Basyoni et al.

Cancer Cell International (2025) 25:150 Cancer Cell International

https://doi.org/10.1186/s12935-025-03731-z

REVIEW Open Access

Harnessing exosomes for targeted drug

delivery systems to combat brain cancer
Abdullah E. Basyoni1, Amira Atta1, Maha M. Salem1*   and Tarek M. Mohamed1

Abstract
Brain cancer remains a significant challenge in the field of oncology, primarily because of its aggressive nature
and the limited treatment options available. Conventional therapies often fall short in effectively targeting tumor
cells, while sparing healthy brain tissue from collateral damage. However, exosomes are now recognized as promis-
ing nanocarriers for targeted drug delivery. These naturally occurring extracellular vesicles can cross the blood–brain
barrier and selectively interact with cancer cells. Utilizing exosomes as drug delivery vehicles offers a novel approach
with significant potential for targeted therapy. By encapsulating therapeutic agents within exosomes, drugs can
be specifically targeted to tumor cells, maximizing their impact whilst minimizing damage to healthy brain tissue.
Furthermore, exosomes can be modified to display molecules that specifically recognize and bind to cancer cells,
further enhancing their precision and efficacy. While exosome-based therapies show potential, scalability, purifica-
tion, and clinical application challenges remain. The scalability of exosome production, purification, and modification
techniques remains a hurdle that must be overcome for clinical translation. Additionally, the intricate interactions
between the tumor microenvironment and exosomes necessitate further research to optimize therapeutic outcomes.
The review explores applications and future perspectives of exosome-based therapies in advancing targeted brain
cancer treatment.
Keywords Exosomes, Nanocarriers, Brain cancer, Targeted drug delivery

Overview of brain cancer looking at the brain of a lifeless individual who had alleg-
Brain cancer, a devastating disease that affects the cen- edly died from a benign tumor. However, he found out
tral nervous system, includes primary brain tumors that that it was indeed a cancerous tumor.
develop within the actual brain tissue and secondary
brain tumors, metastatic tumors originate from can- Primary brain tumors
cers in other parts of the body and spread to the brain
(Table 1) (Fig. 1) [1]. These tumors typically arise from • Gliomas
primary cancers in organs such as the lungs, breasts, or
colon, and spread to the brain through the bloodstream • Astrocytoma: Tumors arising from star-shaped
or lymphatic system. Brain cancer can be benign or astrocyte cells in the cerebrum.
malignant. Gupta Longati, a Russian scientist, made the • Oligodendroglioma: Tumors originating from oli-
initial observation of brain cancer in 1873 [2]. He was godendrocytes, more common in adults [3, 4].
• Glioblastoma: Aggressive tumors classified into
primary (de novo) and secondary glioblastomas.
*Correspondence:
Maha M. Salem • Mixed Glioma: Tumors arising from multiple cell
maha_salem@science.tanta.edu.eg types such as ependymal and oligodendrocyte
1
Biochemistry Division, Chemistry Department, Faculty of Science, Tanta cells. Behavior varies based on tumor grade.
University, Tanta 31527, Egypt

© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Basyoni et al. Cancer Cell International (2025) 25:150 Page 2 of 32

Table 1 Summarizing the key characteristics of various brain tumor types

Tumor type Origin Characteristics Key notes

Astrocytoma Astrocyte cells in the cerebrum Most common glioma; can range Tumor grade determines prognosis
from low-grade to highly malignant forms and treatment approach
Oligodendroglioma Oligodendrocytes Rare, slow-growing tumors; more com- Uncommon in children; associated
mon in young to middle-aged adults with better prognosis
Glioblastoma Glial cells Aggressive and fast-growing; divided Challenges include poor prognosis
into primary (de novo) and secondary and limited treatments
types
Mixed Glioma Multiple glial cell types Tumors arise from combinations Behavior depends on tumor grade
of ependymal, astrocyte, and oligoden-
drocyte cells
Schwannoma Schwann cells in peripheral nerves Typically benign; affects nerve roots Often associated with hearing loss
or peripheral nerves if involving acoustic nerve
Meningioma Meningeal arachnoid matter Most common non-glial brain tumor; Constitutes 38% of primary brain cancers
generally slow-growing
Germ Cell Tumors Germ cells Rare; primarily occurs in ovaries, testicles, Can secrete hormones or proteins detect-
or other locations, including the brain able in blood tests
Craniopharyngioma Near the pituitary gland Slow growth; often impacts hormonal bal- Two types: papillary and adamantinoma
ance due to proximity to the pituitary Tous
Medulloblastoma The cerebellum, near the brainstem Highly malignant; rapid growth Commonly seen in children; responsive
and potential to spread to radiation therapy
Secondary Brain Cancer Cancers from other organs (e.g., Tumors metastasize to the brain Often indicates advanced-stage primary
lung, breast, colon) via bloodstream or lymphatic system cancer

Fig. 1 Brain cancer, includes primary brain tumors develop within the actual brain tissue and secondary brain tumors
Basyoni et al. Cancer Cell International (2025) 25:150 Page 3 of 32

• Schwannoma Risk factors for brain cancer

Benign tumors develop from Schwann cells, typically Brain cancer risk factors can be broadly categorized into
in peripheral nerves. environmental and genetic factors (Fig. 2).
• Other types
Environmental factors
• Meningioma: Tumors originating from meningeal
arachnoid matter, constituting 38% of primary • Lifestyle choices: Unbalanced diet, smoking, exces-
brain cancers. sive alcohol consumption, and physical inactivity
• Germ Cell Tumors: Rare tumors arise from germ have been implicated in various cancers, including
cells. brain cancer, through indirect mechanisms such as
• Craniopharyngioma: Growths near the pituitary increased inflammation and oxidative stress [8, 9].
gland. • Radiation exposure: High levels of ionizing radiation,
• Medulloblastoma: Rapidly growing tumors in the such as from medical imaging or occupational expo-
cerebellum [5]. sure, significantly increase brain cancer risk.
• Chemical exposure: Carcinogenic substances in
industrial chemicals and pollutants are well-docu-
mented contributors to cancer risk [10].
Secondary brain cancer • Dietary factors: Consumption of nitrite-preserved
Secondary (metastatic) brain cancers originate from foods has been linked to an increased risk of certain
malignancies in other parts of the body, such as the lungs, brain tumors due to the potential for nitrosamine
breasts, or colon, and spread to the brain [6, 7]. formation [11].

Fig. 2 Environmental and genetic risk factors of brain cancer

Basyoni et al. Cancer Cell International (2025) 25:150 Page 4 of 32

Genetic factors Mechanism of metastatic brain cancer

The intricate process of cancer cells spreading from a pri-
• Inherited mutations: Genetic predispositions, such as mary tumor to the brain is known as brain metastasis.
mutations in tumor protein 53 (TP53), Phosphatase Circulating tumor cells (CTCs) from the primary tumor
and TENsin homolog (PTEN), or Neurofibromatosis that survive in the bloodstream and go to the brain is
type 1 (NF1) genes, are associated with an increased the major mechanism of brain metastasis from primary
risk of gliomas and other brain tumors [11]. cancers (Fig. 3) [13]. Once within the brain, these cells
• Familial syndromes: Conditions like Li-Fraumeni can go dormant and avoid immune system recognition.
syndrome and neurofibromatosis are linked to higher They can also facilitate their migration in the brain by
brain cancer incidence (Fig. 2). activating signaling pathways such as cathepsin S, L1 cell
adhesion molecule (L1CAM), and Signal transducer and
While certain medications, such as immunosuppres- activator of transcription 3 (STAT3) [13, 14]. Further-
sive drugs used in transplant patients, have been asso- more, they can prevent the activation of the protein Fas
ciated with an increased risk of specific cancers, robust ligand (FasL), which causes cancer cells to undergo apop-
evidence linking common medications (e.g., over- tosis and spread to the brain [15].
the-counter drugs or sleeping pills) to brain cancer is When CTCs are released into the bloodstream from
lacking. Any mention of such associations has been the main tumor, brain metastasis starts. In circulation,
removed to maintain scientific accuracy [10, 12]. these cells are subject to shear pressures and immunolog-
ical monitoring, and they must survive. CTCs can attach
to the brain’s endothelium of blood arteries after they are

Fig. 3 The mechanism of brain metastasis involves the release of Circulating tumor cells (CTCs) from the primary tumor into the bloodstream,
extravasation into the brain tissue through the blood–brain barrier (BBB), and adaptation to the brain microenvironment. [L1cell adhesion molecule
(L1CAM), Signal transducer and activator of transcription 3 (STAT3), Fas ligand (FasL), phosphatidylinositol-3 kinase (PI3K), Plasminogen activator (PA),
the serine proteinase inhibitor (Serpin), Oxidative phosphorylation (OXPHOS)]
Basyoni et al. Cancer Cell International (2025) 25:150 Page 5 of 32

in the bloodstream. This is made possible via interactions medical history, overall health, and types of symptoms,
between the endothelial cells and adhesion molecules on all influence the treatment plan that is selected. The sev-
the CTCs. The CTCs can extravasate and enter the brain eral approaches used to treat brain cancer [21] include.
tissue because of this adhesion.
Upon entering the brain, CTCs face the challenge of Surgery
surviving and proliferating in a foreign microenviron- The major treatment technique in the early stages of
ment [13, 16]. The cellular and molecular makeup of the a benign tumor is surgical excision of the tumor while
brain microenvironment sets it apart from those of other maintaining brain function. In most cases, surgery is the
organs. CTCs need to adjust to this setting to spread only treatment available for low-grade tumors. Cancer
throughout the brain. One way they achieve this is by symptoms are lessened by surgically removing the tumor,
entering a dormant state, where they temporarily cease which also decreases intracranial pressure caused by the
proliferation and evade immune detection. This dor- tumor. With few or no side effects [22], it is the simplest
mancy allows the CTCs to evade therapeutic interven- and safest method of treating brain cancer. Surgery can
tions and remain undetected for extended periods. only be used as a therapeutic option if the tumor is at a
CTCs use a variety of signaling pathways in addition to place that may be reached without endangering vital
dormancy to encourage their migration and proliferation brain functions. This is a limitation of the procedure.
within the brain. One such pathway that aids in cell inva- Headache, weakness, fatigue, brain swelling, or a build-
sion, proliferation, and survival is the signal transducer up of fluid in the brain are common post-operative side
and activator of the STAT3 pathway. To improve their effects. Brain injury can also be a very significant issue.
survival and proliferation in the brain microenviron- Epilepsy surgery can also result in cognitive, verbal, and
ment, CTCs can stimulate STAT3 signaling [17]. Another visual impairments [9].
important pathway is L1CAM, which is implicated in
promoting cell motility and invasion. CTCs can upregu- Radiation
late L1CAM to facilitate their migration within the brain Brain cancer shrinks as a result of radiation therapy,
tissue [18], contributing to the formation of metastatic which uses gamma, x-, and proton beams to kill or
lesions. destroy malignant cells. Typically, radiation therapy is
Furthermore, CTCs can exploit the protease cath- administered five days a week for 6 weeks [23]. In general,
epsin S to facilitate their migration and invasion in the there are two forms of radiation therapy: exterior radia-
brain [13]. Cathepsin S can encourage CTC migra- tion treatment, including whole brain radiation therapy
tion and invasion in the brain and is involved in modi- (WBR), which delivers a lower radiation dose after each
fying the extracellular matrix. Through the cleavage of treatment, and stereo static radiosurgery (SRS), which
blood–brain barrier (BBB) junctional adhesion molecules delivers a high dose of radiation in a single treatment.
(JAMs), such as JAM-B [11], cathepsin S facilitates the Conversely, brachytherapy, or implant-based radiation
migration of CTCs into the brain tissue, contributing to therapy, is a component of internal radiation therapy.
the formation of brain metastases. Radiation therapy side effects can include headaches,
Moreover, CTCs can inhibit the activation of FasL, a nausea, edema, exhaustion, and changes in movement or
protein that induces apoptosis in cancer cells [19]. Essen- feeling [21].
tially, the process of brain metastasis entails the discharge
of CTCs from the original tumor into the bloodstream, Chemotherapy
their endurance and attachment within the brain micro- Chemotherapeutic medications are used in this cancer
vasculature, their infiltration into the brain tissue, their therapy approach. The main ways that therapeutic drugs
adjustment to the brain microenvironment via signaling work are by preventing blood flow to tumor cells or by
pathway activation and dormancy, and the suppression of interfering with the process of cell division, which kills
apoptosis to generate brain metastases. Comprehending aberrant cells and causes tumors to shrink [22, 24]. This
the complex processes associated with brain metastasis therapeutic approach’s drawback is that it could harm
is essential for creating tailored treatments to avoid or healthy tissues or cells as well. Chemotherapeutic treat-
manage this severe cancer consequence [20]. ment commonly causes side effects including exhaustion,
thirst, weakness, nausea, and decreased white blood cell
Treatments for brain cancer counts, all of which raise the risk of infection.
The two main goals of cancer treatment options are to
either remove the tumor or, by decreasing the tumor, Hormonal therapy
provide relief from the symptoms of cancer. The kind, Hormonal therapy is a type of cancer treatment in
location, and size of the tumor, as well as the patient’s age, which the growth of the cancer is slowed or stopped by
Basyoni et al. Cancer Cell International (2025) 25:150 Page 6 of 32

