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Formulation and Delivery - Biomolecular

Category: Poster Abstract

(M1230-02-10) Effect of Homotypic vs Heterotypic Interactions on the Cellular Uptake of Extracellular Vesicles

Monday, October 23, 2023
12:30 PM – 1:30 PM ET


Purpose: Extracellular vesicles (EVs) are cell derived nano-sized particles that play a vital role in intercellular communication. EVs transfer cellular cargo such as proteins, nucleic acids and lipids between donor and recipient cells (1-4). In our studies, we focus on two EV subtypes that differ in size: the 100-1000 nm medium-to-large EVs (m/lEVs) that are also referred to as microvesicles and the 50-200 nm small EVs (sEVs) that are commonly referred to as exosomes (5). Their role in intercellular communication propelled the development of EVs as carriers for drug delivery. Owing to their biological origin and potentially low immunogenicity, EVs may function better as drug delivery carriers (6). Our laboratory has previously demonstrated that m/lEVs, but not sEVs, transfer their innate mitochondrial content to recipient brain endothelial cells (BECs). This mitochondrial transfer resulted in increased mitochondrial function in the recipient BECs. We are currently developing m/lEVs as mitochondria delivery vehicles to BECs lining the BBB as a potential therapy for stroke (7-10). While endocytosis is reported to be a key mechanism via which cells internalize EVs (11), the entry pathway of EVs into BECs is currently not known. In this work, we seek to understand the effect of homotypic (EV donor cells and recipient cells of the same type) vs. heterotypic (EV donor cells and recipient cells are of different types) interactions on EV uptake into the recipient BECs (Figure 1). Specifically, we used BEC-derived and RAW macrophage-derived EVs and determined their uptake into recipient BEC and RAW macrophages, respectively, or vice versa. While BECs are our focus, we used RAW macrophages as an additional cell type. To study the cellular uptake mechanism, we individually blocked different routes of endocytosis using respective pharmacological inhibitors.

Methods: m/lEVs and sEVs were isolated from EV conditioned medium of hCMEC/D3 BECs and RAW 264.7 macrophages using a differential ultracentrifugation method (6, 7, 10). We measured total EV protein content using a MicroBCA assay. We characterized EVs using dynamic light scattering to measure EV particle size, dispersity index and zeta potential, nanoparticle tracking analysis to measure its particle concentration. We used ImageStream imaging flow cytometry to determine the presence of characteristic EV protein markers. EVs were labelled with calcein AM dye. Intracellular uptake of calcein-AM-EVs into recipient BECs and RAW macrophages were measured using flow cytometry to quantify the %EV uptake and the mean fluorescence intensity (MFI). Sucrose, genistein and 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) were used as the endocytosis inhibitors for clathrin-mediated endocytosis, clathrin-independent endocytosis and macropinocytosis pathways, respectively.

Results: BEC-derived sEVs and m/lEVs showed particle diameters of 142.6 ± 2.9 nm and 178.7 ± 3.2 nm with dispersity indices of 0.38 and 0.27, respectively. RAW macrophage-derived sEVs and m/lEVs showed particle diameters of 113 ± 0.8 nm and 144.5 ± 7.6 nm, respectively with dispersity index of 0.25 (Figure 2). We measured the cellular uptake of calcein-labelled EVs using flow cytometry wherein we analysed the following two aspects of uptake: (1) % EV uptake (%cells internalizing EVs, i.e., “frequency of uptake”) and (2) MFI was used as a measure of “intensity of cellular uptake” (Figure 3).
Homotypic pair 1: Recipient BECs treated with BEC EVs
sEVs showed a significant reduction in %uptake and MFI values when different endocytosis pathways were blocked, whereas m/lEVs showed reduction in %uptake and MFI values only when macropinocytosis pathway was blocked.
Homotypic pair 2: Recipient RAW macrophages treated with RAW macrophage EVs
Both sEVs and m/lEVs showed a significant reduction in %uptake and MFI values when clathrin-mediated endocytosis was blocked using sucrose. sEVs and m/lEVs also showed a significant reduction in the MFI values but no changes in uptake was noted when macropinocytosis was inhibited using EIPA.
Heterotypic pair 1: Recipient RAW macrophages treated with BEC EVs
Both sEVs and m/lEVs showed a significant reduction in % uptake and MFI values when clathrin-mediated endocytosis was blocked using sucrose. Both sEVs and m/lEVs also showed a significant reduction in MFI values when macropinocytosis was blocked using EIPA.
Heterotypic pair 2: Recipient BECs treated with RAW macrophage EVs
Both sEVs and m/lEVs showed a significant reduction in %uptake when clathrin-mediated endocytosis was blocked using sucrose. However, we did not note a reduction in MFI values.

