Targeted nano-carriers to combat neurodegeneration

Neurodegenerative diseases are a large burden to the society. They are characterized by a loss of neuronal cells that affect the ability to perform daily activities, and are often caused by environmental or genetic factors. Therapeutics can treat clinical symptoms of chronic disease, but there is a need to additionally treat the underlying mechanisms leading to neuronal atrophy, such as mitochondrial dysfunction and inflammation. Efficacious treatment is very difficult due to the existence of several physiological hurdles, including the blood-brain barrier, diseased neuron, and intracellular organelle. Targeted nano-carriers can enhance local bioavailability by targeting each of these hurdles. Polyanhydride nanoparticles (NPs) in particular are attractive nano-carriers for central nervous system delivery of therapeutics, and can easily be functionalized with targeting ligands to further improve delivery. The goal of the project detailed herein is to rationally design a functionalized polyanhydride NP drug delivery platform addressing all physiological hurdles of the neurovascular unit to combat neurodegeneration. First, functionalized and non-functionalized 20:80 CPH:SA polyanhydride NPs were evaluated for the ability to cross the BBB in vitro. These NPs demonstrated promise in the ability to cross the BBB. Second, bulk-functionalized 20:80 CPH:SA NPs were evaluated for the ability to be internalized by neurons and enhance protective capability of antioxidants against oxidative stress in vitro. It was found that the functionalized NPs demonstrated superior internalization by N27 neurons compared to non-functionalized NPs, and antioxidant-loaded NPs protected against hydrogen peroxide – induced oxidative stress. Collectively, these studies lay the foundation for further investigation of the functionalized NP platform for central nervous system drug delivery

Transcriptomic Analysis of Glioma Cells

Advisors: David Odde (principal advisor), Benjamin Bangasser (graduate student advisor)This research was supported by the Undergraduate Research Opportunities Program (UROP)

Mito-Apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice

Aims: Parkinson\u27s disease (PD) is a neurodegenerative disorder characterized by progressive motor deficits and degeneration of dopaminergic neurons. Caused by a number of genetic and environmental factors, mitochondrial dysfunction and oxidative stress play a role in neurodegeneration in PD. By selectively knocking out mitochondrial transcription factor A (TFAM) in dopaminergic neurons, the transgenic MitoPark mice recapitulate many signature features of the disease, including progressive motor deficits, neuronal loss, and protein inclusions. In the present study, we evaluated the neuroprotective efficacy of a novel mitochondrially targeted antioxidant, Mito-apocynin, in MitoPark mice and cell culture models of neuroinflammation and mitochondrial dysfunction. Results: Oral administration of Mito-apocynin (10 mg/kg, thrice a week) showed excellent central nervous system bioavailability and significantly improved locomotor activity and coordination in MitoPark mice. Importantly, Mito-apocynin also partially attenuated severe nigrostriatal degeneration in MitoPark mice. Mechanistic studies revealed that Mito-apo improves mitochondrial function and inhibits NOX2 activation, oxidative damage, and neuroinflammation. Innovation: The properties of Mito-apocynin identified in the MitoPark transgenic mouse model strongly support potential clinical applications for Mito-apocynin as a viable neuroprotective and anti-neuroinflammatory drug for treating PD when compared to conventional therapeutic approaches. Conclusion: Collectively, our data demonstrate, for the first time, that a novel orally active apocynin derivative improves behavioral, inflammatory, and neurodegenerative processes in a severe progressive dopaminergic neurodegenerative model of PD. Antioxid. Redox Signal. 27, 1048–1066

