Membrane-Disrupting Nanofibrous Peptide Hydrogels | ACS

Article pubs.acs.org/journal/abseba

Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Membrane-Disrupting Nanofibrous Peptide Hydrogels Biplab Sarkar,† Zain Siddiqui,† Peter K. Nguyen,† Namita Dube,‡ Wanyi Fu,§ Steven Park,∥ Shivani Jaisinghani,† Reshma Paul,† Stephen D. Kozuch,⊥ Daiyong Deng,# Patricia Iglesias-Montoro,† Mengyan Li,# David Sabatino,⊥ David S. Perlin,∥ Wen Zhang,§ Jagannath Mondal,‡ and Vivek A. Kumar*,†,¶,□ †

Department of Biomedical Engineering, New Jersey Institute of Technology, Newark, New Jersey 07102-1982, United States Center for Interdisciplinary Sciences, Tata Institute of Fundamental Research, 500075 Hyderabad, India § Department of Civil and Environmental Engineering, New Jersey Institute of Technology, Newark, New Jersey 07102-1982, United States ∥ Public Health Research Institute, Rutgers UniversityNew Jersey Medical School, Newark, New Jersey 07103, United States ⊥ Department of Chemistry and Biochemistry, Seton Hall University, South Orange, New Jersey 07079-2646, United States # Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, New Jersey 07102-1982, United States ¶ Department of Chemical and Materials Engineering, New Jersey Institute of Technology, Newark, New Jersey 07103, United States □ Department of Restorative Dentistry, Rutgers School of Dental Medicine, Newark, New Jersey 07103 United States

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ABSTRACT: Self-assembled peptide nanofibers can form biomimetic hydrogels at physiological pH and ionic strength through noncovalent and reversible interactions. Inspired by natural antimicrobial peptides, we designed a class of cationic amphiphilic self-assembled peptides (CASPs) that self-assemble into thixotropic nanofibrous hydrogels. These constructs employ amphiphilicity and high terminal charge density to disrupt bacterial membranes. Here, we focus on three aspects of the self-assembly of these hydrogels: (a) the material properties of the individual self-assembled nanofibers, (b) emergence of bulk-scale elasticity in the nanofibrous hydrogel, and (c) trade-off between the desirable material properties and antimicrobial efficacy. The design of the supramolecular nanofibers allows for higher-order noncovalent ionic cross-linking of the nanofibers into a viscoelastic network. We determine the stiffness of the self-assembled nanofibers via the peak force quantitative nanomechanical atomic force microscopy and the bulk-scale rheometry. The storage moduli depend on peptide concentration, ionic strength, and concentration of multivalent ionic crosslinker. CASP nanofibers are demonstrated to be effective against Pseudomonas aeruginosa colonies. We use nanomechanical analysis and microsecond-time scale coarse-grained simulation to elucidate the interaction between the peptides and bacterial membranes. We demonstrate that the membranes stiffen, contract, and buckle after binding to peptide nanofibers, allowing disruption of osmotic equilibrium between the intracellular and extracellular matrix. This is further associated with dramatic changes in cell morphology. Our studies suggest that self-assembled peptide nanofibrils can potentially acts as membranedisrupting antimicrobial agents, which can be formulated as injectable hydrogels with tunable material properties. KEYWORDS: self-assembly, hydrogel, peptide nanofibers, antimicrobial peptides, membrane disruption, noncovalent cross-linking

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INTRODUCTION

can form entangled or cross-linked hydrogels in physiologic conditions. There are two distinct levels of self-assembly involved in the formation of the thixotropic hydrogels from self-assembling peptides. First, the peptide monomers self-organize into linear supramolecular polymers (nanofibers) (Figure 1). The molec-

Self-assembling peptide hydrogels are promising biomaterials for potentiating in vivo responses in targeted tissue niches.1 βsheet forming peptides, in particular, are attractive targets for drug delivery and facile functionalization with terminal biofunctional moieties, such as growth factor mimics and immunomodulating domains.2−10 Such peptides can be interesting model systems to study bottom-up hierarchical self-assembly of peptide monomers into nanofibers (supramolecular polymers), which © XXXX American Chemical Society

Received: July 2, 2019 Accepted: July 26, 2019 Published: July 26, 2019 A

DOI: 10.1021/acsbiomaterials.9b00967 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Design and hierarchical self-assembly of the CASP nanofibrils. (A) The peptides have a central domain consisting of alternating hydrophilic (serine, shown in green) and hydrophobic (leucine, shown in red) residues. There are positively charged domains (lysine residues, shown in blue) of varying length at the termini. (B) Fibrillar self-assembly is driven by shielding of hydrophobic residues at the core of the nanofiber, as well as the formation of canonical β-sheet hydrogen bonds. However, the supramolecular polymerization is opposed by electrostatic repulsion among the charged terminal domains. This supramolecular frustration between the favorable and opposing factors determines the dynamic equilibria among the monomers and multimers.

defense against pathogens23 and even many bacterial defense systems against competing strains.24 These natural AMPs have inspired the design of synthetic cationic peptides that induce bacterial membrane permeabilization and lysis.18−22 Amphiphilic peptides have also been used as potent disruptors of viral lipid envelops.25 These peptides may be useful for facilitating healing of chronic wounds.26 One of the drawbacks of peptide therapeutics is rapid enzymatic clearance in vivo.27 A potential means to sustain the efficacy of such peptides is to attach them to self-assembling motifs, increasing the effective molecular weight (reducing susceptibility to enzymes) and slowing down physiological clearance mechanisms.28 Thus, capitalizing on the antimicrobial properties of the cationic peptides and muted degradation selfassembled scaffolds, we can develop implantable antimicrobial biomaterials with sustained efficacy. Self-assembled peptides thus may be a promising new class of antibacterial materials.29−34 These antimicrobial peptides can potentially have a more sustained efficacy profile compared to monomeric peptides. Here, we describe the design and mechanism of action of a set of self-assembled thixotropic nanofibrous hydrogels capable of disrupting bacterial membranes. These peptide-based hydrogels are effective against the Gram-negative bacteria Pseudomonas aeruginosa (PAO1).35 In silico and in vitro analyses suggest that the efficacy of the antimicrobial nanofibers is based on their

ular interactions involved at this level of assembly have been explored.11,12 The principal driving force is the hydrophobic effect mobilizing the hydrophobic side chains away from the aqueous environment into the core of the nanofiber (akin to a one-dimensional surfactant micelle).13 The directionality is driven by the canonical hydrogen bonds that align the building blocks into a semiflexible linear β-sheet assembly with long persistence lengths.1 Thus, the self-assembled nanofibers share characteristics of both semiflexible biopolymers14 and worm-like micelles.13 The next level of self-assembly involving the entanglement and cross-linking of the nanofibers remains less well understood.12,15−17 The repulsion among the like-charged nanofibers opposes the association of the nanofibers.11 Multivalent counterions, such as phosphate, can ionically cross-link these nanofibers.6,11 However, we lack a unified predictive model that can explain the variation and scaling of the bulk-scale mechanical properties of the hydrogel. These properties may depend on factors such as nanofiber stiffness, concentration of the peptide, charge of the building blocks, and the concentration of cross-linkers. Self-assembling peptide hydrogels, because of their polycationic amphiphilic nature, are also promising candidates for antimicrobial applications. Notably, natural antimicrobial peptides (AMPs) act by permeabilization of a pathogen’s membrane, leading to osmotic imbalance and lysis.18−22 Such peptides constitute a significant part of the body’s first-line B


