Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy DOI Creative Commons
Sagar Rayamajhi, Benjamin K. Gibbs, Jared Sipes

et al.

ACS Applied Bio Materials, Journal Year: 2024, Volume and Issue: 7(11), P. 7770 - 7783

Published: Nov. 1, 2024

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of information play an important role in cell communication and alter the physiological process under both normal pathological conditions. As such, tracking EVs can provide valuable regarding basic understanding communication, onset early malignancy, biomarker discovery. Most current EV-tracking strategies invasive, altering natural characteristics by modifying with lipophilic dyes or surface proteins fluorescent reporters. The invasive labeling could processes thereby have major limitations for functional studies. Here, we report alternative minimally EV strategy using PicoGreen (PG), a small molecule fluoresces at 520 nm when bound to dsDNA. We show PG binds dsDNA associated (50–200 nm), forming stable highly PG-DNA complex (PG-EVs). In 2D culture 3D organoid models, PG-EV showed efficient properties, including high signal-to-noise ratio, time- concentration-dependent uptake, ability traverse environment. further validated dual-labeled following two orthogonal strategies: (1) Bioconjugation via amine (2) donor engineering endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown feasibility as effective be applied studying across multiple model systems.

Language: Английский

Extracellular Vesicle Preparation and Analysis: A State‐of‐the‐Art Review DOI Creative Commons
Zesheng Wang, Xiaoyu Zhou, Qinglong Kong

et al.

Advanced Science, Journal Year: 2024, Volume and Issue: 11(30)

Published: June 14, 2024

Abstract In recent decades, research on Extracellular Vesicles (EVs) has gained prominence in the life sciences due to their critical roles both health and disease states, offering promising applications diagnosis, drug delivery, therapy. However, inherent heterogeneity complex origins pose significant challenges preparation, analysis, subsequent clinical application. This review is structured provide an overview of biogenesis, composition, various sources EVs, thereby laying groundwork for a detailed discussion contemporary techniques preparation analysis. Particular focus given state‐of‐the‐art technologies that employ microfluidic non‐microfluidic platforms EV processing. Furthermore, this discourse extends into innovative approaches incorporate artificial intelligence cutting‐edge electrochemical sensors, with particular emphasis single proposes current outlines prospective avenues future research. The objective motivate researchers innovate expand methods analysis fully unlocking biomedical potential.

Language: Английский

Citations

42
Exosome-derived microRNAs: emerging players in vitiligo DOI Creative Commons

Wenquan li,

Yaobin Pang,

Qingying He

et al.

Frontiers in Immunology, Journal Year: 2024, Volume and Issue: 15

Published: July 8, 2024

Exosome-derived microRNAs (miRNAs) are biomacromolecules and nanoscale extracellular vesicles originating from intracellular compartments that secreted by most cells into the space. This review examines formation function of exosomal miRNAs in biological information transfer, explores pathogenesis vitiligo, highlights relationship between vitiligo. The aim is to deepen understanding how influence immune imbalance, oxidative stress damage, melanocyte-keratinocyte interactions, melanogenesis disorders development enhanced may contribute potential diagnostic therapeutic options for

Language: Английский

Citations

7
Spectral flow cytometry for detecting DNA cargo in malaria parasite-derived extracellular vesicles DOI Creative Commons
Ewa Kozela, Ekaterina Petrovich-Kopitman,

Yves Berger

et al.

Journal of Biological Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 108481 - 108481

Published: April 1, 2025

Cells across biological kingdoms release extracellular vesicles (EVs) as a means of communication with other cells, be their friends or foes. This is indeed true for the intracellular malaria parasite Plasmodium falciparum (Pf), which utilizes EVs to transport bioactive molecules various human host systems. Yet, study this mode in research currently constrained due limitations high-resolution tools and absence commercial antibodies. Here, we demonstrate power an advanced spectral flow cytometry approach robustly detect secreted EVs, isolated from Pf-infected red blood cells. By labeling both EV membrane lipids DNA cargo within (non-antibody staining approach), were able subpopulation parasitic-derived enriched DNA. Furthermore, could quantitatively measure DNA-carrying two distinct stages parasite: rings trophozoites. Our findings showcase potential monitor dynamic changes nucleic acid pathogenic EVs.

Language: Английский

Citations

1
A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system DOI Creative Commons
María-Angélica Calderón-Peláez,

Jaime E. Castellanos,

Myriam L. Velandia-Romero

et al.

PLoS ONE, Journal Year: 2025, Volume and Issue: 20(1), P. e0317689 - e0317689

Published: Jan. 24, 2025

Extracellular vesicles (EVs) are membrane-bound structures produced and released into the extracellular space by all types of cells. Due to their characteristics, EVs play crucial roles in cellular communication signaling, holding an immense potential as biomarkers molecular transporters. Various methods have been developed label characterize EVs, however, visualizing remains a process that requires highly specialized expensive equipment, which is not always available laboratories. In this study, we adapted protocol originally designed for analysis flow cytometry using Calcein-AM, convert it useful effective tool epifluorescence microscopy coupled with deconvolution system. This approach can be very basic analyses, enabling researchers verify distribution internalization across Such insights guide decisions on whether advance more detailed confocal or perform additional assays.

Language: Английский

Citations

0
Glycan‐Anchored Fluorescence Labeling of Milk‐Derived Extracellular Vesicles for Investigating Their Cellular Uptake and Intracellular Fate DOI

Xueqi Su,

Siqin Zhang,

Tianyu Zhang

et al.

