Mixing Biomimetic Heterodimers of Nucleopeptides to Generate Biocompatible and Biostable Supramolecular Hydrogels

Abstract

As a new class of biomaterials, most of the supramolecular hydrogels formed by small peptides require the attachment of a long alkyl chain, multiple aromatic groups, or strong electrostatic interactions. Based on the fact that the most abundant protein assemblies in nature are dimeric, we select short peptide sequences from the interface of a heterodimer of proteins with known crystal structure to conjugate with nucleobases to form nucleopeptides. Being driven mainly by hydrogen bonds, the nucleopeptides self-assemble to form nanofibers, which results in supramolecular hydrogels upon simple mixing of two distinct nucleopeptides in water. Moreover, besides being biocompatible to mammalian cells, the heterodimer of the nucleopeptides exhibit excellent proteolytic resistance against proteinase K. This work, as the first report that supramolecular hydrogelation triggered by mixing heterodimeric small nucleopeptides, illustrates a new and rational approach to create soft biomaterials.

Supramolecular hydrogels,^[1] formed by small molecules (referred as hydrogelators) in aqueous phase, share certain properties (e.g., soft, polymorphic, and bioactive) with extracellular matrices in tissue, and have attracted considerable attention as emerging biomaterials for applications such as stem cell delivery,^[2] antibacterial,^[3] cancer cell inhibition,^[4] scaffolds for regenerative medicine^[5] cell culture,^[6] and media for electrophoresis.^[7] Among the building blocks for generating supramolecular hydrogels, peptides^[1c,8] are the most explored ones. The current strategies for designing peptidic hydrogelators are to introduce a long alkyl chain,^[8a,9] aromatic groups,^[8c,10] or residue of opposite charges.^[11] Each of these strategies can effectively generate hydrogels, but not without limitation. For example, it is difficult to choose the length of the alkyl chain,^[9b] too many aromatic groups may inhibit cells in some cases,^[4,12] too strong charge interactions results in poor reversibility.^[13] Thus, it is necessary and beneficial to explore new strategies to complement the existing ones for supramolecular hydrogels.

One attractive approach is to learn from Nature. Intriguingly, most of the cytosol proteins exist as protein assemblies with different symmetry,^[14] yet the most common form of protein assemblies are dimers (e.g., 38% of proteins in E. coli exist as dimers^[14]). The formation of heterodimer implies rather strong non-covalent interactions at the interface of two proteins, thus one should be able to take those complementary sequence to generate hydrogels. This approach, in fact, has been explored by a few groups by using peptides to bind with proteins.^[15] For example, the specific TPR-peptide interaction,^[15a] TIP1-peptide interaction,^[15b] heparin-VEGF interaction,^[15c] allows the formation of polymeric hydrogels. One drawback of this approach is the use of proteins being high cost and susceptible to proteolysis. Interestingly, this approach has yet to be explored in the use of nucleopeptides^[16] for creating supramolecular hydrogels. Based on this principle, our works on supramolecular hydrogels made of homonucleopeptides via pH changes or enzymatic reaction,^[16a] and the biostability of nucleopeptides,^[16a,17] we decide to explore the use of heteronucleopeptides to generate hydrogels via simple mixing of two structurally distinct nucleopeptides that bind with each other.

We choose nucleobase (thymine or adenine) to connect with short peptide sequences (Scheme 1) from the binding interface of two well-characterized proteins,^[18] calcium channel protein (stargazin^[19]) and synapse associated protein 102 (SAP102^[20]). While the homonucleopeptides themselves are unable to self-assemble to form molecular nanofibers that result in a hydrogel, the mix of heteronucleopeptides, 1+3 and 2+3, results in self-assembly to form supramolecular nanofiber/hydrogels. Furthermore, the nucleopeptides show excellent cell compatibilities, and the hydrogels of the heterodimers exhibit enhanced biostability. As the first report of supramolecular hydrogels formed by mixing heterodimeric nucleopeptides, this work illustrates a facile and rational methodology for creating nucleopeptides that act as a new class of supramolecular hydrogelators for developing sophisticated soft materials according to the need of various applications.

Scheme 1.

Molecular structures of the nucleopeptides containing the epitopes from stargazin or SAP102.

