Page 88 - Read Online

Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12 Page 182

publication, the authors displayed rabies viral glycoprotein (RVG), which targets the nicotinic acetylcholine
receptor, on the EV membrane for functional delivery of encapsulated siRNA to the brain following
[76]
systemic injections . The RVG targeting peptide was enriched on the EV surface by fusion to lysosome-
[185]
associated membrane protein 2b (Lamp2b), which is known to associate with the EV membrane . This
parental cell engineering strategy has since then been successfully adapted to display a number of different
[186]
[187]
targeting moieties, including lamp2b fused to T7 peptide , αv integrin-specific iRGD peptide , tLyp-
1 , and to chondrocyte-affinity peptide (CAP) for EV targeting to the brain, breast cancer, lung cancer,
[189]
[188]
and chondrocytes, respectively. In addition to Lamp2b, targeting peptides have been fused to other EV
anchoring proteins, such as different tetraspanins, including CD9 fused to ApoB for brain targeting and
[190]
CD63 fused to ApoA1 for liver cancer (HepG2) targeting. Other examples include the use of the EV-
[191]
associated lactadherin (C1C2 domain), which has been fused to anti-Her2 single-chain variable fragments
to target HER2-positive breast cancer , glycosylphosphatidylinositol (GPI) fused to anti-EGFR
[192]
[193]
nanobodies for cancer targeting , and platelet-derived growth factor receptors (PDGFRs) fused to the
EGFR binding peptide GE11 . There is thus a great body of evidence showing the successful targeting of
[194]
[195]
EVs by the surface display of a variety of targeting peptides fused to different EV sorting proteins .
Additionally, overexpression of TNF-related apoptosis-inducing ligand (TRAIL) has been shown to
function as a homing mechanism for EVs to lymphoma tumour tissue . Another interesting, perhaps
[196]
somewhat overlooked approach is glycoengineering, which involves the manipulation of the dense
glycocalyx of the EVs membrane. In a recent publication, the display of the glycan ligands sialyl Lewis X
(sLeX) or Lewis X on EVs were shown to successfully target them to activated endothelial cells and dendritic
cells, respectively . Albeit the approach of genetic manipulation of the source cell is the most common
[197]
approach for targeted EVs, there are also other promising EV targeting strategies. One alternative to cell
manipulation is to directly modify the purified EVs . For instance, surface functionalization has been
[198]
[199]
shown by click chemistry, which has been used to display brain targeting peptides, e.g., c(RGDyK) and
RGE that showed increased brain accumulation in a brain ischemic mouse model and in glioma-bearing
[200]
mice, respectively. Another targeting strategy is to employ aptamers on the EV surface for cancer targeting
using cholesterol conjugation. Successful in vivo targeting has been shown with EV display of DNA aptamer
(AS1411) that binds nucleolin for breast cancer targeting , RNA aptamer that binds EGFR, as well as RNA
[201]
[202]
aptamer binding prostate-specific membrane . Yet another approach is to functionalize the EV surface by
fusion with liposomes. For instance, EV-liposome hybrids employing cRGD decorated liposomes have
[203]
demonstrated improved tumour targeting . Of note, despite the compelling evidence for EVs targetability,
the magnitude of targeting is still often unclear. The reports often merely show the fold increase in
accumulation of the engineered EVs in the target tissue and do not report the actual percentage of the
injected EV dose, which is needed for improved comparison between different methods and for off-target
considerations.

CONCLUSION
Targeted delivery with EVs is still a challenging task. With the existence of various biological barriers, EVs
are rapidly cleared by the liver from the plasma, which restricts the distribution to diseased tissue. With the
huge overlap of biochemical properties of EVs with other synthetic nanoparticles, understanding and
learning from other fields could be applied to refining EV-based therapeutics. Therefore, for achieving
superior targeted tissue delivery, multiple EV engineering strategies need to be combined to enhance the
plasma half-life of EVs, and engage with endothelial barriers for efficient transcytosis and endosomal escape
enhancement for breaching cellular barriers.