Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive

Abstract

Expanding the bioactivation toolbox of supramolecular materials is of utmost relevance for their broad applicability in regenerative medicines. This study explores the functionality of a peptide mimic of the Notch ligand Jagged1 in a supramolecular system that is based on hydrogen bonding ureido-pyrimidinone (UPy) units. The functionality of the peptide is studied when formulated as an additive in a supramolecular solid material and as a self-assembled system in solution. UPy conjugation of the DSL_JAG1 peptide sequence allows for the supramolecular functionalization of UPy-modified polycaprolactone, an elastomeric material, with UPy-DSL_JAG1. Surface presentation of the UPy-DSL_JAG1 peptide was confirmed by atomic force microscopy and X-ray photoelectron spectroscopy analyses, but no enhancement of Notch activity was detected in cells presenting Notch1 and Notch3 receptors. Nevertheless, a significant increase in Notch-signaling activity was observed when DSL_JAG1 peptides were administered in the soluble form, indicating that the activity of DSL_JAG1 is preserved after UPy functionalization but not after immobilization on a supramolecular solid material. Interestingly, an enhanced activity in solution of the UPy conjugate was detected compared with the unconjugated DSL_JAG1 peptide, suggesting that the self-assembly of supramolecular aggregates in solution ameliorates the functionality of the molecules in a biological context.

Figure 1

Chemical structures of compounds used in this study and schematic representations of the supramolecular platforms and their components: UPy unit (blue), urea-alkyl spacer (red), oligo(ethylene glycol) spacer (white), and peptide sequence (yellow). (A) Chemical structure of UPy-DSL_JAG1. (B) Solid material functionalization by mixing UPy-DSL_JAG1 with UPy-PCL, with detailed zoom of the co-assembly of the two components in the solid state. (C) Supramolecular fibrous stack formed in solution by UPy-DSL_JAG1.

Figure 2

Surface characterization of drop-cast samples of UPy-PCL with the addition of UPy-DSL concentration of 1 and 5 mol % and HEK293 FLN1 cell response. (A) AFM micrographs in the phase mode. (B) XPS spectra with highlighted peaks corresponding to elements of interest. (C) Magnification of the XPS spectrum at the characteristic binding energy interval of S 1s for the different material compositions.

Figure 3

Effect of immobilized 5 mol % UPy-DSL_JAG1 on the activation of 12× CSL-Luc in HEK293 FLN1, compared with the FcJagged1 ligand as the positive control. Data are normalized with respect to the negative control (Fc-fragment coating only).

Figure 4

Effect of Notch ligand-functionalized substrates on HCASMCs. Gene expression of downstream Notch target genes HES1 and HEY1 on (A) control and (B) polymer surfaces. Expression of JAG1 and NOTCH3 genes related to lateral induction on (C) control and (D) polymer surfaces. Phenotypic switch of CASMC with expression of ACTA2 on the mRNA level on (E) control and (F) polymer surfaces. (G) CASMC differentiation to contractile phenotype visualized by immunofluorescence by DAPI (blue) and α-SMA (magenta) staining (scale bar is 100 μm).

Figure 5

(A) Cryo-TEM micrographs of aqueous solutions of DSL_JAG1 (left) and UPy-DSL_JAG1 (right), with white arrowheads indicating fibrous assemblies (scale bar is 50 nm). (B) Nile Red intensity of 50 μM solutions of DSL_JAG1 (dotted line) and UPy-DSL_JAG1 (solid line).

Figure 6

Effect of soluble DSL_JAG1 (pink bars) and UPy-DSL_JAG1 (red bars) on Notch-signaling activity in the HEK293 FLN1 cell line cultured on Fc coating (basal activity—white bar) or FcJagged1 coating (enhanced basal activity—black bar). Soluble molecules are used at concentrations of 10, 50, and 100 μM. Data are normalized with respect to the Fc control.