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and characterization is essential for enabling comparisons between studies and increasing the robustness of
findings. Investigating the specificity and sensitivity of biomarkers in EVPs is crucial for accurate and
reliable results. By focusing on these challenges, the potential of EVPs as powerful tools for cancer detection,
prognosis, and therapeutic guidance can be fully realized, ultimately leading to improved patient outcomes
and more effective treatments.

THE THERAPEUTIC POTENTIAL OF EVPS
The ability of EVPs to act as vehicles for delivering various biomolecules has prompted extensive research
into the therapeutic applications of EVPs [17,163] . The pioneering study aiming to exploit EVPs for therapeutic
purposes used EVPs derived from DCs pulsed with tumor antigen peptides as a vaccine . This study
[164]
demonstrated that EVP-based therapy could prime the function of T cells and exert antitumor effects.
Although the subsequent clinical trials did not achieve the predefined efficacy, this landmark study
demonstrated the potential for using EVPs in cancer therapy and showed that immune cells and other non-
cancerous cells could also be targeted for cancer therapy using EVPs. Subsequently, Alvarez-Erviti et al.
genetically engineered DCs to express a fusion protein of an EVP membrane protein and a neuron-affinity
[165]
protein, resulting in the successful production of neuron-targeted EVPs . Loading these neuron-affinity-
enhanced EVPs with siRNA reduced the expression of Alzheimer's disease target proteins in the brain. The
approach of genetically modifying cells to alter the membrane surface of the produced EVPs and loading
cargo to exert effects on target cells demonstrates the potential of engineering EVPs to selectively deliver
biomolecules to target cells and organs, inducing intended biological effects.

Engineered EVPs carrying proteins, nucleic acids, small molecules, and anticancer drugs can induce various
biological effects in target cells. For example, siRNA targeting the Kras G12D mutation was introduced into
fibroblast-like mesenchymal cell-derived EVPs via electroporation, inhibiting tumor growth in a preclinical
pancreatic cancer mouse model and serving as a basis for clinical trials [166,167] . EVPs loaded with anticancer
[168]
agents, such as doxorubicin, also exhibit antitumor effects . Beyond merely transporting biomolecules,
EVPs can mediate targeted gene editing in cells when loaded with CRISPR-Cas9 , suggesting an increased
[169]
potential for EVP-based therapeutic approaches.

Furthermore, EVPs with engineered surface components can selectively target specific cells. For example,
rabies viral glycoprotein (RVG) has a high affinity for neurons, and expressing RVG on the surface of
genetically modified EVPs targets them to the central nervous system . Additionally, enrichment of
[165]
specific combinations of integrins may enable the development of EVPs that can be taken up by selected
target cells. As such, engineering not only the payload but also the surface of EVPs is expected to be
increasingly utilized to fully harness their properties.

Highly engineered EVPs, such as enveloped protein nanocages (EPNs), which contain artificial proteins
nanocages , have also been developed. The production of protein nanocages mimics viral assembly,
[170]
autonomously assembling within cells and then trafficking through vesicles. EPNs, enveloped by a lipid
bilayer, are then released from the cells. EPNs contain tens of protein nanocages, which can deliver
biomolecules to target cells and exert their effects. Numerous strategies for engineering EVPs are being
developed, and it is expected that they will become mainstream in the future. Combining engineered EVPs
with other treatment modalities, such as chemotherapy, radiotherapy or immunotherapy, may also
represent promising anticancer strategies.

In addition, EVPs derived from specific cells, such as mesenchymal-derived stem cells (MSCs), dendritic
[171]
cells, platelets, adipocytes, and even plants, are being studied as sources of EVPs for treatment .