Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation
| Autor: | C. Vila-Sanjurjo, Bruno M. Moerschbacher, S Kolkenbrock, Stefan Hoffmann, Tobias Weikert, Francisco M. Goycoolea, J. P. Fuenzalida |
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| Rok vydání: | 2014 |
| Předmět: |
Polymers and Plastics
Glycoside Hydrolases Polymers Nanoparticle Bioengineering Madin Darby Canine Kidney Cells Biomaterials Chitosan chemistry.chemical_compound Dogs Materials Chemistry Fluorescence Resonance Energy Transfer Animals chemistry.chemical_classification Chemistry technology industry and agriculture Substrate (chemistry) Acetylation Polymer Fluorescence In vitro Recombinant Proteins Förster resonance energy transfer Biochemistry Biophysics Nanoparticles |
| Zdroj: | Biomacromolecules. 15(7) |
| ISSN: | 1526-4602 |
| Popis: | Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Forster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provide in vitro proof-of-concept that these approaches can be used to label CS-NPs and colocalize them following their interactions with mammalian cells. |
| Databáze: | OpenAIRE |
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