Mesenchymal stem cell-derived extracellular vesicles as next generation drug delivery platforms

Table 1 Comparative summary of mesenchymal stem cell-derived extracellular vesicle cargo loading strategies

Loading strategy	Typical efficiency	Advantages	Limitations	Clinical feasibility	Representative applications	Ref.
Passive loading (incubation)	Approximately 5%-10%	Simple, preserves EV integrity	Low loading efficiency	Moderate (small molecules)	Paclitaxel loading	[49]
Electroporation	Approximately 15%-25%	Effective for nucleic acids	RNA aggregation risk	Moderate	siRNA delivery	[72]
Sonication	Approximately 20%-30%	High encapsulation efficiency	Membrane damage risk	Moderate	Chemotherapy drugs	[49]
Extrusion	Approximately 15%-25%	Uniform vesicle formation	Structural alteration	Moderate	Drug loading	[84]
Genetic engineering	High cargo specificity	Stable loading	Regulatory complexity	High potential	miRNA delivery	[85]
Surface functionalization	Not direct loading	Improved targeting	Immune response risk	High potential	Targeted therapy	[83]

EV: Extracellular vesicle; siRNA: Small interfering RNA; miRNA: MicroRNA.

Table 2 Mesenchymal stem cell-derived extracellular vesicles as platforms for drug delivery

Engineering/delivery strategy	Therapeutic cargo	Target disease/application	Delivery route	Major advantages over synthetic nanoparticles	Challenges	Ref.
Native MSC-EV therapy	Endogenous miRNAs, proteins	Cardiovascular, renal, liver injury	Intravenous	Natural biocompatibility. Low immunogenicity	Biodistribution to liver and spleen	[9,105]
Surface engineered exosomes	Targeting ligands	Tissue-specific targeting	Systemic	Enhanced homing and target specificity	Manufacturing scalability	[106,107]
CRISPR/Cas9 delivery via MSC-EVs	CRISPR/Cas9 gene-editing components	Gene editing applications	Experimental systemic delivery	Ability to cross biological barriers	Cargo loading efficiency	[108,109]
KRAS G12D siRNA-loaded exosomes (iExosomes)	siRNA	Metastatic pancreatic cancer	Intravenous (clinical trial)	Tumor-specific gene silencing	Standardization and dose control	NCT03608631[110]
MSC-exosomes loaded hydrogel	Exosomes-containing growth factors	Full thickness cutaneous wound healing and skin regeneration	Local/topical application	Sustained release, improved stability and retention enhanced wound closure, angiogenesis	Direct injection of exosomes, need for suitable carrier system, limited prior studies on hydrogel encapsulated exosomes	[97]
Bioreactor-based MSC culture	Bulk EV production	Clinical translation	GMP-scale	Increased yield	Phenotypic variability	[111,112]
MSC-EV spray formulation	Regenerative factors	Myocardial infarction (large animals)	Topical/local cardiac	Minimally invasive administration	Stability and storage validation	[113-117]
MSC-EVs vs synthetic nanoparticles comparison	Endogenous therapeutic cargo	Broad therapeutic use	Multiple routes	Reduced toxicity, better barrier penetration	Heterogeneity and pharmacokinetics	[108]

MSC-EV: Mesenchymal stem cell-derived extracellular vesicle; miRNA: MicroRNA; siRNA: Small interfering RNA; GMP: Good manufacturing practice.

Table 3 Therapeutic and mechanistic roles of mesenchymal stem cell-derived extracellular vesicles relevant to drug delivery applications

