight species (about 210000)
were also observed, indicating that the hydrolysis was not
complete. These estimates are in good agreement with the
results of SDS-PAGE electrophoresis of the hydrolyzed soy
protein (M. Robert, M. P. Faure, unpublished data) and outcomes of SEC in denaturing conditions (data not shown
here).
The molecular weight of pea albumin components was
equal to 38000 and 23600, whereas casein was eluted as a
single peak with Mr ) 26000. In both cases, a satisfactory
correlation was found between the molecular weight values
estimated by means of SEC and those reported in the
literature for casein18 and pea albumin fractions.18,19
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Biomacromolecules, Vol. 6, No. 3, 2005
Shingel and Faure
Table 1. Component Compositions, Initial and Equilibrium Water Contents, and Diffusion Characteristics of the Hydrogels Synthesized with
Different Proteins (Mean Value ( SD, n ) 3)
code
protein Mr
[PEG],
mg/g of gel
protein concn,
mg/g of gel
initial water
concn, %
EWC, %
Dcaff,
cm2/s × 10-7
SOYGEL-1
SOYGEL-2
HSOYGEL-1
HSOYGEL-2
PEAGEL
CASGEL-1
CASGEL-2
92800, 34600
92800, 34600
38000
43000
38000, 23600
26000
26000
13.78 ( 2.77
16.27 ( 5.17
19.05 ( 4.83
17.60 ( 3.95
13.37 ( 3.49
14.19 ( 2.12
15.13 ( 3.17
24.85 ( 1.12
29.14 ( 4.20
50.08 ( 1.20
50.14 ( 1.23
22.55 ( 1.95
13.78 ( 1.80
11.91 ( 1.84
68.0
68.0
64.0
64.0
70.0
63.5
63.5
95.78 ( 0.32
95.06 ( 0.24
92.69 ( 0.29
92.68 ( 0.16
95.60 ( 0.24
96.27 ( 0.25
96.16 ( 0.19
6.73 ( 1.25
6.54 ( 1.12
4.57 ( 1.47
4.69 ( 0.83
7.48 ( 2.21
6.78 ( 1.03
7.85 ( 1.64
Figure 2. Optical density of the hydrogels measured at 450 nm as
a characteristic of the transparency of the 2.25 mm thick pastilles of
poly(ethylene glycol)-protein material.
3.2. General Properties of the Hydrogels and Physical
Appearance. To achieve a maximal water-absorbing capacity of the hydrogel as a necessary prerequisite for successful
drug delivery application, proteins with different molecular
weights were cross-linked with activated PEG. The component composition and physical characteristics of these gels
are summarized in Table 1 and Figure 2.
The values of EWC found from hydrogel swelling experiments (EWC ) 92-97%) (see Table 1) are in good
agreement with the values obtained from summation of the
PEG and protein contents, but exceed the values calculated
from the initial amounts of PEG and proteins used for gel
synthesis (initial water content 64-70%). This indicates that
the gels immediately after synthesis are not at the equilibrium
swelling level and are able to absorb solvent upon subsequent
incubation in aqueous solution. It should be noted that the
content of protein and PEG is expressed in Table 1 as a mass
ratio to the weight of fully swollen gel, i.e., when the network
imbibes the maximal volume of water and additional swelling
is precluded by the covalent linkages and/or physical
interactions between the protein and PEG. A statistically
meaningful variation (p < 0.05) in EWC data is observed
for hydrogels synthesized from different proteins, for example, SOYGEL (EWC ) 95-96%) and HSOYGEL (EWC
) 92.7%) samples (Table 1).
Although the concentrations of the proteins taken for
synthesis were close, we found that HSOYGEL samples have
2 times higher protein content than SOYGEL. As is known,
the ability of a hydrogel to swell is dependent on the density
of cross-links and/or occurrence of noncovalent interactions
between components included in a network. Therefore, taking
into account that all studied samples comprise about the same
amount of PEG, the difference in the EWC values (Table 1)
may be attributed to the structural differences of hydrogels
formed by different proteins and their affinity toward water
and also ability to bind solvent.
3.3. Influence of pH on the Swelling of HSOYGELs
and PEAGELs. The solubility of proteins in varied pHs was
found to be another important factor governing the swelling
behavior of the studied materials. The solubility of proteins
is known to be a function of numerous factors, among which
are the net charge of the macromolecules and its distribution
along the backbone and the character of protein-solvent and
protein-protein interactions. For example, at pH 4.5 soy
protein has a lower solubility, while high pH denatures the
protein, facilitating its dissolution.17 This behavior, partially
explained by the presence of a large proportion of acidic
amino acids in the structure of this protein,20,21 was also seen
in this study (see Figure 3). It is interesting to note that the
difference in the solubility of proteins impacts the swelling
capacity of the corresponding hydrogels at varied pHs (see
Figure 3). Generally, the swelling capacity of a HSOYGEL
increases with increasing pH, and the dependence of the
swelling capacity on pH shows a trend similar to that
obtained for the solubility of non-cross-linked protein. Close
to the isoelectric point (pI ) 4.5) soy protein has the
lowest solubility, and respectively, the swelling capacity of
SOYGEL is minimal at this pH. At pH higher than 7.0, the
network is completely ionized, since the pH is greater than
the pK of the carboxylic groups of the biopolymer. This
ionization increases the water-binding capacity of the hydrogel structure, leading to a higher swelling power. The
changes in the ionization of the HSOYGEL are accompanied
by the structural alterations, leading to the pH-controlled
diffusion of the solute through the hydrogel matrix (see
Figure 3a).
