Biomaterials 31 (2010) 3715–3722
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
The adhesive properties of coacervated recombinant hybrid mussel
adhesive proteins
Seonghye Lim 1, Yoo Seong Choi 1, Dong Gyun Kang, Young Hoon Song, Hyung Joon Cha*
National Research Laboratory of Molecular Biotechnology, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 December 2009
Accepted 12 January 2010
Available online 9 February 2010
Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid–liquid phase separation via concentration) might be involved in the highly condensed
and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified
natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation
model. In the present work, we demonstrate complex coacervation in a system including recombinant
MAPs and hyaluronic acid (HA). Our recombinant hybrid MAPs, fp-151 and fp-131, can be produced in
large quantities, and are readily purified. We observed successful complex coacervation using cationic fp151 or fp-131, and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. In
addition, oil droplets were successfully engulfed using a MAP-based interfacial coacervation process, to
form microencapsulated particles. Collectively, our results indicate that a complex coacervation system
based on MAPs shows superior adhesive properties, combined with additional valuable features
including liquid/liquid phase separation and appropriate viscoelasticity. Our microencapsulation system
could be useful in the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers, for use in biotechnological and biomedical applications.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Mussel adhesive protein
Hyaluronic acid
Coacervation
Bulk adhesion
Microencapsulation
1. Introduction
Bioadhesives play essential roles in many living systems, and are
considered to be very promising biomaterials for use in biotechnology and tissue engineering because of their versatile adhesion
properties, biodegradability and biocompatibility [1–3]. Remarkably, mussel adhesive proteins (MAPs), found in byssal adhesive
plaques, have come to be recognized as very attractive biomaterials
for direct use as bioadhesives in medical applications and in the
engineering of new marine-inspired adhesive materials [4–7].
MAPs exhibit both non-toxicity and strong attachment to any type
of inorganic or organic surface in a wet environment [1,8]. Types 1
(fp-1), 3 (fp-3), and 5 (fp-5) MAPs have been extensively studied,
particularly with respect to the high mol% of 3,4-dihydroxyphenylL-alanine (DOPA) content, which enables MAPs to cross-link via
chelates, or covalently, and to adsorb rapidly onto various surfaces
[9–13]. MAP fp-1, with 80 decapeptide repeats, serves as a protective coating, whereas MAPs fp-3 and fp-5 are mainly involved in
* Corresponding author. Tel.: þ82 54 259 2280; fax: þ82 54 279 2699.
E-mail address: [email protected] (H.J. Cha).
1
Equal contribution.
0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.01.063
binding, serving as adhesives between a surface and the adhesion
plaque of mussels [12]. However, the details of the adhesion
process, including condensation of MAPs to levels as high as 30%
(w/v) in vacuoles, and secretion as a watery liquid but with no
dispersion into the surrounding water [14], remain poorly understood. A model of mussel adhesion has been suggested that seeks to
explain the MAP condensation process, the foamy structure of
Mytilus plaques, and the water-resistant adhesion mechanism. The
model was inspired by the adhesion modality of complex coacervation (involving liquid–liquid phase separation) exhibited by the
cement of Phragmatopoma californica, the California sandcastle
worm [14,15]. However, as purified natural MAPs are very difficult
to obtain in quantity, the suggested mechanism of complex coacervation has not yet been demonstrated in the laboratory.
P. californica builds a protective tube made of minerals such as
sand and shell fragments, and these minerals are held together
using a secreted cement composed of three charged proteins, and
divalent cations (Ca2þ and Mg2þ) [15]. Two of the cement components, the basic proteins Pc-1 and Pc-2, contain high levels of lysine
(7–14 mol%) and DOPA (7–10 mol%), whereas the Pc-3 family,
composed of acidic proteins, has at least seven variants with 60–
90 mol% serine, wherein most serines are phosphorylated [16]. The
highly charged acidic and basic proteins are condensed together
3716
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
with Ca2þ and Mg2 into soluble complex coacervates when the net
charges are neutralized. Because complex coacervates have very
low interfacial tension, involve liquid–liquid phase separation
during formation, and behave rather like viscous particles (rather
than as a concentrated viscoelastic polymer solution) [17–19],
robust underwater adhesion to various substrates and the foamlike structure of P. californica were satisfactorily explained by
a model based on complex coacervation [15,16]. Moreover, an
adhesive mimicking P. californica cement was recently developed
based on complex coacervation using DOPA-containing copolymers
and divalent cations, and was applied to bond wet cortical bone
specimens [20].
