Physicochemical Characterization of FRET-labelled Chitosan Nanocapsules and Model Degradation Studies

Abstract

Sub-micron o/w emulsions coated with chitosan have been used for drug delivery, quorum sensing inhibition and vaccine development. To study interactions with biological systems, nanocapsules have been fluorescently labelled in previous works, but it is often difficult to distinguish the released label from intact nanocapsules. In this study, we present advanced labelling strategies based on FRET measurements for chitosan-coated nanocapsules and investigate their dissolution and degradation. We used FRET measurements of nanocapsules loaded with equimolar concentrations of two fluorescent dyes in their oily core and correlated them with DLS count rate measurement and absorbance measurements during their disintegration by dissolution. Using count rate measurements, we also investigated the enzymatic degradation of nanocapsules using pancreatin and how protein corona formation influences their degradation. Of note, nanocapsules dissolved in ethanol, where FRET decreased simultaneously with count rate, and absorbance caused by nanocapsule turbidity, indicating increased distance between dye molecules after their release. Nanocapsules were degradable by pancreatin in a dose dependent manner, and showed a delayed enzymatic degradation after protein corona formation. We present here novel labelling strategies for nanocapsules that allow us to judge their status and an in vitro method to study nanocapsule degradation and the influence of surface characteristics.

Differences of the tumour cell glycocalyx affect binding of capsaicin-loaded chitosan nanocapsules

“…Moreover, reduced cellular binding of negatively charged nanoemulsions (diameter = 135 ± 28 nm, ζ -potential = − 31.4 ± 4.83 mV, Fig. 6 b 25 , 26 ) further confirmed that the interaction between Chi-NCs and the urothelial cells is driven by electrostatic forces (Fig. 6 b).…”

“…Chi-NCs were prepared according to the solvent displacement technique 61 with modifications 26 . Briefly, an organic phase was formed by dissolving 40 mg of lecithin (Cargill, Krefeld, Germany) in 1 mL of ethanol, followed by the addition of 530 µL capsaicin solution (Sigma-Aldrich; 96% pure dissolved in ethanol to 24 mg mL −1 ), 125 μL Miglyol 812 (Peter Cremer Oleo, Hamburg, Germany), and the lipophilic dye DiO (Santa Cruz Biotechnology, Heidelberg, Germany) to obtain capsaicin concentration of 10 mM and dye concentrations of 0.158 mM, in the final formulation after evaporation 25 .…”

“…Unloaded Chi-NCs were prepared by replacing the capsaicin solution with ethanol. The organic phase was made up to 10 mL with ethanol and then immediately poured over 20 mL of an aqueous chitosan solution (0.5 mg mL −1 dissolved in water with a 5% stoichiometric excess of 1 M HCl) containing chitosan HMC 70/5 with a degree of acetylation of 32% (as determined by 1 H NMR) and a molecular weight of 5.01 × 10 4 g mol −1 (Heppe Medical Chitosan, Halle, Germany), the same material as in our previous studies 26 . The Chi-NCs formed spontaneously due to solvent displacement and Marangoni effects 72 .…”

“…The Chi-NCs formed spontaneously due to solvent displacement and Marangoni effects 72 . Finally, the ethanol and part of the water were evaporated at 40 °C under vacuum in a R210 Rotavapor (Büchi Labortechnik, Essen, Germany) and the volume of the formulations was reduced to 6 mL 26 . The nanoemulsions were prepared using the same procedure but without including chitosan.…”

“…In further experiments, we aimed to correlate the extent of the glycocalyx with its ability to interact with positively charged Chi-NCs. The applied Chi-NCs are composed of an oily, lecithin-covered core and a polycationic chitosan shell 25 , 26 . Chitosan is a linear polysaccharide consisting of β(1 → 4)-linked units of glucosamine and N -acetylglucosamine, with a degree of acetylation usually below 40%, thus appearing as a polycation in aqueous media due to the protonation of the glucosamine amine groups 27 – 29 .…”