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assembled nature of the constructs would ensure that the peptides would not diffuse rapidly away from the site of application, injection, or implantation. As a test set, these peptides allow us to control charge of an individual supramolecular building block, while preserving the central fibrillizing domain. We determine the stiffness of the individual supramolecular nanofibers as a function of terminal charge and correlate nanofiber stiffness to elasticity of the bulk material. We elucidate the scaling relation of the storage modulus to factors such as concentration of monomers and solvent parameters (e.g., ionic strength). Finally, we propose an explanation of a curious feature of the ionically cross-linked CASP gels: the relation between their storage modulus and applied strain. The fibrillar self-assembly of the peptides (Figure 1) is driven by the tendency of the hydrophobic residues to be shielded from the surrounding aqueous environment and the ability of the backbone amide groups to form hydrogen bonds along the nanofiber axis. 37,46 These buried hydrogen bonds may contribute more significantly to the stability of the supramolecular construct compared to water-exposed hydrogen bonds.47−49 Although more highly charged nanofibers may have higher antibacterial activity, their fibrillar self-assembly can potentially be inhibited by the like-charge repulsion (Figure 1).37 At high concentration of the monomers and in the presence of multivalent counterions, peptide strands undergo multistep selfassembly into nanofibers. The positively charged nanofibers get entangled and ionically cross-linked by multivalent counterions such as phosphate. Optimization of this fundamental trade-off between the requirement of nanofibrous self-assembly (Table 1) and need for high terminal charge required for antimicrobial efficacy led to our lead peptide CASP-K6, which self-assembles into a nanofibrous hydrogel and demonstrates antimicrobial efficacy. CASP-K6 contains a central β-sheet fibrillizing domain, with flanking N- and C-terminal positively charged lysine arrays. The

amphiphilicity and cationic nature, similar to natural antimicrobial peptides.36

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RATIONAL DESIGN We designed a set of modular cationic amphiphilic selfassembling peptides (CASPs) that share a common selfassembling core and positively charged domains at the termini (Table 1). Similar fiber-forming peptides have been used for Table 1. Sequences and Properties of CASPs peptide

sequence

format

secondary structure

CASP-K2 CASP-K4 CASP-K6 CASP-K8

K2(SL)6K2 K4(SL)6K4 K6(SL)6K6 K8(SL)6K8

hydrogel hydrogel hydrogel solution

β-sheet β-sheet β-sheet random coil

tissue engineering applications.5,7,8,10,37−39 All four peptides are combinations of three amino acid residues: serine (hydrophilic, neutral), leucine (hydrophobic, neutral), and lysine (cationic). These amphiphilic peptides contain varying charge density per monomer (+4 to +16). Single-letter amino acid code has been used for the peptide sequences. CASP-K2 has previously been reported as a scaffold for tissue engineering.12,40 The secondary structure was determined by circular dichroism spectroscopy, discussed later in the article. CASP-K6 is the lead antimicrobial self-assembling peptide (the peptide with the highest charge density that still forms a nanofibrous hydrogel). The central domain of CASPs was selected to promote robust self-assembly into β-sheet nanofibers (Figure 1).12,41−43 CASPs consist of a central domain with alternating serines and leucines, as well as flanking domains of varying lysine residues repeats (Table 1). We hypothesized that a higher positive charge would correlate with more robust antibacterial activity, as they would associate more favorably with negatively charged bacterial cell membrane (similar to natural cationic AMPs).44,45 The self-

Figure 2. Bulk material morphology, nanostructure, and secondary structure of CASPs. (A) Photos of the formulations used for each of the CASPs. CASP-K2 through CASP-K6 formed hydrogels, while CASP-K8 failed to form a gel (at 10 mg/mL). (B) Comparison of CASP nanofibers (1 mg/mL) by AFM quantitative nanomechanical analysis. CASP-K2 (elastic modulus of nanofibers = 279 ± 28 MPa), CASP-K4 (elastic modulus of nanofibers = 164 ± 9 MPa), and CASP-K6 (elastic modulus of nanofibers = 80 ± 5 MPa) formed nanofibers, and CASP-K8 (elastic modulus of aggregates = 68 ± 2 MPa) formed amorphos aggregates. The elastic moduli were calculated by fitting the force−separation curve to the Derjaguin−Muller−Toporov (DMT) model.112 (C) CD spectra of CASPs (0.01 mg/mL). CASP-K2 through CASP-K6 formed β-sheets (characteristic minimum at ∼216 nm), but CASP-K8 remained in a random-coil secondary conformation (characteristic minimum at 197 nm). Molar residual ellipticity (MRE) was calculated from measured ellipticity.97 C


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Figure 3. Rheological properties of the CASP hydrogels. (A) Storage modulus (G′) and loss modulus (G″) of a typical CASP (e.g., 10 mg/mL CASPK2 in 1× PBS) are responsive to shear strain. As the strain is increased from 1% to 100%, G′ first remains constant (up to approximately 10% strain), then decreases steadily and is exceeded by G′′ at a high strain (>80%). (B) Difference in the storage modulus of CASPs at same peptide molar concentration (10 mg/mL CASP-K2, etc.) and under identical solvent conditions (1× PBS). (C) From CASP-K2 to CASP-K8, the storage modulus at 1% strain and 1 rad/s oscillatory frequency (Go) decreases. (D) The dependence of G′ of a CASP (CASP-K2 in this case) on counterion conditions at different strain values. The multivalent counterion phosphate is more capable than monovalent chloride at inducing gelation, even at similar ionic strength conditions (e.g., 1× PBS versus 150 mM NaCl). Dependence of Go of CASP-K2 on (E) peptide concentration (5, 10, 15, and 20 mg/mL at a fixed ratio of peptide and PBS) and (F) concentration of counterion (I, 0.05× to 5× PBS) at a constant peptide concentration 10 mg/mL. Scaling relations obtained, Go ∼ c2.4 (R = 0.99) and Go ∼ I0.6 (R = 0.99) are similar to values reported for fibrillar proteins.59,65 (n = 4, error bars represent standard deviation).