To overcome the problems of low-level production and poor
purification yield of natural MAPs, and thus to expand the practical
applications of MAPs, previously we constructed and successfully
overproduced recombinant hybrid MAP, fp-151, in Escherichia coli
[6]. Our fusion protein is composed of six fp-1 decapeptide repeats
at both termini of fp-5. Recently, another hybrid MAP, fp-131,
containing six fp-1 decapeptide repeats at both termini of fp-3
variant A, was designed and successfully overproduced in E. coli
(unpublished results). We noticed that both fp-151 and fp-131, as
well as fp-1, fp-3, and fp-5, resembled Pc-1 and Pc-2 of P. californica
in terms of both high basicity in the polyanionic state and strong
adhesive properties [6,9,16]. Thus, we surmised that MAPs might
also participate in complex coacervation and considered that this
process might explain the mussel adhesion process. Furthermore,
due to the non-toxic adhesion properties of MAPs and the microencapsulation property associated with coacervation [19,21,22],
MAP-based complex coacervates can be applied as functional bioglues for use in combination with drug carriers for bioactive
compounds in tissue engineering.
In the present work, we investigated the formation of complex
coacervates using hybrid MAPs (fp-151 and fp-131) and hyaluronic
acid (HA). HA is an anionic polysaccharide that contributes to cell
proliferation and migration as one of the major components of the
extracellular matrix, and is found in all tissues and body fluids of
vertebrates, as well as in some bacteria [23,24]. The superior
biocompatibility and biodegradability of HA make it a highly
attractive biomaterial for biomedical and tissue engineering
applications, such as synthesis of drug carriers and artificial scaffolds [25,26]. HA has also been used as an acidic partner in the
preparation of complex coacervates for in vitro drug release and
biomedical applications [27,28]. Condensation of MAPs and the
bulk adhesion properties of complex coacervates were also investigated, to improve our understanding of the mussel condensation
and adhesion process. In addition, microencapsulation of oil
particles was performed using complex coacervation to demonstrate potential applications in the field of adhesive drug carriers.
2. Materials and methods
2.1. Expression and purification of recombinant MAPs
E. coli BL21 (DE3) cells containing plasmid pENG151 encoding fp-151 [6] and
pFP131 encoding fp-131 (unpublished result) were grown in 3 l of Luria–Bertani (LB)
medium with 50 mg/ml ampicillin (Sigma, St. Louis, MO, USA) at 37 C and 250 rpm,
respectively. Isopropyl-b-D-thiogalactopyranoside (IPTG; Calbiochem, Darmstadt,
Germany) was induced at an OD600 value of 0.2–0.5 (1 mM final concentration) and
incubated for 5 h at 37 C. Cells were harvested by centrifugation at 18,000g for
10 min at 4 C, and the pellet was resuspended in 5 ml lysis buffer (10 mM Tris–Cl,
100 mM sodium phosphate; pH 8.0) per gram wet weight. Cells were lysed by cell
disruption system (Constant Systems, Daventry, UK) at 20 kpsi. The lysate was
centrifuged (18,000g, 20 min, 4 C) and cell debris was collected, washed in TTE
buffer (1% TritonX-100, 1 mM EDTA, 0.1 mM PMSF, 50 mM Tris–HCl, pH 8.0), washed
in distilled water, and resuspended in 25% (vol/vol) acetic acid. Extracted proteins
were dialyzed twice in distilled water, and final purified products were freeze-dried
and stored at 80 C. Protein purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined
using the Bradford reagent (Bio-Rad, Hercules, CA, USA) using bovine serum albumin
(BSA) as standard.
2.2. Tyrosinase modification of MAPs
The modification of tyrosine residues into DOPA has been described previously
[6]. Protein samples (2 mg/ml) were incubated overnight at 37 C in 1 phosphatebuffered saline (PBS; 2.68 mM KCl, 13.7 mM NaCl, 1.47 mM KH2PO4, and 0.875 mM
Na2HPO4) with 25 mM ascorbic acid and 50 mg/ml mushroom tyrosinase (Sigma).
Modified fp-151 (mfp-151) and fp-131 (mfp-131) were dialyzed twice against
distilled water. The final modified products were freeze-dried and stored at 80 C.
2.3. Preparation of complex coacervates
Polyelectrolytes (0.02%, wt/vol) of fp-151, mfp-151, fp-131, mfp-131, and HA
(molecular weight 17 kDa, 35 kDa, or 59 kDa; Lifecore Biomedical, Chaska, MN, USA)
were dissolved in various concentrations of sodium acetate buffer (5–500 mM) at
different pH values (pH 3.0–4.6). Various ratios of oppositely charged polyelectrolytes were directly mixed, by pipetting within 1 ml-volume spectroscopic
cuvettes, to form complex coacervates, at fixed pH values. The turbidity of each
mixture was immediately determined by absorbance at 600 nm (Mecasys, Daejeon,
Korea). The morphology of complex coacervates of 1% (wt/vol) MAPs mixed with 1%
(wt/vol) HA (35 kDa) (wt/wt ratio of 8:2) in sodium acetate solution (pH 3.8) was
observed by phase-contrast optical/fluorescence microscopy (Olympus, Tokyo,
Japan).
