Genetically Engineered Polypeptide Adhesive Coacervates for Surgical Applications

Article abstract of DOI:10.1002/anie.202100064

Adhesive hydrogels have been developed for wound healing applications. However, their adhesive performance is impaired dramatically due to their high swelling on wet tissues. To tackle this challenge, we fabricated a new type of non-swelling protein adhesive for underwater and in vivo applications. In this soft material, the electrostatic complexation between supercharged polypeptides with oppositely charged surfactants containing 3,4-dihydroxylphenylalanine or azobenzene moieties plays an important role for the formation of ultra-strong adhesive coacervates. Remarkably, the adhesion capability is superior to commercial cyanoacrylate when tested in ambient conditions. Moreover, the adhesion is stronger than other reported protein-based adhesives in underwater environment. The ex vivo and in vivo experiments demonstrate the persistent adhesive performance and outstanding behaviors for wound sealing and healing.

Full text of DOI:10.1002/anie.202100064

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Genetically Engineered Polypeptide Adhesive Coacervates for
Surgical Applications
Authors: Jing Sun, Lingling Xiao, Bo Li, kelu zhao, Zili Wang, yu zhou,
Chao Ma, Jingjing Li, Hongjie Zhang, Andreas Herrmann, and
Kai Liu
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202100064
Link to VoR: https://doi.org/10.1002/anie.202100064

10.1002/anie.202100064
Angewandte Chemie International Edition
RESEARCH ARTICLE
Genetically Engineered Polypeptide Adhesive Coacervates for
Surgical Applications
Jing Sun1,3+, Lingling Xiao2+, Bo Li2, Kelu Zhao2, Zili Wang2, Yu Zhou4, Chao Ma3, Jingjing Li2, Hongjie
Zhang1,2, Andreas Herrmann3,4,5*, Kai Liu1,2*
Abstract: Adhesive hydrogels have been developed for wound
healing applications. However, their adhesive performance is
impaired dramatically due to their high swelling on wet tissues. To
tackle this challenge, we fabricated a new type of non-swelling protein
adhesive for underwater and in vivo applications. In this soft
material, the electrostatic complexation between supercharged
polypeptides with oppositely charged surfactants containing 3,4-
dihydroxylphenylalanine or azobenzene moieties plays an important
role for the formation of ultra-strong adhesive coacervates.
Remarkably, the adhesion capability is superior to commercial
cyanoacrylate when tested in ambient conditions. Moreover, the
adhesion is stronger than other reported protein-based adhesives in
underwater environment. The ex vivo and in vivo experiments
demonstrate the persistent adhesive performance and outstanding
behaviors for wound sealing and healing. Thus, this new type of
genetically engineered adhesive coacervates is a very promising
alternative for surgical applications.
adhesive properties and high toughness, which can be used to
repair skin defects.[8] However, adhesive hydrogels often suffer
from swelling when applied on wet tissues, which then weakens
their mechanical strength dramatically.[9] This shortcoming leads
to the breakage of adhesive hydrogels during the process of
wound healing. In addition, the swelling of the adhesive hydrogels
can lead to tissue compression and severe complications in the
environment of the wound. Therefore, there is an urgent need to
develop alternatives that exhibit persistent adhesive performance
for surgical applications.
The continuous need for novel adhesives has turned
researchers’ attention to natural systems, e.g. mussel secretions.
In these materials, the interplay of multiple supramolecular
interactions including electrostatic-, π-π-, hydrogen- and van der
Waals bonds is responsible for their superb adhesive
performance. In this context, catechol-based adhesives exhibit
interesting adhesion properties to various surfaces due to the
diverse covalent and noncovalent interactions of catechol binding
moieties.[10–16] Other approaches are focusing on biomimetic
small molecule systems, which exhibit weak adhesive
behaviors.[17-19] Their biomedical applications are thus limited. In
addition, a systematic study of the adhesion performance of
Tissue adhesives and hemostatic agents have attracted
tremendous attention as potential alternatives for tissue
engineering and wound healing.[1] However, those adhesives are
often associated with UV-induced crosslinking or chemical
reactions, which can lead to secondary damage to traumatized
tissues. Furthermore, during the wound healing process, the very
slow degradation of the adhesives prevents the migration of
motile cells and inhibits the remodeling of extracellular matrix
microenvironment, thus impeding wound healing. In addition to
these materials, adhesive hydrogels have been investigated
extensively.[2–7] For example, an adhesive hydrogel consisting of
Ag-Lignin NPs, acrylic acid (AA) and pectin exhibited good
biomacromolecular
supramolecular interactions has rarely been investigated.