Targeted nano-carriers to combat neurodegeneration

Neurodegenerative diseases are a large burden to the society. They are characterized by a loss of neuronal cells that affect the ability to perform daily activities, and are often caused by environmental or genetic factors. Therapeutics can treat clinical symptoms of chronic disease, but there is a need to additionally treat the underlying mechanisms leading to neuronal atrophy, such as mitochondrial dysfunction and inflammation. Efficacious treatment is very difficult due to the existence of several physiological hurdles, including the blood-brain barrier, diseased neuron, and intracellular organelle. Targeted nano-carriers can enhance local bioavailability by targeting each of these hurdles. Polyanhydride nanoparticles (NPs) in particular are attractive nano-carriers for central nervous system delivery of therapeutics, and can easily be functionalized with targeting ligands to further improve delivery. The goal of the project detailed herein is to rationally design a functionalized polyanhydride NP drug delivery platform addressing all physiological hurdles of the neurovascular unit to combat neurodegeneration. First, functionalized and non-functionalized 20:80 CPH:SA polyanhydride NPs were evaluated for the ability to cross the BBB in vitro. These NPs demonstrated promise in the ability to cross the BBB. Second, bulk-functionalized 20:80 CPH:SA NPs were evaluated for the ability to be internalized by neurons and enhance protective capability of antioxidants against oxidative stress in vitro. It was found that the functionalized NPs demonstrated superior internalization by N27 neurons compared to non-functionalized NPs, and antioxidant-loaded NPs protected against hydrogen peroxide – induced oxidative stress. Collectively, these studies lay the foundation for further investigation of the functionalized NP platform for central nervous system drug delivery.</p

Novel Theranostics for Parkinson's Disease

Parkinson’s Disease (PD) is a debilitating neurodegenerative disorder. It is characterized by a substantial and irreversible loss of neurons in the substantia nigra in the brain, which leads to the loss of motor function, and eventually, death. PD is the most common movement-based neurodegenerative disorder and the second-most common cause of dementia in individuals over the age of 65. The diagnosis of PD relies entirely on the observation of the associated motor symptoms, and as such makes a timely diagnosis exceptionally challenging. This leads to a lengthy diagnosis process with the average diagnosis occurring 10 years after the onset of symptoms. By this time, the patient’s quality of life is drastically reduced. There is a need to develop early-stage diagnostics to enable more rapid treatment to improve this quality of life, while also prolonging life expectancy after diagnosis. However, currently approved therapeutics only transiently alleviate motor symptoms. There is a need for developing therapeutic treatment modalities that effectively slow and/or halt disease progression. Among the underlying mechanisms that contribute to disease progression, the buildup of misfolded and aggregated protein in the form of Lewy Bodies (LB) is the leading contributor. As the largest component of LB, aggregated α-synuclein (αSynagg) represents a promising biomarker for both diagnostic and therapeutic thrusts to PD. Accordingly, a large sector of PD research is dedicated towards finding ways to detect αSynagg, and thereby breakdown the aggregates and halt disease progression. Most of these efforts focus on immunological approaches, utilizing antibodies that are specific to only αSynagg, and not the physiological form which is important for neuronal homeostasis. While great strides have been made with these approaches, there is yet to be a fully reliable diagnostic assay, or treatment for that matter, due to the complex nature of aggregates in vivo and the inability to achieve high levels of specificity for the pathogenic form of the protein. In addition to the need for better therapeutics, there is a need to minimize systemic toxicity associated with delivering such disease-slowing therapeutics, while also optimizing brain bioavailability. This can be accomplished by using targeted nano-carriers which encapsulate the therapeutic. Polyanhydride nanoparticles are an attractive option in this field, due to their biocompatibility, sustained release properties, high levels of cellular internalization and ability to easily conjugate targeting ligands in multiple different ways. In this work, a set of early-stage diagnostics is presented. The high levels of specificity for αSynagg using the singularly specific monoclonal antibodies (ssMAbs) in an enzyme-linked immunosorbent assay (ELISA) has led to high levels of discrimination between PD and age-matched control samples, including in an early-age group. Further ELISA optimization is underway to progress to clinical accuracy. Additionally, the ability of these ssMAbs to break down αSynagg in vitro suggests their use as a disease-slowing therapeutic, however there are several shortcomings to the use of full antibody therapeutics, including unfavorable immunogenicity and poor absorption properties. To overcome these issues, recombinant single chain fragment variable antibodies (scFv) based on the chemistry of ssMAb 3A8 (i.e. scFv 3A8) was proposed for therapeutic use to treat PD. The therapeutic potential for this scFv to breakdown αSynagg was observed, and to a greater degree than the ssMAbs themselves. To enhance brain delivery of therapeutics like this scFv, we proposed the use of a polyanhydride nanoparticle delivery platform. After testing select chemistries encapsulating a fluorescent dye and monitoring fluorescence over the course of seven days in biodistribution studies, it was found that the fluorescent dye encapsulated by these nanoparticles was detected in the brain after intravenous delivery. Notably, non-functionalized nanoparticles provided the most sustained release of dye over the course of the study, compared to both a soluble dose and a functionalized polyanhydride chemistry incorporating a peptide of the BBB-targeting transferrin protein. The enhanced brain bioavailability provided by the nanoparticles was also evidenced by more favorable pharmacokinetic parameters. Lastly, how brain bioavailability of this polyanhydride nanoparticle platform is correlated with the ability to enhance therapeutic efficacy was tested in an αSyn overexpression model. Soluble scFv and scFv-encapsulated nanoparticles were administered as treatments and it was found that this scFV-based treatment regimen recovered motor function, which was correlated with higher levels of dopaminergic activity by immunochemistry analysis. Collectively, these data suggest further optimization of this therapeutic platform so that it can be ultimately used as a disease-slowing therapeutic for PD patients

