[Thesis]: Formulation and synthesis of materials with β-glucans

foto-tribunal
Marta Salgado (center left) along with the tribunal members and her thesis supervisors (From left to right: Dr. Ana Rita C. Duarte, Dr. Concepción Domingo, Dr. Ángel Martin, Dr. Marta Salgado, Dr. Marleny D. A. Saldaña, Dr. Jerry W. King, Dr. Jorge Luis Sague, Dr. Soraya Rodriguez-Rojo, Dr. Maria Jose Cocero)

Dr. Marta Salgado defended her thesis on Wednesday 26th October 2016 at the University of Valladolid.

Nowadays, there is a tendency towards the use of natural products in industrial applications, especially in those related with the environment and human health. These renewable raw materials, usually non-toxic, are present in many structures in nature and therefore are able to mimic well their behavior and act in a more efficient way. Some of the most commonly studied products are polyphenols and polysaccharides, the first ones as active compounds mainly due to their anti-oxidant effect, and the seconds as biopolymers instead of some other processed polymers traditionally used in diverse applications.

In this thesis, the ability of β-glucans to be used in formulation of active compounds and in synthesis of materials was evaluated. β-glucans are polysaccharides composed by D-glucose units linked by glycosidic bonds. They are present in different sources in nature, such as cereals, bacteria or algae. Depending on their origin and bonding, they have very different properties regarding 3D-configuration or biological activity, among others. Unlike other polysaccharides like alginate or starch, the use of β-glucans in these fields is not widely studied. The final applications of the products developed with β-glucans were chosen by analysis of the properties of β-glucans and the advantages that they could provide over other biopolymers on those applications. Barley and yeast β-glucans were selected as raw materials (Figure 1).

Figura 1. Structure of barley (a) and yeast β-glucans (b).
Figura 1. Structure of barley (a) and yeast β-glucans (b).

Solid formulations with barley β-glucans were studied in Chapter I. Resveratrol was the selected active compound. It is a phytoalexin spontaneously produced by some plants as a defense mechanism when they are attacked by a fungal infection. Besides, soy lecithin was also used as encapsulating material, both alone and in combination with barley β-glucans, to assess the influence of the encapsulating material on the final product. First, an oil-in-water emulsion was formed by high-shear emulsification, and upon removal of the organic solvent (ethyl acetate), the suspension was dried by PGSS-drying or spray-drying. Also SAS was performed, but it was not a suitable process to produce dry particles of β-glucan from an organic solution (dimethyl sulfoxide), because great quantity of the organic solvent remained trapped into the β-glucan structure. Well-dried particles of barley β-glucans, lecithin and a mixture thereof containing resveratrol were obtained both by spray-drying and by PGSS-drying (Figure 2). The greatest difference between both drying processes was related to particle size, which was found to be dependent on the drying process rather than on the encapsulating material. Smaller particles were produced by PGSS-drying, although they formed bigger agglomerates (in the range of 10 μm by spray-drying and 100 μm by PGSS-drying). Encapsulation efficiency of resveratrol was high (60-96%) and similar between the drying-processes for each encapsulating material since it depends mostly on the emulsification process, which was the same in all cases. According to XRD analysis, amorphous resveratrol was obtained in the final dry product due to the interaction of the active compound with the carriers. Botrytis cinerea was the fungus chosen for assessing the antifungal activity of the different formulations, because it is one of the fungi containing β-glucans on its cell wall. Previous works reported growth inhibition of B. cinerea at 100 ppm of pure resveratrol. This was not observed in our work, but fungal growth was reduced between 50 and 70% with all the formulations developed in this chapter.

Figure 2. SEM images of BBG particles produced by spray-drying (a), PGSS-drying (b) and by SAS (5 g/L in DMSO, precipitation at 10 MPa, 35º C, 2 mL/min; c and d).
Figure 2. SEM images of BBG particles produced by spray-drying (a), PGSS-drying (b) and by SAS (5 g/L in DMSO, precipitation at 10 MPa, 35º C, 2 mL/min; c and d).

Chapter II includes the development of liquid formulations of barley and yeast β-glucans. As in the previous chapter, soy lecithin was also utilized in this one for comparison, and resveratrol as active compound. For the liquid formulation, an oil-in-water emulsion was first developed, and the final suspension was produced by removal of the organic solvent by vacuum evaporation. Three different emulsification techniques were used: high-pressure, high-pressure and temperature and high-shear emulsification. Comparing the emulsification methods, particle size and encapsulation efficiency achieved in the final suspensions were similar for all of them (below 90 nm and between 70-100%, respectively). The encapsulating material affected also the final properties of the product. Nevertheless, the crystallinity of resveratrol depended both on the emulsification procedure and on the encapsulating material. Yeast β-glucans provided higher encapsulation efficiency than barley β-glucans, and also better inhibition of fungal growth of B. cinerea (50% for yeast β-glucans and 20% for barley β-glucans). The formulations with lecithin, both alone and in combination with β-glucans, did not reduced fungal growth, which might be a consequence of the presence of crystalline resveratrol in the final encapsulated particles (Figure 3). Therefore, it was concluded that the formulation of resveratrol with β-glucans improved the action against B. cinerea, probably through an enhanced absorption of the active compound by the fungus.

