Date of Award

2026-05-01

Degree Name

Master of Science

Department

Biomedical Engineering

Advisor(s)

Wilson Poon

Abstract

Lipid nanoparticles (LNPs) have become effective delivery vehicles in nanomedicine, particularly for nucleic acid-based treatments. However, a major limitation remains in the field regarding the simultaneous monitoring of the nanoparticle carrier, its encapsulated payload, and the resulting functional protein production in both in vitro and in vivo systems. Furthermore, traditional lipophilic dyes employed for labeling LNPs frequently exhibit incompatibility with tissue-clearing methods, such as CLARITY, due to signal loss following lipid removal. This research developed and evaluated a new fluorescent labeling method using CM-DiI, a dye that reacts with thiols, for multimodal tracking of LNPs. CM-DiI was included in the LNP formulations to create a stable labeling system for the carriers. To evaluate cargo delivery efficiency and functional protein synthesis, Cy5-labeled eGFP mRNA was utilized. Dynamic light scattering (DLS), the TNS assay, and XTT cell viability assays were employed to examine the physical and chemical characteristics, ionization behavior, and cellular responses to the CM-DiI LNPs respectively. The results indicated that the addition of CM-DiI did not substantially alter the nanoparticles' size, surface charge, apparent pKa, or cell viability. In vitro studies using A549 cells and cardiomyocytes derived from induced pluripotent stem cells showed that CM-DiI LNPs were effectively taken up and maintained strong fluorescence after SDS-mediated delipidation. This was different from what was seen with standard DiI-labeled nanoparticles. Confocal imaging demonstrated the simultaneous detection of the carrier (CM-DiI), cargo (Cy5-labeled mRNA), and functional expression (eGFP), while co-localization with cardiac troponin T (cTnT) immunostaining supported retention in differentiated cardiomyocytes. Flow cytometry analysis corroborated these findings, revealing markedly elevated fluorescence retention and positive cell populations for CM-DiI LNPs in comparison to controls following lipid removal. In vivo studies, combined with three-dimensional (3D) imaging of rendered transparent tissues, have consistently shown the presence of CM-DiI labeled LNPs following CLARITY processing. This method enables the assessment of LNP distribution within intact organs. Initial findings from mouse models that were fed a high-fat diet suggest notable differences in nanoparticle distribution and collagen deposition compared to control groups on a standard diet. This highlights how physiological conditions can influence the behavior of LNPs. Overall, this study demonstrates that CM-DiI is a powerful and flexible method for tracking LNP carriers, their encapsulated cargo, and their functional delivery, using different imaging techniques. This method avoids the limitations of traditional dyes, allowing for both detailed cellular imaging and broad organ-level visualization. As a result, it is a valuable tool for improving nanoparticle-based drug delivery and imaging in nanomedicine.

Language

en

Provenance

Received from ProQuest

File Size

66 p.

File Format

application/pdf

Rights Holder

Hannia Vanessa Balcorta Muñoz

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