Biology subjects

Gololobova, O.

Publications and source records attributed to Gololobova, O..

13 recordsLinked to original sources

bioRxiv2026

Isolation and Characterization of Extracellular Vesicles from Mouse Retina Tissue

This protocol provides a standardized workflow for the isolation of extracellular vesicles (EVs) from mouse retinal tissue, and includes an assessment of EV size and concentration, marker expression, and EV visualization in accordance with the International Society for Extracellular Vesicles Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. Most retinal EV studies rely on cell culture, which may not fully capture in vivo biology. Our approach more accurately reflects physiological and pathological EV states in vivo by enabling the extraction of EVs from intact retinal tissue. This method addresses a key gap in the field by providing a reproducible and rigorous protocol for studying retinal EVs in a biologically relevant context.

cell biology↗

bioRxiv2026

Human endogenous retrovirus envelope proteins alter extracellular vesicle cellular interactions and biodistribution

Extracellular vesicles (EVs) are versatile therapeutic candidates due to biological roles in intercellular communication and amenability to bioengineering. Compared with lipid nanoparticles (LNPs), native or surface-modified EVs may have favorable immunogenicity and biodistribution profiles. However, when administered intravenously (IV), EVs are rapidly cleared and accumulate mostly in the liver and spleen. With the goal of modifying EV biodistribution, we engineered EVs to display the human endogenous retrovirus (HERV) envelope glycoprotein Syncytin-1, an SLC1A5-binding fusogenic viral protein essential for syncytiotrophoblast formation in pregnancy. Here, we comprehensively characterize engineered Syncytin-1+ EVs, examine their interactions with cells in vitro, and assay biodistribution, immunogenicity, and pharmacokinetics ex vivo and in vivo in non-human primates. IV-administered Syncytin-1+ EVs are well tolerated, persist in the blood stream, and have altered organ biodistribution compared with unmodified EVs, suggesting therapeutic potential of Syncytin-1+ EVs at specific sites.

bioengineering↗

bioRxiv2025

Simultaneous Isolation and Characterization of Lipoprotein Classes in Plasma, Including HDL Subclasses and the Uncharacterized Dense HDL

Lipoproteins (LPP) and extracellular vesicles are carriers of extracellular small RNA, with potential applications both in the areas of diagnostics and therapeutics. Lipid nanoparticles overlap across a range of densities and sizes in plasma, making them difficult to isolate intact and without contamination from other plasma components. Accurate characterization of their cargo through efficient isolation from other plasma components is required to understand their function. Here we describe the simultaneous separation of LPP classes using sequential flotation ultracentrifugation followed by size exclusion chromatography from 500{micro}L of starting plasma. Using western blot, denaturing and non-denaturing gel electrophoresis, nuclear magnetic resonance, and electron microscopy, we demonstrate separation of the LPP classes with minimal contamination. We also show unique lipidomic, proteomic and small RNA signatures for each LPP class, including, for the first time, very high density high density lipoprotein particles in the density range of 1.21-1.25 g/mL. MotivationIn plasma, lipid nanoparticles overlap in size and density, making it difficult to separate and isolate intact particles. Additionally, currently available methods do not allow for simultaneous characterization and isolation of these particles. This method resolves both challenges. The results presented also analyze and describe the presence of known lipoprotein particles, as well as those that have been previously under-characterized.

molecular biology↗

bioRxiv2025

Extracellular Vesicles from Multiple Sclerosis White Matter Exhibit Synaptic, Mitochondrial, Complement, and Aging-related Pathway Dysregulation

Extracellular vesicles (EVs) are increasingly recognized as mediators of central nervous system (CNS) function and pathologies, including multiple sclerosis (MS). While plasma-derived EVs have been explored as biomarkers in MS, little is known about EVs in CNS tissue. Here, we characterize EVs from postmortem white matter (WM) of MS and control brains. EVs were separated by differential centrifugation followed by size exclusion chromatography and characterized using nanoflow cytometry, single-particle reflectance imaging sensing (SP-IRIS), and transmission electron microscopy. EV size, yield, and morphology did not differ significantly between MS and control samples. Proteomic analyses revealed downregulation of synaptic and mitochondrial proteins and upregulation of complement and inflammatory proteins and pathways in MS WM EVs. This suggests that EVs reflect ongoing synaptic pathology, metabolic dysfunction, and CNS-compartmentalized inflammation and that they may actively contribute to these pathological processes. Deconvolution analyses suggests a shift in EV cellular origin, with an increased astrocytic and decreased neuronal EV contributions in MS. Several proteomic changes we observed in CNS-derived EVs have also been reported in circulating EVs of people with MS, establishing this CNS tissue EV study as a valuable resource for identifying biomarker candidates for brain-derived plasma EV studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/657509v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f88da5org.highwire.dtl.DTLVardef@11b7ee9org.highwire.dtl.DTLVardef@291d9forg.highwire.dtl.DTLVardef@157cb73_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Magnetically Labeled iPSC-Derived Extracellular Vesicles Enable MRI/MPI-Guided Regenerative Therapy for Myocardial Infarction

Stem cell-derived extracellular vesicles (EVs) offer a promising cell-free approach for cardiovascular regenerative medicine. In this study, we developed magnetically labeled induced pluripotent stem cell-derived EVs (magneto-iPSC-EVs) encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles for image-guided regenerative treatment of myocardial infarction, in which EVs that can be detected by both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). iPSC-EVs were isolated, characterized per MISEV2023 guidelines, and loaded with SuperSPIO20 nanoparticles using optimized electroporation conditions (300 V, 2 x 10 ms pulses), achieving a high loading efficiency of 1.77 ng Fe/106 EVs. In vitro results show that magneto-iPSC-EVs can be sensitively detected by MPI and MRI, with a detectability of approximately 107 EVs. In a mouse myocardial ischemia-reperfusion model, intramyocardially injected magneto-iPSC-EVs (2 x 109) were imaged non-invasively by in vivo MPI for 7 days and ex vivo MRI, with the presence of magneto-iPSC-EVs confirmed by Prussian blue staining. Therapeutically, both native and magneto-iPSC-EVs significantly improved cardiac function, with a 37.3% increase in left ventricular ejection fraction and 61.0% reduction in scar size. This study highlights the potential of magneto-iPSC-EVs as a cell-free approach for cardiovascular regenerative medicine, offering both non-invasive imaging capabilities and therapeutic benefits for myocardial repair.