Small Methods, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 25, 2025

Abstract Milk‐derived extracellular vesicles (mEVs) are promising therapeutic delivery platforms due to their natural bioactivity, biocompatibility, and ability cross biological barriers. However, analyzing cellular uptake trafficking is limited by existing fluorescent labeling methods, which often cause dye leakage disrupt vesicle integrity. Here, a glycan‐anchored fluorescence strategy for mEVs developed, involving periodate oxidation of surface sialic acids followed aniline‐catalyzed ligation hydrazide‐functionalized fluorophores. Nano‐flow cytometry characterization confirmed ≈100% efficiency without compromising integrity or behavior. This approach enabled quantitative analysis internalization, identifying clathrin‐mediated endocytosis macropinocytosis as the primary pathways confirming mEVs’ capacity lysosomal escape. Comparative analyses showed that traditional lipophilic dyes induced aggregation, leakage, transfer, potentially misrepresenting Additionally, co‐labeling with fluorophores FITC‐conjugated paclitaxel real‐time tracking drug delivery, revealing burst release from lysosomes led significant cytotoxicity. Overall, allows precise intracellular fate, paving way further research application in targeted delivery.

Language: Английский

Citations

0
Recent advance on extracellular vesicle labeling: From strategy to probe DOI
Sijung Hu,

Xinwen Chang,

Qiaojiao Ding

et al.

Coordination Chemistry Reviews, Journal Year: 2025, Volume and Issue: 534, P. 216601 - 216601

Published: March 10, 2025

Language: Английский

Citations

0
Overcoming Challenges in MSC-sEV Therapeutics: Insights and Advances After a Decade of Research DOI Creative Commons
Bernd Giebel, Sai Kiang Lim

Cytotherapy, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Language: Английский

Citations

0
Factors to consider before choosing EV labeling method for fluorescence-based techniques DOI Creative Commons
Magdalena Dlugolecka, Malgorzata Czystowska

Frontiers in Bioengineering and Biotechnology, Journal Year: 2024, Volume and Issue: 12

Published: Sept. 18, 2024

A well-designed fluorescence-based analysis of extracellular vesicles (EV) can provide insights into the size, morphology, and biological function EVs, which be used in medical applications. Fluorescent nanoparticle tracking with appropriate controls reliable data for size concentration measurements, while nanoscale flow cytometry is most tool characterizing molecular cargoes. Label selection a crucial element all fluorescence methods. The comprehensive obtained if several labeling approaches given marker are used, as they would complementary information about EV populations interactions cells. In EV-related experiments, influence lipoproteins protein corona on results should considered. By reviewing considering factors affecting methods techniques, we assert that will accurate possible true biology offer precise, clinically applicable future EV-based diagnostic or therapeutic

Language: Английский

Citations

2
Superparamagnetic Iron Oxide Nanoparticle-Labeled Extracellular Vesicles for Magnetic Resonance Imaging of Ischemic Stroke DOI
Shannon Helsper, Xuegang Yuan,

Richard Jeske

et al.

ACS Applied Nano Materials, Journal Year: 2024, Volume and Issue: 7(20), P. 24160 - 24171

Published: Oct. 9, 2024

Stroke is a leading cause of death and disability worldwide. Extracellular vesicles (EVs) derived from human mesenchymal stem cells (hMSCs) offer unique promising alternative to direct cell injection as part cell-based therapy for stroke treatment. The development labeling strategies essential identifying the initial biodistribution clearance EV-based therapeutics. In this study, hMSC-EVs were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles magnetic resonance imaging (MRI). Two methods preparation evaluated after EVs sonicated in presence USPIO nanoparticles. purified by (1) ultracentrifugation only or (2) an extension harvesting approach that employs poly(ethylene glycol) (PEG) enrich EVs. Following vitro assessment, applied ischemic model imaged both immediately longitudinally using MRI. assessment showed EV characteristics nanoparticle labeling. PEG method exhibited 3.6-fold enhancement contrast equivalent concentration 0.5 mg/mL acquisition parameters (TE = 3.5 ms, TR 5 s) when dilution factor considered. Sufficient was achieved visualize assess therapeutic potential. Taken together, simultaneous enrichment enhanced MRI improved outcomes respect delivery recovery.

Language: Английский

Citations

1
GFP Farnesylation as a Suitable Strategy for Selectively Tagging Exosomes DOI
Rebecca Piccarducci, Lorenzo Germelli, Alessandra Falleni

et al.

ACS Applied Bio Materials, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 5, 2024

Exosomes are small extracellular vesicles (EVs) constituting fully biological, cell-derived nanovesicles with great potential in cell-to-cell communication and drug delivery applications. The current gold standard for EV labeling tracking is represented by fluorescent lipophilic dyes which, however, importantly lack selectivity, due to their unconditional affinity lipids. Herein, an alternative approach in-depth evaluated, taking advantage of green protein (GFP) farnesylation (GFP-f), a post-translational modification directly anchor GFP the membrane. performance GFP-f analyzed, terms selectivity efficiency, several typical experimental setups such as recipient cells, surface engineering, cargo loading. First, capability label exosomes was compared, showing significantly higher levels fluorescence intensity GFP-f- than GFP-labeled exosomes, highlighting anchoring cell Then, tag further compared Vybrant DiD dye exosome uptake studies, capturing intracellular time- concentration-dependent manner. internalization assay revealed particular ability monitor tagged into significant peak reached 12 h after administration but not Vybrant-labeled EVs. Finally, challenged presence transfection siRNA Results showed that both procedures can influence naïve although maintained cases. Overall, these data provide direct insight advantages limitations tagging selectively accurately route from isolation also context bioengineering

Language: Английский

Citations

1