Ultracentrifugation (UC) stands as the predominant technology and gold standard for extracting and isolating exosomes. By utilizing variations in size and density, this technique carefully isolates crucial components from a solution. Traditional UC is commonly used in processing biofluids such as urine, serum, aqueous humor, cerebrospinal fluid, amniotic fluid, and breast milk (Helwa et al., 2017). UC is highly efficient in segregating sample components with substantial differences in sedimentation coefficient (Livshits et al., 2015). The initial application of UC for exosome isolation was pioneered by Johnstone et al.in the extraction of exosomes from reticulocyte tissue culture medium (Johnstone, 1992). The UC isolation process involves two main steps. Initially, a sequence of consecutive centrifugation steps at low to moderate speeds is employed to remove dead cells, cellular waste, and sizable EVs. Afterwards, exosomes are separated more quickly by applying a centrifugal force of 100,000 g and then cleansed with PBS to eliminate impurities, including unwanted proteins. By employing this method, the requirement for exosome labeling is eradicated, and the potential for cross-contamination is greatly reduced. A number of factors, including the type of rotor, the centrifugation time, and the viscosity of the sample, impact the yield and purity of exosomes obtained using UC. (Cvjetkovic et al., 2014; Jeppesen et al., 2014). UC protocols that are employed and optimized for specific sample types must consider these parameters. Nonetheless, downstream analysis of it encounters challenges attributed to its time-intensive procedures, elevated cost, potential structural impairments, aggregation issues, and co-separation with lipoproteins. (Böing et al., 2014; Yang et al., 2019a).

Some studies have suggested that repeated ultracentrifugation (UC) results in reduced exosome yields and detrimental effects on exosome quality, rendering them unsuitable for clinical applications. Furthermore, high-speed centrifugation may cause damaged exosomes due to the high shear forces (Mol et al., 2017; Konoshenko et al., 2018).

To enhance exosome purity, density gradient centrifugation is commonly combined with UC. Density gradient centrifugation is a more advanced version of traditional differential ultracentrifugation, using inert centrifugal medium like sucrose and iodixanol to separate exosomes based on density. Sucrose density gradient UC, as a widely used method, utilizes UC and sucrose density gradient to separate exosomes, providing an advantage in the isolation of organelles, proteins, and other biomolecules. While this method improves the purity of exosomes, the higher thickness of the sucrose solution slows down the settling speed of exosomes, consequently prolonging the extraction procedure (Ford et al., 1994; Chen et al., 2019). In contrast, the iodixanol density gradient centrifugation method presents numerous advantages over the sucrose density gradient centrifugation method, such as decreased viscosity, metabolic inertness, non-toxicity to cells, and enhanced preservation of cell integrity and functionality. Konadu et al. separated exosomes from HIV-1 particles in infected individuals’ plasma using iodixanol velocity gradients. Exosomes were found in upper fractions with lower density, while viral particles were in lower fractions with higher density (Konadu et al., 2016).

Density gradient centrifugation improves exosome isolation purity compared to ultracentrifugation, but it is complex, requires high technical skill, has low throughput, is time-consuming, and does not effectively remove lipoproteins and chylomicrons from blood samples (Wang et al., 2024a).

Precipitation techniques use polymers like polyethylene glycol (PEG) to isolate and purify exosomes, with PEG being the most commonly used polymer for this purpose. (Oh et al., 1988). PEG modifies the cell membrane surface structure, decreasing the degradation of endogenous lipids and phospholipids in the sediment. Exosomes aggregate due to cell adhesion and surface tension, forming aggregates that precipitate with PEG (Prabhu and Balasubramanian, 2001). Simple, fast, minimally damaging exosomes, and low equipment requirements make precipitation methods attractive for clinical research. Nevertheless, the level of purity and the rate of recovery are comparatively inadequate, which may result in inaccurate positive outcomes. Additionally, the resulting polymer is challenging to remove, hindering subsequent functional experimental analysis. This challenge can be overcome by adding an extra step of purification or filtration (Afridi et al., 2023).

Contemporary precipitation techniques are advantageous for clinical use due to their requirement of minimal starting material for the manipulation of human biofluids and their compatibility with high-throughput capabilities.

