Mechanobiology is drawing widespread attention for its potential value in the field of biomaterial design for tissue engineering and regenerative medicine [1
Cells are sensitive to the mechanical properties of the extracellular matrix (ECM) in both physiological and pathological conditions (such as stroke, brain trauma, spinal cord injuries, cartilage damage, and tumors) [3
]; it is becoming increasingly clear that the mechanical properties of the ECM are critical in directing cell fate, homeostasis, and survival [6
Hence, synthetic but bio-inspired nanomaterials designed to mimic the ECM should aim to recapitulate most of the features of the native ECM. Scaffold nanoarchitecture and mechanical properties should be tuned in accordance with the targeted tissue and to achieve the precise control of cellular behavior [9
]. Just like the ECM, the chemical structure of the biomaterial should concurrently contain non-covalent and covalent chemical bonds, bringing, respectively, infinite and finite lifetimes of interactions [10
] to obtain a reversibly dynamic matrix. Moreover, the method for incorporating biological epitopes (also named functional motifs) into the material should be applicable to a broad array of bioactive molecules (from small peptides to large proteins) before and after synthesis/processing in order to add new tools to maximize material–cell interactions [11
Biomaterials based on peptide self-assembly meet some of these criteria [12
]. However, despite the fact that self-assembling peptides (SAPs) can self-assemble into high-aspect-ratio nanofibers resembling nanofibrous ECMs [14
], the practical application of such nanomaterials has been limited due to their intrinsic instability, low-performance, and low-strain/stress response [19
]. Further, the incorporation of functional motifs into SAP nanostructures has generally been limited to bioactive peptides tethered to the C- or N-terminal of an SAP sequence during solid-phase peptide synthesis [20
]. Additionally, the presence of functional motifs, bringing additional hydrophobic and charged interactions, could potentially influence the self-assembly process of the SAP molecules, resulting in the formation of an altered nanostructures [29
] and consequently posing limits to their potential functionalization [20
]. Therefore, until now, the multiple functionalizations of SAPs with different bioactive cues have been predominantly obtained via the self-assembly of differently functionalized SAPs sharing the same self-assembling sequence [19
Recently, our group pioneered the use of cross-linked peptide hydrogels with tuned and highly increased mechanical features suiting the needs of different regenerative medicine applications that, at the same time, effectively display bioactive motifs at their nanostructures [33
]. Other groups have also utilized covalent capture to design more robust SAP-biomaterials under a variety of conditions [35
]. However, these synthetic methods are relatively cumbersome and require long reaction times (up to 24 h). In contrast, physical cross-linking can enhance the mechanical properties of self-assembled fibrillar networks by influencing specific intermolecular interactions that modulate fiber intertwining. For example, calcium-mediated ionic bridges can form stronger intra- and inter-fiber cross-links among SAP molecules, and, as a result, cross-linked hydrogels can withstand higher strains [35
Here, we report the use of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide/N
-hydroxysulfosuccinimide (EDC/sulfo–NHS) coupling to readily cross-link LDLK12 (Ac-LDLKLDLKLDLK-CONH2
) SAP molecules (Figure 1
and Figure S1
). The cross-linked LDLK12 features enhanced mechanical features and can be further cross-linked (decorated) with bioactive motifs to expand its range of applications. EDC/sulfo–NHS is one of the most commonly used carbodiimides, and it catalyzes the formation of amide bonds between carboxyl and primary amine groups. Its broad use is derived from its high solubility in water and its ease of removal of the byproduct [38
By using a one-pot and in situ gelation system, we demonstrated, for the first time, that EDC/sulfo–NHS specifically reacts with LDLK12-assembled nanostructures, enhancing their mechanical properties without altering the spontaneous formation of β-sheet-containing nanofilaments. Lastly, we investigated the use of a post-assembly modification of LDLK12 nanostructures using EDC/sulfo–NHS as an alternative approach to add bioactive functional motifs after self-assembling took place, i.e., for all jellified SAP hydrogels. We carried out such a post-assembly functionalization of nanofibers by using a KLPGWSG [22
] phage display-derived epitope as a bioactive cue.