2.4. Measurement of colloid concentration and bulk-scale adhesive strength of
concentrated coacervates
Coacervates of 1% (wt/vol) mfp-151 and mfp-131 with 1% (wt/vol) HA (35 kDa),
at a ratio of 8:2 (wt/wt), were prepared in 100 mM and 50 mM acetate buffer (pH 3.8),
respectively. The coacervates were concentrated and collected by centrifugation
(10,000g, 10 min, 4 C), and dropped into water for measurement of colloid
concentration. Increases in volume and mass of the coacervate/water mixtures were
measured by electronic densitometry (Alfa Mirage, Osaka, Japan).
The bulk adhesive strength of the concentrated coacervate phase was assessed
using aluminum adherends (12 mm in width 150 mm in length), based on
a previously described method [29]. The adherends were etched with 5% (wt/vol)
NaOH solution for 5 min at room temperature and washed with distilled water. The
adherends were immersed in HNO3 solution (30%, vol/vol) for 1 min to eliminate the
smut layer formed by etching. The adherends were cleaned again with distilled
water and dried in air at room temperature. Lyophilized powders of mfp-151 and
mfp-131 dissolved (final concentration; 500 g/l) in 100 mM and 50 mM acetate buffer
(pH 3.8), respectively, were used as controls. BSA (final concentration; 500 g/l) was
also employed as a negative control. Samples were applied to 12 mm 10 mm areas
of aluminum surfaces, and the attached aluminum adherends were incubated for
24 h at room temperature in dry (air) or wet (humid) environment. To create a wet
environment, the attached adherends were wrapped in deionized water-soaked
gauze. Shear strength was directly measured using a universal material testing
machine (Instron, Norwood, MA, USA) with a 2000 N load cell. Adhesion force
expressed in Pascals (Pa) was obtained by dividing the shear force (in Newtons) by
the adherend overlap area (in m2). These adhesion studies were an adaptation of the
ASTM D1002 standard method (ASTM International D1002-05, 2005). Each adhesion
measurement was repeated five times and averaged for a given sample. The errors
shown indicate one standard deviation.
2.5. Microencapsulation of oil by interfacial coacervation
Solutions of 1% (wt/vol) of fp-151 and HA were prepared in 100 mM acetate
buffer (pH 3.8), respectively. Red pepper seed oil (1% (vol/vol); Samyang Co., Yongin,
Korea) was added to the fp-151 solution, and the mixture emulsified by magnetic
stirring (Sibata, Tokyo, Japan) for 10 min. Next, an HA solution was added to the
emulsified solution (the final fp-151/HA ratio was 8:2 wt/wt). Similarly, 1% (wt/vol)
solutions of fp-131 and HA in 50 mM acetate buffer (pH 3.8), at a ratio of 8:2 (wt/wt),
were admixed with red pepper seed oil for evaluation of microencapsulation
potential. Encapsulation resulting from interfacial coacervation was analyzed by
phase-contrast optical/fluorescence microscopy (Olympus). Encapsulated red
pepper seed oil levels were monitored by fluorescence emission from fluorescein
isothiocyanate (FITC) (excitation wavelength 495 nm, emission wavelength 520 nm)
using phase-contrast optical/fluorescence microscopy.
3. Results and discussion
3.1. Formation of a coacervation complex using MAP and HA
Complex coacervation between two oppositely charged polyelectrolytes often occurs principally via attractive electrostatic
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
3717
fp-151/HA
fp-131/HA
0.08
0.20
)
600
Turbidity (Abs
A
pH 3.0
pH 3.8
pH 4.6
0.15
0.06
0.10
0.04
0.05
0.02
0.00
0
20
40
60
80
100
0.20
)
600
Turbidity (Abs
20
40
60
80
0.08
0.04
0.05
0.02
0
20
40
60
80
0.20
HA 17 kDa
HA 35 kDa
HA 59 kDa
0.15
100
E
0.00
0.05
0.02
40
60
80
MAP fraction (wt %)
100
40
60
80
0.00
0
20
100
F
HA 17 kDa
HA 35 kDa
HA 59 kDa
0.06
0.04
20
20
0.08
0.10
0
0
100
D
5 mM
10 mM
50 mM
100 mM
500 mM
0.06
0.10
0.00
0
C
5 mM
10 mM
50 mM
100 mM
500 mM
0.15
0.00
Turbidity (Abs600)
0.00
B
pH 3.0
pH 3.8
pH 4.6
40
60
80
100
MAP fraction (wt %)
Fig. 1. Complex coacervate formation with respect to the mixing percentage of MAPs, as indicated by turbidity measurements. pH effects on coacervation of (A) fp-151 with HA
(35 kDa) and, (B) fp-131 with HA (35 kDa), in 100 mM and 50 mM sodium acetate solutions, respectively, at pH 3.0 (C), pH 3.8 (B), and pH 4.6 (;), are shown. Salt effects on
coacervation of (C) fp-151 and (D) fp-131 with HA (35 kDa), in pH 3.8 sodium acetate solutions at 5 mM (C), 10 mM (B), 50 mM (;), 100 mM (6), and 500 mM (-), are displayed.
