Size Of Exosomes Research Articles

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Isolation and quantification of human urinary exosomes using a Tween-20 elution solvent from polyester, capillary-channeled polymer fiber columns

BackgroundExosomes, a subset of extracellular vesicles (EVs), are a type of membrane-secreted vesicle essential for intercellular communication. There is a great deal of interest in developing methods to isolate and quantify exosomes to study their role in intercellular processes and as potential therapeutic delivery systems. Polyester, capillary-channeled polymer fiber columns and spin-down tips are highly efficient, low-cost means of exosome isolation. As the methodology evolves, there remain questions as to the optimum elution solvent for specific end-uses of the recovered vesicles; fundamental biochemistry, clinical diagnostics, or therapeutic vectors. ResultsWhile both acetonitrile and glycerol have been proven highly successful in terms of EV recoveries in the hydrophobic interaction chromatography workflow, many biological studies entail the use of the non-ionic detergent, Tween-20, as a working solvent. Here we evaluate the use of Tween-20 as the elution solvent for the recovery of exosomes. A novel 10-min, two-step gradient elution method, employing 0.1 % v/v Tween-20, efficiently isolated EVs at a concentration of ∼1011 EV mL−1 from a 100 μL urine injection. Integration of absorbance and multi-angle light scattering detectors in standard HPLC instrumentation enables a comprehensive single-injection determination of eluted exosome concentration and sizes. Transmission electron microscopy verifies the retention of the vesicular structure of the exosomes. The micro-bicinchoninic acid protein quantification assay confirmed high-purity isolations of exosomes (∼99 % removal of background proteins) SignificanceThe effective use of Tween-20 as an elution solvent for exosome isolation/purification using capillary-channeled polymer fiber columns adds greater versatility to the portfolio of the approach. The proposed method holds promise for a wide range of fundamental biochemistry, clinical diagnostics, and therapeutic applications, marking a significant advancement in EV-based methodologies.

Isolation and analysis of the exosomal membrane proteins in bullous pemphigoid

ABSTRACT Background Bullous pemphigoid (BP) is a severe autoimmune sub-epidermal bullous disease. Exosomes are small extracellular vesicles secreted by most cell types. The exosomal membrane proteins are implicated in various biological and pathological pathways. This study aims to explore the potential roles of exosomes in BP pathomechanism. Research design We collected plasma samples from 30 BP patients and 31 healthy controls. Nanoparticle tracking analysis (NTA) was used to analyze the size and concentration of exosomes. The immunogold labelling experiment and extracellular vesicle (EV) array were performed to detect the content and distribution of exosomes. Results The exosomes from both the BP and control groups’ plasma were successfully extracted. EV Array showed that CD63 and CD9 levels were significantly higher in the BP group than in the control group (p < 0.05). Expression levels of the BP180 NC16A and intracellular domain (ICD) were higher in the anti-BP180 positive group versus the controls (p < 0.05). The active BP group exhibits higher CD63 and BP180 ICD protein concentrations than the control or inactive BP groups (p < 0.05). Conclusion BP180 autoantigen fragments were expressed on the exosomal membrane in BP patients. The BP180 ICD and CD63 on exosomes could potentially be novel biomarkers for monitoring disease activity.

Programmable Framework Nucleic Acid-Modified Nanomagnetic Beads for Efficient Isolation of Exosomes and Exosomal Proteomics Analysis.

