As the world population is aging, the cases of Alzheimer’s Disease (AD) are increasing. AD is a disorder of the brain which is characterized by the aggregation of amyloid beta (Aβ) plaques. This leads to the death of numerous brain cells thus affecting the cognitive and motor functions of the individual. Till date, no cure for the disease is available. Aβ are peptides with 40/42 amino acid residues but, their exact mechanism(s) of action in AD is under debate. Having different amino acid residues makes them susceptible to form hydrogen bonds. Dendrimers with sugar units are often referred to as glycopolymers and have been shown to have potential anti-amyloidogenic activity. However, they also have drawbacks, the synthesis involves multiple tedious steps, and dendrimers themselves offer only a limited number of functional units. Pseudodendrimers are another class of branched polymers based on hyperbranched polymers. Unlike the dendrimers, they are easy to synthesize with a dense shell of functional units on the surface. One of the main goals in this dissertation is the synthesis and characterization of pseudodendrimers and dendrimers based on 2,2-bis(hydroxymethyl)-propionic acid (bis-MPA), an aliphatic polyester scaffold, as it offers biocompatibility and easy degradability. Furthermore, they are decorated with mannose units on the surface using a ‘click’ reaction forming glyco-pseudodendrimers and glyco-dendrimers. A detailed characterization of their structures and physical properties was undertaken using techniques such as size exclusion chromatography, asymmetric flow field flow fractionation (AF4), and dynamic light scattering.
The second main focus of this work has been to investigate the interaction of synthesized glyco-pseudodendrimers and glyco-dendrimers with Aβ 40 peptides. For this task, five different concentrations of the synthesized glycopolymers were tested with Aβ 40 using the Thioflavin T assay. The results of the synthesized polymers which produced the best results of showing maximum anti-aggregation behavior against Aβ 40 were confirmed with circular dichroism spectroscopy. AF4 was also used to investigate Aβ 40-glycopolymer aggregates, which has never been done before and constitutes the highlight of this dissertation. Atomic force microscopy was used to image Aβ 40-glycopseudodenrimer aggregates.
A basic but important step in the development of drug delivery platforms is to evaluate the toxicity of the drugs synthesized. In this work, preliminary studies of the cytotoxicity of glyco-pseudodendrimers were performed in two different cell lines. Thus, this study comprises a preliminary investigation of the anti-amyloidogenic activity of glyco-pseudodendrimers synthesized on an aliphatic polyester backbone.:Abstract
List of Tables
List of Figures
Abbreviations
1 Introduction
1.1 Objectives of the work
1.2 Thesis overview
2 Fundamentals and Literature
2.1 Alzheimer’s Disease and its impact
2.1.1 Neurological diagnosis of AD
2.1.2 Histopathology of AD
2.1.3 Amyloid precursor protein (APP) and its role in AD
2.2. Amyloid Beta (Aβ) peptide
2.2.1 Aβ peptide
2.2.2. Location and function
2.2.3 Amyloid hypothesis
2.2.4 The mechanism of Aβ aggregation
2.2.5 Amyloid fibrils
2.2.6 Toxicity of Aβ
2.3 Research methods to study Aβ aggregates
2.3.1 Models to study the mode of action of aggregates
2.3.2 Endogenous Aβ aggregates and synthetic aggregates
2.3.3 Strategies to alter aggregation of amyloids
2.4 Treatment and therapeutics
2.4.1 Current therapeutics
2.4.2 Current therapeutic research
2.4.2.1 Reduction of Aβ production
2.4.2.2 Reduction of Aβ plaque accumulation
2.4.2.2.1 Anti-amyloid aggregation agents
2.4.2.2.2 Metals
2.4.2.2.3 Immunotherapy
2.4.2.2.4 Dendrimers as potential anti-amyloidogenic agent
2.6 Dendrimers
2.6.1 Definition
2.6.2 Structure
Table of Contents
2.6.3 Synthesis
2.6.4 Properties
2.7 Pseudodendrimers - a sub-class of hyperbranched polymer
2.7.1 Definition
2.7.2 Structure
2.7.3 Synthesis
3 Analytical Techniques
3.1 Size Exclusion Chromatography Coupled to Light Scattering (SEC-MALS)
3.2 Asymmetric Flow Field Flow Fractionation (AF4)
3.3 Dynamic Light Scattering
3.4 Molecular Dynamics Simulation
3.5 Nuclear Magnetic Resonance Spectroscopy
3.6 Thioflavin T fluorescence
3.6.1 Kinetic analysis
3.7 Circular Dichroism Spectroscopy
3.8 Atomic Force Microscopy
3.9 Cytotoxic assay
