Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes

Abstract

In the recent years, the possibility of utilizing extracellular vesicles for drug delivery purposes has been investigated in various models, suggesting that these vesicles may have such potential. In addition to the choice of donor cell type for vesicle production, a major obstacle still exists with respect of loading the extracellular vesicles efficiently with the drug of choice. One of the proposed solutions to this problem has been drug loading by electroporation, where small pores are created in the membrane of the extracellular vesicles, hereby allowing for free diffusion of the drug compound into the interior of the vesicle. We investigated the utility of adiposederived stem cells (ASCs) as an efficient exosome donor cell type with a particular focus on the treatment of glioblastoma multiforme (GBM). In addition, we evaluated electroporation-induced effects on the ASC exosomes with respect to their endogenous potential of stimulating GBM proliferation, and morphological changes to single and multiple ASC exosomes. We found that electroporation does not change the endogenous stimulatory capacity of ASC exosomes on GBM cell proliferation, but mediates adverse morphological changes including aggregation of the exosomes. In order to address this issue, we have successfully optimized the use of a trehalose-containing buffer system as a way of maintaining the structural integrity of the exosomes.

Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading

“…However, electroporation may trigger the aggregation of EVs, and change their morphological characteristics. This has been reported by Hood, et al, (69) and Johnsen, et al, (70). Electroporation may also promote aggregation of therapeutic siRNA as reported by Kooijmans, et al, (71).…”

“…In addition to cargo aggregation, EVs could also aggregate by fusing together as reported in the case of liposomes (72). Since the reports showing siRNA aggregate formation, several groups have employed citric acid buffer or the trehalose pulse media to reduce aggregation (68, 70, 71). Trehalose did not appear to affect EVs’ size without electroporation, nor did it hinder pore formation upon electroporation (70).…”

“…Since the reports showing siRNA aggregate formation, several groups have employed citric acid buffer or the trehalose pulse media to reduce aggregation (68, 70, 71). Trehalose did not appear to affect EVs’ size without electroporation, nor did it hinder pore formation upon electroporation (70). In spite of the fact that aggregation could be prevented, electroporation relies upon the passive loading of the cargo.…”

Gold nanoparticle based double-labeling of melanoma extracellular vesicles to determine the specificity of uptake by cells and preferential accumulation in small metastatic lung tumors

“…Indirect labelinginclusion strategies can also alter cell viability and lead to contamination with apoptotic bodies and other extracellular components that can be isolated with the EVs [33]. Other methods, such as electroporation and sonication are well known to disrupt EV membranes and therefore alter their structure, composition as well as distribution [34]. While some attempts to resolve these problems have been reported [32,35,36], to the best of our knowledge, the development of a gold-labeling strategy which does not affect natural EV tropism, as well as the utilization of AuNPs for quantitative tracking of their accumulation/distribution in metastasis has not been described previously.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…EDTA) [166] or membrane stabilizers (e.g. trehalose) [170,171]. Nonetheless, even if transient pores would be formed in the EV membrane and aggregation can be prevented, given that EP likely relies on passive loading it can only be efficient in extremely concentrated EV isolates [169].…”