Exosomes are increasingly being regarded as emerging and promising biomarkers for cancer screening, diagnosis, and therapy. The downstream molecular analyses of exosomes were greatly affected by the isolation efficiency from biosamples. Among the current exosome isolation strategies, affinity nanomaterials performed comparably better with selectivity and specificity. However, these techniques did not take the structure and size of exosomes into account, which may lead to a loss of isolation efficiency. In this article, a framework nucleic acid was employed to prepare a well-designed nanosized bead Fe3O4@pGMA@DNA TET@Ti4+ for enrichment of exosomes. The abundant phosphate groups in the framework nucleic acid provide binding sites to immobilize Ti4+, and its rigid three-dimensional skeleton makes them act as roadblocks to barricade exosomes and provide affinity interactions on a three-dimensional scale, resulting in the improvement of isolation efficiency. The model exosomes can be effectively isolated with 92% recovery in 5 min. From 100 μL of HeLa cell culture supernatant, 34 proteins out of the top 100 commonly identified exosomal proteins were identified from the isolated exosomes by the novel beads, which is obviously more than that by TiO2 (19 proteins), indicating higher isolation efficiency and exosome purity by Fe3O4@pGMA@DNA TET@Ti4+ beads. The nanobeads were finally applied for comparing exosomal proteomics analysis from real clinical serum samples. Twenty-five upregulated and 10 downregulated proteins were identified in the lung cancer patients group compared to the health donors group, indicating that the novel nanobeads have great potential in isolation of exosomes for exosomal proteomics analysis in cancer screening and diagnosis.

Exploration for sorting serum exosome as a measure of spinal cord injury severity

This study aimed to extract exosomes from the serum of rats with spinal cord injury and explore their potential as indicators for assessing the severity of spinal cord injury. Control rats were compared with rats in the mild and severe injury groups. Exosomes were isolated from rat serum using an optimized threshold condition with an ultra-high-speed flow cell sorter. Nanoparticle tracking analysis (NTA) was performed to determine the size distribution of exosomes, transmission electron microscopy (TEM) was used to observe the morphology of exosomes, and Western blot analysis was conducted to assess the expression of CD63(cluster of differentiation antigen 63) and CD9(cluster of differentiation antigen 9) proteins on the surface of exosomes. NanoFlow cytometry was utilized to evaluate the expression of CD63 and CD9. The optimal threshold condition for flow cytometry of exosomes was determined to be 0.002. Exosomes exhibited a cup-shaped morphology with a clean background. The percentages of exosomes in the control, mild injury, and severe injury groups were determined to be 6.4 × 1011 particles/mL, 6.2 × 1011 particles/mL, and 2.3 × 1011 particles/mL, respectively. The mean sizes of exosomes were 129.25 nm ± 18.3, 117.93 nm ± 39.9, and 116.38 nm ± 36.5, respectively. The percentages of CD9 were 15.5% ± 0.037, 10.4% ± 0.082, and 7.4% ± 0.051, while the percentages of CD63 were 1.2% ± 0.025, 2.6% ± 0.067, and 1.5% ± 0.038 for the control, mild injury, and severe injury groups, respectively. Comparison analysis revealed that the diameter of exosomes in rat serum ranged from 80 nm to 150 nm. The percentage of exosomes decreased, and CD9 expression significantly decreased as the severity of spinal cord injury increased. Flow cytometry allows for the isolation of exosomes from serum with high purity and uniform particle size. The percentage of serum exosomes and the expression of CD9 may serve as potential diagnostic indicators for spinal cord injury models.

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

Concussion-Related Biomarker Variations in Retired Rugby Players and Implications for Neurodegenerative Disease Risk: The UK Rugby Health Study.

The health and well-being of retired rugby union and league players, particularly regarding the long-term effects of concussions, are of major concern. Concussion has been identified as a major risk factor for neurodegenerative diseases, such as Alzheimer's and Amyotrophic Lateral Sclerosis (ALS), in athletes engaged in contact sports. This study aimed to assess differences in specific biomarkers between UK-based retired rugby players with a history of concussion and a non-contact sports group, focusing on biomarkers associated with Alzheimer's, ALS, and CTE. We randomly selected a sample of male retired rugby or non-contact sport athletes (n = 56). The mean age was 41.84 ± 6.44, and the mean years since retirement from the sport was 7.76 ± 6.69 for participants with a history of substantial concussions (>5 concussions in their career) (n = 30). The mean age was 45.75 ± 11.52, and the mean years since retirement was 6.75 ± 4.64 for the healthy controls (n = 26). Serum biomarkers (t-tau, RBP-4, SAA, Nf-L, and retinol), plasma cytokines, and biomarkers associated with serum-derived exosomes (Aβ42, p-tau181, p-tau217, and p-tau231) were analyzed using validated commercial ELISA assays. The results of the selected biomarkers were compared between the two groups. Biomarkers including t-tau and p-tau181 were significantly elevated in the history of the substantial concussion group compared to the non-contact sports group (t-tau: p < 0.01; p-tau181: p < 0.05). Although between-group differences in p-tau217, p-tau231, SAA, Nf-L, retinol, and Aβ42 were not significantly different, there was a trend for higher levels of Aβ42, p-tau217, and p-tau231 in the concussed group. Interestingly, the serum-derived exosome sizes were significantly larger (p < 0.01), and serum RBP-4 levels were significantly reduced (p < 0.05) in the highly concussed group. These findings indicate that retired athletes with a history of multiple concussions during their careers have altered serum measurements of exosome size, t-tau, p-tau181, and RBP-4. These biomarkers should be explored further for the prediction of future neurodegenerative outcomes, including ALS, in those with a history of concussion.