This technology primarily focuses on separating exosomes and other elements in biological samples based on their sizes using ultrafiltration and size exclusion chromatography (SEC).

Exosomes are isolated from large volumes of biofluids using ultrafine nanomembranes with different molecular weight cutoffs in ultrafiltration. The separation is based on exosome size and includes three steps: dead-end filtration, tangential flow filtration, and track-etched membrane filtration. (Li et al., 2017). Researchers compared UC and ultrafiltration for isolating exosomes to diagnose lung cancer. Ultrafiltration showed advantages in speed, quantity, and particle size (<100 nm) (Lobb et al., 2015). However, this method has limitations, such as a low recovery rate and purity. Moreover, there is a potential risk of filters becoming clogged with external debris (Li et al., 2017).

Compared to centrifugal and filtration methods, SEC offers several advantages, including cost-effectiveness, non-destructive outcomes, and reproducibility. The underlying principle of SEC is the inability of macromolecules to penetrate gel pores, leading to their elution with the mobile phase through the interstitial spaces between the porous gels. In contrast, smaller molecules are retained within the gel pores and subsequently elute with the mobile phase. Drawbacks associated with this methodology include substantial labor requirements, potential sample contamination with lipoproteins, and the risk of protein aggregation (Witwer et al., 2013).

Although size-based techniques are widely used in various fields, they often have long running times, limiting their usefulness in treatment and research.

ICC utilizes exosome surface biomarkers to capture exosomes by binding antibodies to magnetic beads, plates, chromatographic matrices, or microfluidic devices, forming covalent bonds. These biomarkers specifically bind to antigens or membrane proteins on the exosomes (Yang et al., 2019a; Zhang et al., 2020a). Exosomes are measured in plasma, serum, and urine using ICC-based techniques such as ELISA, magneto-immunoprecipitation, and Western blotting (Mahgoub et al., 2020). Immunoaffinity chromatography isolates and purifies substances from complex mixtures using antibodies and ligands, resulting in strong specificity, exceptional purity, increased sensitivity, and significant yield. The strict storage needs for ICC-obtained exosomes make large-scale isolation impractical due to possible interference adsorption.

In general, this method is appropriate for both qualitative and quantitative analysis of exosomes. Nevertheless, various limitations, including its elevated cost, limited yield, stringent handling protocols, and specific storage prerequisites, restrict its widespread utility (Zhang et al., 2020a).

The membrane of the exosome consists of transmembrane proteins such as Lamp, GPI, and tetraspanins (such as CD63, CD9, CD81), which have the potential to fuse with targeting ligands, thereby improving the accuracy of exosome delivery (Mentkowski et al., 2018). One common method for generating surface-modified exosomes (SMEs) involves using plasmid vectors to genetically modify cells that produce exosomes. This modification entails fusing a targeting ligand with one of the aforementioned transmembrane proteins.

Lamp2b is often used to modify exosomal surface proteins for RNA transport. Liang et al. created chondrocyte-targeted exosomes by fusing Lamp2b with an affinity peptide (CAP). They then used these exosomes to encapsulate CRISPR/Cas9 plasmids by fusing them with liposomes. The hybrid CAP-Exo/Cas9 sgMMP-13 was injected into rat chondrocytes to mimic osteoarthritis. Knocking out MMP-13 reduced its expression and protected cartilage from degradation. (Liang et al., 2022). Lin et al. genetically engineered exosomes by fusing HSTP1 with the exosome-enriched membrane protein Lamp2b, resulting in the creation of engineered exosomes (HSTP1-Exos). These engineered exosomes demonstrated enhanced internalization by HSC-T6 cells and a superior ability to induce the transition of hepatic stellate cells-T6 (HSC-T6) cells to a quiescent phenotype when compared to unmodified exosomes (blank exosomes) and exosomes overexpressing Lamp2b protein (Lamp2b + Exos) (Lin et al., 2022).

