Authors:

Brown Researcher (D10)

Date Created:

Not specified

Course Title:

Professor:

Not specified

About Paper:

No description available.

Source:

Brown / Faculty Mentor: Anita Shukla, School of Engineering Evaluating the mechanism of metabolite-based lipid nanoparticle (LNP) internalization in macrophages Staphylococcus aureus is a leading cause of bacterial death globally. S. aureus can form persistent biofilms that are not susceptible to clearance using our traditional antibiotic approaches. The complex biofilm structure hijacks the immune system by impairing and altering innate immune cells such as macrophages, involved in pathogen clearance. Therefore, there is a crucial need for novel therapeutics that can target and manipulate host immune responses to effectively clear bacterial biofilm infections. We hypothesize that modulating macrophage polarization from an anti-inflammatory (M2) towards a pro-inflammatory (M1) phenotype can enhance bacterial clearance. Lipid Nanoparticles (LNPs) offer a versatile platform with tunable composition, efficient drug encapsulation, and low toxicity that is promising for this application. This project aimed to fabricate metabolite-based LNP formulations and investigate the mechanism of nanoparticle uptake by bone-marrow derived macrophages (BMDMs). LNP formulation and fabrication consisted of ionizable lipid ALC-0315, cholesterol, helper lipid DSPC, PEG-lipid DMG-PEG 2000, and specific metabolites (e.g., cholesteryl hemisuccinate, palmitic acid). Metabolite-based LNPs were characterized by dynamic light scattering (DLS) for hydrodynamic diameter, polydispersity index (PDI), and ζ-potential. The LNPs exhibited hydrodynamic diameters of ~90-100 nm, PDI ~0.1-0.19, and neutral surface charge. LNP uptake by BMDMs was evaluated using laser scanning confocal microscopy and flow cytometry. Specifically, two formulations: a control LNP (with no metabolite) and a cholesteryl hemisuccinate-loaded LNP, both containing DiD perchlorate were tested. BMDMs were labeled with Hoechst 33342 and Vybrant DiO to visualize the nuclei and cell membranes via confocal, respectively. Confocal microscopy showed successful uptake of both LNP formulations by BMDMs within one hour. Moreover, time-dependent flow cytometry uptake studies showed that within the first 10 minutes, the control LNP exhibited 92.1% uptake by BMDMs, while the cholesteryl hemisuccinate-loaded LNP showed 82.3% uptake. After 1 hour, greater than 99.8% of the LNPs were internalized for both formulations. Future work will explore the use of endocytic pathways inhibitors (e.g., chloroquine, amiloride, 7-ketocholesterol, and methyl-β-cyclodextrin) to determine intracellular uptake pathways of the metabolite-based LNPs in macrophages. 200 Rodrigo González Marcano: Poster #D11 Home Institution: University of Puerto Rico at Río Piedras Summer Research Program: Leadership Alliance-Summer Research Early Identification Program (SR-EIP)

Topics:

No topics listed

Co-authors:

Brown Researcher (D10)