Peptide‐based supramolecular hydrogels for delivery of biologics

Abstract The demand for therapeutic biologics has rapidly grown over recent decades, creating a dramatic shift in the pharmaceutical industry from small molecule drugs to biological macromolecular therapeutics. As a result of their large size and innate instability, the systemic, topical, and local delivery of biologic drugs remains a highly challenging task. Although there exist many types of delivery vehicles, peptides and peptide conjugates have received continuously increasing interest as molecular blocks to create a great diversity of supramolecular nanostructures and hydrogels for the effective delivery of biologics, due to their inherent biocompatibility, tunable biodegradability, and responsiveness to various biological stimuli. In this context, we discuss the design principles of supramolecular hydrogels using small molecule peptides and peptide conjugates as molecular building units, and review the recent effort in using these materials for protein delivery and gene delivery.

cause issues for delivery such as limited solubility, chemical and physical instability, rapid renal clearance, low membrane permeability, and poor tissue penetration. 5,8 In addition, for systemically delivered therapeutic biologics, an in-depth understanding of both pharmacokinetics (PK) and pharmacodynamics (PD) is much needed. 9 Due to the high sensitivity of proteins to the gastrointestinal tract environment, the most preferred route for proteins-oral delivery-is such a complex and challenging issue that no orally taken protein drugs are currently approved by the FDA. In light of this, most protein drugs are administered intravenously, intramuscularly, or subcutaneously. To address the issues in protein delivery, controlled chemical modifications such as substitutions, acylation, and PEGylation have been widely explored to optimize the PK properties.
One concern associated with this strategy is the possibilities of altering the bioactivity and potency of the conjugated proteins when auxiliary groups or segments are added. Another methodology that has been extensively investigated is encapsulating therapeutic proteins in welldefined nanostructures or nanostructured materials such as nanoemulsions, liposomes, polymeric micelles, polymersomes, and hydrogels. 8,10 Gene therapy is another promising biological therapy that has drawn considerate attention which focuses on diseases that include cystic fibrosis, hemophilia, cancer, AIDS, cardiovascular pathologies and others. 11,12 As of 2012, more than 1,800 clinical trials on gene therapies had been carried out and the future trial activity is predictably positive. 13 By directly delivering nucleic acids of selected sequences into cells, multiple therapeutic effects could be achieved including correcting genetic defects, replacing or inactivating mutated genes, or overexpressing desired proteins. Apart from DNA, nucleases and several types of ribonucleic acid (RNA) such as small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA), may also be used to modulate gene expression. 12,14 Challenges also remain in delivering the large, fragile, and negatively charged DNA and RNA molecules with short lifetimes. 15,16 Viral vectors were the first and most widely used vehicles to deliver therapeutic genes with high efficiency, but concerns arose due to unwanted issues with pathogenicity, immune response and inflammatory reactions. 14 Therefore, non-viral vectors (e.g., gene gun, liposomes and particlemediated gene transfer) have also been developed as safer alternatives. 17 The continued growth of the biopharmaceutical market necessitates the development of efficient delivery systems that can address the numerous challenges in delivering biologics. For both protein carriers and gene vectors, their introduced delivery systems play a vital role in their PK and drug release profiles, and thus should be at least non-toxic and biocompatible. 18 In most cases of employing a nanocarrier, the PK of the entire system is usually determined by the properties of the drug carrier, rather than the drug itself. Carriers loaded with a considerable amount of therapeutic agents are expected to circulate for a sufficient period of time before preferentially accumulating in targeted sites. 19 For example, polyethylene glycol coated (PEGylated) liposomal carriers can prolong plasma half-life and prevent rapid renal clearance. 20 The loaded drugs are expected to release from the carriers with a desired releasing profile that is controlled by the design of the material properties of the carrier. In the cases of local delivery using hydrogels, the drug release from a hydrogel scaffold can be well-controlled by the mesh size, 5 the interactions between the drug and the scaffolding material, as well as the degradation rates of the hydrogel. Stimuli-responsive hydrogels, often referred to as smart hydrogels, have shown great promise due to their high loading capacity, high stability, and responsiveness to biological stimuli such as pH, temperature, ionic strength, enzymes, and redox state. [21][22][23] The delivery of therapeutics using hydrogels has been extensively explored over the past two decades, mainly focusing on synthetic polymer hydrogels. However, several limiting factors such as component and degradation product toxicity, pains caused by post-gelation swelling, and short-term release, remain to be overcome. Among the recently developed hydrogel platforms, self-assembling peptide nanofibrous hydrogels are particularly fascinating because of their biocompatibility, biodegradability, and low toxicity. [24][25][26] In general, the peptidebased supramolecular hydrogels are formed by physical entanglements of filamentous assemblies as a result of several types of non-covalent interactions among the peptidic building units, which could involve hydrogen bonding, hydrophobic interactions, electrostatic interactions, and p-p interactions (Figure 1a). They display a unique reversibility that most chemically cross-linked hydrogels do not possess. The entangled networks are able to encapsulate large biologics and offer a stimuli-triggered and well-controlled release capability ( Figure 1b).
