Revealing the Microstructure of Binary Solvent Hydrogels: a Novel Cryo-SEM Approach

Hydrogels are viscoelastic networks of hydrophilic polymers capable of absorbing large amounts of water within their structure. Their biocompatibility and resemblance to biological tissues result in diverse applications in tissue engineering, drug delivery, agriculture, and bioelectronics [1-4]. Despite their advantages, hydrogels also face challenges such as improving thermal and environmental stability or incorporating useful hydrophobic compounds. To resolve these issues, researchers have formulated binary-solvent gels, such as those in aqueous dimethyl sulfoxide (DMSO) [5-6], that are more stable and interact more favorably with hydrophobic compounds. Unlike water-only hydrogels, however, the internal structure of these binary solvent hydrogels remains poorly understood due to the lack of sample preparation techniques specific to the materials systems. The natural microstructure of water-only hydrogels (and also gels coagulated with hydrophobic compounds) can be studied utilizing a previously developed preparation method for cryogenic scanning electron microscope (cryo-SEM) characterization [7]. This preparation method is centered on rapid sample freezing in cryogenic media and controlled sublimation to remove water molecules physically trapped within the hydrogel’s polymeric network [8]. This previously-developed preparation method, however, tends to fall short in revealing the true microstructure of binary solvent hydrogels, and therefore, the influence of a secondary solvent on the hydrogel structure is not yet fully understood. The difficulty arises from the co-solvent’s effect on the short-range order of water molecules which inhibits sublimation [9]. In order to observe the true hydrogel structure in a binary solvent system, it is vital to develop a new sample preparation method for cryo-SEM characterization. In this study, a benzimidazolone (BZI) derivative supramolecular hydrogel, synthesized in both single and binary solvent systems, was characterized using cryo-SEM. The single solvent hydrogel was prepared in water while the binary solvent hydrogel was obtained in 75 wt.% water and 25 wt.% DMSO solvent. The DMSO-containing binary solvent hydrogel was developed to improve coagulation of hydrophobic drug compounds within the hydrogel structure. Since the phase diagram of the DMSO/water system at cryogenic temperatures and low pressures is not defined, determining the sublimation conditions for such a system necessitates experimentation [10]. This includes identifying an optimized condition that balances the appropriate sublimation temperature and duration while ensuring specimen integrity. The new method designed for binary solvent hydrogels initiates with a soaking treatment of a small piece (5×5×5 mm3) of hydrogel for 30 min in 50 ml of deionized (DI) water (Figure 1A). This soaking condition was determined by periodic monitoring of the sample in an optical microscope to ensure no change in size, shape or color of the hydrogel occurs. Subsequently, the sample was squeezed between a set of copper rivets to form a thin but continuous gel. While held together, the coupled rivets are plunged in liquid nitrogen (LN2) for rapid freezing; then, the rivets are separated to expose a fractured cross-section (Figure 1B). To eliminate ice and reveal the hydrogel structure, a sublimation step is required [7]. While a mild sublimation (10-20 min at -100oC) suffices to reveal the structure in water-only hydrogels, for a binary solvent hydrogel the time and temperature were carefully re-designed to make the sublimation stronger. The re-optimized process consists of 40 min sublimation at -90°C and 10-6-10-7 mbar in a Leica ACE600 (Figure 1C). The higher sublimation temperature is found to be effective for removing binary solvent from the hydrogel structure while avoiding specimen damage. The duration of sublimation in this recipe was determined by monitoring the pressure profile to ensure ongoing stability. Furthermore, a temperature ramp is introduced to the recipe to impede sample damage due to thermal shocks. This includes 5 minutes of thermal soaking for every 10oC temperature increment between -140 and -90oC. The processing completes with adding a thin platinum layer by sputter coating for 120 s at -140oC [7]. Finally, the sample is transferred to a Zeiss NVision 40 at -140oC for cryo-SEM observation at 3 keV acceleration voltage using an in-lens EsB (energy and angle selective backscattered electron) detector. Figure 2A exhibits the honeycomb matrix in the water-only BZI hydrogel, with pores ∼3.2 μm in diameter, which were defined by thin and flat faces, composed of fine fibrous structures (yellow arrows), resulting from previously reported sublimation protocol [7]. In contrast, following the same sample preparation protocol for the BZI hydrogel developed in the binary solvent system delivers an amorphous structure with no evidence of honeycomb architecture (Figure 2B). Figure 2C details the changes of temperature (red line) and pressure (green line) during the previously developed sublimation. It was hypothesized that the obscure structure of the binary solvent hydrogel could be attributed to stronger intermolecular bonding between water and DMSO molecules, as compared to the bonding in water-only systems [9]. This enhanced bonding was thought to impede the effectiveness of the previously reported sublimation method. The application of a novel preparation method in our research provided evidence demonstrating that the binary solvent's unique interactions indeed play a critical role in maintaining the hydrogel structure. Figure 2D shows the revealed chamber-like structure of the binary solvent BZI hydrogel, emerged from entangled fibers (yellow arrows), when the new protocol including the soaking and modified sublimation (Figure 2E) are applied. Similarly, the inset in Figure 2B presents a vague view of fibers covered with remaining solvent while the inset in Figure 2D details the entangled fibers of the hydrogel network which is analogous to fibers developed in water-only hydrogels (inset in Figure 2A). In summary, we provide a novel preparation method for characterizing binary solvent hydrogels with cryo-SEM. The method includes a controlled soak of the hydrogel in deionized water (30 min, 50 ml) to reduce the DMSO concentration. Additionally, followed by a controlled multi-step temperature reduction under continuous pressure regulation, the temperature and time for an effective sublimation were -90 oC and 40 min respectively. We demonstrated that our new cryo-SEM characterization method can successfully remove the surface solvent to expose the details of hydrogel structure while ensuring structural integrity. This newly developed method also helps to understand the other complicated binary solvent hydrogel structures, and the structure with organic solvents in their system using cryo-SEM characterization. The method will be harnessed in the future to comprehend the microstructural evolution of the binary solvent BZI hydrogel when it is loaded/unloaded with hydrophobic drugs [11]. Sample preparation for binary solvent hydrogels: A) hydrogel soaking in deionized water; B) squeezing the hydrogel between the rivet set, plunging in LN2 and freeze fracturing; C) sublimation and Pt coating in ACE600 cryo-coater; and D) imaging in Zeiss NVision40 cryo-SEM/FIB system. A) Cryo-SEM images of BZI derivative hydrogel for water-only hydrogel prepared with previously developed method; B) morphology and C) sublimation curves for binary solvent hydrogel prepared with previously developed method; D) morphology and E) sublimation curves for binary solvent hydrogel prepared with newly developed method. The insets in 2A, 2B and 2D compare the exposure of fibrous structure of hydrogels in high resolution.

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