The HA molecular weight effect on coacervation of (E) fp-151 and (F) fp-131 with HA of 17 kDa (C), 35 kDa (B), or 59 kDa (;), in 100 mM or 50 mM sodium acetate solutions (pH
3.8), respectively, are depicted. All polyelectrolyte concentrations were 0.02% (wt/vol) and turbidity was measured by absorbance at 600 nm. Each absorbance value is indirectly
representative of the effectiveness of complex coacervate formation.
interactions, which depend on pH, ionic strength, the types of ions
present, mixing ratio, total electrolyte concentration, charge
density, and macromolecule flexibility [19,21]. In view of reports
that pH plays a key role in complex coacervation [19,21,30], we first
investigated the effects of pH on this process. Because hybrid MAP
fp-151 and fp-131 are very basic proteins and precipitate at physiological pH values [31] as do natural MAPs [10], and as the pKa of
HA is pH 2.5 [27], experiments were performed within the pH range
of 3.0–4.6 in sodium acetate buffer. The structural and morphological transitions of complex coacervation can be simply analyzed
by using turbidimetric titration and phase-contrast optical
microscopy to discern the liquid–liquid phase separation process
[30,32]. The results showed that the mixing ratio for maximum
coacervation yield increased slightly with pH for both fp-151/HA
and fp-131/HA (Fig. 1A and B). Because the pI values of both MAPs
are about 10, net positive charges on the proteins will be relatively
well conserved in the pH range of 3.0–4.6. However, the net
negative charge of HA increases with pH, because of the low pKa.
Thus, higher levels of MAPs will be required for neutralization of
two oppositely charged polyelectrolytes at elevated pH. Next, the
3718
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
Fig. 2. Light microscopic morphology of complex coacervates. (A) fp-151, (B) fp-131, (C) mfp-151, and (D) mfp-131 mixed with HA (35 kDa) at a ratio of 8:2 (wt/wt) in 100 mM [for
(m)fp-151] or 50 mM [for (m)fp-131] sodium acetate solutions (pH 3.8). All polyelectrolyte concentrations were 1% (wt/vol). The scale bar is 50 mm.
optimum salt concentration for complex coacervation was determined at pH 3.8 using turbidity measurements (Fig. 1C and D).
Complex coacervates were optimally formed in 100 mM and 50 mM
sodium acetate solutions of fp-151/HA and fp-131/HA, respectively.
In different salt environments, the optimum ratio of MAP to HA
remained steady at 8:2 (wt/wt) (Fig. 1C and D). This optimum ratio
was insensitive to the molecular weight of HA (Fig. 1E and F). We
found negligible variation in maximum coacervation yield in the
HA molecular weight range of 17–59 kDa, in good agreement with
data of a previous study on the effect of polyelectrolyte molecular
weight on coacervation formation using lysozyme and polyacrylic
acid [21].
Phase-contrast optical microscopy yielded clear information on
the morphology of complex coacervates of polyelectrolytes of fp151/HA or fp-131/HA (Fig. 2A and B). In addition, we observed that
complex coacervation of DOPA-containing mfp-151 or mfp-131
with HA was efficient (Fig. 2C and D), similar to what was seen
when fp-151/HA and fp-131/HA were tested in the system. Immediately upon mixing of the oppositely charged polyelectrolytes,
spherical droplets formed as a result of liquid–liquid phase separation; the diameters of these droplets were about 1–5 mm for
(m)fp-151/HA and 5–20 mm for (m)fp-131/HA. Complex coacervation is a kinetic process involving the following sequence of events:
1) stable mixture droplet formation; 2) macroscopic phase separation via coalescence of these droplets; and 3) sedimentation [22].
Size differences between initial droplets formed in (m)fp-151/HA
and (m)fp-131/HA complexes might be attributable to variations in
the driving forces of phase separation of protein and polysaccharide
mixtures, which segregate one type of macromolecule from the
other [22]. Earlier studies on complex coacervation showed that
electrostatic interaction of strongly charged polyelectrolytes tended to result in precipitation rather than coacervation [19,33]. In
fact, we found that precipitates were formed when either fp-151 or
fp-131 was incubated with polystyrene sulfonate (PSS), a polyelectrolyte that bears a strong negative charge (Supplementary
data; Fig. S1A and B). Interestingly, although a precipitate formed
when fp-151 interacted with ferredoxin (from Anabaena sp.; pI ¼ 3–
4), complex coacervates were successfully formed between fp-131
and ferredoxin (Supplementary data; Fig. S1C and D). Thus,
complex coacervation appears to be kinetically favored in the p131/HA system, compared to fp-151/HA mixtures.