the concentrations of the peptide strands and ionic cross-linkers increase, the nanofibers can more effectively form bundles, entanglements, and ionic cross-links (1 mg/mL CASP-K2, Figures S2 and 2B). At a critical concentration of peptide and ionic cross-linker, the fiber network forms a hydrogel. The factors driving the self-assembly involve: (a) hydrophobic shielding of the leucine residues in the central (SL)6 fibrillizing domain, (b) typical cross-strand hydrogen bonding for antiparallel β-sheets, and (c) favorable van der Waals interaction among the strands in the nanofiber.11,12 Note that the repulsion among the charged domains counteract the favorable hydrophobic interactions (Figure 1).11 Effect of Charge of the Building Blocks on the Mechanical Properties of the Nanofibrils. As the length of the terminal charged domain increases from CASP-K2 to CASP-K8, the like-charge ionic repulsion among the building blocks strengthens, leading to less favorable fibrillar selfassembly. Consequently, CASP-K2, CASP-K4, and CASP-K6 form nanofibers but CASP-K8 does not (Figure 2B shows peptide nanofibers at 1 mg/mL, see Figure S3 for the fiber morphology at a higher concentration). The nanofibrous mesh morphology is similar to previously described self-assembling

peptide owes its design roots to similar self-assembling peptides developed for tissue engineering.5,7,8,10,37−39,43,50 We hypothesized that the terminal charged domains would allow favorable attachment of the supramolecular nanofibrils to bacterial membranes, which may lead to membrane permeabilization.44,45

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RESULTS AND DISCUSSION Synthesis and Formulation. This series of CASPs was synthesized through solid-phase peptide synthesis and verified by mass spectrometry (Figure S1). After purification and lyophilization, the peptides were dissolved in isotonic aqueous buffer (298 mM sucrose solution, 1× PBS, 20 mg/mL). CASPK2, CASP-K4, and CASP-K6 formed hydrogels within seconds. However, under identical conditions, CASP-K8 remained in solution (Table 1 and Figure 2A). The inability of CASP-K8 to form a hydrogel can be attributed to the extremely high charge density at its termini and the resultant interpeptide electrostatic repulsion. Fibrillation. These self-assembling peptides can form nanofibers in diluted solutions (0.01 mg/mL CASP-K2, Figure S2). Since the nanofibers are well dispersed, they cannot form extensive physical entanglements required for hydrogelation. As D


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ACS Biomaterials Science & Engineering peptide nanofibers used for tissue engineering applications,7,8,38 and we anticipate that the set of CASPs described here can potentially be integrated with such scaffolds. We determined the nanomechanical properties of the nanofibers through peak force quantitative nanomechanical analysis (PeakForce QNM). The technique enables relatively fast scanning of the nanomechanical landscape of a surface through peak force tapping and can yield a nanometer-level resolution of stiffness, adhesion, dissipation, and deformation (Figure 2B, also see Figure S4).51 The CASP nanofibers in general are quite stiff (elastic modulus of 60−300 MPa, about an order of magnitude lower than that of silk and amyloid fibers),15,51−53 but brittle. The brittleness of the fibers is evident by the fact that just the mechanical energy involved in vortexing and spin-coating the nanofibers on a mica plate can cause the strands to break into smaller pieces (see Figure S2A), that is, the contour length of the nanofibers is sensitive to mechanical stimulation. The stiffness of the nanofibers systematically decreases with increasing terminal charge (from 279 ± 28 MPa for CASP-K2 to 80 ± 5 MPa for CASP-K6, Figures 2A and S4), which can be explained by increasing interpeptide repulsion which destabilizes the β-sheet structure and nanofiber entanglements. Qualitatively, the contour length and the aspect ratio of the nanofibers also decreases from CASP-K2 to CASP-K6. The width of the nanofibers increases from CASP-K2 (10.6 ± 1.2 nm) to CASPK4 (13.7 ± 1.5 nm) to CASP-K6 (19.7 ± 1.1 nm), which is consistent with increasing chain lengths. As mentioned before, CASP-K8 does not form nanofibers with high aspect ratios (Figure S5). Effect of Charge of Building Blocks on Network Properties. The circular dichroism (CD) spectra of the nanofibrous solutions are consistent with the microscopy data outlined above: CASP-K2 through CASP-K6 form β-sheets, CASP-K8 remains in an unfolded coil conformation (Figure 2C). Unsurprisingly, the bulk-scale morphology correlates to underlying fibrillation (CASP-K2 through CASP-K6 form hydrogels, CASP-K8 remains in solution). The storage and loss moduli of the CASPs are shearresponsive (Figure 3A). We observe that the storage moduli (G′) of the hydrogels decrease steadily from CASP-K2 to CASPK6 (CASP-K8 remained a solution) (Figure 3B−C). The affine network model developed by MacKintosh et al. predicts that the storage modulus of a hydrogel consisting of semirigid filaments should decrease with diminishing stiffness of the nanofibers, if peptide concentration and solvent conditions remain invariant.14 Additional factors that may contribute to decreasing network elasticity with respect to increasing monomer charge: (a) the repulsion between the like-charged nanofibers should systematically increase from CASP-K2 to CASP-K8, lowering the likelihood of fibrillar association, and (b) the contour length of the nanofibers should decrease with charge (Figures 2B and S3), rendering nanofiber entanglements less likely. Response to Shear Strain. Semiflexible biopolymer networks, such as actin tend to stiffen in response to higher shear strain. Storm et al. have proposed that the strain response is due to entanglement of filamentous networks and longitudinal stiffening of fibers.54 An alternative explanation of the strain stiffening based on nonaffine network response has also been proposed by Onck et al.55 In contrast, the storage modulus of self-assembled nanofibrous hydrogels decrease at a higher shear strain (Figure 3A−D), in spite of having persistence length, cross-link length, contour length, and network mesh size similar

