Probing neuroactive intrinsically disordered protein and membrane interactions with NMR and nanobowls

Dementia affects 50 million people around the world, with 10 million new cases every year. The hunt for causes and therapies of dementia causing neurodegenerative diseases (NDDs) have gone hand in hand. An important direction for therapeutic development is to combat the aberrant amyloid aggregation of intrinsically disordered proteins (IDPs). The consensus in the field is that oligomeric intermediates formed during amyloid aggregation are the disease-causing species via their lipid membrane interactions. The heterogeneity and transient nature of intermediates have stymied efforts to study structures, membrane interactions and the mechanism by which therapies act. To this end, this dissertation has three objectives, (i)understand structural features and membrane interactions of intermediates; (ii)develop methods to isolate oligomers from cells in their native forms; (iii)develop efficient therapeutic delivery solutions. First, we employ NMR spectroscopy to resolve atomic scale structural features of a synthetic preparation of pre-fibrillar α-synuclein (αS)-intermediate, an IDP implicated in Parkinson’s disease. We find that the αS-intermediate displays a fold very similar to the fibril, although with distinct lipid interactions and side-chain arrangements. This observation is interesting in the context of the anti-parallel transition to parallel cross β-sheets which is perceived as an important step in initiating amyloid aggregation. Next, to establish accurate structure-function correlations, we propose the use of silica nanobowls to scavenge and purify membrane bound amyloid aggregates from neuronal cultures. We demonstrate with amyloid-β (Aβ) aggregates, an IDP implicated in Alzheimer’s disease, that atleast their aggregation driving domains are conserved by this method and the amount of non-amyloid contaminants is minimal. Lastly, to address the concern of minimizing side-effects and increasing efficiency of therapeutic delivery, we explore the use of magnetically modified nanobowls for targeted delivery to neurons. The three projects in this work advance the understanding of the pathological interaction of amyloid oligomers and membranes and aid in efforts to study and modulate oligomers in their native environments

Magnetic Drift Velocity Characterization of Iron Oxide- Silica Nanocarriers for Applications in Targeted Drug Delivery

Nanoparticles (NPs) are promising candidates to penetrate the blood brain barrier for delivering therapeutics to treat diseases affecting the central nervous system. However, obtaining effective doses of therapeutic NPs in diseased locations is challenging due to rapid sequestering by phagocytic organs. A potential solution is to use magnetic nanoparticles (MNPs) and guide them away from undesired organs through blood vessel networks. This can come to fruition if MNPs have large magnetic moments that enable high guiding efficiencies in technologically feasible magnetic field gradients (∇B ⃑). To this end, we designed nanobowls composed of a silica core embedded with magnetic iron oxide-NPs. These nanobowls are nanoparticles featuring a bowl-like pit for drug encapsulation. Nanobowls have a large magnetic moment of 2x〖10〗^(-17) Am^2. Guiding efficiency for nanobowls was determined in vitro using particle trajectories. The mathematical framework for particle trajectories involves the force balance between magnetic (F_M) and Stokes drag force. Magnetic drift velocity was measured as concentration flux toward a magnet to quantify F_M. This framework can be used to predict particle trajectories. Their validity was confirmed by imaging nanobowl cluster trajectories in different convective fluid flow and magnetic conditions. ∇B ⃑ used was larger than the average in commercial MRI machines. As expected, in 15 μm/s fluid velocity, clusters of nanobowls deviate 15° due to magnetic force. In case of physiological convection velocities often >1mm/s, framework calculations predict negligible deviation of nanobowls to the same ∇B ⃑ , insufficient for high guiding efficiency. Further work is thus required to develop larger magnetic moment nanocarriers

Magnetic Drift Velocity Characterization of Iron Oxide- Silica Nanocarriers for Applications in Targeted Drug Delivery

Nanoparticles (NPs) are promising candidates to penetrate the blood brain barrier for delivering therapeutics to treat diseases affecting the central nervous system. However, obtaining effective doses of therapeutic NPs in diseased locations is challenging due to rapid sequestering by phagocytic organs. A potential solution is to use magnetic nanoparticles (MNPs) and guide them away from undesired organs through blood vessel networks. This can come to fruition if MNPs have large magnetic moments that enable high guiding efficiencies in technologically feasible magnetic field gradients (∇B ⃑). To this end, we designed nanobowls composed of a silica core embedded with magnetic iron oxide-NPs. These nanobowls are nanoparticles featuring a bowl-like pit for drug encapsulation. Nanobowls have a large magnetic moment of 2x〖10〗^(-17) Am^2. Guiding efficiency for nanobowls was determined in vitro using particle trajectories. The mathematical framework for particle trajectories involves the force balance between magnetic (F_M) and Stokes drag force. Magnetic drift velocity was measured as concentration flux toward a magnet to quantify F_M. This framework can be used to predict particle trajectories. Their validity was confirmed by imaging nanobowl cluster trajectories in different convective fluid flow and magnetic conditions. ∇B ⃑ used was larger than the average in commercial MRI machines. As expected, in 15 μm/s fluid velocity, clusters of nanobowls deviate 15° due to magnetic force. In case of physiological convection velocities often >1mm/s, framework calculations predict negligible deviation of nanobowls to the same ∇B ⃑ , insufficient for high guiding efficiency. Further work is thus required to develop larger magnetic moment nanocarriers

Scavenging amyloid oligomers from neurons with silica nanobowls: Implications for amyloid diseases.

