Extracellular vesicles and circulating nucleic acids最新文献

Extracellular vesicles (EVs) have been regarded as influential intracellular delivering parcels that possess tremendous potential because of their strict and complex secretion regulation processes. However, traditional detection methods cannot monitor the secretion of EVs due to their small particle diameters. Inspired by their peculiar diverse appearances and lipid membranes ingredients, we developed an innovative strategy to detect EVs in any kind of fluids by using rationally designed peptide probes that particularly recognize the highly curved surface of EVs. These peptide probes also serve as novel tools to selectively target cancerous cells with specific lipid compositions and distributions. With this strategy, we discovered a series of EV-secreting regulation mechanisms and identified their roles within physiological processes. Recently, we found that the transportation of oligodeoxynucleotides and cell division control protein 42 homolog from TLR9-activated macrophages to naïve cells via EVs exerts synergetic effects in the propagation of the intracellular immune response, which suggests a general mechanism for EV-mediated uptake of pathogen-associated molecular patterns.

Extracellular vesicles (EVs), which are nanocarriers with phospholipid bilayer structures released by most cells, play a key role in regulating physiological and pathological processes. EVs have been investigated due to their loading capacity, low toxicity, immunogenicity, and biofunctions. Although EVs have shown good potential as therapeutic vehicles, natural EVs have a poor targeting ability, which substantially reduces the therapeutic effect. Through the addition of a targeting unit into the membrane surface of EVs or inside EVs by engineering technology, the therapeutic agent can accumulate in specific cells and tissues. Here, we focus on mammalian EVs (MEVs) and bacterial EVs (BEVs), which are the two most common types of EVs in the biomedical field. In this review, we describe engineered MEVs and BEVs as promising nanocarriers for targeted therapy and summarize the biogenesis, isolation, and characterization of MEVs and BEVs. We then describe engineering techniques for enhancement of the targeting ability of EVs. Moreover, we focus on the applications of engineered MEVs and BEVs in targeted therapy, including the treatment of cancer and brain and bone disease. We believe that this review will help improve the understanding of engineered MEVs and BEVs, thereby promoting their application and clinical translation.

Aim: Despite intensive research during the last decade, it remains challenging to prepare extracellular vesicles (EVs) of high purity, especially from primary body liquids or protein-rich conditioned media. For now, time-consuming combinations of at least two orthogonal methods, e.g., density and size separation, are required to enrich EVs to high purity, often at the expense of processing time. Therefore, novel technologies are required that allow EV preparation in acceptable time intervals and to fair purities. Free-flow electrophoresis (FFE) constitutes a well-established semi-preparative method to separate and prepare analytes, e.g., by inherent differences in their electric charges. FFE combines a flow-driven longitudinal transport of sample material with vertical electrophoresis and allows the separation of sample components into up to 96 different fractions. It was our aim to evaluate the potential of FFE for the separation of EVs from other sample components of EV-containing protein-rich conditioned cell culture media.

Methods: Exemplarily, conditioned media of mesenchymal stem/stromal cells raised in the presence of EV-containing 10% human platelet lysate were processed. We analyzed the obtained fractions by different technologies, including imaging flow cytometry, western blot and nanoparticle tracking analysis.

Results: We demonstrate that FFE quickly and reproducibly separates EVs from a huge proportion of molecules included in the original sample.

Conclusion: Our results qualify FFE as a feasible, quick and reproducible technology for the preparation of bona fide EVs.

Extracellular vesicles (EVs), as natural carriers, are regarded as a new star in nanomedicine due to their excellent biocompatibility, fascinating physicochemical properties, and unique biological regulatory functions. However, there are still some challenges to using natural EVs, including poor targeting ability and the clearance from circulation, which may limit their further development and clinical use. Nucleic acid has the functions of programmability, targeting, gene therapy, and immune regulation. Owing to the engineering design and modification by integrating functional nucleic acid, EVs offer excellent performances as a therapeutic system in vivo. This review briefly introduces the function and mechanism of nucleic acid in the diagnosis and treatment of diseases. Then, the strategies of nucleic acid-functionalized EVs are summarized and the latest progress of nucleic acid-functionalized EVs in nanomedicine is highlighted. Finally, the challenges and prospects of nucleic acid-functionalized EVs as a promising diagnostic system are proposed.

Liquid biopsy of tumor-derived extracellular vesicles (EVs) has great potential as a biomarker source for prostate cancer (CaP) early diagnosis and predicting the stages of cancer. The contents of EVs play an important role in intercellular communication and have specific expression in blood and urine samples from CaP patients. Powered by high-throughput, next-generation sequencing and proteomic technologies, novel EV biomarkers are easily detected in a non-invasive manner in different stages of CaP patients. These identified potential biomarkers can be further validated with a large sample size, machine learning model, and other different methods to improve the sensitivity and specificity of CaP diagnosis. The EV-based liquid biopsy is a novel and less-invasive alternative to surgical biopsies which would enable clinicians to potentially discover a whole picture of tumor through a simple blood or urine sample. In summary, this approach holds promise for developing personalized medicine to guide treatment decisions precisely for CaP patients.