to semiflexible biological networks that undergo strainstiffening.54 As the shear strain increases, first the storage modulus (G′) maintains a plateau. After a critical strain, G′ drastically decreases and is overtaken by the loss modulus (G″). The value of G′ and its dependence on strain are dependent on solvent conditions such as salt concentrations (Figure 3D). Charge Shielding versus Ionic Cross-linking. The apparent attractive interaction among like-charged protein molecules (and other polyelectrolytes) in the presence of multivalent counterions was first reported by Kirkwood et al.56 and has been explored in detail since then.57,58 These models generally focus on the formation of a counterion layer on the charged subunit for the apparent attraction. However, recently, Lin et al. have conjectured that multivalent counterions can form long-lasting ionic cross-links among neighboring protein fibrils to yield an elastic network.59 The bulk storage modulus of the network increases with the concentration of the cross-linker (for example, from 1× PBS to 5× PBS in Figure 3D). Charge-shielding by a monovalent counterion such as Cl− is not effective at similar ionic strength conditions (Figure 3D) in forming strong hydrogels (based on a phenomenological cutoff of G′ > 100 Pa). The repulsion among the like-charged nanofibers is lessened at higher ionic strength conditions and fiber entanglements become more likely. Thus, only at an extremely high ionic strength conditions (750 mM NaCl), CASP-K2 can form a weak hydrogel in the presence of monovalent salts due to favorable entanglement of nanofibers.16 As the monovalent ions cannot effectively cross-link the nanofibers at a physiological ionic strength (150 mM NaCl), the nanofibers remain in solution and cannot form a hydrogel. The ionically cross-linked nanofiber networks form stronger hydrogels compared to the charge-shielded entanglements under similar ionic strength conditions (e.g., 5× PBS vs 750 mM NaCl, see Figure 3D). An elegant explanation of the apparent like-charge attraction among the like-charged polyelectrolytes has been proposed by Butler et al.58 As the multivalent counterions, such as phosphate attach to the surface of the supramolecular polyelectrolyte, they can create patches of inverse charge, attracting more nanofibers, creating ionic cross-links. Such local charge inversion is not possible for monovalent ionshence, they are not effective for cross-linking the nanofibers. On the basis of this model, we can predict that large multivalent counterions that have large distances among point charges should be suboptimal agents for ionic cross-linking. If the distance among the point charges are longer than the screening length of the system, a multivalent ion may effectively act as multiple monovalent ions.58 Scaling of Bulk Elasticity. Cross-linked networks of semiflexible biopolymers generally obey precise scaling with respect to concentration of the biopolymer, as well as the concentration of the cross-linker. The theoretical scaling of the plateau storage modulus (Go) with biopolymer concentration is predicted by the affine network model to be Go ∼ c2.2.14,60,61 Experimental values of the exponent ranges between 2.0 to 2.7 for fibrillar networks formed by actin, intermediate filament, collagen, fibrin, amyloid proteins, and synthetic peptides.59,62−67 The experimental value of the exponent was 2.4 (Figure 3E) for CASP-K2, which we selected as a model peptide for the CASP series. The scaling with concentration of ionic cross-linker was weaker, Go ∼ I0.6 (Figure 3F), similar to ionically cross-linked networks of neurofilament.59 Thus, at the plateau region of storage modulus, our results are consistent with the affine model. E


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Figure 4. Association of the bacterial cells with the CASP nanofibers and subsequent membrane disruption. (A) Antibacterial activity of CASP hydrogels (10 mg/mL) applied on colonies of Pseudomonas aeruginosa PAO1 grown on agar plates (control = PBS). (B, C). The rod-shaped cells on intact bacterial films (such as the PBS-treated films) can be identified by SEM at different resolutions (false colored in panel C). (D, E) Treatment with CASPs disrupted the bacterial cell morphologies. The fibrillar hydrogel matrix of CASP-K6 is indicated by the blue arrow and the bacterial cell is pointed out by the red dashed arrow. (F) The analysis of four sets of plates such as panel A, showed that CASP-K6 and CASP-K8 were very effective in disrupting the bacterial layer (distinct Greek letters each denote significant differences, n = 4, p < 0.05). The kill rate is defined by (the density of the bacterial colony in the region where the hydrogel is applied)/(the density of the bacterial colony in the region where no hydrogel is applied).

modulus of the semiflexible filaments, network mesh size, and entanglement length. Network elasticity is predicted to be very sensitive to length of the filaments.14 At low shear strain (Figure S6), the mechanical properties of the semiflexible fiber network can be explained by the affine entropic stretching of the nanofiber network, as the scaling relations obtained for Go with respect to the concentration of the peptide and the ionic cross-linker are consistent. As the supramolecular nanofibers of CASPs are held together by relatively weak and reversible interactions, they can fracture at high shear strain into smaller nanofibrils, disentangling the network. Thus, after a critical shear strain (∼10% strain for CASP-K2 hydrogel in Figure S6), the network undergoes a mechanical transition, where the nanofibers calve into smaller strands, disentangling the supramolecular polymer network. In this regime, the network behaves like a worm-like micelle.13,68 Calving of the nanofibers and the resultant disentanglement

Constitutive Model of the Network. Self-assembling peptides have a high propensity to form β-sheet nanofibers, even at a very low concentration of peptides and ionic cross-linkers (Figure S2). With the increase of the peptide concentration, the nanofibers get entangled, while retaining their semiflexible nature. Thus, the characteristic entanglement length for the network and the mesh size for the network decreases with the increasing peptide concentration. These features share similarity to the constitutive affine network model developed by MacKintosh et al.14 and Gardel et al.60 for semiflexible biopolymers. If the concentration of multivalent ionic crosslinker (phosphate ions in PBS) is also increased, the nanofibers form dense cross-links (Figures 2B, S2, and S3). A key requirement for the model is that the nanofibers be almost straight between the entanglements, which is applicable for the self-assembled fiber networks (Figures 2B and S3). The plateau storage modulus (Go) in this model depends on bending F


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Figure 5. PeakForce QNM was used to study the mechanical integrity of bacterial membranes while interacting with peptide nanofibers. (A1) Bacterial cells interacting with CASP-K2 retain their (A2) overall oblong shape and height (A3) membrane stiffness (84.6 ± 4.2 MPa), and (A4) normal adhesiveness. (B1) Bacterial cell membranes are completely disrupted by CASP-K6 nanofibers. Most cells are lysed, demonstrated by loss of characteristic cell morphology. For the rare relatively intact cells (box), (B2) the cell and membrane are flattened, (B3) rigidified with a concomitant increase in stiffness (131.0 ± 7.9 MPa), and (B4) adhesiveness (5.5 nN).

