Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel

Abstract

Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K₂(SL)₆K₂ (abbreviated as K₂), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K₂ hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K₂ hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K₂. This imine interaction was also observed to improve K₂ + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K₂, R-Man loaded in K₂, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K₂ can be used to normalize the release of O-Man to that of R-Man. Further, since K₂ itself is an adjuvant, a combination of O-Man and K₂ could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.

Figure 1.

Schematic showing O-Man (blue) and R-Man (red) loaded into a K₂ hydrogel. Aldehyde groups on O-Man can form imine bonds with the amines on the K₂ peptide fibers whereas the hydroxyl groups present on R-Man cannot form imine bonds with K₂.

Figure 2.

(A) Schematic of oxidation and reduction reactions on a representative portion of the linear mannan polysaccharide showing one mannose monomer being oxidized and then subsequently reduced. (B) Results of the fluorometric aldehyde quantification assay show the percent of mannose monomers oxidized, referred to as aldehyde functionalization, for O-Man and R-Man. Mixing O-Man with K₂ led to a significant decrease in aldehyde content, suggesting the formation of imine bonds between the peptide and the carbohydrate.

Figure 3.

Structural characterization of K₂ loaded with O-Man and R-Man. (A) Area normalized FTIR absorbance spectrum of K₂, K₂ loaded with O-Man (K₂ + O-Man), and K₂ loaded with R-Man (K₂ + R-Man) showing characteristic peaks for antiparallel sheet: 1675 cm⁻¹, 1695 cm⁻¹, and β-sheet: 1620 cm⁻¹. The peak shift observed in K₂ + O-Man is attributed to the presence of imine bonds. (B) CD spectrum of K₂, K₂ + O-Man, and K₂ + R-Man show a characteristic minimum at 218 nm and maximum at 198 nm for β-sheets. TEM negative stain of (C) K₂ + O-Man and (D) K₂ + R-Man demonstrate both have a nanofibrous architecture. The scale bar is 200 nm.

Figure 4.

Oscillatory rheology characterization of the viscoelastic properties of K₂, K₂ loaded with O-Man (K₂ + O-Man), K₂ loaded with O-Man (K₂ + R-Man), and K₂ loaded with mannan (K₂ + Man). (A) Storage modulus of each hydrogel and (B) percent recovery 10 min after a deformation force yielding 200% strain.

Figure 5.

(A) In vitro release of K₂ loaded with unmodified mannan (K₂ + Man), O-Man (K₂ + O-Man), and R-Man (K₂ + R-Man) over a 24 h period demonstrates an overall slower release of K₂ + O-Man. (B) In vivo release IVIS images of O-Man, R-Man, K₂ + O-Man, and K₂ + R-Man for 56 days after injection. (C) Graph of the percent of released carbohydrate from the first 30 days of the in vivo release experiment (n = 4–5). R-Man, K₂ + O-Man, and K₂ + R-Man all release at similar rates while O-Man is cleared from the site of injection at a significantly faster rate.