Exosomes derived from Polygonum multiflorum-treated human dental pulp stem cells (hDPSCs): New approach in regenerative medicine

Extracellular vesicles (EVs) are currently being extensively utilized in clinical trials. Our previous study demonstrated that the conditioned medium of human dental pulp stem cells (hDPSCs) treated by 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) exhibits biological activity and anti-inflammatory responses, which are believed to be regulated by EVs. To date, no study has thoroughly investigated the potential of the exosomes released by THSG-treated hDPSCs. In this study, we utilized the conditioned medium (CM) of hDPSCs with/without THSG stimulation to compare the differences in exosome extraction using ultracentrifugation (UC), tangential flow filtration (TFF), and TFF combined with size-exclusion chromatography (TFF + SEC) methods, aiming to establish an effective purification method. Subsequently, we analyzed the contents of the exosomes using proteomic strategy. The results showed that TFF + SEC was more effective in reducing protein contamination compared to CM and TFF alone. TFF effectively concentrated large-scale CM and maintained exosome particle sizes consistently around 85–100 nm. Flow cytometry analysis revealed that the presence of THSG increased exosome secretion from hDPSCs, concurrently leading to the identification of 219 unique proteins through proteomic analysis, including BMP-1, VEGF, and members of the MMP family. Compared to non-lyophilized exosomes, lyophilized exosomes showed no change in protein activity, indicating that lyophilization was an effective preservation method. Collectively, these findings present a combined extraction strategy for dental stem cell exosomes and reveal the high purity and proteome composition of THSG-exosomes, thereby highlighting their potential applications in regenerative therapy.

De novo synthesis of monounsaturated fatty acids modulates exosome-mediated lipid export from human granulosa cells

BackgroundOvarian somatic cells support the maturation and fertility of oocytes. Metabolic desaturation of fatty acids in these cells has a positive paracrine impact on the maturation of oocytes. We hypothesized that the enzyme stearoyl-CoA desaturase 1 (SCD1) in granulosa cells regulates the lipid cargo of exosomes secreted from these cells by maintaining the balance between saturated and unsaturated lipids. We investigated the effect of SCD1 on exosome lipid content in a cumulus-granulosa cell model under physiologically relevant in vitro conditions. MethodsNon-luteinized human COV434 granulosa cells were subjected to treatment with an inhibitor of SCD1 (SCDinhib) alone, in combination with oleic acid, or under control conditions. Subsequently, the exosomes were isolated and characterized via nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. We used liquid chromatography mass spectrometry to investigate the lipidomic profiles. We used quantitative PCR with TaqMan primers to assess the expression of genes involved in lipogenesis and control of cell cycle progression. ResultsA trend toward exosome production was observed with a shift toward smaller exosome sizes in cells treated with SCD1inhib. This trend reached statistical significance when SCDinhib was combined with oleic acid supplementation. SCD1 inhibition led to the accumulation of saturated omega-6 lipids in exosomes. The latter effect was reversed by oleic acid supplementation, which also improved exosome production and suppressed the expression of fatty acid synthase and Cyclin D2. ConclusionThese findings underscore the critical role of de novo fatty acid desaturation in the regulation of the export of specific lipids through exosomes, with potential implications for controlling intercellular communication within the ovary.

