Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Tissue nanotransfection causes tumor regression by its effect on nanovesicle cargo that alters microenvironmental macrophage state.

Extracellular vesicles (EVs) are nanovesicles released by all eukaryotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angiogenic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macrophages (TAMs). Such delivery resulted in an angiogenic (LYVE+) change of state in TAM that supported tumor formation. Study of the trafficking of tumor-originating fluorescently tagged EV revealed colocalization with TAM demonstrating uptake by these cells. Ex vivo treatment of macrophages with tumor-derived EVs led to gain of tumorigenicity in these isolated cells. Single-cell RNA sequencing of macrophages revealed that EV-borne miR-126 characterized the angiogenic change of state. Unique gene expression signatures of specific macrophage clusters responsive to miR-126-enriched tumor-derived EVs were revealed. Topical tissue nanotransfection (TNT) delivery of an oligonucleotide comprising an anti-miR against miR-126 resulted in significant knockdown of miR-126 in the tumor tissue. miR-126 knockdown resulted in complete involution of the tumor and improved survival rate of tumor-affected mice. This work identifies a novel tumorigenic mechanism that relies on tumorigenic state change of TAM caused by tumor-originating EV-borne angiomiR. This disease process can be effectively targeted by topical TNT of superficial tumors.

Driving adult tissue repair via re-engagement of a pathway required for fetal healing.

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and ß-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†.

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

Analysis of Keratinocytic Exosomes from Diabetic and Nondiabetic Mice by Charge Detection Mass Spectrometry.

Unresolved inflammation compromises diabetic wound healing. Recently, we reported that inadequate RNA packaging in murine wound-edge keratinocyte-originated exosomes (Exoκ) leads to persistent inflammation [Zhou, X. ACS Nano 2020, 14(10), 12732-12748]. Herein, we use charge detection mass spectrometry (CDMS) to analyze intact Exoκ isolated from a 5 day old wound-edge tissue of diabetic mice and a heterozygous nondiabetic littermate control group. In CDMS, the charge (z) and mass-to-charge ratio (m/z) of individual exosome particles are measured simultaneously, enabling the direct analysis of masses in the 1-200 MDa range anticipated for exosomes. These measurements reveal a broad mass range for Exoκ from ∼10 to >100 MDa. The m and z values for these exosomes appear to fall into families (subpopulations); a statistical modeling analysis partially resolves ∼10-20 Exoκ subpopulations. Complementary proteomics, immunofluorescence, and electron microscopy studies support the CDMS results that Exoκ from diabetic and nondiabetic mice vary substantially. Subpopulations having high z (>650) and high m (>44 MDa) are more abundant in nondiabetic animals. We propose that these high m and z particles may arise from differences in cargo packaging. The veracity of this idea is discussed in light of other recent CDMS results involving genome packaging in vaccines, as well as exosome imaging experiments. Characterization of intact exosome particles based on the physical properties of m and z provides a new means of investigating wound healing and suggests that CDMS may be useful for other pathologies.

Staphylococcus aureus Biofilm Infection Compromises Wound Healing by Causing Deficiencies in Granulation Tissue Collagen.

OBJECTIVE: The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. BACKGROUND: Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. METHODS: Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. RESULTS: Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. CONCLUSION: This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism.

Neurogenic tissue nanotransfection in the management of cutaneous diabetic polyneuropathy.

This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNTABM) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNTABM caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNTABM caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNTABM spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes.

Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms.

Objective: Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)-related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov NCT02762032) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females. Methods: The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA®). The expression of the differentially regulated genes identified by IPA® were verified using real-time polymerase chain reaction (RT-PCR). Results: Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerase chain reaction (RT-PCR) analysis. Conclusions: This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.

Topical Lyophilized Targeted Lipid Nanoparticles in the Restoration of Skin Barrier Function following Burn Wound.

Lyophilized keratinocyte-targeted nanocarriers (TLNκ) loaded with locked nucleic acid (LNA) modified anti-miR were developed for topical application to full thickness burn injury. TLNκ were designed to selectively deliver LNA-anti-miR-107 to keratinocytes using the peptide sequence ASKAIQVFLLAG. TLNκ employed DOTAP/DODAP combination pH-responsive lipid components to improve endosomal escape. To minimize interference of clearance by non-targeted cells, especially immune cells in the acute wound microenvironment, surface charge was neutralized. Lyophilization was performed to extend the shelf life of the lipid nanoparticles (LNPs). Encapsulation efficiency of anti-miR in lyophilized TLNκ was estimated to be 96.54%. Cargo stability of lyophilized TLNκ was tested. After 9 days of loading with anti-miR-210, TLNκ was effective in lowering abundance of the hypoxamiR miR-210 in keratinocytes challenged with hypoxia. Keratinocyte uptake of DiD-labeled TLNκ was selective and exceeded 90% within 4 hr. Topical application of hydrogel-dispersed lyophilized TLNκ encapsulating LNA anti-miR-107 twice a week significantly accelerated wound closure and restoration of skin barrier function. TLNκ/anti-miR-107 application depleted miR-107 and upregulated dicer expression, which accelerated differentiation of keratinocytes. Expression of junctional proteins such as claudin-1, loricrin, filaggrin, ZO-1, and ZO-2 were significantly upregulated following TLNκ/anti-miR-107 treatment. These LNPs are promising as topical therapeutic agents in the management of burn injury.

