**Background:** Exosomes are small (30–150 nm), single-membrane extracellular vesicles of endosomal origin, secreted by most cell types and found in all biological fluids. They facilitate intercellular communication by transporting DNA, RNAs (mRNA, miRNA, circRNA, etc.), proteins (tetraspanins CD9/CD63/CD81, heat shock proteins, GTPases), and lipids (cholesterol, sphingomyelin, ceramides). Their biogenesis involves endocytic membrane invagination, formation of intraluminal vesicles within multivesicular bodies (MVBs), and release via exocytosis when MVBs fuse with the plasma membrane. Key regulators include the ESCRT complex (0–3), Rab GTPases, tetraspanins, and sphingomyelinase. In cancer, tumor-derived exosomes promote a favorable microenvironment by boosting proliferation, preventing apoptosis, inducing angiogenesis, enabling metabolic reprogramming, and enhancing migration, invasion, and extracellular matrix remodeling. They also suppress anti-tumor immune responses by cytotoxic T lymphocytes and NK cells, contributing to immune evasion.
**Methods:** The review comprehensively describes isolation techniques: (1) Centrifugation-based methods—differential centrifugation (sequential spins at 300g, 2000g, 10,000g, and 20,000–100,000g) and density gradient centrifugation using sucrose or iodixanol media. Ultracentrifugation is considered the gold standard but is susceptible to contamination from lipoproteins. (2) Size-based techniques—ultrafiltration using nanomembranes with defined molecular weight cut-off, sequential filtration, and size-exclusion chromatography (SEC), which separates exosomes based on hydrodynamic radius. SEC is suitable for small volumes (as low as 15 µL) and yields highly resolved isolation. (3) Polymer precipitation using polyethylene glycol (PEG)—simple and scalable but prone to protein contamination. (4) Immunoaffinity capture chromatography (ICC)—uses antibodies against exosomal surface markers (CD9, CD63, ALIX, Ep-CAM) coupled to magnetic beads or matrices; Tim4@ILI-01 immunoaffinity material achieved 85% capturing efficiency, ~5 times greater than ultracentrifugation. (5) Microfluidic techniques—lab-on-a-chip systems exploiting size, density, or immunoaffinity; acoustofluidic chips achieved 98.4% purity and 99% cell removal from whole blood. Cargo loading methods include: co-incubation (simple diffusion, e.g., doxorubicin loading at 200 µg/mL showed maximal loading), electroporation (transient membrane pores; boosted doxorubicin loading threefold vs. simple diffusion), sonication (loading capacity 11.68% for gemcitabine vs. 2.79% by incubation; 28% for paclitaxel vs. 1.4% incubation and 5.3% electroporation), transfection (endogenous loading via donor cell engineering), extrusion (76% loading efficiency for paclitaxel at 50 µg/mL), freeze-thaw cycles (moderate loading), saponin-assisted loading (11-fold better encapsulation of hydrophilic porphyrins vs. other methods), hypotonic dialysis, and novel approaches like cellular nanoporation (50-fold more exosomes, 1000-fold more exosomal mRNA transcripts vs. electroporation) and EXPLORs (optically reversible protein-protein interaction). Characterization techniques include nanoparticle tracking analysis (NTA; gold standard, range 50–1000 nm), dynamic light scattering (DLS; requires 70 µL, less sensitive in heterogeneous mixtures), flow cytometry (high-throughput, limited to >300 nm), electron microscopy (SEM, TEM, Cryo-TEM for morphology), atomic force microscopy (AFM; 3D geometry), and western blotting for marker proteins (CD9, CD63, CD81, TSG101).
**Key Results:** Exosomal proteins identified as diagnostic biomarkers include CD24 and EpCAM in serum/ascites for early breast cancer; EDIL3 and fibronectin for early breast cancer; survivin for breast and prostate cancer; Glypican-1 (GPC1) for pancreatic cancer with absolute specificity and sensitivity; alpha-2-HS-glycoprotein (AHSG) and ECM1 for non-small cell lung cancer (NSCLC); TP63 and keratin 5 for lung squamous cell carcinoma; and cell adhesion molecule 6 and surfactant protein for lung adenocarcinoma. Exosomal nucleic acid biomarkers include miRNA-17-5p (overexpressed in colorectal and pancreatic cancer), miR-224-5p (NSCLC), miR-34c-3p (low levels in NSCLC), miRNA-3607-3p (pancreatic cancer suppressor), miRNA-23b-3p and miRNA-339-5p (pancreatic cancer), miRNA-217 and miRNA-23b-3p (prostate cancer), miRNA-21 (late-stage breast cancer), miRNA-105 and miRNA-373 (early-stage and triple-negative breast cancer), miRNA-1246 (metastatic breast cancer), and miRNA-222 (Adriamycin resistance in breast cancer). Exosomal lipid biomarkers include phosphatidylserine (elevated in prostate and ovarian cancer), phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, hexosylceramides, and lactosylceramides in prostate cancer. Therapeutic applications include: paclitaxel-loaded MSC-derived exosomes (76% loading efficiency by extrusion); doxorubicin-loaded exosome-sheathed porous silicon nanoparticles; cisplatin-loaded umbilical cord-derived M1 macrophage exosomes (superior inhibition of cisplatin-resistant ovarian cancer cells); gemcitabine-loaded autologous exosomes (sonication method, selective cellular uptake, dose-dependent tumor clearance); and MUC1 aptamer-decorated doxorubicin-loaded MSC exosomes (single-dose IV significantly suppressed tumor growth vs. non-functionalized exosomes and free doxorubicin). Chemoresistance mechanisms include direct drug export via exosomes, horizontal transfer of efflux pumps (P-gp, MRP-1, ABCA-3, ABCG-2), and miRNA exchange (miR-100, miR-222, miR-30a, miR-24, miR-26a, miR-27a associated with resistant phenotype). Exosome-based anti-cancer vaccines utilize dendritic cell-derived exosomes expressing MHC-I/II molecules to induce cytotoxic T lymphocyte priming. The review lists 18 clinical trials, including: NCT03608631 (Phase I, mesenchymal stromal cell-derived exosomes with KRAS G12D siRNA for pancreatic cancer), NCT01294072 (Phase I, curcumin-conjugated plant exosomes for colon cancer), NCT01159288 (Phase II, dendritic cell-derived exosomes for NSCLC—completed, no T cell induction), and NCT01668849 (Phase I, grape extract exosomes for oral mucositis in head and neck cancer).
**Clinical Implications:** Exosomes offer a promising platform for cancer diagnostics through non-invasive liquid biopsy (detection of tumor-specific proteins, miRNAs, and lipids in serum, plasma, urine) and for therapeutics as natural drug delivery vehicles with advantages including long-term stability, active/passive targeting, high drug loading capacity, reduced administration frequency, and low immunogenicity. However, major challenges remain: lack of standardized isolation and purification methods, difficulties in large-scale production, heterogeneity of exosomes (size, content, cellular origin, functional impact), variable storage conditions, unclear pharmacokinetics, risk of off-target capture by liver/lungs, and potential cargo damage during loading. The heterogeneity issue, first raised by Johnstone et al. in 1987, is driven by tumor microenvironment factors (pH, hypoxia, calcium concentration, radiation, chemotherapy, mechanical stress) and differences in cellular origin. Despite these hurdles, exosome-based approaches are actively being investigated in clinical trials across pancreatic, colon, prostate, lung, breast, thyroid, head and neck cancers, and sarcoma, positioning exosomes as a transformative tool in oncology.