(229b) Modulating Nonspecific Uptake of Engineered Extracellular Vesicles

Engineered extracellular vesicle (EV) therapies are emerging treatment modalities for many diseases. An open challenge for clinical use is the susceptibility of EVs to nonspecific uptake by the mononuclear phagocytic system (MPS), leading to fast clearance times¹. Extending circulation time and decreasing nonspecific uptake would provide therapeutics with more opportunities to reach their targets while maintaining the biological advantages of EVs as drug delivery vehicles. While several strategies for reducing nonspecific uptake of EVs exist, there has been minimal exploration of post-isolation modification of EV membrane lipid composition. Altering the composition and arrangement of membrane lipids can lead to changes in membrane biophysical properties like fluidity, rigidity, and elasticity². Because changes in these properties have been shown to alter the uptake of synthetic nanoparticles³, we hypothesized that treating EVs with lipid altering chemical compounds would reduce their nonspecific uptake and increase circulation time. To evaluate our hypothesis, we isolated EVs from dTomato fluorescent human embryonic kidney 293 FT (HEK293 FT) cells using differential ultracentrifugation and nanoluciferase (Nluc) luminescent FreeStyle 293-F cells using tangential flow filtration. While dTomato fluorescent EVs isolated from the workhorse adherent HEK293 FT cell line are suitable for in vitro experiments, Nluc luminescent EVs from the FreeStyle 293-F suspension cell line are more practical for scale up to in vivo circulation and biodistribution studies. EVs were treated with lipid altering compounds post-isolation and purified before evaluating changes in biophysical properties such as fluidity and rigidity via Laurdan generalized polarization and atomic force microscopy. Changes in nonspecific uptake of fluorescent EVs were measured by flow cytometry after a macrophage uptake assay. RAW 264.7 mouse macrophage-like cells were utilized in this assay, as they provide an indication of the potential effects on nonspecific uptake by the MPS in vivo. Circulation time and biodistribution are being measured in vivo, by evaluating Nluc luminescence in mouse organs and from blood draws at several time points. Preliminary uptake study results have indicated a reduction in macrophage uptake compared to control conditions when EVs are treated with the cholesterol-depleting compound methyl-β-cyclodextrin (MβCD) (Figure 1). We theorize that this decrease in cholesterol increases fluidity and decreases rigidity in EVs, making them more challenging for the MPS to take up. Further investigation into other membrane lipid altering compounds is ongoing. Studies investigating the changes in membrane properties which affect uptake will elucidate the underlying mechanisms, and preclinical studies investigating biodistribution and circulation time will advance therapeutic treatments. We anticipate that this study will expand the field of post-isolation modification of EVs therapeutics, accelerating their development to combat diseases.

References: ¹Belhadj, et al., J. Extracell. Vesicles. 2020. 9 (1), 1806444. ²Agarwala, et al, Medicinal Research Reviews. 2021. 42 (2), 1098-1128. ³Hui, Y., et al., Sci. Adv. 2020. 6 (16), eaaz4316.