In this work, new class of bioengineered protein
adhesives
controlled
by
multiple
a
coacervates with robust adhesive performance was developed.
The electrostatic complexation between supercharged
polypeptides (SUPs) and biomimetic synthetic surfactants leads
to liquid-liquid phase separation and the formation of non-swelling
adhesive materials. Moreover, the synergy of catechol chemistry
and electrostatic interactions allows to engineer extraordinary
adhesion performance of the resulting bioglues with adhesion
properties surpassing the ones of other reported systems. In vivo
experiments suggest that this protein-based adhesive coacervate
is very well suited for the acceleration of wound sealing and skin
regeneration.
[1]
Dr. J. Sun, Prof. H. Zhang, Prof. K. Liu
Department of Chemistry, Tsinghua University, Beijing, 100084,
China
E-mail: kailiu@tsinghua.edu.cn
[2]
L. Xiao, B. Li, K. Zhao, Z. Wang, Dr. J. Li, Prof. H. Zhang, Prof. K.
Liu
State Key Laboratory of Rare Earth Resource Utilization,
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences, Changchun, 130022, China
Dr. J. Sun, Dr. C. Ma, Prof. A. Herrmann
Results and Discussion
[3]
[4]
[5]
[+]
The SUPs were derived from elastin-like polypeptides,
consisting dominantly of repetitive pentapeptide sequences with
the primary structure (VPGXG)n, in which the fourth position X is
occupied by lysine (K) or glutamic acid (E). These SUPs were
produced by recombinant DNA technology and expressed in E.
Coli.[20–22] A series of SUPs were produced with different chain
lengths and charges, including K18, K72, K108, E36, E72, and
E144. The letter code denotes positive (K) or negative (E) variants
while the digit indicates the number of charged amino acid
residues (Figures 1A and S1-S2, Table S1). Meanwhile, a series
Zernike Institute for Advanced Materials, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Y. Zhou, Prof. A. Herrmann
DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50,
52056 Aachen, Germany. E-mail: herrmann@dwi.rwth-aachen.de
Prof. A. Herrmann
Institute of Technical and Macromolecular Chemistry, RWTH
Aachen University, Worringerweg 1, 52074, Aachen, Germany
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.202100064
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 1. Preparation and characterization of the SUP-surfactant coacervate glues. (A) Schematic for the expression of supercharged polypeptides
(SUPs) (K72, K108, and E36, E72, E144) and (B) chemical structures of different DOPA-based surfactants (NDP, BnNDP, PDD, PDO, and PDH) and
azo-based surfactant (NAT). The SUP glues were prepared via electrostatic complexation of the SUP and the respective surfactants. (C) Photograph
of SUP-NDP adhesive. An elastic, sticky thread can be stretched between two fingers with freshly prepared protein-based coacervates. Here, K108-
NDP was used as a representative example. (D) Photograph showing two smooth glass bottles adhered together by the SUP glue and bearing a load
of 600 g. The inset is a zoom-in of the contact area of the K108-NAT glue connecting the two smooth glass bottoms of the bottles.
of surfactants containing 3,4-dihydroxylphenylalanine (DOPA) or
azobenzene (Azo) moieties were synthesized (Figure 1B). The
synthetic routes are shown in Schemes S1-S4. Starting from L-
DOPA or 3-(3,4-dihydroxyphenyl) propanoic acid, the
corresponding negatively charged DOPA-surfactant (NDP) and
positively charged DOPA-surfactants with different tail lengths
including decyl- (PDD), octyl- (PDO), and hexyl- (PDH) chains
were readily synthesized through stepwise protection, amination,
and deprotection. Additionally, the negatively charged Azo-based
surfactant with a triethylene glycol unit (NAT) was synthesized in
a one-step procedure from 4-hydroxyazobenzene-4'-sulfonic acid
demonstrates the robust adhesion performance of the SUP
adhesives allowing to glue two glass bottles, bearing a load of 600
g.
