Determination of Structure-Property Relationships for Polysaccharide Hydrogels via Correlated Electron Microscopy and Ultra-Small-Angle Neutron Scattering
Year: 2025
Type: article
Abstract: Polysaccharides are among the most-commonly used family of polymer in the world due to the lower critical solution temperature (LCST) behavior of many cellulose-derivatives. The LCST behavior allows for the viscosity to increase as a function of temperature and has made these class of polymers excellent candidates for additives in cosmetics, food packaging, and the pharmaceutical industry. Despite their extensive use, the origins of their mechanical properties has been widely debated. Specifically, the most heavily studied of these classes of materials are methylcellulose (MC) and hydroxypropylmethylcellulose (HPMC) [1-4]. MC is a water-soluble semi-flexible polymer at low temperatures and transitions into a turbid gel at temperatures greater than the lower critical solution temperature (LCST) (Figure 1A and 1B) [5-8]. Over the last decade, cryogenic TEM (cryo-TEM) and bulk small-angle neutron scattering (SANS) techniques have shown that gelation results from the self-assembly of MC chains into ∼20 nm diameter fibrils, which then arrange into a physical gel (Figure 1C) [2, 4, 9, 10]. Following this discovery, cryo-TEM supported the argument that the rheological properties of these randomly percolated fibrillar networks could be related to the previously established model of semiflexible biopolymer networks [11]. Yet, due to the resolution limitations of conventional cryo-TEM and the disturbance of the fibril network inherent to sample preparation, the mechanism of assembly of the MC chains within the fibrils and the assembly of the fibrils into network have yet to be revealed by real-space techniques [12]. Furthermore, these same artifacts have prevented the direct observation of the native gel structure. To study the sol-gel phase transition of MC and other LCST polysaccharides, we have utilized variable temperature liquid cell transmission electron microscopy (VT-LCTEM), an in situ imaging technique that enables high resolution insight into solvated nanomaterials. We have directly observed, using VT-LCTEM, that the gelation of MC at elevated temperatures is driven by its self-assembly into an ordered fibril network (Figure 1D and 1E). To confirm these observations were not an artifact of the liquid cell geometry, we not only performed VT-SANS and VT-Ultra-SANS to verify higher-order bundle structures, but we also reinvented a freeze-drying methodology established in the 1980’s and imaged the resulting samples via scanning electron microscopy (SEM) to confirm the presence of fibers in the bulk hydrogel [13]. Both the VT-USANS and the freeze-dried SEM micrographs of MC gels confirm the observations originally made via VT-LCTEM. Furthermore, we found that conducting VT-USANS and freeze-dried SEM on other LCST polysaccharide hydrogels, such as HPMC, also reveals previously unobserved higher-order structures. This observed order contradicts previous hypotheses that fibrillar hydrogel networks are composed of randomly percolated fibrils and would lead to substantial changes in the observed rheological properties [14-16]. Moreover, complementary bulk rheological experiments on these polymers have revealed that the previous semiflexible biopolymer network model may not be applicable to accurately describe the relationship between the storage modulus and the polymer concentration [3, 17, 18]. Combining these data, we were able to determine persistence lengths of gel forming units, confirming that sample preparation is critical to determine structure-property relationships (Figure 2A and 2B). We believe that due to the lack of significant temperature and shearing artifacts, VT-LCTEM followed by freeze-drying SEM are superior techniques to reveal the native fibrillar network structure more accurately across all length scales, but the bulk network architecture should be confirmed with VT-USANS. Thus, utilizing the persistence lengths determined from our VT-LCTEM and freeze-dried SEM micrographs of the LCST polysaccharide hydrogels, we are better able to predict the rheological properties as a function of MC and HPMC concentration. These observations confirm the need to readdress the methodologies currently utilized to study fibrillar networks (biomaterials) but may also aid in broadening the application of these types of materials in the pharmaceutical industry and beyond. A. Chemical structure of cellulose-derivatives. B. Photograph illustrating the transition of MC from water-soluble polymer to a turbid gel. C. Cryo-TEM micrographs illustrate a collection of flexible polymer fibrils that form a disorganized network. D. VT-LCTEM micrographs that reveal the formation of aligned bundles of fibrils upon heating the liquid cell above the LCST. E. Zoomed micrograph of an individual 20 nm diameter fibril composing the larger fibril bundle. A. Collection of plots fitting the storage moduli (G’) vs concentration (c) according to the semiflexible cylinder model. Utilizing the persistence lengths determined via image analysis of VT-LCTEM, freeze-dried SEM, and cryo-TEM of MC gels, the rheological properties of MC are most accurately estimated by the models considering the higher persistence lengths (purple and light blue curves) when compared the bulk rheology data (red curve). B. Collection of plots fitting the storage moduli (G’) vs concentration (c) according to the semiflexible cylinder model. Utilizing the persistence lengths determined via image analysis of VT-LCTEM, freeze-dried SEM, and cryo-TEM of HPMC gels, the rheological properties of HPMC are most accurately estimated by the models considering the higher persistence lengths (light blue curve) when compared the bulk rheology data (red curve).
Source: Microscopy and Microanalysis
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4412911042