bioengineering↗

bioRxiv2025

An ex vivo and in vitro investigation of extracellular vesicle interactions with B cells of Macaca nemestrina and humans

AbstractExtracellular vesicles may modify recipient cell behavior through multiple mechanisms, including interacting with the cell surface or internal membrane components and delivering luminal cargo to the cytoplasm. Here, we use a previously established ex vivo approach to investigate the cellular fate of EVs spiked into whole blood samples from nonhuman primate (NHP) and human donors and contrast these findings with results from in vitro assays. We report that EVs are internalized by NHP and human B cells while also associating to some degree with other PBMCs. EVs exhibit greater association with B cells in ex vivo whole blood compared to isolated B cells, suggesting that blood components may promote EV interactions or that cell isolation factors may inhibit this association. Cellular uptake of EVs involves clathrin-dependent endocytosis and may be aided by other pathways, including direct EV-cell membrane fusion. Overall, our data suggest that EV association, including uptake, by B cells occurs in at least two primate species. These findings highlight the potential to develop new strategies to either enhance or inhibit EV tropism toward B cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/637883v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@bd28e3org.highwire.dtl.DTLVardef@c76b28org.highwire.dtl.DTLVardef@4c2e44org.highwire.dtl.DTLVardef@1719771_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Defining the parameters for sorting of different RNA cargo into Extracellular vesicles

Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell-cell communication by transferring bioactive molecules. RNA cargo of EVs, including coding and non-coding RNAs, can change the behavior of recipient cells, affecting processes like gene expression, proliferation, and apoptosis. Circular RNAs (CircRNA) are a newly appreciated class of regulatory RNAs that are stable, resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis. While microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circRNAs as well as long linear RNAs. Here, we take a comprehensive genome-wide approach to investigate the role of GC%, size, exon count, structuredness and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We found that size and structuredness had a significant role in enriching RNAs into EVs which was consistent across all classes of RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analyzing these factors, we aim to better understand the complex mechanisms behind EV-mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.

cell biology↗

bioRxiv2024

In vitro efficacy and in vivo toxicity and retention of targeted nanoformulated carboplatin in a sustained release carrier for treatment of osteosarcoma

Objectiveevaluate 1) if targeting of platinum magnetic nanoclusters will promote uptake in osteosarcoma cells in vitro, 2) targeting will improve uptake and delivery in murine OSA in vivo compared to free carboplatin, 3) incorporation into a sustained release carrier (SRC) will prolong local retention in vivo. MethodsComplex stability and peptide loading was assessed. Drug release was tested at pH 7.4 and 5.5 and cellular uptake and cytotoxity determined for canine, human and mouse osteosarcoma. Subcutaneous murine osteosarcoma was induced and optimal dose and time until tumor growth were established. Tumor bearing mice were equally distributed between 8 treatment (0.5mg carboplatin/mouse) and 1 control group and sacrificed at 8 predetermined time points between 1 hour and 8 days. Blood, tumor site and organs were harvested for tissue ferron and platinum content analysis (ICP-MS). ResultsCarboplatin was preferentially released at pH5.5. Targeting increased cellular uptake for carboplatin 15.2-fold, and decreased IC50 at 24h and 48h. At 2 weeks, a SC injection of 1-1.56 live cells/mouse reliably resulted in a palpable tumor. Plasma platinum peaked prior to 6 hours while plasma ferron peaked at 24-48 hours. Intratumoral delivery did not lead to a sustained local presence while local delivery in a SRC after surgery did. ConclusionsTargeting of MNC-carboplatin is possible with an increased osteosarcoma cell uptake in vitro. In vivo metastatic uptake could not be assessed due to lack of metastases, but local delivery in a SRC yielded high local, and low systemic platinum concentrations in mice.

cancer biology↗

bioRxiv2024

Simultaneous protein and RNA analysis in single extracellular vesicles, including viruses: SPIRFISH

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles. Teaser: A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

molecular biology↗

bioRxiv2023

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 hr post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labeled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

immunology↗

bioRxiv2022

miR-210 expression is strongly hypoxia-induced in anaplastic thyroid cancer cell lines and is associated with extracellular vesicles & Argonaute-2

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.

cancer biology↗

bioRxiv2021

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina

Extracellular vesicles (EVs) have great potential as novel drug carriers for the treatment of various diseases. These lipid bilayer vesicles are naturally abundant in mammalian tissues and circulation, can be loaded with therapeutic small molecule drugs, (si)RNA, proteins and CRISPR/Cas9, and may be engineered for retention by specific tissues. However, many questions remain on the optimal dosing, administration route, and pharmacokinetics of EVs. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available from larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had markedly longer circulation times in plasma than previously reported in mice, and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. Already after one minute following IV administration, we observed EV uptake by PBMCs, most notably B-cells. EVs were detected in liver and spleen within one hour of IV administration. None of the IN doses resulted in readily detectable EV levels in plasma, CSF, or organs, suggesting that IN delivery of EVs in large animals including humans may require reconsideration or pretreatment approaches. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

pharmacology and toxicology↗

bioRxiv2021

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

immunology↗