Synthetic extracellular matrices with function-encoding peptides

Abstract

The communication of cells with their surroundings is mostly encoded in the epitopes of structural and signalling proteins present in the extracellular matrix (ECM). These peptide epitopes can be incorporated in biomaterials to serve as function-encoding molecules to modulate cell–cell and cell–ECM interactions. In this Review, we discuss natural and synthetic peptide epitopes as molecular tools to bioengineer bioactive hydrogel materials. We present a library of functional peptide sequences that selectively communicate with cells and the ECM to coordinate biological processes, including epitopes that directly signal to cells, that bind ECM components that subsequently signal to cells, and that regulate ECM turnover. We highlight how these epitopes can be incorporated in different biomaterials as individual or multiple signals, working synergistically or additively. This molecular toolbox can be applied in the design of biomaterials aimed at regulating or controlling cellular and tissue function, repair and regeneration.

Peptide epitopes of structural and signalling proteins constitute the language through which cells communicate and orchestrate biological processes. This Review provides a library of peptide epitopes that can serve as a molecular toolbox for the design of bioactive synthetic matrices.

Key points

• The communication between cells and their extracellular matrix (ECM) is encoded in peptide epitopes of structural and signalling ECM proteins, acting as molecular ‘orchestrators’ of multiple cell functions.

• Peptide epitopes can be incorporated in biomaterials to achieve selective and specific communication with cells and tissues.

• Biomaterials designed with specific peptide epitopes can control cell adhesion, differentiation, immunomodulation and extracellular matrix turnover.

• A peptide epitope molecular toolbox can be applied for the design of functional synthetic ECMs in tissue engineering and bioengineering applications.

Introduction

Molecular and cellular building blocks communicate with each other in a coordinated manner to define the structure and function of tissues. In particular, the extracellular matrix (ECM) is a key regulator of cell communication and regulation. The ECM is a meshwork composed of water, proteins (including collagens, fibronectin, elastin and laminins), proteoglycans, signalling cues (such as growth factors and cytokines), minerals and ions¹. ECM composition varies among tissues and provides an adaptive microenvironment², exhibiting precise, often co-existing, mechanisms, including feedback loops, to control molecule concentration³, protein disorder–order transitions to temporally control signalling⁴, and enzymatic degradation⁵ to allow turnover. For example, in bone healing, evolving and coordinated signalling events finely encode and regulate inflammation, immunomodulation, recruitment of cells, osteogenesis, angiogenesis and ultimately tissue repair⁶. Therefore, the ECM not only provides a rich resource of signals to interact with cells, but can also serve as inspiration for the design of biomaterials aimed at communicating with cells.

Biomaterials are used in vaccines, drug delivery systems, imaging tools, prostheses, biosensors, bioelectrodes, tissue engineering scaffolds and surgical equipment⁷. Most biomaterials rely on physiochemical properties, such as surface charge, chemical composition, topography and stiffness, to interact with biomolecules and cells, resulting in a limited capacity to guide biological responses. To design biomaterials that precisely communicate with biological structures, selectivity, specificity, dynamicity, hierarchy and multifunctionality can be implemented; however, to achieve clinical translation, regulatory and financial constraints should be considered in biomaterial design⁸.

In particular, protein epitopes can be used as building blocks to engineer cell-instructive biomaterials⁹. Proteins are composed of amino acid chains called peptides, which are referred to as epitopes if they contain a bioactive sequence capable of interacting with specific cell receptors or antibodies. These short amino acid sequences are the main ‘function-encoding language’ of proteins and can be used as building blocks of biomaterials. In particular, self-assembling peptides are molecular building blocks with high propensity to self-assemble into ECM-like nanofibres and hydrogels in aqueous environments¹⁰. This material platform enables the presentation of peptide epitopes on the surface of nanofibres to actively interact with cells, initiate specific cell signalling pathways, serve as therapeutic agents, or target cells and tissues in vivo¹¹. The versatile molecular design of self-assembling peptides also allows the engineering of 3D nanofibrillar hydrogels with a range of mechanical, chemical and biological properties for applications in cell culture, cell delivery and biofabrication^12,13.

Compared with full-length proteins, peptides show less degradation, more long-term functionality, higher purity, and less sensitivity to pH and temperature, and, in some cases, they can achieve more precise cell signalling¹⁴. In addition to ECM epitopes, synthetic amino acid sequences can be designed by artificial intelligence (AI) and by taking information from phage display¹⁵, complementing natural peptides as structural and signalling building blocks of biomaterials that can interact, communicate with and respond to their cellular surroundings.

In this Review, we discuss the design of biomaterials based on peptides as signalling building blocks. We provide a library of peptide epitopes that can serve as a molecular toolbox for the bioengineering of synthetic hydrogels with biological functions such as structural support, signalling and dynamicity (Fig. 1). Based on the function encoded in the hydrogel, we discuss peptides exploited for direct signalling, which foster cell adhesion, differentiation and immunomodulatory effects; peptides binding signalling or structural cues, such as growth factors, proteins and minerals; and enzymatically cleavable peptides to create degradable hydrogels. Finally, we describe the combination of the aforementioned peptides to induce synergistic or additive effects.

Fig. 1. Function-encoding peptide epitopes.

a, Classification  and examples of peptide epitopes. b, Synthetic hydrogels endowed with function-encoding epitopes can induce multiple biological effects, such as adhesion, differentiation, immunomodulation and matrix turnover in encapsulated cells. ECM, extracellular matrix; MMP, matrix metalloproteinase.

Peptide sequences for direct signalling

Cell adhesion receptors, in particular, integrins and cadherins¹⁶, are pivotal for cell–cell and cell–ECM communication, transferring environmental information into intracellular pathways. An integrin receptor is a heterodimer structure containing an α and a β subunit, whose pairings are specific for ligand-binding interactions. For example, RGD peptides, which are present in many ECM proteins, including vitronectin, fibrinogen, osteopontin, bone sialoprotein and fibronectin¹⁷, are recognized and bound by multiple integrin subunits¹⁸, such as αvβ3, αvβ5, αvβ1, αvβ6, αvβ8, α5β1, α8β1 and αIIbβ3¹⁹. Integrins can engage with elements of the cytoskeleton, such as actin, vinculin and talin molecules, transducing external information into changes of cell shape, cell architecture, motility and differentiation²⁰. Above a certain threshold adhesion area, integrin adhesion strength increases exponentially with the number of bound integrins, and with recruited vinculin and talin²¹. Moreover, adhesion strength is modulated by integrin bonds, bond force and distribution along the cell contact area²².

Cadherins display extracellular tandem repeats in cell-to-cell junctions to create linkages with actin-containing cytoskeletons, while they interact intracellularly with catenins to modulate differentiation and morphogenesis^23–25. Alongside cell adhesion, different transmembrane receptors are exploited by cells to bind specific growth factors and cytokines. Here, ‘direct signalling’ refers to the direct interaction of cell surface receptors with peptide epitopes to trigger intracellular signalling. Peptides bound by cell receptors include peptides promoting cell adhesion, mimicking growth factor epitopes, or regulating immunomodulatory effects (Fig. 2, Table 1 and Supplementary Table 1).

Fig. 2. Peptide epitopes in synthetic hydrogels for direct signalling.

a,b, Peptide epitopes inducing direct signalling include: (a) adhesion peptides operating, for example, through integrin binding, and (b) growth factor-mimetic peptides mimicking, for example, transforming growth factor-β (TGFβ) molecules that bind to TGFβ receptors (TGFβR). c,d, Other peptides have been explored for immunomodulatory effects, including (c) antimicrobial peptides able to disrupt bacteria cell membranes and (d) immunomodulatory peptides that elicit immune-cell responses. MHC, major histocompatibility complex; Smad, small mothers against decapentaplegic protein.

Table 1.

