Standardization Approaches for Extracellular Vesicle Loading with Oligonucleotides and Biologics.

Extracellular vesicles (EVs) are widely recognized for their potential as drug delivery systems. EVs are membranous nanoparticles shed from cells. Among their natural features are their ability to shield cargo molecules against degradation and enable their functional internalization into target cells. Especially biological or bio-inspired large molecules (LMs), like nucleic acids, proteins, peptides, and others, may profit from encapsulation in EVs for drug delivery purposes. In the last years, a variety of loading protocols are explored for different LMs. The lack of standardization in the EV drug delivery field has impeded their comparability so far. Currently, the first reporting frameworks and workflows for EV drug loading are proposed. The aim of this review is to summarize these evolving standardization approaches and set recently developed methods into context. This will allow for enhanced comparability of future work on EV drug loading with LMs.

Synergistic siRNA Loading of Extracellular Vesicles Enables Functional Delivery into Cells.

RNA interference opened new approaches for disease treatment but safe and efficient cell delivery remains a bottleneck. Extracellular vesicles (EVs) are known to naturally shuttle RNA. Due to their potent cell internalization and low-cost scalability, milk-derived EVs in particular are considered promising RNA delivery systems. However, low drug loading currently impedes their use. Here, innovative exogenous loading strategies for small interfering RNA (siRNA) are explored and systematically compared regarding encapsulation efficiency, loading capacity, and loading concentration. Firstly, siRNA is pre-accumulated in liposomes or stabilized calcium phosphate nanoparticles (CaP-NP). The selected systems, which exhibited neutral or negative zeta potentials, are then applied for EV loading. Secondly, EVs are concentrated and applied to protocols known for liposome loading: dehydration-rehydration of vesicles, based on freeze-drying, and mixing by dual asymmetric centrifugation (DAC) after ultracentrifugation. Additionally, DAC after EV ultracentrifugation is combined with CaP-NP leading to a synergistic loading performance. The balance between energy input for siRNA loading and EV integrity is evaluated by monitoring the EV size, marker proteins, and morphology. For the EV-based siRNA formulation via DAC plus CaP-NP, EV properties are sufficiently maintained to protect the siRNA from degradation and deliver cell-death siRNA dose-dependently in Caco-2 cells.

A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the Caco-2 intestinal barrier model.

Bovine milk-derived extracellular vesicles (EVs) hold promises as oral drug delivery systems. Since EV bioavailability studies are difficult to compare, key factors regarding EV uptake and intestinal permeability remain little understood. This work aims to critically study uptake and transport properties of milk-derived EVs across the intestinal barrier in vitro by standardization approaches. Therefore, uptake properties were directly compared to liposomes in intestinal Caco-2 cells. Reliable staining results were obtained by the choice of three distinct EV labeling sites, while non-specific dye transfer and excess dye removal were carefully controlled. A novel fluorescence correction factor was implemented to account for different labeling efficiencies. Both EV and liposome uptake occurred mainly energy dependent with the neonatal Fc receptor (FcRn) providing an exclusive active pathway for EVs. Confocal microscopy revealed higher internalization of EVs whereas liposomes rather remained attached to the cell surface. Internalization could be improved when changing the liposomal formulation to resemble the EV lipid composition. In a Caco-2/HT29-MTX co-culture liposomes and EVs showed partial mucus penetration. For transport studies across Caco-2 monolayers we further established a standardized protocol considering the distinct requirements for EVs. Especially insert pore sizes were systematically compared with 3 µm inserts found obligatory. Obtained apparent permeability coefficients (Papp) reflecting the transport rate will allow for better comparison of future bioavailability testing.