Evaluation of the biophysical characteristics of upconversion nanoparticle-based nano-bio hybrids for blood-brain barrier crossing
thesisposted on 29.03.2022, 02:27 by Libing Fu
Brain diseases including Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis (ALS) are fatal diseases without effective treatments. In past decades, some potentially effective therapeutics have been developed and demonstrated promising effects for brain diseases in pre-clinical cell culture and animal model evaluation. However, most of these developed drugs cannot reach the brain for therapy due to their inability to cross the blood-brain barrier (BBB). To overcome the BBB, nanotechnology is emerging as a promising approach to mediate and increase BBB penetration of drugs to the specific site of the brain. However, the efficiency of nanoparticle-based BBB penetration is still very low (<1%), with much to learn about how the biophysical properties of nanoparticles such as which size, surface, shape of nanoparticles facilitate BBB penetration. This project takes advantage of the specific properties of upconversion nanoparticles (UCNPs) including low auto-fluorescence, non-photobleaching, deep tissue penetration and adjustable size and shape, to develop a versatile platform to systematically evaluate the effects of nanoparticle surface, shape on BBB crossing in vitro and in vivo for future construction of multifunctional nano-carrier for theranostic applications in neurodegenerative diseases. In the first chapter, this thesis introduced the BBB and currently understood mechanisms of BBB penetration, and reviewed recent advances in nanoparticle-based BBB penetration strategies and approaches for brain disease therapy. In the second chapter, I described the key experimental methods that were developed and optimized for use in this project. Typically, the UCNPs employed in this project were fabricated via modified and optimized standard protocols for specific application as required. Importantly, the approaches of zebrafish imaging and microinjection were developed brand new in this project. In the third chapter, a UCNPs-based evaluation platform was synthesized for bio-nano surface selection in vitro and in vivo to systemically evaluate the suitability of various surface modifications for theranostic applications in neurodegenerative diseases. First, high lanthanide-doped UCNPs was designed, which provide strong tissue penetrable emission at 800nm. Then, these as-prepared UCNPs were further modified with four popular surfaces (OA-free, DNA-modified, silica coated and PEG-COOH capped) for comparison. The result showed that PEG-COOH performed superior cell internalization and excellent uptake capability into spinal motor neurons in zebrafish. Our work provides a versatile strategy via systemically surface evaluation for future construction of multifunctional nano-systems for therapeutic delivery to the central nervous system. In the fourth chapter, the effect of nanoparticle shape upon cell uptake and BBB penetration was further studied in vivo and in vitro. Firstly, a series of UCNPs with different shapes (including spherical, rod, disk and dumbbell) which retained similar size with each other were fabricated. Thereafter, these UCNPs were further modified with transferrin to make them specifically target the BBB. The results revealed that rod - shaped Tf - UCNPs displayed excellent brain endothelial cell uptake and brain accumulation in living zebrafish. Importantly, this study provides promising morphology information for design of efficient nano-carriers to cross the BBB for treatment of brain disease. In the fifth chapter, the optimal aspect ratio (AR, width/length) of upconversion nanorods (UCNRs) for BBB penetration were investigated. A series of high-lanthanide doped UCNRs with various aspect ratios (1, 2, 3 and 4) were developed. Then those UCNRs were further modified with PEG-COOH, since PEG-COOH modification was found to have superior cell internalization and excellent uptake capability for central nervous system from the results of Chapter 3. It was found that the aspect ratio of 2 PEGylated UCNRs provides the best cell uptake efficiency in neuron cells (NSC-34 neuron like cells, primary neuron cells and glial cells) compared to nanoparticles with other aspect ratios. This study demonstrates that aspect ratio has a significant influence upon cell uptake of neuron cells, which provides an alternative opportunity for further design of nanoparticle-based therapies for drug delivery of nanoparticles into the brain. In the sixth chapter, result of this thesis was summarized and perspective for the future applications for UCNPs-based drug delivery across BBB for CNS diseases was presented. To summarise, a transferrin-coated UCNRs with respect ratio of 2 was investigated as a potential drug carrier for treatment of neurodegenerative disease. These nanoparticles were capable of readily crossing the BBB and accumulating within the brains of living zebrafish, highlighting the therapeutic potential of this specific nanoparticle design. Overall, this study demonstrated a novel method to identify preferable biophysical characteristics of upconversion nanoparticle-based nano-bio hybrids to increase targeted nanoparticle accumulation in the brain.