REVIEW

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Bio-inspired Protein-Based and Activatable Adhesion

Systems
Christina Heinritz, Xuen J. Ng, and Thomas Scheibel*

intrusive and invasive procedure. Dur-

Adhesives are in general chemically or physically sticky substances used to ing the last century, adhesives have also
join surfaces. In case of gluing biological and living substrates, there is a need been employed for medical purposes. How-
for bioadhesives that meet requirements such as biocompatibility, ever, to date, both for internal and ex-
non-toxicity, and degradability. Inspiration for bioadhesives is found in nature, ternal wounds, sutures still describe the
gold standard for surgical procedures. Al-
where distinct mussels, sandcastle worms, barnacles, caddisfly larvae,
though there have been efforts to develop
spiders, and glowworms amongst others, use mainly protein-based glues for degradable and anti-microbial sutures,[3]
various purposes. There is a great selection of reviews and books covering the the use of sutures, in general, is time-
use of various bioadhesives in various applications, but here the focus lies on consuming and requires high precision
advances in the development of bio-inspired protein-based adhesives for and well-trained personnel.[4] In this re-
gard, staples and clips offer a quicker
biomedical applications.
solution to closing wounds. In either
case, however, the physical penetration of
healthy tissue creates a possible gate for
pathogens to enter the body, thus increasing
1. Introduction the risk of infection. Pathogen intrusion then might require ad-
Adhesives have been used by humans for thousands of years, at- ditional intervention after wound healing, in case the materials
tested by wall carvings in Theben dating 3300 years back depict- used are non-absorbable.[5] In this context, and with the emer-
ing the gluing of veneer, which are sheets of wood.[1] From mak- gence of the first commercial cyanoacrylate-based synthetic tis-
ing tools to the use for constructions and buildings in the form sue adhesive known as Eastman 910 in the late 1960s, the appli-
of cement, adhesives are a versatile and maybe even indispens- cation of adhesives for wound treatment has gained increasing
able tool to bond surfaces of a variety of materials, geometries, interest.
and topographies, which is otherwise not possible by other join- Cyanoacrylates (CA) are a class of synthetic adhesives that un-
ing methods.[2] The use of adhesives is also associated with a less dergo rapid polymerization in contact with weak bases such as
water or blood, which can cause trouble in case of the treat-
ment of internal wounds, as the rapid hardening will form a
C. Heinritz, X. J. Ng, T. Scheibel layer with weak adhesive surface properties, limiting its appli-
Chair of Biomaterials cation to external wound treatment, but not inside the body.[6]
Engineering Faculty The biocompatibility of CAs is low but can be improved by in-
University of Bayreuth
Prof.-Rüdiger-Bormann-Straße 1, 95447 Bayreuth, Germany creasing the length of ester side chains,[7] yielding derivatives
E-mail: [email protected] such as n-butyl-cyanoacrylates (Histoacryl and Glubran2) and 2-
T. Scheibel octyl-cyanoacrylates (Dermabond), which are nowadays mostly
Bayreuth Center for Colloids and Interfaces (BZKG) used for wound sealing. Still, there are valid concerns concerning
Bavarian Polymer Institute (BPI) toxic degradation products such as cyanoacetate and formalde-
Bayreuth Center for Molecular Biosciences (BZMB)
Bayreuth Center for Material Science (BayMAT)
hyde, causing inflammatory responses.[8] Evading the toxicity is-
University of Bayreuth sue, polyethylene-glycol (PEG)-based adhesives have increasingly
Universitätsstraße 30, 95447 Bayreuth, Germany been used, such as the commercially available ProGel, CoSeal,
T. Scheibel DuraSeal, or FocalSeal. The highly modifiable chemistry of PEG-
Faculty of Medicine based systems allows tuning of the adhesive and cohesive prop-
University of Würzburg erties, and those materials are further biocompatible and mostly
Pleicherwall 2, 97070 Würzburg, Germany
biodegradable but display a strong swelling behavior, which can
The ORCID identification number(s) for the author(s) of this article compromise their mechanical properties due to a decrease in
can be found under https://doi.org/10.1002/adfm.202303609 crosslinking density or result in delamination of the gel made
© 2023 The Authors. Advanced Functional Materials published by thereof caused by stress of the hydrogel interface.[9]
Wiley-VCH GmbH. This is an open access article under the terms of the Biocompatibility and biodegradability issues have shifted the
Creative Commons Attribution-NonCommercial License, which permits course for exploring and developing new tissue adhesives toward
use, distribution and reproduction in any medium, provided the original
work is properly cited and is not used for commercial purposes.
natural and semi-synthetic systems. The global market size for
tissue sealants and adhesives was valued at roughly $2.7 billion in
DOI: 10.1002/adfm.202303609

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Figure 1. Mechanisms of bioadhesion. A) mechanical interlocking; B) chain entanglement; C) intermolecular bonding; and D) electrostatic bonding.
Reproduced (Adapted) with permission.[12] Copyright 2018, Wiley–VCH GmbH.

2020 and is estimated to reach ≈$5.2 billion by 2030 with a com- does not degrade, it could create a barrier between tissue inter-
pound annual growth rate of 6.7%. Natural biological sealants faces and prevent the tissue to interconnect. As adhesives are also
and adhesives make a larger share in this section compared to aimed to replace suturing processes, they further require ease of
synthetic and semi-synthetic systems, with this trend expected to application to reduce surgery time and should be applicable in
continue in the future, considering the ongoing development of physiological conditions, including wet surfaces, which is partic-
surgical sealants and adhesives.[10] Due to the increasing atten- ularly challenging. Interfacial water prevents the contact of func-
tion to these new materials, the term bioadhesive has been intro- tional groups of adhesive and substrate, hence significantly re-
duced. A bioadhesive is defined as a material used for joining two ducing the adhesive strength if not even completely preventing
surfaces by interfacial forces, where one or both constitute living it. The removal or displacement of interfacial water is a key el-
tissue, over an extended period of time.[11] ement of bioadhesion and the main to-be-solved issue for most
For the successful development of new adhesives, a compre- common glues, as their performance is significantly decreased in
hensive understanding of the underlying mechanisms is indis- a wet environment.[14]
pensable. In general, there are four mechanisms by which ad- Nature can teach us how the demanding (bio)adhesion re-
hesion can occur (Figure 1).[12] There is A) non-specific physical quirements could be met. Natural glues are used by many an-
adhesion such as mechanical interlocking, by which the adhe- imals and plants for various purposes like attachment, loco-
sive infiltrates microscopic pores and cavities of the substrate, motion, home construction, mating, defense, and prey hunting
and the surface roughness or rather surface area is determining (Figure 2).[15] Differences are observed regarding the adhesive
the adhesive strength. B) Chain entanglement is based on poly- mechanism, as some organisms use permanent adhesion, like
mer chain mobility and respective physical entanglement, often mussels and barnacles that secrete a hardening cement, whereas
seen among two polymer interfaces such as those of self-healing other adhesive strategies are non-permanent, for instance, used
hydrogels, hence requiring amorphous polymer networks with by starfish or geckos for attachment and locomotion.[16] More-
low crystallinity. Then there is specific adhesion mediated by C) over, physical adhesion can take place in a dry environment,
intermolecular chemical bonds between molecules of the adhe- which, according to many studies, is caused by very fine hairy
sive and the surface. The forces arising from covalent, ionic, or structures in the nano- or micrometer-scale, maximizing the sur-
metal-ion coordinated bonds are the strongest, while hydrogen face area of contact and, thus, is derived from Van der Waals
bonds, dipole-dipole interactions, and Van der Waals forces pro- interactions.[17] In wet environments, other adhesion mecha-
vide less energy, however, can also play a significant role in case nisms are necessary, and many adhesives, although showing a
of high enough quantities. Lastly, there are D) electrostatic inter- strong adhesion on dry surfaces, fail underwater, because the in-
actions mediated by oppositely charged surfaces. While all these terfacial hydration layer needs to be absorbed or repelled prior to
interactions at the substrate-adhesive interface provide the ba- emergence of adhesive interactions.[18]
sis of adhesion, an effective joint between two surfaces requires Mimicking the multifaceted natural adhesion mechanisms en-
furthermore an adequate level of cohesion, denoting the inter- ables the exploration of bio-inspired adhesives, displaying an at-
nal strength of the adhesive material. The network can be held tractive alternative to synthetic glues with respect to mechani-
together by covalent bonding or non-covalent strategies, such as cal and medical requirements. For a broad overview of bio-based
mechanical reinforcement by particles or fibrillar structures, self- and bio-inspired adhesives from animals and plants, we would
assembly, or phase transitions, and should be able to withstand like to recommend the recent review from Lutz et al.[15] More-
movement and shear stress.[13] The performance of an adhesive over, Rathi et al. provide a good overview of protein adhesives and
is determined substantially by a balanced combination of adhe- glues, which are commercially available or in clinical trials, most
sive and cohesive forces. of which are based on either fibrin, collagen, or albumin from hu-
There is general consent that the ideal bioadhesive has to reli- man or bovine sources.[12] The focus of this review is on protein-
ably hold tissue in place and aid in healing and regeneration of based adhesives inspired by various animals. First, possible nat-
the tissue. Hence, it has to be biocompatible, non-immunogenic, ural adhesive mechanisms will be elucidated as basis for the cur-
and should undergo enzymatic or hydrolytic degradation within rent state of research on bio-inspired adhesives. Then, triggered
several weeks, at which point it should have been infiltrated and adhesion-on-demand will be highlighted to provide insights into
gradually replaced by body-own tissue or cells. If the material specifically activatable bioadhesives.

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Figure 2. Schematic representation of applications of sticky substances produced by animals and plants for attachment (purple), defense (yellow),
locomotion (green), hunting (pink), home construction (orange), and mating (blue). Reproduced under the terms of the Attribution-NonCommercial-
NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.[15] Copyright 2022, the Authors, published by Elsevier.