We choose thymine and adenine as the complementary nucleobases for producing the nucleopeptides because of their application in design supramolecular materials.^[21] Among many available heterodimeric proteins that have well-characterized structures, we select a pentapeptidic sequence, leucine-glycine-phenylalanine-asparagine-isoleucine, LGFNI, from the binding loop of PDZ domain,^[22] which is a common modular domain for protein-protein interactions in many organism.^[22–23] To match with the LGFNI sequence, we use another pentapeptide, lysinethreonine-threonine-proline-valine, KTTPV, for generating the nucleopeptides because the recent crystal structure of the binding of TTPV with a PDZ domain^[18] has provided atomistic details of the non-covalent interactions (e.g., hydrogen bonding (shown in Scheme 1)) between LGFNI and KTTPV, which offers the molecular base that warrants adequate binding between the designed heterodimeric nucleopeptides. We choose to connect the nucleobase at the N-terminal of the peptides because the attachment of nucleobase at the C-terminal of small peptides unlikely would result in effective molecular self-assembly.^[24] According to these nucleobases and pentapeptides, we plan to examine the gelation properties of 1, 2, 3 and their mixtures (Scheme 1).

After their synthesis and characterizations, we test the ability of the nucleopeptides to form hydrogels. The dissolution of 3 (12 mg) in PBS (1 mL) to give a clear solution of 3 (16.4 mM and pH = 6.2). So we prepare the solutions of 1 or 2 in PBS (pH = 6.2) at 16.4 mM as well. The simple mixing of 1 (or 2) with equal volume of 3 affords the mixture, 1+3 (or 2+3), with each component to be 8.2 mM in concentration. After 48h at room temperature, the mixture of 1+3 (or 2+3) transforms from a clear solution to a transparent hydrogel (Figure 1), while the stock solutions of 1, 2, and 3 (at 16.4 mM) remain as transparent solutions (Figure S4), so does the mixture of 1+2 (Figure S6). Rheometry shows that dynamic storage moduli (G′) dominate the dynamic loss moduli (G″) for the mixture of 1+3 (or 2+3), confirming that 1+3 (or 2+3) forms a hydrogel. On the contrary, the G′ values overlap with the G″ values for the solution of 1, 2, or 3 (Figure S5), confirming that 1, 2, or 3 itself at 16.4 mM remains as a liquid. Moreover, the maximum storage modulus of the hydrogel of 2+3 (G' = 8928 Pa) is about an order of magnitude higher than that of the hydrogel of 1+3 (G' = 942 Pa). This result is consistent with the structure difference between 1 and 2 and indicates that the interactions between the Waston-Crick base pair (from adenine in 2 & thymine in 3) results in higher mechanical strength of the heterodimeric nucleopeptides than the interactions between thymines (from 1 & 3) do.

Figure 1.

TEM images of (A) hydrogel of 1+3; (B) hydrogel of 2+3. All compounds are dissolved in PBS buffer (pH = 6.2, and [1] = [2] = [3] = 8.2 mM). Inserts: optical images of the hydrogels. Scale bar = 100 nm.

We use transmission electron micrograph (TEM) to characterize the hydrogels of the heterodimeric nucleopeptides. The hydrogel of 1+3 contains long and aligned bundles that consist of thin nanofibers with an average width about 4 ± 2 nm (Figure 1A). The TEM image of the hydrogel of 2+3 shows long, flexible, and entangled nanofibers with an average width about 9 ± 2 nm (Figure 1B). The extensive entanglement of the nanofibers in the hydrogel of 2+3 agrees with its higher storage moduli than that of the hydrogel of 1+3. This result suggests that the interactions between A-T pairs and the interactions between T-T pairs provide quite different interfibrillar interactions in the hydrogels. There are hardly any ordered nanostructures in the solutions of 1, 2, and 3 (Figure S4), agreeing with the character of a solution.

To investigate the interaction between 1 (or 2) with 3, we examine the mixture of 1+3 (or 2+3) from 0.13 mM to 8.2 mM by circular dichroism (CD). The CDs of 1+3 display a decrease of the intensity of the peak originated from ππ* transition (around 200 nm) with the increase the concentration of mixture (Figure 2A), agreeing with that the association of the heterodimer of 1 and 3 results in antiparallel dipole interactions to reduce the CD signals. The negative peaks shift from 204 nm to 234 nm from 0.13 to 4.1 mM, which is consistent with that the increase of dimerization places the ππ* transition in less aqueous environment.^[25] The sudden peak-shift back to 200 nm at 8.2 mM agrees with the phase transition due to hydrogelation, implying that the weak peaks at 200 nm likely originate from small amount of monomers of 1 or 3 (Figure 2C). For the mixture of 2+3, the ππ* transitions also exhibit red-shift (227 nm to 236 nm) from 0.13 to 2.1 mM (Figure 2B), indicating the increase of dimerization. But the sudden blue-shift back to around 200 nm occurs at 4.1 mM (Figure 2D), which is consistent with that the interaction between 2 and 3 is stronger than that between 1 and 3 to favor self-assembly at lower threshold concentration. The most notable feature of the CD signals of the mixture of 1+3 or 2+3 is α-helix and random-coil like characters of the heterodimeric self-assembly in the hydrogels, which differs from the β-sheet like features usually observed in other types of supramolecular hydrogels.