This versatile approach may be potentially applied to biomaterials containing aspartic acid (D) or glutamic acid (E), and lysine (K) residues to enhance their biomechanics and biomimetic properties, hence leading to a new precious tool for biomedical applications.
2. Results and Discussion
2.1. EDC/Sulfo–NHS Cross-Linked Peptide Preparation
Water-soluble EDC carbodiimide reacts with carboxylic acids and forms reactive O
-acylisourea intermediates, which are then linked to a nucleophile (i.e., a primary amine) to create an amide bond. However, O
-acylisourea intermediates are labile in the presence of polar solvents and must react immediately after dissolution in water. Indeed, oxygen atoms from water can also act as a nucleophile, cleaving off the intermediate and releasing isourea, thus inactivating EDC molecules [39
To improve the coupling efficiency, EDC can be used in conjunction with sulfo–NHS to form an active ester with the carboxylic acid. This type of intermediate ester is hydrophilic, stable, and hydrolyzes relatively slowly in water (circa 4–5 h), offering an advantage for coupling reactions. In the presence of amine nucleophiles (e.g., lysine side-chains in peptide molecules), the sulfo–NHS ester is rapidly hydrolyzed, allowing for the formation of an amide bond.
To investigate the EDC/sulfo–NHS coupling reaction, we selected the previously characterized SAP LDLK12 [17
], which spontaneously self-assembles into nanofibers. When pH is triggered from acidic (pH~4.5) to neutral values (pH~7.4), nanofibers in the LDLK12 solution form bulk hydrogels that act as a biodegradable artificial ECM with proven neuroregenerative potential in vitro and in vivo [41
The LDLK12 peptide was dissolved in distilled water (pH 5.5) to achieve a final concentration of 1% (w
), and it was then left overnight at +4 °C in order to guarantee the spontaneous formation of pre-assembled nanofibers. Afterwards, EDC (0.2 M, 5 μL) in Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4) was added dropwise to the pre-assembled peptide bundles to obtain O
-acylisourea intermediate peptides. Immediately, sulfo–NHS (0.2 M, 10 μL) was added to the EDC pre-activated peptide solution. Sulfo–NHS promptly reacted with the water-exposed lysine in the peptide bundles, thus forming a stable amide cross-link among the pre-assembled peptide molecules (Figure 1
). When the sulfo–NHS solution was added to the solution of O
-acylisourea pre-activated SAP, we observed the formation of self-supporting cross-linked scaffolds in less the 5 min.
Coupling yield was minimal when EDC/sulfo–NHS was added to SAPs immediately after their solubilization in water, while, after the chosen overnight stay, cross-linking showed a rapid peptide coupling after EDC/sulfo–NHS addition. This suggests an interesting correlation between the degree of coupling and the physico-chemical conditions of peptide molecules.
Since this reaction takes place in situ, at physiological conditions (pH 7.4), relatively fast, and without the need of external stimuli (such as temperature or ionic strength), it could be useful as filler in traumatic brain injury (TBI) [42
], acute spinal cord injury (SCI) [41
], or as sprayable hemostatic solution in combination with common clotting bandages during uncontrolled bleeding in surgeries [47
2.2. Mechanical Properties of Peptide Nanostructures
To investigate the impact of EDC/sulfo–NHS cross-linking, rheological studies were conducted to evaluate the mechanical properties of the LDLK12 hydrogel. First, we analyzed the viscosity of peptide solutions (Figure 2
A). Both SAPs with (red dots) and without (blue dots) EDC/sulfo–NHS cross-linking displayed non-Newtonian shear-thinning behavior with a decrease of viscosity that was concomitant with the shear-rate increase. Even if the cross-linked LDLK12 showed an increased viscosity (1.9 Pa.s) compared to the standard LDLK12 (0.06 Pa.s), both hydrogels had negligible differences at higher shear rate values (500–1000 s−1
). The non-Newtonian shear-thinning behavior of both SAPs was also confirmed by assessing the shear stress (σ) trend alongside shear-rate increments (Figure S2
Next, we investigated the storage and loss moduli (G′ and G″, respectively) in the function of angular frequency (1–100 Hz). The G′ value (full dots) of both peptides was found to be higher than G″ (empty dots), indicating the formation of a hydrogel with a predominant solid-elastic behavior (Figure 2
B). However, the cross-linked SAP displayed a small change in elastic shear modulus (2.2 kPa) compared to the standard LDLK12 (1.5 kPa), which still indicated a slight increment of the gelation propensity of the cross-linked LDLK12 due to the EDC/sulfo–NHS coupling.
