Miriam Anfaiha-Sanchez, Aranzazu Santiago-Hernandez, Juan Antonio Lopez, Nerea Lago-Baameiro, Maria Pardo, Ariadna Martin-Blazquez, Jesus Vazquez, Gema Ruiz-Hurtado, Maria G. Barderas, Julian Segura, Luis M. Ruilope, Marta Martin-Lorenzo, Gloria Alvarez-Llamas
{"title":"Urinary extracellular vesicles as a monitoring tool for renal damage in patients not meeting criteria for chronic kidney disease","authors":"Miriam Anfaiha-Sanchez, Aranzazu Santiago-Hernandez, Juan Antonio Lopez, Nerea Lago-Baameiro, Maria Pardo, Ariadna Martin-Blazquez, Jesus Vazquez, Gema Ruiz-Hurtado, Maria G. Barderas, Julian Segura, Luis M. Ruilope, Marta Martin-Lorenzo, Gloria Alvarez-Llamas","doi":"10.1002/jex2.170","DOIUrl":"https://doi.org/10.1002/jex2.170","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Current definition of chronic kidney disease (CKD) identifies only advanced stages, but effective management demands early detection. Urinary albumin-to-creatinine ratio (ACR) 30 mg/g is a cut-off point for CKD clinical diagnosis. Patients with lower values (normoalbuminuria) and eGFR > 60 mL/min/1.73 m<sup>2</sup> are considered at no increased cardiorenal risk. However, higher incidence of renal function decline and cardiovascular events have been shown within the normoalbuminuria range. Novel subclinical indicators may help to identify higher-risk patients. Urinary extracellular vesicles (uEVs) are sentinels of renal function non-invasively. Here we aimed to approach the early assessment of cardiorenal risk by investigating the protein cargo of uEVs.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Hypertensive patients were classified in control group (C) with ACR < 10 mg/g, and high-normal group (HN) with ACR 10–30 mg/g. Isolated uEVs were characterized by western blotting and electron microscopy and the protein cargo was analyzed by untargeted proteomics (LC-MS/MS) in a first discovery cohort. Protein confirmation was performed in a different cohort by ExoView. Immunohistochemistry of human kidney biopsies was also performed to evaluate the potential of uEVs to reflect renal damage.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>HN albuminuria does not affect the uEVs concentration, size, or tetraspanin profile. Among >6200 uEVs proteins identified, 43 define a panel significantly altered in HN patients without variation in urine, mostly annotated in the tubule (39 out of 43). The tubular transporter long-chain fatty acid transport protein 2 (SLC27A2) and the apical membrane protein amnionless (AMN) confirmed their alteration in HN patients evidencing impaired tubular reabsorption. SLC27A2 showed tubular expression and significantly reduced levels in patients with diagnostic criteria for CKD.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Alterations in the EV-mediated molecular profile are evident before pathological ACR levels are reached. Direct quantitation of SLC27A2 and AMN in uEVs helps identifying normoalbuminuric subjects with higher cardiorenal risk in early monitoring of CKD.</p>\u0000 </section>\u0000 </div>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.170","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142244874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dimitrios Pollalis, Gopa Kumar Gopinadhan Nair, Justin Leung, Clarisa Marie Bloemhof, Jeffrey K. Bailey, Britney O. Pennington, Kaitlin R. Kelly, Amir I. Khan, Ashley K. Yeh, Kartik S. Sundaram, Dennis O. Clegg, Chen-Ching Peng, Liya Xu, Constantin Georgescu, Jonathan D. Wren, Sun Young Lee
{"title":"Dynamics of microRNA secreted via extracellular vesicles during the maturation of embryonic stem cell-derived retinal pigment epithelium","authors":"Dimitrios Pollalis, Gopa Kumar Gopinadhan Nair, Justin Leung, Clarisa Marie Bloemhof, Jeffrey K. Bailey, Britney O. Pennington, Kaitlin R. Kelly, Amir I. Khan, Ashley K. Yeh, Kartik S. Sundaram, Dennis O. Clegg, Chen-Ching Peng, Liya Xu, Constantin Georgescu, Jonathan D. Wren, Sun Young Lee","doi":"10.1002/jex2.70001","DOIUrl":"https://doi.org/10.1002/jex2.70001","url":null,"abstract":"<p>Retinal pigment epithelial (RPE) cells are exclusive to the retina, critically multifunctional in maintaining the visual functions and health of photoreceptors and the retina. Despite their vital functions throughout lifetime, RPE cells lack regenerative capacity, rendering them vulnerable which can lead