Subsequently, the adhesion performance of SUP glues was
evaluated by uniaxial tensile testing. A typical adhesion test setup
is shown in Figure S6. All the tensile testing was performed under
ambient conditions with different substrates, including steel,
polyethylene (PE), and polyvinyl chloride (PVC). It should be
noted that the SUP glues and commercial cyanoacrylate-based
glue (super glue) were tested in parallel for comparison.
The charge ratio between lysine and NDP surfactant (Figure
S7-S8) (1:0.5, 1:1, 1:2, and 1:5) was investigated firstly to
determine the optimal molecular composition regarding the
adhesion performance. It was found that there’s a substantial
increase in the breaking strength as the molar ratio of
lysine/surfactant is increased until a maximum value of 6.3 MPa
at a 1:1 charge ratio was reached. Afterwards, the breaking
strength decreased when the ratio was further increased from 1:1
to 1:5. In particular, the fracture strength decreased by about one
order of magnitude when fabricating the SUP-NDP complex in a
1:5 lysine/surfactant stoichiometry. Under this condition, the 1H
NMR analysis of the glue showed that the stoichiometry of 4.9
NDP surfactant molecules to one lysine of the SUP was found in
the SUP-NDP complex (Figure S9). In this case, cationic lysines
are neutralized by too many sulfates of NDP, leading to the
difficulty in the formation of cation-π interaction. This behavior
strongly suggests that cation-π interactions are important for the
interfacial adhesion.[31] Therefore, this supramolecular interaction,
resulting from the recombinant positively charged protein and
aromatic surfactants, contribute significantly to the overall
adhesion performance.
sodium
salt
hydrate
and
1-(2-bromoethoxy)-2-(2-
methoxyethoxy)ethane. All surfactants were characterized by
nuclear magnetic resonance spectroscopy (NMR) and mass
spectrometry (MS).
The SUP-surfactant coacervates were prepared by
electrostatic complexation of SUPs and the corresponding
surfactants.[23,24] Typically, SUPs and surfactants were mixed in
an aqueous solution with the initial molar ratio of lysine or glutamic
acid to surfactant at 1:1. As a result, the solution became turbid
and after centrifugation a viscous SUP-surfactant complex was
obtained. This liquid-liquid phase separation behavior leads to the
formation of protein-surfactant coacervates.[25,26] The water
content was ~ 30% (w/w) in the SUP-surfactant complexes as
determined by thermogravimetric analysis (TGA) (Figure S3).
After lyophilizing for ~30 min, ~14 % of water remained in the
coacervate (Figure S4), resulting in the formation of the protein-
based adhesive (Figure 1C). To determine the composition of the
SUP-surfactant complexes quantitatively, the K18-NDP complex
was characterized by 1H NMR spectroscopy (Figure S5). The
analysis revealed the stoichiometry of K18 to NDP surfactant to
be 1:17 (i.e., ca. 0.95 NDP surfactant molecules per lysine of the
SUP), indicating that ~5% of lysine moieties were not complexed
with the surfactant molecules. Therefore, we hypothesized that
cation-π interactions between these free lysine residues and the
DOPA phenyl groups may be present in the system, as observed
in other polypeptide systems.[13,19,27–30] Furthermore, Figure 1D
The adhesion performance of the K-NDP glue was then
further evaluated on different substrates (steel, PE, and PVC)
under dry conditions. The K-NDP glue exhibited stronger
adhesion performance on a metal substrate (steel) than non-
metal substrates (PE and PVC) (Figures 2A and S10). Taking
K108-NDP as an example, a fracture strength of 6.3 MPa on steel
2
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Figure 2. Adhesion investigation of the SUP coacervate glues. (A) Lap shear strength for the SUP-NDP adhesives on three different substrates
(Steel, PE, and PVC). K72-NDP and K108-NDP were chosen as representative examples and cyanoacrylate was used as a control. P-values were
calculated using the student’s t-test. ns, not significant difference; *p = 0.013 < 0.05, *** p = 0.0003 and 0.0006 < 0.001 (in the group of steel), **p =
0.0017 and 0.0023 < 0.01 (in the group of PE), * p = 0.049 < 0.05, ** p = 0.0077 < 0.01 (in the group of PVC). (B) The effect of different metal ions
(Fe3+, Tb3+) on the bonding strength of SUP-NDP adhesives on steel (K72-NDP and K108-NDP). *p = 0.012 < 0.05, ***p = 0.00039 < 0.001 (in the
