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Targeting Fibrillar Structures Associated with Ocular Diseases
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- Author / Creator
- Ghaffari Sharaf, Mehdi
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Impaired vision and blindness are serious global health problems that affect vast numbers of the population. The impact of ocular diseases is more severe in under-developed countries where, due to the lack of or limited access to vision care, many preventable or curable ocular diseases lead to vision loss and blindness. Exfoliation syndrome (XFS) and cataract are two ocular diseases that both strongly correlate with age and are associated with irreversible aggregation of proteins. These aggregates are mainly composed of fibrillar structures that cannot be removed through normal regulatory machinery that dictate ocular homeostasis. The exact pathomechanism of these diseases is unknown, and curative pharmacotherapy to prevent or eliminate protein fibrillation has not been achieved to date. The scope of this thesis rests on the thought that the effectiveness of future pharmacotherapy strategies for these diseases may be directly related to an ability to target these fibrillar aggregates. That said, the objectives of the study were as follow: to develop an in vitro model for cataract and to identify targeting peptides having specific binding ability to fibrillar aggregates of developed model, moreover to adopt an ex vivo strategy to identify targeting peptides having specific affinity to fibrillar aggregates associated with XFS and use identified peptides to investigate on development of a new therapeutic approach for XFS and exfoliation glaucoma (i.e. XFS induced glaucoma).
The in vitro model of cataract was developed using recombinantly expressed human βB2-crystallin protein. Fibrillation of βB2-crystallin was conducted under acidic denaturing conditions at different temperatures. It was demonstrated that formed fibrillar aggregates follow common structural pattern of amyloid fibrils. The phage display method was used to screen a random cyclic peptide library against model fibrils in vitro. The binding ability of selected phage-displayed peptides was analyzed using enzyme-linked immunosorbent assay (ELISA). A disulfide-constrained cyclic heptapeptide (CKQFKDTTC) was identified with the highest binding affinity. The specific binding ability of identified peptides was confirmed via competitive inhibition assay.
A phage-displayed random peptide library was used to develop an ex vivo panning method over XFS materials on lens capsules excised from the XFS eyes. Two 12-mer peptides (LPSYNLHPHVPP, and IPLLNPGSMQLS) were identified over three rounds of biopanning. Further investigations using phage-displayed peptides and free peptides showed their specific targeting ability towards XFS materials. Fluorescently labeled phages displaying identified peptides showed specific binding to XFS materials on the surface of human lens capsule. Peptides were conjugated to magnetic particles through copper-catalyzed azide-alkyne cycloaddition reaction and chemical bonding was analyzed by different techniques including surface zeta potential measurement, Fourier-transform infrared spectroscopy (FTIR), and competitive labeling of magnetic particles. Upon interacting with XFS lens capsules in a solution containing aqueous humor, magnetic particle-peptide conjugates showed specific binding to XFS materials. The behavior of the fibrillar materials upon binding to these magnetic particles was assessed using magnetic pins and rotating magnetic fields of various strengths. Ex vivo studies showed that the magnetic particle-peptide conjugates could generate enough mechanical force to remove large aggregates of exfoliation materials from the lens capsule when exposed to a low-frequency rotating magnetic field (5000 G, 20 Hz). Biocompatibility of magnetic particle-peptide conjugates was investigated using different methods including, MTT cell proliferation assay, live/dead cell viability assay, and DNA fragmentation analysis. Peptides in either free or conjugated form had no cytotoxicity effect. Electron microscopy was used to analyze possible internalization of magnetic particle-peptide conjugates into cultured trabecular meshwork cells, and it was shown that cellular uptake of aggregated particles could be limited. This is a novel, minimally invasive, therapeutic approach for the treatment of exfoliation glaucoma via the targeting and removal of exfoliation materials that could be applied to all tissues within the anterior segment of the eye.
Potential patient-to-patient structural polymorphism was investigated in fibrillar aggregates isolated from lens capsules of different XFS patients. Negative stain transmission electron microscopy was used to analyze the structural diversity of XFS fibrils. Fibrillar aggregates associated with XFS showed a wide range of fibril morphologies. It was speculated that this variation might arise from patient-specific fibril composition and/or formation mechanisms. These findings could open the door to further biophysical studies on XFS fibrils which might reveal more information about varying biological phenomena underlying that structural diversity.
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- Graduation date
- Spring 2021
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.