2. Protein-Based Natural Adhesives the strength of the anchorage.[24] Each thread is produced indi-
vidually by the foot of the mussel within 30 s to 8 min, one at a
Natural protein-based adhesives are used for different purposes. time, and connects with an adhesive plaque at the distal end to the
For example, the permanent underwater adhesion of adult mus- substrate surface.[19a] Further, mussel byssus provides a mechan-
sels and barnacles is mediated by a proteinaceous secrete.[19] ical gradient to connect the soft mussel tissue with hard surfaces
Sandcastle worms build their protective retreat using protein- (e.g. rocks), with increasing stiffness toward the substrate in or-
based cement.[20] Spiders and glowworms rely on sticky silk der to avoid deformational damage of the softer tissue.[25] This
threads for capture of food,[21] whereas caddisfly larvae have particular property has also inspired the design of new materials
adapted their adhesive silk to their habitat in the freshwa- and is discussed elsewhere.[26]
ter environment.[22] These and more adhesive strategies can The formation of a byssus thread starts with the mussel foot
be found under water and ashore, underlining the complexity pressing against the target surface and temporarily sticking itself
and diversity of bioadhesion. Li et al. summarized the protein- using suction force by lifting the ceiling of the pad, thus creating
mediated bioadhesion, especially in marine organisms, consider- a cavity with negative pressure.[19a] Shielded from the surround-
ing some shared generic characteristics:[23] 1) Protein biosynthe- ing seawater, the pH, ionic strength, and redox conditions are
sis, packaging, and release of adhesives are clearly localized in se- adjusted prior to deposition of the thread components within a
cretory cells. 2) Proteins display a distinctive amino acid composi- ventral groove.[27] Three major glands, the phenol (plaque for-
tion, which might be closely related to their adhesive function. 3) mation), collagen (thread core formation), and accessory gland
Functional domains – functional structural units in a protein se- (cuticle formation) are subsequently involved in production of
quence – are often conserved. 4) Proteins contain abundant post- the byssus filament.[28] The filament is composed of collagen-like
translational modifications (like hydroxylation, phosphorylation, proteins (preCols) embedded in several matrix proteins includ-
and glycosylation) and oxidative crosslinking. 5) Often enzymes ing the so-called mussel foot protein (Mfp), of which new vari-
are involved for efficient adhesion. ants are still occasionally identified.[28] Six of the Mfps are found
Importantly, adhesives of most organisms show different ad- in the adhesive plaque.[29] Common to all is the abundance of
hesive mechanisms (Figure 3), including rather complex combi- post-translational modifications, which occur to variable degrees
nations thereof to produce the most suitable bioadhesive adapted depending on the species and protein, like hydroxylation of ty-
to the particular needs. rosine (l-3,4-dihydroxyphenylalanine, DOPA; highest content in
Mfp-5 with ≈30 mol%), sometimes paired with phosphorylated
2.1. DOPA-Based Adhesion Used by Marine Mussels serine (pSer) or 4-hydroxylated arginine (Table 1).[30] As DOPA
contributes particularly to bioadhesion,[31] its mechanism will be
The most prominent and extensively studied model system for discussed in more detail below.
protein-based bioadhesion in nature and a frequently used model DOPA is a catechol-derivate and comprises a benzene ring
system derives from mussels, which are able to anchor to di- with two neighboring hydroxy groups.[32] Its molecular interac-
verse surfaces even under wet ambient conditions. Several mus- tions are diverse and range from electrostatic interactions, hy-
sels form so-called byssus filaments (threads), which prevent drogen bonds, hydrophobic interactions, 𝜋–𝜋 stacking, cation-𝜋
dislodgement by wave-induced water motion as well as hungry interactions, and metal coordination to covalent bonds (Figure
predators and adhere the mussel effectively to almost any under- 4).[33] The catechol-metal complexation is of high strength, close
water surface. Among different mussel species, the number, size, to a covalent bond, and enhances adhesion on metal-containing
and mechanical properties of byssal threads can vary, influencing surfaces. It is also connected to high cohesion, since metal ions

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Figure 3. Schematic overview of described animals and their main adhesion mechanism. Mussels predominantly use adhesive interactions based on
post-translational modifications like L-3,4-dihydroxyphenylalanine (DOPA) or phosphorylated serine (pSer). Those are also found in sandcastle worms,
whose polyelectrolytic adhesive proteins form coacervates to successfully apply the glue to the target area. The proteinaceous cement of barnacles
comprises amyloid-like structures. Fibrillar protein assembly is also observed in the silk of several animals, for instance, the silk of caddisfly larvae. Rich
in pSer, the phosphorylated sites interact with Calcium ions to form 𝛽-sheet crystalline regions. Ecribellate spiders modify their silk web with adhesive
droplets from their aggregate gland, consisting of specific glycoproteins, including spidroins.

like Ca2+ and Fe3+ are present in the mussel byssus and, depend- catechol group has a high affinity to form bidentate hydrogen
ing on the concentration, contribute to crosslinking due to the bonds to oxide and amine groups on a surface, which is pre-
interaction of multiple DOPA residues with one metal ion.[34] sumably the dominating adhesion force at acidic pH on sur-
Metal complexation is a reversible interaction, leading to good faces like mica, minerals, metal oxides, and oxygen-containing
self-healing ability and high extensibility of the byssus cuticle. polymers. Hydrogen atoms are donated to acceptors such as
While metal coordination is favored at neutral to basic pH, the oxygen, nitrogen, and fluorine, or in summation to hydrophilic

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Table 1. Primary sequences and structural elements of exemplary adhesive proteins found in natural organisms.

Species Protein Sequence (consensus or complete) a) Ref.

M. edulis Mfp-1 [AKPSYPPTYK]n [85]

M. Mfp-2 [TDKAYKPNPCVVSKPCKNRGKCIWNGKAYRCKCAYGYGGRHC]n [29,86]

galloprovincialis
M. edulis Mfp-3f ADYYGPNYGPPRRYGGGNYNRYNRYGRRYGGYKGWNNGWNRGRRGKYW [87]

M. californianus Mfp-4 [HVHTHQVLHG]36 [88]

[DDHVNDIAQTA]16

M. edulis Mfp-5 SSEEYKGGYYPGNAYHYSGGSYHGSGYHGGYKGKYYGKAKKYYY [30a]

KYKNSGKYKYLKKARKYHRKGYKYYGGSS

M. californianus Mfp-6 GGGNYRGYCSNKGCRSGYIFYDNRGFCKYGSSSYKYDCGNYACLPRNP [89]

YGRVKYYCTKKYSCPDDFYYYNNKGYYYYNDKDYGCFNCGSYNGCCLRSGY

P. californica Pc-1 [VGGYGYGGKK]15 [45]

P. californica Pc-2 [HPAVXHKALGGYG]8 [45]

P. californica Pc-3A DSSSSSYSSSSSYSSSSSSSSSSSSSYSSSSSYSSSSSSSYSSSSSYSSSSSY [45]

SSSSYSSSSYSSSSYSSSSILTSTSSSDWKRKVPARRVLRTRRFLKCTRCTRC
ILFRSAKTCARKCSRRCLKRVF

P. californica Pc-3B CCKRYSSSSYSSSSSSSSSSYSSSSSSSSYSSSSSSSSSYSSSSSSSSSSYSSSSS [45]

SYSSSSSSSYSSSSSSSSSSYSSSSSSYSSSSSSSSSYSSSSSSSSSYSSSSSSSSS
SYSSSSSSYSSSSSSSYSSSSSSSSSYSSSSSSYSSSSSSSSSYSSSSSSSSSYSSS
SSSYSSSSSSYSSSSSSSSSSSYSSSSSSSSSSYSSSSSSYSSSSSSSSSYSSSSSS
SSSYSSSSSSSSSSYSSSSSSSSSSSYSSSSSSYSSSSSSSYSSSSSSSSSSYSSSS
SSSYSSSSSSSSSSYSSSSSSSSSSSSSYSSS

L. decipiens H- [VSISRSVSIERIVTPGVYTQISRSSSVSVEGGRRRGPWGRGYG [90]

Fibroin
PTGSVSVSVSVEGGRRRGPWGYGRRLGG
LSGSGDLDGLGGVGGLGGLGGLGGRRGPWVRGYG]n

M. rosa CP-20 AHEEDGVCNSNAPCYHCDANGENCSCNCELFDCEAKKPDGSYAHPCRRC [91]

DANNICKCSCTAIPCNEDHPCHHCHEEDDGDTHCHCSCEHSHDHHDDDTH
GECTKKAPCWRCEYNADLKHDVCGCECSKLPCNDEHPCYRKEGGVVSCDC
KTITCNEDHPCYHSYEEDGVTKSDCDCEHSPGPSE

A. diadematus ASG1 TPTTTTTPTTTTTPTTTTTPTTTTIPTTTTTPTTTTTPTTTTTPTTTTTP [21b]

TTTTTPTTTTTPVPTTTETTKVTKPNTPPPTDCDENDLDCIIDDTGDVTG
WFDCPKRYGHFPHGSSHQLFIHCDNWHPFVKKCADDTVFSPKYLVCMWKK
EPGLKSDA

A. diadematus AgSp1 [GPGTTPGANTDSDGDISEILLPSTPSPPAAPQPANPTTPTDVRAPQGSPI [21b]

LIVPAGPGTTPGTITGSDGNPTKFIVPLGAFTTPGSIPGPDGRPIHVQPA]n
a)
Symbols for standard amino acids follow convention. Anionic amino acids are colored in blue, cationic in red. Known post-translational modifications (highlighted) are
hydroxylation (orange, bold) and phosphorylation (green, underlined) to obtain L-3,4-dihydroxyphenylalanine (DOPA), hydroxyproline, hydroxyarginine, and phosphoserine,
respectively. Some cysteine residues are known to form disulfide bonds (purple, italic).

surfaces. On the other hand, more hydrophobic surfaces lead tions. The interaction with surfaces results from the hydrophobic
to an increased number of mussel byssus threads in Mytilus aromatic ring of DOPA (or other aromatic amino acids), which
edulis,[35] as such surfaces are better suited for Mfp adsorption orients planar to a surface or, in the case of organic compo-
compared to hydrophilic surfaces due to hydrophobic interac- nents deposited onto a surface, is attracted to hydrophobic alkyl

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Figure 4. Overview of the proposed different adhesive and cohesive molecular interactions found in proteins from e.g. mussels and sandcastle worms.
Reproduced (Adapted) under the terms of the Attribution-NonCommercial (CC-BY-NC) license.[39] Copyright 2018, John Wiley and Sons.

residues.[33] Furthermore, the aromatic moiety can form 𝜋–𝜋 in- The mussel overcomes these unfavorable effects by the afore-
teractions: organic surfaces with other electron-rich 𝜋-systems mentioned shielding from the environment during the plaque
evoke stacking of the aromatic rings, e.g. graphite or graphene.[32] formation process and the initial adjustment of the conditions.
Even stronger than these 𝜋–𝜋 interactions are cation-𝜋 interac- In addition, the cysteine-rich Mfp-6 is secreted at the inter-
tions between the aromatic system and cations (e.g. Li+ , Na+ , face, which converts DOPA-quinone back to DOPA via oxida-
K+ , NH4 + ). Analysis of the amino acid content of Mfps also tion of its thiol groups and, at the same time, partially forms
revealed an increased occurrence of lysine residues, which led disulfide bridges through oxidation, which cross-links the pro-
to the hypothesis of a synergistic effect of lysine and DOPA tein glue.[37,39] Mfp-6 can partially rescue DOPA, but cannot
in close proximity.[36] Importantly, in addition to lysine, other completely prevent the formation of DOPA-quinone. However,
charged amino acid residues and phosphorylation may support this is not necessary, because oxidized DOPA can form cova-
adhesion through electrostatic interactions with charged surface lent bonds with a second DOPA molecule and, thus, acts as a
groups.[19a] cross-linker contributing to cohesion. The mussel thereby mas-
A hallmark characteristic of DOPA-chemistry is its depen- ters the balance between cohesion and adhesion, as too high
dence on redox conditions. The loss of one electron leads to levels of oxidation of DOPA lead to failure of surface adhesion,
DOPA-semiquinone, and further loss of a second one to DOPA- while too little oxidation-mediated cross-linking leads to failure
quinone, resulting in reduced surface adhesion.[31,37] According of cohesive forces.[40] At the same time, control of the oxida-
to Cooper, seawater with high oxygen content has a positive redox tion level enables controlled adhesion and detachment of the
potential and, therefore, a high affinity to accept electrons.[19a,38] mussel.
Moreover, spontaneous auto-oxidation of DOPA is promoted by The abundance of different interaction mechanisms of DOPA
alkaline conditions around pH 8, but can also be initiated by makes it a highly versatile system, allowing mussels to in-
chemical oxidants like periodate and catechol oxidase enzymes. teract with almost any type of surface, both hydrophobic and