hormones. It is a non-toxic treatment for prostate and Oral tiny medicines or monoclonal antibodies are used
estrogen receptor-positive breast cancers. The two pri- in targeted therapy. Cancer cells’ surface proteins and
mary approaches to hormonal therapy are blocking the receptors are bound by monoclonal antibodies. By block-
body’s ability to produce hormones and interfering with ing substances that signal angiogenesis, or the growth
the hormones’ ability to function in the body [24, 25]. of cancer cells, these large molecules prevent the spread
of cancer. Small-molecule drugs are another type of tar-
Photodynamic therapy geted therapy; because of their low molecular weight,
A photosensitizer is used in photodynamic therapy to kill they can penetrate the cell surface and either stop the
the malignancy. A photosensitizer is a type of photosen- growth of tumor cells or kill them [31].
sitive material that, when it absorbs light with a certain
wavelength that is concentrated on the target cell, starts a Challenges in targeting therapy for brain cancer
photochemical or photophysical response [26]. The pho- The most difficult obstacle to the effective transport of
tosensitizer converts light energy into molecular oxygen medicinal medicines into the brain is the blood–brain
upon absorption, resulting in the formation of reactive barrier (BBB). It functions as a physiological and physi-
oxygen species (ROS), which include free radicals and cal barrier to the transfer of medications to the brain. The
singlet oxygen. Cellular toxicity is caused by the harmful capillary endothelial cells’ tight connections are what give
effects of ROS. the blood–brain barrier its impermeability. According to
reports, the majority of small molecular-weight medici-
Immunotherapy nal compounds and nearly all high molecular-weight
Biotherapy known as immunotherapy works by sensitiz- therapeutic drugs lack the innate ability to pass the
ing the immune system to cancer, hence reducing side blood–brain barrier. For a drug ingredient or formulation
effects and increasing selectivity. Malignancy growth and to penetrate the blood–brain barrier [32], it is ideal if it
metastasis can use immunosuppressive mechanisms to has a tiny molecular weight (500 Da), is lipid-soluble, log
prevent the immune system from recognizing the malig- P < 5, has electrically neutral molecules, and weak bases
nancy. Cancer immunotherapy aims to strengthen the that can diffuse passively across the barrier. Efflux trans-
immune system to fight these malignant cells. In cancer porters are engaged in the transfer of solutes out of the
immunotherapy, there are four main approaches: immu- brain endothelial cells and are additional barriers to the
nomodulation, targeted antibodies, adoptive cell treat- delivery of medicine throughout the brain. The drug sub-
ment, and cancer vaccines. Priming the host immune stance that has entered the brain is removed by the efflux
system enhances the immunological response through transporters. A feeble Enhanced Permeability and Reten-
immunomodulation. Essentially [27], it works by stimu- tion (EPR) effect is a further obstacle to the treatment
lating T cells to kill more tumor cells through the pres- of brain tumors [33]. By adding lipophilic or amphiphi-
entation of antigens to T cells. Although there are other lic groups to the blood–brain barrier at specific points,
drugs available, cytokines are the most popular ones for it is possible to increase drug delivery to the brain while
immunomodulation. By modifying the patient’s T cells in preserving the equilibrium between hydrophilic and lipo-
a lab to target the cancer cells more effectively, adoptive philic conjugates.
cell therapy is an immunotherapy technique used to sup-
port the immune system’s defense against cancer cells. Conventional nanocarriers
One form of active immunotherapy that aims to stimu- Conventional nanocarriers can be broadly classified into
late the immune system’s effector activities is the cancer two groups as shown in Tables 2 and 3. Liposomes and
vaccine. For the immune system to develop memory cells micelles are examples of organic nanocarriers, On the
resistant to these antigens, cancer vaccines are responsi- other hand, inorganic nanocarriers include layered dou-
ble for exposing the immune system to particular anti- ble hydroxides, graphene, nanoparticles that are mag-
gens produced on the surface of cancer cells [28, 29]. netic, nanoparticles of gold, mesoporous silicon dioxide
nanoparticles, and quantum dots. Exosomes possess
Targeted cancer therapy numerous benefits over traditional nanocarriers and
The delivery of medications to genes or proteins unique hold great promise for the treatment of tumors in clinical
to cancer cells or the tissue milieu that fosters the growth settings. First off, exosomes’ phospholipid bilayer shape
of cancer is known as targeted treatment. Targeted ther- allowed them to deliver medications steadily, avoid-
apy includes developing drugs that stop cancer cells from ing the breakdown of drugs by enzymes and prolonging
growing, aid in the regulation of the cell cycle, cause their half-lives. Additionally, their membranes interacted
apoptosis or autophagy, or target specific cancer cells well with the target cells. As a result, the loaded drug’s
with toxic compounds in an attempt to kill them [30]. bioavailability also increased. The immunogenicity
Basyoni et al. Cancer Cell International (2025) 25:150 Page 7 of 32

Table 2 Inorganic nanocarriers

Nanocarrier Advantages Disadvantages

Mesoporous Silica Highly stable; easily functionalized for targeted delivery (e.g., Limited biocompatibility without surface modification; poten-
across the blood–brain barrier) tial for toxicity
Magnetic Nanoparticles Excellent biocompatibility; enable magnetic field-based Size and heating control during hyperthermia are challenging;
targeting; effective for hyperthermia treatments toxicity concerns
Gold Nanoparticles High electron density; easy conjugation with biomolecules; Expensive synthesis; potential biosafety concerns; limited
surface-enhanced Raman scattering capabilities scalability
Carbon Nanotubes High drug-loading capacity; superior transmembrane Cytotoxicity; poor dispersibility; surface functionalization
capabilities challenges

Table 3 Organic nanocarriers

Nanocarrier Advantages Disadvantages

Liposomes Biodegradable; biocompatible; can encapsulate hydrophilic, Low stability; prone to oxidation; challenging sterilization
hydrophobic, and amphiphilic drugs
Micelles High drug-loading capacity for hydrophobic drugs; good stability Restricted to hydrophobic drugs; limited application for hydrophilic
in biological fluids molecules
Dendrimers High surface functionality; monodisperse size; adaptable internal High synthesis cost; complex purification; scalability issues
cavities
Exosomes Low immunogenicity; high bioavailability; effective for crossing Difficult large-scale production; challenges in isolation and purifica-
the blood–brain barrier tion

and toxicity of exosomes were also inferior to those of a hot topic in the study. To serve as a guide for cancer
liposomes, a conventional drug carrier. Furthermore, treatment in the future, this paper defines exosomes in
we can not disregard their small frame. Exosomes can detail and provides an overview of their use as nanocar-
move throughout tumor tissue and extravasate in tumor rier-loaded medications in tumor therapy.
vasculature to treat malignancies because of their nano
size. Exosomes can therefore also pass across some Origin and structure
physiological barriers, such as the blood–brain barrier. Exosomes are a kind of lipid bilayer vesicles that have
An additional benefit is that the medication’s potency is a round shape and a diameter that ranges from 30 to
increased when it is placed into exosomes. 150 nm. Their monodisperse distribution and round
shape are seen in the electron microscope image of
Exosomes exosomes [37]. In 1981, Trams et al. discovered that
A class of naturally occurring nanoscale membrane exosomes exist. By using electron microscopy, Pan et al.
vesicles called exosomes is produced by live cells via a described the creation of exosomes in 1985. The term
sequence of regulatory mechanisms called “endocytosis- “exosome” was not formally introduced until 1987, by
fusion-efflux”. To put it succinctly, exosomes were ini- Johnstone et al. Exosomes are composed of various com-
tially identified about 40 years ago. The understanding ponents. They are typically secreted by nearly all mam-
of exosomes has substantially increased in the last few malian cells (Fig. 4), including tumor cells, mesenchymal
decades. Exosomes were first believed to be a route for stem cells, epithelial cells, endothelial cells, dendritic
cell excretion, but more investigation showed that they cells, and T and B lymphocytes, and operate as messen-
are also a medium involved in material and information gers and transmitters in cellular crosstalk [38].
transfer between cells, transporting host cell proteins,
lipids, nucleic acids, and other materials. As a result, Exosome biogenesis
exosomes are employed as a type of nanocarrier to trans- Exosomes were generated by the endocytic pathway.
fer medications like paclitaxel or nucleic acids to cure a According to Fig. 5, the particular generation process
variety of illnesses including cancers [34–36]. Exosome is as follows: In the early endosomal (EE) stage, endo-
utilization and production of minimal or harmless gran- cytosis forms early endosomes in the cell membrane;
ules having high-efficiency exosome loading, which were in the late endosomal stage, Endosomal Sorting Com-
used in the treatment of cancer, has quietly emerged as plex Required Transport (ESCRT-0) binds to the early
Basyoni et al. Cancer Cell International (2025) 25:150 Page 8 of 32

Fig. 4 Exosome secretion by various cells

endometrial membrane’s specific receptors via the ubiq- Exosome composition

uitination binding site, and selectively splices a section The size and cargo of exosomes might vary even when
of the cytoplasm, causing ESCRT-I to attach to ESCRT- they originate from the same cell. Exosomes originating
0, which in turn allows ESCRT-II to attach to ESCRT-I, from different sources do share some payloads, though.
forming intraluminal particles through budding. After- Several investigations on exosomes have reported the
ward, ESCRT-III chops the neck bud, and ESCRT-I and presence of a wide range of biomolecules, including pro-
ESCRT-II collaborate to promote the formation of intra- teins, lipids, RNAs, and DNAs (Fig. 6). The majority of
luminal vesicles (ILVs) [39]. As a result of the ILVs’ sepa- lipids found in exosomes, including cholesterol, sphin-
ration from the endosomal membrane, other substances gomyelin, and phosphatidylserine, are recognized to be
are released into the endosomal cavity. Additionally, the elements of plasma membranes. Heat shock proteins
budding process is completed, resulting in the forma- (HSP70, HSP90), integrins myosin heavy chain (MHC)
tion of mature late endosomes, which are also known as class II proteins, proteins such as (Rab GTPases, annex-
multi-vesicle bodies (MVBs) because they contain mul- ins), etc. are among the proteins found in exosomes
tiple ILVs. Exocytosis: after that, some MVBs are broken [41]. Furthermore, exosomes have proteins transcribed
down by fusing with lysosomes, while some of them fuse on their outer layer, where they interact with recipient
with the plasma membrane, releasing the MVBs’ intracel- cell surface receptors to trigger intracellular signaling.
lular vesicles into the extracellular media in the form of Exosomes have been found to include all RNA species,
exosomes. In addition, some MVBs are paired with the including transfer RNA, long noncoding RNA, messenger
Golgi apparatus to recycle [40]. RNA (mRNA), and microRNA (miRNA). Certain RNAs
Basyoni et al. Cancer Cell International (2025) 25:150 Page 9 of 32