to degenerative retinal diseases. With advancements in stem cell technology enabling the differentiation of functional cells from pluripotent stem cells and leveraging the robust autocrine and paracrine functions of RPE cells, extracellular vesicles (EVs) secreted by RPE cells hold significant therapeutic potential in supplementing RPE cell activity. While previous research has primarily focused on the trophic factors secreted by RPE cells, there is a lack of studies investigating miRNA, which serves as a master regulator of gene expression. Profiling and defining the functional role of miRNA contained within RPE-secreted EVs is critical as it constitutes a necessary step in identifying the optimal phenotype of the EV-secreting cell and understanding the biological cargo of EVs to develop EV-based therapeutics. In this study, we present a comprehensive profile of miRNA in small extracellular vesicles (sEVs) secreted during RPE maturation following differentiation from human embryonic stem cells (hESCs); <i>early</i>-<i>stage</i> hESC-RPE (20–21 days in culture), <i>mid</i>-<i>stage</i> hESC-RPE (30–31 days in culture) and <i>late</i>-<i>stage</i> hESC-RPE (60–61 days in culture). This exploration is essential for ongoing efforts to develop and optimize EV-based intraocular therapeutics utilizing RPE-secreted EVs, which may significantly impact the function of dysfunctional RPE cells in retinal diseases.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142230999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shimian Qu, Hannah M. Nelson, Xiao Liu, Yu Wang, Elizabeth M. Semler, Danielle L. Michell, Clark Massick, Jeffrey L. Franklin, John Karijolich, Alissa M. Weaver, Robert J. Coffey, Qi Liu, Kasey C. Vickers, James G. Patton
{"title":"5-Fluorouracil treatment represses pseudouridine-containing miRNA export into extracellular vesicles","authors":"Shimian Qu, Hannah M. Nelson, Xiao Liu, Yu Wang, Elizabeth M. Semler, Danielle L. Michell, Clark Massick, Jeffrey L. Franklin, John Karijolich, Alissa M. Weaver, Robert J. Coffey, Qi Liu, Kasey C. Vickers, James G. Patton","doi":"10.1002/jex2.70010","DOIUrl":"https://doi.org/10.1002/jex2.70010","url":null,"abstract":"<p>5-Fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. We examined the impact of 5-FU on post-transcriptional small RNA modifications (PTxMs) and the expression and export of RNA into small extracellular vesicles (sEVs). EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. We found that treatment of colorectal cancer (CRC) cells with 5-FU represses sEV export of miRNA and snRNA-derived RNAs, but promotes export of snoRNA-derived RNAs. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and sEV small RNA profiles. In contrast, 5-FU exposure led to increased levels of cellular small RNAs containing a variety of methyl-modified bases. These unexpected findings show that 5-FU exposure leads to altered RNA expression, base modification, and aberrant trafficking and localization of small RNAs.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142230998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Géraldine C. Genard, Luca Tirinato, Francesca Pagliari, Jessica Da Silva, Alessandro Giammona, Fatema Alquraish, Maria Parra Reyes, Marie Bordas, Maria Grazia Marafioti, Simone Di Franco, Jeannette Janssen, Daniel Garcia-Calderón, Rachel Hanley, Clelia Nistico, Yoshinori Fukasawa, Torsten Müller, Jeroen Krijgsveld, Matilde Todaro, Francesco Saverio Costanzo, Giorgio Stassi, Michelle Nessling, Karsten Richter, Kendra K. Maass, Carlo Liberale, Joao Seco
{"title":"Lipid droplets and small extracellular vesicles: More than two independent entities","authors":"Géraldine C. Genard, Luca Tirinato, Francesca Pagliari, Jessica Da Silva, Alessandro Giammona, Fatema Alquraish, Maria Parra Reyes, Marie Bordas, Maria Grazia Marafioti, Simone Di Franco, Jeannette Janssen, Daniel Garcia-Calderón, Rachel Hanley, Clelia Nistico, Yoshinori Fukasawa, Torsten Müller, Jeroen Krijgsveld, Matilde Todaro, Francesco Saverio Costanzo, Giorgio Stassi, Michelle Nessling, Karsten Richter, Kendra K. Maass, Carlo Liberale, Joao Seco","doi":"10.1002/jex2.162","DOIUrl":"https://doi.org/10.1002/jex2.162","url":null,"abstract":"<p>Despite increasing knowledge about small extracellular vesicle (sEV) composition and functions in cell–cell communication, the mechanism behind their biogenesis