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hydrophilic, and at both acidic and basic pH.[41] The mussel foot packed phase-separated fluid – a phenomenon known as complex
proteins and their DOPA-based adhesion mechanism, hence, coacervation – that consists of a polymer-enriched liquid phase
have inspired the design of a multitude of biomimetic adhesives. (the coacervate phase), and a polymer-depleted dilute phase (the
remaining solution).[20,49–51] If, as in case of the sandcastle worm,
two or more individual polyelectrolytes of opposite charge inter-
2.2. Coacervation in Sandcastle Worms act with each other, the process is called complex coacervation.
However, liquid-liquid phase separation may also occur with only
Another prominent example of wet adhesion derives from P. cal- one, amphipathic polymer, which is then referred to as simple
ifornica, commonly known as sandcastle worm. The worm, as coacervation. The components of the sandcastle worm cement
well as other relatives of the species of polychaetes, uses a pro- stay in the complex coacervated state, and components of homo-
teinaceous glue to assemble mineral particles and build tubular geneous and heterogeneous granules are strictly separated un-
shells, which in their entity form almost reef-like structures in til secretion from the gland. After release, the granules rupture,
the coastal region and provide the worms with protection from probably caused by mechanical stress, leading the contents to
predators as well as environmental stress caused by waves and fuse with little mixing and harden into a porous solid foam.[49]
desiccation.[42] Mineral particles like sand grains and calcareous The transition from the coacervated fluid phase into a solid is
shell fragments floating in the marine water are captured and promoted by small changes in the solution conditions, like ionic
transported by the animal with tentacles inwards to the so-called strength and the pH jumping from acidic in the packaging sys-
building organ, where a protein-based adhesive fluid is secreted tem to basic in the seawater.[43] At a seawater pH of 8.2, histidine
to join the bits, also termed cement.[43] Molecular analysis of the residues (pKa ≈6.5) would become deprotonated and, thus, result
proteins revealed that the sandcastle worm, like the mussel, ex- in a loss of positive charge, setting free their counter phosphate
ploits the post-translational modified DOPA.[44] More prominent groups. The phosphate groups in turn enter into new interac-
than the overall 2–3 mol% DOPA, the whole glue shows a high tions with the remaining positively charged Ca2+ and Mg2+ ions,
content of phosphorylated serine (pSer, ≈30 mol%), with indi- with the interactions being more similar to ionic bonds, resulting
vidual proteins even comprising >80% serine in their primary in spontaneous hardening of the glue.[43] Additionally, a catechol
sequence (Table 1).[42b,44–45] pSer is a second post-translational oxidase enzyme is also found in both homogeneous and hetero-
modification widely found in marine adhesive proteins and oc- geneous granules and catalyzes the oxidation of the DOPA-side
curs moreover in sequences, which have similarities to mineral- chains into DOPA-quinone, which are subsequently cross-linked
binding motifs in proteins of the bone extracellular matrix,[46] covalently during curing of the glue.[20] In summary, the coac-
suggesting an adaption to adhesion on calcareous surfaces.[30a,47] ervate processing provides an intermediate for the storage and
The consequence of the high phosphorylation level, which oc- delivery of aqueous underwater bioadhesives without dispersion,
curs predominantly in Pc3 proteins, is the anionic net charge. which is also used by mussels.[52] The state is accompanied by ex-
On the other hand, a closer look at the residual proteins Pc1, cellent wet coating and adhesive properties, caused by the low in-
Pc2, Pc4, and Pc5 reveals a polycationic character, due to the en- terfacial tension of the coacervate and good surface wetting ability
richment in arginine, lysine, and histidine.[48] Overall, ≈50% of in water.[53]
amino acids are charged at physiological pH, and the separation
of charges into macromolecules led to the theory that complex
coacervation – liquid-liquid phase separation of components of 2.3. Phosphorylated Serine in the Glue of Caddisfly Larvae
opposite charges – plays a role in the adhesion process of sand-
castle worms,[49] which is highlighted in more detail below. Caddisflies (order Trichoptera) spend most of their lifecycle in the
The gland tissue producing the glue components and storing larval stage. Caddisfly larvae live in freshwater habitats where
them in granular secretory vesicles is composed of two major cell they feed, mature, and pupate underwater.[30b] All suborders use
types: one type is producing homogeneous granules, the other adhesive silk for the construction of protective tube-like shelters,
heterogeneous granules, a designation that is due to the different like the sandcastle worm, and for food procurement.[22] Related
morphologies and staining behavior.[50] The result is a local sepa- to moths like the domesticated silkworm, homologies of the silk
ration of the glue components into two adhesive modules, which have been identified.[54] In particular, the silk fiber is made of a
constitutes an important feature of the adhesive system of sand- nanofibrous core comprising two major fibroins: the heavy chain
castle worms in addition to the high polyelectrolyte content.[49] fibroin (H-fibroin) and light chain fibroin (l-fibroin), which are
The oppositely charged macromolecules are stored as pairs: The most likely connected by disulfide bridges.[55] H-fibroin makes
cationic proteins Pc2 and 5 are combined with negatively charged up the largest proportion and consists of nonrepetitive N- and C-
sulfated polysaccharides in the homogeneous granules, while the termini flanking a long, conserved core region. Other than silk
heterogeneous granules comprise the polyphosphate proteins from silkworms or spiders, caddisfly silk is not abundant in ala-
Pc3B and polyampholytic proteins Pc3A along with the polyca- nine but enriched in serine, which is extensively phosphorylated
tionic proteins Pc1 and 4.[49] Additional high concentrations of (13 mol% pSer in H-fibroin).[56] Since phosphoserines are com-
Mg2+ ions in the heterogeneous granules are thought to neutral- monly found in the adhesive secretes from marine organisms
ize the negative charge of the phosphate groups.[49] In each of the like mussels, sandcastle worms, and sea cucumbers, this obvi-
granules, the electrostatic association of oppositely charged poly- ously represents an adaptation to the aquatic environment.[57]
electrolytes causes partial expulsion of water and small bound Repeats of (pSX)n E motifs (pS = phosphorylated serine, X =
counter ions driven by the associated gain in entropy (Figure hydrophobic or basic residue, n = 4 or 5, E = glutamic acid)
5). This leads to a concentration of the polymers into a densely and glycine-rich motifs conferring elasticity are located in the

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Figure 5. Schematic diagram of complex coacervation resulting from a two-step process, namely the charge neutralization of polyelectrolytes by attractive
interactions (such as electrostatic, van der Waals, hydrophobic interactions, and hydrogen bonding) and the gain in entropy by the delocalization and
release of bound counter ions. The result is macrophase separation yielding a concentrated polymer phase (the coacervate) and the equilibrium solution
(the diluted phase). Reproduced with permission.[51a] Copyright 2005, Royal Society of Chemistry.

central core region (Table 1).[22,58] Multiple studies support the thus, drives up fuel consumption, resulting in economic loss.[62]
proposed nanofibril core structure of Ca2+ -bridged phosphoser- For their adhesion, barnacles use a type of cement with ≈90%
ines, forming a rigid 𝛽-sheet-like crystalline region.[22,56,59] An- protein content,[63] and not all of the proteins have yet been fully
tiparallel 𝛽-hairpins are thought to be electrostatically stabilized characterized. No evidence has been found so far for the above-
by calcium ions, resulting in mechanical stiffness and tensile mentioned post-translational modifications (neither DOPA[63b,64]
strength of the silk fibers.[60] Furthermore, the silk fibers are nor pSer[65] ), but the proteins comprise lots of polar amino acids
coated with a layer of glycoproteins and peroxidase.[56] Initial sur- and a varying amount of cysteine (Cys) depending on the species.
face adhesion is mediated by the fuzzy glycoprotein layer via in- In addition to its primary protein composition, the barnacle ad-
termolecular interactions such as chain entanglement, electro- hesion mechanism differs from other marine organisms like the
static interactions, or hydrogen bonds, but then the peroxidase previously discussed mussel and sandcastle worm in its secre-
can catalyze di-tyrosine crosslinking to covalently connect tyro- tion process. A single cell type in the cement gland is responsible
sine residues from the peripheral fiber layer with phenolic groups for the production and packaging of glue components in secre-
from surface absorbed organic compounds and, thus, support tory granules.[66] Secretion is a slow process, and solidification
both cohesion and adhesion.[61] of the cement, which has been discussed to include enzymatic
crosslinking,[67] takes several hours reducing the risk of prema-
ture curing.[68] Since the current understanding of barnacle ad-
2.4. Amyloid-Like Proteins in Barnacle Adhesives hesion still includes controversial aspects such as the absence of
intermolecular covalent crosslinking,[19b,67] the question arises as
Barnacles as sessile adults exhibit a strong permanent attach- to the origin of the adhesive and cohesive force.
ment and form a protective calcified shell. They adhere strongly One component possibly involved could be highly insoluble
to any suitable underwater surface, which constitutes a severe amyloid-like protein fibrils,[63b] which were detected through spe-
problem in shipping. Barnacle settlement, also termed barnacle cific staining methods for their characteristic cross-𝛽 structures
fouling, significantly increases frictional resistance of ships and, (using the dyes Congo red and Thioflavin T). Moreover, atomic