Enhanced brain-targeting and efficacy of cannabidiol via RVG-Exo/CBD nanodelivery system

This study introduces an innovative brain-targeted drug delivery system, RVG-Exo/CBD, utilizing rabies virus glycoprotein (RVG)-engineered exosomes for encapsulating cannabidiol (CBD). The novel delivery system was meticulously characterized, confirming the maintenance of exosomal integrity, size, and successful drug encapsulation with a high drug loading rate of 83.0 %. Evaluation of the RVG-Exo/CBD's brain-targeting capability demonstrated superior distribution and retention in brain tissue compared to unmodified exosomes, primarily validated through in vivo fluorescence imaging. The efficacy of this delivery system was assessed using a behavioral sensitization model in mice, where RVG-Exo/CBD notably suppressed methamphetamine-induced hyperactivity more effectively than CBD alone, indicating a reduction in effective dose and enhanced bioavailability. Overall, the RVG-Exo/CBD system emerges as a promising strategy for enhancing the therapeutic efficacy and safety of CBD, particularly for neurological applications, highlighting its potential for addressing the limitations associated with traditional CBD administration in clinical settings.

Exploring the role of Müller cells-derived exosomes in diabetic retinopathy

Exosomes are nanosized vesicles that have been reported as cargo-delivering vehicles between cells. Müller cells play a crucial role in the pathogenesis of diabetic retinopathy (DR). Activated Müller cells in the diabetic retina mediate disruption of barrier integrity and neovascularization. Endothelial cells constitute the inner blood-retinal barrier (BRB). Herein, we aim to evaluate the effect of Müller cell-derived exosomes on endothelial cell viability and barrier function under normal and hyperglycemic conditions. Müller cell-derived exosomes were isolated and characterized using Western blotting, nanoparticle tracking, and electron microscopy. The uptake of Müller cells-derived exosomes by the human retinal endothelial cells (HRECs) was monitored by labeling exosomes with PKH67. Endothelial cell vitality after treatment by exosomes under normo- and hypoglycemic conditions was checked by MTT assay and Western blot for apoptotic proteins. The barrier function of HRECs was evaluated by analysis of ZO-1 and transcellular electrical resistance (TER) using ECIS. Additionally, intracellular Ca+2 in HRECs was assessed by spectrofluorimetry. Analysis of the isolated exosomes showed a non-significant change in the number of exosomes isolated from both normal and hyperglycemic condition media, however, the average size of exosomes isolated from the hyperglycemic group showed a significant rise when compared to that of the normoglycemic group. Müller cells derived exosomes from hyperglycemic condition media markedly reduced HRECs cell count, increased caspase-3 and Annexin V, decreased ZO-1 levels and TER, and increased intracellular Ca+ when compared to other groups. However, treatment of HRECs under hyperglycemia with normo-glycemic Müller cells-derived exosomes significantly decreased cell death, preserved cellular integrity and barrier function, and reduced intracellular Ca+2. Collectively, Müller cell-derived exosomes play a remarkable role in the pathological changes associated with hyperglycemia-induced inner barrier dysfunction in DR. Further in vivo research will help in understanding the role of exosomes as therapeutic targets and/or delivery systems for DR.

Suppression of gastric cancer cell proliferation by miR-494-3p inhibitor-loaded engineered exosomes

