Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. METHODS: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-ß)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. RESULTS: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-ß-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. CONCLUSION: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Joule heating and electroosmotic flow in cellular micro/nano electroporation.

Localized micro/nano-electroporation (MEP/NEP) shows tremendous potential in cell transfection with high cell viability, precise dose control, and good transfection efficacy. In MEP/NEP, micro or nanochannels are used to tailor the electric field distribution. Cells are positioned tightly by a micron or nanochannel, and the cargoes are delivered into the cell via the channel by electrophoresis (EP). Such confined geometries with micro and nanochannels are also widely used in sorting, isolation, and condensing of biomolecules and cells. Theoretical studies on the electrokinetic phenomena in these applications have been well established. However, for MEP/NEP applications, electrokinetic phenomena and their impact on the cell transfection efficiency and cell survival rate have not been studied comprehensively. In this work, we reveal the coupling between electric field, Joule heating, electroosmosis (EO), and EP in MEP/NEP at different channel sizes. A microfluidic biochip is used to investigate the electrokinetic phenomena in MEP/NEP on a single cell level. Bubble formation is observed at a threshold voltage due to Joule heating. The bubble is pushed to the cargo side due to EO and grows at the outlet of the nanochannel. As the voltage increases, the cargo transport efficiency decreases due to more intense EO, particularly for plasmid DNAs (3.5 kbp) with a low EP mobility. An 'electroporation zone' is defined for NEP/MEP systems with different channel sizes to avoid bubble formation and excessive EO velocity that may reduce the cargo delivery efficiency.

Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, it is developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of the coronavirus disease of 2019 (COVID-19). BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.

Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis.

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late-stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes-rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles-rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early-stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late-stage PDAC patients undergoing chemotherapy, lower GPC1 tMV-mProtein and Exo-mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.