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force microscopy imaging and spectroscopic methods confirmed ity, and strength. The flagelliform fibers are coated during spin-
nanofibrils and major 𝛽-sheet content.[69] Amyloid fibrils are ning with the aqueous glue solution secreted by framing aggre-
highly ordered, typically unbranched, and structurally consist gate glands, thus also called aggregate glue.[78] The aqueous ad-
of 𝛽-strands aligned perpendicular to the fiber axis, which self- hesive is secreted as a continuous coating of the fiber, absorbs
assemble from soluble proteins.[70] This particular type of pro- water, and breaks spontaneously into regularly spaced droplets.
tein assembly is associated in the human body with pathogenic When in contact with caught prey, the glue shows also viscoelas-
conditions like Alzheimer’s or Parkinson’s disease.[71] As a bio– tic solid properties, meaning that the droplets elongate in order
based material, amyloid fibrils are characterized, among oth- to maintain a physical connection to the substrate.[79]
ers, to possess high mechanical strength and stability against Chemical analysis revealed that the glue droplets consist of an
degradation.[69a] This specific structural fold is also effectively anchoring granule, surrounded by a glycoprotein-containing in-
used by other organisms for adhesion (e.g. prokaryotic curli ner layer and a more aqueous outer layer, which comprises low
fibers).[72] molecular weight compounds and salts.[80] The salts in the se-
Best studied is the cement of the acorn barnacle M. rosa, which creted droplets are hygroscopic and absorb water from the atmo-
consists of five different proteins.[19b] One of the examined pro- sphere and, thereby, support solvation of the glycoproteins via
teins, CP-20, which is thought to interact at the substrate inter- a salting-in effect.[80c,81] Consequently, spider silk glues are hu-
face alongside a second protein, is enriched in charged amino midity responsive: high humidity increases adhesive strength,
acids (42 mol%) as well as Cys (17 mol%) (Table 1). Using a re- which is probably an adaption related to the humid natural habi-
combinantly produced CP-20, So et al. proposed a dynamic inter- tat of spiders.[82] The inner layer consists of two extensively
action between the protein and calcite. They observed a disorder- O-glycosylated protein subunits, aggregate spider glue proteins
to-order transition of CP-20 upon contact with crystalline cal- (ASG) 1 and 2. Evidence was found that ASG2 belongs to the
cite, which could initiate fibril self-assembly.[73] Structural pro- spidroin family with its characteristic repetitive amino acid se-
tein characterization also revealed that 12 out of 32 Cys residues quence region (Table 1) flanked by conserved non-repetitive ter-
form intramolecular disulfide bonds to stabilize molecular fold- minal domains and contributes to cohesion of the glue. Due
ing. The remaining thiol groups are free to engage in other inter- to this finding the protein was re-named aggregate spidroin 1
molecular interactions as a functional group and might assist in (AgSp1).[21a,b] AGS1 displays an adhesive function, as the protein
the barnacle adhesive mechanism on various surfaces. includes regions similar to chitin-binding domains, revealing op-
timal prerequisites for insect attachment,[21a,81] but there are still
many unanswered questions regarding the exact adhesion mech-
2.5. Aggregate Silk from Orb-Weaving Spiders and Glowworms anism.
as Adhesives The bioluminescent larvae of certain fungus gnats, commonly
referred to as glowworms, have developed a similar strategy to
The web of orb-weaving spiders is essentially a trap for small catch prey.[21c] In their natural habitats, preferably dark and hu-
insects used for catching prey.[74] Orb-wavers (Araneoidea) rely mid places like caves, glowworms construct a tubular nest of silk
on the strength of their major ampullate silk fibers used for the and an adhesive mucus. Attracted by their luminous abdomen,
webs’ frame and radii, and the spiral architecture of the flagel- small insects are caught using long elastic silk threads hang-
liform silk (forming the capture spiral) to absorb the kinetic en- ing down from the nest. The glowworm silk reveals a crystalline
ergy from incoming insects, or, in other words, slow down or stop cross-𝛽-sheet structure akin to amyloids and different from the
the insect in mid-flight.[74] From a mechanical point of view, the antiparallel 𝛽-sheets in spider silk.[83] The adhesive mucus, in
major ampullate silk fibers represent a unique combination of contrast to spiders where the flagelliform fibers are continuously
high tensile strength and elasticity, caused by the primary protein coated, is placed in droplets selectively every 1–1.5 mm onto the
structure of its spidroins (MaSp1 and MaSp2). The proteins’ core fiber during fiber spinning. The glue droplets consist of ≈1% of
domains consist of highly repetitive motifs, of which poly-alanine hygroscopic macromolecules and 99% volatile substances and,
regions are forming crystalline antiparallel 𝛽-sheets and are re- therefore, are more sensitive to humidity, as relative humidity
sponsible for the high toughness, whereas less ordered glycine- of <80% leads to drying of the droplet and crystallization of the
rich sections contribute to the elasticity.[75] To hinder the prey components.[84] However, the mechanism behind the adhesive
from escaping the web, spiders originally produced dry cribellar property of the glowworm secretes still needs to be investigated.
capture threads, which were fixed in between major ampullate
silks on top of thick core fibers, featuring thousands of fine fib- 3. Bio-Inspired Protein-Based Adhesive Systems
rils on the surface and, thus, achieving adhesion more by phys-
ical entanglement paired with Van der Waals and hygroscopic Given the variety of available natural mechanisms for effective
interactions.[76] While this type of adhesion is still used by mem- dry and wet adhesion, a straightforward approach for the prepa-
bers of Deinopoidea, the modern orb-weaving spiders (>95% of ration of a bioadhesive would be the direct extraction of compo-
orb weavers) have transitioned over 135 million years ago to use nents from biological sources. Mussel foot proteins are a charac-
a liquid viscous glue placed onto the above-mentioned flagelli- teristic example, which has been extensively studied to date and
form silk, featuring chemical adhesion, hence yielding a greater exhibit strong adhesion ideally adapted to the aqueous environ-
stickiness relative to volume and increased extensibility at lower ment. However, direct retrieval of Mfps by isolation and purifi-
costs for the spider.[77] This co-called viscid silk is a composite ma- cation is very laborious with a low yield estimated at 1 g protein
terial of highly hydrated flagelliform silk and adhesive droplets, per 10 000 mussels, despite advanced processes.[92] Therefore,
leading to a combination of adhesiveness, extensibility, elastic- commercially available mussel adhesive protein (MAP)-based

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Figure 6. Overview of the described aspects of bio-inspired protein-based adhesives. Extracted natural proteins can be chemically or enzymatically
functionalized (top). Bio-inspired adhesive proteins can be designed akin to the natural sequence, chimeric/hybrid, or de novo. The proteins are then
produced in recombinant expression systems to either yield an already functional protein adhesive, or an intermediate, that can further undergo post-
translation modifications (PTM) to obtain those functions (bottom).

adhesives comprise both extracted natural and recombinantly enabling photo-induced crosslinking, all-protein-based hydro-
produced Mfps.[93] gels have been developed by Zhao et al. using enzymatic
Next to bioadhesives derived from natural sources, bio- crosslinking of intrinsic tyrosine residues.[100] Horse radish per-
inspired adhesives have clearly been on the rise in recent years. oxidase introduced covalent bonds and, thus, promoted the for-
The design of new materials using natural adhesive motifs and mation of hydrogels, while the addition of CaCl2 led to elec-
structures enables control and fine-tuning of material properties trostatic and chelation interactions of the silk fibroin with Ca2+
as desired. The vast amount of literature discussing MAP and the ions. The resulting hydrogel was reported to be conductive, self-
development of mussel-inspired proteins reflects the main focus adhesive, and stretchable leading to possible applications as wear-
on DOPA and catechol groups, as it has been suggested to be able electronic sensors.
the key factor for strong and rapid underwater adhesion not only As reflected in the previous description of the natural adhe-
in mussels. Research reports range from polydopamine materi- sive systems, the implementation of the non-canonical amino
als (e.g. coatings)[94] and DOPA-containing short polypeptides to acid DOPA into proteins is an attractive way to significantly in-
DOPA-functionalized polymers[95] and whole recombinant Mfps. crease surface adhesion. Chemical modification of proteins can
A selection of these and other protein-based adhesive systems be achieved using cross-linking between N-hydroxysuccinimide
is discussed in the following section, divided into functionalized (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt
natural proteins and bioengineered recombinant protein adhe- (EDC), reported in multiple works.[101–103] In this manner, DOPA-
sives (Figure 6). The adhesive strengths are typically not deter- functionalized silk fibroin was produced by Liu et al. and blended
mined using standardized procedures. Thus, a direct compari- with PEG to promote gelation into a hydrogel.[101] Adhesion stud-
son of the values should be treated with caution due to varying ies on porcine skin revealed a high tensile strength in dry and
test set-ups with variable parameters, like amount and concen- wet conditions, which increased within 42 h due to cross-linking
tration of applied adhesive, used substrate, contact area, curing of oxidized DOPA-quinone during curing up to 0.5 MPa, and
time and environment, and testing strain rate (Table 2). in vitro degradability of the glue was demonstrated using a pro-
tease. Another group improved the adhesive properties of its
chemically DOPA-modified silk fibroin and accelerated the gela-
3.1. Modified Proteins for Adhesives tion process through the addition of genipin and Fe3+ .[102] The
chelation of metal ions by catechol hydroxyl groups forming co-
Regenerated silk fibroin from B. mori is an FDA-approved pro- ordination bonds increased the adhesion using Fe3+ (248 kPa
tein, which is a well-suited biomaterial due to its biocompati- on porcine skin) more than using Cu2+ , which is explained by
bility, biodegradability, and mechanical properties. Silk fibroin, the ability of Fe3+ to coordinate with more hydroxyl groups. The
like other silk proteins, forms 𝛽-sheet-rich fibrils and com- other compound, genipin, acts as a cost-effective cross-linking
prises several amino acids with hydroxyl and acidic groups pro- agent. Both additives had a negligible negative impact on in
viding potential reaction sides for chemical modifications or vitro cell viability and had no effect at all on cell migration,
crosslinking.[100] Therefore, it has been used to some extent thus indicating good biocompatibility as well as degradability
for the development of several adhesive systems. Next to semi- of such adhesives, two important aspects for future biomedical
synthetic approaches like the methacrylation of silk fibroin[115] applications.

Adv. Funct. Mater. 2023, 2303609 2303609 (10 of 25) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH
 Table 2. Overview of reported adhesive systems with a focus on adhesive properties.

Adhesive system Method Test conditions Substrate Curing conditions adhesion strength/ Ref.
adhesion energy

Reference materials
Cyanoacrylate Lap-shear test ASTM F2255 (modified), 10 porcine skin 20 min, humid ≈0.075 MPa [96]

Adv. Funct. Mater. 2023, 2303609

× 10 mm2
Lap-shear test 10 mm min−1 or 40 mm steel 12 h, dry 10.4 ± 0.6 MPa [97]
www.advancedsciencenews.com

min−1 , 5 × 5 mm2
End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, dry 2.01 ± 0.58 MPa [98]
End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, humid 0.96 ± 0.24 MPa
Fibrin Lap-shear test ASTM F2255 (modified), 1×1 porcine skin 20 min – 24 h, humid ≈0.02 MPa [96]
cm2
End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, dry 0.54 ± 0.39 MPa [98]
End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, humid 0.43 ± 0.19 MPa

2303609 (11 of 25)

natural adhesives
Mussel extracts (M. edulis) End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, dry 0.33 ± 0.17 MPa [98]
End-to-end joint tensile test 10 mm min−1 , 35 mm2 porcine skin 24 h, humid 0.93 ± 0.32 MPa
Sandcastle worm cement (P. Estimation based on “pull - - - ≈0.35 MPa [99]
californica) out” set-up
Modified natural proteins
Silk fibroin + CaCl2 + horse radish T-peel test 50 mm min−1 , 10 × 20 mm2 porcine skin 12 h, 50% relative humidity (RH) 200 N m−1 [100]
peroxidase
DOPA-functionalized silk fibroin + Lap-shear test ASTM F2255, 10 × 10 mm2 porcine skin 30 h, dry 0.448 ± 0.017 MPa [101]
PEG Lap-shear test ASTM F2255, 10 × 10 mm2 porcine skin 42 h, wet 0.503 ± 0.017 MPa
DOPA-functionalized silk fibroin + Lap-shear test ASTM F2255, 1 mm min−1 , porcine skin 12 h, dry, 37 °C 0.248 ± 0.029 MPa [102]
genipin + Fe3+ 10 × 10 cm2
DOPA-functionalized pectin + Lap-shear test 50 mm min−1 , 8 × 8 mm2 porcine skin 12 h, dry ≈1.48 MPa [103]
𝛽-lactoglobulin coacervate Lap-shear test 50 mm min−1 , 8 × 8 mm2 glass 12 h, dry ≈0.4 MPa
Lap-shear test 50 mm min−1 , 8 × 8 mm2 porcine skin 12 h, wet ≈0.78 MPa
Tensile test 5 mm min−1 , 8 × 8 mm2 porcine skin 12 h, dry ≈0.72 MPa
Tensile test 5 mm min−1 , 8 × 8 mm2 glass 12 h, dry ≈0.54 MPa
T-peel test 5 mm min−1 porcine skin 12 h, dry 880 J m− 2
Silk fibroin + tannic acid Lap-shear test ASTM F2255 (modified), 1 × porcine skin 20 min, wet 0.1341 ± 0.0052 MPa [96]
1 cm2
T-peel test ASTM F2255 (modified) porcine skin 20 min, wet ≈50 N m−1
Recombinant proteins
(Continued)

© 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH

www.afm-journal.de

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 Table 2. (Continued).

