Rational design of MMP degradable peptide-based supramolecular filaments

Abstract

One-dimensional nanostructures formed by self-assembly of small molecule peptides have been extensively explored for use as biomaterials in various biomedical contexts. However, unlike individual peptides that can be designed to be specifically degradable by enzymes/proteases of interest, their self-assembled nanostructures, particularly those rich in β-sheets, are generally resistant to enzymatic degradation because the specific cleavage sites are often embedded inside the nanostructures. We report here on the rational design of β-sheet rich supramolecular filaments that can specifically dissociate into less stable micellar assemblies and monomers upon treatment with matrix metalloproteases-2 (MMP-2). Through linkage of an oligoproline segment to an amyloid-derived peptide sequence, we first synthesized an amphiphilic peptide that can undergo a rapid morphological transition in response to pH variations. We then used MMP-2 specific peptide substrates as multivalent cross-linkers to covalently fix the amyloid-like filaments in the self-assembled state at pH 4.5. Our results show that the cross-linked filaments are stable at pH 7.5 but gradually break down into much shorter filaments upon cleavage of the peptidic cross-linkers by MMP-2. We believe that the reported work presents a new design platform for the creation of amyloid-like supramolecular filaments responsive to enzymatic degradation.

Figure 1

(a) Chemical structure of the studied molecule and (b) the key design feature of the molecular assembly principle: the reversible nature of dissociating the self-assembled 1D filaments in response to pH variation. (c) Illustration of the cross-linking strategy and the expected degradation pathway by targeted MMP. PCL represents peptide cross-linkers (PCL) containing MMP specific substrates. The designed amphiphilic peptide is expected to self-assemble into stable 1D nanostructures at a lower pH value, followed by a postcross-linking process that employs EDC/NHS chemistry and utilizes PCLs as linkers. Chemical structures of the PCL linkers are presented in Scheme 1.

Scheme 1. Chemical Structures of the Four Cross-Linkers Designed/Used in the Study

Figure 2

Molecular assembly and characterization of the design peptide. TEM (a) and cryo-TEM (b) micrographs of supramolecular filaments formed by MASP1 in MES buffer at 2.1 mM. Both bars = 200 nm. (c) Photograph of 2.1 mM MASP1 in MES buffer (pH 4.5; a self-supporting gel marked with a white arrow) and in Tris buffer (pH 7.5; a fluidic liquid). (d) Fluorescence spectra of Thioflavin T when incubated with 500 μM MASP1 in MES and Tris buffer. Concentration of Thioflavin T = 100 μM; excitation: 442 nm. (e) CD spectra of 400 μM MASP1 in MES and Tris buffer, showing strong characteristics of PPII helical secondary structure of the oligoproline.

Figure 3

(a) Chemical structure of C₈-Sup35. Cryo-TEM (b) and TEM (c) micrographs of the filamentous structures formed by C₈-Sup35 in acidic conditions (pH 4.5) at 2.1 mM. (d) TEM micrograph of the filaments formed by C₈-Sup35 in pH ∼ 7 at 2.1 mM. All bars = 200 nm.

Figure 4

TEM micrographs of filaments after the postcross-linking treatment and qualitative analysis of cross-linked filaments. TEM micrographs of cross-linked filaments by PCL1 (a), PCL2 (b), EDBE (c), and spermine (d) at pH ∼ 7. All scale bars = 200 nm. (e) Analytical HPLC traces of cross-linked filaments by PCL1 (blue) and PCL2 (orange; solid lines). In all cases, the corresponding peaks remain sharp and monodisperse in the chromatograms if EDC/NHS were not introduced to initiate the cross-linking chemistry (dashed lines).

Figure 5

Enzymatic degradation studies on the cross-linked filaments in the presence of MMP-2. MALDI-ToF mass spectra of cross-linked filaments by PCL1 (a), PCL2 (b), and EDBE (c) before the enzymatic treatment (top spectrum), after the addition of Tris buffer (middle spectrum), and after 6 h of incubation with MMP-2 (bottom spectrum). Peaks are labeled with the (m,n) format, in which m represents the number of MASP1 molecules and n represents the number of the cross-linking molecules. (d) Schematic illustration of a representative pentamer of MASP1 linked with five PCLs and their possible degradation products after enzymatic cleavage of the PCLs.

Figure 6

(a) TEM micrographs for the PCL2-cross-linked (top panels) and EDBE-cross-linked (bottom panels) filaments after 6 h incubation with MMP-2. The lengths of PCL2-cross-linked filaments were significantly reduced (top left panels) after incubation with MMP-2 for 6 h, while the EDBE-cross-linked filaments did not reveal any noticeable changes in filament length (bottom left panels). Both PCL2-cross-linked (top right panels) and EDBE-cross-linked filaments (bottom right panels) are very stable in the absence of MMP-2, and did not display any noticeable changes in filamentous structures 6 h after Tris buffer was added. (b) Schematic illustration of degradation pathways of supramolecular filaments cross-linked by MMP degradable and nondegradable linkers.