The COVID-19 pandemic has challenged researchers to rapidly understand the capabilities of the SARS-CoV-2 virus and investigate potential therapeutics for SARS-CoV-2 infection. COVID-19 has been associated with devastating lung and cardiac injury, profound inflammation, and a heightened coagulopathic state, which may, in part, be driven by cellular crosstalk facilitated by extracellular vesicles (EVs). In recent years, EVs have emerged as important biomarkers of disease, and while extracellular vesicles may contribute to the spread of COVID-19 infection from one cell to the next, they also may be engineered to play a protective or therapeutic role as decoys or "delivery drivers" for therapeutic agents. This review explores these roles and areas for future study.

Aim: Activation of microglial NLRP3 inflammasome is an essential contributor to neuroinflammation underlying HIV-associated neurological disorders (HAND). Under pathological conditions, microglia-derived-EVs (MDEVs) can affect neuronal functions by delivering neurotoxic mediators to recipient cells. However, the role of microglial NLRP3 in mediating neuronal synaptodendritic injury has remained unexplored to date. In the present study, we sought to assess the regulatory role of HIV-1 Tat induced microglial NLRP3 in neuronal synaptodendritic injury. We hypothesized that HIV-1 Tat mediated microglia EVs carrying significant levels of NLRP3 contribute to the synaptodendritic injury, thereby affecting the maturation of neurons.

Methods: To understand the cross-talk between microglia and neuron, we isolated EVs from BV2 and human primary microglia (HPM) cells with or without NLRP3 depletion using siNLRP3 RNA. EVs were isolated by differential centrifugation, characterized by ZetaView nanoparticle tracking analysis, electron microscopy, and western blot analysis for exosome markers. Purified EVs were exposed to primary rat neurons isolated from E18 rats. Along with green fluorescent protein (GFP) plasmid transfection, immunocytochemistry was performed to visualize neuronal synaptodendritic injury. Western blotting was employed to measure siRNA transfection efficiency and the extent of neuronal synaptodegeneration. Images were captured in confocal microscopy, and subsequently, Sholl analysis was performed for analyzing dendritic spines using neuronal reconstruction software Neurolucida 360. Electrophysiology was performed on hippocampal neurons for functional assessment.

Results: Our findings demonstrated that HIV-1 Tat induced expression of microglial NLRP3 and IL1β, and further that these were packaged in microglial exosomes (MDEV) and were also taken up by the neurons. Exposure of rat primary neurons to microglial Tat-MDEVs resulted in downregulation of synaptic proteins- PSD95, synaptophysin, excitatory vGLUT1, as well as upregulation of inhibitory proteins- Gephyrin, GAD65, thereby implicating impaired neuronal transmissibility. Our findings also showed that Tat-MDEVs not only caused loss of dendritic spines but also affected numbers of spine sub-types- mushroom and stubby. Synaptodendritic injury further affected functional impairment as evidenced by the decrease in miniature excitatory postsynaptic currents (mEPSCs). To assess the regulatory role of NLRP3 in this process, neurons were also exposed to Tat-MDEVs from NLRP3 silenced microglia. Tat-MDEVs from NLRP3 silenced microglia exerted a protective role on neuronal synaptic proteins, spine density as well as mEPSCs.

Conclusion: In summary, our study underscores the role of microglial NLRP3 as an important contributor to Tat-MDEV mediated synaptodendritic injury. While the role of NLRP3 in inflammation is we

Aim: Diabetic nephropathy (DN) has become the most common cause of end-stage renal disease (ESRD) in most countries. Elucidating novel epigenetic contributors to DN can not only enhance our understanding of this complex disorder, but also lay the foundation for developing more effective monitoring tools and preventive interventions in the future, thus contributing to our ultimate goal of improving patient care.

Methods: The 5hmC-Seal, a highly selective, chemical labeling technique, was used to profile genome-wide 5-hydroxymethylcytosines (5hmC), a stable cytosine modification type marking gene activation, in circulating cell-free DNA (cfDNA) samples from a cohort of patients recruited at Zhongnan Hospital, including T2D patients with nephropathy (DN, n = 12), T2D patients with non-DN vascular complications (non-DN, n = 29), and T2D patients without any complication (controls, n = 14). Differentially analysis was performed to find DN-associated 5hmC features, followed by the exploration of biomarker potential of 5hmC in cfDNA for DN using a machine learning approach.

Results: Genome-wide analyses of 5hmC in cfDNA detected 427 and 336 differential 5hmC modifications associated with DN, compared with non-DN individuals and controls, and suggested relevant pathways such as NOD-like receptor signaling pathway and tyrosine metabolism. Our exploration using a machine learning approach revealed an exploratory model comprised of ten 5hmC genes showing the possibility to distinguish DN from non-DN individuals or controls.

Conclusion: Genome-wide analysis suggests the possibility of exploiting novel 5hmC in patient-derived cfDNA as a non-invasive tool for monitoring DN in high risk T2D patients in the future.