Herein, we review the recent progress in the development of peptidebased supramolecular hydrogels designed for delivery of biologics. The functions and applications of peptide-based hydrogels in protein drug delivery and gene therapy will be highlighted.
When dissolved in solvents, amphiphiles such as surfactants, lipids, and amphiphilic block copolymers are able to self-assemble into various nanostructures (e.g., micelles, nanofibers, nanotubes, vesicles, etc.), with the solvophilic moieties facing towards the solvent and solvophobic moieties packed internally. The final morphology is determined by a balance of the hydrophobic attraction with hydrophilic or ionic repulsion, the system entropy, as well as some directional interactions such as hydrogen bonding or p-p stacking. Among nanostructures of diverse morphologies, one dimensional filaments show great tendency to form supramolecular networks through entanglement or inter filament interactions. Rapid responses to diverse external stimuli are easy to achieve because of the highly reversible property of non-covalent interactions.
With their hierarchically organized structures and the diversity of amino acid function, peptide-based materials are suitable as building blocks for forming bioresponsive hydrogels. Typical nanofibers LI ET AL. | 307 formed by self-assembling peptides have diameters between 5 nm and 15 nm and lengths on the micro-meter scale, and are able to further entangle to form a 3D network. The inherent biocompatibility and biodegradability are the most fascinating properties for peptidic hydrogels, because the degradation products can be metabolized and reused by cells. Moreover, the well-established solid-phase peptide synthesis (SPPS) protocols further contribute to the efficient and easy synthesis of peptides by researchers who have not had rigorous chemistry trainings. The varying functionality of the constituent amino acid side chains within peptides provide a broad basis for non-covalent interactions including hydrogen bonding (polar amino acids like glutamine), p-p stacking (aromatic amino acids like phenylalanine), hydrophobic collapse (non-polar amino acids like valine), and electrostatic interactions (acidic and basic amino acids like glutamic acid and lysine) (Figure 1c). 26 Meanwhile, each type of interaction may respond to different environmental stimuli. Thus one simple sequence-specific molecular modification may have a huge impact on the physical and chemical properties of the bulk hydrogels. 32 For example, the solubility of acidic and basic amino acids is determined by the degree of proton dissociation, a property that is pH and ionic strength dependent. 33 The self-assembly process of charged peptides can thus be facilitated by tuning the pH or adding salts to reduce the electrostatic repulsions and promote aggregation.
Many studies have been carried out on the bioresponsive properties of peptidic systems. 24,26,34 The design and development of self-assembling peptide nanofiber hydrogels will be discussed according to the classification of peptides and peptide conjugates. Some representative selfassembling peptide hydrogel systems are illustrated in Figure 2.

| P E P T I D E S
A variety of native peptides are able to self-assemble into filamentous networks. 25,35,36 Ulijn and coworkers have recently summarized the bioresponsive elements incorporated in short oligopeptide systems based on different secondary structural motifs including, b-sheets, b-hairpins, and helices and coiled-coils. 26 For example, the design principles for responsive b-sheet peptides usually consist of peptide chains with alternating cationic, hydrophobic, and anionic amino acid residues.