Moreover, due to their classification as transmembrane proteins and high levels of expression, the tetraspanin superfamily CD63/CD9/CD81 are frequently selected for genetic modification and protein fusion (Li et al., 2019). A recent study has documented the development of a new engineered exosome, which contains membrane proteins or soluble protein cargoes that are actively bound. This exosome, known as Gene-Injected Functionalized Exosomes (GIFTed-Exos), is created by incorporating the exosome-associated tetraspanin CD9 gene, resulting in the display of transmembrane proteins CD70 and glucocorticoid-induced tumor necrosis factor receptor family-related ligand (GITRL) on the exosome surface. This modification leads to the formation of GIFTed-Exos with strong T-cell co-stimulatory capabilities. The GIFTed-Exos produced exhibit significant in vitro and in vivo efficacy in the delivery of various protein cargos to specific target cells (Cheng et al., 2021). Lee et al. present a novel in situ labeling technique for identifying exosome cargo, which eliminates the need for exosome isolation procedures. This method involves the expression of a modified form of the engineered ascorbate peroxidase conjugated (APEX), fused with an exosome cargo protein like CD63, specifically in exosome-producing vesicles within live cells or in exosomes released into the conditioned medium. This results in the biotinylation of proteins near the APEX variant for a brief period. Mass spectrometry analysis of the biotinylated proteins in exosomes secreted by kidney proximal tu is then conducted (Lee et al., 2022).

And Kooijmann et al. conducted an experiment in which they engineered exosomes presenting GPI-anchored anti-EGFR nanobodies, resulting in a notable increase in binding affinity of these exosomes to tumor cells with varying levels of EGFR expression. Furthermore, under flow conditions, the exosomes decorated with nanobodies significantly enhanced the adhesion of cells to tumor cells that express EGFR (Kooijmans et al., 2016).

Nonetheless, altering the genes of cells that produce exosomes presents a considerable obstacle. The introduction of a targeting moiety into the exosome membrane may potentially disrupt the normal functionality of exosome membrane proteins. Furthermore, a modified purification technique must be employed to isolate specifically modified exosomes compared to the technique currently utilized for nonfunctionalized or unmodified exosomes.

Exosomes often undergo covalent modification using techniques such as bioconjugation, amidation, aldehyde-amine condensation, and the widely employed method known as ‘click chemistry’. By utilizing these methods, the rapid formation of chemical bonds between external functional groups and bioactive compounds is facilitated (Wu et al., 2021). Smyth et al. devised a technique for attaching ligands to exosomes through the application of click chemistry. Exosomes functionalized with alkyne groups via carbodiimide chemistry were linked to a model azide, specifically azide-fluor 545. The conjugation process did not result in any significant changes in the size of exosomes, nor did it impact the ability of exosomes to adhere to or be internalized by recipient cells. These findings suggest that the reaction conditions employed had a minimal effect on the structure and function of exosomes (Smyth et al., 2014). A recent study introduced a novel nanoagent, referred to as MEXI, which was developed for the treatment of spinal cord injury (SCI) through the conjugation of bioactive IKVAV peptides onto the surface of M2 macrophage-derived exosomes using a facile and expeditious click chemistry approach. In vitro experiments demonstrated that MEXI effectively attenuated inflammation by modulating macrophage activity and facilitated the differentiation of neural stem cells into neurons. Furthermore, in vivo administration of engineered exosomes via tail vein injection resulted in targeted delivery to the site of spinal cord injury (Zeng et al., 2023a).

However, the deployment of covalent bonds in therapeutic applications poses inherent challenges, primarily attributed to their elevated stability and the requisite use of potentially toxic chemicals to induce such bonds. Hence, it is crucial to exercise caution when considering the utilization of covalent alteration techniques in therapeutic settings (Choi et al., 2021). Modifications of the exosomal membrane can occur through non-covalent methods, including binding of receptors and ligands, electrostatic interactions, and hydrophobic insertions (Nel et al., 2009; Armstrong et al., 2017). Hua et al. developed a multi-site targeting polymer with fluorescence and targeted recognition properties, capable of binding to the hydrophilic groups of CD63 through hydrogen bonding. In vitro, the suggested polymer demonstrated enhanced cellular uptake following its conjugation with exosomes through CD63; in vivo, it exhibited stable exosome binding and targeted tumor implants effectively (Hua et al., 2022). Zhu et al. collected exosomes from LECs, loaded them with doxorubicin using electroporation, and then immobilized them on IOL surfaces. In vitro tests showed that Dox@Exos were taken up more by LECs than free Dox, leading to better anti-proliferative effects. Animal tests showed that Dox@Exos-IOLs effectively prevented posterior capsule opacification (PCO) and were well-tolerated in the eye. (Zhu et al., 2022). Jing et al. created a nanoprobe using tumor cell exosomes (Tex) and tested its ability to image colon cancer using single-photon emission computed tomography (SPECT) and near-infrared fluorescence (NIRF). The findings confirmed the enhanced tumor-targeting ability of TEx compared to adipose stem cell-derived exosomes (AEx), highlighting the potential of exosomes in biomedical applications (Jing et al., 2021).