Conclusion: Regardless of homotypic vs. heterotypic interactions, blocking the endocytosis pathways did not alter the frequency of cellular uptake (% EV uptake) but rather, we noticed a significant decrease in the intensity of uptake as noted by reduced MFI values. Our results suggest that both BEC- and RAW macrophage-derived EVs utilize multiple endocytosis pathway to enter the recipient BECs and macrophage cells. We do not see complete reduction in uptake (100% reduction) when blocking different endocytosis pathways likely suggesting that EVs may also undergo membrane fusion to enter the recipient cells.

References: 1. Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3.
2. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. Journal of Cell Biology. 2013;200(4):373-83.
3. Hong C-S, Funk S, Muller L, Boyiadzis M, Whiteside TL. Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. Journal of extracellular vesicles. 2016;5(1):29289.
4. Bonsergent E, Grisard E, Buchrieser J, Schwartz O, Thery C, Lavieu G. Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells. Nat Commun. 2021;12(1):1864.
5. Vader P, Mol EA, Pasterkamp G, Schiffelers RM. Extracellular vesicles for drug delivery. Advanced drug delivery reviews. 2016;106:148-56.
6. Dave KM, Zhao W, Hoover C, D'Souza A, D SM. Extracellular Vesicles Derived from a Human Brain Endothelial Cell Line Increase Cellular ATP Levels. AAPS PharmSciTech. 2021;22(1):18.
7. D'Souza A, Dave KM, Stetler RA, Manickam DS. Targeting the blood-brain barrier for the delivery of stroke therapies. Advanced drug delivery reviews. 2021;171:332-51.
8. Manickam DS. Delivery of mitochondria via extracellular vesicles–A new horizon in drug delivery. Journal of Controlled Release. 2022;343:400-7.
9. D'Souza A, Burch A, Dave KM, Sreeram A, Reynolds MJ, Dobbins DX, Kamte YS, Zhao W, Sabatelle C, Joy GM. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. Journal of Controlled Release. 2021;338:505-26.
10. Dave KM, Stolz DB, Venna VR, Quaicoe VA, Maniskas ME, Reynolds MJ, Babidhan R, Dobbins DX, Farinelli MN, Sullivan A. Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures. Journal of Controlled Release. 2023;354:368-93.
11. Costa Verdera H, Gitz-Francois JJ, Schiffelers RM, Vader P. Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis. J Control Release. 2017;266:100-8.

Figure 1: Scheme representing the homotypic vs. heterotypic experimental groups used in the cellular uptake studies. EVs refer to both sEVs and m/lEVs.

Figure 2: (a, b) Average particle diameters; dispersity indices; zeta potentials and EV particle concentration of BEC and RAW-derived EVs were determined using dynamic light scattering on a Malvern Zetasizer Pro-Red and nanoparticle tracking analysis on a multiple-laser ZetaView f-NTA Nanoparticle Tracking Analyzer (Particle Metrix Inc., Mebane, NC). m/lEVs and sEVs were prepared using 1X PBS at pH 7.4 at a final concentration of 0.1 mg/mL for particle size measurement and DI water at pH 7.4 for zeta potential measurement. Data are presented as mean ± SD of n = 3 measurements. For NTA, stock samples of sEV and m/lEV were diluted in 1X PBS. Three 60 s videos were acquired at 520 nm laser wavelengths for particle concentration measurements. Average particle concentrations were reported as mean ± SD of n = 5 measurements. (c) Representative images of CD9 and CD63-positive BEC-derived EVs detected on a Amnis ImageStreamx MkII equipped with IDEAS software.

Figure 3: Effect of homotypic vs. heterotypic interactions on EV cellular uptake.
Recipient macrophage/BECs were pre-incubated for 0.5 h with endocytosis inhibitors, removed and were then treated with macrophage/BEC-derived m/lEVs and sEVs for 4 h in the presence of fresh endocytosis inhibitors. The % EV uptake and MFI values are determined using flow cytometry and was normalized to the untreated cells. m/lEV- and sEV-treated cells (no inhibitors) were used as controls. Unpaired t test was used for statistical comparisons. Data are presented as mean±SD (n=3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.