group of K72), * p = 0.031 < 0.05, ** p = 0.0019 < 0.01 (in the group of K108). (C) Lap shear strength for SUP-NAT glues (K72-NAT and K108-NAT)
on three different substrates (Steel, PE, and PVC). *p = 0.016 < 0.05 (in the group of steel), **p = 0.002 < 0.01 (in the group of PE), *p = 0.0015 <
0.05, ** p = 0.009 < 0.01, **** p = 0.00008 < 0.0001 (in the group of PVC). (D) Lap shear strength for SUP-PDD glues (E36-PDD, E72-PDD, and
E144-PDD) on three different substrates (Steel, PE, and PVC. *p = 0.018 < 0.05, ** p = 0.0066 < 0.01, *** p = 0.00041 and 0.00061 < 0.001 (in the
group of steel), * p = 0.022 < 0.05, **p = 0.0032, 0.0014, and 0.0042 < 0.01 (in the group of PE), * p = 0.013 < 0.05, ** p = 0.0049 < 0.01, ***p =
0.00028 < 0.001 (in the group of PVC). (E-H) Adhesion energy of SUP adhesives under different conditions. (I-J) Lap shear strength and adhesion
energy of SUP-BnNDP on steel before/after Fe3+ ion treatment. (K) Schematic for the adhesion mechanism of SUP-surfactant adhesives. Besides
electrostatic interaction, van der Waals forces, hydrophobic interactions, and hydrogen bonds both inside the complex and on the interface of samples
and substrates, cation-π, and metal coordination bonding between catechol units of surfactant and metal ions are important to enhance the adhesion
effect of SUP-surfactant complexes. The brown cylinder and cyan fiber represent surfactant and SUP, respectively. The deep blue sphere represents
metal ions, including Fe3+ and Tb3+
.
was observed. In contrast, the fracture strength of K108-NDP is
only 2.1 MPa on PE and 1.2 MPa on PVC, respectively. The
adhesion energies of K-NDP on different substrates are depicted
in Figure 2E. This behavior might be caused by the strong
interactions between lysines from the SUPs and catechol
moieties of the surfactants and the metal surface. Moreover, the
adhesion performance of K-NDP glues became significantly
stronger when increasing the molar mass of the SUP (Figure 2).
Next, the metal coordination effect on the adhesion property
of K-NDP glue was investigated (Figure 2B). To mimic the ratio
between Fe3+ ions and DOPA from mussel adhesive, a molar ratio
of metal ions to NDP of 1:3 was chosen.[32] Upon adding Fe3+ ions
in aqueous solution to the K-NDP coacervate, the color of the
complex turned black immediately, indicating the formation of
catecholate complexes. The absorbance peak at ~ 545 nm in the
UV-vis spectrum (Figure S12) confirmed the formation of bis Fe-
catecholate.[33] As a result, the bonding strength of SUP glue
increased another 2.5 times in a dry environment. It should be
mentioned that the adhesive performance of K108-NDP-Fe glue
(13.5 ± 1.6 MPa) is even superior to cyanoacrylate-based glue
(10.4 ± 0.6 MPa) on steel.[34] This behavior indicates that the Fe3+-
catechol coordination bonds are of great importance for the
overall performance of SUP-glue. In addition to Fe3+ ions, Tb3+
ions were also used to modulate the adhesion behavior of SUP-
glues (Figures 2B and S13). Similarly, the adhesion
performances of K72-NDP and K108-NDP increased to 6.1 ± 1.4
MPa and 10.0 ± 2.1 MPa after treatment with Tb3+ ions,
respectively. Similarly, high values for the adhesion energies
were obtained for the addition of metal ions (Figure 2F). To the
best of our knowledge, the adhesion performance of K108-NDP
mediated by metal ions were higher than any other reported
protein-based and DOPA-mimicking adhesive (Table S2).[35,36]
To further prove the importance of metal ions in the SUP
coacervate glues, benzyl protection groups were installed at the
catechol units in the NDP surfactant (BnNDP). The bulk adhesion
strength of K-BnNDP glue on steel was measured under the same
conditions as K-NDP. It turned out that the breaking strength of
K72-BnNDP glue was 5.7 ± 1.4 MPa, which is comparable to the
Fe3+-mediated K72-BnNDPglue (5.3 ± 0.6 MPa) (Figure 2I and
S14). This indicates that the metal coordination force was absent
3
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Figure 3. Quantitative evaluation of the SUP glues in ex vivo and in vivo applications. (A-B) The underwater adhesion test and adhesion energy of
SUP adhesive on steel and glass, respectively. (C) The ex vivo application of SUP glue on porcine skin and muscle. Three parallel experiments
were performed for each group of specimens. (D) Schematic illustration of the in vivo hemostasis model, i.e. use of SUP glue on pig liver and kidney.
