(M1430-12-80) Encapsulation in Glucan Particles Promotes Solubility and Anti-biofilm Activity of Natural Poly-actives In-vitro

Purpose: The ability of microorganisms to cause chronic infections is often linked with their ability to form biofilms. Open wounds contain various types of microorganisms, given the right conditions, microbial clusters mature into biofilms, covering the wound's surface, hindering closure and healing. The wound then becomes chronic and very complex to treat. Natural extracts represent a potential route to the treatment of chronic infections. Polyphenols from natural extracts have been extensively studied not only for their antioxidant and anti-inflammatory properties but also for their ability to act as antimicrobial agents against a broad spectrum of microorganisms. Two natural extracts of particular interest in this respect are propolis and silymarin. The advantage of propolis and silymarin lies in their composition. Both are a mixture of multiple compounds, mainly polyphenols, that can lead to a synergistic effect on bioactivity. Nevertheless, a common problem for many natural extracts is their low solubility in water, which leads to decreased bioactivity. A natural cost-effective solution could be an encapsulation into glucan particles, porous hollow shells obtained from Saccharomyces cerevisiae. Their own bioactivity and natural origin make them a suitable carrier for propolis and silymarin extracts. We encapsulated propolis and silymarin extracts in glucan particles to increase their solubility and determine their anti-biofilm activity in vitro on the different strains: C. albicans, P. aeruginosa, and S. aureus.

Methods: Preparation of glucan particles - Glucan particles were obtained from baker's yeast by multiple washing steps using: sodium hydroxide solution, acidified water, acetone, and isopropanol. The obtained product was lyophilized. Active ingredient encapsulation in glucan particles. Encapsulation was performed using a Mini Spray Dryer B-290 (Buchi AG, Switzerland) with an ultrasonic nozzle under a nitrogen atmosphere. The inlet gas temperature was 120 °C, the flow rate of the suspension was 5 ml/min and the ultrasonic nozzle was set to 2.4 W. The active compound was dissolved in ethanol and GPs were added. The concentration of GPs in suspension was 20 mg/ml. The aim was to ensure that the resulting particles contained 10, 20, and 30 % (w/w) of active substance. Characterization of glucan particles - Characterization of the surface morphology and shape was done by scanning electron microscopy (SEM) using a Joel JCM-5700 microscope. The crystallinity of the samples was determined by powder X-ray diffraction using Panalytical X’Pert PRO with a High Score Plus diffractometer. Dissolution kinetics - The release kinetics was studied using a USP II dissolution test apparatus with 200 ml vessels in phosphate buffer (pH 7.4), at a stirring speed of 150 rpm and a temperature of 37 °C. The concentration of active ingredient at pre-defined time points was evaluated by UV-VIS spectrophotometer Analytik Jena Specord 205. Anti-biofilm activity - Biofilm formation was tested in multispecies consisting of S. aureus, P. aeruginosa and C. albicans. Anti-adhesion tests and anti-biofilm activity tests were performed. The viability of biofilms was evaluated by resazurin assay.

Results: Complete encapsulation of active ingredient in glucan particles was confirmed by SEM images, where no mass outside of glucan particles was observed for all prepared composites. Additionally, XRPD spectra showed an amorphization of originally crystalline silymarin. For the following experiments, only composites with the highest loading were used. The dissolution tests showed a rapid release in the case of silymarin particles, where 50.9% of the dose (40 mg) was dissolved after the first 2 minutes and reached the highest concentration 70.5% of the dose at 5 minutes point. Compared to silymarin extract, where only 16.4% of the dose was released in the first 5 minutes and continued with a slight increase over the testing period. Dissolution profiles of propolis extract and encapsulated propolis showed a significant improvement in propolis solubility after encapsulation in glucan particles. While only around 2% of crude extract was dissolved over the testing period, 73.1% of the dose was released from composites after 15 minutes. Encapsulation of extracts in glucan particles significantly reduced the adherence on all tested strains. Compare to raw extracts, which didn’t show any activity, propolis composites showed activity in the disruption of preformed biofilms in all tested strains, while silymarin composites were active against S. aureus and C. albicans.

Conclusion: We have successfully improved the solubility and anti-biofilm activity of propolis and silymarin by encapsulation in glucan particles. Using this encapsulation method, we were able to achieve a 30% loading with complete amorphization of silymarin and a great increase in surface area in the case of propolis. These aspects highly contribute to solubility improvement, even reaching supersaturation. Encapsulation in glucan particles also proved to enhance inhibition of adherence and formation of C. albicans, P. aeruginosa, and S. aureus biofilms up to 10 times. Propolis loaded in glucan particles presented a great improvement in the disruption of mature biofilms of all tested strains. Our results indicate a possibility for a natural but effective solution for chronic wound treatment.

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Acknowledgements: The financial support from the Specific University Research (MSMT), of the Czech Science Foundation (project No. GX19-26127X)

Dissolution profiles

Anti-biofilm activity