Adhesive system Method Test conditions Substrate Curing conditions adhesion strength/ Ref.
adhesion energy

recombinant MAP fp-151 Lap-shear test ASTM D1002 (modified), 10 aluminum 3 h, dry, 37 °C 0.8 ± 0.2 MPa [104]
× 10 mm2
co-expressed fp-151 + tyrosinase Lap-shear test ASTM D1002 (modified), 10 aluminum 3 h, dry, 37 °C 3.01 ± 0.62 MPa
× 10 mm2

Adv. Funct. Mater. 2023, 2303609

fp-151 + hyaluronic acid coacervate Lap-shear test 1 mm min−1 , 10 × 10 mm2 porcine skin 24 h, 37 °C, humid 0.0572 ± 0.0097 MPa [105]
recombinant barnacle cement Atomic force microscopy contact time 2 s, 1 μm s−1 mica - ≈3 mN m−1 ; 0.7 mJ m−2 [106]
protein rBalcp19k (colloidal probe)
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DOPA-modified recombinant Lap-shear test 10 mm min−1 , 50 × 10 mm2 aluminum 24 h, dry ≈1.38 – 1.58 MPa [107]
spider silk proteins Lap-shear test 10 mm min−1 , 50 × 10 mm2 pig bone 24 h, dry 1.2 to 2.1 MPa
Spider silk-MAP fusion protein Surface force apparatus - mica wet 55 mN m−1 ; 12 mJ m− 2 [108]
Amyloid-silk-MAP fusion protein Lap-shear test 2 mm min−1 , 10 × 10 mm2 porcine skin 18 h, 37 °C, wet 0.078 ± 0.011 MPa [109]
Lap-shear test 2 mm min−1 , 10 × 10 mm2 glass 18 h, 37 °C, wet 1.0 ± 0.3 MPa
Lap-shear test 2 mm min−1 , 10 × 10 mm2 glass 18 h 37 °C wet, then FeCl3 ≈0.3 MPa
solution or buffer pH 11 for 8 h

2303609 (12 of 25)

Lap-shear test 2 mm min−1 , 10 × 10 mm2 aluminum 18 h 37 °C wet, then FeCl3 0.25 ± 0.158 MPa
solution or buffer pH 11 for 8 h
Spider silk-cellulose binding Lap-shear test 5 μm s−1 , 10 × 10 mm2 bacterial cellulose 10 min, 50% RH 75 N cm−2 [110]
domain fusion protein
de novo repetitive peptides/sodium Lap-shear test 10 mm min−1 or 40 mm glass, steel 12 h, dry 11.0 to 14.0 MPa [111]
dodecylbenzene sulfonate min−1 , 5 × 5 mm2 aluminum
(SDBS) coacervate Lap-shear test 10 mm min−1 or 40 mm glass, steel 12 h dry, then 1 h wet 0.33 MPa, 0.49 MPa
min−1 , 5 × 5 mm2
de novo repetitive Lap-shear test 10 mm min−1 or 40 mm steel 12 h, dry 13.5 ± 1.6 MPa [97]
peptides/negatively charged min−1 , 5 × 5 mm2
DOPA-surfactant + Fe3+
de novo repetitive Lap-shear test 10 mm min−1 or 40 mm steel 12 h, dry ≈16 MPa
peptides/negatively charged min−1 , 5 × 5 mm2
azobenzene-surfactant
de novo repetitive Lap-shear test 10 mm min−1 or 40 mm steel, glass 30 min dry, then wet overnight ≈0.4 ± 0.07 MPa, ≈0.36
peptides/negatively charged min−1 , 5 × 5 mm2 ± 0.08 MPa
DOPA-surfactant
de novo repetitive peptides/DNA Lap-shear test 10 mm min−1 glass, steel, Cu, 10 h, dry 15.4 to 21.3 MPa [112]
coacervates ceramics
Lap-shear test 50 mm min−1 porcine skin 10 h, dry 172.2 ± 13.0 J m-2
de novo repetitive peptides/crown Lap-shear test 10 mm min−1 , 3 × 5 mm2 steel 5 days, 33 °C, dry 22.3 ± 2.1 MPa [113]
ether coacervates
de novo repetitive peptides/SDBS Lap-shear test 10 mm min−1 , 3 × 5 mm2 glass, steel, 4 days, dry 13.0 to 20.0 MPa [114]
coacervate + gold nanorods aluminum
Lap-shear test 10 mm min−1 , 3.5 × 9 mm2 porcine skin 1 h, dry 0.015 ± 0.0025 MPa
Lap-shear test 10 mm min−1 , 3.5 × 9 mm2 porcine skin 808 nm irradiation, 30 s intervals ≈0.065 MPa
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Figure 7. Bio-inspired adhesive comprising catechol-modified silk fibroin. A) Molecular dynamics simulations support the hypothesis of the impact of
tannic acid (red) on the conformational transition of the silk protein (green) into a ß-sheet-rich fold. B) Schematic representation of the hierarchically
assembled nanofibrillar structure, responsible for the cohesion properties. C) Proposed schematic adhesion mechanism on tissue through binding to
nucleophilic surface molecules. Reproduced (Adapted) with permission.[96] Copyright 2016, Royal Society of Chemistry.

Even more powerful adhesion can be obtained by combining TA and hydroxyl or amino groups of the protein upon mixing,
two or more adhesion mechanisms. In a recent study, Bashir et al. followed by 𝜋–𝜋-stacking interactions as the folding proceeds.
reported a glue with wet adhesion based on chemical DOPA mod- This co-assembly of silk and TA led to spontaneous gelation,
ification of one component and cohesion promoted by liquid- or in other words, to the formation of a nanofibrillar structure,
liquid phase separation.[103] The use of 𝛽-lactoglobulin and op- which resulted in improved toughness (Figure 7B). Wet adhesion
positely charged DOPA-functionalized pectin led to formation (in water and blood) was confirmed on substrates like porcine
of a coacervate, which was assumed injectable through a sy- skin and exceeded the adhesion strength of other clinically used
ringe without clogging due to shear-thinning behavior. Adhesive adhesives like cyanoacrylate or fibrin glue. The tannic acid was
strength was determined to a maximum of 1.5 MPa in lap-shear explained to strongly interact with nucleophilic molecules dis-
tests using porcine skin, and after 1 min an adhesion of already played on the tissue surface (Figure 7C). Sealing effects and good
0.27 MPa was measured. The rise in adhesive strength within cytocompatibility were demonstrated in vivo in rat model hearts
several hours is a generally commonly observed phenomenon, and intestines, and the silk glue showed hemostatic ability as well
which could be attributed to the slow curing process via DOPA- as antibacterial effects. Furthermore, 45 days after application,
quinone oxidation and crosslinking. However, a rapid increase the glue was degraded to 80% and replaced by the rat’s own cells.
in adhesive strength up to 134 kPa within 20 min has been ob-
served by Bai et al.[96] Instead of DOPA, the group reached back to
tannic acid (TA), a natural polyphenol that can be extracted from 3.2. Recombinant Adhesive Proteins
plants. TA contains five catechol groups and has a high affinity to
nucleophiles (like amines and thiols), thus interacting with the 3.2.1. Natural-Like Designed Adhesive Proteins
amino acids of silk fibroin. The addition of TA to an aqueous silk
fibroin solution induced the conformational transition from ini- With the technology of genetic engineering and protein design,
tially mostly random coil to mainly 𝛽-sheet (Figure 7A). The ex- new bioadhesives can be generated in high purity and consis-
perimental observations were supported by molecular dynamics tent quality along with good manufacturing practice (GMP) stan-
simulations, which suggested strong hydrogen bonds between dards, ensuring also biological safety for in vivo applications. On

