Advanced fluorescence methods for nanoscale systems: from HIV-1 dynamics to drug delivery nanoparticles

Gialdini, Irene (2026): Advanced fluorescence methods for nanoscale systems: from HIV-1 dynamics to drug delivery nanoparticles. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

Over the last few decades, fluorescence microscopy and spectroscopy have emerged as powerful techniques to investigate nanoscale systems. Thanks to their high sensitivity and molecular specificity coupled with high spatial and temporal resolution, fluorescence methods provide access to direct quantification of dynamic processes at the single-molecule level. Yet, their potential remains underused in the study of complex biological systems, where heterogeneity and environmental interactions play critical roles and need to be addressed at the single-cell and single-particle level. In this thesis, advanced fluorescence methods including fluorescence correlation spectroscopy, raster image correlation spectroscopy, fluorescence lifetime imaging and single particle tracking were applied to two main nanosystems: biological nanoparticles, i.e. HIV-1, and synthetic particles, i.e. drug delivery nanocarriers. First, the early cytosolic stages of HIV-1 assembly were investigated with focus on the role of GagPol, the HIV-1 polyprotein containing the viral enzymes. By generating a fluorescently labeled variant of GagPol in which both Gag and GagPol are simultaneously tagged, Gag and GagPol relative concentrations and intermolecular interactions were quantified at the single-cell level. The results revealed the formation of RNA-interacting Gag/GagPol cytosolic complexes that suggest GagPol’s participation in the early, cytoplasmatic steps of HIV-1 assembly driven by Gag multimerization, providing new insights into the regulation of this fundamental process. In a second research direction, the stability and mobility of fluorescently labeled drug delivery nanoparticles were examined under in-vivo mimicking conditions, such as high concentration serum and mucus. Multiple formulations and surface functionalization were evaluated, revealing that the nanoparticle behavior is highly affected by the local environment and that stability observed in simplified models may not be maintained under more realistic conditions. Thanks to their specificity and minimal sample requirements, advanced fluorescence methods have the potential to complement standard nanoparticle characterization with a systematic evaluation of particle behavior in-vitro in complex, biologically relevant environments, increasing the clinical success rate of newly designed drug delivery systems. Overall, fluorescence-based methods are powerful, versatile tools for the investigation of nanoscale systems in complex environments, ranging from the study of viral processes to the evaluation of new drug design and can therefore support both fundamental research and translational applications.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 3. July 2026
• 1. Referee: Lamb, Don C.
• Signature of the printed copy: 0001/UMC 32168