Peptide sequences for direct signalling

Function	Source	Epitope
Cell adhesion	Fibronectin	RGDS26,30–33
		KQAGDV71
		PRARI71
	Laminin	IKVAV36,39,45,227
		YIGSR38,44,228
		PDSGR228
		RKRLQVQLSIRT229
		CGGRKRLQVQLSIRT45
		RYVVLPR35
		RNIAEIIKDI230
		AGQWHRVSVRWG65
		IKLLI71
		SINNNR71
		LRGDN71
	Tenascin	VFDNFVLK48–50
	Collagen	POG53,54
		DGEA55,208
		(PKG)4-(POG)4-(DOG)452
		GFOGER56
		FPGERGVEGPGP57
		GFPGER62,63
		NYYSNS71
	Nidogen	FRGDGQ71
	CAM	QIDS72
	CAM	LDT72
	Netrin	QWRDTWARRLRKFQREKKGKCRKA67
	VCAM	DELPQLVTLPHPNLHGPEILDVPST68
	Thrombospondin	CSVTCG69,70
	Cadherins	HAVDI58,59,231
	De novo	RRETAWA64
Growth factor-mimetic	BMP-2	DWIVA79
		KIPKA-SSVPT-ELSAISTLYL79,80,232
		SDVGWNDWIV233
	BMP-7	KPSSAPTQLN77,78
	Cytomodulin and TGFβ1	LIANAK81
	VEGF	KLTWQELYQLKYKGI83,84,234
	Osteonectin	GHK88
	Osteopontin	SVVYGLR90
	Angiopoietin	QHREDG91
	FGF-2	YRSRKYSSWYVALKR86
	Phage-display	KLPGWSG235,236
		FAQRVPP237
		QHLPRDH237
		SSLSVND236
	BDNF	RGIDKRHWNSQ92,95
	NGF	CTDIKGKCTGACDGKQC95
	NCAM	EVYVVAENQQGKSKA96
Antimicrobial	Myxinidin	WGIRRILKYGKRS99
	Melittin	GIGAVLKVLTTGLPA LISWIKRKRQQ102,103
	Melittin/cecropin	KWKLFKKIGIGAVLKVL TTGLPALISC104
	De novo	K6-(SL)6-K6105
		NAVSIQKKK106
		PEP6R107
		MAX1108
		MARG1109
		FFKK, X-FFKK (X = ibuprofen, indomethacin, naproxen)111,112
		PA-1 (+ AgNPs)113
		FFECG (+ AgNPs)114
Immunomodulatory	Ovalbumin	ISQAV-HAAHAEINEAGR116,119,238, SIINFEKL118
	Staphylococcus aureus T cell epitope	PADRE + KFEGTEDAVETIIQAIEA239
	H1N1 epitope	SSLENFRAY115
	Plasmodium falciparum	(NANP)3117
	EV71 capsid 1	YPTFGEHKQEKDLEY121
	EV71 capsid 3	HYRAHARDGVFDYYT121
	HIV-1 T cell epitope	SLYNTVATL122
	West Nile virus	EIII protein domain123
	De novo	FLIVIGSIIGPGGDGPGGD124,125

AgNP, silver nanoparticle; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CAM, cell adhesion molecule; EV71, enterovirus 71; FGF, fibroblast growth factor; H1N1, haemagglutinin type 1 and neuraminidase type 1; HIV, human immunodeficiency virus; NCAM, neural cell adhesion molecule; NGF, nerve growth factor; PADRE, Pan hlA DR-binding epitope; TGFβ, transforming growth factor-β; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor.

Peptide sequences promoting cell adhesion and growth

Cell adhesion epitopes derived from fibronectin

The RGD peptide, present in the fibronectin domain FNIII_9-10, is one of the most studied peptide sequences for cell adhesion. Synthetic polymers, such as polyethylene glycol (PEG) diacrylate (PEGDA), polyvinyl alcohol and poly-2-hydroxyethylmethacrylate, can be functionalized with RGD-containing motifs to control the adhesion and spreading of mammalian cells²⁶. RGD-containing peptides can also be implemented in self-assembling peptide systems as structural and signalling building blocks, for example, in peptide amphiphile (PA) hydrogels to induce cell adhesion²⁷, enamelogenesis²⁸ and bone formation²⁹; in RADA16-I hydrogels to differentiate human tendon stem and progenitor cells³⁰; and in FEFEFKFK hydrogels for delivery and recovery of cardiac progenitor cells³¹. In these supramolecular hydrogels, RGD display can be controlled through co-assembly to modulate RGD density³², by host–guest moieties for dynamic display³³, or to integrate surface topographies³⁴. Thus, incorporation of RGD endows synthetic hydrogels with cell adhesion features.

Cell adhesion epitopes derived from laminins

Laminin-derived peptides, including IKVAV from the laminin α-chain, YIGSR, PDSGR, RYVVLPR from the laminin β1-chain, and RNIAEIIKDI from the laminin γ-chain, can be applied to induce cell aggregation and cluster formation in angiogenesis and neurogenesis³⁵. For example, peptide-decorated synthetic polymers, such as poly(HEMA-co-AEMA) with YIGSR/IKVAV, and poly(tetrafluoroethylene-co-hexafluoropropylene) with YIGSR/IKVAV/RGD, have been explored in neural tissue engineering^36,37. PEGDA with IKVAV and poly(urethane urea)/PEG with YIGSR can promote in vitro endothelialization³⁸ and in vivo angiogenesis in a mouse model³⁹. Self-assembling RADA16 hydrogels containing IKVAV, YIGSR and PDSGR promote cell attachment, and increase the expression of the neural marker βIII-tubulin in vitro and neurite network formation of mouse embryonic stem cells⁴⁰; for example, IKVAV epitopes incorporated within PA hydrogels promote in vitro differentiation of neural progenitor cells and prevent differentiation of astrocytes, which are involved in scar formation after injury⁴¹. Thus, IKVAV-displaying PAs have been explored to reduce astrocyte-derived scars and aid regeneration of descending motor fibres and ascending sensory fibres in a mouse model^42,43. Laminin is also an important component of the liver, and therefore, RADA16-I hydrogels comprising YIGSR can be used to culture hepatocyte for drug screening⁴⁴. In addition, laminins are present in the pericellular matrix of nucleus pulposus cells, regulating nucleus pulposus cell attachment. PEG-based hydrogels containing IKVAV and AG73 peptides (full sequences are CSRARKQAASIKVAVSADR and CGGRKRLQVQLSIRT, respectively, from laminin-111 globular domains) can induce a juvenile phenotype in human degenerate nucleus pulposus cells through modulation of hydrogel stiffness and epitope density⁴⁵. Therefore, laminin-mimetic peptides can serve as building blocks to enhance cell attachment and improve tissue regeneration in endothelial and neural tissues.

Cell adhesion epitopes derived from tenascins

Peptides derived from tenascins, a class of glycoproteins involved in neurogenesis, skeletogenesis and vasculogenesis, can be used as function-encoding tools to promote cell adhesion; for example, tenascin C is involved in spinal-cord regeneration, axon ingrowth and brain development⁴⁶, and thus, hydrogels containing peptides from tenascin C have been explored for neural tissue engineering. In particular, binding of the tenascin-derived octapeptide VFDNFVLK to α7β1 integrins increases neurite length and promotes cell attachment in cerebral granule and cortical neurons⁴⁷. Cell migration, differentiation and neurite formation can be promoted in neurospheres cultured on tenascin-C-mimicking PAs⁴⁸. Similarly, a tenascin-C-derived peptide can direct mesenchymal stem cell (MSC) differentiation towards the osteogenic lineage, promoting cell viability, cell differentiation and mineralization in vitro⁴⁹. Moreover, acetyl-VFDNFVLK, which is the shortest bioactive sequence derived from tenascin C, can form biocompatible and injectable hydrogels at physiological pH⁵⁰. This hydrogel was tested in vitro as cell substrate for various neural and non-neural-derived cell lines (L929, C6, NIH3T3 and LN18), exhibiting low cytotoxicity and promoting cell adhesion and migration⁵⁰.

Cell adhesion epitopes derived from collagens

Collagens provide structural support to soft and hard tissues, binding to integrins to support cell attachment. The collagen superfamily is made of 46 different polypeptide chains that supramolecularly assemble into 28 collagen types, depending on their hierarchical organization and function. As such, collagen types can be found as fibrillar components, such as type I, II, III and V, in bone and cartilage; as organized meshwork, including type IV, VI, VIII and X, in the cornea and dermis; and as anchoring fibrils, such as type VII, in the bladder⁵¹. The collagen-mimetic peptide KOD ((PKG)₄-(POG)₄-(DOG)₄) establishes strong lysine–aspartate salt-bridged hydrogen bonds and can hierarchically assemble into stable triple helices⁵². In a thrombosis in vitro model using whole human blood, KOD-based hydrogels promote platelet adhesion, with blood clotting and degradation rates similar to those triggered by animal-derived collagen⁵³. Furthermore, injection of POG-containing PA hydrogels into a degenerate rabbit intervertebral-disc (IVD) model leads to increased deposition of ECM components of the IVD, including glycosaminoglycans (GAGs) and collagen type II⁵⁴, promoting recovery of the intervertebral disc within 2 weeks. The collagen-type-I-derived peptides DGEA and GFOGER have been investigated in bone tissue engineering. DGEA-displaying PA hydrogels increase the expression of the osteogenic markers alkaline phosphatase, RUNX2 and osteocalcin (OCN) in vitro⁵⁵, and GFOGER-presenting peptides stimulate osteoblast adhesion and differentiation, and bone formation when used as coatings of polymeric plugs press-fitted into rat femoral defects⁵⁶.

In addition, RADA16 hydrogels comprising the collagen type I epitope FPGER as part of FPGERGVEGPGP peptides can modulate keratinocyte and dermal fibroblast migration and proliferation in vitro⁵⁷. Collagen mimicry can involve peptides that self-assemble into collagen-like bundles or collagen-mimetic motifs, to recreate signalling and structural functionalities of collagens.