Correction of MFG-E8 Resolves Inflammation and Promotes Cutaneous Wound Healing in Diabetes.

Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8(-/-) mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8(-/-) bone marrow to MFG-E8(+/+) mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8(-/-) mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8-directed therapeutics in diabetic wound care.

Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

Epidermal E-Cadherin Dependent ß-Catenin Pathway Is Phytochemical Inducible and Accelerates Anagen Hair Cycling.

Unlike the epidermis, which regenerates continually, hair follicles anchored in the subcutis periodically regenerate by spontaneous repetitive cycles of growth (anagen), degeneration (catagen), and rest (telogen). The loss of hair follicles in response to injuries or pathologies such as alopecia endangers certain inherent functions of the skin. Thus, it is of interest to understand mechanisms underlying follicular regeneration in adults. In this work, a phytochemical rich in the natural vitamin E tocotrienol (TRF) served as a productive tool to unveil a novel epidermal pathway of hair follicular regeneration. Topical TRF application markedly induced epidermal hair follicle development akin to that during fetal skin development. This was observed in the skin of healthy as well as diabetic mice, which are known to be resistant to anagen hair cycling. TRF suppressed epidermal E-cadherin followed by 4-fold induction of ß-catenin and its nuclear translocation. Nuclear ß-catenin interacted with Tcf3. Such sequestration of Tcf3 from its otherwise known function to repress pluripotent factors induced the plasticity factors Oct4, Sox9, Klf4, c-Myc, and Nanog. Pharmacological inhibition of ß-catenin arrested anagen hair cycling by TRF. This work reports epidermal E-cadherin/ß-catenin as a novel pathway capable of inducing developmental folliculogenesis in the adult skin.

miRNA control of tissue repair and regeneration.

Tissue repair and regeneration rely on the function of miRNA, molecular silencers that enact post-transcriptional gene silencing of coding genes. Disruption of miRNA homeostasis is developmentally lethal, indicating that fetal tissue development is tightly controlled by miRNAs. Multiple critical facets of adult tissue repair are subject to control by miRNAs, as well. Sources of cell pool for tissue repair and regeneration are diverse and provided by processes including cellular dedifferentiation, transdifferentiation, and reprogramming. Each of these processes is regulated by miRNAs. Furthermore, induced pluripotency may be achieved by miRNA-based strategies independent of transcription factor manipulation. The observation that miRNA does not integrate into the genome makes miRNA-based therapeutic strategies translationally valuable. Tools to manipulate cellular and tissue miRNA levels include mimics and inhibitors that may be specifically targeted to cells of interest at the injury site. Here, we discuss the extraordinary importance of miRNAs in tissue repair and regeneration based on emergent reports and rapid advances in miRNA-based therapeutics.

Barrier Function of the Repaired Skin Is Disrupted Following Arrest of Dicer in Keratinocytes.

Tissue injury transiently silences miRNA-dependent posttranscriptional gene silencing in its effort to unleash adult tissue repair. Once the wound is closed, miRNA biogenesis is induced averting neoplasia. In this work, we report that Dicer plays an important role in reestablishing the barrier function of the skin post-wounding via a miRNA-dependent mechanism. MicroRNA expression profiling of skin and wound-edge tissue revealed global upregulation of miRNAs following wound closure at day 14 post-wounding with significant induction of Dicer expression. Barrier function of the skin, as measured by trans-epidermal water loss, was compromised in keratinocyte-specific conditional (K14/Lox-Cre) Dicer-ablated mice because of malformed cornified epithelium lacking loricrin expression. Studies on human keratinocytes recognized that loricrin expression was inversely related to the expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Compared to healthy epidermis, wound-edge keratinocytes from Dicer-ablated skin epidermis revealed elevated p21(Waf1/Cip1) expression. Adenoviral and pharmacological suppression of p21(Waf1/Cip1) in keratinocyte-specific conditional Dicer-ablated mice improved wound healing indicating a role of Dicer in the suppression of p21(Waf1/Cip1). This work upholds p21(Waf1/Cip1) as a druggable target to restore barrier function of skin suffering from loss of Dicer function as would be expected in diabetes and other forms of oxidant insult.