BackgroundGastric cancer necessitates novel treatments, and exosomes are promising therapeutic carriers. We created miR-494-3p inhibitor exosomes to assess their effects on gastric cancer cells. MethodsWe conducted a comprehensive investigation into the expression of the oncogenic miR-494-3p in gastric cancer tissues from patients. Subsequently, we engineered miR-494-3p inhibitor-loaded exosomes and characterized their morphology and size through transmission electron microscopy and nanoparticle tracking analysis. We next determined the encapsulation efficiency of the miR-494-3p inhibitor within these exosomes and evaluated the exosomes' structural integrity by quantifying the presence of exosomal markers. Following these validations, we co-cultured miR-494-3p inhibitor exosomes with cancer cells and employed PKH26 staining to visualize the efficient endocytosis of engineered exosomes by gastric cancer cells and assess the impact of these modified exosomes on gastric cancer cell proliferation, apoptosis, migration, and invasion. ResultsIncreased expression of miR-494-3p was observed in gastric cancer tissues as compared to controls. Significant low miR-494-3p levels were found within miR-494-3p inhibitor exosomes, signifying effective encapsulation. The incorporation of miR-494-3p inhibitor into engineered exosomes did not alter exosome morphology or size. Finally, PKH26-stained exosomes clearly demonstrated efficient endocytosis by gastric cancer cells, leading to reduced proliferation, migration, invasion, and increased apoptosis. ConclusionOur study identifies elevated miR-494-3p in gastric cancer tissues prompting the development of miR-494-3p inhibitor-loaded exosomes with efficient encapsulation. These engineered exosomes demonstrate successful endocytosis by cancer cells. This highlights their potential for therapeutic use in gastric cancer treatment by suppressing proliferation, migration, and invasion while enhancing apoptosis.

Exosome-mediated secretion of miR-127-3p regulated by RAB27A accelerates metastasis in renal cell carcinoma

BackgroundThe exosome-mediated extracellular secretion of miRNAs occurs in many cancers, and RAB27A is a potent regulator of exosome secretion. For metastatic renal cell carcinoma (RCC), this study examines the mechanisms of cancer metastasis via the RAB27A-regulated secretion of specific miRNAs.MethodsRAB27A knockdown (KD) and overexpressing (OE) RCC cells were used to examine cell migration and adhesion. The particle counts and sizes of exosomes in RAB27A OE cells were analyzed using Exoview, and those of intraluminal vesicles (ILV) and multivesicular bodies (MVB) were measured using an electron microscope. Analysis of RNA sequences, protein–protein interaction networks, and the competing endogenous RNA (ceRNA) network were used to identify representative downregulated miRNAs that are likely to undergo cargo-sorting into exosomes and subsequent secretion. A molecular beacon of miR-137-3p, one of the most representatively downregulated genes with a fold change of 339, was produced, and its secretion was analyzed using Exoview. RAB27A OE and control cells were incubated in an exosome-containing media to determine the uptake of tumor suppressor miRNAs that affect cancer cell metastasis.ResultsMigration and cell adhesion were higher in RAB27A OE cells than in RAB27A KD cells. Electron microscopy revealed that the numbers of multivesicular bodies and intraluminal vesicles per cell were higher in RAB27A OE cells than in control cells, suggesting their secretion. The finding revealed that miR-127-3p was sorted into exosomes and disposed of extracellularly. Protein–protein interaction analysis revealed MYCN to be the most significant hub for RAB27A-OE RCC cells. ceRNA network analysis revealed that MAPK4 interacted strongly with miR-127-3p.ConclusionThe disposal of miR-127-3p through exosome secretion in RAB27A overexpressing cells may not inhibit the MAPK pathway to gain metastatic potential by activating MYCN. The exosomes containing miRNAs are valuable therapeutic targets for cancer treatment.

Abstract 6973: Investigates the role of exosomes in dasatinib treated HT29 colon cancer cells