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the basis of natural Mfps, a multitude of recombinant MAPs were sion, proposing a more robust adhesion mechanism than the
created, like the recombinant hybrid MAP called fp-151, which DOPA-based one. Li et al. demonstrated recombinant spider ag-
combines the sequences of Mfp-1 and Mfp-5 (Figure 8).[116] This gregate glue proteins (AgSp1), which allowed biomimetic wet
particular recombinant protein is a good example of the mani- spinning into fibers. Structure and mechanical properties were
fold possibilities related to genetic engineering. Starting with a analyzed, but no data on adhesive properties were shown.[122]
recombinant natural-like Mfp-5, difficulties such as low yields Some recombinant spider silk materials possess antimicrobial or
and low solubility in aqueous buffers became apparent along microbe-repellent properties, and are biocompatible, degradable,
with protein production in E. coli and purification.[116] These lim- and non-immunogenic, thus representing a predestined mate-
itations were overcome by design of the hybrid protein, fusing rial for biomedical applications.[123] Further, some silks display
Mfp-5 with Mfp-1-derived decapeptide repeats at the termini. intrinsic adhesive properties.[123–124] Two recombinant spider silk
The strategy for bacterial expression and purification was more- proteins were demonstrated recently by Bogush et al. to possess
over optimized toward biosafety, obtaining an endotoxin-free and good wettability and adhesive strength on organic and inorganic
safe product.[117] One remaining challenge was the incorpora- substrates.[107] Differences in breaking shear stress between both
tion of the non-canonical amino acid DOPA into recombinant proteins were attributed to the different proline content, as pro-
MAP, due to the expression host lacking the post-translational line with its pyrrolidine ring introduces steric constraints into the
modification system of mussels. It can be achieved either by protein backbone, disturbs the crystalline 𝛽-sheet regions and in-
in vitro modification of tyrosine residues using tyrosinase enzy- duces 𝛽-turns, which generally contributes to a higher elasticity
matic treatment after or during protein purification, or in vivo of silk.[125] Adhesion was furthermore observed during lap-shear
during protein translation. Through co-expression of fp-151 with experiments to be higher in a wet environment, and breaking of
a recombinant tyrosinase in E. coli for in vivo DOPA modifi- the adhesive bond was caused by cohesive failure of the material.
cation, the group of Cha detected a 4-fold increased adhesive The group then compared these results to the performance of
strength (3 MPa) compared to that of the same protein modi- their proteins after in vitro DOPA modification using a recom-
fied in vitro (0.8 MPa).[104] This was explained by increased pro- binant tyrosinase. The enzymatic tyrosine-to-DOPA conversion
tein solubility and better exposure of tyrosine residues. However, with a maximum yield of 57% of total tyrosine residues was not
this pathway represents an additional effort compared to conven- complete, but could not be further increased. Nevertheless, it led
tional modifications by commercially available mushroom tyrosi- to an increase in the protein adhesion capacity by 26–75%.
nase and produced mainly the oxidized DOPA-quinone. A sim-
pler in vivo modification method uses the endogenous tyrosyl-
tRNA synthetase (TyrRS) of the expression host. The strategy is 3.2.2. Chimeric/Hybrid Fusion Adhesive Proteins
based on the binding affinity for DOPA being competitive with
that for tyrosine. Supplemented DOPA is loaded to the tRNA The intermediate level of tyrosine residues in for example spi-
intended for tyrosine as soon as no tyrosine is available in re- der silk (3–4%)[107] can be increased by genetic engineering for
spective auxotrophic cells and is residue-specifically incorporated DOPA-based adhesion. Aich et al. designed a fusion protein be-
with a detected yield of >90% of tyrosine residues.[118] To fur- tween a mussel foot protein motif (Mfp-3) and a spider dragline
ther improve cell-interaction with the bioadhesive, a version of silk protein (Figure 8).[108] After recombinant expression, the fu-
fp-151 with the cellular recognition motif RGD was generated sion protein’s tyrosine residues were in part enzymatically con-
and demonstrated to be suitable for tissue engineering.[119] A verted into DOPA by tyrosinase during the purification process
more recent study describes a complex coacervate glue using the yielding 20% of conversion. More precisely, modification was per-
cationic fp-151 and negatively charged hyaluronic acid, a com- formed on the resin-bound Hexa-histidine tagged protein during
ponent of the extracellular matrix.[105] After in vitro DOPA mod- immobilized metal ion affinity chromatography (IMAC), simpli-
ification, the coacervate was loaded with two drugs, epidermal fying the subsequent removal of the enzyme. Structural analysis
growth factor, and allantoin, and was applied through a syringe of the fusion protein showed the ability to self-assemble into fib-
to fix autologous skin grafts in place. The drug-loaded coacervate rils, leading to the suggestion that the silk portion determines
exhibited a wet adhesion strength of 57 kPa and, compared to fib- the protein structure, and fibril assembly was not hindered by
rin glue (25 kPa) and black silk suture threads, promoted faster the additional MAP sequence. This might be advantageous for
in vivo wound regeneration with less scaring and inflammatory an adhesive since reduced cohesion and adhesion stresses have
response, caused by the steady release of the drugs. been suggested in highly flexible self-assembled fibrillar struc-
Several other recombinant adhesive proteins, which are tures, caused by a more effective dissipation of energy across the
natural-like by design, have been reported to date. For a com- protein molecules. Moreover, the fibrillar assembly enlarges the
prehensive overview of recombinant Mfps, we would like to re- surface area of the material, enabling a higher degree of adhe-
fer to Wang and Scheibel (2018).[120] Nevertheless, a recombi- sive interactions in contact with the substrate.[13] The MAP por-
nant Mfp-6-based protein is worth noting, as it is able to res- tion, in turn, contributes significantly to wet surface adhesion,
cue DOPA activity after oxidation to DOPA-quinone.[121] Consid- illustrated by the detected 3-fold higher surface adhesion energy
ering other natural-like engineered proteins besides the mussel of the fusion protein compared to the pure silk without DOPA
as a model, a recombinant barnacle cement protein rBalcp19k modification (Figure 9).
was reported, which self-assembled into nanofibers at acidic and The benefit of a nanofibrillar structure is also evident when
low-ionic conditions.[106] Fibrillar structures were highly stable analyzing amyloid-like proteins. A silk-amyloid-Mfp hybrid pro-
and showed a lower adhesion capacity than a commercial DOPA- tein was generated by Kim et al. by alternatingly combining
based adhesive, but also less pH-dependent decline in adhe- sequences from a domain of the A𝛽-amyloid protein, which

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Figure 8. Recombinant engineering enables the combination of different natural motifs or domains (upper part) into new adhesive proteins (lower part).

tends to self-assemble into crystalline 𝛽-sheets, with the flexible by the amyloid-silk moiety, as well as a strong underwater adhe-
glycine-rich region of a MaSp1 spider silk protein.[109] A mus- sion on various substrates (78 kPa on porcine skin, 1 MPa on
sel foot protein (Mfp-5) was added at the C-terminus of the 8- glass). Oxidation of DOPA induced by a basic pH or the addi-
fold repetitive sequence (Figure 8). The group used a tyrosine- tion of FeCl3 solution for catechol-Fe3+ chelation was observed to
auxotrophic E. coli strain for protein expression and added DOPA reduce the wet adhesive interactions significantly and was pro-
to the culture medium, resulting in the in vivo incorporation of posed to be used for controllable debonding of the adhesive.
DOPA instead of tyrosine residues with 79–86% efficiency. Hy- Despite E. coli being the working horse of recombinant pro-
drogels made of the hybrid protein exhibited a high stretchability tein production, due to drawbacks of bacterial expression sys-
(up to three times the length), indicating good cohesion caused tems like protein aggregation or misfolding when reaching

Figure 9. Proposed adhesive and cohesive interactions of hybrids combining structural proteins, like amyloids or spidroins, with DOPA-containing
proteins. Adhesion results from bidentate hydrogen bonding with metal oxide surfaces as well as 𝜋–𝜋-stacking on plastic and covalent bonds via
Michael-type addition with lysines or cysteines within a tissue. Cohesive interactions include 1) intermolecular hydrogen bonds, 2) interactions between
parallel ß-sheets, 3) 𝜋–𝜋-stacking, 4) bidentate hydrogen bonding, 5) cation-𝜋 interactions, and 6) covalent bonds between two DOPA groups via aryloxyl
radicalization. Reproduced (Adapted) with permission.[109] Copyright 2021, American Chemical Society.

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high expression levels and the limitations in post-translational mini of proteins, allowing complexation with gold nanorods via
modifications,[126] first efforts were made to switch to eukary- gold-sulfur bonds. Because gold nanorods strongly absorb near-
otic expression hosts for new bio-inspired adhesives.[127] Two infrared light, the resulting heating of the patch upon laser ir-
chimeric Mfp-amyloid proteins combining Mfp-3 with amyloid- radiation leads to protein denaturation, DNA breakage, and ul-
forming gas vesicle protein A (GvpA) from a cyanobacterium, as timately death of adjacent cancer cells, demonstrated in vivo by
well as Mfp-5 with the E coli curli protein CsgA, were produced inhibition of tumor growth paired with improved wound heal-
in the yeast P. pastoris.[127b] Adhesion in a wet environment was ing and skin regeneration. The adhesion of the coacervate was
improved in both cases upon addition of the amyloid moiety. not affected by the modification; in fact, adhesion strength in-
Lastly, spontaneous simple coacervation was the inspiration of creased with longer curing time and after short laser irradi-
choice for the design of a recombinant hybrid protein, compris- ation, probably caused by the photothermal effect leading to
ing a spider silk repetitive core domain engineered with terminal accelerated evaporation of water and therefore strengthening
cellulose binding domains.[110] The block-architecture of intrin- molecular interactions. Although the de novo design of protein-
sically disordered silk domains and the folded, thermally stable based adhesives is still in its infancy, this strategy represents
terminal domains led to self-coacervation at high concentrations a promising approach for the future to generate tailor-made
and low ionic strength. The coacervate displayed increased ad- biomaterials.
hesion on several cellulose-based materials like paper, bacterial
cellulose, cotton, and wood.
4. Activatable Adhesives
3.2.3. De Novo Designed Adhesive Proteins Extending the application of an adhesive toward usage inside a
human body comes with several challenges. While with exter-
The emerging field of de novo protein design has also reached nal wounds, depending on the site of application, an adhesive
biomaterials research, reflected in the recently presented de has to withstand motion-induced shear forces, the treatment of
novo protein adhesives inspired by coacervation.[111–112] Imitat- internal wounds additionally presents a dynamic, wet environ-
ing a very short elastin-like polypeptide sequence, positively ment often accompanied by the presence of blood. This can re-
charged repetitive proteins with various lengths were constructed sult in undesired pre-mature polymerization or reactions limit-
((VPGXG)n , X = cationic amino acid). Mixing with salmon ing the adhesive strength. Further, rearrangement of misplaced
sperm DNA or sodium dodecylbenzene sulfonate (SDBS) as adhesive patches is practically only possible with activatable sur-
negative counterparts yielded complex coacervates with a high faces or adhesives.[128] The following discusses some of the cur-
lap-shear strength (up to 21.3 MPa, dry conditions) compara- rently studied activatable wound sealing and adhesive systems to
ble to cyanoacrylate superglues, depending on polymer length overcome these challenges (Table 3).
and type of positive charge. Stronger adhesion was detected
in case of proteins with a higher number of repetitions, and
when the positively charged amino acid was arginine instead 4.1. Photo-Activation to Induce Adhesion
of lysine, stronger cation-𝜋 interactions were gained. In addi-
tion, the coacervate including the DNA exhibited a more ro- The curing of polymer adhesives is an important aspect toward
bust interfacial adhesion, probably due to 𝜋–𝜋, cation-𝜋, and hy- a triggerable mode of action and can be defined as the induced
drogen bonding provided by the nucleobases. Adhesive proper- polymerization using a catalytic or co-reactive agent, which is also
ties could even be more increased by the combination of the termed hardening.[142] A widely applied mechanism for adhesion
repetitive proteins with other negatively charged molecules like activation described in literature is photo-curing with or without
crown-ether, which introduce additional host-guest interactions the use of photo-initiators due to convenient and efficient spa-
through their ring-like structure and result in a strong adhesive tiotemporal activation control.[143] Methacrylic-group functional-
withstanding also extreme temperature conditions.[113] More- ized polymers account for the most prominent examples found in
over, as negative counterparts synthetic surfactants containing literature. However, among protein-based adhesives, the canoni-
DOPA or aromatic-rich azobenzene moieties were tested, also cal amino acid tyrosine has a particularly intriguing potential as
resulting in high adhesion strength due to additional DOPA- it can be either used to introduce catechol groups through enzy-
mediated or 𝜋–𝜋 stacking and cation-𝜋 interactions.[97] Com- matic hydroxylation or used in its natural state to form di-tyrosine
bining two natural adhesion motifs, the DOPA-containing coac- bonds through photo-oxidative reactions. Especially the use of
ervate adhesion could be reinforced by the chelation of metal white-light photo-initiator systems employing ruthenium com-
ions through hydroxyl groups and was found also functioning plexes in combination with an electron acceptor such as sodium
in wet conditions. The coacervate glues were found biocom- persulfate presents a promising strategy to form tyrosyl radicals,
patible (90% cell viability after 24 h), biodegradable, antibacte- which then react with other proximate tyrosine residues of either
rial, and showed reduced inflammatory responses.[112] There- the adhesive protein or the ones presented by the tissue surface,
fore, it was suggested suitable for biomedical applications in- hence, increasing the adhesive strength.[144] The use of visible-
cluding wound closure devices and tissue regeneration set-ups. light curable systems circumvents issues concerning secondary
One proposed possible application was demonstrated to be pho- damage through UV-light traditionally used in photo-activatable
tothermal therapy for skin tumors like melanoma, enabling a materials. Recombinant MAPs make a substantial contribution
non-invasive strategy with an adhesive bioplaster.[114] The adhe- in this particular niche, as they contain a high mole percentage
sive coacervate was designed with cysteine residues at the ter- of tyrosine residues.

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Table 3. Selection of activatable protein and protein-based adhesives.