Since recent work has demonstrated that G′ is not the sole determining factor in cell mechanobiology [1
], so the shear strain–stress response was also determined to assess the cross-linked LDLK12 failure when subjected to a linear strain–stress progression. Strain–sweep experiments (Figure 2
C) demonstrated a wide linear viscoelastic regime (LVR) of the cross-linked LDLK12 (red dots) and an unusual bi-modal failure at 5% and 60% of strain, respectively, that had never been observed before in SAP-based biomaterials. Indeed, standard LDLK12 (blue dots), belonging to fragile and soft-hydrogels, showed a one-step strain failure at 6.6%. As a consequence, in its stress–failure curves (Figure 2
D), the cross-linked LDLK12 displayed a two-step unusual stress–failure process. This characteristic only being observed on the cross-linked LDLK12 suggests that the two-step shear strain–stress behavior may be strictly dependent on the EDC/sulfo–NHS coupling and could be correlated with the morphological transitions caused by the supramolecular rearrangement of LDLK12 reacted with EDC/sulfo–NHS. Moreover, it is worth noting that these stain–stress rates fell within a regime that has been shown to be conducive to matrix reorganization [48
], implying that the EDC/sulfo–NHS coupling system could be suited for 3D cell cultures.
We also observed a spontaneous self-healing propensity of the cross-linked LDLK12 (Figure 2
E). After a large strain failure (γ = 1,000%; cyan dots), the G′ of the cross-linked LDLK12 returned to its original values after 30 min (blue dots), demonstrating a recovery of the mechanical properties in the LVR. A possible explanation for this observation may be the simultaneous presence of non-covalent and covalent bonds, which led to the formation of a dynamic supramolecular system capable of self-healing without the need of external stimuli. On the contrary, as we previously described [53
], when exerting larger amplitude oscillatory deformation (γ > 100%), LDLK12-based SAPs show a negligible mechanical recovery, implying that they lose parts of mechanical stability because of their weak and brittle nature.
Since injectable hydrogels have gained increasing amounts of attention in the fields of tissue engineering and drug, cells, or growth factor delivery due to their minimally invasive way of delivery [54
], to evaluate the propensity of LDLK12-materials to recover their initial viscosity after injection, the thixotropy of all solutions was investigated ex vivo.
In this test, the injection conditions were simulated through a series of constant shear rate tests (see Materials and Methods for further details). Both SAPs with (red dots) and without (blue dots) EDC/sulfo–NHS cross-linking exhibited a fast recovery after injection simulation (Figure 2
F). This fast viscosity recovery hinted a space-filling propensity of all solutions after injection, highlighting that both LDLK12 scaffolds could readily provide a good fit and interface between the hydrogel and damage tissues.
Our data demonstrated that the EDC/sulfo–NHS coupling could be applied to the LDLK12 systems without hampering their predominant solid-elastic behavior (Figure 2
G). In addition, the cross-linked LDLK12 peptide demonstrated a wide LVR with an unusual two-step shear strain–stress behavior, which was unmatched by other SAP scaffolds, typically relying on non-covalent interactions for their biomechanics.
2.3. Supramolecular Organizations of Peptide Nanostructures
To gain insights into the supramolecular and global arrangement of the cross-linked peptide hydrogel, we used a Thioflavin-T (ThT)-binding assay, FT-IR spectroscopy, circular dichroism (CD), and atomic force microscopy (AFM) morphological analysis.
