Thesis
The development of a pipeline for the analysis of polymeric nanoparticle interactions with protein-containing media
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2024
- Thesis identifier
- T17100
- Person Identifier (Local)
- 202069218
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Nanoparticles are increasingly implemented in biomedical applications, including the diagnosis and treatment of disease. When exposed to complex biological media, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small and bio- macromolecules- termed the “corona”. Corona composition is governed by nanoparticle properties and incubation parameters. While the focus of most studies examining nanoparticle interactions with biological systems is on the protein signature of the nanoparticle corona, the impact of experimental protocols on nanoparticle size in the presence of complex biological media, and the impact of nanoparticle recovery from biological media remains under reported in the literature. Therefore, the principal hypothesis of this thesis is that the methods used to isolate nanoparticles during the screening of bio-nano interactions in biological media, can significantly alter the results obtained from these studies. To further probe this hypothesis, in this thesis polystyrene latex nanoparticles were used as a robust and non-biodegradable model to investigate the impact of different nanoparticle isolation pipelines on subsequent nanoparticle physical parameters. To validate this hypothesis, I examined the routinely implemented pipeline for nanoparticle isolation, which is the centrifugation-resuspension protocol for nanoparticle isolation from biological media. In Chapter 2 1 , I showed that the commonly used centrifugation-wash protocol leads to a significant increase in the mean particle size, of nanoparticle-protein samples when compared with in-situ samples analysed using Particle Tracking Analysis. This is likely due to protein aggregation, and particle agglomeration caused by high-speed centrifugation. The centrifugation-wash protocol was typically accompanied by a significant decrease in sample concentration. Furthermore, nanomedicines are typically intended for intravenous administration (IV). Therefore, it was crucial to understand the impact of physiologically-relevant shear flow conditions on protein corona formation. Results showed that there were significant differences in measured parameters following incubation at shear flow rates which mimicked the median cubital vein, and arteries compared to static incubation conditions with SDS-PAGE analysis further showing changes in protein corona composition. Subsequently, I explored the use of asymmetric flow field flow fractionation multiplexed with a range of multiple inline optical inline detectors (AF4-MD) to optimise the separation of model polystyrene latex nanoparticles from bulk incubation protein media, following exposure to physiologically-relevant temperature conditions and protein composition mimicking cell culture conditions. I studied the impact of AF4 flow parameters and decay profiles on the quality of fractogram obtained and applied these design principles to the analysis of polystyrene latex nanoparticles exposed to media containing 10% vol FBS for 2 and 24 hours (Chapter 3). Results from this chapter indicate that AF-MD offers a potential promising tool for both the separation of nanoparticles from the bulk protein content in the media, and inline analysis of physical changes occurring in nanoparticle systems following exposure to biological media. An added benefit of using AF4-MD as a technique for studying nano-bio interactions is that it is possible to simultaneously study the physical properties of different intermediates formed during the sample elution step. There is also the scope for downstream recovery of pooled fractions collected from different elution peaks for offline analysis using other techniques such as liquid chromatography-mass spectrometry-based proteomics analysis of the nanoparticle protein corona composition. In Chapter 3, I also found that through combining inline multiangle light scattering with dynamic light scattering measurements, the shape factor can be determined for particles- an indicator of nanoparticle morphology. Peaks eluting at later timepoints were found to have altered morphology from a spherical shape to more extended elongated shapes following incubation with protein-containing media, with the extent being more pronounced for samples incubated for up to 24 hours. Findings from chapter 3 overall showed that frit-inlet based AF4 was more appropriate for the resolution and analysis of nanoparticle-protein corona complexes and the signal quality was consistently low across all three polystyrene latex nanoparticle types studied. Therefore, the use of additional AF4 based methodologies based on charge separation was explored in later chapters to examine whether better efficiency of resolution and mass recovery could be achieved. In chapter 4, I further explored the utility of simultaneous diffusion and charge-based separation through the implementation of electric flow-field flow fractionation (EAF4) multiplexed with online light scattering, UV and fluorescence detectors. In addition to simultaneous separation and online analysis via multiple detectors, I demonstrate the resolution of different charged species in response to incubation with protein-containing media, where I also compared the different approaches used in this thesis in terms of their ability to resolve nanoparticle from complex biological media use to screen nano-bio interactions in drug discovery efforts. EAF4 demonstrated more significant promise for the resolution of nanoparticles from bulk protein content in comparison to conventional and frit-inlet AF4, and the simultaneous inline analysis of nanoparticle-protein complex surface electrostatic properties (zeta potential) and changes occurring in particle size and geometry (shape factor) in response to incubation with media containing protein. Overall, this thesis has examined the current state of the art in the separation and physical analysis of nanoparticles from biological media. For future efforts, I recommend that pipelines studying bio-nano interactions during early nanomedicine development consider more biologically relevant shear flow conditions and media composition that can significantly alter nanoparticle physical parameters and subsequent conclusions from these studies. Moreover, in this thesis I demonstrate the need for case-by-case optimisation of AF4 based protocols, an area which at the time of this thesis remains underreported in the literature.
- Advisor / supervisor
- Rattray, Zahra
- Perrie, Yvonne
- Resource Type
- DOI
Relations
Contenu
| La vignette | Titre | Date de téléchargement | Visibilité | actes |
|---|---|---|---|---|
|
|
PDF of Thesis T17100 | 2024-10-24 | Public | Télécharger |