remains unclear. Here, we reveal for the first time that sEV biogenesis and release into the microenvironment are tightly connected with another important organelle, Lipid Droplets (LDs). The correlation was observed in several human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results demonstrated that external stimuli such as radiation, pH, hypoxia or lipid-interfering drugs, known to affect the number of LDs/cell, similarly influenced sEV secretion. Importantly, through multiple omics data, at both mRNA and protein levels, we revealed RAB5C as a potential important molecular player behind this organelle connection. Altogether, the potential to fine-tune sEV biogenesis by targeting LDs could significantly impact the amount, cargos and properties of these sEVs, opening new clinical perspectives.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.162","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142160293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jose G. Marchan-Alvarez, Loes Teeuwen, Doste R. Mamand, Susanne Gabrielsson, Klas Blomgren, Oscar P. B. Wiklander, Phillip T. Newton
{"title":"A protocol to differentiate the chondrogenic ATDC5 cell-line for the collection of chondrocyte-derived extracellular vesicles","authors":"Jose G. Marchan-Alvarez, Loes Teeuwen, Doste R. Mamand, Susanne Gabrielsson, Klas Blomgren, Oscar P. B. Wiklander, Phillip T. Newton","doi":"10.1002/jex2.70004","DOIUrl":"10.1002/jex2.70004","url":null,"abstract":"<p>Skeletal growth and fracture healing rely on the mineralization of cartilage in a process called endochondral ossification. Chondrocytes firstly synthesize and then modify cartilage by the release of a wide range of particles into their extracellular space. Extracellular vesicles (EVs) are one type of such particles, but their roles in endochondral ossification are yet to be fully understood. It remains a challenge to obtain representative populations of chondrocyte-derived EVs, owing to difficulties both in preserving the function of primary chondrocytes in culture and in applying the serum-free conditions required for EV production. Here, we used the ATDC5 cell-line to recover chondrocyte-derived EVs from early- and late-differentiation stages, representing chondrocytes before and during cartilage mineralization. After screening different culture conditions, our data indicate that a serum-free Opti-MEM-based culture medium preserves chondrocyte identity and function, matrix mineralization and cell viability. We subsequently scaled-up production and isolated EVs from conditioned medium by size-exclusion chromatography. The obtained chondrocyte-derived EVs had typical ultrastructure and expression of classical EV markers, at quantities suitable for downstream experiments. Importantly, chondrocyte-derived EVs from late-differentiation stages had elevated levels of alkaline phosphatase activity. Hence, we established a method to obtain functional chondrocyte-derived EVs before and during cartilage mineralization that may aid the further understanding of their roles in endochondral bone growth and fracture healing.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11375531/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142141939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Roksan Franko, Marcia de Almeida Monteiro Melo Ferraz
{"title":"Exploring the potential of in vitro extracellular vesicle generation in reproductive biology","authors":"Roksan Franko, Marcia de Almeida Monteiro Melo Ferraz","doi":"10.1002/jex2.70007","DOIUrl":"10.1002/jex2.70007","url":null,"abstract":"<p>The interest in the growing field of extracellular vesicle (EV) research highlights their significance in intercellular signalling and the selective transfer of biological information between donor and recipient cells. EV studies have provided valuable insights into intercellular communication mechanisms, signal identification and their involvement in disease states, offering potential avenues for manipulating pathological conditions, detecting biomarkers and developing drug-delivery systems. While our understanding of EV functions in reproductive tissues has significantly progressed, exploring their potential as biomarkers for infertility, therapeutic interventions and enhancements in assisted reproductive technologies remains to be investigated. This knowledge gap stems partly from the difficulties associated with large-scale EV production relevant to clinical applications. Most existing studies on EV production rely on conventional 2D cell culture systems, characterized by suboptimal EV yields and a failure to replicate in vivo conditions. This results in the generation of EVs that differ from their in vivo counterparts. Hence, this review firstly delves into the importance of EVs in reproduction to then expand on current techniques for in vitro EV production, specifically examining diverse methods of culture and the potential of bioengineering technologies to establish innovative systems for enhanced EV production.