Novel Theranostics for Parkinson's Disease

Parkinson’s Disease (PD) is a debilitating neurodegenerative disorder. It is characterized by a substantial and irreversible loss of neurons in the substantia nigra in the brain, which leads to the loss of motor function, and eventually, death. PD is the most common movement-based neurodegenerative disorder and the second-most common cause of dementia in individuals over the age of 65. The diagnosis of PD relies entirely on the observation of the associated motor symptoms, and as such makes a timely diagnosis exceptionally challenging. This leads to a lengthy diagnosis process with the average diagnosis occurring 10 years after the onset of symptoms. By this time, the patient’s quality of life is drastically reduced. There is a need to develop early-stage diagnostics to enable more rapid treatment to improve this quality of life, while also prolonging life expectancy after diagnosis. However, currently approved therapeutics only transiently alleviate motor symptoms. There is a need for developing therapeutic treatment modalities that effectively slow and/or halt disease progression. Among the underlying mechanisms that contribute to disease progression, the buildup of misfolded and aggregated protein in the form of Lewy Bodies (LB) is the leading contributor. As the largest component of LB, aggregated α-synuclein (αSynagg) represents a promising biomarker for both diagnostic and therapeutic thrusts to PD. Accordingly, a large sector of PD research is dedicated towards finding ways to detect αSynagg, and thereby breakdown the aggregates and halt disease progression. Most of these efforts focus on immunological approaches, utilizing antibodies that are specific to only αSynagg, and not the physiological form which is important for neuronal homeostasis. While great strides have been made with these approaches, there is yet to be a fully reliable diagnostic assay, or treatment for that matter, due to the complex nature of aggregates in vivo and the inability to achieve high levels of specificity for the pathogenic form of the protein. In addition to the need for better therapeutics, there is a need to minimize systemic toxicity associated with delivering such disease-slowing therapeutics, while also optimizing brain bioavailability. This can be accomplished by using targeted nano-carriers which encapsulate the therapeutic. Polyanhydride nanoparticles are an attractive option in this field, due to their biocompatibility, sustained release properties, high levels of cellular internalization and ability to easily conjugate targeting ligands in multiple different ways. In this work, a set of early-stage diagnostics is presented. The high levels of specificity for αSynagg using the singularly specific monoclonal antibodies (ssMAbs) in an enzyme-linked immunosorbent assay (ELISA) has led to high levels of discrimination between PD and age-matched control samples, including in an early-age group. Further ELISA optimization is underway to progress to clinical accuracy. Additionally, the ability of these ssMAbs to break down αSynagg in vitro suggests their use as a disease-slowing therapeutic, however there are several shortcomings to the use of full antibody therapeutics, including unfavorable immunogenicity and poor absorption properties. To overcome these issues, recombinant single chain fragment variable antibodies (scFv) based on the chemistry of ssMAb 3A8 (i.e. scFv 3A8) was proposed for therapeutic use to treat PD. The therapeutic potential for this scFv to breakdown αSynagg was observed, and to a greater degree than the ssMAbs themselves. To enhance brain delivery of therapeutics like this scFv, we proposed the use of a polyanhydride nanoparticle delivery platform. After testing select chemistries encapsulating a fluorescent dye and monitoring fluorescence over the course of seven days in biodistribution studies, it was found that the fluorescent dye encapsulated by these nanoparticles was detected in the brain after intravenous delivery. Notably, non-functionalized nanoparticles provided the most sustained release of dye over the course of the study, compared to both a soluble dose and a functionalized polyanhydride chemistry incorporating a peptide of the BBB-targeting transferrin protein. The enhanced brain bioavailability provided by the nanoparticles was also evidenced by more favorable pharmacokinetic parameters. Lastly, how brain bioavailability of this polyanhydride nanoparticle platform is correlated with the ability to enhance therapeutic efficacy was tested in an αSyn overexpression model. Soluble scFv and scFv-encapsulated nanoparticles were administered as treatments and it was found that this scFV-based treatment regimen recovered motor function, which was correlated with higher levels of dopaminergic activity by immunochemistry analysis. Collectively, these data suggest further optimization of this therapeutic platform so that it can be ultimately used as a disease-slowing therapeutic for PD patients