Figure 2.

Circular dichroism (CD) spectra of (A) 1+3 and (B) 2+3 from 0.13 mM to 8.2 mM. Relationship between wavelength and concentration in CD spectra of (C) 1+3 and (D) 2+3.

We examine the FTIR of the solutions of 1, 3, and 1+3 in DMSO containing different ratios of D₂O (Figure S8, Table S1). After mixing of 1 and 3, the weak N-H stretch vibration peaks turn to a broadened peak, and the broadened peak becomes more expander with increasing the D₂O content. The strong C=O stretch vibration peaks of 1 and 3 also disappear in 1+3, showing two weak peaks at 1687 cm⁻¹ and 1674 cm⁻¹. The weak peaks shift with adding different ratios of D₂O. In addition, N-H bending vibration was weaker in 1+3 compared to that of 1 and 3, and weaker with higher contents of D₂O. These changes of amide vibrations suggest the formation of hydrogen bonds between 1 and 3.

To assess the biostability of the nucleopeptides (1, 2 and 3), we incubate 1, 2, 3, 1+3, or 2+3 with proteinase K, a powerful protease, for 24 h at 37 °C. As shown in Figure 3A, 1 or 2 resists protease K (96.5 ± 1.8 % of 1 or 91.5 ± 1.9 % of 2 remains after 24 h), and 3 hydrolyzes completely after 24 h. Since the pentapeptide (KTTPV) resists proteolysis (Figure S9), the proteolytic stability of 1 and 2 likely originates from the KTTPV. For the solution mixture of 1+3 (or 2+3), 98.9 ± 1.6% of 1 (or 92.1 ± 1.0 % of 2) remains, but 3 still degrades completely after 24 h (Figure 3B). Contrary to the properties of the solution mixtures, the hydrogelation of the heterodimers drastically improve the proteolytic stability of these nucleopeptides, and 98% of 3 remains in the hydrogel of 1+3 or 2+3 after the incubation with proteinase K for 24h (Figure 3C). This result suggests that the strong interactions between nucleopeptides in the gel phase significantly improve the biostability of 3, thus providing a new approach to enhance the resistance of small peptide hydrogels against proteolysis.

Figure 3.

Compounds remained after incubating with proteinase K (3.2 U/mL) for 24 h at 37 °C. (A) Single component of 1, 2, or 3 at the concentration of 0.2 mg/mL in HEPES buffer (pH = 7.4); (B) solution mixture of 1+3 or 2+3 in PBS buffer ([1] = [2] = [3] = 4.1 mM, pH = 6.2); (C) gel mixture of 1+3 or 2+3 in PBS buffer ([1] = [2] = [3] = 8.2 mM, pH = 6.2).

We also investigate their biocompatibility by incubating 1, 2, 3, 1+3, or 2+3 with two types of mammalian cells, HeLa cells (24 h doubling time^[26]) for 3 days and PC12 cells (92 h doubling time^[27]) for 7 days. As shown in Figure S9, at the concentration range of 20~500 μM, the cell viabilities of HeLa are about 100 % after the treatment of 1+3 or 2+3 for 3 days; 1+3 is innocuous to PC12 cells; 2+3 hardly inhibits PC12 cell proliferation (Figure S10). In addition, 1, 2, or 3 shows no toxicity to HeLa cells and PC12 cells (Figures S11 and S12). These results indicate that these nucleopeptides are cell compatible.

In conclusion, this work illustrates a novel and rationale methodology to produce supramolecular hydrogels that integrates known non-covalent interactions between heteropeptidic motifs of proteins and the interactions between nucleobases. The well-established reversibility of the interactions between nucleobases also warrants the reversibility of the interaction between the heterodimeric nucleopeptides. The facile synthesis, biostabililty, and non-β-sheet secondary structures of nucleopeptides in the gel phase render the supramolecular hydrogels of nucleopeptides to have many unexplored features that warrant further development. For example, our result indicates that the use of nucleopeptides promises the development of the supramolecular hydrogels that require the preservation of α-helical and random-coli motifs to exhibit specific functions, which is a direction of our on-going research.