Fig. 5 The endocytic process, the endosome process, and exocytosis are the three phases of the exosome process. When endocytosis occurs,
the endosome gets tapped and matures into an older endosome that has numerous intraluminal vesicles (ILVs), or multi-vesicle bodies (MVBs).
Exosomes represent the tiny vesicles that MVBs discharge into the external membranes

are reportedly actively, rather than passively, sorted and caveolin-mediated endocytosis, macropinocytosis,
transported into exosomes, where they eventually affect clathrin-mediated endocytosis, and lipid raft-mediated
host cells’ transcripts, according to numerous studies. endocytosis. The result is cell-specificity due to surface
Remarkably, Batagov et al. also discovered that a particu- chemicals interacting with particular cells.
lar RNA fragment might allow the 3′-untranslated region
(UTR) of mRNA to reach the exosome. Furthermore, Tumor microenvironment
single-stranded DNA was found in exosomes by Balaj Exosomes transport chemicals and are essential for inter-
et al., while whole genome sequencing by Kalluri and col- cellular communication. Biomolecules produced from
leagues demonstrated the presence of double-stranded exosomes secreted by donor cells have the potential to
genomic DNA pieces of at least 10 kb in exosomes [42]. significantly alter recipient cells’ biological response.
Zhang et al. discovered that the human monocytic leu-
Intercellular communication mediated kemia cell line (THP-1) secretes a sizable amount of
by exosomes miRNA-150, which may facilitate target cell migration
Uptake of exosomes by suppressing target gene expression. In a different
Cosseti et al. discovered that in brain stem/precur- investigation, Wang et al. showed that the administra-
sor cells, interferon (IFN) binds to IFN-receptor 1 to tion of transient receptor potential polycystic 2 (TRPP2)
form a complex that then triggers signaling transmis- siRNA via exosomes dramatically suppresses the expres-
sion through this complex. Furthermore, research has sion of TRPP2 and the epithelial-mesenchymal transi-
indicated that exosomes may fuse with recipient cell tion in FaDu cells. Additionally, several investigations
membranes to deliver their contents to target cells [43], demonstrated that exosomes with functional proteins
while the precise molecular process is yet unknown. can facilitate cell-to-cell contact and exert significant
Many studies conducted recently indicate that the pri- influence over the target cell’s signaling pathway, which
mary mechanism of exosome uptake is internaliza- is linked to the advancement of cancer. Consequently,
tion by endocytic pathways, including phagocytosis, exosome-mediated cell-to-cell contact is a useful method
Basyoni et al. Cancer Cell International (2025) 25:150 Page 10 of 32

Fig. 6 Exosome components with lipids, integrins, amino acids, nucleic acid, and metabolites

for delivering various cellular macromolecules and influ- modify macrophage physiology, inhibit NK cell activity,
encing target cell phenotypes and activities [44]. Natural- and promote tumor growth by controlling T cell function
killer (NK) cells play a crucial role in immune surveillance (Fig. 7) [43]. Through their ability to control the tumor
and act as the primary defense in controlling the growth microenvironment (TME), TEXs are also crucial in the
and metastasis of cancer in our bodies. Through the spread of tumors. Exosomes generated from breast can-
delivery of killer proteins along with characteristic NK cer cells, for instance, carry miRNAs and oncogenic pro-
and exosome signals, NK cell-derived exosomes confer teins like miRNA-130a and miRNA-328 that promote
lethal action to tumor cells. Like NK cells, macrophages tumor growth and spread. Exosomes produced from
produce pro-inflammatory cytokines like interleukin 6 human pancreatic cells play a crucial role in immune-
(IL-6) and tumor necrosis factor-alpha (TNF-α), as well suppressive processes by promoting metastasis. In a
as reactive oxygen/nitrogen species, which are crucial for similar vein, Wang and colleagues discovered that TEXs
both innate host defense and tumor cell death. Tumor- in the gastric tumor cell microenvironment promote the
associated macrophages (TAMs) in the surroundings of development of primary tumor growth by eliciting a pop-
the tumor have been linked to the display of an M2-like ulation of immunosuppressive PD1+ tumor-associated
phenotype. Macrophages are generally classed into M1 macrophages that impede the function of CD8+ T cells
or M2 kinds. These TAM-derived exosomes mediate Numerous accounts have also been published regard-
cell-to-cell contact with other immune cells to induce ing the investigation of TEXs’ role in angiogenesis [42].
immune suppression for tumor development. Malignant The primary indicator of the advancement of a tumor is
cells generate tumor-derived exosomes (TEXs), which angiogenesis, which provides the blood vessels linked to
Basyoni et al. Cancer Cell International (2025) 25:150 Page 11 of 32

Fig. 7 Intercellular communication mediated by exosomes. [Dendritic cells (DCs), Natural-killer (NK), and Tumor-associated macrophages (TAMs)]

the tumor and serves as the target of cancer immuno- stimulate angiogenesis. Exosomes generated from tumors
therapy. TEXs play a role in the induction of new arteries can inhibit immune cells, including dendritic cells (DCs),
during the early stages of cancer development in a vari- T cells, and NK cells, from responding to tumors.
ety of tumor forms, such as hypoxic lung cancer, malig-
nant mesothelioma, and glioblastoma [5]. Furthermore, Microenvironment and therapeutic exosome
because of changes in tumor metabolism, there are ten interactions
times more of these TEXs than exosomes generated from The interactions between exosomes and the tumor
healthy cells. Consequently, because of their role in TME. microenvironment are paramount to understanding how
Numerous recent research have examined the function of these interactions can influence exosome functionality
TEXs as a biomarker [45]. In a different study, Mousavi and therapeutic outcomes. The complexity of exosome
et al. looked into the possibility that TEXs regulating the absorption, involving various proteins and cellular pro-
development of cancer could serve as possible biomark- cesses like clathrin-dependent endocytosis and macro-
ers for colorectal cancer. These results show that TEXs pinocytosis, highlights the intricate mechanisms at play
have a variety of effects on the pathophysiology of can- when exosomes interface with recipient cells. Numerous
cer. An overview of exosomes produced from tumors and exosomal proteins interact with receptors on the receiv-
their function in the tumor microenvironment. Cancer ing cells to enable exosome absorption [46]. Cell uptake
cells can produce exosomes, which can stimulate the of exosomes may be enhanced by radiotherapy-like mod-
development of mesenchymal stem cells (MSCs) and ulation of CD29/CD81 complex formation. It has been
cause other cancer cells to undergo the epithelial-mesen- proposed that using a CD9 antibody against tetraspanin
chymal transition [45]. To polarize macrophages toward to prevent DCs from absorbing exosomes works just
TAM, a phenotype that supports tumors, cancer cells can as well. To regulate exosome binding and absorption,
Basyoni et al. Cancer Cell International (2025) 25:150 Page 12 of 32

integrins (integrins v and 3, i.e., CD51 and CD61), pro- Cell‑secreted exosomes
teoglycans (heparan sulfate proteoglycans), and lectins Exosomes are nanoscale in size and can be discharged by
(C-type lectins DEC-205, galectin-5) collaborate. We nearly any kind of cell. Since extracellular vesicles (EVs)
still do not fully understand the mechanisms behind vary widely in composition and function. Character-
exosome-cell interactions, even though an increasing izing exosomes recovered from conditioned cell culture
number of protein interactions are known to influence media is relatively well-established in the present EV
exosome attachment and absorption. The process by field, as opposed to exosomes derived from complicated
which cells absorb molecules through clathrin-coated biological fluids like plasma [53]. Differential centrifuga-
vesicles in response to certain membrane receptors and tion is the known “gold standard” for isolating exosomes
their ligands is known as clathrin-dependent endocytosis. as a subset of EVs. Moreover, several techniques are fre-
As vesicle buds develop and expel their clathrin-coated quently employed in exosome applications, including
blisters, the plasma membrane may become distorted. immunoaffinity chromatography, polymer precipitation,
After clathrin breakdown, the contents of a vesicle and ultrafiltration. Exosomes from human embryonic
merged with an endosome were released chlorproma- kidney (HEK) cells, cancer cells, immune cells, and stem
zine may impede this enzymatic pathway [47, 48]. Clath- cells have been extracted by numerous research groups;
rin-dependent endocytosis is promoted by increases in these exosomes differ depending on where they come
membrane fusion, membrane curvature, and membrane from [54].
fission brought on by the GTPase dynamin2. Unlike
dynamin-positive cells, dynamin-negative cells dra- (HEK)‑derived exosomes
matically suppress the exosome internalization process. Because of its many benefits, including easy growth, low
Therefore, dynamin inhibitor therapy or a reduction in care requirements, and high transfection efficiency, the
caveolin-1 significantly reduces exosome internalization. HEK cell line (HEK293T) is the most often applied in
On the other side, the mechanism by which vesicles rang- the biopharmaceutical production industry. Exosomes
ing in diameter from 0.2 to 10 nm take up external flu- isolated from HEK293T contain membrane similarities
ids and solutes is known as macropinocytosis. This often to a variety of human tissues, including the epithelium,
occurs in the plasma membrane’s most tightly wrapped lung, muscle, lymph, and hepatocytes, according to some
regions. Cells employ the macropinocytosis process to prior investigations. This implies that medication distri-
absorb certain exosomes. Micropinocytosis is the mecha- bution to diverse target organs is made possible by HEK-
nism by which oligodendrocyte-produced exosomes are derived exosomes. Furthermore, Zhu et al. found that
delivered to microglia [49]. To ascertain if exosomes are giving mice repeated doses of HEK293T exosomes for
internalized for intercellular communication or removed three weeks did not significantly alter the mice’s immune
by phagocytosis, more investigation is required. At last, system or cause harm. Apart from its safety-related char-
it is important to mention that exosomes aid in organo- acteristics, HEK-derived exosomes have the potential to
trophic metastasis, and other crucial oncogenic signals enhance medication delivery and therapeutic efficacy
also contribute to balancing the functioning and selectiv- by supplying cancer cells with membrane proteins. Kim
ity of exosome payloads engaged in this process [50]. et al. [54] used HEK exosomes that had been geneti-
cally altered into “xenogeneic” tumor cells. The authors
Exosome types generated minimal vesicular stomatitis virus glycopro-
The field of research on the creation of effective drug tein (mVSVG)-engineered Exosomes (mVSVG-Exo) by
delivery systems (DDS) has expanded rapidly, and transfecting HEK293T cells with plasmids containing
exosomes that have a high rate of bioabsorption and min- mutant vesicular stomatitis virus glycoprotein (mVSVG).
imal immunogenicity are being targeted as powerful drug The phagocytosis of xenogenized cancer cells by bone
carriers. To enable the drug to cross biological barriers marrow-derived macrophages (BMDMs) and bone
(such as cell membranes [39, 41], efflux transporters, and marrow-derived dendritic cells (BMDCs) was enhanced
metabolic enzymes) and having high bioavailability in by mVSVG-Exo. Furthermore, it was discovered that
target regions is the primary goal of the DDS. Depending exosomes expressing therapeutic membrane proteins
on the kind of exosome source, different physicochemi- could enhance tumor penetration and antitumor activity.
cal characteristics may influence the pharmacokinetics of The fundamental component of the tumor microenviron-
the material [51]. As a result, it’s critical to research how ment, the tumor extracellular matrix (ECM), is degraded
exosomes’ diverse biochemical characteristics originate by native PH20 hyaluronidase-expressing exosomes
from distinct sources. This section will be devoted to derived from HEK293T cells, which inhibits tumor
classifying the many exosome kinds that come from vari- growth. These findings are demonstrated in experimental
ous sources [52]. studies by Hong et al. Furthermore, in the tumor-bearing
Basyoni et al. Cancer Cell International (2025) 25:150 Page 13 of 32

