Biodistribution of extracellular vesicles in physiological and pathological conditions

EVs are membrane-enclosed nanostructures released by various cell types and serve as critical mediators of intercellular communication. Their ability to transport proteins, nucleic acids, and lipids has positioned EVs as promising biomarkers and delivery vehicles in diagnostics and therapeutics. However, a comprehensive understanding of their in vivo biodistribution, clearance, and immunomodulatory potential remains limited, hindering translational progress. The work described in this thesis summarizes recent advances in EV engineering, labeling, biodistribution analysis, and their roles under physiological and pathological conditions.

To address limitations in EV analysis, luciferase-based endogenous labeling systems were optimized for sensitive and specific EV detection. Among several luciferases tested, NanoLuc and ThermoLuc demonstrated superior luminescence, pH stability, and suitability for in vivo applications. Fusion of these luciferases to tetraspanins enabled efficient EV incorporation without altering vesicle morphology, size, or producer cell viability. Importantly, CD63-NanoLuc provided high signal intensity and sensitivity. In contrast, CD63-ThermoLuc exhibited stronger EV specificity with minimal soluble background signal, thus offering advantages in unpurified systems. Luciferase-labelled EVs enabled quantification of vesicle secretion kinetics under different media conditions and facilitated assessment of cellular uptake in various recipient cell lines.

In vivo tracking of luciferase-labelled EVs revealed rapid EV clearance from circulation following intravenous administration, with a half-life of less than 2 minutes in plasma. The liver and spleen represented major uptake sites, with over 90% of EVs accumulating in these organs within minutes. Systemic biodistribution was route-dependent; intravenous and intraperitoneal administration effectively delivered EVs to peripheral organs, while subcutaneous and oral routes showed limited systemic dispersion.

To further investigate the fate of injected EVs, we employed high-resolution imaging flow cytometry methods and discovered that EV clearance is mediated, in part, by transient binding to platelets and red blood cells. These interactions peaked within 5 minutes post-injection and coincided with a drop in circulating blood cell counts, suggesting clearance via phagocytic uptake in the liver and spleen. Co-localization of EVs with platelets and red blood cells in macrophages supports a hitchhiking-based mechanism for EV sequestration. We hypothesized that by changing the affinity of EVs to different blood components, their clearance will be drastically altered. To test this, EVs were engineered to display albumin- binding domains. This modification significantly reduced their interaction with blood components, thereby extending circulation time and offering a strategy to enhance delivery efficiency.

Having established a foundational understanding of EV pharmacokinetics in physiological conditions, we wanted to determine how inflammatory conditions impacted these properties. In LPS-primed mice, circulating EV levels remained elevated up to 24 hours post-injection, a stark contrast to non-inflammatory conditions. Enhanced accumulation was observed in immune-rich organs, including the liver, spleen, lungs, kidneys, and brain. Flow cytometric analysis demonstrated increased EV association with myeloid cells, particularly with macrophages and neutrophils, and adaptive immune cells under inflammatory conditions. However, this increased uptake did not translate to improved cytosolic delivery of functional protein cargo, suggesting that endosomal escape remains a major bottleneck in EV-mediated delivery.

Given the clear differences in the fate of injected EVs between physiological and inflammatory conditions, we were interested in studying the functional properties of EVs derived from pathological sources. Tumor-derived EVs (tEVs) from melanoma cells play a key role in hematopoietic dysregulation and immune suppression. These tEVs contain angiogenesis-related factors like VEGF and immunomodulatory chemokines. tEV injection in mice induced splenomegaly, extramedullary hematopoiesis, and expansion of myeloid-derived suppressor cells (MDSCs) and erythroid progenitors, mimicking tumor-bearing profiles. Heat inactivation or VEGF pathway blockade prevented these effects, highlighting VEGF as a critical driver of immunosuppressive remodeling.

These findings emphasize the complexity of EV biodistribution, the influence of inflammatory and tumor microenvironments, and the utility of engineered luciferase-EVs in quantitative and mechanistic studies. The dual challenges of rapid clearance and limited functional cargo release highlight the need for continued development of EV modifications to enhance stability, targeting, and delivery efficiency. Understanding and manipulating EV interactions with the immune system and blood components is essential for their successful application in therapeutic settings.

List of scientific papers

I. Gupta, Dhanu, Xiuming Liang, Svetlana Pavlova, Oscar P. B. Wiklander, Giulia Corso, Ying Zhao, Osama Saher, Jeremy Bost, Antje M. Zickler, Andras Piffko, Cecile L. Maire, Franz L. Ricklefs, Oskar Gustafsson, Virginia Castilla Llorente, Manuela O. Gustafsson, R. Beklem Bostancioglu, Doste R. Mamand, Daniel W. Hagey, André Görgens, Joel Z. Nordin, and Samir EL Andaloussi. 2020. "Quantification of Extracellular Vesicles in Vitro and in Vivo Using Sensitive Bioluminescence Imaging." Journal of Extracellular Vesicles 9(1). https://doi.org/10.1080/20013078.2020.1800222

II. Andre Görgens*, Svetlana Pavlova*, Doste R. Mamand, Daniel W. Hagey, Xiuming Liang, Yesid Estupiñan Velasquez, Wenyi Zheng, Guannan Zhou, Risul Amin, Antje M. Zickler, Scott Bonner-Harris, Miina Ojansivu, Manuela O. Gustafsson, Oliver G. Hayes, Oscar P. B. Wiklander, Manuchehr Abedi-Valugerdi, Samantha Roudi, Molly M. Stevens, and Samir EL Andaloussi. Injected Extracellular Vesicles and other nanoparticles hitchhike on Erythrocytes and Platelets from circulation towards organ clearance. [Manuscript]
* shared first authorship

III. Svetlana Pavlova, Doste R. Mamand, André Görgens, Antje M.Zickler, Wenyi Zheng, Xiuming Liang, Oscar Wiklander, Manuchehr Abedi- Valugerdi, Elien Van Wonterghem, Junhua Xie, Zheyu Niu, Guannan Zhou, Roosmarjin E. Vanderbroucke, Dhanu Gupta, Samir El Andaloussi Systemic Inflammation Modulates Clearance and drives Extra-Hepatic Distribution of Extracellular Vesicles. [Manuscript]

IV. Doste R. Mamand, Safa Bazaz, Dara K. Mohammad, Xiuming Liang, Svetlana Pavlova, Carsten Mim, Susanne Gabrielsson, Joel Z. Nordin, Oscar P. B. Wiklander, Manuchehr Abedi-Valugerdi, Samir El- Andaloussi. Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting. J Extracell Vesicles. 2024 Jul;13(7):e12471. https://doi.org/10.1002/jev2.12471