Inspired by Z-DNA binding protein zuotin, a number of ionic selfcomplementary peptides such as RADA16-I, RAD16-II, EAK-I, and EAK16-II that can self-assemble into well-defined nanofiber scaffolds were designed by Zhang et al. 37,38 These peptides contain both hydrophilic (charged) side chains and hydrophobic side chains on the different sides of self-assembled b-sheet structures. 37 Under physiological conditions of neutral pH and millimolar salt concentration, millions of b-sheets pack in parallel and assemble into individual nanofibers that can further form a transparent hydrogel with 99.5-99.9% water content (Figures 2a, b). 37 Both small molecules and proteins were utilized to show the sustained release through these peptide scaffolds depending on the molecular characteristics (e.g., size and shape) and scaffold properties (e.g., density of nanofibers). 39,40 These designer selfassembling peptide nanofiber scaffolds have been widely used in cell culture, reparative and regenerative medicine, and tissue engineering.
Recently, two-layered injectable self-assembling peptide hydrogels were used as a carrier for therapeutic antibodies. 41 The self-assembling Ac-(RADA) 4 -CONH 2 and Ac-(KLDL) 3 -CONH 2 peptide hydrogels were FIG URE 1 Schematic illustration of (a) the formation of peptide-based supramolecular hydrogels, (b) the encapsulation and release of biologics using supramolecular hydrogels as carriers, and (c) possible non-covalent interactions among peptide-based building blocks, including hydrophobic collapse, Van der Waals interactions, hydrogen bonding, p-p stacking, and electrostatic interactions shown to sustained release human antibodies for a period of over 3 months.
Schneider, Pochan, and coworkers developed a series of b-hairpin peptides that undergo fibril formation and triggered hydrogelation for various biomedical applications. [42][43][44][45][46] For example, MAX1(VKVKVKV KV D PPTKVKVKVKV-NH 2 ) is composed of high b-sheet propensity valine and charged lysine residues in an alternating manner. 47 An intermittent tetrapeptide (-V D PPT-) was designed to adopt a type II 0 turn structure that leads to b-hairpin formation. 48 The ability of MAX1 to assemble into a b-hairpin relies on a pH-promoted intramolecular folding event. 46 Under the conditions in which the lysine side chains of MAX1 are largely deprotonated, the intramolecular folding occurs with hydrophobic valine residues and hydrophilic lysine residues lined on the opposite face of the hairpin (Figure 2c). The b-hairpin structure can be stabilized through sub-sequent self-assembly of monomeric hairpins into nanofibers as a result of H-bond formation between distinct hairpins and hydrophobic association of the valine-rich faces of hairpins. 46 Importantly, the b-hairpin structure can be reversibly unfolded by charge repulsion between ionized lysine side chains through lowering of the pH. These (VK) n V D PPT(VK) nbased peptide hydrogels can be engineered to be transparent, shear recoverable, injectable and pH or ionic strength responsive, all important properties for biomedical applications. 44,45,49 The mesh size of the hydrogel could be readily controlled by tuning the peptide concentration and the rate of gel formation. In a typical example, by replacing one lysine in MAX1 at position 15 with a glutamic acid, MAX8 (VKVKVKV KV D PPTKVEVKVKV-NH 2 ) was shown to self-assemble and gelate at a much faster rate than MAX1 and form more rigid gels with smaller mesh sizes. 44 Release studies were carried out using model dextran and protein probes, indicating the well-controlled release of macromolecules from b-hairpin peptide hydrogels. 43 Recently, MAX8 was shown to be able to deliver nerve growth factor and brain-derived neurotrophic factor (BDNF), as well as active chemotherapeutics (vincristine). 46,50 Biomimetic collagen-inspired hydrogels have been widely developed and applied to cell scaffolding applications. 51,[56][57][58][59] Conticello and coworkers used a synthetic peptide system based on a Gly-Xaa-Yaa repeat sequence that can self-assemble into collagen-like homotrimeric helices driven through electrostatic interactions. 60 Hartgerink and coworkers utilized the short collagen-like peptides (Pro-Lys-Gly) 4 (Pro-Hyp-Gly) 4 (Asp-Hyp-Gly) 4 to mimic the multi-hierarchical self-assembly of a collagen fiber from triple helix to nanofiber and hydrogel (Figures 2d, e). 51,58 The collagen mimetic peptide is able to exhibit triple helical packing and assemble into nanofiber morphologies in a quasihexagonal fashion under a wide range of buffers and ionic strengths, stabilized by salt-bridged hydrogen bonds between lysine and aspartate on an adjacent lagging peptide offset by three amino acids. 51 The collagen mimetic hydrogels formed by the triple helix nanofibers were shown to have similar storage modulus to that typically observed for a natural collagen hydrogel and was temperature sensitive due to the unfolding of the triple helix at 40-418C. 51 Later on, by comparing two classes of collagen mimetic peptides with the same composition but different domain arrangements, they found that larger sticky-ended nucleation domains result in rapid fiber formation and eventual precipitation or gelation. 58  sufficiently large, they can adopt a-helices, b-sheets, or random coil structures depending on the peptide sequence and self-associate to form fibrillar assemblies that further entangle to form 3D networks. 67 A detailed understanding of DCH structure-property relationships was established to achieve a high level of control over the gel properties. 67,68 Polypeptides have shown many advantages including temperature stability, structural tunability, rapid healing after stress, and injectability. 53 Recently, Sofroniew and coworkers demonstrated that a DCH system can provide significantly longer delivery of nerve growth factor maintaining activity for at least 4 weeks, suggesting that DCH have promise as delivery vehicles for therapeutic applications. 69

| P E P T I D E C O N J U G A T E S
Amino acids or short peptides conjugated to alkyl chains or aromatic groups have been extensively investigated over the recent decades.