A ThT-binding assay, an amyloid-specific fluorescent dye [57
] (see Materials and Methods), was used to examine the amyloidogenic nature of the cross-linked LDLK12 fibers (Figure 3
A). Staining the fibers with ThT resulted in high fluorescence levels with a typical amyloid-binding emission signal (centered at ∼500 nm): this highlighted that both SAPs with (red dots) and without (blue dots) EDC/sulfo–NHS cross-linking featured a similar amyloid-like nature.
To study the secondary structure of the peptide in solution, we carried out FT-IR spectroscopy tests (Figure 3
B). FT-IR spectra exhibited a sharp Amide I band at ∼1630 cm−1
with a shoulder at ∼1695 cm−1
, indicating predominantly anti-parallel β-sheet features. The band at ∼1540 cm−1
in the Amide II region also confirmed the β-sheet aggregation of both peptide samples. In the cross-linked LDLK12, these peaks were slightly red-shifted compared to the β-sheet signal of the standard LDLK12; this might have been ascribable to the interference of sulfo–NHS in the formation of the amide bonds between the O
-acylisourea pre-activated SAP chains.
To explore the effect of EDC/sulfo–NHS on the formation of β-sheet conformation of LDLK12 assemblies, CD spectra were recorded (Figure 3
C). Positive and negative peaks at, respectively, 197 and 217 nm, typical of β-sheet rich structures, were observed for both SAPs with (red dots) and without (blue dots) EDC/sulfo–NHS cross-linking. No assembly was observed for EDC/sulfo–NHS alone (Figure S3
AFM was used to elucidate the fiber morphology of the cross-linked LDLK12. After EDC/sulfo–NHS cross-linking, the LDLK12 peptide revealed the presence of entangled fibers (Figure 4
A). The cross-linked peptide adopted an elongated and unbranched nanofiber network morphology with a width of ~15 ± 4 nm (Figure 4
B). The height distribution ranged from 1.06 to 8.76 nm (Figure 4
C) and peaked at 3.65 ± 0.7 nm, as depicted in the 2D interpolation maps (Figure 4
D and Figure S4
In addition, we characterized the curvature of the cross-linked LDLK12 nanofibers (Figure 4
E). The AFM images of the cross-linked LDLK12 revealed a wide range of nanofibrils curvature values, with a maximum curvature value of 0.17 nm−1
. This was unusual, since standard LDLK12-based fibers usually appear rigid and flat [19
]. These data are in good agreement with the rheological analysis and could explain the reason for the large shear strain/stress resistance of the cross-linked LDLK12 bestowed by cross-linking with EDC/sulfo–NHS. Lastly, the cross-linked LDLK12 fibrils showed an orientation distribution with a clear peak along one direction (Figure 4
F), and they also exhibited a strongly aligned nematic fibril domain [60
] (Figure S5
In summary, the spectroscopic characterization (ThT-binding assay and FT-IR) and AFM analysis confirmed the amyloid-like nature and self-assembling propensity into β-sheets of the cross-linked LDLK12, suggesting that the introduction of EDC/sulfo–NHS does not impair the formation of β-sheet-containing nano-filaments, but it does lead to the formation of mainly aligned curved supramolecular nanostructures that are more resistant to shear strains.
2.4. Post-Assembly Functionalization of KLPGWSG Peptide to the Surface of SAP Nanofibers
Next, we sought to extend the scope of the EDC/sulfo–NHS coupling to the functionalization of SAP nanofibers. To demonstrate that the EDC/sulfo–NHS reaction can be used to ligate bioactive cues to the LDLK12 nanofibers, we utilized a phage display-derived KLPGWSG bioactive peptide that has been shown to interact with neural stem cells and to significantly shift their differentiation toward the neuronal phenotype. [22
] Indeed, we previously demonstrated that, by using BLAST analysis, the major consensus sequences for KLPGWSG are found on three proteins known to affect stem cells behaviors: namely Notch1, Dll4, and MEGF10 [22
The EDC/sulfo–NHS post-assembly reaction was performed upon the simple mixing of water-soluble KLPGWSG (see Materials and Methods for further details) on the top surface of the pre-activated EDC/sulfo–NHS-assembled LDLK12 peptide hydrogel (Figure 5
A). The post-assembly conjugation of the KLPGWSG molecule added highly hydrophobic aliphatic side-chains and one positively charged lysine residue to the LDLK12 fibers, resulting in an increase in surface charge and in a shift of the isoelectric point (from pH 7.19 to 10.19).