Treatment of Neurodegenerative Disorders through the Blood-brain Barrier using Nanocarriers

Neurodegenerative diseases refer to disorders of the central nervous system (CNS) that are caused by neuronal degradations, dysfunctions, or death. Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (APHD) are regarded as the three major neurodegenerative diseases. There is a vast body of literature on the causes and treatments of these neurodegenerative diseases. However, the main obstacle in developing an effective treatment strategy is the permeability of the treatment components to the blood-brain barrier (BBB). Several strategies have been developed to improve this obstruction. For example, nanomaterials facilitate drug delivery to the BBB due to their size. They have been used widely in nanomedicine and as nanoprobes for diagnosis purposes among others in neuroscience. Nanomaterials in different forms, such as nanoparticles, nanoemulsions, solid lipid nanoparticles (SLN), and liposomes, have been used to treat the neurodegenerative diseases. This review will cover the basic concepts and applications of nanomaterials in the therapy of APHD.This is a manuscript of an article published as Poovaiah, Nitya, Zahra Davoudi, Haisheng Peng, Benjamin Schlichtmann, Surya K. Mallapragada, Balaji Narasimhan, and Qun Wang. "Treatment of Neurodegenerative Disorders through the Blood-brain Barrier using Nanocarriers." Nanoscale (2018). DOI: 10.1039/C8NR04073G. Posted with permission.</p

Aggregation-Inhibiting scFv-Based Therapies Protect Mice against AAV1/2-Induced A53T-α-Synuclein Overexpression

To date, there is no cure for Parkinson’s disease (PD). There is a pressing need for anti-neurodegenerative therapeutics that can slow or halt PD progression by targeting underlying disease mechanisms. Specifically, preventing the build-up of alpha-synuclein (αSyn) and its aggregated and mutated forms is a key therapeutic target. In this study, an adeno-associated viral vector loaded with the A53T gene mutation was used to induce rapid αSyn-associated PD pathogenesis in C57BL/6 mice. We tested the ability of a novel therapeutic, a single chain fragment variable (scFv) antibody with specificity only for pathologic forms of αSyn, to protect against αSyn-induced neurodegeneration, after unilateral viral vector injection in the substantia nigra. Additionally, polyanhydride nanoparticles, which provide sustained release of therapeutics with dose-sparing properties, were used as a delivery platform for the scFv. Through bi-weekly behavioral assessments and across multiple post-mortem immunochemical analyses, we found that the scFv-based therapies allowed the mice to recover motor activity and reduce overall αSyn expression in the substantia nigra. In summary, these novel scFv-based therapies, which are specific exclusively for pathological aggregates of αSyn, show early promise in blocking PD progression in a surrogate mouse PD model.This article is published as Schlichtmann, Benjamin W., Bharathi N. Palanisamy, Emir Malovic, Susheel K. Nethi, Piyush Padhi, Monica Hepker, Joseph Wurtz, Manohar John, Bhupal Ban, Vellareddy Anantharam, and et al. 2023. "Aggregation-Inhibiting scFv-Based Therapies Protect Mice against AAV1/2-Induced A53T-α-Synuclein Overexpression" Biomolecules 13, no. 8: 1203. https://doi.org/10.3390/biom13081203. Posted with permission. © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)