3.2. Bulk adhesive strength of concentrated complex coacervates
In nature, mussels accumulate adhesive proteins as highly
concentrated condensates in large vacuoles [33]. Thus, both
condensation and DOPA content should be considered when evaluating mussel adhesion ability. From this viewpoint, fp-151 and
fp-131 were modified in vitro to obtain DOPA-containing MAPs
(mfp-151 and mfp-131), using mushroom tyrosinase [29], and
then highly condensed complex coacervates of mfp-151/HA and
mfp-131/HA were prepared to investigate bulk-scale adhesive
properties. Liquid–liquid phase separation between concentrated
coacervates and an equilibrium solution was accelerated by centrifugation, and the resulting coacervate phases of mfp-151/HA and
mfp-131/HA were very viscous (Fig. 3) and not dispersed in the
equilibrium solution. The concentrations of colloids determined by
electronic densitometry were w800 g/l and w960 g/l for mfp-151/HA
and mfp-131/HA, respectively. Bulk adhesive strengths of concentrated complex coacervates (mfp-151/HA and mfp-131/HA), and
individual MAPs, were compared by shear strength measurements.
An aluminum surface was used as a model adhesive material because
of good surface reproducibility and the possibility of rapid assessment. To attain full adhesive strength, samples were incubated at
room temperature for 24 h. As control experiments, 500 g/l solutions
of mfp-151, mfp-131, and BSA were employed (it was not possible to
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
3719
Fig. 3. Concentrated coacervate phases of (A) mfp-151/HA and (B) mfp-131/HA, on a spatula. Proteins mfp-151 and mfp-131 were mixed with HA (35 kDa) at a ratio of 8:2 (wt/wt) in
100 mM or 50 mM sodium acetate solutions (pH 3.8), respectively. All polyelectrolyte concentrations were 1% (wt/vol). The concentrated coacervate phases were collected by
centrifugation.
First, we could condense our hybrid mussel proteins to 650–750 g/
l, from the coacervation phase. Compared to the w300 g/l
condensation of natural MAPs at pH 5.5 in mussel vacuoles [14],
the observed concentration is sufficiently high to suggest the
possibility of condensation via coacervation in mussel vacuoles.
Complex coacervation typically exhibits several special features
including phase separation from water, remarkably low interfacial
5
Adhesive strength (MPa)
handle solutions over 500 g/l). The results showed that the bulk
adhesive strengths of coacervates (3.17 0.51 MPa for mfp-151/HA
and 4.00 0.53 MPa for mfp-131/HA) were about twofold higher
than those of adhesive proteins employed alone (1.98 0.40 MPa for
mfp-151 and 1.87 0.24 MPa for mfp-131) (Fig. 4). Interestingly, the
coacervate of mfp-131/HA showed a higher adhesion strength than
did mfp-151/HA, although the protein levels used for complex coacervation, and the adhesion strengths of the individual MAPs, were
similar. An examination of the mixing ratios of MAPs and HA allowed
us to estimate that about 640 g/l and 770 g/l of mfp-151 and mfp-131,
respectively, were included in the colloids. Because the coacervate of
mfp-131/HA appears to be kinetically favored compared to that of
mfp-151/HA, we hypothesize that better wettability (the extent of
liquid spreading on a solid surface [35]) of MAP/HA complex coacervates, resulting from both low interfacial tension and increased
adhesive concentration, might improve adhesive strength [34].
Therefore, we assume that complex coacervation may improve all
of adhesive strength, condensation, and secretion, in the process
of mussel adhesion. We further suggest that such adhesive coacervates may be valuable as efficient and strong bulk adhesive
biomaterials.
Our results suggest that the process of complex coacervation
employing MAPs can be a tool for condensation of MAPs in mussel
vacuoles, although no anionic polyelectrolyte partner has yet been
discovered in mussels. Processing concepts of mussel adhesion
based on a coacervation model might explain the condensation of
MAPs in vacuoles, secretion of MAPs as a watery solution with no
dispersion in seawater, and the relatively easy attachment mediated by MAPs to various surfaces in an aqueous environment.
However, no experimental data on coacervation after condensation and secretion of natural MAPs have yet been reported,
because of limitations in natural MAP availability [14]. We expect
that the behaviors of fp-151 and fp-131, fusion proteins of natural
MAPs, reflect those of natural MAPs including fp-1, fp-3, and fp-5,
because of close similarities in amino acid sequences, basicities,
pH-dependent solubilities, and strong adhesive properties to
various surface types [6,10,11,29,31]. The condensation and secretion processing of natural MAPs can be explained on the basis of
the complex coacervation properties of fp-151/HA and fp-131/HA.
mfp-151
mfp-131
P = 3.5 x 10-6
P = 3.5 x 10-3
4
3
2
1
0
BSA
Sole MAP
MAP/HA
coacervates
Fig. 4. Bulk adhesive strengths of concentrated coacervate phases of mfp-151/HA and
mfp-131/HA. Proteins mfp-151 and mfp-131 were mixed with HA (35 kDa) at ratios of
8:2 (wt/wt) in 100 mM or 50 mM sodium acetate solutions (pH 3.8), respectively. All
polyelectrolyte concentrations were 1% (wt/vol). The concentrated coacervate phases
were collected by centrifugation. Proteins mfp-151 and mfp-131 alone, as controls,
were dissolved at a concentration of 500 g/l in 100 mM or 50 mM sodium acetate
solutions, respectively. BSA (500 g/l) was used as a negative control. Samples were
applied to aluminum surfaces and adherends were cured for 24 h at room temperature. Each adhesion measurement was repeated five times and averaged for a given
sample. The errors indicate one standard deviation.
