The Effect of Ethanol on Gelation, Nanoscopic, and Macroscopic Properties of Serum Albumin Hydrogels

Abstract

Serum albumin has shown great potential in the development of new biomaterials for drug delivery systems. Different methods have been proposed to synthesis hydrogels out of serum albumin. It has been observed that ethanol can also act as a trigger for serum albumin denaturation and subsequent gelation. In this study, we focus on basic mechanisms of the albumin gelation process at 37 °C when using the chemical denaturant ethanol. The temperature of 37 °C was chosen to resemble human body temperature, and as under physiological conditions, albumin is in a non-denatured N conformation. As established in our previous publication for the triggers of pH and temperature (and time), we here explore the conformational and physical properties space of albumin hydrogels when they are ethanol-induced and show that the use of ethanol can be advisable for certain gel properties on the nanoscopic and macroscopic scale. To this end, we combine spectroscopic and mechanically (rheology) based data for characterizing the gels. We also study the gels′ binding capacities for fatty acids with electron paramagnetic resonance (EPR) spectroscopy, which implies observing the effects of bound stearic acids on gelation. Ethanol reduces the fraction of the strongly bound FAs in bovine serum albumin (BSA) hydrogels up to 52% and induces BSA hydrogels with a maximum storage modulus of 5000 Pa. The loosely bound FAs in ethanol-based hydrogels, besides their relatively weak mechanical properties, introduce interesting new materials for fast drug delivery systems and beyond.

Molecular-Level Release of Coumarin-3-Carboxylic Acid and Warfarin-Derivatives from BSA-Based Hydrogels

“…Thermally induced gels were obtained by keeping the vials in a thermomixer at 65 °C and 59 °C, above and below the denaturation temperature of BSA, while some hydrogels were prepared at 37 °C by the addition of 2 M HCl and lowering the pH to 3.5 by the so-called pH induced method. The mechanism of hydrogel formation from albumin by heating and chemical crosslinking has been previously established [27]. Thermally induced gel formation requires conformational changes and unfolding of polypeptide segments induced by heating above denaturation temperature of the albumin, which results in the availability of functional groups present in intramolecular hydrogen bonding for intermolecular interactions, which are essential in the aggregation and build-up of the gel network [28].…”

“…We recently elucidated the ability to form hydrogels from bovine and human serum albumin below their denaturation temperature and wide pH range [19]. Moreover, ethanol induced hydrogels can be formed at 37 • C by mixing different concentrations of BSA solution and chemical denaturant ethanol [27].…”

“…Thus, by spin labeling or spin probing techniques, which are based on the covalent or non-covalent corporation of paramagnetic substances such as organic nitroxide radicals into the drug, the protein, or both, it is possible to study drug-protein interactions in drug delivery systems. Furthermore, through this method, information about the local environment, motional parameters, and ligand binding of the protein can be obtained, which allow for deep insights into the release properties [16,25,27,33]. Spectral information as well as spatial distribution of free radicals can be studied by EPR imaging.…”

Hydrogels from serum albumin in a molten globule‐like state

“…[9][10][11][12] Albumin hydrogels have previously been formed by thermal denaturation and chemical cross-linking. [13][14][15][16][17] These hydrogel systems typically require either extensive protein denaturation (thermal) or chemical modification, which can hamper protein functionality and compromise biocompatibility. Another method called electrostatically triggered albumin self-assembly introduced by Baler et al 18,19 takes advantage of the fact that albumin has the ability to reversibly change its conformation when exposed to changes in solution pH while at least partly preserving secondary structure elements that may act as functional binding domains.…”

“…The reason behind this insolubility is that the polymer is physically and/or chemically cross‐linked 9–12 . Albumin hydrogels have previously been formed by thermal denaturation and chemical cross‐linking 13–17 . These hydrogel systems typically require either extensive protein denaturation (thermal) or chemical modification, which can hamper protein functionality and compromise biocompatibility.…”