Table 2:

Studies in which EVs have been used for their intrinsic medicinal effects (EVs as drugs).

Study type	EV type	EV source	Parent cell modification	Responsible natural mechanisms / contents	Context/model	Outcomes	Ref.
In vitro, in vivo	EVs (NS)	Endothelial progenitor cells	NA	• ↑ Regeneration • ↑ Angiogenesis	Mouse experimental MI; HUVECs	→ Improved ventricular contractility → Preserved ventricular geometry → Cardiovascular recovery post-MI	(64)
In vitro, in vivo	Exosomes	MSCs	NA	• ↓ Cardiomyocyte apoptosis • ↑ Angiogenesis via altering VEGF, bFGF, and HGF	Rat MI model; NRCMs	→ Improved cardiac function → Myocardium protection → Cardiovascular recovery post-MI	(65)
In vivo	Exosomes	MSCs	NA	• ↓ TNF-α and IL-1β • ↓ Apoptosis • ↓ Atg5 cleavage Contents: A wide range of anti-inflammatory, anti-apoptotic and neuroprotective molecules	Rat RD model	→ Maintaining normal retinal structure	(67)
In vivo	Exosomes	MSCs	NA	• ↓ Inflammatory cytokines • ↓ M1 and ↑ M2 macrophages; Contents: (TLR4/NF-κB)-targeting miRNAs	Diabetic mice	→ Promoted functional recovery in mice with neuropathy → Alleviated neurovascular function	(68)
In vitro, in vivo	Exosomes	MSCs	miR-20a reinforcement using its mimics	• Beclin-I and FAS inhibition > apoptosis inhibition • Improved cardiac function; Contents: miR-20a	Rat IR-induced injury	→ Improved IR-induced injury  Outcomes post-modification:   • Almost full alleviation of the injury	(66)
In vitro, in vivo	Exosomes	MSCs	miR-20b-3p overexpression (transfection)	• ↓ Oxalate-induced autophagy • ↓ Inflammation (ATG7 and TLR4 inhibition) Contents: miR-20b-3p	CaOx-induced rat kidney stone model	→ Protection against kidney stones	(70)
In vitro	EV fraction of total secretome	MSCs	Stimulation via culture condition modification including starvation, IL-1β addition and dexamethasone addition	• Encompassing natural contents of antimicrobial secretome including AAT	To combat lung pathogens	→ Demonstrated antimicrobial efficacy against Gram-negative bacteria → Elastase inhibition → Demonstrated lung regeneration properties  Outcomes post-modification:   • Increased protein content and AAT production   • Increased AAT gene expression	(57)
In vitro, in vivo	Exosomes	Cancer cells	NA	• Presence of immune activating molecules (HSP70, etc.) • Presence of native tumor antigens • Overexpressed receptor and molecules involved in antigen sampling by DCs	Mice and cancer cell lines	→ Efficient cross-presentation of shared tumor antigens → Tumor rejection	(80)
In vitro, in vivo	EVs (NS)	Cancer cells	Transfection with anti-miR-21	• Surface tumor-targeting properties of the T-EVs • Capability of co-delivery  Anti-miR-21	Mice and cancer cell lines: 4T1, HepG2, and SKBR3	→ Efficient targeted delivery to tumor sites	(81)
In vitro, in vivo	Exosomes	MQs & monocytes	NA	• Circumventing elimination of the therapeutic EVs owing to the natural origin of EVs (the host immune system)	Mouse model of PD	→ Efficient accumulation in brains of PD mouse models → Efficient delivery of the loaded agent (catalase)	(85)
In vitro, in vivo	Exosomes	DCs	NA	• Exploiting the natural antigen presentation-related molecules of DC-EVs combined with antigen loading • Cell-free EV-based tumor vaccine	Mouse tumor model	→ Successful CTL priming → Successful tumor eradication	(34)
In vitro; In vivo	EVs (NS)	IC21 macrophages	NA	• Exploiting the rich LFA-1 surface content of MQ-EVs (Abundance of LFA-1 on EVs results in their affinity to inflamed sites with ICAM-1 overexpression)	Batten disease models: LINCL mice and TPP1 enzyme-deficient cells (CLN2)	→ Targeted delivery of the therapeutic EVs to inflamed brain	(87)
In vivo	EVs (NS)	Bacteria (S. aureus)	NA	• Vaccine design exploiting antigenicity and adjuvant properties of microbial EVs	S. aureus lung infection	→ Activation of the Th1 response → Protection against S. aureus-induced lethal pneumonia	(93)
In vitro	Exosomes	Porcine milk	NA	• ↓ Expression of p53, p21, caspase 3 and 9 • Regulation of β-catenin and cycline D1 • ↑ Cell viability • ↓ mRNA levels of p21, fas and Tp53 • ↑ mRNA levels of ZO-1, OCLN and CLDN1	Deoxynivalenol-induced damage/IPEC-J2	→ Decreasing DON-induced damage by promoting cell proliferation and reducing apoptosis	(140)
In vitro	EVs	Human Breast Milk	NA	• ↑ Cellular proliferation after H/R • ↓ Apoptosis after H/R	Necrotizing Enterocolitis/IEC-6	→ Decreased histological damage → Decreased incidence of NEC	(97)
In vivo	P35K EVs P100K EVs	Commercial cow’s milk	NA	• After P35K EVs feeding:  ↑ G-CSF, GM-CSF, IL-7, CCL3, CCL4, IFN-ɣ and IFN-α  ↓ IL-12-p40, IL-23, IL-4 • After P100K EVs feeding:  ↓ IL-3, IL-6, IL-10, IL-12-p40, IL-17 and TNFα ↑ G-CSF, GM-CSF  ↑ M-CSF, GM-CSF, IL-5 and IL-4  ↑ Expression of anti-inflammatory A20  Normalized levels of COX-2 and ZO-1  ↓ Colitis-associated microRNAs: miR-21, miR-29b and miR-125b	Murine colitis	→ Improve colitis via decreasing inflammation	(141)
In vitro	Exosomes	Cow Milk	NA	• ↑ Macrophage proliferation • ↑ β-catenin expression • ↑ p21 and p53 expression • ↓ Cyclin D1 expression	Cisplatin-Induced Cytotoxicity RAW 264.7	→ Protective effect against cisplatin cytotoxicity through boosting immune system and increasing proliferation markers	(142)
In vivo In vitro	EVs	Cow milk	NA	• In vivo:  ↓Serum levels of MCP-1 and IL-6 • In vitro:  ↓ TNF-α and MCP-1  ↑ Expression of GATA-3 (Th2), IL-17 (Th17), and Foxp3	Murine arthritis Splenocytes	→ Ameliorating arthritis via cartilage pathology and bone marrow inflammation	(100)
In vitro	Exosomes	Camel Milk	NA	• Significant anti-proliferative activity • Suppression of migration • ↑ DNA damage • ↑ Caspase-3 activity • Bax upregulation and Bcl2 downregulation • ↓ MDA levels and iNOS mRNA • ↑ SOD, GPX, and CAT activities • ↓ Expression levels of IL1β, NF-κB, VEGF, MMP9	Breast cancer: MCF7	→ Anticancer effect through induction of apoptosis and inhibition of oxidative stress, inflammation, angiogenesis, and metastasis	(101)
In vitro	Exosomes	Porcine Milk	NA	• ↓ Expression of IL-1β, IL-6, and TNF-α • ↑ Cell viability • ↓ mRNA levels of Tp53, Fas, and Caspase-3 • ↓ Phosphorylation of IκBα and NF-κB	LPS-Induced Apoptosis IPEC-J2	→ Decreasing LPS-induced injury by inhibiting inflammation and apoptosis	(143)