The blue box indicates the pasting position on pig organs. (E) The bleeding wound was ceased in 10 s after SUP glue was applied. The scale bar
is 0.5 cm.
in the K-BnNDP system. Comparison with the breaking strength
of K72-NDP on steel (4.1 ± 0.7 MPa, Figure 2B), the results
confirmed the synergistic effect of catechol moieties and metal
ions (Fe3+ ions and Tb3+ ions) for the robust adhesion performance
(Figure 2J). It is important to note that the breaking strength of
K72-BnNDP was higher than that of K72-NDP adhesive,
suggesting additional contributions from the aromatic rings of
BnNDP to the adhesion performance.
In addition to the DOPA-inspired NDP system, the scope of
surfactants was further broadened to investigate further structure
property relationships of the glue. As a typical aromatic rich photo-
switchable molecule, azobenzene was selected to study the effect
of an increased π-system on the adhesive properties. The
negatively charged azo-based surfactant (NAT) was investigated
under similar conditions as the NDP-based glues (Figures 2C,
2G and S15). To our surprise, the K-NAT glues exhibited even
better adhesion performance on all different substrates. In
particular, the bonding strength of ~16 MPa of K108-NAT was
higher than any other reported protein-based adhesive and even
cyanoacrylate super glue (Table S2).[35,37] We speculate that the
robust adhesion performance of K-NAT glues might be related to
the presence of strong π-π stacking and cation-π
interactions.[13,29,30,38] We also investigated photo-modulation of
the adhesion performance by switching the azobenzene moiety
from trans- to cis configuration. However, it turned out that the
light irradiation with different wavelength didn’t change the
adhesion performance of the K-NAT adhesive.
Furthermore,
a
family of positively charged DOPA
surfactants including PDD, PDO, and PDH was used to fabricate
SUP glues by the complexation with anionic SUPs (E type). As
shown in Figure 2D and 2H, the E-PDD glues exhibited enhanced
adhesion performance with increasing the SUP chain length
(Figures S16-S18). The higher molecular weight of SUP would
lead to the stronger supramolecular network formation of the glue
system, thus enhancing the adhesion properties. Based on these
results, it can be concluded that the nature of SUP does not
greatly affect the adhesion properties of the glue system.
Important contributions to the strong adhesion behavior of SUP
glues can be ascribed to the multiple supramolecular interactions
in the system, especially the cation-π, π-π, and metal
coordination interactions (Figure 2K).
Aside from dry surfaces, wet adhesion for SUP glues was
investigated. The substrates were glued together and cured in air
for 30 min and then put into water bath overnight before lap shear
tensile test. The SUP glue exhibited high underwater adhesion
strength of ~ 0.4 ± 0.07 MPa and ~ 0.36 ± 0.08 MPa on steel and
glass, respectively (Figure 3A-B and S19), which are higher than
underwater protein-based systems reported to date.[39,40]
Furthermore, the non-covalent adhesive behavior endows the
SUP glues with additional attractive features. Firstly, SUP glues
are cleanable and recyclable. The SUP glues on surfaces can be
removed under sufficiently high external shear due to the non-
covalent interactions between SUP and surfaces (Figure S20).