Cell adhesion epitopes derived from cadherins

Cadherins are transmembrane cell receptors responsible for the transmission of signals between cells, cell migration and maintenance of tissue structure. For example, N-cadherins are essential for MSC condensation, which is a crucial step in cartilage morphogenesis. Cadherins contain the evolutionary-conserved tripeptide HAV, which is an important cell-to-cell adhesion mediator. This motif mediates both MSC–MSC (‘homotypic’) and MSCs–osteoblast (‘orthotypic’) interactions; for example, methacrylated hyaluronic acid hydrogels with HAV sequences promote early chondrogenesis of MSCs in vitro and neocartilage deposition in vivo when implanted in subchondral pockets of nude mice⁵⁸. Similarly, HAVDI, a pentapeptide located in the extracellular domain-1 of N-cadherins, attached to the N-terminus of KLD12 peptide hydrogels triggers chondrogenesis of human MSCs in vitro by suppressing canonical Wnt/β-catenin signalling, a master regulator of osteogenesis and chondrogenesis⁵⁹. Methacrylated gelatin (GelMA) conjugated to N-cadherin-mimetic peptides promote the differentiation of induced pluripotent stem cells into neurons, functionally better than Matrigel⁶⁰.

Other cell adhesion epitopes targeting integrins

Given the pivotal role of integrin binding in mediating angiogenesis, osteogenesis and wound healing, integrin targeting is a key design consideration for many biomaterials⁶¹. For example, hydrogels based on streptococcal collagen-like proteins containing the sequence GFPGER, derived from collagens interacting with α1β1 and α2β1 integrins, promote in vitro endothelial cell adhesion on tissue-engineered vascular grafts⁶² and in vitro osteogenesis in 3D hydrogels⁶³. Osteogenic differentiation can also be promoted with the phage-display-derived sequence RRETAWA that specifically interacts with α5β1 integrin⁶⁴. Wound healing of a splinted excisional wound model in type II diabetic mice can be accelerated using PEG that contains the laminin-derived dodecapeptide A5G81 (AGQWHRVSVRWG), which targets α3β1 and α6β1 integrins⁶⁵. Derived from the fibrinogen γC and βC chains, the peptides P1 (GWTVFQKRLDGS), P2 (YSMKKTTMKIIPFNRLTIG) and GWTVIQNRQ can also target integrins (αMβ2 binding sites) to support cell adhesion and migration⁶⁶. Integrin-targeting peptides have also been derived from netrin-1 (QWRDTWARRLRKFQREKKGKCRKA), targeting α6β4 and α3β1 integrins⁶⁷, from fibronectin and vascular cell adhesion molecule (CAM)-1 (DELPQLVTLPHPNLHGPEILDVPST) targeting α4β1 integrin⁶⁸, and from thrombospondin (for example, the domain CSVTCG) binding β1 integrins^69,70. However, within the majority of published studies, RGD remains the best studied integrin-binding peptide (89%), followed by IKVAV (6%), YIGSR (4%) and other sequences (1%)⁷¹. In addition, laminin-derived IKLLI, LRGDN and SINNNR, fibronectin-derived KQAGDV and PRARI, collagen-type-IV-derived NYYSNS, nidogen-1-derived FRGDGQ⁷¹, and CAMs-derived QIDS and LDT are being explored⁷².

Peptide sequences mimicking growth factors

Cells sense their microenvironment through cell receptors that sequester and bind growth factors from this microenvironment. Growth factor ligands bind to serine/threonine kinase receptor complexes, which in turn become phosphorylated and initiate a downstream intracellular cascade that reaches the nucleus and regulates transcription. Growth factor receptors can also move along the plasma membrane and cluster into small domains to enhance signalling⁷³. Moreover, growth factors can cross-talk with integrins⁷³. Growth factor-binding and unbinding are regulated by feedback controls; growth factor ligands can be sequestered in their latent form to prevent growth factor signalling. Growth factors are involved in numerous pivotal cellular processes, such as cell proliferation, migration and differentiation, and they orchestrate embryonic development, tissue homeostasis and repair by influencing and directing stem cell phenotype; for example, bone morphogenic proteins (BMPs) have a key role in osteogenesis; vascular endothelial growth factors (VEGFs) enable angiogenesis; and transforming growth factors (TGFβs) are important regulators of chondrogenesis⁷⁴. Growth factor-mimetic peptides can be incorporated in 3D synthetic hydrogels to avoid rapid denaturation and clearance of full-length growth factors, yet ensuring reactivity, specificity and efficiency.

Growth factor-mimetic peptides for osteogenesis, chondrogenesis and discogenesis

Peptides derived from BMPs can be used in osteogenesis applications. BMPs are multifunctional growth factors from the TGFβ superfamily, which play a crucial role in heart, brain, cartilage and bone development⁷⁵. BMPs have been investigated for multiple therapeutic interventions; for example, BMP-7 has shown great regenerative chondrogenic potential and anti-apoptotic effects in vitro and in vivo⁷⁶. The BMP-7-derived peptide epitope KPSSAPTQLN can be conjugated to the C-terminus of a RADA16-I hydrogel to create hydrogels (RADA-KPSS) with anti-inflammatory and anti-apoptosis effects⁷⁷; for example, this system reduces the expression of catabolic enzymes and increases secretion of GAGs in intervertebral-disc nucleus pulposus cells⁷⁷, and induces migration, proliferation and ECM deposition of human nucleus-pulposus-derived stem cells in vitro⁷⁸. The BMP-2-derived peptides DWIVA and ‘knuckle epitope’ KIPKASSVPTELSAISTLYL⁷⁹ can be implemented in SSVPT-containing hydrogels, which show high osteogenic potential by increasing alkaline phosphatase, osteopontin production, and in vitro mineral deposition when cultured with murine osteoblasts and MSCs⁷⁹. Furthermore, SSVPT-displaying PA hydrogels can induce osteogenic MSC differentiation in vitro and promote rat skull defect repair in vivo⁸⁰. LIANAK, a TGFβ1-simulating peptide, can be conjugated to a self-assembling RADA16 hydrogel to increase the expression of chondrogenic genes and ECM deposition⁸¹. Combined with decellularized cartilage matrix, the hydrogel promotes neocartilage formation and restoration of the subchondral unit in a rabbit model of full-thickness knee cartilage defect⁸¹.

Growth factor-mimetic peptides for angiogenesis

Biomaterials that promote angiogenesis are of great interest for various applications, including tissue engineering to promote vascularization of tissue-engineered constructs and to mimic native levels of oxygenation, nutrients and waste removal, as well as in vitro models to perform drug screening studies. In particular, VEGF has a key role in angiogenesis and endothelial cell proliferation. The VEGF-mimicking peptide KLTWQELYQLKYKGI (KLT) from the VEGF helix region 17–35 binds to VEGF receptors, activates cell signalling and promotes capillary formation⁸². A KLT-decorated RADA16-I hydrogel promotes endothelial cell growth and induces capillary-like tubule formation in 2D and 3D, as well as neovascularization in a chicken embryo chorioallantoic membrane assay⁸³. Moreover, an injectable KLT-containing self-assembling peptide hydrogel promotes angiogenesis, infiltration of haematopoietic stem cells and MSCs, and rapid host–gel integration in a rat model⁸⁴. Basic fibroblast growth factor (bFGF), also known as FGF-2, also has pleiotropic effects, including angiogenesis and wound healing. In particular, the peptide domain 106–120 in FGF-2 is a partial agonist of FGF receptors⁸⁵. The FGF-2-mimicking peptide YRSRKYSSWYVALKR can be applied to formulate angiogenic peptide nanoribbon hydrogels, which can promote the proliferation and migration of human umbilical vein endothelial cells in vitro, with similar bioactivity as native FGF-2⁸⁶. The osteonectin-derived GHK tripeptide⁸⁷ is also pro-angiogenic, and can be implemented in a hyaluronic acid tyramine (HA-Tyr)/Laponite/PA) nanocomposite hydrogel to stimulate osteoblastic differentiation of human MSCs and neovessel formation in vitro, as well as bone formation in a rabbit maxillary sinus floor bone model⁸⁸. Derived from osteopontin, the heptapeptide SVVYGLR coupled to RADA16 hydrogels can stimulate in vitro angiogenesis of rat lung endothelial cells with a comparable level to VEGF⁸⁹, which may serve as treatment for myocardial infarction⁹⁰. Similarly, MSC-laden RADA16-I hydrogels grafted with QHREDG, derived from an integrin-binding motif of angiopoietin-1 (Ang-1), reduces infarct size in a rat model owing to a paracrine effect that results in downregulation of the pro-inflammatory cytokines interleukin (IL)-6 and IL-1⁹¹.

Growth factor-mimetic peptides for neurogenesis

Synthetic hydrogels can be designed to promote nerve regeneration. Nerve growth factor (NGF) promotes neurite outgrowth and regulates peripheral sensory and sympathetic cell function, and brain-derived growth factor (BDNF) provides neuroprotection and regeneration of injured neurons. The peptides CTDIKGKCTGACDGKQC and RGIDKRHWNSQ, derived from NGF and BDNF, respectively, can promote neurite proliferation and neuroprotection similar to the full-length growth factors^92,93. In a RADA16-RGIDKRHWNSQ hydrogel, MSCs can be differentiated into neuron-like cells, with neurites and dendrites extending in 3D both in vitro and in vivo⁹². The cyclic tetrapeptide RKKA^DP is derived from loop 4 of native BDNF, and can activate BDNF-associated tyrosine kinase B (TrkB) signalling, thereby promoting primary cortical neuron assembly into an electrically conductive network⁹⁴. Similarly, RADA16-NGF hydrogels can be used as bioactive ‘fillers’ of nerve conduits to bridge sciatic nerve defects, triggering nerve-conduction and re-myelinization in rats⁹⁵. In addition, the fibroblast growth factor ligand motif EVYVVAENQQGKSKA, derived from the neural cell adhesion molecule (NCAM), promotes in vitro neurite sprouting andspinal-cord-derived neural stem cell adhesion and migration⁹⁶.