Both alkyl chains and aromatic groups serve as the hydrophobic segment to promote the aggregation. Bhattacharya et al. reported the gelation of oil using N-alkanoyl-L-alanine amphiphiles in an immiscible system. 70 Recently, they presented a two-component hydrogelator composed of an N-C 16 H 33 -chain-appended L-alanine amphiphile and a redox-active viologen-based partner. 71 A lamellar-type of morphology formed by this two-component system can lead to redox-active 3Dfibrous networks. Another phenylglycine-based amphiphilic gelator was found to exhibit phase selective gelation within 90 s. 72 With the increased interactions and stability that p-p stacking can provide, aryl motifs such as ferrocenyl, fluorenyl, naphthyl and phenylalanine have become prevalent components of hydrogelators. 25 Fmoc, a common amino-protecting group, has been shown to facilitate the formation of self-assembled hydrogels (Figures 2g, h). Fmoc-protected diphenylalanine was one of the earliest reported dipeptides that were able to selfassembled into fibrillar structures and form a rigid hydrogel with less than 1% peptide material in aqueous solution. 73 Noting the vital function of the Fmoc group in the gelation, Ikeda et al. proposed to replace this Fmoc group with a stimuli-triggered degradation unit, such as 6bromo-7-hydroxycoumarin-4-yl methoxycarbonyl (Bhcmoc), to achieve an oxidation and pH responsive hydrogel, BPmoc-FF. 74 The Xu Lab has made significant contributions to Fmoc protected amino acids such as Fmoc-Lys and Fmoc-phenylalanine. [75][76][77] with Fmoc, the self-assembly and hydrogelation of naphthyl-protected amino acids and peptides hydrogelators were also widely explored. [78][79][80][81] Ulijn and coworkers have investigated a library of Fmoc-dipeptides including Fmoc-diglycine, Fmoc-dialanine, Fmocdiphenylanaline, and so on. 52,82,83 Self-assembled nanofibers can be easily prepared by suspending the Fmoc-dipeptides in water, with the aromatic groups sitting in the core driven by p-p stacking. It was found that the conditions under which gelation took place varied with the peptide type and some gels were stable under physiological conditions. 52 Later, a novel model comprising p-p interlocked b-sheets architecture was constructed, a design based on the anti-parallel b-sheets and anti-parallel p-stacking shown by spectroscopy in the self-assembly of Fmoc-FF. 82 Recently, by investigating a series of aromatic short peptides, Ulijn and coworkers demonstrated the nanofibrous networks can form at the organic/aqueous interface of a biphasic solvent system. Hand shaking of this mixture leads to formation of micro-sized emulsion droplets with exceptional stability and potential applications for drug encapsulation and delivery. 84 Tovar and coworkers developed a series of "peptide-p-peptide" triblock molecules that self-assemble into fairly uniform tape-like nanostructures by embedding a diverse range of p-electron units directly within peptide backbones. 85,86 These assembled peptide networks can further aggregate into entangled fibrillar superstructures through intimate p-p contacts and electronic delocalization. 85 Xu and coworkers pioneered the design of b-amino acid and Damino acid derivatives for creating supramolecular hydrogels. [87][88][89][90][91][92] Such b-amino acid derived hydrogels were shown to be proteolytically resistant with prolonged bioavailability, yet retained their sensitivity to the enzyme (phosphatase) that triggers their formation. 87,88 Although the unique stereochemistry of D-amino acids imparts similar stability with b-amino acids towards most of the endogenous enzymes, its low cellular uptake remains challenging for biomedical applications. 91 Xu and coworker have also designed a novel class of supramolecular hydrogelators based on conjugates of nucleobases and short peptides. 93,94 These nucleopeptides such as nucleobase-FRGD were shown to exhibit long-term biostability, resisting degradation by proteinase K. In a recent study, the first use of heteronucleopeptides to generate biocompatible and biostable hydrogels was reported. 95 Two structurally distinct peptides were selected from the interface of a heterodimer of proteins and then conjugated with nucleobases. Supramolecular hydrogels formed by simple mixing of the heteronucleopeptides in water displayed non-b-sheet secondary structures, which may preserve the specific functions of a-helical and random-coil motifs.
