ABSTRACT
We investigated two ways for fabricating 1, 3, 4, 6-tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (Ac42AzGlc)-loaded poly (lactic-co-glycolic acid) PLGA nanoparticles in this article : 1) single emulsion solvent evaporation and 2) the nanoprecipitation method. Among the available methods of collecting nanoparticles using an ultra-high-speed centrifuge, we improvised a less-known method for collecting synthesized nanoparticles without a high-speed centrifuge, based on molecular weight (MW)-dependent centrifugal filters. These nanoparticles were collected in a tabletop centrifuge at a meager centrifugal force in the range of 200-300 xg whereas the conventional high-speed centrifuge method for nanoparticle recovery results in a hard nanoparticle pellet with poor resuspendability which hampers the yield and outcomes of the product. The Ac42AzGlc-loaded PLGA nanoparticles were spherical in shape with consistent and reliable nanometric particle size. The polydispersity indices were well within the acceptable limits. The preliminary studies in RAW 264.7 cell and C57BL/6 mice advocated efficient engineering in the former; however, the latter needs further confirmatory investigations. Preliminary in vivo studies with un-encapsulated Ac42AzGlc showed poor engineering of cardiac glycoproteins, opening up avenues for Ac42AzGlc-loaded nanoparticles for improved bioavailability and efficient metabolic engineering.
ABSTRACT
Owing to its poly-anionic charge and large hydrodynamic volume, polysialic acid (polySia) attached to neural cell adhesion molecule regulates axon-axon and axon-substratum interactions and signalling, particularly, in the development of the central nervous system (CNS). Expression of polySia is spatiotemporally regulated by the action of two polysialyl transferases, namely ST8SiaII and ST8SiaIV. PolySia expression peaks during late embryonic and early post-natal period and maintained at a steady state in adulthood in neurogenic niche of the brain. Aberrant polySia expression is associated with neurological disorders and brain tumours. Investigations on the structure and functions, over the past four decades, have shed light on the physiology of polySia. This review focuses on the biological, biochemical, and chemical tools available for polySia engineering. Genetic knockouts, endo-neuraminidases that cleave polySia, antibodies, exogenous expression, and neuroblastoma cells have provided deep insights into the ability of polySia to guide migration of neuronal precursors in neonatal brain development, neuronal clustering, axonal pathway guidance, and axonal targeting. Advent of metabolic sialic acid engineering using ManNAc analogues has enabled reversible and dose-dependent modulation polySia in vitro and ex vivo. In vivo, ManNAc analogues readily engineer the sialoglycans in peripheral tissues, but show no effect in the brain. A recently developed carbohydrate-neuroactive hybrid strategy enables a non-invasive access to the brain in living animals across the blood-brain barrier. A combination of recent advances in CNS drugs and imaging with ManNAc analogues for polySia modulation would pave novel avenues for understanding intricacies of brain development and tackling the challenges of neurological disorders.
ABSTRACT
Sialic acids are abundant in the central nervous system (CNS) and are essential for brain development, learning, and memory. Dysregulation in biosynthesis of sialo-glycoconjugates is known to be associated with neurological disorders, CNS injury, and brain cancer. Metabolic glycan engineering (MGE) and bioorthogonal ligation have enabled study of biological roles of glycans in vivo; however, direct investigations of sialoglycans in brain have been intractable. We report a simple strategy utilizing carbohydrate-neuroactive hybrid (CNH) molecules, which exploit carrier-mediated transport systems available at the blood-brain barrier, to access brain via tail vein injection in mice. Peracetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) conjugated with neuroactive carriers, namely, nicotinic acid, valproic acid, theophylline-7-acetic acid, and choline, were synthesized and evaluated in SH-SY5Y (human neuroblastoma) cells for MGE. Intravenous administration of CNH molecules in mice (C57BL/6J and BALB/cByJ) resulted in robust expression of N-azidoacetyl-neuraminic acid (NeuAz)-carrying glycoproteins in both brain and heart, while the nonhybrid molecule Ac4ManNAz showed NeuAz expression in heart but not in brain. Successful neuroactive carriers were then conjugated with N-butanoyl-d-mannosamine (ManNBut) with a goal to achieve modulation of polysialic acid (polySia) on neural cell adhesion molecules (NCAM). PolySia levels on NCAM in adult mice were reduced significantly upon administration of Ac3ManNBut-nicotinate hybrid, but not with Ac4ManNBut. This novel application of MGE not only offers a noninvasive tool for investigating brain glycosylation, which could be developed in to brain mapping applications, but also serves as a potential drug by which modulation of neural glycan biosynthesis and thus function can be achieved in vivo.