Peptide sequences inducing immunomodulatory effects

Peptide hydrogels with naturally derived antimicrobial effects

Cationic amphipathic peptides possess intrinsic antimicrobial properties owing to the presence of positive charges that promote interactions and interdigitation within negatively charged bacteria membranes, causing bacterial cell death⁹⁷. Taking advantage of their inherent antimicrobial properties, antimicrobial self-assembling hydrogels can be designed using the antimicrobial peptide (AMP) WGIRRILKYGKRS (WMR), which is derived from the native sequence of the marine peptide myxinidin present in the epidermal mucus of hagfishes (Myxine glutinosa)⁹⁸. Compared with the myxinidin sequence, WMR peptides possess an extra tryptophan amino acid at the N terminus and a higher number of positively charged arginine residues, which confers strong antimicrobial activity on the sequence⁹⁹. WMR-grafted PAs self-assembled into nanofibres can be used as substrates to inhibit biofilm formation of the Gram-negative bacteria Pseudomonas aeruginosa and the pathogenic fungus Candida albicans¹⁰⁰, which are often involved in clinical infections. Antimicrobial activity and high cytocompatibility can also be achieved by presenting melittin¹⁰¹, a natural honeybee-venom-derived AMP, on the surface of synthetic multidomain peptides. In this design, upon assembly on peptides, melittin conformation is altered to selectively inhibit bacterial growth (Escherichia coli), while preserving biocompatibility towards mammalian cell membranes (mouse fibroblasts)¹⁰². Melittin can also be applied as antimicrobial coating; for example, a melittin-derived peptide (GIGAVLKVLTTGLPALISWIKRKRQQ) film layered on chitosan-antibiotic coatings of titanium bone implants results in the rapid killing and prevention of biofilm formation of methicillin- and vancomycin-resistant Staphylococcus aureus¹⁰³. Melittin can further be used in combination with a cecropin peptide, derived from the haemolymph of Hyalophora cecropia moth, to create a hybrid melittin–cecropin epitope (KWKLFKKIGIGAVLKVLTTGLPALISC) with high stability and antimicrobial effect against E. coli when immobilized on polymeric films¹⁰⁴. A biocompatible bacterial-membrane-disrupting self-assembled peptide system (CASP-K6), containing multiple lysine residues, shows strong bactericidal effect against P. aeruginosa¹⁰⁵. Similarly, the presence of lysines in β-sheet-rich hexapeptides linked to lysine tripeptides (NAVSIQKKK) leads to antimicrobial activity against E. coli and S. aureus. Peptide hydrogels with designed antimicrobial effects are biocompatible in human lung fibroblasts and red blood cells¹⁰⁶.

Peptide hydrogels with inherent  antimicrobial effects

Injectable hydrogels can be made antimicrobial by incorporating peptides with antimicrobial properties. For example, β-hairpin self-assembling AMPs, such as PEP6R¹⁰⁷, MAX1¹⁰⁸ and MARG1¹⁰⁹, inhibit growth of P. aeruginosa, Gram-positive and Gram-negative pathogens (for example, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae and E. coli)¹⁰⁸, and methicillin-resistant S. aureus¹⁰⁹. Low-molecular-weight peptide hydrogels based on Fmoc-FFE and Nap-FFE peptides also have bactericidal effects against S. aureus and S. epidermidis, when functionalized with the cationic group spermine¹¹⁰. Furthermore, NapFFKK and NapFFFKK show anti-biofilm activity against Gram-positive and Gram-negative strains, and co-assembly of FFKK with the drug naproxen leads to anti-inflammatory properties against cyclooxygenase enzymes, which are associated with chronic wound scar-tissue formation^111,112. PA nanofibres, synthesized through mineralization of Fmoc-FFECG with silver nanoparticles, harness the antimicrobial properties of silver¹¹³ and elicit long-term in vitro antimicrobial activity against E. coli and Bacillus subtilis¹¹⁴. Such antimicrobial peptide-based biomaterials may be applied to treat bacterial infections after surgery or to overcome antibiotic resistance.

Peptide hydrogels that enhance T cell and B cell immune responses

Peptides can also serve as chemically defined immune adjuvants; for example, a peptide system (O-Q11) based on the Q11 self-assembling domain in tandem with an ovalbumin region (that is, OVA_323–339) that contains antigenic epitopes recognized by T and B cells self-assembles into nanofibres in physiological conditions. Intranasally or subcutaneously administered nanofibres elicit a strong immune response in mice without adjuvants, causing high levels of immunoglobulins and promoting T cell activation and an antigenic response if conjugated to an H1N1 influenza epitope¹¹⁵. Furthermore, displaying Q11 on a virulence factor from tuberculosis¹¹⁶ or conjugating Q11 to (NANP)₃, a malaria peptide epitope¹¹⁷, also results in an immune response in mice. In general, OVA sequences offer useful immunogenic epitopes; for example, peptide amphiphilic nanofibres biotinylated to OVA proteins cause antigen-specific splenocyte proliferation and CD8⁺ T cell responses when administered subcutaneously in mice¹¹⁸. OVA_323–339 can also be conjugated to β-sheet-rich FKFEFKFE hydrogels; the chirality of specific amino acids in these hydrogels has a crucial role in the stability of T cell immune responses in mice¹¹⁹. Moreover, the OVA epitope SIINFEKL has self-assembling capabilities and may thus be used as hydrogel immune adjuvant system¹²⁰.

Peptide hydrogels eliciting immunity against viruses

Peptide vaccines are based on peptide epitopes that elicit immune responses against specific viruses; for example, a self-assembled multimeric peptide amphiphilic system enhances immunity against enterovirus 71 (EV71), a Picornaviridae virus that causes hand, foot and mouth diseases¹²¹. Here, the peptide amphiphilic nanofibres display the two antigenic epitopes, virus particles 1 (YPTFGEHKQEKDLEY) and 3 (HYRAHARDGVFDYYT) from the enterovirus 71, leading to humoral and cell immune responses in mice. The serum from these immunized mice can neutralize EV71 and protect host cells¹²¹. CD8⁺ T cell epitopes and toll-like receptor 7/8 agonists R848 and R837 can be co-assembled in EAK16-II peptide hydrogels to elicit a cytotoxic T cell response against human immunodeficiency virus-1 (HIV-1) when they are administered subcutaneously in mice¹²². KFE8 peptide hydrogels can be used as adjuvants in combination with envelope protein domain III (EIII) and injected subcutanously, conferring immune protection against the West Nile virus in mice, similar to gold-standard adjuvants¹²³. A shear-thinning peptide (FLIVIGSIIGPGGDGPGGD, known as h9e) can induce immunity against porcine reproductive and respiratory syndrome virus¹²⁴ and H1N1 influenza¹²⁵. Finally, the conserved peptide sequence RSAIEDLLFDKV, derived from coronavirus spike proteins, assembles and disassembles into β-sheet amyloid tapes depending on the pH, which is being explored for peptide vaccine design¹²⁶.

Peptide sequences for binding ECM components

Peptide motifs can also bind and sequester ECM components, in particular, ECM molecules, and ions (Fig. 3a–c; Table 2; Supplementary Table 2).

Fig. 3. Peptide epitopes in synthetic hydrogels that bind to extracellular matrix components.

a–c, Peptide sequences that can bind extracellular matrix (ECM) components to allow matrix degradation include epitopes that bind (a) growth factors, (b) ECM components and (c) ions. d, Enzymatically cleavable peptides can be exploited to create dynamic matrices. MMP, matrix metalloproteinase.

Table 2.