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11375532/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142141940","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Aliona Wöhler, Sabine K. Gries, Rebekka J. S. Salzmann, Christina Krötz, Bingduo Wang, Paula Müller, Angelina Klein, Ingo G. H. Schmidt-Wolf, Sebastian Schaaf, Robert Schwab, Veronika Lukacs-Kornek, Arnulf G. Willms, Miroslaw T. Kornek
{"title":"Monocyte derived large extracellular vesicles in polytrauma","authors":"Aliona Wöhler, Sabine K. Gries, Rebekka J. S. Salzmann, Christina Krötz, Bingduo Wang, Paula Müller, Angelina Klein, Ingo G. H. Schmidt-Wolf, Sebastian Schaaf, Robert Schwab, Veronika Lukacs-Kornek, Arnulf G. Willms, Miroslaw T. Kornek","doi":"10.1002/jex2.70005","DOIUrl":"10.1002/jex2.70005","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>Despite significant progress in the medical field, there is still a pressing need for minimal-invasive tools to assist with decision-making, especially in cases of polytrauma. Our team explored the potential of serum-derived large extracellular vesicles, so called microparticles/microvesicles/ectosomes, to serve as a supportive tool in decision-making in polytrauma situations. We focused on whether monocyte derived large EVs may differentiate between polytrauma patients with internal organ injury (ISS > 15) and those without. Thus, we compared our EV data to soluble biomarkers such as tumour necrosis factor alpha (TNF alpha) and Interleukin-8 (IL-8). From the blood of 25 healthy and 26 patients with polytrauma large EVs were isolated, purified, and characterized. TNF alpha and IL-8 levels were quantified. We found that levels of these monocyte derived large EVs were significantly higher in polytrauma patients with internal organ damage and correlated with the ISS. Interestingly, we also observed a decline in AnnV<sup>+</sup>CD14<sup>+</sup> large EVs during normal recovery after trauma. Thus, inflammatory serological markers as TNF alpha and as IL-8 demonstrated an inability to discriminate between polytrauma patients with or without internal organ damage, such as spleen, kidney, or liver lacerations/ruptures. However, TNF and IL-8 levels were elevated in polytrauma cases overall when contrasted with healthy non-traumatic controls. These findings suggest that delving deeper into the potential of AnnV<sup>+</sup> large EVs derived from monocytes could highly beneficial in the managment of polytrauma, potentially surpassing the efficacy of commonly used serum markers.</p>\u0000 </section>\u0000 </div>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11367151/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142121272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chang Liu, Li Sun, Hannah Worden, Justice Ene, Olivia Z. Zeng, Jamini Bhagu, Samuel C. Grant, Xiaoping Bao, Sunghoon Jung, Yan Li
{"title":"Profiling biomanufactured extracellular vesicles of human forebrain spheroids in a Vertical-Wheel Bioreactor","authors":"Chang Liu, Li Sun, Hannah Worden, Justice Ene, Olivia Z. Zeng, Jamini Bhagu, Samuel C. Grant, Xiaoping Bao, Sunghoon Jung, Yan Li","doi":"10.1002/jex2.70002","DOIUrl":"https://doi.org/10.1002/jex2.70002","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV application is still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <p>In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <p>Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <p>Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240–750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.</p>\u0000 </section>\u0000 </div>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142089842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Usri H. Ibrahim, Mohammed A. Gafar, Rene Khan, Abdelrahman Tageldin, Thirumala Govender, Irene Mackraj