3720
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
Fig. 5. Light (A, B) and fluorescence (C, D) microscopic images of oil droplets microencapsulated by (A, C) fp-151/HA and (B, D) fp-131/HA interfacial coacervation. Solutions of 1%
(wt/vol) of fp-151 and fp-131 in 100 mM and 50 mM sodium acetate solutions (pH 3.8), respectively, were mixed with 1% (vol/vol, final concentration) red pepper seed oil and
emulsified, and 1% (wt/vol) HA (35 kDa) solution was added at a ratio of 8:2 (wt/wt). The scale bar is 50 mm.
tension, and both a relatively lower viscosity and a higher diffusivity compared with non-coacervated solutions [36–38], all of
which are features invaluable for effective underwater adhesion,
as emphasized in the proposed coacervation model of the California sandcastle worm [15]. Thus, it may be suggested that the
viscous particle dispersion properties of complex coacervates
facilitate secretion of highly concentrated mussel MAPs through
the narrow mussel thread. Secreted concentrated coacervates are
possibly well-spread over, or adsorbed to, diverse substrates
underwater, using the extraordinary properties of liquid–liquid
phase separation and very low interfacial tension. Next, metal ions
and the high pH (pH 8.2) of seawater might intervene to induce
metal chelation and DOPA oxidation to affect strong and stable
adhesion [39,40]. We explored the attachment of aluminum
adherends in a wet environment using complex coacervates of
mfp-151/HA. Whereas non-coacervated mfp-151 did not show wet
adhesion, mfp-151/HA coacervates showed relatively good bulk
adhesive strength (0.24 0.13 MPa) in wet environments, even
though the interaction strength level was much lower than that
seen in a dry environment. Ideally, non-coacervated modified
MAPs should demonstrate adhesive abilities even in wet situations. However, current in vitro DOPA modification procedures
affect under 20% of all tyrosine residues because of limited access
of recombinant MAP tyrosines to the tyrosinase active site [29].
This degree of access seems to be inadequate to affect waterresistant bulk adhesion (most tyrosine residues in natural MAPs
are modified to DOPA residues in the mussel [6]). Therefore,
enhancement of DOPA content in MAPs will be very important in
achieving strong water-resistant adhesion. Although the current
adhesive strength is not impressive in a wet environment, because
of limited DOPA content, the mechanical properties of colloids
based on MAPs are very promising for improvement of underwater adhesion. In addition, the fact that MAPs can demonstrate
coacervation under acidic conditions suggests that a condensation
and secretion model based on coacervation remains plausible in
the development of an understanding of mussel adhesion.
3.3. Microencapsulation of oil by interfacial coacervation
Complex coacervation has been conventionally utilized for
microencapsulation, to initially protect and subsequently release
encapsulated biologically active compounds such as hydrophobic
drugs and food ingredients in a controlled manner, for applications
in diverse industries including biotechnology, medicine, and the
cosmetic and food-preparation domains [21,41]. Especially,
dispersed oil encapsulation via interfacial coacervation is widely
regarded as efficient when the oil preparation includes oil-soluble
hydrophobic active molecules. In the present work, we used red
pepper seed oil as a model of microencapsulation because of facile
monitoring by fluorescence emission. We observed that interfacial
coacervations of about 1–30 mm in diameter were spontaneously
formed using either tested MAP (Fig. 5A and C). Importantly, fluorescence from red pepper seed oil was observed only from the cores
of microcapsules (Fig. 5B and D). Thus, it may be expected that
biologically active compounds dissolved in oil can be encapsulated
using our coacervation procedure, and that such adhesive microspheres may be employed in drug delivery procedures requiring
adhesive ability [42,43]. Also, the low stability of complex coacervates has been a major bottleneck in the development of useful
applications [44]. Toxic chemical cross-linkers such as formaldehyde and glutaraldehyde are conventionally used to stabilize such
complexes, and particular non-toxic compounds including glycerol
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
and gelatin, and microwave energy, have also been used to this end
[21,45,46]. We expect that complex coacervates based on MAPs can
overcome the current limitations of coacervation systems, because
of the non-toxic and strong adhesive properties of MAPs. Although
the long-term stability of our microencapsulated coacervates has
not yet been quantitatively analyzed, MAP-based colloids with
inner fluorescence were well observed for at least 8 days in PBS
buffer (pH 7.6) (Supplementary data; Fig. S2). In addition, MAP
coacervates could be employed as surface coatings mediating efficient cell adhesion (Supplementary data; Fig. S3). Potentially, MAPbased encapsulated coacervates can be used as smart adhesive
biomaterials with drug carrier ability. In this way, organ-adhesive
properties can be combined with target-oriented drug delivery
protocols for orthopedic surgery or surgical disclosure in medical
applications.