Abstract The mortality rate due to metastasis in cancer is as high as 90%. The underlying mechanisms have been studied, however, the process of metastasis is complicated, current therapies still cannot inhibit metastasis efficiently. In recent years, studies have mentioned that extracellular vesicles play an important role in cancer cell metastasis and tumor microenvironment. Extracellular vesicles can be divided into microvesicles, apoptotic bodies and exosomes, which can carry different substances into the tumor microenvironment. Especially exosomes, whose diameter is around 30-160nm, can carry DNA, RNA, miRNA, membrane proteins, lipids and other intracellular molecules into the cell through endocytosis for regulation. The process of metastasis and the drug resistance were also been demonstrated can be regulated by exosomes. At present, the physiological functions of exosomes are not clear. HT29 colon cancer cells previously discovered in our laboratory can have different responses to dasatinib treatment, and cells can be isolated to dasatinib selected cells(DSTS) and resistant cells(DSTR). In this study, the role of exosome upon dasatinib treatment in HT29 cell is investigated. In the experiment, exosomes from HT29, DSTS, and DSTR were isolated by ExoQuick-TC. Transmission electron microscope was used to observe the difference in exosome size. Exosomes from different cells were treated in HT29 cells, and differences at the protein and mRNA levels were observed using western blot and reverse transcription-polymerase chain reaction(RT-PCR). In my results, the diameter of exosomes in DSTS cells was smaller than that in DSTR and HT29 cells. The protein expression levels of Cavoelin-1 and Clathrin in HT29 were also increased in the treatment of exosome that from three types of cells. In addition, the DSTR derived-exosomes can induce the expression of pFAK and Clathrin mRNA in HT29 cells. The preliminary results suggest that DSTR derived-exosomes may affect cell migration. This study provides the clues that through understanding exosomes that secreted from different cells, cancer diagnosis and treatment can be conducted according to their characteristics, so as to reduce the possibility of cell metastasis precisely. Citation Format: Hui Min Koo, Yi-Wen Lu, Xiang-Ling Hou, Wei-Ting Chao. Investigates the role of exosomes in dasatinib treated HT29 colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6973.

Abstract 1376: Engineered exosomes for the delivery of PD-L1 siRNA for lung cancer immunotherapy

Abstract Cancer immunosurveillance involves a dynamic interplay between the host’s innate and adaptive immunity. The regulation of programmed death ligand-1 (PD-L1) expression on cancer cells has emerged as an attractive approach for cancer treatment. However, challenges such as the immunosuppressive tumor microenvironment, ineffective trafficking, and tumor antigen heterogeneity limit the applications of cancer immunotherapy for solid tumors. Small interfering RNA (siRNA) against PD-L1 presents a compelling alternative to antibody treatment to reduce PD-L1 presentation on cancer cells, thereby enhancing T-cell detection and preventing tumor progression. Bovine milk/colostrum serve as a biocompatible source for exosomes for delivery biologics such as siRNA. Here, we demonstrate exosomes mediated delivery of siPD-L1 for lung cancer immunotherapy. Exosomes were isolated from bovine colostrum powder using ultracentrifugation. The average particle size of exosomes was 66 ± 2.5 nm, with polydispersity index (pdi) 0.27 ± 0.01 and a zeta potential -9.2 ± 0.70 mV, as analyzed by Zetasizer and validated by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Formulations of exosomes with polyethyleneimine (EPM) and siRNA (1 - 20 μg), when mixed with 5’-32P-labeled siRNA as a tracer resulted in over 90% entrapment of siRNA. Biodistribution studies of exosomes and EPM, with or without the tumor-targeting ligand folic acid (FA), revealed predominantly similar distribution patterns. Notably, FA-EPM exhibited enhanced tumor accumulation compared to EPM, attributed to the overexpression of folate receptors on tumor cells.To assess the functionality of EPM-siRNA, we screened 4 different siPD-L1 sequences in LLC-1 and A549 lung cancer cells. Following 48 h of EPM-siPD-L1 treatment, cells were harvested. Analysis of protein lysates by western blot showed a significant downregulation of the PD-L1 expression, ranging from 55% - 80% in LLC-1 cells and over 80% in A549 cells. In vivo mouse study with orthotopic Lewis lung carcinoma (LL/2-Luc2) tumors is underway to validate the effective silencing of PD-L1 and its effect on tumor inhibition and immune markers. In summary, our targeted EPM technology represents a novel nanoplatform for siRNA therapeutics delivery. As milk/colostrum are the most abundant sources of exosomes, clinical translatability of this novel approach is eminent. Funding: This research work was supported from the Kentucky Lung Cancer Research Program (GB170558) and Immunotherapy CCII CoBRE pilot project (P20GM135004). Keywords: Exosomes, siPD-L1, siRNA delivery, Lung cancer, Immunotherapy Citation Format: Raghuram Kandimalla, Disha N. Moholkar, Margaret Wallen, Chuanlin Ding, Ramesh C. Gupta, Farrukh Aqil. Engineered exosomes for the delivery of PD-L1 siRNA for lung cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1376.

Isolation and usage of exosomes in central nervous system diseases.