KLPGWSG-functionalized LDLK12 hydrogels were analyzed using oscillatory stress rheology. In the gel phase, phage-derived functionalized LDLK12 showed an almost unchanged G′ profile along the tested frequency range (Figure 5
B), and the thixotropic and space-filling propensity of the LDLK12 based-hydrogel was still preserved (Figure 5
Again, the amyloidogenic nature and the secondary structures of nanofibers functionalized with KLPGWS were investigated using the ThT-binding assay, FT-IR, and CD spectroscopy. The ThT-binding assay exhibited a typical amyloid-binding emission signal centered at ∼500 nm (Figure 5
D), and the FT-IR spectrum of the LDLK12 peptide after the EDC/sulfo–NHS reaction with the KLPGWSG peptide suggested a predominant β-sheet secondary structure, with a minimal change caused by the conjugation of epitope (Figure 5
E). Additionally, CD spectra displaying positive and negative peaks at, respectively, 200 and 217 nm (Figure S6
) confirmed the presence of β-sheet secondary structures in the LDLK12 peptide after EDC/sulfo–NHS cross-linking with KLPGWSG. In contrast, the KLPGWSG alone showed an unstructured conformation with a minor negative band at 196 nm.
These results indicated that the presence of the KLPGWS epitope on the LDLK12 molecules did not influence their self-assembly process, biomechanics, and resulting secondary structures.
To confirm that the ligated KLPGWSG epitope was present on the surface of the LDLK12 nanofibers, we added a fluorescein isothiocyanate (FITC) group to the N-terminus of the KLPGWSG (see Materials and Methods) and used EDC/sulfo–NHS to attach this peptide to the LDLK12 nanofibers. A higher fluorescence intensity was observed following the EDC/sulfo–NHS-mediated conjugation of the FITC-KLPGWSG peptide to LDLK12 nanofibers (Figure 5
F). Conversely, adding the same bioactive peptide without EDC/sulfo–NHS conjugation produced a significantly lower fluorescence intensity (Figure S7
), indicating that the observed fluorescence was due to the retention of the cross-linked FITC-KLPGWSG by the specific EDC/sulfo–NHS reaction with LDLK12 and not a non-specific adsorption to SAP nanofibers.
In summary, this post-assembly functionalization approach mediated by EDC/sulfo–NHS can be applied to any primary amine-containing SAPs. It is possible to combine different bioactive and fluorescent peptides to potentially decorate SAP hydrogel surfaces with multiple functions (e.g., for sensing, adhesion, or cell signaling) while maintaining their desirable structural, biomechanical, and biocompatibility properties typical of SAP biomaterials.
Here, we reported the design of cross-linked SAPs via a one-pot and in situ gelation system based on EDC/sulfo–NHS coupling, yielding self-healing and functionalized hydrogels. EDC/sulfo–NHS cross-linked SAP also exhibited an unusual two-step shear strain–stress behavior strictly correlated to the EDC/sulfo–NHS coupling.
Furthermore, we extended the scope of the EDC/sulfo–NHS coupling for the post-assembly functionalization of SAP nanofibers. Bioactive peptides thoroughly decorated the hydrogels without compromising their self-assembly propensity, molecular packing, and biomechanics. Our results point out that EDC/sulfo–NHS post-assembly cross-linking could be a useful tool to tailor the bioactivity and biomechanics of supramolecular nanostructures.
Overall, our approach may offer new additional tools to optimize the design and biomimetic properties of peptide biomaterials for biomedical applications independently from other key variables of supramolecular peptide hydrogels such as nanotopography and porosity.