mice model, co-administration of doxorubicin (Dox) Stem cell‑derived exosomes

and PH20 demonstrated significantly higher anticancer One of the cell types known to secrete exosomes is also
effects in comparison to Dox-only delivery groups [55]. thought to be a perfect source to manufacture exosomes
for therapeutic application because they can be isolated
Cancer cell‑derived exosomes from a range of human tissues and have a high growth
Cancer cells are also thought to be effective exosome potential. The Kalluri group created a scalable isolation
makers such as subtypes of Ras-associated binding (Rab) method to produce exosomes of good manufacturing
proteins (Rab27a and Rab27b), which are involved in practice (GMP) grade from MSCs produced from bone
the exosome release mechanism. A trait that sets them marrow for therapeutic applications [62]. Furthermore,
is their affinity for their parent cells. Exosomes of tumor therapeutic siRNA targeting oncogenic Kirsten rat sar-
cell lines HT1080, and HeLa, a cervical cancer cell line coma virus (KRAS) can be delivered via GMP-grade
were extracted by Qiao and colleagues, who also found MSC exosomes. The tumor size and metastasis level
that HT1080 exosomes exhibited twice as much absorp- in the tumor-bearing mice model were considerably
tion in HT1080 cells as HeLa exosomes. In addition, reduced [63].
the researchers used HT1080 exosomes that were drug-
loaded with anticancer drugs to perform an in vivo effi- Red blood cell‑derived exosomes
cacy test [56]. They discovered that this resulted in a Exosomes essentially contain a wide variety of biological
considerably higher concentration of HT1080 exosomes components, including lipids, proteins, and nucleic acids,
at the HT1080 tumor location when compared to HeLa that are specific to their parent cells. Exosomes that have
exosomes. Exosomes produced from cancer cells have been separated from bodily fluids such as blood plasma,
demonstrated encouraging potential as drug delivery sys- urine, and amniotic fluids have thus been used in the
tems; nevertheless, there are still several issues that need diagnosis of several illnesses [64]. Exosomes generated
to be resolved before using them to treat cancer. First, the from red blood cells, one of these several bodily fluids,
pharmacokinetic profile of naive exosomes produced by have been utilized to deliver treatments based on nucleic
cancer cells is not optimal. Second, while employing can- acids. They were proposed as a flexible delivery vehicle
cer exosomes as medication carriers, it is important to for therapeutic RNAs in the Le group’s prior investiga-
carefully evaluate any potential side effects, as numerous tion. Exosomes generated from red blood cells offer many
studies have suggested that cancer exosomes may play a benefits for use in clinical settings. In a nutshell, blood
role in tumor spreading. Exosomes obtained from cancer units, which are the source of exosomes, are easily acces-
patients should be a useful tool for treating the disease if sible from blood banks and patients as needed. Because
the flaws mentioned above are fixed [57–60]. each blood unit contains a comparatively large volume
of red blood cells (~ 1012 cells/L), there is less chance of
Immune cell‑derived exosomes unanticipated in vitro mutations during cell cultivation.
T cell-mediated immunotherapy frequently uses den- RBCs are enucleated cell types, as opposed to other cell
dritic cells (DCs), which are utilized to convey tumor types that have a nucleus. This implies that exosomes that
antigens to naive T cells. However, DCs have a short have been separated from red blood cells are not suscep-
half-life following activation. Nevertheless, because DC- tible to dangers associated with genes, such as horizon-
derived exosomes (DEX) retain the immune stimulation- tal gene transfer. RBC-derived exosomes can be made
related capabilities of their source, DEX is suggested as a to avoid triggering harmful and immunogenic reactions,
crucial molecule to supplement the shortcomings of DC- just like blood transfusions can. Through the matching of
based immunotherapy [61]. First, the exact molecular donors’ and recipients’ blood types. A better transfection
makeup of each patient’s DEX determines the molecular efficiency is offered by RBC exosomes [65].
criteria for biological materials quality control. Second,
the DEX surface expresses the ligand peptides that stimu- Food‑derived exosomes
late NK cells. Furthermore, compared to DC which ena- Apart from the customary constraints of exosomes pro-
bles DEX therapy to last up to six months with just one duced from cells, such as low yield and potential for
leukapheresis. DEX extracted from immature DCs gener- triggering immunogenicity [66], the restricted adminis-
ally displayed a lack of expression of immunostimulatory tration method is proposed as an area for enhancement.
(e.g., CD86), which can avert unanticipated immunologi- In light of these advantages, food such as milk and edible
cal reactions brought on by the activation of naive T cells plants is suggested as a source of exosomes for use in
[53]. clinical settings [65].
Basyoni et al. Cancer Cell International (2025) 25:150 Page 14 of 32

Exosomes derived from milk Ultracentrifugation

Milk is a sort of bodily fluid that adults as well as babies It is said that the traditional and gold-standard technique
drink because it includes numerous elements that stim- for separating exosomes is ultracentrifugation. Centrifu-
ulate growth. Specifically, milk derived from cows is gal force is used in this technique to condition biological
looking more and more like a viable alternative to exo- fluids or cell culture conditions to remove big cell debris
some sources because it can be produced in large. The and cells based on their size, density, and form [72]. The
primary benefit of exosomes generated from milk is following is an experimental methodology for ultracen-
that, because of their durability in low pH stomach cir- trifugation described by Théry et al. to collect and iso-
cumstances, they can effectively transfer medicinal com- late exosomes: To extract or separate the live cells, the
pounds that have been encapsulated through the mouth culture-conditioned media is first centrifuged at 300×g
canal. Through binding in the upper gastrointestinal for 10 min. After the collected supernatant has been cen-
tract, exosomes generated from bovine milk can induce trifuged for 10 min at a centrifugation force of 2000×g to
cross-species transit via the conserved IgG-neonatal precipitate the dead cells, the supernatant is centrifuged
Fc receptor (FcRn). According to Agrawal et al., oral for 30 min at 10,000×g to remove cell debris and then for
administration of paclitaxel-loaded exosomes (ExoPAC) 70 min at 100,000×g to precipitate the exosomes. To get
significantly reduced tumor growth in the tumor-bear- rid of the tainted proteins, a lot of phosphate-buffered
ing animal model while having no negative impacts on saline (PBS) is used to wash the collected pellet. The
immune responses or systemic toxicity [67]. Apart from resulting solution is centrifuged at 100,000×g for 70 min.
biocompatibility and safety considerations, post-isola- The effects of centrifugal force on exosomes can cause
tion modification can also be used to functionalize milk the exosome membrane to rupture, even though ultra-
exosomes. Using polyethylene glycol (PEG), a research centrifugation is a well-established technique for isolat-
team under the direction of Bajpayee et al. has modified ing exosomes [73].
milk exosomes to enhance their integrity in acidic gastro-
intestinal conditions. As predicted, the mucus permeabil-
Density gradient ultracentrifugation
ity of PEG-modified milk exosomes was around 3.2 times
The foundation of this technique is a rise in the solution
higher than that of untreated milk exosomes [68].
density gradient from the tube’s top to bottom. Contami-
nants whose densities differ from that of exosomes will
be layered after centrifugation, and exosomes will settle
Edible plant‑derived exosomes into other layers that correspond to their density. The
Plant-derived exosomes (PDEs) are also regarded as pro- most widely utilized gradient media for exosome isola-
spective options as an exosome source because they are tion are sucrose, iodixanol in water, and ice-cold PBS.
obtained from plants and can be modified with func- A theoretically pure fraction of exosomes can be pro-
tional moieties like folate [69]. Exosomes extracted from duced by separating impurities whose density differs
plants can also be delivered orally. First, it has been docu- from that of the exosomes using the gradient density of
mented that PDEs in and of themselves protect against sucrose. When compared to other physical exosome iso-
inflammatory diseases. Ju et al., for example, discovered lation techniques, density gradient ultracentrifugation is
that grape exosomes can regulate intestinal homeostasis thought to be among the best because of its purity, yield,
and provide protection against colitis produced by dex- and ability to preserve vesicular structure. Nonetheless,
tran sulfate sodium (DSS) when taken orally [67]. Even it has been noted that low-density lipoprotein (LDL) and
though the in vivo experiments in this study were carried high-density lipoprotein (HDL) might contaminate the
out via intravenous injection, given PDE’s therapeutic exosome-containing fraction. The ultracentrifugation-
properties following oral administration [69]. derived exosomes have the potential to disrupt non-exo-
some microvesicles such as apoptotic bodies and protein
aggregates. Ultracentrifugation with a density gradient
Isolation of exosomes can be used to get around this [74].
It is essential to separate exosomes from interfering
chemicals and fragments of cells to achieve ultrapure
exosomes. Exosomes can be isolated using a variety of Ultrafiltration
methods, such as polymer precipitation, size exclusion Standard membrane filters with specified molecular
chromatography (SEC), filtration, and differential centrif- weight or size exclusion criteria can be used to sepa-
ugation [70]. The methodologies, mechanisms, benefits, rate exosomes from other extracellular vesicles, solu-
and drawbacks of exosome isolation techniques are dis- ble protein aggregates, and cell detritus. Exosomes can
played in Table 4 [71]. be separated based on their size because they are tiny.
 Basyoni et al. Cancer Cell International

Table 4 Isolation methods of exosomes

Isolation methods Principle Advantages Disadvantages References

Size exclusion liquid chromatography Separation is based on size using porous Preserves exosome integrity; separates Limited throughput; lengthy process [76]
polymeric beads; large molecules elute first exosomes from lipoprotein and protein
while smaller ones enter pores contaminants
(2025) 25:150

Density Gradient Ultracentrifugation Separates components based on differ- High purity; preserves vesicular structure Time-consuming; requires expensive [74]
ences in density within a sucrose or iodix- equipment
anol gradient
Ultracentrifugation Uses centrifugal force to isolate exosomes Capable of producing large quantities; Potential structural damage to exosomes; [71, 72]
based on size, density, and shape widely used contamination with protein aggregates
or microvesicles
Ultrafiltration Filters exosomes using membranes Faster and simpler than ultracentrifugation; Risk of membrane fouling; limited size- [72]
with defined molecular weight cutoffs suitable for large-scale isolation specific separation
or pore sizes
Immunoaffinity capture-based techniques Uses antibodies immobilized on beads High specificity for target exosomes; effec- Expensive; not suitable for bulk isolation [79, 80]
or surfaces to capture specific exosome tive for low-abundance samples
surface markers
Microfluidic technologies Combines physical (size, density) and bio- High sensitivity; allows high-throughput Requires specialized equipment; limited [72, 81, 82]
logical (surface marker) properties for exo- analysis and automation scalability
some isolation
Precipitation technique Utilizes polymers (e.g., polyethylene glycol) Simple, inexpensive, and scalable Co-precipitate non-exosome contaminants [77, 78]
to reduce exosome solubility and precipi-
tate vesicles
Page 15 of 32
Basyoni et al. Cancer Cell International (2025) 25:150 Page 16 of 32