Mechanism Composition Activation time Test method Substrate Adhesion strength/ Proposed application in vivo Ref.
adhesion energy studies

Light
radical polymerization AlgMA + GelMA 4 min UV, 30 Tensile test, 10 porcine ≈0.09 MPa (wet) sealant No [9]
min CaCl2 mm min−1 ureter
dityrosine cross-linking, recombinant 1 min Lap-shear-test, porcine skin 0.048 ± 0.01 MPa various (e.g. brain, liver, Yes [129]
photo-oxidation via MAP 100 mm2 , (wet) breast)
Ru(II)(bpy2+ )3 ; 𝜆 = 452 (“LAMBA”) 5mm min−1
nm (10kN)
dityrosine cross-linking, MAP-Substance 1 min Lap-shear-test porcine skin 0.042 ± 0.001 MPa hemostatic, peripheral Yes [130]
photo-oxidation via P fusion (wet) nerve injuries
Ru(II)(bpy2+ )3 ; 𝜆 = 460 protein
nm
dityrosine cross-linking, recombinant 1 min Lap-shear-test, porcine 0.028 ± 0.002 MPa tissue grafting (e.g. Yes [131]
photo-oxidation via MAP 100 mm2 , 1.2 scleral (wet) amniotic membrane
Ru(II)(bpy2+ )3 ; 𝜆 = 460 (“FixLight”) mm min−1 tissue transplantation)
nm (10kN)
cleavage of restrictive recombinant 45 min Atomic force mica - prove of principle No [132]
ortho-nitrobenzyl (ONB) MAP microscopy
group; 𝜆 = 365 nm containing
non-canonical
ONB-DOPA
radical polymerization poly(glycerol 0.5 min Pull-off porcine ≈ 2 N cm−2 (wet) hemostatic, treatment Yes [128]
sebacate adhesion test epicardial of e.g. ventricular and
acrylate) (90°), 8 mm tissue vascular defects
(PGSA) min−1
radical polymerization GelMA + 4 min Lap-shear-test, glass 0.093 ± 0.009 MPa hemostatic, treatment Yes [133]
hemocoagulase 1 mm/ min (dry) of strong bleeding
4 min End-to-end joint porcine skin 0.035 ± 0.003 MPa wounds
tensile test, (wet)
300 mm2 , 1
mm/ min
Temperature
thermal phase-transition, DOPA-modified 24 h at 37 °C Lap-shear-test, aluminum 0.24 ± 0.09 MPa not specified No [134]
coacervation elastin-like 2 mm min−1 (wet)
protein (2kN)
(ELY16 )
thermal phase-transition, elastin-like 30 min at 37 Lap-shear-test, glass/ PVC 0.5 – 0.6 MPa (wet) not specified No [135]
coacervation protein + °C/ 4 °C 25mm2 , 10
sodium mm min−1
dodecyl 30 min at 37 Lap-shear-test, porcine skin ≈0.008 MPa (wet)
benzene °C/ 4 °C 84 mm2 , 50,
sulfonate and 200 mm
(SDBS) min−1
thermal phase-transition, MAP-PNIPAM 5 min at 37 °C Pull-off porcine skin ≈0.006 MPa (wet) adipose tissue Yes [136]
coacervation conjugate + adhesion test
decellularized (90°), 0.3
adipose tissue mm min−1
dynamic Schiff base bonds Gelatine and 20 min at 37 Lap-shear-test, porcine skin ≈0.03 MPa (wet) hemostatic, various Yes [137]
chondroitin °C/ 20 °C 100 mm2 , 1
sulfate mm min−1
aldehyde +
borax
(Continued)

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Table 3. (Continued).

Mechanism Composition Activation time Test method Substrate Adhesion strength/ Proposed application in vivo Ref.
adhesion energy studies

pH
controlled ß-sheet formation recombinant pH 5.5 to pH Atomic force silica 2.64 ± 0.15 mJ m−2 prove of principle No [138]
via depsi switch defects MAP/ mfp 6.8 microscopy (wet)
controlled oxidation by dopamine pH 3 to pH 9 Atomic force borosilicate ≈2 J m−2 (pH 3) prove of principle [139]
network-bound boronic methacry- microscopy glass (wet)
acid lamide and
3-acrylamido
phenylboronic
acid
Chemical
induced oxidation hyaluronic acid instant Lap-shear-test, glass ≈0.04 MPa (dry) gastric tissue Yes [140]
prepolymers 6.25 mm2 , 1
with either mm min−1
catechol or instant Lap-shear-test, glass ≈0.015 MPa (wet)
NCSN group 6.25 mm2 , 1
mm min−1
Contact
absorbance of interfacial PAAc-NHS ester 5s Tensile test, various 0.02 – 0.12 MPa skin, small intestine, Yes [141]
water cross-linked 6.25 mm2 , 50 tissues (wet) stomach, muscle,
with GelMA + mm min−1 heart, and liver
gelatine (2.5 kN)
5s Pull-off various 190 – 700 J m−2 skin, small intestine,
adhesion test tissues (wet) stomach, muscle,
(90°/180°), heart, and liver
50 mm min−1

Jeon et al. described a light-activated, mussel protein-based and showed a wet adhesive strength of ≈28 kPa comparable
bioadhesive (LAMBA) (Figure 10A), which could be cured within to common fibrin glues.[131] Further underlining the utility of
60 s and exhibited wet-adhesive strength higher than that of com- recombinant protein-based adhesives, an intriguing study by
mon fibrin-glues (≈50 kPa). LAMBA further showed rapid re- Hauf et al. describes the development of a photocaged DOPA-
epithelialization and significantly faster reduction in wound ar- derivative termed ortho-nitrobenzyl DOPA (ONB-DOPA), which
eas in rats compared to cyanoacrylate-based glues.[129] More re- could be incorporated into MAPs through an engineered M. jan-
cently, Cheong et al. extended this approach and described the naschii tyrosyl-tRNA synthetase (MjTyrRS).[132] Using engineered
development of a MAP-based hydrogel, activated by visible-light aminoacyl-tRNA synthetases (aaRS), site-specific incorporation
and genetically fused with the neurotransmitter Substance P of non-canonical amino acids such as ONB-DOPA can be per-
(MAP-SP) for sutureless neurorrhaphy (Figure 10B). SP was rea- formed in living cells with higher specificity and yields. The ONB
soned to mediate polarization of macrophage subtypes, which are group is then cleaved upon irradiation (𝜆 = 365 nm) allowing
involved in the transition between the inflammatory response for spatiotemporal control of the adhesive catechol side chains
and repair/remodeling phase of nerves. While neurorrhaphy (Figure 10D). However, this method only achieved >50% depro-
with sutures can inflict traumatic damage and severe inflamma- tection upon irradiation for 45 minutes, making it rather unprac-
tion around the injured nerve tissues, MAP-SP exhibited good tical for surgical use at this stage.
wet-adhesion and induced genotypic and phenotypic character- While purely natural adhesives exhibit limitations in adhe-
istic M2 macrophage polarization promoting functional nerve sive strength and toughness, and purely synthetic adhesives
regeneration.[130] Maeng et al. developed a MAP-based adhesive raise concerns regarding toxic degradation products, attention
termed FixLight with proposed application for effective suture- has been directed toward semisynthetic multifunctional sys-
less amniotic membrane transplantation (AMT) (Figure 10C). tems. For example, a hybrid system combining methacrylate-
The amniotic membrane is a tissue derived from the placenta modified alginate (AlgMA) and gelatin-methacryloyl (GelMA)
and has attracted attention as a promising material for use in has been developed by Tavafoghi et al., which could be ap-
tissue regeneration since it modulates adult wound healing by plied to a wound and cross-linked by two mechanisms. The
suppressing stromal inflammation, angiogenesis, and scarring first cross-linking occurred under visible-light by the reaction
while promoting epithelialization.[145] However, this approach of methacrylate and methacryloyl-groups followed by metal ion-
still requires suturing to attach the membrane. In this regard, induced physical cross-linking of mannuronic- and guluronic-
FixLight could be used to rapidly perform AMT in rabbit con- acid blocks present in the alginate compound. While the chemi-
junctiva defect models compared to time-consuming suturing cally cross-linked MA-groups of GelMA and AlgMA maintained

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Figure 10. Examples of photo-activatable protein-based adhesive systems and their advantages in surgery. A) Di-tyrosine crosslinking can be in-
duced by UV–vis light irradiation in tyrosine-rich proteins such as MAPs. This approach resembles the mechanism found in insects. Reproduced with
permission.[129] Copyright 2015, Elsevier. B) Incorporation of small molecules such as neurotransmitters into bio-inspired protein-glue systems enables
rapid and sutureless neurorrhaphy of sensible tissue such as nerves, in which macrophage-polarizing properties improve regeneration. Reproduced with
permission.[130] Copyright 2022, Elsevier. C) Rapid light-induced polymerization allows relatively easy and quick transplantation of regenerative tissue
grafts, for instance, the amniotic membrane, to otherwise difficult-to-operate organs such as the eye. Reproduced with permission.[131] Copyright 2021,
Wiley–VCH GmbH. D) A more elaborate route for photo-activation is the use of protective groups such as photocaged ortho-nitrobenzyl (ONB)-DOPA,
a non-canonical amino acid that is introduced into the amino acid sequence upon recombinant production using engineered tRNA synthetases. The
DOPA group is deprotected upon exposure to UV light. Reproduced with permission.[132] Copyright 2017, Wiley–VCH GmbH.

