Intracellular nanoparticle diffusion analysis through quantum dot imaging and tracking

Arogundade, Opeyemi H.

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https://hdl.handle.net/2142/125756 Copy

Description

Title

Intracellular nanoparticle diffusion analysis through quantum dot imaging and tracking

Author(s)

Arogundade, Opeyemi H.

Issue Date

2024-06-24

Director of Research (if dissertation) or Advisor (if thesis)

Smith, Andrew M

Doctoral Committee Chair(s)

Smith, Andrew M

Committee Member(s)

• Nie, Shuming
• Selvin, Paul R
• Perez-Pinera, Pablo

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Quantum dots
• imaging
• single particle tracking

Abstract

Many pharmaceutical agents such as biomacromolecules and drug-loaded nanocarriers need to be delivered intracellularly for therapeutic action in specific organelles or in the cytoplasm. Success of these agents in the cell can be hindered by multiple factors including endolysosomal sequestration or low intracellular mobility which prevents access to specific targets of interest. Parameters of these macromolecular agents such as size and chemical functionalization play a major role in ultimate success. However, design rules for preparing these materials are not well established. Current approaches for monitoring transport in cells often produce ensemble average results that do not reflect heterogeneous processes underlying intracellular transport. Therefore, there is a need to develop quantitative single particle approaches for monitoring intracellular transport that can lead to rapid development, assessment, and improvement of macromolecular pharmaceuticals. Toward this goal, single particle tracking (SPT) has become a valuable method for directly observing macromolecular motion including diffusion and active transport in live cells. However, accurately inferring diffusion information from SPT studies remains challenging due to experimentally and analytically generated artifacts. Recent developments that improve the accuracy of diffusion measurement in SPT measurements require sparse labeling which might not be biologically relevant for applications like lipid nanoparticle mediated gene delivery for which a high density of materials may be delivered. My PhD thesis is focused on developing a new analytical process for accurately measuring diffusion of high density of cargo and applying this to study the impact of physiochemical properties on intracellular diffusion. I also develop an approach for measuring mixed diffusion in cells to serve as a tool for assessing endosomal disruption. As SPT requires probes with high signal intensity, I use quantum dots (QDs) which are fluorescent nanocrystals that are exceptional probes for single particle imaging and tracking. Highly homogeneous QDs are often prepared in organic solution and are transferred into aqueous dispersion through surface polymer coatings. Heterogeneity in QD dispersion is often observed in the resulting aqueous dispersions. Homogeneity is an important factor for accurate SPT measurements. In the first part of my thesis, I characterize aqueous dispersed QDs to identify sources of heterogeneity deriving from the transfer process to aqueous phase and establish approaches for improving homogeneity of QDs. In the second part of this thesis, I assess the limitations of commonly used single particle tracking algorithms particularly for accurately measuring diffusion parameters for fast-moving dense fields of particles and introduce a new analysis approach to improve accuracy by analyzing diffusion across varying maximal allowed displacement in connecting single particle trajectories. Using this analysis approach, I validate an existing intracellular diffusion model which points to a size-dependent sieving of particles in the cell cytoplasm. Finally, I use a variation of the new analysis approach to measure the fraction of mobile subpopulation in single particle videos of a cell as a metric for assessing endosomal rupture. This improved analysis approach allows me to compare endosomal disruption degrees across three types of lipid nanoparticle formulations. High rates of cell uptake and endosomal release are particularly observed for cationic lipid nanoparticles. Overall, this thesis describes a new method for accurately measuring intracellular diffusion of fast-moving densely packed particles in cells. I expect that this new approach can be applied as an analytical method for the evaluation nanoparticle formulations to establish generalizable design rules for intracellular drug delivery.

Graduation Semester

2024-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/125756

Copyright and License Information

Copyright 2024 Opeyemi Arogundade

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