Figure 3. Growth area of B. cinerea with the centrifuged suspensions of resveratrol. Light grey: high-shear emulsification. Dots: high-pressure emulsification. Dark grey: high pressure and temperature emulsification. *: non-centrifuged suspensions.
Figure 3. Growth area of B. cinerea with the centrifuged suspensions of resveratrol. Light grey: high-shear emulsification. Dots: high-pressure emulsification. Dark grey: high pressure and temperature emulsification. *: non-centrifuged suspensions.

In Chapter III, barley and yeast β-glucans aerogels were produced by supercritical drying with CO2. First hydrogels were created with both β-glucans, and their rheological behavior was studied. It was determined that the ones produced with yeast β-glucans were more stable, resistant to shear stress and elastic than the ones with barley β-glucans because of their different structure and 3D-configuration in the gel. On the one hand, barley β-glucan is formed by linear chains of polymer that arrange parallel creating a more compact material. On the other hand, yeast β-glucan is a branched polymer which upon gelation creates a structure with more void space due to the crosslinking of the chains. Then, solvent was changed from water to ethanol, and ethanol was further extracted with supercritical CO2. The noticed differences between both β-glucans in the hydrogels were also observed in the aerogels. Thus, yeast β-glucan aerogels had higher density, were stronger against compression stress and were able to absorb more water. Nevertheless, all the aerogels had similar morphology, with mean pore size between 13-16 nm and BET surface area around 180 m2/g. Furthermore, supercritical impregnation of acetylsalicylic acid was also performed at the same time as the drying of the alcogels, at different temperature and pressure conditions. Up to 15% impregnation was achieved, with an increasing tendency with CO2 density. Finally, release of the drug in PBS was analyzed. An initial delay for the first 3h of analysis was noticed, which was indicative of a good impregnation of acetylsalicylic acid into the β-glucan matrix by supercritical impregnation. After that time, 60% of the drug was released for the following 5h, and this value was maintained for 16h more. By analysis of the release profile, it was determined that the release was controlled by relaxation of polymer chains and swelling of the matrix (Figure 4).

Figure 4.- (a) Quantity of acetylsalicylic acid per mass of aerogel at different conditions of pressure and temperature. Black: 50ºC; Dark grey: 40ºC; Light grey: 35ºC. (b) Cumulative release of acetylsalicylic acid per mass of aerogel. Lines are added to guide the eye.
Figure 4.- (a) Quantity of acetylsalicylic acid per mass of aerogel at different conditions of pressure and temperature. Black: 50ºC; Dark grey: 40ºC; Light grey: 35ºC. (b) Cumulative release of acetylsalicylic acid per mass of aerogel. Lines are added to guide the eye.

The materials produced in chapter III were not porous enough to be used as scaffolds in tissue engineering. Therefore, Chapter IV explores the possibility of creating more porous materials with barley and yeast β-glucans by hydrogel foaming with supercritical CO2. In this process, the hydrogels created with the β-glucans were subjected to high-pressure CO2, that was dissolved in them in those conditions. Upon depressurization, supersaturation of CO2 in the hydrogels promoted the formation of pores inside. After complete water removal by freeze-drying, the structures produced were highly porous, achieving up to 80% porosity, mean pore size of 250 μm and 75% of interconnected pores (Figure 5). These parameters increased with foaming pressure, although at the highest pressure tested (20 MPa) the porosity of the resulting materials was non-homogeneously distributed, having an external crust of polymer while the inner part was mostly hollow. Despite the good porosity achieved, the scaffolds produced with both β-glucans were brittle and had low resistance to compression stress. Scaffolds were loaded also with dexamethasone, which is a compound extensively used because of its anti-inflammatory properties and its ability to induce differentiation of stem cells towards osteogenic linage. The scaffolds provided a controlled release until total dissolution up to 4 days, with higher release rate during the first 8h. Analysis of the mechanism governing the release revealed that it was mainly (above 75%) controlled by diffusion of the drug in the liquid medium, with slight contribution of relaxation of polymer chains.

Figure 5: Porosity (a), interconnectivity (b) and mean pore size (c) of the scaffolds at different foaming pressure. Closed symbols: BBG. Open symbols: YBG. Lines are added to guide the eye.
Figure 5: Porosity (a), interconnectivity (b) and mean pore size (c) of the scaffolds at different foaming pressure. Closed symbols: BBG. Open symbols: YBG. Lines are added to guide the eye.

Thus, in this thesis barley and yeast β-glucans were effectively used as encapsulating agents in antifungal products. Also, they were processed and studied as biomaterials for medical and pharmaceutical applications. In both cases, supercritical fluids were utilized as green technology to achieve the final products.

Thesis Supervisors:

  • Soraya Rodríguez-Rojo
  • M. José Cocero
  • Ana Rita C. Duarte (3B’s Research Group, University of Minho).

Members of the committee:

  • Dr. Jerry W. King, University of Arkansas
  • Dr. Marleny D. A. Saldaña, University of Alberta
  • Dr. Concepción Domingo, University of Barcelona
  • Dr. Jorge Luis Sague, RMS Foundation
  • Dr. Ángel Martín, University of Valladolid
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