A contrasting trend of pH-dependent solubility was
observed for pea albumin (see Figure 3b). The solubility of
this protein increases with decreasing pH; however, the
corresponding gel showed no pH responsiveness for swelling
capacity. To obtain this gel, the suspension obtained by
dispersing pea albumin at pH 11.5 was centrifuged so that
only the alkaline-soluble fraction of pea protein was crosslinked with PEG. It is interesting to note that this alkalinesoluble fraction precipitates from the solution at neutral pH,
but the corresponding hydrogel shows no pH-dependent
structural changes (Figure 3b).
3.4. Hydrophobicity of Proteins and Transparency of
the Gels. Transparency of the hydrogel is important both
esthetically and practically, as this property permits visual
inspection of the site of application in various biomedical
PEG-Protein Hydrogels
Biomacromolecules, Vol. 6, No. 3, 2005 1639
Figure 4. Reversed-phase chromatograms of soy protein (a) and
hydrolyzed soy protein (b) on a C4 reversed-phase column in a linear
gradient of acetonitrile concentration.
Figure 3. Solubility of hydrolyzed soy protein (a) and pea albumin
(b), and the swelling capacity of the hydrogels synthesized thereof
as a function of pH. The rate constants of p-nitrophenol release, kpNP,
are shown to demonstrate the pH-dependent mechanism of the solute
diffusion in hydrogel.
applications. The colorimetric determination of the intensity
of the light scattered by the protein aggregates is shown in
Figure 2. PEAGEL is completely transparent, as it has the
lowest index of absorbance at 450 nm, whereas other samples
are characterized by an opalescence, which increases in the
order PEAGEL > CASGEL > HSOYGEL > SOYGEL (see
Figure 2).
The poor transparency of the soy protein gels can be
explained on the basis of the inherent ability of this protein
for self-association and self-assembly in solution due to
hydrophobic interaction and formation of disulfide bridges
(-S-S-) between cysteine residues in response to different
conditions including pH, temperature, ionic strength, etc.17
Hence, the degree to which these macromolecules are prone
to form aggregates could be evaluated by measuring the
surface hydrophobicity of the protein.
In this study the hydrophobicity of soy proteins was
evaluated from RP-HPLC experiments. Typical elution
profiles of native and hydrolyzed soy proteins are shown in
Figure 4. The major components of SPI are eluted at 13 and
22-26 min, whereas, in contrast, the peaks found for
hydrolyzed soy protein are shifted toward shorter retention
times and appear between 18 and 23 min, indicating that
these macromolecules after hydrolysis are less retentive on
the reversed-phase column. The retention time of soy protein
fragments was found to increase with increasing hydrophobic
moiety content (not shown), and this strongly evidences the
occurrence of a hydrophobicity-based mechanism of protein
separation. Thus, the chromatographic behavior found for
hydrolyzed soy proteins suggests a lowered hydrophobicity
of the biopolymer as a result of the acid treatment. Hydrolysis
of highly aggregative native soy protein19 is believed to
reduce the ability for self-association, which results in the
formation of a more transparent hydrogel upon polymerization with PEG (Figure 2). These findings illustrate the
existence of the relationships between the processes of the
self-association and the physical appearance of the hydrogels,
in particular their visual transparency.
3.5. Mechanical and Drug Release Properties. Typical
stress-relaxation curves obtained for all studied hydrogels
are depicted in Figure 5. In these experiments, a load cell is
brought into contact with the surface of the gel, thus
generating the load data as a function of the cell displacement. A set of 10-17 compression ramps were performed
thus that once a certain displacement is achieved, the load
recedes and the material starts to relax to an equilibrium level
(see Figure 5). For all 10 cycles, the relaxation time did not
exceed 30 s; however, the amplitude of the load generated
by the constant 100 µm displacement was found to increase
with increasing number of compression steps (see Figure 5).