Ultrafiltration is commonly employed as the last stage samples are co-incubated with a PEG solution (molecu-
in chromatography and as a step after ultracentrifuga- lar weight: 8000 Da). After being incubated at 4 °C for
tion. The following is the standard ultrafiltration proto- an entire night. This approach is comparatively simple
col: Tangential flow filtration (TFF) of the filtrate through to use and does not take a lot of running time [77]. It
a filter with a molecular weight cutoff of 500 kDa at a also does not require specific equipment. According to
process temperature of 4 °C; dead-end filtering with Kanchi et al., the polymeric networks of Tamm-Horsfall
a 0.1 mm filter at 22 °C to extract floating cells and cell protein can be removed or separated by employing a dl-
wastes from the cell supernatant of cells; additional filtra- dithiothreitol solution to isolate exosomes from urine
tion of the deposits from step 2 using a sterilized 100 nm fluid using the precipitation approach. Exosomes were
filter. Direct flow filtration or tangential flow filtration then precipitated at 25 °C for 30 min using a 10,000 cen-
can be used for ultrafiltration. Nevertheless, it has draw- trifugal force. Exosomes separated using the precipita-
backs, including membrane fouling and poor particle tion approach are more easily recovered and resuspended
separation. Large-scale exosome isolation can be accom- than those isolated using the ultracentrifugation method
plished more quickly, easily, and efficiently with tangen- [78].
tial flow filtration also referred to as crossflow filtering.
To prevent clogging or cake formation, the sample fluid Immuno‑isolation
in TFF travels tangentially across the filter membrane. To Using magnetic beads covered with antibodies, the
remove impurities smaller than 500 kDa, the retentate is immune-isolation or immunoaffinity approach distin-
further serially reconcentrated using TFF [75]. The puri- guishes between certain proteins on the lipid bilayer
fied exosomes are kept at 80 °C in 0.1 M sucrose. After membrane of exosomes and other substances. Accord-
ultrafiltration is used for isolation, it can be supple- ing to reports, exosomes from tumors, human exosomes,
mented with additional methods like chromatography to and acute myeloid leukemia blasts are frequently iden-
improve the quality of the exosomes. tified using biomarkers like CD34, CD63, and CD326.
Exosomes can be isolated using an immunoaffinity isola-
Size exclusion chromatography (SEC) tion kit on the tetraspanin proteins and exosome surface
Depending on the size of the exosome, big molecules and indicators, which are thought to be determining factors
other particulate materials are separated in SEC by using for the immune isolation technique [79]. The immune-
a porous stationary phase. The sample of interest’s small isolation approach is better at selectively catching small
hydrodynamic radius components can pass through the amounts of plasma than ultracentrifugation. As a result,
pores, causing late elution. On the other hand, early elu- it is frequently employed to further isolate the particu-
tion results from components (exosomes) having a com- lar Exosomes that have already been separated using
paratively higher hydrodynamic radius finding it difficult alternative methods. However, this methodology can be
to enter the pores. Transmission electron microscopy employed for the extraction of exosomes specific to the
has verified reports that exosomes isolated from the SEC individual biomarkers [80].
can retain their exosome structure. Moreover, unlike the
centrifugal approach, shear force won’t compromise the Chip isolation techniques derived from microfluidics
structure and integrity of the exosomes after their sepa- Microfluidics-based chip isolation approaches have
ration from the SEC [76]. This is because SEC may be emerged as a viable method for exosome separation in
carried out at low pressure, preserving exosome integ- recent years [81]. These techniques rely on the distinc-
rity while they are isolated. This method mostly aids in tions between the exosomes’ physical and biological
the extraction of lipoprotein or protein contaminants characteristics, including their size, density, and immu-
from the extracted exosomes. This procedure has also noaffinity. Three methodologies may be distinguished
been applied as an ultrafiltration and ultracentrifugation between the purification and separation processes that
method’s subsequent isolation technique. However, the use microfluidics-based chip isolation techniques: the
SEC technique’s applicability for sample isolation is lim- immunoaffinity-based exosome trapping strategy, the
ited by its comparatively long operating time. sieving approach, and the exosomes being adsorbed into
the porous structure approach [72]. The sample prepara-
Precipitation technique tion processes for all three approaches require off-chip
This method uses hydrophilic polymers, including poly- operations, which increase processing complexity. This
ethylene glycol (PEG), to precipitate exosomes based method selectively entraps exosomes with a size range of
on charge. By stealing the water molecules and forcing 40–100 nm, and the exosomes have high selectivity, espe-
exosomes, PEG reduces the solubility of exosomes. In a cially when used in conjunction with the microfluidic-
nutshell, exosomes precipitate when exosome-containing chip-based immunoaffinity capture method. The clinical
Basyoni et al. Cancer Cell International (2025) 25:150 Page 17 of 32

market entrance of this technology may be restricted, Calcium chloride (­ CaCl2)

nonetheless, by the need to economically produce and Calcium ­(Ca2+) stands out as a prominently researched
efficiently separate the exosomes in large enough quanti- element known to influence the release of extracellu-
ties [82]. lar vehicles [87]. The effects of intracellular and external
­Ca2+ concentrations on exosome secretion in various bio-
Promising strategies for high exosome scalability logical settings have been the subject of several studies.
The intrinsic complexities of exosome production gift More specifically, research investigating the incubation of
hurdles that prevent smooth scaling. The problematic glioblastoma cells with increasing ­CaCl2 concentrations
nature of cell processes concerned with exosome biogen- found that the emission of EVs from the tumor increased
esis, coupled with the want for excessive purity and yield, in tandem, highlighting the stimulatory effect of elevated
demands tailored solutions to propel exosome manufac- extracellular ­CaCl2 levels [88].
turing performance to new heights. A comprehensive,
multifaceted approach is needed to address the scaling Cytokines
issues. In contemporary biomedical research, the utilization of
cytokine pretreatment has emerged as a standard prac-
Pretreating manipulations tice to augment both the yield and therapeutic efficacy
Pretreat parent cells is stimulating exosome production of exosomes, thereby holding significant promise in the
through different environmental stimulators or medium realm of brain cancer therapeutics. Interestingly, a com-
composition, such as pH, oxidative stress, and glucose parison of untreated MSC-derived exosomes and IFN-γ-
starvation [83]. pretreated MSC-EXO revealed that the latter had better
effects in preventing peripheral blood mononuclear cell
Hypoxia and oxygen stress proliferation, perhaps due to the action of indoleamine
Several studies have described the fascinating phenom- 2, 3-dioxygenase (IDO). Alongside this improvement,
enon whereby mesenchymal stem cells (MSCs) secrete pro-inflammatory cytokines were decreased, Treg induc-
more exosomes and enhance the therapeutic effects of tion was facilitated in vitro, and neuroinflammation and
exosomes in hypoxic environments. Hypoxia-treated demyelination in experimental autoimmune encepha-
MSC-derived exosomes (MSC-EXO) demonstrated lomyelitis (EAE) mice were lessened. Furthermore, sev-
greater amelioration of myocardial infarction com- eral studies highlight how cytokine-induced changes in
pared to untreated MSC-EXOs, according to studies exosome payloads might improve treatment results in
conducted in an infarcted heart model. This was dem- the setting of brain cancer [89]. Investigating how IL-1β
onstrated by reduced myocardial apoptosis, reduced pretreatment affected BMSC-derived exosomes (BMSC-
fibrosis, and increased vascular density. The elevation of EXO) showed that miR-146a expression was upregulated,
vascular endothelial growth factor (VEGF) expression which caused macrophages to differentiate into an anti-
and the promotion of angiogenesis were also linked to inflammatory M2 phenotype and therefore reduce sepsis.
the hypoxic environment [84]. Likewise, in osteoarthritis SW982 cells, IL-1β-primed
Additionally, after ethanol-induced stimulation of reti- MSC-EXOs demonstrated enhanced anti-inflammatory
nal pigment epithelial cells, oxidative stress promotes the capabilities, mostly due to miRNAs such as miR-147b
production of tiny extracellular vehicles (EVs). Notably, that functioned by blocking the Nuclear factor kappa β
these circumstances were linked to an increase in the (NF-κβ) pathway [90].
released EVs’ vascular endothelial growth factor receptor
mRNA cargo [85]. The interaction between inflamma- Genetic manipulation
tion and the tumor microenvironment was highlighted The total amount of exosomes produced can be greatly
as a significant component influencing the kinetics of impacted by altering important genes involved in exo-
EV release, in addition to hypoxia and oxidative stress. some synthesis and recycling. The primary issue with
Dendritic cells treated with Interferon-γ (IFN-γ) showed this approach is the degree to which the exosomes gener-
increased production of exosomes and tiny EVs in the ated in the modified routes differ from those generated
setting of inflammation. These exosomes were enriched in the non-manipulated pathways; more investigation
with miRNAs linked to oligodendrocyte re-myelination. is required to elucidate these distinctions. Addition-
Furthermore, a large body of data indicates that tumor ally, some scientists hypothesize that the biological roles
cells coordinate significant extracellular vesicle secretion, of these recently generated exosomes are comparable
in which these vesicles carry a variety of protein cargos to those of natural exosomes. To influence cellular exo-
and tumor-specific antigens, therefore promoting tumor some secretion, genetic engineering approaches target
growth [86]. important genes involved in exosome biosynthesis (e.g.,
Basyoni et al. Cancer Cell International (2025) 25:150 Page 18 of 32