Exosomes are vesicles secreted by all types of mammalian cells. They are characterized by a double-layered lipid membrane structure. They serve as carriers for a plethora of signal molecules, including DNA, RNA, proteins, and lipids. Their unique capability of effortlessly crossing the blood-brain barrier underscores their critical role in the progression of various neurological disorders. This includes, but is not limited to, diseases such as Alzheimer's, Parkinson's, and ischemic stroke. Establishing stable and mature methods for isolating exosomes is a prerequisite for the study of exosomes and their biomedical significance. The extraction technologies of exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer coprecipitation, immunoaffinity capture, microfluidic, and so forth. Each extraction technology has its own advantages and disadvantages, and the extraction standards of exosomes have not been unified internationally. This review aimed to showcase the recent advancements in exosome isolation techniques and thoroughly compare the advantages and disadvantages of different methods. Furthermore, the significant research progress made in using exosomes for diagnosing and treating central nervous system (CNS) diseases has been emphasized. The varying isolation methods result in differences in the concentration, purity, and size of exosomes. The efficient separation of exosomes facilitates their widespread application, particularly in the diagnosis and treatment of CNS diseases.

Curcumin Changed the Number, Particle Size, and miRNA Profile of Serum Exosomes in Roman Laying Hens under Heat Stress.

Exosomes have the ability to transport RNA/miRNAs and possess immune modulatory functions. Heat stress, a significant limiting factor in the poultry industry, can induce oxidative stress and suppress the immune responses of laying hens. In this study, we investigated the expression profiles of serum exosomes and their miRNAs in Roman laying hens who were fed a diet with either 0 or 200 mg/kg curcumin under heat stress conditions. The numbers of exosomes were significantly higher in both the HC (heat stress) and HT (heat stress with 200 mg/kg curcumin) groups compared to the NC (control) group and NT (control with 200 mg/kg curcumin) group (p < 0.05). Additionally, we observed that the most prevalent particle diameters were 68.75 nm, 68.25 nm, 54.25 nm, and 60.25 nm in the NC, NT, HC, and HT groups, respectively. From our sRNA library analysis, we identified a total of 863 unique miRNAs; among them, we screened out for subsequent bioinformatics analysis a total of 328 gga-miRNAs(chicken miRNA from the miRbase database). The KEGG pathways that are associated with target genes which are regulated by differentially expressed miRNAs across all four groups at a p-value < 0.01 included oxidative phosphorylation, protein export, cysteine and methionine metabolism, fatty acid degradation, ubiquitin-mediated proteolysis, and cardiac muscle contraction. The above findings suggest that curcumin could mitigate heat-induced effects on laying hens by altering the miRNA expression profiles of serum exosomes along with related regulatory pathways.

Circulating plasma exosomes reflect the severity of myocardial damage in STEMI patients

Exosomes are small extracellular vesicles involved in intercellular communication and they contribute to inflammation, coagulation and vascular injury. Exosomes have demonstrated a great potential as diagnostic markers of disease, however their ability to reflect myocardial damage assessed by Cardiac Magnetic Resonance (CMR) in ST-segment elevation myocardial infarction (STEMI) is still unknown. To fill this gap, plasma exosomes were isolated from 42 STEMI patients treated by primary percutaneous coronary intervention (pPCI) and evaluated by CMR between days 3 and 6. Exosome concentration and size were measured by Nanoparticle Tracking Analysis, surface epitopes by flow cytometry, and platelet marker expression by ELISA kit. Exosome levels were greater in patients with anterior STEMI (p = 0.0001), with the culprit lesion located in LAD (p = 0.045), and in those who underwent late revascularization (p = 0.038). A smaller exosome size was observed in patients with a low myocardial salvage index (MSI, p = 0.014). Exosomes of patients with microvascular obstruction (MVO) had smaller dimension (p &lt; 0.002) and lower expression of the platelet marker CD41–CD61 (p = 0.039). Exosome size and CD41–CD61 expression were independent predictors of MVO/MSI (OR [95% CI]: 0.93 [0.87–0.98] and 0.04 [0–0.61], respectively). In conclusion, we reported for the first time the ability of exosomes isolated a few days after STEMI to reflect myocardial damage. In particular, the exosome size and expression of the platelet marker CD41–CD61, likely reflecting the level of circulating platelet-derived exosome, were independent predictors of MVO and low MSI that are both predictors of short-term prognosis of acute STEMI after pPCI treatment and are key variables for risk-stratification of patients after STEMI. This finding paves the way for the development of a new strategy for the timely identification of high-risk patients and their treatment optimization.

