Functionalization

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Functionalization

Extracellular vesicles (EVs) possess remarkable qualities, including high cellular uptake, low immunogenicity, excellent biocompatibility, and the ability to easily cross the blood-brain barrier, making them a promising generation of drug delivery platforms.

Nevertheless, the inherent lack of specificity in EVs presents a notable challenge. While EVs are acclaimed for their ability to transport biological cargo to target cells, our specialization lies in customizing EVs to enhance their therapeutic impact.

Functionalization Actions Methods
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  • Biopharmaceuticals
  • ASOs, RNA, DNA
  • Plasmid
  • Peptide
  • Transfection
  • Electroporation
  • Sonication
Surface modification
  • Any molecules of choice
  • Antibody
  • Nanobody
  • Peptide
  • Click chemistry
  • Enzymatic ligation
  • Avidin-biotin

Case study

EV-nanobody: specific uptake into target cells

Nanobody-conjugated RBCEVs (EGFR-VHH-EVs) exhibit increased uptake in target cells during co-culture experiments with mouse 4T1 cells that co-express tdTomato and human EGFR, and parental 4T1 cells. RBCEVs, labeled with CFSE (green), show enhanced intracellular accumulation in tdTomato-hEGFR double-positive 4T1 cells (red), while no accumulation is observed in tdTomato-hEGFR double-negative parental 4T1 cells (blue).

EV-nanobody-nucleic acid: in vitro therapeutic effect

EGFR-targeted RBCEVs (EGFR-VHH-EVs) loaded with GFP siRNA achieved precise knockdown of destabilized GFP in CA1a-dGFP cells (top image). RBCEVs loaded with antisense oligonucleotides (ASOs) against miR-125b, a tumorigenic microRNA, demonstrated a ~10-fold greater reduction with EGFR-targeted RBCEVs compared to non-targeted ones (bottom left image). Furthermore, EGFR-targeted RBCEVs loaded with miR-125b ASOs significantly reduced 4T1 cell viability to ~20% compared to the untreated control (bottom right image)

EV-nanobody-nucleic acid: in vivo therapeutic effect

EGFR-targeted miR-125b ASO loaded EVs efficiently suppress tumor progression. Establishment of an hEGFR-positive lung metastatic breast cancer model by injecting 4T1-tdTomato-hEGFR cells systemically into NSG-SGM3 mice for intratracheal EV treatment (Top image). RT-qPCR analysis showed that EGFR-targeted miR-125b ASO-loaded EVs resulted in significantly higher knockdown only in tumor cells, but not in lung cells (Bottom left image). Flow cytometric analysis of lung cells from each treatment revealed that EGFR-VHH-EVs loaded with miR-125b ASO demonstrated the best tumor suppressive effects (Bottom middle image). This trend extended to the % of lung area occupied by tumor cells, assessed through tdTomato fluorescence in lung sections (Bottom right image).