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structural integrity, the physical crosslinking provided a polymer using formulations with high gelatin content and CS with a high
network that dissipated energy under stress.[9] However, the re- degree of oxidation. Remarkably, a misplaced adhesive gel could
activity with blood was not shown in this study and is a consid- be detached by simply applying cold water and the position
erable draw back. To encounter this issue, materials with hemo- readjusted in vivo, further also showing hemostatic properties
static properties are of great interest. For example, Lang et al. de- underlining the utility of such systems.
scribe a hydrophobic UV-light-activated adhesive (HLAA) com-
posed of poly(glycerol sebacate acrylate) (PGSA) tested in small
and large animal models. The hydrophobic character prevented 4.3. pH-Activation to Induce Adhesion
washing-off and retained its adhesive strength even after pro-
longed contact with blood. Therefore, the adhesive was able to Although mostly underrepresented among protein-based sys-
effectively seal left ventricular wall defects in rats for as long as tems, possibly due to the sensitivity of these to pH in regard to
180 days.[128] Taking photo-activatable, hemostatic systems to an- protein structure and solubility, pH-induced adhesion has also
other level, a study presented by Guo et al. used hemocoagulase been described in literature for highly specific applications such
derived from snake venom together with GelMA to develop a ma- as the treatment of gastric wounds.[149] The pH could be used to
terial that could rapidly seal bleeding wounds within seconds of alter the cohesive forces in recombinant MAPs as demonstrated
application and subsequent curing for the potential treatment of by Arias and colleagues, showing a significant change in adhe-
heavily bleeding wounds.[133] sive energy when switching from pH 5.5 to pH 6.8. This was
hypothesized to relate to suppressed ß-sheet formation, which
was then facilitated by O→N-acyl transfer rearrangement tak-
4.2. Temperature-Activation to Induce Adhesion ing place when shifting toward a more neutral pH.[138] Further-
more, Krogsgaard et al. for instance utilized the cationic nature
Another accessible path for adhesion activation is temperature, of DOPA in DOPA-functionalized polyallylamine in combina-
naturally emitted by the body or possibly also applied externally. tion with Fe3+ -ions to produce a self-healing multi-pH respon-
Elastin and elastin-like proteins (ELP) harbor a particularly inter- sive hydrogel. The coordination of DOPA per ferrous-ion de-
esting characteristic in this regard, as they show a temperature- pends on pH and formed mono-, bis-, and tris-catechol-Fe3+ com-
dependent inverse phase transition, meaning that these proteins plexes with increasing pH exhibiting the highest shear modu-
are rendered into a higher ordered state upon increasing tem- lus of 7 kPa at ≈pH 9.3. The adhesive capacity was subsequently
perature and vice versa, readily forming coacervates.[146] ELP- dropped at higher pH values closer to the pI as the amine groups
based hydrogels with thermo-responsive behavior have been cre- started to deprotonate and the hydrogel structure collapsed.[150]
ated with tunable lower critical solution temperature (LCST) Narkar et al. presented a hydrogel formed by copolymerizing
and have been used, for example, in drug and gene delivery dopamine methacrylamide and 3-acrylamido phenylboronic acid.
applications.[147] Brennan et al. developed an ELP adhesive, in The network-bound phenylboronic acid formed a pH-dependent,
which tyrosine residues were enzymatically converted to DOPA reversible complex with the catechol-groups protecting it against
to enable tunable LCST and, hence, reversible coacervation al- irreversible oxidation and allowing repeated adhesion and de-
lowing moderate adhesion in humid environments.[134] Further, tachment at pH 3 and pH 9 respectively.[139]
the above-discussed de novo-designed repetitive ELP-like pro-
teins able to form adhesive coacervates with the anionic surfac-
tant sodium dodecyl benzene sulfonate (SDBS) were modified 4.4. Other Activation Systems to Induce Adhesion
to further incorporate a temperature-dependent adhesive behav-
ior. Upon addition of the repetitive module VPGVG to the se- To conclude the chapter on activatable adhesives, we further pro-
quence consisting of repetitions of VPGKG, phase transition was vide some selected examples of adhesives, which do not quite fit
observed at temperatures above 25 °C, which was accompanied into the previous classifications but are worth mentioning. As
by switching the adhesion strength from ≈600 kPa to a 10-fold elaborated previously, the oxidation of DOPA to DOPA-quinone
decrease.[135] significantly reduces adhesiveness, hence, mechanisms to pre-
Another strategy is the conjugation of proteins with vent auto-oxidation (e.g. caged-DOPA) have been used to con-
synthetic thermo-responsive polymers such as poly(N- trol the function and rely on the triggered release or deprotec-
isopropylacrylamide) (PNIPAM) or Pluronic.[148] For instance, tion. In a similar approach, Xu et al. describe the use of reduc-
MAP can be grafted with low molecular weight thermo- ing thiourea groups (NCSN, “nitrogen-carbon-sulfur-nitrogen”)
responsive PNIPAM (MAP-PNIPAM) through EDC/NHS to preserve the adhesiveness of DOPA by preparing two HA-
chemistry as described by Jeon et al., forming gels with LCST based pre-polymers, of which one contained NCSN and the other
≈30 °C, depending on the degree of grafted PNIPAM, displaying catechol-groups.[140] Upon mixing, the NCSN reduced already
an adhesive strength of ≈6 kPa.[136] A hydrogel developed by oxidized catechol groups preserving and restoring the adhesive
Zhou et al. consisting of gelatin and aldehyde-functionalized properties. However, by then further applying an oxidation agent,
chondroitin sulfate (CS) in the presence of borax allowed dy- such as simply spraying sodium periodate (NaIO4 ) on top, a
namic Schiff base bonds enabling a reversible adhesion with rapid pH-independent polymerization could be induced in HA-
higher adhesive strength.[137] The injectable and self-healing catechol gels within seconds at ratios of NaIO4 :catechol 10-times
hydrogel underwent rapid and repeated gel-transition when lower than without HA-NCSN. The gels formed by this method
switching between 37 °C and 20 °C. An adhesive strength of up exhibited a wet adhesive strength of ≈15 kPa even after incu-
to ≈30 kPa was reached withstanding up to 800% strain when bation in aqueous environments for 24 h and were shown to

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promote ulcer healing, enhanced neovascularization, and inflam- adhesives are still often struggling to reach the rather unmatched
matory suppression in a porcine model. Thus, the adhesive was adhesive strength of synthetic and semi-synthetic glues, their bio-
proposed as suitable for gastric tissue treatments due to the pH- compatible nature and the capacity to biodegrade without the re-
independent mode of action. lease of toxic components is a major advantage, as it also allows
While interfacial water has been established to be a critical fac- the invasion of body’s own cells into the glued site promoting re-
tor for adhesion, a study presented by Yuk et al. used poly(acrylic epithelialization, neovascularization and rapid, close to seamless
acid) grafted with N-hydroxysuccinimide ester (PAAc-NHS ester) wound healing. Adding small molecules, drugs, and factors or
cross-linked using GelMA in combination with an interchange- activatable features to such materials further extends their range
able biopolymer (e.g. gelatin or chitosan) to incorporate, rather of applicability, making them more complex and versatile, allow-
than displace, the usually unfavorable interfacial water at the ap- ing for instance good spatiotemporal control over the mode of ac-
plication site.[141] Applied in form of a so-called dry double-sided tion or the body’s response. However, when looking at developing
tape, the quick hydration and swelling result in drying of the sur- activatable protein adhesives there are also downsides apart from
face due to the negatively charged carboxylic acid groups of PAAc- weaker adhesives forces. While there is a multitude of photo-
NHS. Subsequently, covalent bonds can be formed via primary activatable systems already successfully employed in small and
amine groups of the tissue, exhibiting rapid interfacial toughness large animal models in vivo, there are only a few temperature-
of more than 600 Jm−2 within 5 s and a tensile strength of up to activatable and even less pH-activatable protein adhesives, which
160 kPa.[141] The storable dry tape has an extensibility of 16 times have currently exceeded in vitro or even conceptional experimen-
its original length and has been successfully used to seal several tation. This is most likely due to the fact that especially pH is a
types of tissue both in vitro and in vivo. common factor directly related to structure and function among
all proteins, hence limiting them to a narrow pH range and spe-
5. Conclusion and Future Perspective cific proteins only. Furthermore, feasible production costs, long-
term stability, and shelf-time durability are considerable factors,
Bioadhesives can be a useful tool for wound treatment, as they which can limit recombinant protein adhesives for commercial
not only provide an often easier and safer form of connecting tis- use.
sue but also reduce surgical time. The tunable chemistry and, A novel direction in this research field, possibly resolv-
hence, also mechanical and adhesive properties allow their ad- ing many of these issues, is known as engineered living
justment to the respective needs and can be adapted to meet the materials,[151] which among other topics also covers so-called
properties of a multitude of different tissues. The use of a bioad- autonomous living glues, aiming to not only create adhesives
hesive can become particularly interesting for fragile tissues such that can respond to external factors but also display other hall-
as nerves, which can be prone to trauma caused by suturing. In mark features of life such as cell-environment interactions, au-
light of the studies on protein-based adhesives presented here, it tonomy, self-replication and -regeneration to create virtually liv-
also becomes apparent that nature can still teach us some tricks, ing materials.[151] Herein, a dual-strain-strategy of prokaryotes is
especially when it comes to the design of adhesives used in wet employed to produce an adhesive on-demand, e.g. induced by
and dynamic environments, which is a particular challenge in light or the presence of heme/blood,[152] by the separate expres-
surgical applications. A clear trend toward the use of injectable sion of a glue protein in one strain and an adhesion-enhancing
or patch-like hydrogels is obvious as hydrogels facilitate the han- protein in another, which only in combination form the adhesive
dling, reduce application time down to seconds, and can infiltrate matter.[151,153] Using this strategy, the “living glue” by An et al.
cavities, uneven surfaces and tissue, further enhancing the adhe- was demonstrated to perform blood-sensing autonomous repair
sive force by mechanism of mechanical interlocking. Especially of a microfluidic device.[152] Although the pathogenicity of bacte-
hydrogels formed by complex coacervation allow strong wet ad- ria strains limits the application in the biomedical field, a transfer
hesion by displacing or adsorbing interfacial water, which is a of the set-up to non-pathogenic organisms or even mammalian
major advantage over common cyanoacrylate adhesives. A good cells could overcome this drawback.
bioadhesive should have an adhesive strength in the range of tens
of / several kilopascals depending on the tissue or the site of ap-
Acknowledgements
plication. While synthetic adhesives with high adhesive strength
often suffer from toxic degradation products and poor biodegrad- The authors acknowledge the funding from the Deutsche Forschungs-
ability, bioadhesives made of proteins and polypeptides represent gemeinschaft (DFG, German Research Foundation) – project number
a highly versatile alternative. Foremost, the amino acid sequence 326998133 in the framework of the Collaborative Research Centre SFB-
TRR225 (funded sub-project: A08 and C01). Support from the Elite Net-
of protein-based adhesives naturally contains an abundance of work of Bavaria was also acknowledged.
available amine and carboxylic groups, which can be readily func- Open access funding enabled and organized by Projekt DEAL.
tionalized or used for conjugation to expand their function. Es-
pecially tyrosine-residues play an outstanding role as they can
be directly utilized for enzymatic cross-linking or transformation Conflict of Interest
to catechol-groups resembling the naturally occurring DOPA, The authors declare no conflict of interest.
which in turn has vast modes of action. Recombinant proteins
further can be tailored and optimized by rational design and ge-
netic engineering, hence, an in-depth understanding of mecha- Keywords
nisms found in natural protein-based bioadhesives is of high im- amyloids, bioadhesions, biomedical applications, coacervation, DOPA,
portance for their development. Although purely protein-based genetic engineering, silks

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Christina Heinritz received both her B.Sc. degree in biochemistry (2018) and her M.Sc. in biochemistry
and molecular biology (2021) from the University of Bayreuth (Germany). She is currently a Ph.D.
candidate at University of Bayreuth (Germany) under the supervision of Thomas Scheibel and her
current research focuses on spider silk-based recombinant proteins and their use in the development
of engineered tissues with vascular supply.

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Xuen J. Ng received his B.Sc. degree in biology (2017) and his M.Sc. in biochemistry and molecular bi-
ology (2020) in cooperation with CSIRO (Clayton Melbourne, Australia) at the University of Bayreuth.
He is currently a Ph.D. candidate at the Department of Biomaterials under the supervision of Prof. Dr.
Thomas Scheibel and investigates the use and development of recombinant spider silk-based bioinks
for application in cardiac tissue engineering.

Thomas Scheibel is a full professor for Biomaterials as well as Vice President for research and junior
scholars at the University of Bayreuth. Since 2014 he is a member of the German National Academy of
Science and Engineering (Acatech). He initiated and chairs the topical committee on “Bioinspired and
interactive materials” at the German Materials Society (DGM), is chairman of the Bavarian Excellence
Network “Cellular Hybrids”, co-chairman of the TranregioSFB TRR225 “Biofabrication”, and Member
of the review board “biomaterials” of the DFG.

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