disruption of transmembrane osmotic equilibrium and cell rupture.22,36 Colonies of the Gram-negative bacteria Pseudomonas aeruginosa PAO1 were grown on agar and CASP gels were applied to assess their colony-disruption ability (Figures 4 and S8−S10, methods in the SI). Cationic amphiphilic peptide nanofibers show noticeable bactericidal effect. This effect was correlated with the terminal charge on the cationic nanofibers (Figure 4A and F), consistent with our hypothesis that a greater terminal charge facilitates the association of the peptide nanofibers with the bacterial cell membrane. The effect of the antibacterial hydrogel was localized to an extent that we were able to print our school abbreviation (NJIT)50 on a bacterial film using CASP-K6 hydrogel (Figures S9). CASP-K6 nanofibers associated with bacterial cells, which led to membrane rupture (within 4 h), as captured via scanning electron microscopy (SEM) (Figure 4D−E, also see Figure S10−S11). However, because of the processing required to image a sample by SEM (covalent cross-linking, solvent exchange, critical point drying, sputter coating, etc.), it is difficult to precisely determine the interaction between the nanofibers and the bacterial membrane. Membrane Disruption. Atomic force microscopy obviates these drawbacks enabling sample characterization without extensive processing or solvent exchange.74 Quantitative nanomechanical analysis enables high-resolution determination of the mechanical properties a wide range of biomaterials, such as amyloid nanofibers.51 Here, we explore the interaction of the peptide nanofibers with bacterial membranes using peak force quantitative nanomechanical atomic force microscopy.75 The technology enables the determination of material properties, morphology, and adhesiveness of a substrate at pixel-level resolution and thus is a vital technique to probe the nanoscale changes in the bacterial membrane caused by the peptide.

drastically lowers the storage modulus of the network, as the supramolecular polymers can access a wider configuration space.69 Role of Secondary Nucleation and Fiber Elongation in Shear Recovery. An interesting property of CASP hydrogels is their resilience with respect to shear strain cycles (Figure S7). The storage modulus decreases as the strain is increased (1% to 100%) and recovers after the strain is lowered (100% to 1%). The reversibility of the storage modulus presents a puzzle regarding the mechanism of reassembly of the hydrogel. For the hydrogel to reform after strain lowering, the nanofibers have to re-self-assemble, get entangled, and form ionic cross-links through the multivalent counterions. If the shear-thinning of the hydrogel is caused by the nanofibers themselves breaking under shear (calving), leading to shorter seed nanofibers,70 the prompt reassembly may be facilitated by autocatalytic secondary nucleation processes, elongation of the fractured nanofibers via an Ostwald ripening type mechanism, or reattachment of broken nanofibers into longer nanofibers.17,71,72 These secondary processes have been demonstrated to be important for the assembly of amyloid nanofibers.17,71,72 The analogous mechanistic implications of the self-assembled peptide hydrogels should be probed further in the future. Antimicrobial Efficacy. We tested the peptides against colonies formed by Pseudomonas aeruginosa PAO1, an archetypical biofilm-forming bacterial strain.35 These bacteria can stay in planktonic form or attach onto solid surfaces, depending on environmental cues.73 When the hydrogel interacts with an established bacterial colony, we predicted that the nanofibers may associate with the bacterial membrane via electrostatic attraction. Subsequent integration of the peptides into the bacterial membrane may be enabled by peptide’s amphiphilic nature, leading to membrane destabilization. Permeabilization of the bacterial membrane can lead to a G


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Figure 6. Coarse-grained simulation of the interaction of CASP-K6 strands with a model bilayer membrane comprising 3:1 POPE: POPG (POPE = palmitoyloleoylphosphatidylethanolamine, POPG = palmitoyloleoylphosphatidylglycerol). (A) The membrane, by itself, is stable within 5 μs time scale (area per lipid = 61.8 Å2). (B) Cationic amphiphilic peptide strands bind to the negatively charged bilayer, leading to buckling and contraction of the bilayer (area per lipid = 38.4 Å2). Peptides are shown in licorice representation (red: lysine; green: serine and leucine). The membrane is also shown in licorice representation (cyan, carbon; blue, nitrogen; tan, phosphate). (C) Density profile of the phosphate head groups along membrane interface suggests that, in the presence of the peptide strands, the lipid head groups become more smeared and diffused. (D) Density profile of water molecules across the membrane interface demonstrate that the peptides induce water leakage in the membrane interior. (E) Order parameter values for the acyl chains of the lipid bilayer with and without CASP-K6, for the last 500 ns of the 5 μs simulation. The chains become more disordered in the presence of the membrane-disrupting peptide.

membrane, we employed the commonly used model membrane consisting of a lipid bilayer membrane comprising 3:1 ratio of the lipids POPE (palmitoyloleoylphosphatidyl-ethanolamine) and POPG (palmitoyloleoylphosphatidyl-glycerol).77 The model membrane by itself is found to be stable over multimicrosecond-long MD simulation, with well-ordered phosphate head groups at the inner and outer surfaces and lipid tail groups at the core of the membrane (Figure 6A). We did not detect any transport of water molecules through the membrane. Once the peptide molecules are added to the system, the corresponding molecular dynamics simulated trajectory suggests that the bilayer shrinks and buckles, causing the polar head groups to become smeared, allowing water molecules to pass through the membrane, and making the core lipid tails to be more disordered (Figure 6B−D). These disruptions threaten the integrity of the cellular architecture, explaining the results we observed in AFM (Figures 5B1−B4).

QNM measurements suggest CASP-K6, in contrast to CASPK2, induces bacterial cell lysis, an increase in the rigidity and adhesiveness of the membrane (Figure 5). The bacterial membranes lose their three-dimensional shape (from rod-like appearance to flat sheet-like morphology) when the cells are exposed to CASP-K6 hydrogel coated on mica. These results demonstrate that the cationic amphiphilic nanofibers can destabilize bacterial membranes. Coarse-Grained (CG) Simulation of Membrane Disruption. We hypothesized that the cationic amphiphilic peptides are first attracted to the negatively charged bacterial membrane where they increase disorder in the core of the bilayer as well as the charged head groups of the lipids. Such processes may lead to leakage of water molecules across the bilayer, disrupting the cellular homeostasis. To probe such membranepeptide interactions, we conducted a coarse-grained molecular dynamics (MD) simulation of the event (using MARTINI force field) in explicit aqueous medium.76 As a model of the bacterial H