AntihypoxamiR functionalized gramicidin lipid nanoparticles rescue against ischemic memory improving cutaneous wound healing.

Peripheral vasculopathies cause severe wound hypoxia inducing the hypoxamiR miR-210. High level of miR-210, persisting in wound-edge tissue as ischemic memory, suppresses oxidative metabolism and inhibits cell proliferation necessary for healing. In wound-edge tissue of chronic wound patients, elevated miR-210 was tightly associated with inhibition of epidermal cell proliferation as evident by lowered Ki67 immunoreactivity. To inhibit miR-210 in murine ischemic wound-edge tissue, we report the formulation of antihypoxamiR functionalized gramicidin lipid nanoparticles (AFGLN). A single intradermal delivery of AFGLN encapsulating LNA-conjugated anti-hypoximiR-210 (AFGLNmiR-210) lowered miR-210 level in the ischemic wound-edge tissue. In repTOP™mitoIRE mice, AFGLNmiR-210 rescued keratinocyte proliferation as visualized by in vivo imaging system (IVIS). 31P NMR studies showed elevated ATP content at the ischemic wound-edge tissue following AFGLNmiR-210 treatment indicating recovering bioenergetics necessary for healing. Consistently, AFGLNmiR-210 improved ischemic wound closure. The nanoparticle based approach reported herein is effective for miR-directed wound therapeutics warranting further translational development.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

Standardized Reporting of Research on Exosomes to Ensure Rigor and Reproducibility.

Significance: The study of extracellular vesicles (EVs), especially exosomes, has unlocked new avenues in understanding cellular communication and potential therapeutic applications. Recent Advances: Advancements in EV research have shown significant contributions from the International Society for Extracellular Vesicles (ISEV), in establishing methodological standards. The evolution of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from 2014 to 2023 reflects enhanced research rigor and reproducibility. The launch of EV-TRACK platform promotes uniformity and reproducibility by providing a centralized repository for data sharing and standardization practices. Furthermore, databases like EVpedia and ExoCarta have facilitated data sharing and collaboration within the scientific community. Concurrently, exosome-based therapies have emerged as a forefront area within regenerative medicine and targeted drug delivery, showcasing the potential of exosomes in promoting tissue regeneration. Critical Issues: Despite advancements, the field grapples with challenges such as vesicular heterogeneity, EV isolation complexity, and standardization. These issues impact research reproducibility and clinical applications. The inconsistency in exosomal preparations in clinical trials poses significant challenges to therapeutic efficacy and safety. Future Directions: The review outlines critical areas for future research, including the need for technological innovation in EV isolation and characterization, the establishment of standardized protocols, and a deeper understanding of exosome biology. The review also highlights the need to reassess guidelines, develop new EV isolation and characterization technologies, and establish standardized protocols to overcome current limitations. Emphasis is placed on interdisciplinary research and collaboration to address the complexities of EV biology, improve clinical trial design, and ultimately realize exosome's therapeutic and diagnostic potential. Continued evaluation and rigorous scientific validation are essential for successful exosome integration.

Insight into the Functional Dynamics and Challenges of Exosomes in Pharmaceutical Innovation and Precision Medicine.

Of all the numerous nanosized extracellular vesicles released by a cell, the endosomal-originated exosomes are increasingly recognized as potential therapeutics, owing to their inherent stability, low immunogenicity, and targeted delivery capabilities. This review critically evaluates the transformative potential of exosome-based modalities across pharmaceutical and precision medicine landscapes. Because of their precise targeted biomolecular cargo delivery, exosomes are posited as ideal candidates in drug delivery, enhancing regenerative medicine strategies, and advancing diagnostic technologies. Despite the significant market growth projections of exosome therapy, its utilization is encumbered by substantial scientific and regulatory challenges. These include the lack of universally accepted protocols for exosome isolation and the complexities associated with navigating the regulatory environment, particularly the guidelines set forth by the U.S. Food and Drug Administration (FDA). This review presents a comprehensive overview of current research trajectories aimed at addressing these impediments and discusses prospective advancements that could substantiate the clinical translation of exosomal therapies. By providing a comprehensive analysis of both the capabilities and hurdles inherent to exosome therapeutic applications, this article aims to inform and direct future research paradigms, thereby fostering the integration of exosomal systems into mainstream clinical practice.