During genetic engineering, the combination of the targeting ligand and the exosome membrane protein gene results in the overexpression of this fusion in the recipient cells. As a result, donor cells engage in the production of genetically modified exosomes expressing the intended targeting ligands. The use of genetic engineering to modify exosomes emerges as a promising approach for incorporating functional ligands onto their membranes. Nonetheless, this approach necessitates the fabrication of plasmids and subsequent amplification of proteins in the donor cells. The exosome membrane can be targeted by both lipid and bioconjugation reactions, which have the ability to attach the targeting moiety. Chemical methods involve the bioconjugation of targeting ligands with surface proteins; nevertheless, there is a potential risk of surface protein inactivation or exosome aggregation during chemical manipulation. Despite these limitations, both strategies have demonstrated effective application (Liang et al., 2021).

As human life expectancy rises and global aging intensifies, the prevalence of OA is steadily rising. Timely diagnosis of early-stage OA is crucial for effective management and control of its progression. Nevertheless, there is currently a lack of a sensitive diagnostic modality for early OA. In clinical practice, the diagnosis of OA is typically made through a combination of imaging studies and physical examinations. However, these methods may be less reliable in detecting early-stage OA due to the subtle nature of symptoms and the lack of clear radiographic evidence, such as joint space narrowing and cartilage thinning (Crossley et al., 2008). The identification of a potential biomarker for early diagnosis in osteoarthritis has garnered interest in synovial fluid. Synovial fluid, produced by synovial membranes in joints, may contain early signs of osteoarthritis like exosomes. Studying exosomes and their contents in synovial fluid shows potential for identifying different stages of osteoarthritis (Skriner et al., 2006; Chen A. et al., 2023).

The Homeobox (Hox) gene encodes a transcription factor that plays a role in regulating limb morphogenesis and bone formation. Dysregulation of Hox has been linked to the onset and advancement of early-stage OA (Pelttari et al., 2015). Gene expression of Hox in chondrocyte-derived exosomes in synovial fluid could be a diagnostic tool for osteoarthritis. Exosomes containing miRNA can also indicate early-stage osteoarthritis. The mesenchymal stroma of the umbilical cord has high levels of miRNA140-5P, which aids cartilage regeneration (Geng et al., 2020). The impaired interaction between miRNA-140 and Let-7 has been identified as a key factor contributing to substantial growth abnormalities in chondrocytes in OA (Li et al., 2018). CircRNA_0005105 contributes to cartilage breakdown, while circ_0008365 is a potential diagnostic marker for OA found in exosomes from patient serum (Wu et al., 2017; Shuai et al., 2022). Exosomal lncRNA PCGEM1 levels were higher in people with OA, varying with disease stage, suggesting it could be used as a diagnostic marker and to distinguish between different stages of OA. (Zhao and Xu, 2018). In conclusion, the phenotypic characteristics of synovial fluid exosomes have the potential to serve as precise and efficient biomarkers for the early detection of osteoarthritis.