4. Conclusions
We show that complex coacervates form when either of two
recombinant hybrid MAPs, fp-151 or fp-131, used as cationic polyelectrolytes, is combined with HA as an anionic partner. These
findings could be obtained because recombinant MAPs are available to us in high amounts. We found that the optimal mixing ratio
of MAP:HA was 8:2 (wt/wt) for both MAPs regardless of salt
concentration or HA molecular weight. The sizes of coacervate
droplets were on the order of micrometers, and fp-131/HA coacervates were generally larger than those of fp-151/HA, regardless of
DOPA modification. Whereas the bulk shear strengths of modified
individual MAPs were in the vicinity of 2 MPa in a dry environment,
the coacervation process enhanced adhesive strength approximately twofold (to 3–4 MPa) when aluminum adherends were
employed. This improvement in bulk adhesive strength explained
the significant density increment seen in MAP-based complex
coacervates. We also performed oil microencapsulation using
interfacial coacervation with fp-151/HA and fp-131/HA, and
confirmed the formation of oil-encapsulated coacervates by fluorescence monitoring. Collectively, the observation of strong bulk
adhesion and oil microencapsulation allows us to propose that
complex coacervation systems based on combinations of MAPs and
HA can be successfully used as superior adhesive biomaterials,
including self-adhesive drug carriers. In addition, our successful
demonstration of coacervation employing recombinant MAPs
supports the idea of coacervation-based condensation and secretion model which was suggested for understanding mussel adhesion process.
Acknowledgments
This work was supported by the National Research Laboratory
program (ROA-2007-000-20066-0) and the Brain Korea 21
program from the Ministry of Education, Science and Technology,
Korea.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.biomaterials.2010.01.063.
Appendix
Figures with essential color discrimination. Figs. 3 and 5 in this
article may be difficult to interpret in black and white. The full color
images can be found in the online version, at doi:10.1016/j.
biomaterials.2010.01.063.
3721
References
[1] Silverman HG, Roberto FF. Understanding marine mussel adhesion. Marine
Biotechnol 2007;9:661–81.
[2] Mooney DJ, Silva EA. Tissue engineering: a glue for biomaterials. Nat Mater
2007;6:327–8.
[3] Hammer DA, Tirrell M. Biological adhesion at interfaces. Annu Rev Mater Sci
1996;26:651–91.
[4] Wang J, Liu C, Lu X, Yin M. Co-polypeptides of 3,4-dihydroxyphenylalanine
and L-lysine to mimic marine adhesive protein. Biomaterials 2007;28:
3456–68.
[5] Lee H, Dellatore SM, Miller WM, Messersmith PB. Mussel-inspired surface
chemistry for multifunctional coatings. Science 2007;318:426–30.
[6] Hwang DS, Gim Y, Yoo HJ, Cha HJ. Practical recombinant hybrid mussel
bloadhesive fp-151. Biomaterials 2007;28:3560–8.
[7] Strausberg RL, Link RP. Protein-based medical adhesives. Trends Biotechnol
1990;8:53–7.
[8] Young GA, Crisp DJ. Marine animals and adhesion. In: Allen KW, editor.
Adhesion. London: Applied Science; 1982. p. 19–39.
[9] Waite JH. Adhesion a’ la Moule. Integr Comp Biol 2002;42:1172–80.
[10] Lin Q, Gourdon D, Sun CJ, Holten-Andersen N, Anderson TH, Waite JH, et al.
Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3. Proc Natl
Acad Sci U S A 2007;104:3782–6.
[11] Cha HJ, Hwang DS, Lim S. Development of bioadhesives from marine mussels.
Biotechnol J 2008;3:631–8.
[12] Hwang DS, Gim Y, Cha HJ. Expression of functional recombinant mussel
adhesive protein type 3A in Escherichia coli. Biotechnol Prog 2005;21:965–70.
[13] Hwang DS, Yoo HJ, Jun JH, Kim YK, Moon WK, Cha HJ. Expression of functional
recombinant mussel adhesive protein Mgfp-5 in Escherichia coli. Appl Environ
Microbiol 2004;70:3352–9.
[14] Waite JH, Andersen NH, Jewhurst S, Sun C. Mussel adhesion: finding the tricks
worth mimicking. J Adhes 2005;81:297–317.
[15] Stewart RJ, Weaver JC, Morse DE, Waite JH. The tube cement of Phragmatopoma californica: a solid foam. J Exp Biol 2004;207:4727–34.
[16] Zhao H, Sun CJ, Stewart RJ, Waite JH. Cement proteins of the tube-building
polychaete Phragmatopoma californica. J Biol Chem 2005;280:42938–44.
[17] Bungenberg de Jong HG. Morphology of coacervates. In: Kruyt HR, editor.
Colloid science. Amsterdam: Elsevier Publishing Company; 1949. p. 431–82.
[18] Overbeek JT, Voorn MJ. Phase separation in polyelectrolyte solutions: theory of
complex coacervation. J Cell Physiol 1957;49:7–22.
[19] de Kruif CG, Weinbreck F, de Vries R. Complex coacervation of proteins and
anionic polysaccharides. Curr Opin Colloid In 2004;9:340–9.