Isolation of three different sizes of exosomes in an Asian population with different retinal diseases before and after treatment: preliminary results

ABSTRACT Exosomes are membranous structures measuring between 40–120 nm that are secreted by various cells of the human body into the body fluid system. Exosomes contain proteins, mRNA, miRNA, and signaling molecules, and physiologically they assist in the intercellular transport of proteins and RNA molecules. In this study, we used an immunoaffinity filter paper platform combined with scanning electron microscopy and microfluidic systems to detect the size of exosomes within the aqueous humor. Eight aqueous humor samples showed three distinct sizes of exosomes that were significantly different on scanning electron microscopy(P < 0.01). We further used nanoparticle tracking analysis to assess the size distribution of exosomes within the aqueous humor. We found significantly different distributions of exosomes between patients with three different ocular diseases and patients with normal cataracts as controls. An obvious peak of exomeres(size around 35 nm)was found in the patients with central retinal vein occlusion and vitreous hemorrhage. Flare-ups of large exosomes(size 90–120 nm)were found in the patients with the inflammatory ocular disease pars planitis. No obvious peaks in exomeres or large exosomes were found in the control group. There was a high association between the distribution of exosomes and the pathogenesis of ocular diseases. After intravitreal anti-vascular endothelial growth factor treatment, the aqueous humor from the patients with neovascular diseases showed a significant reduction in exosomes in nanoparticle tracking analysis. These findings suggest that at least three distinct sizes of exosomes exist in the aqueous humor:(1)exomeres:<35 nm;(2)small exosomes:60–80 nm; and (3)large exosomes:90–120 nm. Different sizes of exosomes may have different implications in normal or diseased eyes.

Early-Pregnancy Serum Maternal and Placenta-Derived Exosomes miRNAs Vary Based on Pancreatic β-Cell Function in GDM.

Pancreatic β-cell function impairment is a key mechanism for developing gestational diabetes mellitus (GDM). Maternal and placental exosomes regulate maternal and placental responses during hyperglycemia. Studies have associated exosomal micro-RNAs (miRNAs) with GDM development. To date, no studies have been reported that evaluate the profile of miRNAs present in maternal and placental exosomes in the early stages of gestation from pregnancies that develop GDM. We assessed whether early-pregnancy serum maternal and placenta-derived exosomes miRNA profiles vary according to pancreatic β-cell function in women who will develop GDM. A prospective nested case-control study was used to identify exosomal miRNAs that vary in early-pregnancy stages (<18 weeks of gestation) from women with normoglycemia and those who developed GDM based on their pancreatic β-cell function using the homeostasis model assessment of pancreatic β-cell function (HOMA-%β) index. Early-pregnancy serum maternal and placenta-derived exosomes were isolated to obtain miRNA profiles. Potential target and pathway analyses were performed to identify molecular and metabolic pathways associated with the exosomal miRNAs identified. In early-pregnancy stages, serum maternal exosome size and concentration are modified in GDM group and fluctuate according to HOMA-%β index. Serum maternal exosomal hsa-miR-149-3p and hsa-miR-455-3p in GDM are related to insulin secretion and signaling, lipolysis, and adipocytokine signaling. Early-pregnancy serum placenta-derived exosomes hsa-miR-3665 and hsa-miR-6727-5p in GDM are related to regulating genes involved in response to immunological tolerance of pregnancy and pathways associated with placental dysfunction. Early serum exosomal miRNAs differ depending on their origin (maternal or placental) and pancreatic β-cell function. This research provides insights into the interactions between maternal and placental exosomal miRNAs and may have implications for identifying potential biomarkers or therapeutic targets for GDM.