Inulin crystal initiation via a glucose-fructose cross-link of adjacent polymer chains: Atomic force microscopy and static molecular modelling

Abstract

Semi-crystalline microparticles of inulin (MPI) have clinical utility as potent human vaccine adjuvants but their relevant surface structure and crystal assembly remain undefined. We show inulin crystal surfaces to resemble multi-layered, discoid radial spherulites resulting from very rapid formation of complex tertiary structures, implying directed crystal initiation. Physical and in silico molecular modelling of unit cells confirm steric feasibility of initiation by hydrogen-bonded cross-linking of terminal glucose to a fructose of another chain, mimicking bonding in sucrose crystals. A strong, chelate-like dual H-bond is proposed to compel the known antiparallel alignment of inulin chains. Such cross-linking would require one extra fructose per chain in the native inulin crystal, as observed. Completion of five H-bonded internal ring-domains would ‘lock in’ each new 6-fructose structural unit of each antiparallel helix pair to create a new isoform. All known properties of inulin isoforms follow readily from these concepts.

Physical characterization and in silico modeling of inulin polymer conformation during vaccine adjuvant particle formation

“…For polyfructose, the highest peak force occurs when fructose residues at the end of one chain are hydrogen bonded with the 4 th and the 5 th fructose residues located in the middle of the adjacent chain (Supplementary Information, Figure S12). Thereby, taking both the umbrella calculations and the force versus time profiles into account, overall the MD simulations support our previously theorized importance of the glucose and fructose end groups in the formation of stable semi-crystalline structures for inulin (Cooper, Rajapaksha, Barclay, Ginic-Markovic, Gerson & Petrovsky, 2015). …”

“…The changing nature of the Porod region upon precipitation is illustrated in Figure 3b, which shows an increase in the R g to 29 Å between 150 and 300 min, attributable to the formation of nanoscale structures. As this change in R g occurs, the changing Porod exponent (Figure 3c) indicates that the randomly coiled inulin (t < 125 min; p = 1.68–1.66) aggregates into high-axial-ratio ribbons (t = 215; p = 1.36) previously identified in time-resolved AFM studies (Cooper, Rajapaksha, Barclay, Ginic-Markovic, Gerson & Petrovsky, 2015), which then transform to fully-formed nanostructured inulin microparticles (t > 290 min; p > 2) (Hammouda, 2010). …”

“…Time-resolved AFM studies of the formation of inulin particles have been described recently by us (Cooper, Rajapaksha, Barclay, Ginic-Markovic, Gerson & Petrovsky, 2015). Early morphologies include small, round structures that have an average height of 5.5 nm (std = 0.9 nm) and diameter of 53 nm (std = 12 nm) when dried on a flat surface (Figure 4a).…”

“…For computational efficiency, inulin with a degree of polymerization of eight sugar rings was used as this is the minimum size able to assume a helical arrangement comprising a six fructose unit cell with additional glucose and fructose end groups (Cooper, Rajapaksha, Barclay, Ginic-Markovic, Gerson & Petrovsky, 2015; Oka, Ota, Mino, Iwashita & Komura, 1992). The simulation commenced with either two antiparallel inulin helices or two antiparallel polyfructose helices.…”

“…Samples were taken over time, diluted to 0.1 mg/ml and then 5 μL was air-dried on a silicon wafer for imaging. Full details of the imaging process have been published previously (Cooper, Rajapaksha, Barclay, Ginic-Markovic, Gerson & Petrovsky, 2015) and are also presented in the Supplementary Information.…”