Peptide sequences binding growth factors, extracellular matrix components and ions

Function	Target	Epitope
Growth factor-binding	TGFβ1	HSNGLPL128–131
	BMP-2	TSPHVPYGGGS133
		YPVHST128
	IGF-1	Biotin-IGF-1134,135
	PDGF-BB	PDGF-BB136
	BMP-2, HGF, VEGF, NGF, FGF-2	VVAGEGDK(p-sulfobenzoyl)S138–140
	BMP-2, BMP-4, FGF-1, FGF-2, VEGF, Shh, Noggin	GlcNAc-PAs137
ECM-component binding	Heparin (in turn FGF-2 and VEGF)	LRKKLGKA142–145
	Collagen I	LRELHLNNN240
	Hyaluronic acid	K3153
	Elastin-like polypeptides	K2, K3, K4, E3154
	Keratin	VPGIGK3, H2K, K3, E3155,158
	Keratin and fibronectin	H2K158,159
	Resilin	E3CY160
	β-lactoglobulin and BSA	YN, YS, YL,VL149
	Haemoglobin, myoglobin, serum albumin and fibrinogen	FF150
Ion and crystal binding	Hydroxyapatite	FF, R162, FF/S, RGD163
		PD-(FD)5-P241
		QQRFEWEFEQQ167
		NPYHPTIPQSVH242
		MLPHHGA165
		P26, P32168
		TKREEVD169,170
		DSS171
	Fluorapatite	DDDEEKFLRRIGRFG172

BMP, bone morphogenetic protein; BSA, bovine serum albumin; FGF, fibroblast growth factor; GlcNAc, N-acetylglucosamine; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor; PA, peptide amphiphile; PDGF-BB, platelet-derived growth factor-BB; Shh, Sonic hedgehog; TGFβ, transforming growth factor-β; VEGF, vascular endothelial growth factor.

Peptide sequences binding growth factors

Peptides directly binding growth factors

Soluble growth factors are commonly used in vitro and in the clinic to direct cell fate. However, they are often supplied at supraphysiological doses to ensure high concentration and efficacy over a long time period, which causes off-target effects, such as ectopic bone, neurological morbidities and cancer¹²⁷. Alternatively, to avoid side effects, rapid clearance and degradation of exogenous growth factors in vivo, biomaterials can deliver growth factors to maximize growth factor retention, delivery and signalling. Growth factors bound to a solid phase closely recapitulate how growth factors are presented in native ECMs, mimicking sequestering and release dynamics, and improving their efficacy as therapeutics. In particular, growth factor-binding peptides can be implemented in 3D scaffolds to sequester and create growth factor reservoirs and gradients.

Growth factor-binding peptides are typically derived from cell receptors that bind growth factors; for example, a hydrogel based on a thermosensitive copolymer, consisting of poly(N-isopropylacrylamide)-grafted hyaluronic acid (Hyal-pN) functionalized with functional dendrimers, can bind BMP-2 (YPVHST) and TGFβ1 (HSNGLPL). The copolymer undergoes sol–gel transitions, which allows injection and progressive release of loaded growth factors¹²⁸. Similarly, TGFβ1-binding PAs (HSNGLPL) induce chondrogenic differentiation of MSCs in vitro and cartilage regeneration in a rabbit model¹²⁹. The same heptapeptide grafted on photo-crosslinkable GelMA also promotes chondrogenic differentiation of MSCs in vitro and accelerates cartilage tissue repair by recruiting TGFβ1 in situ in a full-thickness cartilage defect rabbit model¹³⁰. A fully deamidated version of HSNGLPL retains 25% more TGFβ1 molecules than the amidated version, with a significant increase in chondrogenic markers and matrix deposition by ATDC5 chondrogenic cells encapsulated in the TGFβ-binding peptide hydrogels¹³¹. Similar peptide amphiphilic hydrogels based on the peptide motif TSPHVPYGGGS can retain endogenous and exogenous BMP-2¹³², increasing the expression of osteogenic markers by C2C12 pre-myoblast cells in vitro and achieving complete spinal fusion in a rat model. Remarkably, this effect is achieved with low doses (1 μg BMP-2), compared with collagen controls (10 μg BMP-2)¹³³.

High growth factor retention and prolonged delivery are also important in cell therapies of myocardial infarction. Biotinylated nanofibres based on a ‘biotin sandwich’ of biotinylated insulin-like growth factor-1 (IGF-1) complexed with streptavidin bound to biotinylated peptides decreases caspase-3 cleavage, increases myocyte cross-sectional area, and improves systolic function after injection into the infarct zone of rat myocardium¹³⁴. Biotin–streptavidin bonds can also be exploited to tether IGF-1 and TGFβ1 to the surface of self-assembling (KLDL)₃ nanofibres to controllably deliver these growth factors in vitro for potential applications in cartilage tissue regeneration¹³⁵. Through non-specific peptide–growth factor interactions, a RADA16-II hydrogel can sustainably deliver platelet-derived growth factor (PDGF) into the myocardium of rats, decreasing infarct size after ischaemia and reperfusion¹³⁶.

Peptides binding growth factors through heparin-mimetic and heparin-binding domains

Glycan-mimetic peptides provide a versatile toolbox to bind multiple growth factors. Glycans bind to more than 300 secreted or membrane-bound proteins, such as growth factors, enzymes and chemokines, thereby regulating various biological processes. For example, the GAGs heparan sulfate and heparin promiscuously bind several growth factors. PAs can be designed to display dominant motifs of sulfated polysaccharides that bind and activate multiple growth factors¹³⁷; here, the PA nanofibres are glycoconjugated with N-acetylglucosamine (GlcNAc) monosaccharides to interact with heparan-sulfate-binding growth factors, such as BMP-2, BMP-4, FGF-1, FGF-2, VEGF, Shh, and the growth factor inhibitor Noggin, to promote full spinal fusion in a rat model with low sub-therapeutic doses of BMP-2 (100 ng)¹³⁷. GAG-mimetic peptide amphiphilic systems, based on the peptide epitope VVAGEGDK(p-sulfobenzoyl)S can also sequester BMP-2 and promote in vitro osteogenesis of Saos-2 osteosarcoma cells¹³⁸. Similarly, heparin-mimetic peptide nanofibres bind VEGF and induce angiogenesis in vitro and in vivo¹³⁹. Other similar systems show moderate-to-high binding affinity to growth factors, including hepatocyte growth factor (HGF), FGF-2, BMP-2 and NGF¹⁴⁰.

Heparin, in particular, has high binding affinity to growth factors, and thus, peptides can be explored that bind heparin to subsequently bind growth factors. For example, the sequence LRKKLGKA, derived from the Cardin–Weintraub heparin-binding consensus sequence XBBBXXBX (where X and B are hydrophobic and basic amino acids, respectively), can be applied to design heparin-binding peptide amphiphilics. Owing to its anionic nature, heparin co-assembles with positively charged peptides, generating hydrogels that bind VEGF and FGF-2 to promote vascularization in a rat corneal assay¹⁴¹. These heparin-binding peptide amphiphilics also promote angiogenesis in the dorsal skinfold chamber¹⁴² and infarction¹⁴³ models, and they can infilitrate the interior of islet cells in vitro, which may be explored for the treatment of diabetes type 1¹⁴⁴. Retention and delivery of VEGF can also be achieved in a RADA16-I hydrogel¹⁴⁵ that contains GAG-mimetic and GAG-binding peptides.

Peptide sequences binding other ECM components

Peptides binding ECM molecules through selective binding

The capacity to bind multiple molecular and biological components is key to emulate the compositional complexity of native tissues. In particular, selective binding between peptides and ECM components may enable the design of hydrogels that can controllably present or release these macromolecules. For example, a poly(allyl amine hydrochloride) hydrogel selectively binds a carcinoembryonic antigen, a glycosylated surface protein overexpressed by several carcinomas¹⁴⁶. Similarly, poly(methacrylic acid) hydrogels decorated with hepsin-targeting peptides (IPLVVPL) selectively bind hepsin-overexpressing tumour cells¹⁴⁷. Nap-FFG peptides are low-molecular-weight gelators that selectively bind and self-assemble into coatings at the surface of platelets, preventing human platelet aggregation in vitro¹⁴⁸. Tunable assembly of Fmoc-dipeptides (that is, Fmoc-YN, Fmoc-YS, Fmoc-YL and Fmoc-VL) with β-lactoglobulin and bovine serum albumin (BSA) can be achieved by varying amino acid polarity¹⁴⁹. FF-based hydrogels can also be designed to self-assemble in the presence of haemoglobin, myoglobin, serum albumin and fibrinogen, by exploiting covalent Schiff base bonding¹⁵⁰. In addition, supramolecular systems can be engineered based on tyrosine and pyridine-containing gelators to selectively bind nickel (Ni²⁺)¹⁵¹ and chloride ions (Cl⁻)¹⁵².

Peptides binding ECM molecules through non-selective binding

Peptides that enable non-selective binding of ECM components based on complementary charges and hydrophobic forces allow the incorporation of multiple macromolecules and the assembly of materials with a high level of structural hierarchy and functionality. For example, non-specific interactions at liquid–liquid interfaces between a positively charged PA (V₃A₃K₃) and negatively charged hyaluronic acid results in the formation of a diffusion barrier and subsequent co-assembly of the peptide and hyaluronic acid into structures of different sizes¹⁵³. Similar co-assembling approaches by non-specific interactions can be applied to organize proteins into hierarchical ensembles; for example, by tuning the charge of PAs (K2, K3, K4), the conformation of disordered elastin-like polypeptides (ELPs) can be modulated to trigger co-assembly mechanisms that result in different hierarchical material structures and properties, including the capacity to grow and heal¹⁵⁴. This approach enables the use of hydrodynamic forces to hierarchically guide assembly¹⁵⁵, organize exogenous components, such as graphene oxide¹⁵⁶, or tune peptide structures (Fmoc-FFK) to increase material stability¹⁵⁷. Further modulation of the charge density and epitope presentation of PAs allows co-assembly with multiple ECM components to generate complex, yet controlled, cell-instructive matrices; for example, to model the tumour microenvironment (TME) of ovarian cancer, keratin can be co-assembled with multiple peptides (VPGIGH₂K for hydrogel stability, RGDS for cell adhesion, GHK for angiogenesis)¹⁵⁸, and to recreate the TME of pancreatic cancer, fibronectin, collagen, laminin and hyaluronan can be integrated in an EEE-containing PA¹⁵⁹. Tough hydrogels can be created by merging PAs with polypeptides mimicking resilin¹⁶⁰, a tough and extensible rubber-like protein found in insects, or Laponite¹⁶¹.