Signal-transducing adaptor molecule (STAM1), Tumor precipitation method, the microfluidic device demon-
susceptibility gene 101 (TSG101), CD63, CD82, CD9, strated comparable exosome sizes and purity levels.
hepatocyte growth factor-regulated substrate (Hrs), and However, notably, exosomes isolated through the micro-
HSP70) and release [e.g., the Rab family, Vesicle Asso- fluidic device exhibited an earlier miRNA detection com-
ciated Membrane Protein 7 (VAMP7), VAMP8, Syn- pared to those obtained through the PEG-based isolation
taxin-5 (STX5), and synaptosome-associated protein-23 technique. This underscores the potential of microfluid-
(SNAP23)]. The goal is to comprehend the distinctions ics in expediting exosome analysis and underscores its
between natural and genetically modified exosomes. efficacy in enhancing biomarker detection speed and
Using certain genes such as six‐transmembrane epithe- accuracy [95].
lial antigen of the prostate 3 (STEAP3), syndecan-4, and
L-aspartate oxidase, Kojima et al. developed the EXO Exosomes drug loading techniques
device for effective exosome manufacturing, increas- Exosome acts as a natural defense against cargo destruc-
ing output by 40 times without changing the size of the tion during blood circulation. However, medication
exosomes [91]. loading into exosomes is difficult due to its endogenous
composition and lipid bilayers. In general, the medi-
3D‑Culturing cine can be sorted into exosomes using both passive and
Emerging evidence indicates that 3D-derived exosomes active loading techniques (Table 5) [96]. Active load-
(3D-EXO) exhibit higher abundance and activity com- ing, sometimes referred to as remote or post-drug load-
pared to 2D-derived exosomes (2D-EXO), leading to ing, involves incubating the medication with separate
enhanced therapeutic effects By supporting the paracrine exosomes. Exosomes sorted by drug are secreted from
actions of mesenchymal stem cells and increasing mac- donor or source cells that have been pretreated. This pro-
rophage recruitment and polarization towards a healing- cess is known as passive drug loading, or the preloading
enhancing phenotype, the use of macroporous or fibrous approach. Adding medication to the exosome vesicle is
scaffolds can eventually advance tissue regeneration and not necessary with this technique. Because of its active
repair [92]. Notably, 3D-EXO was produced at a rate that pumping mechanisms, the active loading technique is
was 7.5 times greater than 2D-EXO by cultivating cells in more successful in achieving a greater drug/vesicle ratio.
a hollow-fiber bioreactor. Additionally, 3D-EXO showed Hydrophobic medicines respond better to the post-load-
improved efficacy in encouraging cartilage regenera- ing strategy than hydrophilic ones [97].
tion. Additionally, 3D-EXO has demonstrated improved
results in several applications, such as improving mem- Passive loading method
ory and cognitive impairments in mice with Alzheimer’s Incubate exosome with drug
disease, protecting the kidneys, increasing the osteogenic In the passive drug incorporation technique, the drug
capacity of BMSCs, promoting cell migration and prolif- and exosomes are incubated together. Because hydropho-
eration, and preventing apoptosis. The distinctive spheri- bic pharmaceuticals can interact with the lipid bilayer,
cal structure of cells induced by 3D culture reshapes the the efficiency achieved with this approach is directly
cellular microenvironment, augmenting exosome activity correlated with the hydrophobicity of the drug mol-
and yield. Because of the restricted oxygen supply, grow- ecules. In one investigation, Dongmel et al. incubated
ing BMSC in 3D spheroids causes a hypoxic condition in curcumin-treated mouse lymphoma-derived exosomes
the center, which eventually increases exosome secretion in PBS for five minutes at 22 °C. The mixture was sub-
and highlights the potential of 3D culture settings for sequently centrifuged using a different sucrose gradi-
improving exosome functioning [93]. ent. When compared to free curcumin, the solubility,
stability, and bioavailability of curcumin were increased
Utilization of microfluidic devices upon encapsulation into exosomes. Similar to this, Vash-
The integration of microfluidics offers a promising solu- isht et al. found that a loading efficiency of 70.46% was
tion to address these challenges by enabling streamlined obtained when curcumin was incubated with exosomes
isolation and purification processes, leading to enhanced [98] (Fig. 8).
scalability and operational efficiency [94]. Utilizing a dou-
ble filtration microfluidic device that leverages size exclu- Incubation of drugs with donor cells
sion principles allows for rapid isolation of exosomes in This method involves treating the targeted exosome
point-of-care (POC) settings. This innovative device can donor cells with an interesting pharmacological mole-
effectively extract exosomes from 50 to 100 μL of plasma cule, after which the pretreatment cells release exosomes
within a mere 50-min timespan [80]. In a comparative that contain the drug. Using this method, the goal for
study with the conventional polyethylene glycol-based decent cells is to gather medicinal or bioactive substances
 Basyoni et al. Cancer Cell International

Table 5 Exosomes drug loading techniques

Type Drug loading methods Advantage Disadvantage Principe References

Passive loading Incubation of exosomes Straight forward process. Does not jeopard- Loading effectiveness. Medications may be Cargo diffusion across an exosomal mem- [98, 99]
and free drugs ize the integrity of the membrane cytotoxic brane or inside a cell
(2025) 25:150

Incubation of tumor cells

with free drugs
Passive loading Sonication Greater loading capacity compared Sonication-induced membrane damage Formation of micropores using physical [100]
to the basic incubation technique is a roadblock for large-scale applications shear stress for diffusion
Active loading Extrusion High effectiveness of freight loading. Exosomes’ immune-privileged status may Membrane recombination [101, 102]
Consistent extrusion results in a uniform be jeopardized by recombination of their
mixture of cargo-loaded exosomes surface structure, which would reveal
exosomes to immune cells
Active loading Freeze–thaw cycles Easy and efficient method for directly load- Exosome accumulation and protein degra- Membrane fusion [103]
ing several cargos into exosomes dation may result from repeated
cycles of freezing and thawing
Active loading Electroporation High effectiveness of packing The principal constraints include loading Formation of micropores for the electrical [104, 105]
efficiency and baggage aggregation field’s dispersion
Active loading Membrane permeabilizers Greater loading capacity when com- Because saponin is hemolytically active Dissolves cholesterol-containing [106, 107]
pared to the straightforward incubation in vivo, its toxicity at higher concentrations, membrane molecules and leaves pores
technique when utilized for drug loading, is con- on the surface of exosomes
strained
Page 19 of 32
Basyoni et al. Cancer Cell International (2025) 25:150 Page 20 of 32

Fig. 8 Exosomal drug loading by passive method

and release exosomes that can hold those substances. Sonication

Because this technique is not targeted, the yield of A medicine or protein of interest is combined with
exosomes may be minimal. Pascucci et al. administered exosomes produced from donor or target cells, and the
and cultured SR4987 mesenchymal stromal cells for a full mixture is sonicated using a homogenizer probe. The
day at a low dose of paclitaxel. Following that, the cells integrity of the exosome membrane is perturbed by the
were cleaned and reseeded in a brand-new flask with shearing force produced during sonication, which also
fresh media [99]. The paclitaxel-loaded exosomes were deforms the membrane and permits the diffusion of
separated and extracted from the cell-conditioned media bioactive substances into the exosome. According to
during a 48-h culture period (Fig. 8). Kim et al., sonication dramatically reduces the exosome
membrane’s micro-viscosity. Nevertheless, this mem-
Active drug loading approaches brane deformation mechanism does not affect the lipid
To facilitate the easy diffusion of active cargo into the contents or membrane-bound proteins of the exosome.
vesicles, active drug loading entails momentarily dis- It was discovered that when incubated at 37 °C, the exo-
rupting the exosome membrane. Extrusion, freeze–thaw some’s membrane integrity can be restored in less than
cycles, and sonication are some of the methods utilized an hour. Furthermore, when the medications are con-
to damage the exosomes’ membranes. The active drug tained within the exosomes and also adhered to the outer
loading strategy enhanced the drug loading capacity by membrane layer of the exosome vesicle, biphasic drug
up to 11 times when compared to passive drug loading. release is occasionally seen from the exosomes [100].
The primary issue with this strategy is that during the Drugs that are enclosed inside exosomes release slowly,
membrane disruption process, it may harm exosomes’ while drugs that are connected to the outside layer of
original structure and target characteristics. exosomes explode explosively.
Basyoni et al. Cancer Cell International (2025) 25:150 Page 21 of 32

Extrusion hydrophilic substances into exosomes by 11-fold com-

Extrusion is a post-loading technique that loads drugs pared to the inactive loading approach without saponin.
using a lipid extruder based on a syringe. After being This method should be used to isolate exosomes after
separated from the donor cells, exosomes are combined saponin incubation and to determine the optimal amount
with a specific medication and put into a lipid extruder of saponin for drug addition [107].
that is syringe-based and has a porous membrane (100–
400 nm) at a temperature that is controlled. The medica- Cancer treatment via exosome cargo loading
tion is thoroughly combined with the damaged exosome Anti‑cancer drugs
membrane during the extrusion process. The benefits of It has been observed that both hydrophilic and hydro-
the extrusion technique for drug-loading exosomes were phobic chemotherapeutic medications, such as pacli-
documented by Fuhrmann et al. Porphyrin was loaded taxel (PTX) and Dox, are loaded into exosomes. A
into exosomes made from MDA-MB231 breast cancer growing body of research has demonstrated that
cells utilizing the extrusion technique. The cytotoxic chemotherapeutic administration via exosomes can
effect of extrusion loading was higher than that of the augment anti-cancer effects. One of the best anti-
incubation approach [101]. Moreover, the zeta potential cancer medications, Dox, is used to treat lymphoma,
of the initial exosomes is changed by the extrusion pro- solid tumors of various kinds, and leukemia. However,
cess, and the modification of the vesicle constitution is because of its low biocompatibility and severe side
brought about by a higher number of extrusions which effects such as suppressing bone marrow function and
can aid in the efficiency [102]. cardiac toxicity, the therapeutic usage of Dox is highly
limited. While numerous attempts are being made
Freeze–thaw cycles to improve Dox’s biocompatibility and anti-cancer
When utilizing the freeze–thaw method for drug load- properties using different nanoparticle technologies,
ing, exosomes are incubated with a specific medica- there are certain adverse effects related to nanoparti-
tion at room temperature for a predetermined duration cles that need to be addressed, namely oxidative stress
before being quickly frozen at – 80 °C or in liquid nitro- and immunological response [108, 109]. Dox has been
gen. After that, the mixture is left to thaw at the ambient thoroughly investigated in exosome-mediated anti-
temperature. For the best drug encapsulation, cycles of cancer therapy because of its inherent fluorescence,
freeze–thaw should be performed a minimum of 3 times. which makes it easy to follow. In the colon adenocar-
Comparing this procedure to sonication techniques cinoma mouse model, exosomes produced by serial
reveals a reduced drug-loading capacity [103]. extrusion from macrophages pretreated with doxoru-
bicin exhibit stronger anti-cancer effects than groups
Electroporation of liposomes loaded with Dox or free Dox. Exosomes
Using an electric field during the electroporation process have a far higher capacity to target cancer cells than
causes the phospholipid bilayer of the exosomes to rup- liposomes because of their enhanced method of endo-
ture, allowing drug molecules to enter the lumen of the cytosis by cholesterol and the phospholipid makeup of
exosomes and form holes more easily [104]. Drug mole- their membranes [110]. Dox-loaded exosomes lessen
cules permeate via the pores that are created on the lipid cardiotoxicity, a common side effect of Dox, by pre-
bilayer membrane of the exosome after electroporation; venting Dox from reaching cardiac endothelial cells.
in the meantime, the membrane’s integrity is restored More recently, it was discovered that the anticancer
following loading. This technique is frequently used to impact of Dox in osteosarcoma may be improved by
load big molecules into exosomes, including nucleotides exosomes made from mesenchymal stem cells. This
(siRNA or miRNA). The limited loading capacity of the could be attributed to mesenchymal stem cells’ prefer-
electroporation technique can be attributed to problems ence for tumor tissues, highlighting the significance of
with exosome instability and RNA aggregation [105]. choosing exosome sources carefully. Another popular
anti-mitotic medication for cancerous tumors like glio-
Incubation with membrane permeabilizers blastoma multiforme and breast cancer is PTX. When a
Exosomal membrane permeability can result from inter- patient develops cisplatin resistance, PTX is frequently
actions between membrane permeabilizers and sur- utilized to overcome medication resistance. PTX’s poor
factants like saponin, which can generate pores. When bioavailability and dose-dependent toxic impact, how-
compared to the incubation approach, the membrane ever, pose a significant challenge to its clinical utiliza-
permeability method can improve the catalase load- tion. Additionally, According to multiple investigations,
ing efficiency into exosomes [106]. A previous study PTX was unable to cross the BBB. Strong anti-cancer
showed that the use of saponin increased the loading of effects were demonstrated by PTX-loaded exosomes,
Basyoni et al. Cancer Cell International (2025) 25:150 Page 22 of 32