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G″) when the force is removed (Figure S12). The shear-thinning and rapid recovery of the hydrogel can allow the gel to be syringe aspirated and injected into infection sites in vivo. The underlying fibrillar architecture (Figure S13) may also aid physiological wound-healing response.42 The CASP-K6 peptide shares its core antibacterial features (cationic nature, amphiphilicity) with natural antimicrobial peptides.78 Such peptides can be susceptible to enzymatic degradation in vivo within a time period of hours, limiting their therapeutic potential.79 In contrast, CASP-K6 persists for several days in vivo (Figure S14−S15), ensuring lack of prompt enzymatic degradation. In this work, we have demonstrated that CASP nanofibers can self-assemble into robust hydrogels and are potent disruptors of bacterial colonies. These hydrogels can be syringe aspirated and injected onto or into a target site. We have devised a selfassembling peptide, CASP-K6, that is optimized for fibrillar selfassembly, injectability, biocompatibility, in vivo persistence, and antibacterial efficacy. This combination of desirable properties places this class of antimicrobial peptides as worthy successors to polymer-based hydrogels developed by the Schneider group80 and soluble protein micronets developed by the Ryadnov group.81 Self-assembled nanostructures have been shown to have higher antimicrobial efficacy compared to the corresponding unassembled peptide due to higher local density of functional bactericidal domains.82,83 Combined with the favorable material properties, these class of self-assembled materials could become excellent tools for design hybrid biomaterials that inherently resist microbial colonization. Limitations of the Current Study and Avenues for Future Development. We plan to explore the specificity of the antimicrobial nanofibers to pathogen membranes (compared to different mammalian stromal cell membranes) and the biodistribution of the peptides in the body. Building on this work, we next aim to test this platform against a range of multidrug resistant bacterial biofilms in a dose-dependent manner and test the antibacterial efficacy changes in the presence of body fluids. Peptide amphiphiles have demonstrated uniform coating of substrates that resist formation bacterial biofilms.84,85 Thus, it would be valuable to determine the interaction of the peptide nanofibers described here (especially CASP-K6) with various substrates and its effect on the formation of biofilms by ESKAPE pathogens under static and flow conditions.86 The nanofibrous nature of the self-assembled peptides can potentially help CASP hydrogels coat a surface more uniformly, compared to monomers in solution (as shown by Ravi et al.84 and Beter et al.85). The nanofibrous nature of CASP-K6 thus may make it a better candidate than CASP-K8 for coating a biomaterial for persistent prevention of biofilm formation. Supramolecular nanofibers also have higher local cationic charge densities on the fibrils, enabling lower minimum inhibitory concentration for antimicrobial efficacy. We would also like to analyze possible routes of resistance development in bacteria in response to the peptide nanofibers, as well as long-term sequelae of the treatment.87 Several membrane rupture mechanisms have been reported for natural AMPs,44,88,89 as well as synthetic antibiotic peptides.22,90,91 A promising route for further prolonging in vivo persistence of the peptides may be the replacement of the levorotatory (L) amino acids in the primary sequence of the peptides with dextrorotatory (D) amino acids.25,92

By thorough visualization of trajectory, as well as the initial and final snapshots for each system, we observe that CASP-K6 induces significant buckling of the bacterial mimicking membrane (Figure 6B). The buckling increases as the peptide: lipid ratio is increased (Table 2). Table 2. Bilayer Propertiesa system

peptide: lipid

APL for top layer (Å2)

APL for bottom layer (Å2)

PEPG 24DPEPG

N/A 1:25

61.8 38.4

61.8 55.6

a

APL = average area per lipid; PEPG = short patch of 3:1 POPE:POPG bilayer; 24DPEPG = PEPG + 24 peptides.

The final density profiles for all simulations along the z-axis of the simulation box (i.e., membrane normal axis) are shown in Figure 6C−D. Comparison of the density profile of phosphate groups suggests that the lipid head groups (Figure 6C) become more smeared and diffused in the presence of peptide strands. Moreover, a close-up view of water density profile (Figure 6D) shows non-negligible increase in water density in the membrane center in the presence of peptides, suggesting peptide-mediated water leakage in the membrane interior. We calculated mean order parameters for the acyl chains of the POPE and POPG lipid tails and plotted against the position in the chain (Figure 6E). We observed that the presence of the peptide lowered the lipid ordering in the lower acyl tail regions. Thus, the peptide perturbs the membrane structure deeper in the hydrophobic region. These observations collectively validate that the presence of peptides causes the membrane disruption. The average area per lipid (APL) is a useful indicator for monitoring the equilibrium properties of the membrane under different conditions. Table 3 shows the area per lipid for the Table 3. List of Simulation Systems system PEPG 24DPEPG

component of the systems PE: PG (3:1) 24 peptides + PE: PG (3:1)

peptide: lipid ratio

total number of atoms

N/A 1:25

25621 30413

POPE: POPG (3:1) mixed lipid membrane over last 100 ns of simulation time for each system. The final average value of APL for the top layer and the bottom layer were lower than the simulated values for pure POPE: POPG (3:1) mixed lipid system (61.8 Å2 at 310 K), suggesting the significant membrane deformation takes place in the presence of these peptides. Importance of Favorable Material Properties. Hydrogelation of the CASP system could be useful for topical applications on infected wounds, similar to previous applications of self-assembling peptide hydrogels for rapid hemostasis41 and wound healing.40 Comparable antibacterial peptides are often mixed with a gel base, such as hypromellose, to obtain a more viscoelastic hydrogel formulation.22 The supramolecular design of our peptides allows for the formation of an antibacterial hydrogel at physiological pH and ionic strength without the addition of a gel base. Rheometry suggests that the CASP-K6 hydrogel has a storage modulus of >100 Pa, comparable to previous self-assembled therapeutic peptide hydrogels (Figure 3).7,38 Fibrillar assembly of CASP-K6 is driven by noncovalent cross-linkingthus, the hydrogel can liquefy under high shear strains (>30%), as well as rapidly recover its viscoelasticity (G′/ I