This can be explained by structural alterations of the original
network, which become denser as the load cell advances
1640
Biomacromolecules, Vol. 6, No. 3, 2005
Shingel and Faure
Figure 5. Stress-relaxation curves obtained for HSOYGEL-1 (a) and
PEAGEL (b) samples upon the application of multiple compression
ramps with an amplitude of the load cell displacement of 100 µm and
a compression velocity of 50.0 µm/s. The values of the elasticity
modulus of HSOYGEL-1 (1) and PEAGEL (4), as calculated for each
compression step with the use of eq 1, are plotted in the inset.
progressively into the gel. The elasticity modulus also
increased with increasing number of compression steps,
demonstrating nonlinear elasticity of the material (see the
inset in Figure 5) upon compression. The relatively short
relaxation times are symptomatic for the loosely cross-linked
network, where the motion of macromolecules is only lightly
restricted by cross-links. Indeed, for the hydrogels with such
high water content the theory predicts a relatively low crosslinking density.22 Interestingly, a linear elastic response of
the material was observed recently in the experiments with
the hydrogel samples undergoing tensile deformation (unpublished results). The disparity observed between compressive and tensile properties of the protein-containing hydrogel
requires further investigations, being supposedly associated
with the specific microstructure of the network.
Water or aqueous solution represents a primary transport
vehicle in the studied systems. Therefore, an analysis of
solvent-network interactions is important for understanding
the mechanism of drug release. The affinity of the studied
hydrogels toward aqueous solvent was studied in osmotic
deswelling experiments, which allowed calculation of the
osmotic pressure of the hydrogel, Πsw, as a function of the
polymer volume fraction φ (see Figure 6). Dependences of
Πsw on φ were then analyzed by using the equation23
∏sw ) Aφn - Aφn-1/3φ1/3
(2)
where n is a constant showing a thermodynamic quality of
the solvent. The calculated values of n are summarized in
Table 2 for several gels in pure water and in a 1% (w/v)
solution of caffeine. The values of n in the range of
2.24-2.42 obtained in pure solvent are indicative of good
solvent conditions,23 and this serves as strong evidence of
the high affinity of the network toward an aqueous solvent.
It appears that the protein component of the hydrogel has
a strong influence on its diffusion barrier properties. This
effect is illustrated in Table 1 by the apparent diffusion
coefficient of caffeine, which was taken as an example of a
Figure 6. Osmotic swelling pressure of the hydrogels as a function
of the polymer volume fraction. The dashed lines represent the best
fit approximating experimental points, as calculated by eq 2.
Table 2. Values of Exponent n and Correlation Coefficient R
Obtained from the Least-Squares Fits to Eq 2 for Hydrogels in
Pure Solvent (naq) and 1% (w/v) Caffeine Solution (ncaff)
sample
naq
R
ncaff
R
Soygel-1
Hsoygel-1
Casgel-1
Peagel
2.241
2.424
2.295
2.252
0.998
0.982
0.999
0.999
3.097
2.550
2.408
2.352
0.996
0.997
0.997
0.999
nonionic drug. HSOYGEL samples are characterized by the
smallest diffusion coefficients, as can be expected from the
structural peculiarities discussed above and the EWC values
of this hydrogel (Table 1). Another factor responsible for
the observed retardation of the diffusion in this gel is believed
to be the interaction between caffeine and hydrophobic
moieties of the protein. This was confirmed by measuring
the permeability of the HSOYGEL for streptomycin, caffeine,
and methylparaben. Interestingly, the value of the permeability coefficient for streptomycin (Pstr ) 6.49 × 10-5
cm2/s) exceeds those of methylparaben (Pmp ) 0.96 × 10-5
cm2/s) or caffeine (Pcaff ) 2.51 × 10-5 cm2/s) despite the
larger hydrodynamic size of the streptomycin molecule.
These results thus demonstrate that there are probably strong
interactions between the solutes and the protein network
which are favored by the pronounced hydrophobic character
of both caffeine and methylparaben.
For all studied systems the diffusion coefficients are much
smaller than the self-diffusion coefficient of bulk water, D
) 2.8 × 10-5 cm2/s. This indicates that, in the studied gels,
the diffusion/transport of both water and drug is governed
by a diffusive flow mechanism,24 which is possible owing
to the high porosity of the matrixes.
4. Conclusion
In this study we synthesized a series of PEG-protein
hydrogels using soy protein, hydrolyzed soy protein, pea
albumin, and casein. The component composition of the
hydrogel was determined, and the composites obtained were
PEG-Protein Hydrogels
characterized in terms of swelling behavior and mechanical
and drug release properties. From the experimental results
it appeared that the individual molecular properties of
proteins influence the physicochemical characteristics of the
resultant hydrogel. Studied systems were found to represent
a lightly cross-linked network, where both PEG and protein
are completely solvated, enabling as high as 96% equilibrium
water content. As was found for HSOYGEL and SOYGEL
samples, the polyelectrolyte nature of the biopolymer governs
the pH responsiveness of the corresponding network, which
manifested itself in a pH-dependent mechanism of swelling
and drug release. This property of the soy protein gels makes
them excellent candidates as pH-controlled drug release
material. The disadvantages of this system, such as fragility
and poor transparency, can be overcome by utilization of
other proteins such as pea albumin or casein in combination
with soy protein in polymerization with PEG, since both pea
albumin and casein were shown to form a transparent
hydrogel. Thus, the result of this work demonstrated that
there is a great opportunity to modulate the final characteristics of the system to fit the need of specific biomedical
applications.
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