Drug Delivery into CNS-Resident Cells using Cubosome Lipid Nanoparticles

Abstract

Nanoparticle-based therapies are recent developments in the field of medicine for their ability to deliver drugs to targets that otherwise would be inaccessible on their own and also to improve their performance. The focus of this study is to examine promising nanoparticle formulations that may assist in the delivery of therapeutics both into CNS-resident cell populations and across the blood-brain barrier. Specifically, this body of work examines the suitability of polysaccharide, polymer, and lipid composite nanoparticles for this purpose. Extensive characterization of the candidate particle solutions was carried out to determine which particle type may be more suited to achieve drug delivery. The particles chosen for this body of work are chitosan, O-carboxymethyl chitosan, PLGA, and Monoolein 9.9MAG based cubosomes. The size and dispersity of the particles within the formulations were measured using dynamic light scattering. Additionally, the effective surface charge of the particles in these solutions, known as the Zeta-potential, was also measured. Given that smaller nanoparticles tend to perform better at breaching tissues and tumour masses, attempts were made to adjust parameters so as reduce particle size before selection for biological testing. Stability of the candidate particles was also examined over time to ascertain whether the particle solutions as treatments could be stored. Focus was shifted to cubosomes for biological testing due to superior stability in serum conditions. Extensive toxicity and metabolic testing was carried out on in Vitro models of 3 major CNS resident cell populations to determine the effects of loaded and non-loaded cubosomes, and to determine their tolerance to these formulations. To this end, induced Brain Microvasculature Endothelial cells derived from human induced pluripotent stem cells, immortalized murine microglia and primary cortical neurons were examined for responses to these cubosome formulations. Toxicity thresholds and IC50 values for cubosome formulations were calculated for each of these CNS-resident cell types in this body of work. Additionally, the effect of the cubosome solutions on endothelial barrier integrity model was also measured by recording the Trans-Endothelial Electrical Resistance (TEER) across a cell monolayer transwell system as a model of the blood-brain barrier. We found that cubosomes disrupt barrier integrity temporarily at high concentrations, but this recovers 4h after treatment. Extensive metabolic testing in response to the cubosomes was also carried out, and we show that certain cubosome formulations can alter respiratory and glycolytic function in CNS-resident cells. Transport of cell-impermeable cargo in the form of fluorescein was then examined using the cubosome formulations to investigate the ability of these particles to deliver cell impermeable cargo to CNS-resident cell populations. We found that cubosomes can facilitate the entry of fluorescein into CNS-resident cell populations with repeated treatments without inducing cellular toxicity. To expand on this, in vivo testing of fluorescein uptake was also carried out after intraventricular injection of Fluorescein-loaded cubosomes, where we found that cubosomes appear to facilitate entry of fluorescein along the surface of the lateral ventricle surface. Following this, we examined the ability of the cubosomes when loaded with therapeutic cargo in the form of methotrexate, intravenous immunoglobulin, and human milk oligosaccharides to treat models of CNS based disease conditions. Specifically, we investigate the capacity of cubosomes to provide an improvement in efficacy for the killing of CTA2 glioblastomas using loaded methotrexate. We also investigate the capacity of the cubosomes to protect cortical neurons from rotenone-induced toxicity using human milk oligosaccharides, and for the regulation of microglial activation using intravenous immunoglobulin and human milk oligosaccharides. We found that through loading of methotrexate into cubosomes, anti-CTA2 glioblastoma activity was potentiated. We also found that loading of human milk oligosaccharides and intravenous immunoglobulin potentiate the anti-inflammatory effects of these cargo on immortalized murine microglia. These results collectively further research into cubosomes as a method for the delivery of therapeutics into CNS-resident cell populations and across the blood-brain barrier. We demonstrate that loading of cargo into cubosomes allows for entry into otherwise inaccessible regions and can also potentiate therapeutic performance against models of CNS-based disease.