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(MRE) [θ] according to the formula [θ] = (θ·m)/(10·c·l·n), [m = molecular weight of the peptide, c = concentration of the peptide solution in mg/mL, l = path length of the cuvette in cm, and n = no. of residues in the peptide].97 Simulation. We employed a coarse-grained (CG) simulation approach to study the mechanism of bacterial membrane disruption by CASP-K6 peptides in POPE: POPG (3:1) membrane mimicking the inner bacterial membrane98 and its interaction with membrane [POPE = palmitoyl-oleoylphosphatidyl-ethanolamine, POPG = palmitoyloleoylphosphatidyl-glycerol]. A series of systems focused on CASPK6, including 12DPEPG and 24DPEPG, were investigated (Table 3). The data were compared to simulated measurements obtained by studying the system only with lipids (POPE: POPG) system, in order to investigate the possible mechanism by which the CASP-K6 may disrupt bacterial membrane processes that is still not understood at the molecular level. The sequences used to model each monomer is shown in Table 1. The model was built using PyMol.99 The β-strand secondary structure initially was enforced on residues 7−18 of as these residues were found to be important in the formation of the nanofibers.43,100,101 All coarse-grained simulations were run using GROMACS 5.1.4 package102−104 with the martini force field version 2.6.105 The CG peptide models were parametrized using the Martini script martinize.py, version 2.6.105 Overall two systems were prepared, where the peptide concentration was increased from zero to 24 (Table 3). For the 24-peptide system, the monomers of 12 peptides were arranged in single layer while other 12 peptides were arranged in the single layer above the first one (24DPEPG). Control simulation system (PEPG) was prepared by using the peptide free system, where the POPE: POPG (3:1) were used (Table 3). The “standard” approach (using only dihedral potentials based on the secondary structure of the atomistic model) of MARTINI coarsegraining was opted for all subsequent simulations. Protein structure was converted into the CG model generating the suitable Martini topology. The python script “Insane.py” was used to build the membrane model (in solution, in the presence of counterions to ensure charge neutrality). Short steepest descents minimization was employed, followed by 5 ns equilibration phase with position restraints of 1000 kJ mol−1 applied to peptide strands, using a 20 fs time step at 310 K. Unrestrained production run of 5 μs was carried out at 310 K. A 20 fs time step was used during production molecular dynamics simulation. The coarse-grained peptide-membrane simulations were carried out using CG MARTINI FF version 26.105 The CG lipid parameters were built using the “insane.py” python script of MARTINI.105 The atomistic model of the peptide was translated to CG representation using the MARTINI “martinize” script. Angles and dihedrals were used to conserve the secondary structure according to initial atomic models. A leapfrog time propagator for integrating Newton’s equations of motion was used. A time step of 20 fs for membrane systems containing embedded proteins was used. The LINCS algorithm was used to constrain all CG simulations.106 The electrostatic interactions were calculated using a reaction field with a cutoff of 1.1 nm, and a distancedependent dielectric constant of 15. Vdwtype cutoff and vdw-modifier potential shift verlet is used.107 The temperature was maintained at 310 K, well above the phase transition temperature for POPE (298 K) and for POPG (269 K). Temperature coupling with velocity rescaling108 thermostat and a coupling time of 1.0 ps, were used. Isotropic pressure coupling employing the Parrinello−Rahman barostat with coupling constant of 5 ps was used (compressibility of 3.0 × 10−4).109 Statistical Analysis. Results are shown as mean ± standard deviation unless otherwise noted. Statistical analysis was performed using a one-way ANOVA with follow-up Tukey posthoc test when necessary. Any nonparametric data was analyzed using the Kruskal− Wallis ANOVA with follow-up Dunnʼs posthoc test when appropriate. The p-value for statistical significance was p < 0.05.

To explore the interaction of the peptide nanofibers with the membrane of the pathogens in detail, we intend to determine amino acid side chain specific interactions with the phospholipid molecules. As the hydrophobic interior of a bilayer membrane has drastically different chemical properties compared to the hydrophilic interior or exterior of a cell, it is possible that the folding and self-assembly of the peptides will be different than in aqueous phase.93−95 We intend to investigate the stepwise mechanism inside the membrane in a future work through biophysical studies, complemented by atomistic simulations. We anticipate that the CASP system may be used in conjunction with other self-assembling peptide scaffolds8,38 to design multicomponent tissue-engineering constructs.96 Such hybrid cytocompatible scaffolds may become useful tools in our arsenal for the repair and regeneration of tissues. Establishment of this self-assembled nanofibrous platform may lead to clinical advances in pathogen-resistant biomaterials, as well as injectable/topical antibacterial hydrogels.

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EXPERIMENTAL SECTION

Peptide Synthesis. The series of CASPs were synthesized through solid-phase peptide synthesis using a CEM Liberty Blue microwave peptide synthesizer (acetyl N-terminal and amide C-terminal protective groups).8,10,37 The reagents and starting materials were purchased from CEM Corporation and Fisher Scientific. We used Rink amide resin with 0.18 mmol/g loading. The peptides were purified by dialysis (against deionized water with 2000 Da molecular weight cutoff dialysis tubing) with purity >90% (HPLC) prior to use. Then, the peptides were then lyophilized and identified with matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) or electrospray ionization mass spectrometry (ESI-MS) in an Apex-ultra 70 hybrid Fourier transform mass spectrometer (Bruker Daltonics). Peptide Characterization. Atomic force microscopy (AFM) and rheology methods have been described previously.8,10,37,38 To conduct AFM analysis, the peptide hydrogel was diluted 10-fold in water (0.1%). The diluted solution was deposited on a freshly cleaved mica disc. We performed spin-coating on a purpose-built spin-coater. The coated mica disc was air-dried for 30 min prior to imaging. We used PeakForce tapping (ScanAsyst) mode on a Bruker Dimension FastScan AFM machine to measure the mechanical properties of individual nanofibers. The quantitative nanomechanical analysis is sensitive in the 0.7 MPa−70 GPa range and can achieve lateral resolution of less than 5 nm. In contrast to the tapping mode AFM usually used to characterize similar samples, PeakForce tapping method keeps the maximum force experienced by the AFM tip (when it approaches the sample in the zdimension) constant. It is important to note that individual fibers are tested in this technique. The fiber sample is deformed only a few angstroms, minimizing the risk of sample tearing or breaking of the tip. The dependence of the force experienced by the tip on the tip−sample separation yields elastic modulus, adhesion, dissipation, and deformation of the sample at any given point. As the nanofibers entangle and solvate, they trap significant amounts of water that allow unique bulk viscoelastic properties investigated using rheometry. For rheology, 40 μL of 1% peptide hydrogel was transferred between a plate and a 4 mm parallel plate geometry with a gap of 250 μm, on a Malvern Kinexus Ultra+ rheometer. The storage and loss moduli of the hydrogels were measured at constant strain (1%) and angular frequency (1 rad/s) for 5 min and then averaged. Four independent runs were averaged to obtain plateau storage modulus (Go) for each sample. Strain sweep (0.1−100% strain at 1 Hz) and shear recovery (1% strain at 1 Hz for 5 min, 100% strain at 1 Hz for 1 min, and 1% strain at 1 Hz for 5 min) were also performed. Shear recovery was repeated several times to demonstrate hydrogel resilience. For circular dichroism (CD), we used an Olis Rapid Scanning Monochromator (RSM) to measure the ellipticity of a 0.01 mg/mL peptide solution from 190 to 240 nm in a 1 cm cuvette. The ellipticity (θ, measured in millidegrees) was converted to molar residual ellipticity

