Optimization of the Microscopic Method of Observing of the Oleogel Structure

An example of a two-component mixture of gelators is lecithin–STS oleogels. None of these substances can independently formulate a gel from oil. However, when their mixture is used in the right proportions, a synergistic connection occurs, leading to successful oleogelation. If this is the minimum concentration of the mixture inducing the formation of a gel, a mass concentration of 4% is sufficient.

The subject of the research was the oleogel structure of the oral suspension OraMAF, containing a mixture of active substances, in which the oleogel environment is created by purified olive oil in combination with SL and STS, performing the role of gelling agents. Other components of the investigated mixture were glycosaminoglycans, fructooligosaccharides, and heteropolysaccharides, low-molecular-weight CS, F, inulin, dextrose, cholecalciferol, and live bacterial strains of Lactobacillus delbrueckii, subsp. bulgaricus and Streptococcus thermophilus. The oleogel environment protects suspended substances from oxygen and moisture, thus providing increased microbiological stability (Zima, 2021).

The goal of the research was the development of an optimal methodology for the work process, enabling microscopic observation of the structure of the oleogel suspension.

We tried to separate the insoluble lecithin residues by the filtration method. Gelators STS and SL were dissolved in olive oil by heating to a temperature of 80°C. The mixture was stirred during heating until the target temperature was reached with a common laboratory stirrer to prevent the formation of air bubbles. Subsequently, the mixture was allowed to cool to room temperature. To separate the insoluble residues of SL, filtration under ordinary pressure using a cellulose filter partition was subsequently used. The mentioned preparation method did not prove successful as the dispersion showed considerable viscosity and insoluble residues of SL and the gel structure clogged the filter partition, which made normal filtration impossible.

Batches of STS and SL were dissolved in olive oil by heating to a temperature of 80°C. The mixture was stirred with a high-speed stirrer during heating until the target temperature was reached. Subsequently, the dispersion was allowed to slowly cool to room temperature. The dispersion prepared in this way, containing air bubbles and crushed insoluble residues of SL, was allowed to settle for 14 days at room temperature. The clear supernatant of the dispersion was separated from the sediment containing the remains of insoluble SL by the mentioned procedure. This method proved to be technically undemanding, practical, and repeatable and was suitable for the preparation of clear oleogel dispersion as an optimal environment for microscopic observation of the gel structure and the way solid particles are trapped in the gel structure.

To increase the contrast of the observed dispersion and capture of solid particles, colorless powder samples of low-molecular-weight CS and F were colored using organic dyes. Both investigated substances are well soluble in water. It follows that it was not possible to use an organic dye soluble in water, since after mixing an aqueous solution of the dye with a solid substance, the solid substance itself would dissolve. Therefore, dyes soluble in an ethanol/propanol mixture in a volume ratio of 1:2 were used, while solid substances did not dissolve in the said mixture. Solutions of methylene blue and organic red were used to stain solids. The dye solution was mixed with the solid and then the solvent was evaporated. Using this procedure, colored samples of solids suitable for microscopic observation were obtained.

The sedimentation method of separation of the oleogel medium and the coloring of the solid phase of the suspension with organic dyes brought the expected results, allowing the observation of the network structure of the gel capturing solid particles (Fig. 3).

Figure 3.

Investigation of the dispersion formed by SL and STS brought insight into the direct mechanism of gel network formation. The most interesting structures were clearly the star-like shapes created from the microtubules of the STS and lecithin complex, colored blue. The coloring was provided by the added dye methylene blue, which was partially adsorbed on the gelling substances, thus enhancing the contrast and enabling the observation itself. The results of the observation confirm that a combination of STS and SL forms fibrous three-dimensional structures of various lengths, intertwined and connected in some places into typical star-shaped structures (Fig. 3). It can be assumed that the growth of fibers starts at central points and further branches out in several directions. The initial step is thus the nucleation of nuclei. From the point of view of the capture of solid particles, it was possible to identify individual particles of F and CS, distributed over the entire surface of the sample. Macroscopically, it was also possible to observe that these solid components do not sediment because they are stabilized in the formed solid gel network. The stability is disturbed when the temperature increases when the gel melts.

The main goal of the experiment was to prepare and optimize the procedure for preparing an oleogel suspension using combined gelators suitable for microscopic observation. Sedimentation separation of the medium and staining of the solid phase of the suspension enabled the microscopic observation of the three-dimensional structure of the oleogel, which consisted of interlaced gelator fibers assembling into star formations. Solid connections also provide an excellent basis for maintaining solid particles, which can be represented by similar bioactive molecules. In pharmaceutical technology, the full potential of oleogelation is still not sufficiently utilized and the transfer of knowledge from the theoretical level to common practice is lacking. However, in the researched area, new findings on the incorporation of drugs are increasing very rapidly and ways of influencing the liquid oil environment continue to be an attractive topic for many scientific researchers.