Peptides binding ions to tailor organic–inorganic interactions

ECMs of some tissues, such as bone and enamel, are nanocomposites of organic and inorganic phases, which are hierarchically organized to provide high strength and fracture toughness. In these tissues, the local distribution and concentration of ions and minerals, such as calcium and phosphate deposits, are essential to ensure structure and function. For example, in bone, the organic phase is mainly collagen type I, whereas the inorganic phase is hydroxyapatite, a naturally occurring mineral form of calcium apatite. Peptides have an important role in biomineralization, guiding organic–inorganic interactions. In particular, hydroxyapatite aggregates and its crystal formation can be tuned by interaction between negatively and positively charged amino acids (for example, glutamate and arginine) with Ca²⁺ and PO₄³⁻ ions. Fmoc-FF, Fmoc-R and hydroxyapatite phases can be combined to create a multicomponent peptide-based scaffold for bone regeneration. In this system, Fmoc-FF provides structural rigidity, and Fmoc-R serves as template for biomineralization owing to the presence of arginine amino acids¹⁶². These hydroxyapatite-containing hydrogels are biocompatible and mechanically robust (stiffness up to 30 kPa) and can thus be applied in cell culture as biomimetic scaffolds for bone regeneration studies¹⁶². Fmoc-FF/S can also be combined with Fmoc-RGD and hydroxyapatite nanocrystals to deposit hydroxyapatite on RGD motifs, from R and D amino acids, and stimulate the differentiation of Raw 264.7 macrophages into osteoclasts¹⁶³. In addition, peptide sequences can be rationally designed to direct biomineralization; for example, the dodecapeptide NPYHPTIPQSVH binds hydroxyapatite crystal surfaces¹⁶⁴, and the heptapeptide MLPHHGA, conjugated to a β-hairpin-forming sequence (MDG1), directs hydroxyapatite formation in the presence of cementoblast cells¹⁶⁵.

In dental enamel, the organic matrix plays a crucial role in the development of its hierarchical inorganic structure and mechanical properties. Negatively charged macromolecules of the enamel ECM, including glycoproteins and proteins, such as amelogenin, enamelin and amelotin¹⁶⁶, bind Ca²⁺ ions and initiate apatite crystal nucleation and growth. To mimic this process, a low-viscosity solution of an anionic P11-4 self-assembling peptide can be designed that infiltrates and polymerizes into caries lesions, providing a biomimetic scaffold for hydroxyapatite nucleation. This peptide increases enamel mineralization and de novo crystalline hydroxyapatite depositions in vitro, and achieved mineralization of enamel in a clinical safety trial (UK National Research Ethics System; project number [10/H1207/75]) conducted in healthy adults with class V ‘white spot’ lesions¹⁶⁷. Mimicking enamel glycoproteins, 26 and 32 amino acid amelogenin-inspired peptides (P26 and P32) can be engineered as hydrogels to promote the bottom-up formation of multilayered hydroxyapatite crystals on sectioned human molar teeth¹⁶⁸. Similarly, enamel remineralization can be achieved with shorter amelogenin mimics, such as LEAWPATDKTKREEVD¹⁶⁹ and TKREEVD¹⁷⁰, and with the tripeptide motif (DSS) derived from dentin phosphoprotein¹⁷¹. Moreover, the peptide DDDEEKFLRRIGRFG, derived from statherin, a protein present in saliva, can control fluorapatite formation¹⁷².

Peptide sequences to create dynamic matrices

Peptides cleaved by matrix metalloproteinases

In healthy tissues, ECM deposition and degradation are tightly regulated to ensure homeostasis and matrix turnover. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are often overexpressed during tissue remodelling and in pathological conditions such as cancer¹⁷³. The MMP-sensitive sequence GPX₁G*LX₂G (in which * denotes the cleavage site, X₁ is A or L, and X₂ is G) can be incorporated into hydrogels to control their enzymatic degradation and to create cleavable synthetic matrices; for example, the MMP-2-sensitive sequence GTAGLIGQ¹⁷⁴ can be implemented as cleavable backbone of PA hydrogels to deliver cisplatin and test cancer treatment in vitro. Similarly, the MMP-9-sensitive sequence GL within the peptide FFAGLDD allows the in vitro release of doxorubicin from phenylacetyl-PAs, able to switch from micellar aggregates to fibres upon degradation of the MMP-sensitive site¹⁷⁵. A diphenylalanine-based peptide hydrogel, comprising an MMP-sensitive tetrapeptide (PLGL) linked to a neuroprotective hexapeptide (NAVSIQ), can be injected to the brain of mice and release the hexapeptide upon cleavage by MMP-9¹⁷⁶. Self-assembling PA–hyaluronic acid membranes that contain an MMP-1-cleavable epitope (GPQGIWGQ) allow the study of cell–ECM remodelling interactions¹⁷⁷.

Many MMP-sensitive peptides have been optimized to ensure high enzymatic sensitivity and specificity, including sequences derived from phage-display libraries, such as SGESPAYYTA (optimized for MMP-2)¹⁷⁸, GAPFALRLV (optimized for MMP-3 and MMP-7)¹⁷⁹, GGYAELRMGG (optimized for MMP-11)¹⁸⁰ and GPLGLWAR (optimized for MMP-13)¹⁸¹, GPQGIAGQ (sensitive to MMP-1, -2, -3, -7, -8 and -9¹⁸²), GPLGMRGL (cleavable by MMP-13¹⁸¹), and the sequences VPMSMRGG and IPESLRAG, which have high degradation rates with MMP-1 and MMP-2¹⁸³. MMP-sensitive epitopes have also been applied for the design of dynamic matrices, induction of morphogenesis, and enhanced cell invasion; for example, implementing the MMP-sensitive GPQGIWGQ epitope in PEG hydrogels allows proteolytic remodelling and organogenesis of intestinal stem cells in vitro¹⁸⁴. Similarly, renal tubulogenesis can be achieved in vitro by tuning the mechanics and degradability of heparin-based hydrogels containing the MMP-sensitive GPQGIWGQ sequence¹⁸⁵. Alginate gels modified with the MMP-sensitive PVGLIG epitope can deliver human MSCs and ensure cell invasion upon subcutaneous implantation in a mouse model and subsequent degradation¹⁸⁶.

Peptides cleaved by other enzymes

In addition to MMPs, other enzymes have been explored to create degradable peptide-based hydrogels; for example, PAs that display the consensus sequence RRX₁SX₂, where X₁ can be any residue and X₂ must be a hydrophobic residue, are sensitive to cleavage by protein kinase A (PKA)¹⁸⁷. Accordingly, PA nanofibres displaying RRASV assemble and disassemble in response to a specific enzyme. When exposed to PKA, the nanofibres are phosphorylated and disassemble, whereas in the presence of ALP, the phosphate groups are cleaved, restoring the assembly capabilities of the system¹⁸⁷. The serine protease α-chymotrypsin cleaves the sequence KKFFVLK next to the F residues, resulting in products with different tendencies to form nanotubes or spherical micelles¹⁸⁸. Similarly, cathepsin-sensitive sequences can be exploited to create degradable matrices; for example, PEGDA hydrogels grafted with the cathepsin-K-sensitive peptide GGGMGPSGPWGGK specifically degrade in response to osteoclasts, but not in response to osteoblasts, in bone resorption studies using porcine cortical bone slices as substrates¹⁸⁹. The cathepsin-B-cleavable peptide AVPIAQFRRG can be conjugated to a pro-apoptotic peptide drug and doxorubicin to achieve high cancer cell specificity in tumour-bearing mice¹⁹⁰. In addition, by incorporating ester backbones in β-sheet nanofibres and gels, they can be made degradable by hydrolysis¹⁹¹. Therefore, degradation can be tailored using specific peptides and by varying their secondary structure¹⁹² or amino acids composition¹⁹³ to create cell-responsive, dynamic biomaterials conducive to cell remodelling and matrix turnover (Fig. 3d, Table 3 and Supplementary Table 3).

Table 3.