{"title":"Engineered extracellular vesicles coated with an antimicrobial peptide for advanced control of bacterial sepsis","authors":"Usri H. Ibrahim, Mohammed A. Gafar, Rene Khan, Abdelrahman Tageldin, Thirumala Govender, Irene Mackraj","doi":"10.1002/jex2.70000","DOIUrl":"https://doi.org/10.1002/jex2.70000","url":null,"abstract":"<p>Alarming sepsis-related mortality rates present significant challenges to healthcare services globally. Despite advances made in the field, there is still an urgent need to develop innovative approaches that could improve survival rates and reduce the overall cost of treatment for sepsis patients. Therefore, this study aimed to develop a novel multifunctional therapeutic agent for advanced control of bacterial sepsis. Extracellular vesicles (EVs) isolated from lipopolysaccharide (LPS) induced HepG2 (hepatocellular carcinoma cells) (iEV) displayed an average particle size of 171.63 ± 2.77 nm, a poly dispersion index (PDI) of 0.32 ± 0.0, and a zeta potential (ZP) of −11.87 ± 0.18 mV. Compared to HepG2 EV, LPS induction significantly increases the EV protein concentration, PDI and ZP, reduces the average size and promotes cell proliferation and cytoprotective effects of the isolated EVs (iEVs) against LPS-induced cytotoxicity. Coating of iEV with a cationic antimicrobial peptide (AMP) to form PC-iEV slightly changed their physical properties and shifted their surface charge toward neutral values. This modification improved the antibacterial activity (2-fold lower minimum bactericidal concentration [MBC] values) and biocompatibility of the conjugated peptide while maintaining iEV cytoprotective and anti-inflammatory activities. Our findings indicate the superior anti-inflammatory and antibacterial dual activity of PC-iEV against pathogens associated with sepsis.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 8","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70000","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142041570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Neona M. Lowe, Rachel R. Mizenko, Bryan B. Nguyen, Kwan Lun Chiu, Vishalakshi Arun, Alyssa Panitch, Randy P. Carney
{"title":"Orthogonal analysis reveals inconsistencies in cargo loading of extracellular vesicles","authors":"Neona M. Lowe, Rachel R. Mizenko, Bryan B. Nguyen, Kwan Lun Chiu, Vishalakshi Arun, Alyssa Panitch, Randy P. Carney","doi":"10.1002/jex2.70003","DOIUrl":"https://doi.org/10.1002/jex2.70003","url":null,"abstract":"<p>Since extracellular vesicles (EVs) have emerged as a promising drug delivery system, diverse methods have been used to load them with active pharmaceutical ingredients (API) in preclinical and clinical studies. However, there is yet to be an engineered EV formulation approved for human use, a barrier driven in part by the intrinsic heterogeneity of EVs. API loading is rarely assessed in the context of single vesicle measurements of physicochemical properties but is likely administered in a heterogeneous fashion to the detriment of a consistent product. Here, we applied a suite of single-particle resolution methods to determine the loading of rhodamine 6G (R6G) surrogate cargo mimicking hydrophilic small molecule drugs across four common API loading methods: sonication, electroporation, freeze-thaw cycling and passive incubation. Loading efficiencies and alterations in the physical properties of EVs were assessed, as well as co-localization with common EV-associated tetraspanins (i.e., CD63, CD81 and CD9) for insight into EV subpopulations. Sonication had the highest loading efficiency, yet significantly decreased particle yield, while electroporation led to the greatest number of loaded API particles, albeit at a lower efficiency. Moreover, results were often inconsistent between repeated runs within a given method, demonstrating the difficulty in developing a rigorous loading method that consistently loaded EVs across their heterogeneous subpopulations. This work highlights the significance of how chosen quantification metrics can impact apparent conclusions and the importance of single-particle characterization of EV loading.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 8","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142041569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}