3.9.1 MTT assay
3.9.2 Determining the level of reactive oxygen species
3.9.3 Changes in mitochondrial transmembrane potential
3.9.4 Flow cytometric detection of phosphatidyl serine exposure
4 Experimental Details and Methodology
4.1 Details of chemicals/components used
4.1.1 Other materials
4.1.2 Peptide preparation
4.1.3 Buffer preparation
4.1.4 Fibril growth conditions
4.2 Synthesis and characterization of polymers
4.2.1 Synthesis and characterization of pseudodendrimers and dendrimers
4.2.1.1 Synthesis of hyperbranched polymer (1)
4.2.1.2 Synthesis of protected monomer
4.2.1.2.1 bis-MPA acetonide (2)
4.2.1.2.2 bis-MPA-acetonide anhydride (3)
4.2.1.3 Synthesis of protected pseudodendrimers (4, 6 and 8) and
protected dendrimers (10, 12, and 14)
4.2.1.4 Deprotection of pseudodendrimers (5,7, and 9) and dendrimers
(11,13 and 15)
4.2.2 Synthesis of glyco-pseudodendrimers and glyco-dendrimers
4.2.2.1 Pentynoic anhydride (16)
4.2.2.2 Synthesis of pentinate modified pseudodendrimers (17, 18
and 19) and dendrimers (20, 21 and 22)
4.2.2.3 3-Azido-1-propanol (23)
4.2.2.4 Mannose propyl azide tetraacetate (24)
Table of Contents
4.2.2.5 Mannosepropylazide (25)
4.2.2.6 Glyco-pseudodendrimers (Gl-P) (26, 27 and 28) and glyco-
dendrimers (Gl-D) (29, 30 and 31)
4.3 Analytical techniques and their general details
4.3.1 SEC-MALS - Instrumentation, software and analysis
4.3.2 AF4 - Instrumentation, software and analysis
4.3.2.1 Sample preparation
4.3.2.2 Method development for analysis of Gl-P and Gl-D
4.3.2.3 Method development for analysis of Aβ 40 and its interaction
with Gl-P and Gl-D
4.3.3 Batch DLS - Instrumentation, software and analysis
4.3.3.1 Sample preparation
4.3.4 Theoretical calculations and molecular dynamics simulations
4.3.4.1 Ab-initio calculations
4.3.4.2 Modelling of the polymer structures
4.3.4.2.1 Pseudodendrimers
4.3.4.2.2 Dendrimers
4.3.4.2.3 Modification of the polymers with special end groups
4.3.4.2.4 Preparing of the THF solvent box
4.3.4.2.5 Solvation of the polymer structures
4.3.4.3 Molecular dynamics simulations
4.3.4.3.1 Evaluation of the simulation trajectories
4.4 Investigation of interaction of Gl-P and Gl-D with amyloid beta (Aβ 40)
4.4.1 ThT Assay - Instrumentation and software
4.4.1.1 Sample preparation
4.4.1.2 Kinetics based on ThT assay- software and data analysis
4.4.2 CD spectroscopy - Instrumentation and software
4.4.2.1 Sample preparation
4.4.3 AFM - Instrumentation and software
4.4.3.1 Substrate and sample preparation
4.4.3.2 Height determination and counting procedures
4.4.3.3 Topography and diameter
4.5 Cytotoxicity
4.5.1 Zeta potential
4.5.2 Cell culturing
4.5.3 Sample preparation
4.5.4 MTT assay
4.5.5 Changes in mitochondrial transmembrane potential (JC-1 method)
4.5.6 Flow cytometric detection of phosphatidyl serine exposure
(Annexin V and PI method)
5 Results and Discussion
5.1 Synthesis and characterization of glyco-pseudodendrimers and glyco-
dendrimers
5.1.1 Synthesis and characterization of hyperbranched polyester
Table of Contents
5.1.2 Synthesis and characterization of pseudodendrimers P-G1-OH,
P-G2-OH and P-G3-OH
5.1.3 Synthesis and characterization of dendrimers D-G4-OH, D-G5-OH
and D-G6-OH
5.1.4 Synthesis and characterization of Gl-P and Gl-D
5.1.4.1 Molecular size determination of Gl-P and Gl-D using SEC
5.1.4.2 Particle size determination using batch DLS
5.1.4.3 Apparent densities
5.1.4.4 Molecular size determination of Gl-P and Gl-D using AF4 .....
5.1.5 Molecular dynamics simulation
5.2 Investigation of interaction of Gl-P and Gl-D with amyloid beta (Aβ 40) ......
5.2.1 ThT Assay
5.2.1.1 Kinetics based on ThT assay
5.2.2 CD spectroscopy
5.2.3 Time dependent AF4
5.3.2.1 Separation of Aβ 40 by AF4
5.3.2.2 Aβ 40 amyloid aggregation in the presence of Gl-P and Gl-D
5.2.4 AFM
5.2.4.1 Height
5.2.4.2 Topography and diameter
5.2.4.3 Length
5.2.4.4 Morphology
5.2.5 Cytotoxicity
5.2.5.1 MTT assay
5.2.5.2 Changes in mitochondrial transmembrane potential
5.2.5.3 Flow cytometric detection of phosphatidyl serine exposure
6 Conclusions and Outlook
7 Bibliography
Appendix
Acknowledgements
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:82943 |
| Date | 16 January 2023 |
| Creators | Firdaus, Shamila |
| Contributors | Voit, Brigitte, Klajnert-Maculewicz, Barbara, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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