Peptide amphiphiles (PAs) are peptide conjugates containing one or more linear alkyl chains that structurally resemble small molecule surfactants (Figures 2i-l). 54,96-101 Stupp and coworkers have designed a series of PAs that can self-assemble into nanofibers that enmesh into hydrogels under the physiological condition. The nanofibers are composed of b-sheets with alkyl chains packing in the core of the fibers and peptide segment exposed to the aqueous environment. 54,96,97,99 A variety of peptide sequences can be ideally engineered into the peptide region of the PA to realize desired functions such as solubility enhancement or cell adhesion. 54,97 Self-assembly of PA molecules into nanofiber matrices could be mediated by metal ions 102 or aided by charged amino acid residues. 103 In another work, tubular hydrogels formed by circumferentially aligned peptide amphiphile nanofibers were shown to encapsulate vascular cells and direct cellular organization. 104 Recently, C 16 -V 2 A 2 E 2 -NH 2 PA hydrogels were used as a sonic hedgehog protein delivery system for the treatment of erectile dysfunction, suggesting the PA hydrogels have potentially broad applications as protein vehicles. 105 Tirrell and coworkers designed pH-responsive branched peptide amphiphile composed of histidine and serine amino acids conjugated to a palmitoyl tail. 106 These PA solutions are able to switch from viscoelastic liquids to an injectable tissue scaffold above pH 6.5 as a result of the protonation of histidine. More recently, a PA hydrogel consisting of C 16 GSH was optimized to have greater utility for peripheral nerve repair compared with a commercially available collagen gel. 107 The peptides or polypeptides conjugated to a polymer have been shown to be effective molecular gelators. Peptide-polymer conjugates combine the desired functionality of peptides and synthetic polymers. 108,109 For example, a diblock or triblock peptide-PEG hydrogel system with peptide segment were designed from the coiled coil region of fibrin. 110 By studying the FEFEFKFK and poly(N-isopropylacrylamide) conjugates, Maslovskis et al. found that the fibrillar network and the lower critical solution temperature of the polymer were not affected by each other. 55 However, the interaction between the polymer and peptide will significantly influence the polymer conformation and the mechanical properties of the hydrogels. Recently, Chmielewski and coworkers designed a thermosensitively tunable hydrogel using collagen-mimetic peptides and PEG star polymer, suggesting potential applications in protein delivery and tissue regeneration. 111 Although all of these above-mentioned peptide-based hydrogel systems have unique design principles and properties, what remains essentially the same is the spontaneous or triggered hydrogelation after self-assembly driven by supramolecular interactions, which provides the foundation for delivery of biologics. Of particular note is that these peptide systems themselves can be used as bioactive agents by incorporating or directly using therapeutic or other functional peptide sequences into the hydrogel networks. [112][113][114][115][116] The incorporation of the well-known cell adhesion motif RGD into the peptide nanofibers has been investigated by many research groups. [117][118][119] In one example, self-assembled nanofibers with bioactive signal displayed on the surface are shown to promote adhesion of encapsulated dermal fibroblasts with subsequent cell spreading and proliferation. 120 Studies on the significant immunogenicity of the self-assembling peptide (such as OVA-Q11) as well as other nonimmunogenic peptide by Collier and coworkers showed broad applications in modulating adaptive immune responses. 121,122 Other protein-derived sequences such as IKVAV sequence have also received much attention to functionalize the peptide networks. 123,124 These peptide-based active groups may not be classified as "biologics" in terms of their chemical origins, relatively smaller size, simpler structures, and easily controlled chemistry. In fact, peptides bridge the gap between small molecules (typically <500 Da) and biologics (typically >5,000 Da) and can potentially combine the advantages of small molecules and biologics, offering specificity and potency similar to larger biologics but ease for synthesis. 125 However, the discussion on the small peptide-based therapeutics is beyond the scope of this review.