[20] Shao H, Bachus KN, Stewart RJ. A water-borne adhesive modeled after the
sandcastle glue of P. californica. Macromol Biosci 2009;9:464–71.
[21] Schmitt C, Sanchez C, Desobry-Banon S, Hardy J. Structure and technofunctional properties of protein–polysaccharide complexes: a review. Crit Rev Food
Sci 1998;38:689–753.
[22] Turgeon SL, Beaulieu M, Schmitt C, Sanchez C. Protein–polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Curr
Opin Coll In 2003;8:401–14.
[23] Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997;242:27–33.
[24] Laurent TC, Fraser JR. Hyaluronan. Faseb J 1992;6:2397–404.
[25] Luo Y, Kirker KR, Prestwich GD. Cross-linked hyaluronic acid hydrogel films:
new biomaterials for drug delivery. J Control Release 2000;69:169–84.
[26] Park SN, Park JC, Kim HO, Song MJ, Suh H. Characterization of porous collagen/
hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Biomaterials 2002;23:1205–12.
[27] Malay G, Bayraktar O, Batıgün A. Complex coacervation of silk fibroin and
hyaluronic acid. Int J Biol Macromol 2007;40:387–93.
[28] Lim ST, Martin GP, Berry DJ, Brown MB. Preparation and evaluation of the in
vitro drug release properties and mucoadhesion of novel microspheres of
hyaluronic acid and chitosan. J Control Release 2000;66:281–92.
[29] Cha HJ, Hwang DS, Lim S, White JD, Matos-Perez CR, Wilker JJ. Bulk adhesive
strength of recombinant hybrid mussel adhesive protein. Biofouling
2009;25:99–107.
[30] Cooper CL, Dubin PL, Kayitmazer AB, Turksen S. Polyelectrolyte–protein
complexes. Curr Opin Colloid In 2005;10:52–78.
[31] Lim S, Choi YS, Song YH, Cha HJ. Salt effects on aggregation and adsorption
characteristics of recombinant mussel adhesive protein fp-151. J Adhes
2009;85:812–24.
[32] Ducel V, Richard J, Saulnier P, Popineau Y, Boury F. Evidence and characterization of complex coacervates containing plant proteins: application to the
microencapsulation of oil droplets. Colloid Surf A 2004;232:239–47.
[33] Weinbreck F, Nieuwenhuijse H, Robijn GW, De Kruif CG. Complex formation of
whey proteins: exocellular polysaccharide EPS B40. Langmuir 2003;19:
9404–10.
[34] Blazquez M, Shennan KIJ. Basic mechanisms of secretion: sorting into the
regulated secretory pathway. Biochem Cell Biol 2000;78:181–91.
[35] Michalski MC, Desobry S, Hardy J. Food materials adhesion: a review. Crit Rev
Food Sci 1997;37:591–619.
[36] Bungenberg de Jong HG. Crystallisation–coacervation–flocculation. In:
Kruyt HR, editor. Colloid science. Amsterdam: Elsevier Publishing Company;
1949. p. 232–58.
3722
S. Lim et al. / Biomaterials 31 (2010) 3715–3722
[37] Nairn J. Coacervation-phase separation technology. In: Ganderton D, Jones T,
McGinity J, editors. Advances in pharmaceutical science. London: Academic
Press; 1995.
[38] Weinbreck F, Rollema HS, Tromp RH, de Kruif CG. Diffusivity of whey protein
and gum arabic in their coacervates. Langmuir 2004;20:6389–95.
[39] Sever MJ, Weisser JT, Monahan J, Srinivasan S, Wilker JJ. Metal-mediated crosslinking in the generation of a marine-mussel adhesive. Angew Chem Int Ed
2004;43:448–50.
[40] Monahan J, Wilker JJ. Cross-linking the protein precursor of marine mussel
adhesives: bulk measurements and reagents for curing. Langmuir 2004;
20:3724–9.
[41] Arshady R. Microspheres and microcapsules, a survey of manufacturing
techniques. Part II. Coacervation. Polym Eng Sci 1990;30:905–14.
[42] Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, et al.
Biologically erodable microspheres as potential oral drug delivery systems.
Nature 1997;386:410–4.
[43] Mathiowitz E, Chickering DE, Lehr C-M. Bioadhesive drug delivery systems: fundamentals, novel approaches, and development. New York: Marcel Dekker; 1999.
[44] Desai KGH, Park HJ. Recent developments in microencapsulation of food
ingredients. Dry Technol 2005;23:1361–94.
[45] Vanin FM, Sobral PJA, Menegalli FC, Carvalho RA, Habitante AMQB. Effects of
plasticizers and their concentrations on thermal and functional properties of
gelatin-based films. Food Hydrocolloid 2005;19:899–907.
[46] Huang YI, Cheng YH, Yu CC, Tsai TR, Cham TM. Microencapsulation of extract
containing shikonin using gelatin-acacia coacervation method: a formaldehyde-free approach. Colloid Surf B 2007;58:290–7.