which were produced by mesenchymal stromal cells these miR-122-loaded exosomes demonstrated enhanced
that had been pretreated with PTX. Additionally, PTX- anticancer effects.
encapsulated cancer-derived exosomes may specifi-
cally target drug-resistant Cancer stem cells (CSCs), Proteins
enhancing their cytotoxicity against cancer cells. MDR Using exosomes is the most promising method for deliv-
stands for multiple drug resistance, and it is the main ering proteins. Exosomes can be directly loaded with pro-
challenge to effective cancer treatment. It has been teins by physical loading techniques like electroporation,
demonstrated that exosomes can effectively defeat mul- or they can be created by genetically modifying donor
tiple drug resistance (MDR) in cancers. The P-glyco- cells. An interest protein’s gene is transfected into donor
protein drug efflux transporter could be circumvented cells. Consequently, the inserted genes cause the cell to
by PTX-loaded macrophage-derived exosomes [111]. produce proteins that are then secreted into exosomes.
Exosomes made from U-87 MG cells can carry PTX Many cancer cells depend on the anti-apoptotic protein
across the blood–brain barrier and overcome MDR, survivin for their continued survival. This survivin is
improving the therapeutic efficacy against glioblastoma inhibited by the dormant mutant survivin-T34A, which
multiforme. causes cancer cells’ mitochondrial apoptotic cascade
to begin. It was shown that in several adenocarcinoma
of the pancreas cell lines. Because of their advantage in
Nucleic acids transporting bioactive compounds and their physiologi-
Utilizing nucleic acids like DNA and RNA for gene cal significance in the immune system, exosomes offer
therapy is a compelling and exciting approach to treat- significant potential as vaccine vectors [115]. Dendritic
ing cancer. Specifically, to control the expression of cell-derived exosomes (DEX) are one immune system-
genes. Liposomes and inorganic nanoparticles are derived exosome that can elicit immunological responses
examples of nano-based delivery methods that have like that of parental DCs. Tissue-specific antigens, pep-
been designed to transport nucleic acids to tumors tides, and immune stimulants that can trigger the host
while shielding them from endonuclease degradation. immune system’s attack on tumor cells have recently
However, obstacles to delivery efficiency, stability, and been tried to be loaded into DEX. A mouse DC cell line
safety must be removed before these gene delivery was infected with a lentivirus encoding the AFP gene
methods may be used in real-world clinical settings to employ the fetal liver protein-fetoprotein (AFP) as a
[112]. These nanocomplexes are typically produced hepatocellular cancer antigen [116]. Tyrosinase-related
by interaction electrostatics between strongly nega- protein-2 (TRP2) was electroporated or loaded into
tively charged phosphate backbone RNA and positively serum-derived Exosomes using a detergent like saponin
charged carriers. Though it is more difficult to release to improve membrane permeability. Immunotherapy
short RNAs for gene control, the stability is due to can be used to treat cancer by using fluorescently labeled
charge interaction strengthening the protection for exosomes that demonstrate high signals in lymph nodes
RNA. Furthermore, these nanocarriers’ cationic surface and efficiently absorb TRP2-containing exosomes into
charges could be harmful. Therefore, a key component macrophages.
of effective short RNA distribution is striking a balance
between their release and protection [113]. Potential therapeutic strategies for treatment
Exosomes have gained interest recently as gene deliv- Exosomes for stimulating immune response
ery vehicles because of their special qualities that allow An innovative immunotherapy approach involving
them to get around these challenges. Exosomes were the engineering of exosomes generated from a fusion
shown to contain a large number of miRNAs that are between dendritic cells and tumor cells has been crafted.
engaged in intercellular communication, some of which Stimulators of Interferon Genes (STNG) ligands are the
have anti-cancer characteristics. It has been suggested to substances that these specialized exosomes are packed
pre-overexpress candidate RNAs in parental cells to load with. DT-Exo-STING is a delivery system that is intended
desired RNAs into exosomes. To create miR-122 encap- to increase the defense system’s T-cell attack against can-
sulated exosomes, adipose tissue-derived mesenchymal cer cells in particular (Fig. 9) [117].
stem cells were transfected with the miR-122 expression This immunotherapeutic approach is particularly
plasmid. The hepatocellular carcinoma cells’ chemosen- novel when it comes to brain cancer. Traditional cancer
sitivity was enhanced by the miR-122-loaded exosomes, treatments like radiation or chemotherapy can cause
which changed genes like cyclin G1 and metalloprotein- serious collateral harm to the healthy brain because of
ase domain-containing protein 10 [114]. Furthermore, in the delicate and intricate nature of brain tissue. With
the xenograft mouse model, intratumorally injection of precisely engineered chimeric exosomes that activate
Basyoni et al. Cancer Cell International (2025) 25:150 Page 23 of 32

Fig. 9 DC-tumor hybrid cell-derived chimeric exosomes loaded with STING agonists (DT-Exo-STING) delivery system that is intended to increase
the defense system’s T-cell attack against cancer cells

T-cell responses with little effect on surrounding brain the signaling pathways that further activate the dendritic
cells, DT-Exo-STING offers a glimmer of hope by ena- cells’ immunostimulatory functions. This two-pronged
bling the activation of tumor-specific antigens CD8+ T approach is essential to successfully treating tumors.
cell lymphocytes that trigger an effective immune reac- Sustained investigation and clinical studies are required
tion against cancer cells [118]. The personalized DT- to improve this method, maximize its effectiveness, and
Exo-STING nano vaccine was used in conjunction with guarantee patient safety [117].
immune checkpoint inhibitor therapy in a preclinical
investigation that used a mouse model of brain cancer to Exosomes for carrying anti‑tumor drugs
avoid post-operative glioblastoma recurrence. The study’s Numerous immune-related chemicals that control the
results were encouraging, demonstrating that mice immune response are carried by exosomes. Tumor
given the nano vaccine had better survival rates and an necrosis factor-related apoptosis-inducing ligands
increased immune response [119]. (TRAIL) can bind TRAIL receptors like TRAIL-R1 (DR4)
Furthermore, there are two sides to the inclusion and TRAIL-R2 (DR5) within tumor cells, which can trig-
of STING agonists in these exosomes. They not only ger target cell apoptosis. TNF family proteins, such as
encourage the direct activation of T cells but also boost Fas ligands, can fight brain cancer through the Fas/FasL
Basyoni et al. Cancer Cell International (2025) 25:150 Page 24 of 32

Fig. 10 Exosomes for carrying anti-tumor drugs

pathway. Beyond these ligands that cause apoptosis [120], containing metformin and cPLA2 siRNA were evalu-
exosomes can transfer chemotherapy medications like ated for cellular absorption, primary GBM cells showed
Paclitaxel and Doxorubicin (Fig. 10) [121]. encouraging therapeutic benefits. The efficacy of the exo-
some-mediated cPLA2 siRNA/metformin strategy and
Exosomes for carrying siRNA the efficiency of GBM-targeted delivery were assessed
Compared to the existing systemic gene therapy deliv- in vivo in a patient-derived xenograft (PDX) model. Both
ery techniques, exosome-mediated delivery shows great genomic analysis and experimental validation empha-
promise and benefits. When loaded with therapeutic sized the significance of polymerase 1 and transcript
nucleic acids, targeted exosomes effectively and safely release factor (PTRF) in improving exosome uptake by
reduce the rate at which tumors proliferate [122]. This GBM cells, proving the feasibility of this delivery strategy
tactic one possible treatment method for glioblastoma [123].
(GBM) is to target the energy metabolism of the dis-
ease by inhibiting both mitochondrial and phospho- Classification as a therapeutic product
lipid pathways. One novel strategy for treating GBM Exosomes can be classified differently depending on
is to simultaneously target mitochondrial metabolism their origin, composition, and intended use; Biologics:
and cytoplasmic phospholipase A2 (cPLA2) by treat- Exosomes derived from cells (e.g., stem cells) are often
ing it with metformin and knocking down cPLA2. Blood regulated as biologics, requiring compliance with strin-
exosomes were found to be the best vehicles for dis- gent guidelines, such as the FDA’s regulations under
tributing this medicinal concoction. When exosomes the public health service (PHS). Drugs: Exosome-based
Basyoni et al. Cancer Cell International (2025) 25:150 Page 25 of 32

products with a defined pharmacological activity may Regulatory requirements differ across regions (e.g., FDA
be treated as drugs, necessitating preclinical and clinical in the U.S., EMA in Europe, and PMDA in Japan), com-
trials. Cell or Tissue-based Products: If the exosomes are plicating international development. Emerging Stand-
derived from human cells or tissues, additional rules may ards: Regulatory agencies are still developing guidance
apply under the human cells, tissues, cellular, and tissue- on exosome-based therapies. For example, defining what
based products (HCT/Ps) regulations. The classification constitutes “substantial manipulation” or “homologous
determines the regulatory pathway, including preclinical use” in the context of exosome therapies is evolving.
and clinical requirements [124]. Compliance with good manufacturing practices (GMPs):
Manufacturers must meet GMP standards, which require
Manufacturing and quality control robust documentation and validation of processes, facili-
The production of exosome-based therapies presents ties, and personnel [129].
challenges in ensuring consistency and quality as; source
material variability: Exosomes derived from different cell Ethical and legal issues
types or donors may vary in composition and efficacy. Donor material: Ethical concerns arise when exosomes
Scalability: Large-scale manufacturing while maintain- are derived from human donors, including consent,
ing product consistency is a significant challenge. Purity traceability, and the potential for exploitation. Intellec-
and safety: Rigorous methods to purify exosomes and tual property (IP): Protecting IP while complying with
remove contaminants (e.g., residual cells, proteins, or regulatory standards can be challenging, especially for
genetic material) are essential to minimize risks such as novel therapies with overlapping patents [130].
immune reactions or off-target effects. Characterization:
There is a need for standardized methods to characterize Addressing these challenges
exosomes, including size, content (RNA, proteins, lipids), To facilitate clinical translation, the following strategies
and biological activity. Regulatory agencies often require can help; development of regulatory guidelines: Col-
detailed documentation of the manufacturing process laboration between researchers, regulatory agencies, and
and robust quality control measures [125]. industry to establish standardized guidelines and frame-
works. Standardization of methods: Developing universal
Preclinical studies standards for exosome isolation, characterization, and
To demonstrate safety and efficacy, exosome-based ther- quality control. Education and communication: Enhanc-
apies must undergo rigorous preclinical testing as; bio- ing dialogue between developers and regulators to clarify
distribution and pharmacokinetics: Understanding how expectations and resolve uncertainties early in the devel-
exosomes distribute in the body, their half-life, and clear- opment process [131]. By addressing these regulatory
ance mechanisms is essential. considerations comprehensively, developers of exosome-
Toxicology: Comprehensive toxicological studies are based therapies can navigate the path to clinical applica-
required to assess potential risks, such as unintended tion more effectively.
immune responses or tumorigenicity. Mechanism of
action: Clear elucidation of the therapeutic mechanism is Influence of patient characteristics
often required, though this remains challenging given the on exosome‑based therapies
complexity of exosome content [126, 127]. Exosome-based therapies hold great promise for preci-
sion medicine, as they can be engineered to address spe-
Clinical trials cific pathological conditions. However, patient-specific
Exosome-based therapies face unique challenges in clini- factors, such as genetic variants and pre-existing condi-
cal trial design such as Patient safety: Given their novel tions, may significantly influence the efficacy and safety
nature, extensive safety data are needed to address poten- of these therapies.
tial immunogenicity or adverse effects. Dose standardi-
zation: Establishing an appropriate dosing regimen can Genetic variants and exosome dynamics
be challenging due to variability in exosome content and Patient-specific genetic variations can affect multiple
activity. Endpoints: Clear, measurable clinical endpoints aspects of exosome biology, including Exosome produc-
are necessary, particularly if exosomes are intended for tion: Variants in genes involved in exosome biogenesis,
regenerative medicine or as drug delivery systems [128]. such as those encoding Rab GTPases and tetraspanin,
may alter the quantity or quality of exosomes produced
Regulatory guidance and standards by the patient’s cells [132]. Cargo loading and composi-
There is a lack of comprehensive regulatory frameworks tion: Genetic polymorphisms in regulatory pathways,
specific to exosome-based therapies as; global variability: such as those affecting miRNA or protein expression,
Basyoni et al. Cancer Cell International (2025) 25:150 Page 26 of 32