Enzymatically cleavable and degradable peptide sequences

Function	Enzyme	Epitope
Enzymatically cleavable	MMP-1	GPQGIWGQ177,184,185
		VPMSMRGG183
		IPESLRAG183
	MMP-2	GTAGLIGQ174,243
		PVGLIG186,244,245
		SLGK246
		LDLPVGLIGKLD247
		GPQGIAGQ248
		IPVSLRSG248
		SGESPAYYTA178
		VPMSMRGG183
		IPESLRAG183
	MMP-3	GAPFALRLV179
	MMP-7	GAPFALRLV179
	MMP-9	FFAGLDD175
		PLGL176
	MMP-11	GGYAELRMGG180
	MMP-13	PTG-XKV (X = I, L, F, A)193
		GPLGYLWAR181
		GPLGMRGL181
	PKA	RRASV187
	Cathepsin B	GFLG249
		AVPIAQFRRG190
	Cathepsin K	GGMGPSGPWGGK189
	Thermolysin	FF, X (X = G, A, V, L, F, P)250
	α-chymotrypsin	KKFFVLK188
		FEFK251
		FKFE252
		F, W, OMe, OEt253
Degradable peptide	No enzyme — hydrolysis	QQKFQXQFEQQ (X = glycolic acid, lactic acid, 2-hydroxycaproic acid, 3-phenyllactic acid)191

MMP, matrix metalloproteinase; PKA, protein kinase A.

Hydrogels with synergistic peptide motifs

To recapitulate the dynamic complexity of the ECM, biomaterial designs should allow spatiotemporal control, avoid epitope redundancies and consider feedback loops. The incorporation of cleavable peptides enables matrix turnover, spatiotemporal changes and feedback controls driven by specific conditions (for example, pH changes, enzymes). Furthermore, peptides can be designed to target specific cell receptors and biological effects to avoid redundancies. In addition, self-assembling peptides allow the implementation of multiple signals within the same biomaterial through interpenetration of different epitope-displaying peptide nanofibres. Such 3D synthetic hydrogels containing multiple peptide epitopes can achieve synergistic and additive effects (Supplementary Fig. 1, Table 4 and Supplementary Table 4).

Table 4.

Peptide sequences with synergistic and additive effects

Function	Source	Epitope
Cell adhesion	Fibronectin	RGDS, PHSRN196,254
	Fibronectin, laminin	RGDS, IKVAV, YIGSR255
	Fibronectin, laminin	RGDS, REDV, IKVAV, YIGSR197
	Fibronectin, laminin	FRGDF, DIKVAV198
	Laminin	IKVAV, YIGSR199
	Fibronectin, cadherin	RGD, HAVDIGGGK256
	Fibronectin	FNIII9-10 + FNIII12-14200
	Fibronectin, laminin, collagen	GRGDSP, YIGSR, TAGSCLRKFST44
	Fibronectin, tenascin C	RGD, Tenascin C257
Cell adhesion and growth factor-mimetic/binding	Marrow homing-, heparin-binding motif, fibronectin	SKPPGTSS + FHRRIKA + PRGDSGYRGDS202
	Fibronectin, VEGF	GPRGDSGYRGDS + KLTWQELYQLKYKGI203
	Fibronectin, BMP-2	GGRGDS + KIPKASSVPTELS-AISTLYL258
	Fibronectin, vitronectin, laminin, IGF-1	(GRGDSP)4K + FRHRNRKGY + IKVAV, IGF-1204
	Fibronectin, collagen, VEGF	RGDS + DGEA + SVVYGLR208
	Laminin, BDNF	IKVAV + RGIDKRHWNSQ205
Growth factor-mimetic	NGF, BDNF	CTDIKGKCTGACDGKQC + RGIDKRHWNSQ95
	VEGF, BDNF	KLTWQELYQLKYKGI + RGIDKRHWNSQ209
Other combinations	Fibronectin, MMP-2-cleavable	RGDS + LRG210
		RGD + TPGPQGIAGQ211
	Fibronectin, statherin	RGDS + DDDEEKFLRRIGRFG207
	Fibronectin, hydroxyapatite	RGDS + REDV + HAP206
	Entactin/nidogen, vitronectin, vWF, netrin-1, fibronectin, collagen I/IX, fibrinogen, osteopontin, thrombospondin, tenascin-C, laminins, MMP-1, -2, -3, -7, -9, -13, -14.	RGD + QWRDTWAR + PHSRN-RGD + GFOGER + GPR + SVVYLR + YSMKKTTM + VTCG + DGEA + AEIDGIEL + IKVAV + LRE + YIGSR + LRG + IPESLRAG + GPLGLWAR + VPLSLYSG + VPLSLTMG + RPFSMIMG + SGESPAYYTA + VPMSMRGG212
	Collagen I, IV, XVIII, laminin α, laminin β, thrombospondin, tenascin C, periostin, nidogen, fibulin	DGEA + FYFDLR + IVRRADRAAVP + IKVAV + YIGSR + VTCG + EIDGIEL + ALMKYHILNTLQCSE + RGD + IPVSLRSG + RPFSMIMG + VPLSLYSG + VPLSLTMG + IPESLRAG213

BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; IGF, insulin-like growth factor; MMP, matrix metalloproteinase; NGF, nerve growth factor; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

Mixture of cell adhesion epitopes

ECM components often work synergistically; for example, fibronectin contains an RGDS loop and the pentapeptide PHSRN, which are located close to each other on the folded protein¹⁹⁴ and synergistically bind to α5β1 integrin if their distance is 3.2 nm (ref. ¹⁹⁵). Accordingly, a multidomain peptide system that displays RGDS and PHSRN at a distance between the epitopes of approximately 3.2 nm allows control over endothelial cell adhesion and integrin upregulation in vitro¹⁹⁶. By co-assembling different epitope-displaying chains, multiple peptide epitopes can be incorporated within the same hydrogel; for example, Q11 bearing RGDS, IKVAV, REDV and YIGSR can be co-assembled in various combinations to formulate homogeneous gels with different mechanical and biological properties, depending on the concentrations at which the peptide epitopes are mixed¹⁹⁷. Interestingly, RGDS and YIGSR exhibit an antagonist effect, whereas RGDS and IKVAV show an additive, synergistic effect¹⁹⁷. In addition, RGD and IKVAV can be mixed for cardiac tissue engineering¹⁹⁸, IKVAV and YIGSR for neurogenesis¹⁹⁹, and RGDS with phosphoserine for bone regeneration studies²⁹.

Mixture of cell adhesion and growth factor-binding epitopes

Cell adhesion peptides can also be combined with growth factor-binding epitopes; for example, polyethylene acrylate can be used to unfold fibronectin, by adsorption and assembly of the protein on the methyl groups of polyethylene acrylate monomers, which cause conformational changes in fibronectin and expose its epitopes (FNIII_9-10 and FNIII_12-14) to enable synergistic adhesion and growth factor signalling⁷³. 3D fibrin hydrogels functionalized with recombinant fibronectin that contains two bioactive fragments (FNIII_9-10 and FNIII_12-14) can be loaded with VEGF to promote wound healing in diabetic mice. The same platform can be loaded with BMP-2 for bone regeneration in non-healing bone defects²⁰⁰. A PEG hydrogel that incorporates full-length fibronectin and has tunable physical properties can retain BMP-2 and VEGF to aid in bone regeneration and vascularization in vivo²⁰¹. RAD16-I can be combined with fibronectin- and laminin-derived motifs, and the heparin-binding sequence TAGSCLRKFSTM, to culture hepatocytes⁴⁴. Moreover, synthetic stem cell niches can be designed using RADA16-I functionalized with SKPPGTSS, a bone-marrow homing motif, FHRRIKA, a heparin-binding motif, and PRGDSGYRGDS, a two-unit RGDS cell adhesion motif²⁰². In addition, RADA16 hydrogels can be decorated with RGD- and VEGF-derived epitopes to promote dentin-pulp regeneration²⁰³, and EAK16-II hydrogels can be functionalized with RGD and (GRGDSP)₄K from fibronectin, FRHRNRKGY from vitronectin, IKVAV from laminin, and IGF-1 for nervous tissue engineering²⁰⁴. Similarly, a hydrogel containing a BDNF-mimicking motif and the IKVAV sequence may achieve peripheral nerve regeneration when used as filler of hollow chitosan tubes to bridge 10-mm-long sciatic nerve defects in rats²⁰⁵.

A multi-epitope approach can also be pursued to stimulate osteogenesis and bone formation; for example, ELP-based membranes containing REDV, RGDS and the statherin-derived peptide DDDEEKFLRRIGRFG improve endothelialization²⁰⁶, cell adhesion and calcium phosphate binding, respectively, to increase osteoblastic differentiation in vitro and bone deposition in a critical-size rat calvarial defect model²⁰⁷. Similarly, a co-assembling PA hydrogel comprising RGDS for cell adhesion, SVVYGLR for angiogenesis, and DGEA for osteoblastic differentiation promotes growth of bone spheroids in a co-culture with endothelial cells and MSCs²⁰⁸.

