Development and characterization of nano-micro structures as carrier for bioactive compounds

Abstract

New biopolymers are in high demand due to their excellent biocompatibility, biodegradability, and natural origin. In this PhD project, water soluble fish sarcoplasmic proteins (FSPs) from the North Atlantic cod (Gadus morhua) have been studied as a potential new biopolymer for development of nano-micro structures. Two kinds of nano-micro structures have been explored: electrospun fibers (Paper I, Paper II, and Paper III) and self-assembled nanocomplexes (NCXs) (Paper IV). FSP was observed to be highly suitable for electrospinning. The fiber morphology varied significantly with FSP concentration, from beads to fibers. Moreover, the morphology within one FSP concentration was very diverse, as evident from the fiber diameter ranging from nanosized to micronsized (Paper I). The size distribution of the fiber diameter was decreased by removal of low molecular weight compounds (< 8 kDa). Despite the water-soluble nature of FSP, the fibers were insoluble in aquatic media (except at high sodium dodecyl sulfate concentrations) (Paper I, Paper II, and Paper III). Contact angle measurements indicated that the FSP fibers were hydrophobic, and incubation with the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), confirmed the presence of hydrophobic pockets inside or at the surface of the fibers (Paper III). Interestingly, the physical properties of the fibers significantly changed after incubation with surfactants as well as with the surfactant type; the FSP fibers were dense after incubation with cationic surfactant, whereas inner porosity of the fibers was observed after incubation with anionic or neutral surfactants. Moreover, the contact angle changed from being large for anionic surfactants, to being small for neutral and cationic surfactants. Lastly, the cationic and neutral surfactants decreased the amount of hydrophobic pockets available for dye interaction (Paper III). The inherent property of FSP as consumed food made the fibers degradable by proteolytic enzymes, and the degradation products were observed to inhibit the diabetes related enzyme dipeptidyl peptidase-4 (Paper I). The FSP fibers showed potential as carrier system for delivery of drugs, bioactive agents, and nutraceuticals. The dipeptide Ala-Trp, rhodamine B, or insulin was encapsulated into the fibers, and the release was studied in biorelevant media (Paper I, Paper II, and Paper III). Release of Ala-Trp was slightly decreased in gastric environments compared to pH 6.8, whereas release of insulin was independent of pH. Instead, insulin release was affected by the presence of biorelevant compounds, i.e. surfactants and proteins encountered in the intestinal system. Anionic surfactants increased the release of insulin from FSP fibers in a dose-dependent manner, neutral surfactants had no effect, and cationic surfactants decreased the insulin release to negligible amounts (Paper III). Encapsulation of insulin into the FSP fibers provided protection against chymotrypsin degradation, and interactions between the fibers and epithelial cells led to opening of the tight junction, which promoted an increased transepithelial transport of insulin without compromising cellular viability (Paper II). The FSPs were also suitable for development of self-assembled NCXs. By gentle bulk mixing of FSP and alginate, stable NCXs were formed (Paper IV). The NCXs were 293 ± 3 nm and anionic (zeta potential was −42 ± 0.3 mV). The zeta potential as a function of pH revealed that the NCX surface was dominated by alginate. The NCXs were stable in biorelevant media, and at pH values from 2 to 9, except at pH 3 where the NCXs aggregated. Proteolytic enzymes were capable of degrading the NCXs. The viability of HeLa and U2OS cell lines was only decreased by high concentrations of NCXs (Paper IV). It was concluded that FSP is highly suitable for the production of functional nano-micro structures for food and biomedical applications qua the ability of the FSPs to form electrospun fibers and self-assembled NCXs. The inherent properties of FSP of being biocompatible, biodegradable, and bioactive further promote the use of FSP as a biopolymer.