may influence the cargo packaged into exosomes. For exosomes. These may involve proteins that show signs of
example, single nucleotide polymorphisms (SNPs) in apoptosis or cell damage [138].
miRNA-binding sites could alter the therapeutic efficacy
of miRNA-loaded exosomes [133, 134]. Uptake efficiency: Precision medicine approaches
Variations in receptor genes, such as integrins or lectins, To address these challenges, exosome-based therapies
may affect the binding and internalization of exosomes, can be tailored to individual patient profiles: Genetic
potentially influencing the therapeutic outcomes [135]. screening: Screening for genetic variants associated with
Genetic diseases: Exosome profiles and biological exosome production or uptake could help predict patient
responses may be different in people with underlying responsiveness to therapy. For instance, patients with
medical illnesses, such as cardiovascular or neurologi- altered receptor expression may benefit from exosomes
cal diseases, than in healthy people. For instance, they engineered to target alternative pathways [135]. Condi-
are linked to persistent inflammation, which can lead to tion-specific exosomes: Developing exosomes derived
the development of a pro-inflammatory milieu inside the from patient-specific cell types, such as mesenchymal
brain. During the therapy of brain cancer, this inflamma- stem cells or immune cells, may ensure better compat-
tory environment may change the behavior of exosomes, ibility and therapeutic efficacy [136]. Adaptive engineer-
impacting their stability, cargo content, and interactions ing: Customizing exosome surface markers or cargo
with recipient cells [133, 134]. Furthermore, genetic based on a patient’s disease state and genetic background
mutations affecting components of the endosomal-lys- could enhance targeting and reduce off-target effects.
osomal-exosomal pathways, such as charged multive-
sicular body protein 6 (CHMP6), TSG101, Rab35, and Challenges and perspectives
Rab7A, may influence vesicle cargoes routing, Tau traffic, Because of their special characteristics, including their
degradation, and secretion. These mutations could inter- innate source exosomes present exciting opportunities
fere with the proper sorting and trafficking of therapeutic to improve the administration of medicines. Recogniz-
cargoes within exosomes, thereby compromising the tar- ing the many benefits exosomes offer. It is vital to bear
geted delivery of anticancer agents to tumor cells [136]. in mind that exosomes presently lack approval from the
Food and Drug Administration (FDA) for the treatment
Impact of pre‑existing conditions or diagnosis of any disease. Consequently, their use needs
Pre-existing conditions, particularly those involving to be limited to clinical research or FDA biologics licens-
inflammation or altered metabolic states, can signifi- ing applications.
cantly modify exosome interactions and therapeutic One significant step was taken in 2020 when the FDA
efficacy: Chronic inflammation: Conditions such as released a consumer advisory warning people about the
autoimmune diseases or cancer-associated inflamma- possible risks and unsupported claims related to unregu-
tion can change the composition of circulating exosomes. lated exosome treatments. In particular, individuals in
These “primed” exosomes may compete with therapeutic Nebraska who received unapproved medicines allegedly
exosomes, reducing their effectiveness [135, 137]. Dia- containing exosomes reported experiencing serious side
betes and obesity: Metabolic disorders can alter the lipid consequences. Interestingly, no FDA-approved exosome
and protein composition of exosomes, potentially impair- products are currently on the market; thus, clinical stud-
ing their ability to deliver therapeutic cargo efficiently. ies must be planned, developed, coordinated, and carried
Immune status: Patients with immunodeficiencies or out immediately [139]. Notwithstanding the encourag-
hyperactive immune responses may experience altered ing results of clinical studies, concerns have been raised
biodistribution or clearance of therapeutic exosomes about new data showing that three clinical trials are now
[138]. Environmental contaminants: Exosomes’ pro- underway that focus on altering mesenchymal stem cells
tein composition can change in a variety of ways due to to increase the production of exosomes. However, as
environmental pollutants, which might reveal how cells these studies have not yet been completed, the results are
are responding to stress, inflammation, or unfavorable still pending publication [140].
conditions. For example, exosomes carrying inflamma- The ongoing clinical trials (six trials registered on
tory cytokines including interleukins, tumor necrosis clinical trials.gov, accessed on 23 March 2023) on the
factor-alpha, and chemokines can be released in response utilization of exosomes for the delivery of medicinal sub-
to exposure to pollutants, especially those that have pro- stances have addressed this problem [140]. Concerning
inflammatory properties [137]. As a result, these proteins the employment of exosomes for delivering therapeu-
may intensify immunological and inflammatory reac- tic agents, resolved or rebutted this issue. Nonetheless,
tions, and exposure to toxins that harm cells may result it will be sufficiently tough to assess the therapeutic
in the presence of molecular patterns linked to damage in potential of utilization of autologous or non-autologous
Basyoni et al. Cancer Cell International (2025) 25:150 Page 27 of 32

exosomes for the treatment of brain cancer, investigated Cas9 and then treated them with exosomes loaded with
through experiments on human brain organoids and sgRNA that were recovered from HET293 cells. The
subsequent clinical trial applications [141]. The selection CRISPR/Cas9 system is delivered to MSCs by these
of the exosome source is crucial for therapeutic applica- hybrid exosome-like vesicles, which also successfully
tions. Exosomes generated from tumors have a remark- cleave the target genes. In comparison to traditional
able ability to target cancer cells, and they carry bioactive drug delivery methods, hybrid EMNVs and milk-derived
cargo that may either directly or indirectly encourage the exosomes offer a range of advantages. These advanced
progression of cancer. To address the challenges posed vesicles are derived from natural sources, enhancing bio-
by varied exosome subpopulations in the future, meth- compatibility and reducing the risk of immune reactions
ods for identifying, removing, or adding exosomal com- associated with synthetic nanoparticles. Moreover, they
ponents are essential for exosome-based drug delivery can be precisely engineered to target specific tissues or
for cancer treatment with concern that the uniformity cells, enabling accurate drug delivery while minimizing
of storage conditions has an impact on exosome perfor- off-target effects. Additionally, milk-derived exosomes
mance as well. contain bioactive molecules and genetic material that can
Several techniques, including sonication, transfection, modulate biological processes, providing a natural and
electroporation, and incubation, have been developed potent approach to therapeutic intervention [135]. Addi-
recently to load therapeutic payloads into exosomes. tionally, several studies are attempting to circumvent the
But exosome-cargo-loading methods as they exist now low-yield hurdle by employing exosomes derived from
are insufficient to meet the loading efficiency needed for different diets. It makes sense that the superior cellular
clinical applications. Specifically, there are many restric- absorption efficiency and general safety of these food-
tions on the kind of cargo that may be loaded using the derived Exosomes are drawing attention. Specifically,
basic incubation approach, and its low efficiency makes exosomes generated from milk demonstrated a yield
it unsuitable for use in clinical settings. The cost of mass that was 1000 times higher than those derived from ani-
production should be decreased and the process further mal cell cultures. Furthermore, the intestinal absorption
simplified by transformation techniques. Currently, avail- of milk exosomes administered orally was found to be
able physical treatments like electroporation are the most enhanced.
effective way to incorporate nucleic acids like miRNA Exosomes offer limitless potential as biomarkers for
or siRNA into exosomes. However, new strategies are cancer detection and prognosis, in addition to their
required because this process has the potential to alter use as drug carriers [143]. To help with the clinical uses
the characteristics of exosomes and to cause the aggrega- of exosomes and to investigate their diverse profiles
tion and destruction of charged nucleic acids [142]. Their and roles, a great deal of research has been conducted.
low yields are a significant barrier to the clinical utiliza- Exosomes that are separated from diverse body fluids,
tion of exosomes. The majority of preclinical experimen- like blood, saliva, and urine, have the potential to be used
tal research uses cell culture to produce exosomes. Less as cancer biomarkers since they can detect aberrant cell
than 1 mg of exosomal protein is generated per milliliter physiology. However, the exact identification of distinct
of culture, despite some variations based on the type signals that vary depending on the source presents a sig-
of donor cells. Making exosome-mimetic nanovesicles nificant problem (Fig. 11).
(EMNVs) may be a different tactic to get around this
restriction. Extruding cells through successive microme- Conclusion
ter-sized filtering results in EMNVs. The yield of EMNVs Exosomes represent a promising avenue for targeted
is improved by about 100 times using these serial extru- drug delivery in the treatment of brain cancer. Thus, this
sion procedures, and anticancer medications can be review elucidated the potential of exosome-based thera-
encapsulated at the same time. It is imperative to eluci- pies to revolutionize the way to approach brain cancer
date the alterations in vivo (PK/PD) due to the potential treatment, by leveraging exosomes as nanocarriers, there
impact on the vesicle membrane composition during the is an opportunity to maximize treatment impact while
cell extrusion procedure. Furthermore, research teams minimizing damage to healthy brain tissue. However,
have created hybrid EMNVs known as exosome-lipo- it is important to acknowledge the existing challenges,
some hybrids, which combine exosomes with artificial such as scalability and the intricate interplay between
liposomes. In brief, the following three processes serve as exosomes and the tumor microenvironment. Address-
examples for creating hybrid EMNVs: freeze–thaw, sim- ing these challenges will be crucial for the successful
ple incubation, and extrusion [115]. clinical translation of exosome-based therapies. Overall,
To create hybrid EMNVs, the authors of a work by Lin the potential of exosomes in brain cancer treatment is
et al. loaded liposomes with plasmid vectors carrying immense, and continued research and innovation in this
Basyoni et al. Cancer Cell International (2025) 25:150 Page 28 of 32

Fig. 11 Summary of transportation of drug-loading exosomes through the blood–brain barrier (BBB)

area holds great promise for improving patient outcomes THP-1 Human monocytic leukemia cell line
TRPP2 Transient receptor potential polycystic 2
and quality of life. NK Natural-killer
IL-6 Interleukin 6
Abbreviations
TNF-α Tumor necrosis factor-alpha
TP53 Tumor protein 53
TAMs Tumor-associated macrophages
PTEN Phosphatase and TENsin homolog
TEXs Tumor-derived exosomes
NF1 Neurofibromatosis type 1 genes
TME Tumor microenvironment
CTCs Circulating tumor cells
MSCs Mesenchymal stem cells
L1CAM L1 cell adhesion molecule
DCs Dendritic cells
STAT3 Signal transducer and activator of transcription 3
DDS Drug delivery systems
FasL Fas ligand
EVs Extracellular vesicles
BBB Blood–brain barrier
HEK Human embryonic kidney
JAMs Junctional adhesion molecules
mVSVG Minimal vesicular stomatitis virus glycoprotein
PI3K Phosphatidylinositol-3 kinase
BMDMs Bone marrow-derived macrophages
PA Plasminogen activator
BMDCs Bone marrow-derived dendritic cells
serpin The serine proteinase inhibitor
ECM Extracellular matrix
OXPHOS Oxidative phosphorylation
Dox Doxorubicin
WBR Whole brain radiation therapy
Rab Ras-associated binding
SRS Stereo static radiosurgery
DEX DC-derived exosomes
ROS Reactive oxygen species
KRAS Kirsten rat sarcoma virus
EPR Enhanced permeability and retention
FcRn IgG-neonatal Fc receptor
EE Early endosomal
ExoPAC Paclitaxel-loaded exosomes
ESCRT-0 Endosomal Sorting Complex Required Transport
PEG Polyethylene glycol
ILVs Intraluminal vesicles
PDEs Plant-derived exosomes
MVBs Multi-vesicle bodies
DSS Dextran sulfate sodium
HSP70, HSP90 Heat shock proteins
PBS Phosphate-buffered saline
MHC Integrins myosin heavy chain
LDL Low-density lipoprotein
mRNA Messenger RNA
HDL High-density lipoprotein
miRNA MicroRNA
TFF Tangential flow filtration
UTR​ 3’-Untranslated region
Basyoni et al. Cancer Cell International (2025) 25:150 Page 29 of 32

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