Mixture of multiple growth factor-mimicking epitopes

Epitopes that signal multiple growth factors can be integrated in the same hydrogel; for example, a combination of the peptides CTDIKGKCTGACDGKQC (mimicking NGF) and RGIDKRHWNSQ (mimicking BDNF) can be used as filler of nerve conduits to synergistically promote peripheral nerve regeneration of 10-mm-long sciatic nerve defects in rats⁹⁵. Similarly, a BDNF-mimicking peptide can be combined with a VEGF-mimicking motif in a RADA16-based hydrogel to promote peripheral nerve regeneration and bridge a critical-size sciatic nerve gap in rats²⁰⁹. In addition to its angiogenic properties, VEGF has neurotrophic and neuroprotective effects. Therefore, rat Schwann cells cultured on the BDNF/VEGF-functionalized hydrogels show adhesion, spreading and markers of myelinization²⁰⁹. Furthermore, the functionalized hydrogel induces sprouting of myelinated fibres, regeneration of axons and functional motor recovery in vivo²⁰⁹.

Mixture of cell adhesion, growth factor-mimicking and enzyme-cleavable epitopes

Matrix dynamicity, cell adhesion and growth factor signalling can also be achieved within the same system. For example, the multidomain peptide system ‘SLanc’ incorporates the cell adhesion sequence RGDS, an MMP-2 sensitive peptide, and a VEGF-mimicking peptide. Subcutaneous injection of SLanc hydrogels in rats promotes the formation of a large number of blood vessels surrounding the implant in 7 days, with rapid host cell infiltration and lack of fibrous encapsulation⁸⁴. RGD can also be combined with an MMP-2-sensitive peptide sequence within a hydrogel carrier for the delivery of MSCs to increase MSC attachment and cell remodelling of the scaffold²¹⁰. A PA system containing RGD and a MMP-2-sensitive cleavage site (TPGPQGIAGQ) enables the biofabrication of tissue templates that ‘self-detach’ during cell culture for downstream applications²¹¹. A 3D PEG ECM-inspired hydrogel that contains 13 cell adhesion integrin-binding and 7 MMP-degradable peptides has been designed to mimic the diversity of proteins in human bone marrow. Here, the peptides were selected from native tissue through a proteomic-based and biomechanics method²¹². The specific combination of bone-marrow-derived adhesion cues and cleavable sites recreates the bulk modulus of native bone marrow and provides a biomimetic niche for MSC growth and differentiation²¹². Similarly, a PEG-based hydrogel containing nine integrin-binding and five MMP-degradable peptides present in native brain enables in vitro control and maintenance of astrocyte quiescence with minimal activation²¹³.

Peptide hydrogels on the market

Some peptide-based or peptide-displaying hydrogels have started to reach commercial and medical applications (Supplementary Table 5). For example, as reagents for biomedical research, PuraMatrix from 3-D Matrix (based on the RADARADARADARADARADA (RADA16) sequence), elastin-like recombinamers from Technical Proteins Nanobiotechnology S.L., and HydroMatrix from Sigma-Aldrich provide ECM-mimicking, biocompatible, reproducible and chemically defined platforms with tunable properties. Commercially available peptide hydrogels are also often used in tissue engineering for multiple cell types, including primary cells, cell lines and stem cells²¹⁴. In addition, peptide-based hydrogels are gaining interest as medical products; for example, RADA16 hydrogels are sold as injectable haemostatic agents, known as PuraStat, PuraBond and PuraSinus, for topical haemostatic control of postoperative bleeding in vascular anamostoses and endoscopic operations²¹⁵. Similarly, a chiral version of EAK16 (Sciobio) is used to treat severe bedsore and chronic diabetic ulcers, and to promote wound healing²¹⁶. Other peptide-based systems that have been tested in clinical trials are available on the market, including the mineralizing peptide P11-4 (Curolox) for the treatment of dental caries²¹⁷, and T45K (AC5)²¹⁸ for haemostatic control in patients undergoing antiplatelet therapy. Of note, clinical trials investigating peptide hydrogels are currently mainly focused on the treatment of dental caries, tooth diseases, haemorrhages and skin diseases.

Outlook

In the ECM, integrated biomolecules work together to communicate with cells, and regulate cell and tissue function. Bioengineered platforms aiming to recreate complex biological scenarios or develop efficient regenerative therapies must communicate with biological systems in a similar manner. Despite regulatory challenges (Box 1), mimicry of ECM functionalities in biomaterial design would enable the generation of biomaterials that closely recreate in vivo mechanisms⁸. Peptide epitopes can be used as active molecular building blocks of hydrogel materials, operating as cell-instructive supramolecular ensembles both in vitro and in vivo. Peptide epitopes can be designed to increase cell adhesion and migration, promote cell differentiation, trigger immune response, or regulate ECM turnover. Furthermore, multiple epitopes can be combined to trigger functional effects in a synergistic or additive manner, closely resembling how living systems operate^219,220. Moreover, peptides can serve as modular molecular building blocks capable of binding or co-assembling with ECM or synthetic macromolecules to improve the compositional diversity, structural complexity and functionality of biomaterials.

New functional peptide sequences can be discovered by phage display, in which a range of peptide-displaying phage particles are screened to identify peptides that bind to a specific target¹⁵. This approach does not require knowledge of full-length proteins¹⁵ and allows testing of 10⁹ peptide combinations, in addition to revealing the encoding genes a posteriori. Moreover, artificial intelligence and machine learning may offer an inverse (but complementary) approach, through which full-length protein structures can be analysed to identify new peptides. For example, secondary structures of nearly 2 million protein helices have been compared by a pattern-based search engine to identify and locate antimicrobial motifs and to provide design rules to control protein-protein interactions²²¹. Similarly, a combination of machine learning and multivariate outlier detection models has helped to discover new non-haemolytic peptides and provide de novo design guidelines²²². The list of functional peptides yet to be discovered is expected to grow rapidly. Indeed, in 2022, the artificial intelligence network AlphaFold predicted the structure of >200 million proteins from ~1 million species, covering almost every known protein²²³. From these proteins, peptide epitopes can be identified, in particular from disordered regions of proteins, which are thought to play a key role in protein structure and signalling²²⁴. These peptide discovery tools could provide new sequences that can be implemented in biomaterials to investigate disease mechanisms and cell–biomaterial interactions, and to design therapies²²⁵.

Furthermore, co-assembling peptides with structural proteins or inorganic fillers may enable the engineering of hydrogel nanocomposites; for example, co-assembly of short self-assembling peptides with macromolecules, such as resilin-like polypeptides¹⁶⁰, elastin-like proteins^154,156, or hyaluronic acid^88,153,177, increases the mechanical properties and bioactivity of the peptides. Similarly, peptides can be co-assembled with rigid 2D nanomaterials, such as graphene oxide²²⁶ and the synthetic clay Laponite⁸⁸, to formulate peptide-based constructs for load-bearing applications. Moreover, manufacturing strategies, such as inkjet printing, electrospinning and bioprinting, can be applied to create peptide hydrogel nanostructures with defined hierarchical organization and high geometrical complexity across multiple scales, mimicking the structural and functional hierarchy of biological systems¹².

Box 1 Translational considerations.

A key challenge in translating biomaterials that include peptide epitopes is the lack of affordable technologies and regulatory infrastructures for the scaled-up production of peptides. Currently, costs associated with peptide-based products depend on the chemical production and quality control requirements for products intended for medical use. Chemical peptide synthesis yields highly pure products (>90%) with minimal batch-to-batch variability and high consistency. However, the costs associated with large-scale production remain demanding. Peptide-based hydrogels can be designed to be functional at the nanoscale, but they exhibit low strength and can be difficult to control hierarchically across multiple size scales, which can represent a practical hurdle. Recognition of peptide-based hydrogels as medical devices and standardization of production would decrease production times and costs for material production, which may accelerate clinical translation of peptide-based hydrogels. In the framework of the International Council for Harmonization (ICH), medical regulatory authorities, including the US Food and Drug Administration (FDA), the European Medicines Agency (EMA) and the Medicines and Healthcare products Regulatory Agency (MHRA), have agreed on a set of regulatory criteria based on safety, quality and efficacy for the clinical trial authorization of a medicinal product or device for human use.

The clinical translation of peptide-based hydrogels will require careful consideration of regulatory classifications during the design stage. Indeed, hydrogels are considered medical devices when their therapeutic effect is ascribed solely to their intrinsic structures and inherent mechanical and physiochemical properties. This classification would include synthetic hydrogels displaying peptide epitopes along their structures (that is, nanofibres), because they inherently signal to cells, owing to their molecular design. Seeding cells or including cell-stimulating agents, such as growth factors, within peptide hydrogels raises the regulatory classification of hydrogels to medicinal products, as the mode of action is pharmacological. In such cases, a more extensive investigation of biocompatibility and therapeutic effects is needed before clinical translation. In particular, the route towards clinical translation would include characterization and biocompatibility tests in vivo (that is, ISO 10993-18, ISO 10993-5), and clinical trials (ISO 10993-6, ISO 14155) to evaluate systemic responses²⁵⁹.

Author contributions

C.L. and A.M. developed the manuscript outline. C.L. researched data for the article, made substantial contributions to discussions of the content and wrote the manuscript. C.L. and A.M. drafted, reviewed and edited the manuscript before submission.

Peer review

Peer review information

Nature Reviews Bioengineering thanks Seung-Woo Cho and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s44222-023-00055-3.