Briefly, during the second reporting period, we have engineered a library of modified glycogen nanoparticles. We engineered glycogen nanoparticles with tuneable structural and functional properties for the fabrication of advanced materials
First, we developed a simple and cost-effective method to reduce the size of glycogen nanoparticles in a controlled manner, achieving diameters ranging from 80 nm to 15 nm. These nanoparticles were subsequently functionalized with amine and carboxyl groups, as well as Protein A and Protein G, to enable antibody conjugation and the loading of RNA molecules, peptides, and small hydrophobic drugs. To investigate the interactions between glycogen and nucleic acids, molecular dynamics (MD) simulations were performed. These simulations characterized the binding between 20-nucleotide segments of luciferase-coding mRNA and amine-modified glycogen branches under both physiological (pH 7.4) and endosomal (pH 5) conditions. Next, we utilized biodegradable phytoglycogen nanoparticles (PG NPs)—highly branched glucose-based polysaccharides—as scaffolds for coupling adhesive dopamine motifs (for use as biodegradable underwater adhesives) and gold nanoparticles (for potential biosensing applications). The extent of chemical modification of PG NPs was assessed by NMR spectroscopy. The size distribution and colloidal stability of PG NPs in aqueous suspension were evaluated using dynamic light scattering (DLS), particle tracking, and chromatography. The morphology of the modified PG NPs was examined by electron microscopy (EM), atomic force microscopy (AFM), and super-resolution microscopy, while surface charge was determined via electrophoretic mobility measurements. Therapeutic peptides with anticancer and antimicrobial activity were synthesized using solid-phase peptide synthesis protocols and characterized by fluorescence spectroscopy and circular dichroism (CD). These positively charged peptides were either chemically conjugated to glycogen nanoparticles or encapsulated within negatively charged glycogen nanoparticles via electrostatic interactions. In parallel, porous microparticles based on hyaluronic acid microsponges (MSPs) were fabricated and loaded with therapeutic peptides for oral delivery. The stability of the microsponges and the retention of the peptide payload were evaluated in biological media mimicking the gastrointestinal tract, using fluorescence spectroscopy. Finally, glycogen nanoparticles were loaded with hydrophobic drugs, including GSA-10 and resveratrol, which are molecules under investigation for the potential treatment of multiple sclerosis and HIV, respectively. The interactions and bioactivities of these drug-loaded glycogen nanoparticles were assessed in T cells, cancer cells, and oligodendrocytes, focusing on cellular uptake kinetics and intracellular trafficking, using confocal microscopy and flow cytometry.