Additionally, it was found that the regenerated SUP-NDP glue
4
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RESEARCH ARTICLE
Figure 4. Wound sealing and healing investigated in a rat model treated with SUP glues. (A) Schematic representation of SUP glue application in
vivo for healing of linear wounds. K-NDP and K-NAT glues were used for the experiments. (B) Photographs of the wounds after 1, 5, and 9 d on rat
skin. Different treatments were used for in vivo wound healing experiments. (i) blank (no treatment), (ii) suture closure, (iii) commercial medical
adhesive COMPONT®, (iv) K72-NDP glue, and (v) K72-NAT glue. SUP glue facilitates wound healing and tissue regeneration in a 9-day wound-
healing experiment. The scale bar is 10 mm. (C) Quantitative analysis of SUP glue treatment over time monitoring the wound closure area. Three
trials were performed for each measurement. Statistics were evaluated by t-test. (* p = 0.022 < 0.05). (D) H&E staining to investigate tissue
regeneration. (E) Masson’s trichrome staining indicating collagen deposited within the defects. (F-G) Immunofluorescence analysis of IL-6 and TNF-
α. Red immunofluorescent staining and green immunofluorescent staining as an indicator of the levels of IL-6 and TNF-α (marked by white arrows),
respectively. The scale bar is 100 μm.
exhibited only little less adhesion bonding strength as the original
sample both in dry and wet conditions (Figures S21-22).
To explore the biocompatibility of SUP glues, we co-cultured
SUP-surfactant complexes with A549 cancer cells and two normal
cell lines including L929 cells (mouse fibroblast) and 293t cells
(embryonic kidney, epithelial) in a concentration regime from 5 -
100 μgmL-1. After 24 h of culturing in the presence of the SUP
complexes in DMEM medium, the cell viability did not drop below
80% even at a concentration of 100 µgmL-1, confirming the non-
toxic nature of the SUP glues (Figures S23-24).
5
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RESEARCH ARTICLE
Motivated by the extraordinary performance and non-toxic
nature, ex vivo and in vivo applications of SUP glues including
wound healing and adhesion on wet tissues (porcine skin and
muscle) were investigated. Due to the excellent performance of
SUP adhesives, two pieces of muscle and porcine skin were
glued together. As shown in Figure S25, the corresponding force-
displacement curves showed that the peak force is ~ 180 mN, F/w
equals ~ 26 N m-1, and the adhesion energy is 4393 ± 1144 mJm-
necrosis factor-α (TNF-α) in the wound site were chosen as an
evaluation criterion in this study. As shown in Figure 4F, a high
level of IL-6 (red fluorescence) was detected in groups of suture
closure, commercial medical glue, and the control, indicating a
severe inflammatory response in the traumatized area. Moreover,
intensive green fluorescence was detected in these control
groups, suggesting high levels of secreted TNF-α (Figure 4G).
Encouragingly, only minor green fluorescence could be detected
for SUP glue treated groups, which indicated low signs of
inflammation compared with the other samples. These results
suggested that the biocompatible SUP glues exhibit superior anti-
inflammation properties. Therefore, SUP glues are a potential
candidate as a wound repair material, providing ideal wound
healing ability while avoiding inflammation.
2
on muscle, which is comparable to covalently cross-linked
adhesives on soft tissues reported earlier.[41] Adhesion
performance of SUP glue on porcine skin was further evaluated
exhibiting a peak force of ~ 150 mN, F/w equals ~ 20 N m-1, and
adhesion energy is 857 ± 218 mJm-2 (Figure 3C). Such
behaviors might be attributed to the non-covalent and possible
covalent interactions between adhesives and tissue surfaces.
Subsequently, the hemostatic properties of SUP glue were
investigated in vivo. It was found that SUP glue exhibited tissue
adhering and hemostatic features on bleeding models of pig liver
and kidney inhibiting blood leakage within 10 s (Figures 3D-E).
At the same time, there was no swelling observed in the SUP-
surfactant coacervate glues, indicating their superior tissue
adhesion behavior compared to hydrogel systems (Figure
S26).[2,3,42,43] Regarding the control experiment employing
commercial cyanoacrylate, this glue solidified quickly and the
bleeding continued when the wound was treated with this material
indicating that it is not suitable for hemostasis on tissues and
organs (Figure S27). All the experiments above showed that the
SUP glues are suitable for hemostatic application.
