Poster Y1 - Developing FAP 2286 Conjugated MTX Liposomes targeting Fibroblast like Synoviocytes in Rheumatoid Arthritis Model In vitro.

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Abishek Kamalakkannan

Graduate Student
University of Illinois Chicago College of Medicine Rockford
Rockford, Illinois, United States

Co-Author(s)

IA

Iqra Ahmed

Graduate Student
University Of Illinois College of Medicine Rockford, United States
Neeraja Revi

Postdoctoral research associate
University of Illinois at Chicago, United States

Primary Investigator(s)

Divya Bijukumar (she/her/hers)

Assistant Professor
UIC Rockford
Rockford, Illinois, United States

Introduction: Over the years of research, several therapeutic strategies have been developed to treat Rheumatoid Arthritis. Conventional strategies that include the usage of DMARDs, biological therapies, and macrophage depletion therapies have led to systemic immune suppression and prolonged inflamed conditions. Therefore, it is essential to develop a different targeted drug delivery approach. Fibroblast-like synoviocytes (FLS) are pro-inflammatory and anti-apoptotic, contributing to the pathophysiology of RA. They are also aggressive phenotypes in RA that are involved in pannus production.

Materials and

Methods: Liposomes containing both MTX loaded and empty liposomes were synthesized using the solvent evaporation method. FAP2286 conjugation was done via EDC-NHS click chemistry. Particles were characterized by DLS, SEM, UV-Vis Spectroscopy, and Release Kinetics. In this work, NIH3T3 cells and CAFs were used as RA models. Separately, TGF-β was used to stimulate FAP receptor expression in NIH3T3 fibroblast cells. Cytotoxicity of formulation at different concentrations was evaluated using MTT assay and apoptosis studies. Using confocal microscopy and flow cytometry, the cellular uptake of the conjugated liposomes was assessed in both FAP-activated NIH3T3 and CAF cells. Further, the fate of Nile-Red loaded FAP/liposomes was evaluated using Lysotracker dye to visualize the colocalization with lysosomes via confocal microscopy. One-way ANOVA was used to evaluate the statistical significance.

Results, Conclusions, and Discussions: Based on the structural characterization data, the conjugated liposomes exhibited a polydispersity index of 0.567 and an average size of 241 nm, compared to the unconjugated liposomes' average size of 88.3 nm and 0.263. The IC50 of MTX/liposomes was reached at 100 nm in 24 hours, whereas for free MTX, it took 48 hours to reach that point. Furthermore, at 48 h, compared to free MTX, FAP/MTX-Lipo induced significantly higher toxicity at 500 nM concentration. This suggests a slow-release kinetics of effective concentration of MTX from liposomes for an extended period. MTX and MTX/liposome damage the actin cytoskeleton inducing toxicity to cells. There is a significantly higher particle uptake of FAP/Lipo-MTX to FAP-activated NIH3T3 cells compared to unconjugated liposomes (p < 0.01). We further confirmed the targeting efficiency with FAP-expressing CAF cells. Compared to NIH3T3 cells, FAP upregulated CAF cells had significantly higher Liposomal uptake (p < 0.001). We observed a particle colocalization with lysosomes at different time points with clear lysosomal dynamics from perinuclear to the cell periphery. However, the colocalization was reduced at time increases from 2 h to 4h and 24 h with a significant amount of Nile red presence in the cytoplasm.

Acknowledgements (Optional): Blazer Foundation, MBT Program at UIC.