| P R O T E I N D E LI V ER Y
Challenges in the delivery of therapeutic proteins derive from their large size and complicated structures that lead to limited solubility, stability, and membrane permeability. Desirable properties for the ideal protein delivery vehicles include high protein loading and biocompatibility; the ability to shield the proteins from enzymatic degradation and rapid clearance; the function to provide sustained release in a desired and therapeutically effective way. Among many kinds of delivery systems, the self-assembling peptide nanofibrous hydrogels are one of the most fascinating candidates for protein delivery applications. The highwater content in peptide-based supramolecular hydrogels provides the open space to store biologics. Either hydrophilic or hydrophobic molecules could be entrapped in the inter-or intrafiber areas, greatly increasing the solubility of biologics to be delivered. Generally, biologics can be directly encapsulated into the hydrogel during the selfassembly and gelation process by simply mixing with hydrogelators under mild conditions. The peptide-based supramolecular hydrogels are supposed to ideally mimic the extracellular matrix and expected to be more biocompatible than polymer hydrogels. This provides the foundation for protection and stabilization of biologics to avoid their inactivation before they reach the desired location. Based on their potential in presenting sustained release, self-assembling peptide hydrogels are of high interest to be engineered to optimize the release profile of proteins in a desirable fashion. Although, the delivery of small molecule drugs using supramolecular hydrogels have been largely LI ET AL.

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reported, peptidic hydrogel based delivery of macromolecules such as proteins and gene therapeutics is still in its infancy. Recent advances in protein delivery using self-assembling peptide hydrogels and corresponding applications are summarized in Table 1.
The largest and fastest growing protein therapeutics in the United States are antibody-related drugs for the treatment of many diseases such as cancer, chronic inflammatory disease, cardiovascular, and infectious diseases. 6 Local and sustained antibody release by hydrogels can reduce both the associated toxicity and the frequent dosages necessitated by the limited life time. The self-assembling peptide hydrogels are of great promise to serve as the carriers of therapeutic antibodies.
However, very few peptide-based hydrogels have been reported for this application thus far. Lysozyme, trypsin inhibitor, BSA, and IgG were utilized to study the release from RADA16 systems developed in the Zhang Lab. 40 The release rate was sensitive to the physical size of the proteins, resulting in the slowest release of IgG (more than 60 h before reaching a steady concentration) among the examined proteins due to possessing the largest molecular weight. CD spectra confirmed the nearly identical structures of IgG released from the hydrogel with the native IgG. The monoclonal IgG used here is specific for the 1D4 sequence at the C-terminus of native bovine rhodopsin. A comparison of the association constant k a or dissociation constant k d , the binding affinity between the monoclonal IgG and rhodopsin (antigen) did not change significantly before and after being released through the peptide hydrogel for 48 h. This result suggested that the functionality of IgG is maintained during the release from the peptide hydrogel. In a subsequent study, Zhang and coworkers reported a 3-month study on the release kinetics for human IgG through a two-layered hydrogel structure with an Ac-(RADA) 4 -CONH 2 core and Ac-(KLDL) 3 -CONH 2 shell. 41 This two-layered hydrogel system formed by a two-step gelation process allows for 100% IgG loading efficiency because of the high-water content (up to 99.5%). Results showed a sustained release of human IgG over 3 months without compromising its biological activity.

| GROWTH FACTORS
Growth factors are proteins that are able to stimulate cellular growth and differentiation and regulate a variety of cellular processes, making them useful in tissue engineering and regenerative medicine. For this reason, they are also one of the most commonly investigated protein classes delivered by self-assembling peptide hydrogels, given the widespread use of hydrogels in this field.