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CONCLUSIONS Gelation is fundamental to life. From the intracellular supramolecular assemblies formed by actin, intermediate filaments, J


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ACS Biomaterials Science & Engineering and microtubules, to the extracellular viscoelastic matrix produced by collagens, elastin, and proteoglycans, hydrogels form the constitutive matrix in our body. As biomimetic supramolecular peptide hydrogels have become increasingly important for tissue engineering, the need for a bottom-up understanding for network formation is paramount. Cells in culture or in the body dynamically interact with hydrogels, sensing material properties, and reacting accordingly.110,111 This results in a consequent pressing need for custom hydrogels with tunable mechanical properties at the nanostructural and the bulk-network levels. These cationic amphiphilic peptides we describe here lay a foundation for understanding the hierarchical assembly of such tunable supramolecular networks. Self-assembled peptide nanofibers based on β-sheet motifs share structural similarities to worm-like surfactant micelles (because of their amphiphilic sequences) and semiflexible biopolymers (because of long persistence lengths, high linear order due to canonical hydrogen bonds, etc.). We elucidate multiscale hierarchical self-assembly of these peptides into nanofibers and hydrogels. We demonstrate that the nanofiber network can be modeled as a semiflexible biopolymer network at low shear strains and as worm-like micelles at high shear strains. Our study links the nanomechanical properties of the nanofibers, higher-order entanglements and ionic cross-linking, and the effect of solvent conditions into a coherent predictive model applicable at a wide range of shear strains. Here, we show that the physical properties of the individual nanofibers can be correlated with the material properties of the bulk hydrogel. We demonstrate how the storage modulus depends on factors such as concentration of the monomers, solvent parameters, and applied strain. The reversible selfassembly of the nanofibers enables the hydrogels to liquefy under shear strain and recover its hydrogel-like properties afterward. These physical properties of the hydrogel have important implications for tissue engineering. We describe a selfassembling peptide hydrogel that causes bacterial membrane disruption. The mechanism of action was probed through atomic force microscopy and in silico simulation. Such general antimicrobial scaffolds can be useful additions in our toolkit for tissue engineering.

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Vivek A. Kumar: 0000-0001-7536-9281 Author Contributions

B.S., Z.S., and P.K.N. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

We would like to acknowledge the NJIT startup funds (for V.A.K.), as well as NJIT Undergraduate Research and Innovation (URI) program. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the Materials Characterization Core at NJIT and Dr. Roman Brukh (Rutgers) for materials characterization.

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ABBREVIATIONS CASP, cationic amphiphilic self-assembling peptide; AMP, antimicrobial peptide; AFM, atomic force microscopy; QNM, quantitative nanomechanical; HPLC, high-performance liquid chromatography; ESI, electrospray ionization; PBS, phosphate buffered saline

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(1) Webber, M. J.; Appel, E. A.; Meijer, E. W.; Langer, R. Supramolecular biomaterials. Nat. Mater. 2016, 15 (1), 13−26. (2) Cui, H.; Webber, M. J.; Stupp, S. I. Self-assembly of peptide amphiphiles: from molecules to nanostructures to biomaterials. Biopolymers 2010, 94 (1), 1−18. (3) Webber, M. J.; Tongers, J.; Newcomb, C. J.; Marquardt, K. T.; Bauersachs, J.; Losordo, D. W.; Stupp, S. I. Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (33), 13438−43. (4) Hudalla, G. A.; Sun, T.; Gasiorowski, J. Z.; Han, H.; Tian, Y. F.; Chong, A. S.; Collier, J. H. Gradated assembly of multiple proteins into supramolecular nanomaterials. Nat. Mater. 2014, 13 (8), 829−36. (5) Wickremasinghe, N. C.; Kumar, V. A.; Hartgerink, J. D. Two-step self-assembly of liposome-multidomain peptide nanofiber hydrogel for time-controlled release. Biomacromolecules 2014, 15 (10), 3587−95. (6) Kumar, V. A.; Shi, S.; Wang, B. K.; Li, I. C.; Jalan, A. A.; Sarkar, B.; Wickremasinghe, N. C.; Hartgerink, J. D. Drug-triggered and crosslinked self-assembling nanofibrous hydrogels. J. Am. Chem. Soc. 2015, 137 (14), 4823−30. (7) Kumar, V. A.; Taylor, N. L.; Shi, S.; Wang, B. K.; Jalan, A. A.; Kang, M. K.; Wickremasinghe, N. C.; Hartgerink, J. D. Highly angiogenic peptide nanofibers. ACS Nano 2015, 9 (1), 860−8. (8) Kumar, V. A.; Taylor, N. L.; Shi, S.; Wickremasinghe, N. C.; D’Souza, R. N.; Hartgerink, J. D. Self-assembling multidomain peptides tailor biological responses through biphasic release. Biomaterials 2015, 52, 71−8. (9) Wickremasinghe, N. C.; Kumar, V. A.; Shi, S.; Hartgerink, J. D. Controlled Angiogenesis in Peptide Nanofiber Composite Hydrogels. ACS Biomater. Sci. Eng. 2015, 1 (9), 845−854. (10) Kumar, V. A.; Liu, Q.; Wickremasinghe, N. C.; Shi, S.; Cornwright, T. T.; Deng, Y.; Azares, A.; Moore, A. N.; Acevedo-Jake, A. M.; Agudo, N. R.; Pan, S.; Woodside, D. G.; Vanderslice, P.; Willerson, J. T.; Dixon, R. A.; Hartgerink, J. D. Treatment of hind limb ischemia using angiogenic peptide nanofibers. Biomaterials 2016, 98, 113−9. (11) Dong, H.; Paramonov, S. E.; Aulisa, L.; Bakota, E. L.; Hartgerink, J. D. Self-assembly of multidomain peptides: balancing molecular frustration controls conformation and nanostructure. J. Am. Chem. Soc. 2007, 129 (41), 12468−72.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00967.

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REFERENCES

Detailed methods and data regarding the characterization of the peptides (MS, AFM, rheology, SEM, cytocompatibility and histology) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Mailing address: 138 Warren St., LSEB 316, Newark, NJ, 07102. ORCID

Biplab Sarkar: 0000-0002-9298-2392 Peter K. Nguyen: 0000-0001-9867-887X Wanyi Fu: 0000-0001-9653-4012 David Sabatino: 0000-0002-9797-0833 Wen Zhang: 0000-0001-8413-0598 Jagannath Mondal: 0000-0003-1090-5199 K


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