EV, Extracellular vesicle; NS, Not specified; NA, Not applicable; MI, Myocardial infarction; HUVECs, Human umbilical vein endothelial cells, MOA, Mechanism of action; AAT, Alpha-1-antitrypsin; NRCMs, Neonatal rat cardiomyocytes; VEGF, Vascular endothelial growth factor; bFGF, Basic fibroblast growth factor, HGF, Hepatocyte growth factor; IR, Ischemia-reperfusion; TNF-α, Tumor necrosis factor alpha; IL-1β, Interleukin-1 beta; TLR4, Toll-like receptor 4; NF-κB, Nuclear factor κappa B; CaOx, Calcium oxalate; HEK293, Human embryonic kidney cell line; HSP70, Heat Shock Protein 70; DCs, Dendritic cells; PD, Parkinson’s disease; MQ, Macrophages; DCs, Dendritic cells; S. aureus, Staphylococcus aureus; Th1, Helper T lymphocyte type 1; IPEC-J2, intestinal porcine enterocytes isolated from the jejunum; IEC-6, intestinal epithelium cell; H/R, hypoxia/reoxygenation; A20, anti-inflammatory protein TNFAIP3; IL, Interleukin; RAW 264.7, Murine macrophage cell line; MCF7, Human breast cancer cells; LPS, Lipopolysaccharide; Ref., References