Supramolecular Hydrogels from a Tripeptide and Carbon Nano-Onions for Biological Applications

Abstract

Nanocomposite hydrogels have attracted researchers' attention in recent years to achieve superior performances in a variety of materials applications. In this work, we describe the outcome of three different strategies to combine a self-assembling tripeptide and carbon nano-onions (CNOs), through covalent and non-covalent approaches, into supramolecular and nanostructured hydrogels. Importantly, the tripeptide coated the nano-onions and extended their aqueous dispersions' stability by several hours. Furthermore, CNOs could be loaded in the tripeptide hydrogels at the highest level ever reported for nanocarbons, indicating high compatibility between the components. The materials were formed in phosphate-buffered solutions, thus paving the way for biological applications, and were characterized by several spectroscopic, microscopic, thermogravimetric, and rheological techniques. In vitro experiments demonstrated excellent cytocompatibility.

Keywords: D-amino acids; biomaterials; carbon nano-onions; carbon nanomaterials; gels; hydrogelation; nanocomposites; peptides; self-assembly; supramolecular chemistry.

Figure 1

Nanocomposite supramolecular hydrogels from CNOs and the self-assembling tripeptide (Lff) were envisaged through three strategies.

Figure 2

TGA data in (a) air and (b) nitrogen atmosphere of p-CNOs, oxi-CNOs, and amino-PEG-CNOs. Weight loss (red lines) and its first derivative over temperature (grey dashed lines) were consistent with the covalent functionalization of CNOs.

Figure 3

Spectra of p-CNOs, oxi-CNOs and amino-PEG-CNOs from analysis by (a) ATR-FTIR and (b) Raman spectroscopy.

Figure 4

Photographs of (a) oxi-CNOs without (left) and with (right) the self-assembling tripeptide Lff in alkaline (pH 11.8) sodium phosphate precursor solution after 4 h post sonication (15 min) to prepare gels with 1.0 mg/mL oxi-CNOs, and (b) nanocomposite supramolecular hydrogels with 2.5 mg/mL of Lff and increasing loadings of oxi-CNOs (0.5, 1.0 and 2.0 mg/mL) at pH 7.4.

Figure 5

TEM micrograph of the nanocomposite supramolecular hydrogel formed by Lff in the presence of oxi-CNOs through the non-covalent approach. Inset figure shows aggregate of oxi-CNOs in the absence of the Lff peptide.

Figure 6

(a) Raman spectrum of the nanocomposite supramolecular hydrogel formed by Lff in the presence of oxi-CNOs. (b) Raman mapping of a non-covalent hydrogel fiber; intensity scalebar: the brighter cyan areas correspond to higher levels of oxi-CNOs.

Figure 7

Oscillatory rheology of the nanocomposite supramolecular hydrogels obtained through the non-covalent approach. (a) Time sweeps. (b) Frequency sweeps. (c) Stress sweeps.

Figure 8

Photographs of: (a) Lff-PEG-CNOs (N-terminus approach) with the self-assembling tripeptide Lff in alkaline (pH 11.8) sodium-phosphate precursor solution; (b) nanocomposite supramolecular hydrogels with Lff-PEG-CNOs (1.0 mg/mL) and 2.5 mg/mL of Lff at pH 7.4. (c) Raman mapping of a hydrogel fiber; intensity scalebar: brighter cyan areas correspond to higher levels of Lff-PEG-CNOs.

Figure 9

Oscillatory rheology of the nanocomposite supramolecular hydrogels obtained through the covalent approach. (a) Time sweeps. (b) Frequency sweeps. (c) Stress sweeps.

Figure 10

TEM micrograph of the nanocomposite supramolecular hydrogel formed by Lff in the presence of Lff-PEG-CNOs through the covalent approach.

Figure 11

Live (green)/dead (red) fluorescence staining of fibroblasts grown in contact with the peptide hydrogel without (a) or with (b–d) oxi-CNOs. Scalebars represent 50 μm (top panels) or 10 μm (bottom panels).

Figure 12

MTT metabolic assay on fibroblast cells grown on the plastic control (ctrl), the peptide gel without (Lff gel) or with increasing concentrations of oxi-CNOs (+0.1, +1.0, or +10 μg/mL oxi-CNOs). No statistically significant (n.s.) difference was found between tested conditions (p > 0.05).