Delivery of siRNA using cationic polymeric nanoparticles to understand the localization and function of GABAergic neurotransmission in planaria

Abstract

According to the World Health Organization (WHO), epilepsy affects about 50 million people worldwide, with nearly 80% of cases occurring in developing countries. Existing medications help alleviate epileptic symptoms yet there is no known cure for this debilitating disorder. In addition, 30% of epilepsy patients exhibit refractory (drug resistant) seizures to conventional epilepsy medication. Invertebrate flatworms (planaria) possess a primitive brain (considered as an ancestor to the human brain) and a bilaterally symmetric nervous system, which is analogous to the vertebrate nervous system. These planaria (Dugesia tigrina) also possess components of GABAergic neurotransmission, the fundamental molecular machinery targeted by the majority of anti‐epileptic drug development research. The planarian flatworms have shown significant seizure‐like movements upon exposure to convulsive drugs such as picrotoxin (selective GABAA receptor inhibitor) and others; however, the structure and function of the GABAergic protein targets of these drugs have not yet been studied in the flatworms.The polymeric nanoparticles were fabricated from alanine‐based bio‐compatible polymers (p(H2N‐Ala‐EMA)‐b‐PMMA) through the use of dialysis and size and charge characterization was carried out using Scanning Electron Microscopy (SEM) and dynamic light scattering. Biological toxicity tests were also carried out for varying concentrations of polymeric nanoparticle solutions in D. tigrina worms. In order to determine the functionality of the expression and localization of the GABA protein targets, a proof of concept experiment has been conducted. Small interfering ribonucleic acid (siRNA) which targets green fluorescent protein (GFP) is introduced into Human Embryonic Kidney 293 (HEK 293) cells using polymeric nanoparticles (p(H2N‐Ala‐EMA)‐b‐PMMA). This will allow investigation of whether polymeric nanoparticles can be viable modes of transport of siRNA, across the lipid bilayer which surrounds cells. Examining the silencing of GFP mRNA by siRNA delivery will allow analysis of whether GABA protein targets in planaria can also be inhibited by the use of the polymer nanoparticle complexed siRNA. Preliminary results from this proof concept study will be discussed at the meeting. The success of this proof of concept study will allow investigation of how GABAergic proteins are expressed and localized in the flatworms' simple nervous system. Studies elucidating the functional properties of GABA in the planarian flatworm will provide clues in identifying the evolutionary mechanisms of GABAergic neurotransmission from flatworms to higher vertebrates. Further, these studies will help us to understand the molecular basis of seizures‐like movements exhibited by the worms upon exposure to convulsive drugs.Support or Funding InformationThis work was made possible by funds provided by National Science Foundation's Catalyzing New International Collaboration grant to Dr. Ramakrishnan (1322626). The authors would like to acknowledge Dr. De and his research group at Indian Institute of Science Education and Research (IISER), at Kolkata, India for making the cationic polymers used in this study. The authors would also like to acknowledge the support and matching funds provided by the Office of Research and Sponsored Programs at St. Cloud State University. The Authors would also like to thank Dr. Brian Olson for his support, help and equipment along with advice from graduate students Stuart Fogarty and Brock Cash of the Biology Department.