The peptide amphiphile (PA) system developed in the Stupp lab serves as a natural and powerful platform for delivery of growth factors and related cells. 54,102,105,112,113,129,[133][134][135][136][137] In one example, heparin-binding peptide amphiphile (HBPA) was designed with a heparin-binding domain to specifically bind heparan sulfate-like gylcosaminoglycans (HSGAG). 114 This particular design provides the

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The Zhang Lab designed the biotinylated RAD16-II peptide to deliver the cardiomyocyte growth and differentiation factor IGF-1 to the myocardium to improve cell therapy. 126 The tetravalent streptavidin was used to bind with both biotinylated RAD16-II peptides and biotinylated IGF-1 via a biotin sandwich strategy (Figure 4a), which was found to have no influence on the self-assembly process of the peptides. The IGF-1 was observed to be sustainably released for more than 28 days without compromising bioactivity, and increased the cross-sectional area of cardiomyocytes by 25% compared with cells embedded in hydrogels alone or with untethered IGF-1 (Figures 4b, c). To achieve better control over the pro-   -g). 153 The peptide sequence was carefully selected to non-covalently interact with the viral membrane and contain viral activity to the site of injection. mCherry lentivirus-loaded self-assembling Fmoc-peptide hydrogels that were injected into the mouse brain showed the least number and volume of mCherry1 cells in the host striatum delivery of virus alone. However, the similar density of mCherry1 cells proved that the transduction efficiency of the delivered virus was remained after releasing from the hydrogel networks. This novel method that combines the advantages of viral vectors and peptide hydrogels will enable the localized and efficient delivery of gene therapeutics. Although little progress has been made to use self-assembling peptide hydrogels for gene delivery, the pioneering works described above demonstrate their promising potential as nonviral gene vectors to delivery more types of gene therapeutics and promote gene therapies.

| F U T U R E PE R S P E C T IV E
In this review, we discussed the design principles of peptide-based supramolecular hydrogels in the context of protein delivery and gene First, the toxicity and biocompatibility of peptide-based hydrogels should be evaluated in a comprehensive manner since the biocompatibility is the basic requirement for in vivo applications. 154 It is well known that assembled peptides are much more resistant to enzymatic degradation than the monomeric ones. 155 There might also be a difference in toxicology between peptides in the assembled form and peptides in the soluble form. Second, an in-depth understanding of the biologics' release profile is essential for further optimization of the therapeutic outcomes. This would need rational and quantitative correlations of the physicochemical properties of the biologics (surface charge, water dispersity) with the network structures (mesh size, filament alignment, etc.) and materials properties (degradation rates, stiffness, surface chemistry, etc.). More efforts should also be devoted to developing hydrogels systems capable of stimuli-specific release of biologics as well as enzyme-specific degradation of the hydrogels. 155,156 Third, it is important to incorporate biologically active peptides into the hydrogel design to explore synergistic combination of peptide drugs/ epitopes with biologics. Such bioactive peptides could be used to identify a particular type of cells such as cancer cells, to co-stimulate cells for proliferation, migration, or differentiation, or simply to allow for cell adhesion.
Lastly, self-assembling peptide hydrogels have been mostly used in local delivery due to their high viscosity and low mobility. Although local delivery is able to attain higher concentrations of the biologics at the desired sites without necessarily involving a targeting strategy, systemic delivery is sometimes the more preferred approach due to its ease of administration, low invasive property, and better patient compliance. For this purpose, more effort should be devoted to investigating the peptide-based nanogel system for systemic delivery, as inspired by the recently developed polymeric nanogels. The nanogels are nanosized crosslinked networks mostly composed of synthetic polymers and are very promising as biologics carriers because of their high loading capacity, high stability, and responsiveness to environmental factors. [157][158][159][160] They are mostly produced by polymerization of monomers in nanoscale heterogeneous environments stabilized by surfactants or cross-linking of preformed polymers. These polymer-based nanogels have been largely investigated for protein and gene delivery. [161][162][163] The self-assembling peptide nanogels may be formed by self-assembly and gelation in a confined heterogeneous nanoenvironment and are expected to maintain their advantageous properties in systemic delivery of biologics. Further noteworthy studies include the development of self-assembling peptide nanogels and incorporation of targeting properties by adding specific peptide sequence for targeting or bioresponsive release. Although a long way ahead, we believe that self-assembling peptide hydrogels will exert significant influence on the efficient delivery of biologics.