Disease condition	MSC source	Major EV cargo	Mechanistic pathways modulated	Therapeutic outcome	Experimental model	Ref.
Acute myocardial infarction	Bone marrow MSC-EVs	miR-125b	Suppression of cardiomyocyte apoptosis	↑ Cardiac repair	Mouse	[164]
Myocardial infarction	Bone Marrow MSC-EVs	miR-146a-5p	IL-1 receptor-associated kinase 1/NF-κB signaling	↓ Inflammation, ↑ cardiac function	Rat	[165]
Myocardial ischemia/reperfusion	Bone marrow-MSC-EVs	miR-29c	Phosphatase and tensin homolog/protein kinase B/mammalian target of rapamycin pathway	↓ Autophagy, ↓ infarct size	Mouse	[166]
Acute kidney injury	MSC-EVs	mRNA associated with mesenchymal phenotype	Renal tubular cell survival pathways	↑ Renal recovery	Mouse	[167]
Liver fibrosis	MSC-EVs	Anti-fibrotic mediators	Decreased fibrosis signaling	↓ Reduced liver fibrosis	Mouse	[168]
Osteoarthritis	Bone marrow-MSCs	Regulatory miRNAs	Inhibition of NF-κB signaling, downregulation of cyclooxygenase-2 and inflammatory cytokines	↑ Chondrocyte proliferation and matrix production	In vitro (human cartilage tissues)	[169]
Rheumatoid arthritis	Bone marrow-MSCs-EVs	miR-34a	Cyclin I-activated Ataxia telangiectasia mutated/Ataxia telangiectasia and Rad3-related/p53 pathway	↓ Synovial inflammation	Rat	[170]
Type 1 diabetes mellitus	Adipose-MSCs	Immunomodulatory factors	Increased IL-10 and transforming growth factor-β. Decreased IL-17 and interferon-γ	↑ Glycemic control, ↑ immune balance	Mouse	[171]
Cerebral ischemic/reperfusion injury	Bone marrow-MSCs-EVs	Long non-coding RNA KLF3-AS1	Sirtuin-1 stabilization, miR-206/USP22 axis	↓ Cerebral infarction, ↑ neurological function	Mouse	[172]
Traumatic brain injury	Umbilical cord-MSC-EVs	miRNAs	Suppression of neuronal apoptosis and microglial activation	↑ Neurological function	Rat	[173]
Bacterial pneumonia	Bone marrow-MSC-EVs	Paracrine mediators	Increased alveolar fluid clearance. Reduced bacterial load	Attenuated lung injury	Mouse	[174]
Myocardial ischemia/reperfusion injury	MSC-EVs	Pro-angiogenic factors	Activation of protein kinase B and glycogen synthase kinase-3β pathways	↓ Oxidative stress, ↑ angiogenesis	Mouse	[175]

MSC: Mesenchymal stem cell; EV: Extracellular vesicle; IL: Interleukin; NF-κB: Nuclear factor-kappa B; MSC-EV: Mesenchymal stem cell-derived extracellular vesicle.

Table 4 Translational bottlenecks and mitigation approaches

Translational challenge	Impact on clinical translation	Proposed mitigation strategy	Ref.
Batch heterogeneity	Variable efficacy	Standardized MSC banking	[180]
Donor variability	Product heterogeneity	Defined donor selection criteria	[21]
Low scalability	Limited clinical production	Bioreactor + TFF systems-based manufacture	[181]
Stability issues	Cold-chain dependence	Lyophilization with trehalose	[182]
Regulatory ambiguity	Delayed approval	Early regulator	[183]
Potency assay standardization	Difficulty assessing therapeutic activities	Functional bioassay validation	[180]
Comparability after process change	Regulatory approval risk	Analytical comparability testing	[177]
Storage stability limitations	Reduced shelf-life	Cryopreservation optimization	[182]
Cost effectiveness	Manufacturing feasibility concerns	Process automation	[181]

MSC: Mesenchymal stem cell; TFF: Tangential flow filtration.

Table 5 Regulatory and quality characteristics required for clinical translation of mesenchymal stem cell-derived extracellular vesicles pharmaceutical products

Quality characteristics	Regulatory expectation	Analytical method	Clinical relevance	Ref.
Identity	EV marker confirmation	CD63/CD81, WB, ELISA	Ensures correct product characterization and prevents misidentification of EV preparations	[21]
Potency	Mechanism-linked assay	Angiogenesis, T-cell suppression	Demonstrates therapeutic efficacy and supports dose selection	[180]
Purity	Removal of proteins/DNA	SEC, TFF, PicoGreen	Prevents contamination-related toxicity and improves product safety	[181]
Sterility	GMP requirement	USP <71>: Sterility tests	Required to ensure patient safety during systemic administration	[192]
Stability	Shelf-life	Freeze-thaw, lyophilization	Enables long-term storage and consistent therapeutic dosing	[182]
Process comparability	Manufacturing consistency	CMC validation	Required when manufacturing changes occur during scale-up or optimization	[193]
GMP manufacturing	Quality assurance	Process validation	Ensures consistent large-scale production suitable for clinical trials	[194,195]

EV: Extracellular vesicle; WB: Western blotting; ELISA: Enzyme-linked immunosorbent assay; SEC: Size-exclusion chromatography; TFF: Tangential flow filtration; GMP: Good manufacturing practice; USP: United States Pharmacopeia; CMC: Chemistry, manufacturing and controls.