Amyloid-polysaccharide interfacial coacervates as therapeutic materials

Abstract

Coacervation via liquid-liquid phase separation provides an excellent opportunity to address the challenges of designing nanostructured biomaterials with multiple functionalities. Protein-polysaccharide coacervates, in particular, offer an appealing strategy to target biomaterial scaffolds, but these systems suffer from the low mechanical and chemical stabilities of protein-based condensates. Here we overcome these limitations by transforming native proteins into amyloid fibrils and demonstrate that the coacervation of cationic protein amyloids and anionic linear polysaccharides results in the interfacial self-assembly of biomaterials with precise control of their structure and properties. The coacervates present a highly ordered asymmetric architecture with amyloid fibrils on one side and the polysaccharide on the other. We demonstrate the excellent performance of these coacervates for gastric ulcer protection by validating via an in vivo assay their therapeutic effect as engineered microparticles. These results point at amyloid-polysaccharides coacervates as an original and effective biomaterial for multiple uses in internal medicine.

Fig. 1. Schematic of AF and HA interfacial coacervation for developing advanced materials.

a films, b fibers and c capsules.

Fig. 2. Various amyloid fibrils (AF) - hyaluronic acid (HA) coacervate products and the effect of major parameters on coacervate capsules.

Photographs of a films, b fibers and c capsules formed by coacervation of AF and HA (The colorful image in panel c shows the direction of the fibrils in the plane, see the colormaps in the top-right corner). d Concentration phase diagram for producing capsules (pH 3). AF as the inner phase and HA as the outer phase and e HA as the inner phase and AF as the outer phase. f pH phase diagram for producing capsules (AF 2wt.% and HA 1wt.%). AF as the inner phase and HA as the outer phase and g HA as the inner phase and AF as the outer phase. Yellow square and shading refer to no sac, green circle and shading refer to unstable sac, blue up-triangle and shading refer to stable sac and purple down-triangle and shading refer to AF/HA gelation. Confocal microscopy of h rhodamine-labeled AF, i the fluorescein-labeled HA within the capsule wall, j merged image (confirming colocalization of the AF and HA within the capsule’s wall) and k) transmission image. The confocal microscopy experiment was repeated at least three times and representative images were shown.

Fig. 3. Characterization of amyloid fibrils (AF) - hyaluronic acid (HA) coacervate films at different sides and interfaces.

a The scheme of AFM-IR experimental setup while scanning on the film. b Morphological images (left), maps of infrared (IR) absorption in the Amide I at 1654 cm⁻¹ (middle) and stiffness that indicates the nanomechanical properties of the top (upper) and the bottom (lower) side of the film. The crosses refer to the locations for performing IR spectroscopy measurements. The scale bar is 500 nm. The AFM-IR experiment was repeated at least three times and representative images were shown. c Nanoscale localized infrared spectrum of the top and bottom side of the film collected at the crossed location in panel b. SEM images (d) surface of AF side, (e) AF-HA interface and f. surface of HA side. The SEM experiment was repeated at least three times and representative images were shown. g XPS C1s and O1s chemical state spectra of pure AF and HA, as well as top and bottom surfaces of coacervate films. 2D WAXS scattering patterns of (h) pure AF and (i) AF + HA films. j Intensity I as a function of the azimuthal angle (χ) of AF and AF + HA films. k Intensity I as a function of scattering vector q of AF and AF + HA films, as well as HA powder. p₁ and p₂ are the two identified peaks.

Fig. 4. AF-HA coacervate therapeutic application.

a Schematic representation of the development of microparticles and their application in the in vivo assay. b FEG-SEM images of spray-dried AF-HA microparticles. The FEG-SEM experiment was repeated at least three times and a representative image was shown. c lesion area of the gastric ulcer in rats and (d) macroscopic photo images of representative stomach tissue with or without gastric ulcer. Data are expressed as the mean±SD (n = 8); a one-way analysis of variance (ANOVA), followed by Fisher’s LSD test, p = 0.0003, compared with the positive control group. NC negative control; PC positive control; OME omeprazole; MP microparticles. e Schematic of mechanisms for gastric ulcer protection by AF-HA microparticles.