Preparation and characterization of propolis-loaded polymeric nanoparticles, investigation of their immunomodulatory effects on macrophage cells

Studies in cell cultures and animal models have shown that various bee products (honey, propolis, bee pollen, etc.) and some of the compounds they contain have important pharmacological activities. For this reason, bee products find intensive use in various fields such as healthy nutrition, apitherapy and biocosmetics production. Propolis (bee glue), which is one of the important honey bee products and contains more than 300 bioactive substances, becomes more important with its excellent natural medicine properties discovered in the current century. Depending on its origin, propolis contains various compounds such as sugars, amino acids, flavonoids, phenolic acids, fatty acids, alcohols, aldehydes, and steroids. Biological activities of propolis are generally associated with its rich phenolic content. Propolis, and one of its flavonoids, quercetin, have various biological properties such as anti-allergic, anti-microbial, anti-inflammatory, anti-cancer, anti-oxidant. Its strong sticky behavior, pungent taste and low water solubility limit the use of propolis as a health-supporting product. To overcome these disadvantages and increase bioavailability, nanotechnological encapsulation techniques can be used. In this thesis, the effects of propolis ethanolic extract, commercial quercetin and their polycaprolactone (PCL)-encapsulated forms on some immune response-related cytokines (IL-4, IFN-γ and IL-1β) and nitric oxide (NO) levels were investigated in J774 macrophage cell line. First, an ethanolic extract was prepared from local propolis and this extract was used as a Propolis sample in the experiments. Then, PCL-encapsulated nanoforms of propolis extract and quercetin (Propolis-PCL and Quercetin-PCL) were produced using the dual emulsion solvent evaporation method. The structure of the nanoforms was characterized by UV-VIS spectrophotometry, zeta potential measurement, FTIR, SEM, and XRD analyses. In the next stage of the study, the cytotoxic effects of the prepared samples (Propolis, Quercetin, Propolis-PCL and Quercetin-PCL) on J774 macrophage cells were examined by MTT method, and their non-toxic concentrations were determined. Expression of cytokines and release of NO in J774 macrophage cells were induced by administering a non-toxic dose of lipopolysaccharide (LPS) from Escherichia coli (serotype O111:B4). Finally, the levels of immune response-related cytokines (IL-4, IFN-γ and IL-1β) in Propolis, Quercetin, Propolis-PCL and Quercetin-PCL treated and untreated cells were determined by ELISA method. In addition, the release of nitric oxide (NO), one of the markers of immune response, was analyzed by the Griess reaction. Propolis-PCL and Quercetin-PCL nanoformulations were successfully produced and characterized, and their immunomodulatory effects were compared with those of Propolis and Quercetin. It was determined that all samples at the tested concentrations (5, 25, 50 and 100 µg/mL) inhibited NO release in LPS-induced J774 cells. While 100 µg/mL concentrations of all samples increased the IL-4 level, the inductive effect of nanoforms, most notably Propolis-PCL, was higher. Only 50 µg/mL concentration of Propolis-PCL increased the IFN-γ level. In addition, it was determined that the only sample that reduced IL-1β level was 100 µg/mL Propolis. As a result, in this study, important data were obtained regarding the immunomodulatory effects of propolis and one of its components, quercetin, and polymeric nanoparticles loaded with these substances. In the future, these data may contribute to the development of more bioavailable propolis nanoformulations and new bee products that target the expression of immune response-related cytokines and NO release.