Amyloid-β (Aβ) oligomers are toxic species implicated in Alzheimer's disease (AD). The prevailing hypothesis implicates a major role of membrane-associated amyloid oligomers in AD pathology. Our silica nanobowls (NB) coated with lipid-polymer have submicromolar affinity for Aβ binding. We demonstrate that NB scavenges distinct fractions of Aβs in a time-resolved manner from amyloid precursor protein-null neuronal cells after incubation with Aβ. At short incubation times in cell culture, NB-Aβ seeds have aggregation kinetics resembling that of extracellular fraction of Aβ, whereas at longer incubation times, NB-Aβ seeds scavenge membrane-associated Aβ. Aβ aggregates can be eluted from NB surfaces by mechanical agitation and appear to retain their aggregation driving domains as seen in seeding aggregation experiments. These results demonstrate that the NB system can be used for time-resolved separation of toxic Aβ species from biological samples for characterization and in diagnostics. Scavenging membrane-associated amyloids using lipid-functionalized NB without chemical manipulation has wide applications in the diagnosis and therapy of AD and other neurodegenerative diseases, cancer, and cardiovascular conditions

Multifunctional stimuli responsive polymer-gated iron and gold-embedded silica nano golf balls: Nanoshuttles for targeted on-demand theranostics.

Multi-functional nanoshuttles for remotely targeted and on-demand delivery of therapeutic molecules and imaging to defined tissues and organs hold great potentials in personalized medicine, including precise early diagnosis, efficient prevention and therapy without toxicity. Yet, in spite of 25 years of research, there are still no such shuttles available. To this end, we have designed magnetic and gold nanoparticles (NP)-embedded silica nanoshuttles (MGNSs) with nanopores on their surface. Fluorescently labeled Doxorubicin (DOX), a cancer drug, was loaded in the MGNSs as a payload. DOX loaded MGNSs were encapsulated in heat and pH sensitive polymer P(NIPAM-co-MAA) to enable controlled release of the payload. Magnetically-guided transport of MGNSs was examined in: (a) a glass capillary tube to simulate their delivery via blood vessels; and (b) porous hydrogels to simulate their transport in composite human tissues, including bone, cartilage, tendon, muscles and blood-brain barrier (BBB). The viscoelastic properties of hydrogels were examined by atomic force microscopy (AFM). Cellular uptake of DOX-loaded MGNSs and the subsequent pH and temperature-mediated release were demonstrated in differentiated human neurons derived from induced pluripotent stem cells (iPSCs) as well as epithelial HeLa cells. The presence of embedded iron and gold NPs in silica shells and polymer-coating are supported by SEM and TEM. Fluorescence spectroscopy and microscopy documented DOX loading in the MGNSs. Time-dependent transport of MGNSs guided by an external magnetic field was observed in both glass capillary tubes and in the porous hydrogel. AFM results affirmed that the stiffness of the hydrogels model the rigidity range from soft tissues to bone. pH and temperature-dependent drug release analysis showed stimuli responsive and gradual drug release. Cells' viability MTT assays showed that MGNSs are non-toxic. The cell death from on-demand DOX release was observed in both neurons and epithelial cells even though the drug release efficiency was higher in neurons. Therefore, development of smart nanoshuttles have significant translational potential for controlled delivery of theranostics' payloads and precisely guided transport in specified tissues and organs (for example, bone, cartilage, tendon, bone marrow, heart, lung, liver, kidney, and brain) for highly efficient personalized medicine applications

Magnetically-responsive silica-gold nanobowls for targeted delivery and SERS-based sensing.

Composite colloidal structures with multi-functional properties have wide applications in targeted delivery of therapeutics and imaging contrast molecules and high-throughput molecular bio-sensing. We have constructed a multifunctional composite magnetic nanobowl using the bottom-up approach on an asymmetric silica/polystyrene Janus template consisting of a silica shell around a partially exposed polystyrene core. The nanobowl consists of a silica bowl and a gold exterior shell with iron oxide magnetic nanoparticles sandwiched between the silica and gold shells. The nanobowls were characterized by electron microscopy, atomic force microscopy, magnetometry, vis-NIR and FTIR spectroscopy. Magnetically vectored transport of these nanobowls was ascertained by time-lapsed imaging of their flow in fluid through a porous hydrogel under a defined magnetic field. These magnetically-responsive nanobowls show distinct surface enhanced Raman spectroscopy (SERS) imaging capability. The PEGylated magnetically-responsive nanobowls show size-dependent cellular uptake in vitro

Dual-Functionalized Theranostic Nanocarriers

Nanocarriers with the ability to spatially organize chemically distinct multiple bioactive moieties will have wide combinatory therapeutic and diagnostic (theranostic) applications. We have designed dual-functionalized, 100 nm to 1 μm sized scalable nanocarriers comprising a silica golf ball with amine or quaternary ammonium functional groups located in its pits and hydroxyl groups located on its nonpit surface. These functionalized golf balls selectively captured 10–40 nm charged gold nanoparticles (GNPs) into their pits. The selective capture of GNPs in the golf ball pits is visualized by scanning electron microscopy. ζ potential measurements and analytical modeling indicate that the GNP capture involves its proximity to and the electric charge on the surface of the golf balls. Potential applications of these dual-functionalized carriers include distinct attachment of multiple agents for multifunctional theranostic applications, selective scavenging, and clearance of harmful substances