Conclusion
In summary, a new type of genetically engineered protein
adhesive involving phase-separated coacervates was developed.
Mediated exclusively by multiple supramolecular interactions
especially electrostatic forces, cation-π interactions, and metal
coordination, the proteinaceous adhesives exhibit ultra-high
adhesion performance on various substrates, soft tissues, and
inner organs, which renders them superior to other reported
bioinspired protein-based adhesives. In stark contrast to adhesive
hydrogels, our SUP glues do not swell and exhibit persistent
adhesion performance on wounds. Notably, the biocompatibility
and biodegradability of SUP-based adhesives endows them with
the appealing features to remodel the wound microenvironment
and to promote rapid wound repair.
The simple fabrication strategy, allowing to electrostatically
bond different surfactants equipped with various functionalities to
a charged polypeptide chain, represents a novel paradigm to
fabricate new bioinspired glues without large synthetic chemical
efforts or lengthy cloning protocols or expression optimization.
Besides, the substitution of arginine for lysine may further
modulate the adhesive’s performance due to stronger cation-π
and electrostatic interactions.[44,45] Hence, this electrostatic
mediated two component glue will pave the way for promising
applications in the field of surgery and soft tissue regeneration. In
addition, fusion of antimicrobial peptides or growth factors in this
coacervate-based adhesive represent further means to increase
the functionality of the glue.
To further explore the potential of SUP glues for in vivo
applications, wound sealing and healing experiments were
conducted on rat skin with linear openings of 1 cm in length
(Figure 4A). The coacervates were used directly without further
freeze-drying for in vivo applications. It should be noted that the
K72-surfactant adhesives were chosen as a representative
sample for in vivo tests due to the higher expression yield of K72
compared to K108. The K72-NDP, K72-NAT, suture closure, and
commercial
available
cyanoacrylate-based
adhesive
COMPONT® were selected as the experimental groups, and an
untreated blank defect was used as a control group (Figure 4B).
The quantitative analysis of the healing progress over 9 days
showed that the group treated with SUP glues outperformed the
other groups (Figure 4C) indicating the suitability of SUP glues
for skin regeneration and application for topical wound healing. In
stark contrast to suture closure and commercial chemical
adhesives, the intrinsic biodegradability and the fact that the glue
relies solely on multiple supramolecular interactions probably
renders the SUP glue very well suited to accelerate the healing
and regeneration of skin defects.
The histological analyses, including Hematoxylin and Eosin
(H&E) staining as well as Masson’s trichrome staining further
verified these results. The H&E staining demonstrated that
wounds treated with K72-NDP glue and K72-NAT glue formed
healthy epithelial tissue and new blood vessels, outperforming the
recovery of the control groups (Figure 4D). In addition, Masson’s
trichrome staining showed more collagen content with an intense
blue color in the SUP glue treated groups compared to the other
groups (Figure 4E). It is well known that wound inflammation is
one of the major causes of death among injured patients.
Therefore, immunofluorescence analysis was used to assess the
efficiency of the SUP glue in preventing wound inflammation. Two
typical proinflammatory cytokines, interleukin-6 (IL-6) and tumor
Acknowledgements
This research was supported by the National Key R&D
Program of China (2020YFA0908900 and 2018YFA0902600), K.
C. Wong Education Foundation (GJTD-2018-09), the National
Natural Science Foundation of China (Grant No. 21877104,
21834007, and 22020102003), and the European Research
Council (ERC Advanced Grant SUPRABIOTICS, Grant No.
694610 awarded to AH). J. S. is grateful for financial support from
the China Scholarship Council (grant number 201507720025).
Keywords: bioadhesive, coacervate, supercharged polypeptides,
supramolecular interactions, wound healing
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10.1002/anie.202100064
Angewandte Chemie International Edition
RESEARCH ARTICLE
A new type of non-swelling protein-based coacervate adhesives for in vivo applications was fabricated by employing electrostatic
complexation of supercharged polypetides (SUPs) and oppositely charged surfactants. The obtained protein adhesives exhibited
superior adhesion performance compared to other reported protein-based adhesives. The outstanding adhesion performance, good
biocompatibility and biodegradability of SUP adhesives facilitate fast wound healing and skin regeneration.
8
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