OA is a chronic degenerative condition significantly impacting patients’ quality of life through inflammation and progressive joint cartilage deterioration (Hu et al., 2021a; Liu et al., 2023). The etiology of OA remains elusive, posing a challenge for the development of effective therapeutic interventions (Goldring and Otero, 2011). Current treatment modalities, encompassing physical therapy and anti-inflammatory medications, focus on pain relief and disease progression deceleration. Joint replacement surgery is reserved for severe cases (Carr et al., 2012; Bowden et al., 2020). The incorporation of transcription factors within exosome encapsulation, alongside their adaptable engineering structure, holds considerable promise in anti-inflammatory therapy (Wu et al., 2018). The traditional method involves administering exosomes derived from stem cells directly to the affected region, with the goal of promoting tissue regeneration. Recently, to address constraints associated with natural exosomes, diverse modification techniques have emerged, primarily involving the integration of exogenous substances and exosome transportation through association with bioactive materials (Figure 3).

Currently, the diagnosis of IDD predominantly depends on imaging techniques. Nevertheless, the onset of early IDD is often gradual, and there is a notable absence of clinical biomarkers that accurately indicate the initial stages of the disease (Wang et al., 2024b). Recently, exosomes have attracted the attention of researchers.

Sun et al. found that annulus fibrosus (AF) cells can release AF-derived exosomes (AF-exo) that are taken up by human umbilical vein endothelial cells (HUVECs). Degenerated AF-exo promoted cell migration and increased expression of inflammatory factors in HUVECs. In contrast, non-degenerated AF-exo had different effects, suggesting that AF-exo could be a potential biomarker for early IDD detection (Sun et al., 2021). Ren et al. identified the gene pairs angio-associated migratory cell protein (AAMP) and 4-aminobutyrate aminotransferase (ABAT) as potential biomarkers for IDD due to their correlation with increased CD8⁺ T cell infiltration. (Ren et al., 2023). In summary, exosomes hold promise as accurate and effective biomarkers for the early identification of IDD.7.2.2 Exploration of Exosomes in the Clinical Treatment of IDD.

IDD can result in various physical manifestations, encompassing, but not confined to, low back pain, dysfunction, and spinal stenosis, leading to considerable social and economic burdens. The etiology of IDD involves multiple factors, including genetic predisposition, aging, mechanical trauma, and malnutrition. Pathological alterations linked to IDD primarily entail the aging and apoptosis of nucleus pulposus cells (NPCs), progressive degeneration of the extracellular matrix (ECM), fibrosis of the AF, and an inflammatory response. Currently, conservative and surgical approaches are employed for managing IDD, contingent upon the patient’s symptoms. However, these interventions solely offer pain relief, lacking the capacity to reverse IDD or restore the mechanical functionality of the spinal structure (Xin et al., 2022).

Exosomes therapy holds significant promise as a therapeutic approach for IDD, effectively regulating metabolic processes, cellular homeostasis, and the microenvironment. This leads to the sustained release of microRNAs, proteins, and transcription factors, ultimately resulting in favorable therapeutic outcomes.

Due to the multifactorial etiology of OP, it is challenging to rely on a single diagnostic component for predicting the condition. Therefore, risk score systems integrating a range of proteins, lipids, mRNA, and ncRNAs present in exosomes may serve as promising diagnostic tools for OP.

Researchers discovered abnormal tRF expression in the plasma of OP patients through small RNA sequencing on plasma exosomes. Specifically, tRF-38, tRF-25, and tRF-18 showed elevated levels in individuals with osteoporosis, suggesting they could be used as diagnostic markers for the condition (Zhang et al., 2018a). Shao and colleagues found abnormal miRNAs in exosomes of menopausal women with osteoporosis, which could be used as a biomarker for the condition (Shao et al., 2020). Chen et al. analyzed plasma exosomes from 60 patients with different bone conditions to identify new diagnostic markers. They discovered 45 proteins that were expressed differently, with four proteins confirmed. They created an exosomal protein index to distinguish between individuals with OP and those without, achieving an AUC of 0.805 for classification accuracy (Chen et al., 2020b). Hence, exosomes exhibit potential as precise and efficient biomarkers for the early detection of OP.7.3.2 Exploration of Exosomes in the Clinical Treatment of OP.

OP, a pathological condition characterized by a gradual decline in bone mineral density, results in the degradation of bone tissue microarchitecture and an increased susceptibility to fractures. This condition is significantly associated with the aging process (Ensrud and Crandall, 2017). Postmenopausal individuals, especially those with estrogen deficiency, face a heightened risk of OP development and fractures (Eastell et al., 2016).