Green Nanotechnology and Phytochemical Mediated Production of Ketone Encapsulated Protein Nanoparticles—in vitro and in vivo Bioavailability Investigations

Alice Raphael Karikachery,1 Kavita K Katti,1 Velaphi C Thipe,1 Prajna Hegde,2 Deepa Prakash,2 Anantkumar Hegde,2 Alton Michael Chesne,3,4 Kattesh V Katti1,4– 6 1Department of Radiology, Institute of Green Nanotechnology, University of Missouri, Columbia, MO, 65212, USA; 2Kadamba Intrac Private Ltd, Bangalore, KA, 560011, India; 3Tecton Group, LLC, Alexandria, LA, 71302, USA; 4Indus Advance Green Nanotechnology Institute, Indus University, Gujarat, 382115, India; 5Departments of Physics, Medical Pharmacology and Physiology, University of Missouri, Columbia, MO, 65211, USA; 6 6Department of Biotechnology and Food Technology, University of Johannesburg, Doornfontein, 2028, South AfricaCorrespondence: Kattesh V Katti, Department of Radiology, Institute of Green Nanotechnology, University of Missouri, Columbia, MO, 65212, USA, Email [email protected]: Low carbohydrate, ketogenic foods have shown convincing evidence for their metabolic role in mitigating severe adversities due to obesity and other chronic diseases. They induce systemic ketosis: a process where ketone bodies, namely β-hydroxybutyrate, acetoacetate and acetone are produced in vivo. Beyond serving as an alternative source of energy besides glucose, various analogs of ketones present unprecedented opportunities for therapeutic interventions in the management of numerous chronic diseases and neurological disorders. The profound benefits of ketone bodies to human health, unquestionably, demand exogenous administration of ketone molecules in doses that promote and enhance energy levels in the human body. Hence, it is of paramount importance to develop sophisticated delivery vehicles wherein ketones are made bioavailable in a sustainable fashion in vivo. Engineering nano-formulations of ketone molecules allow efficient cellular penetration of ketones, thus presenting prospects for enhanced bioavailability of energy molecules in vivo. In this article, we report nanoencapsulation of (R)-3-hydroxybutyrate monoglyceride, a Ketone Molecule (KM) within biocompatible pea protein nano-framework utilizing natural phytochemical crosslinking.Purpose: The goal was to develop a sophisticated delivery vehicle wherein ketones are made bioavailable in a sustainable and biocompatible fashion.Methods: We present full details on the production of well-defined Ketone Molecule (KM) encapsulated nanoparticles of pea protein using naturally available crosslinking agents such as mangiferin, epigallocatechin 3-O-gallate (EGCG) and quercetin from their respective plant extracts. The Ketone Molecule (KM) encapsulated Pea Protein Nanoparticles by phytochemical crosslinking was fully characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) size and zeta potential (ZP) measurements. The KM concentration was estimated using gas chromatography–mass spectrometry (GC-MS). Phytochemical and water-soluble pea protein interaction was comprehensively studied using nuclear magnetic resonance (NMR) spectroscopy.Results: Green nanotechnology offers the most effective means to encapsulate and transform small molecules into pea protein nanoparticles with optimum size for effective cell-specific delivery, thus offering an attractive delivery vehicle to enhance bioavailability. The Ketone Molecule (KM) encapsulated Pea Protein Nanoparticles, by phytochemical crosslinking importantly, demonstrated the most favorable in vivo pharmacokinetics with sustained (R)-3- hydroxybutyrate (BHB) levels and higher area under the curve (AUC) relative to free KM.Conclusion: Novel pathways toward the design and development of protein nanoparticle-encapsulated ketone molecules were explored utilizing plant-based proteins from a biocompatibility, biodegradability, and biosafety perspective.Keywords: green nanotechnology, nanoparticle delivery, ketone molecule, nutraceutical applications