Creating Designer Engineered Extracellular Vesicles for Diverse Ligand Display, Target Recognition, and Controlled Protein Loading and Delivery

Abstract

Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag‐fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein‐engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles.

Loading of extracellular vesicles with nucleic acids via hybridization with sponge-like lipid nanoparticles

“…In alignment with previous studies, EVs did not induce endosomal damage, in contrast to the Lipofectamine TM positive control, where a clear enhancement of the punctate mRuby3 signal could be observed (Fig. 6B) ( 62, 63 ). Notably, HEVs displayed no endosomolytic effects whereas LCNPs induced bright mRuby3 punctate signals (Fig.…”

Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan‐Vesicular Affinity Probes

“…It is paradigmatic that a recent work reported on the loss of up to the 80% of EVs with diagnostic potential when affinity isolation by single tetraspanin protein is used, and a loss of 36-47% when a tetraspanin cocktail is employed. [22] This issue reverberate also on the exponentially growing field of engineered, [23] synthetic [24] or hybrid EV-mimetics and analogs [25,26] and other bacterial [27,28] or plant-derived bionanoparticles, [29] which are under extensive investigation as the next generation nano delivery systems in the therapeutic arena. Indeed, their successful translation depends, among other factors, also on the availability of high-sensitivity characterization methods to be applied to robust QC protocols, and to assess biodistribution parameters including their concentration and clearance in biofluids.…”

Addressing Heterogeneity in direct analysis of Extracellular Vesicles and analogues using Membrane-Sensing Peptides as Pan-Affinity Probes

“…This issue reverberate also on the exponentially growing field of engineered 20 , synthetic 21 or hybrid EV-mimetics 22, 23 , and other bacterial 24, 25 or plant derived bio-nanoparticles 26 , which are under extensive investigation as the next generation nanodelivery systems in the therapeutic arena. Indeed, their successful translation depends, among other factors, also on the availability of high-sensitivity characterization methods to be applied into robust QC protocols, and to assess biodistribution parameters including their concentration and clearance in biofluids.…”