exosome-mediated delivery of sirna in vitro and in vivo pdf writer

Exosome-mediated delivery of sirna in vitro and in vivo pdf writer

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Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages

Exosome‑delivered TRPP2 siRNA inhibits the epithelial‑mesenchymal transition of FaDu cells

Exosome-mimetic nanoplatforms for targeted cancer drug delivery

In the s an unexpected gene-silencing phenomena in plants, the later called RNA interference RNAi , perplexed scientists. Following the proof of activity in mammalian cells, small interfering RNAs siRNAs have quickly crept into biomedical research as a new powerful tool for the potential treatment of different human diseases based on altered gene expression. In the past decades, several promising data from ongoing clinical trials have been reported. However, despite surprising successes in many pre-clinical studies, concrete obstacles still need to be overcome to translate therapeutic siRNAs into clinical reality.

Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages

Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. Exosomes, membranous nanovesicles, naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis.

Here, we showed that B cell-derived exosomes can function as vehicles to deliver exogenous miRNA mimic or inhibitor into hepatocytes or macrophages, respectively. Stimulation of B cells significantly increased exosome production. Unlike in parental cells, baseline level of miRNA was very low in exosomes derived from stimulated B cells. Furthermore, exosome-mediated miRNA inhibitor delivery resulted in functionally more efficient inhibition and less cellular toxicity compared to conventional transfection methods.

Similar approaches could be useful in modification of target biomolecules in vitro and in vivo. Extracellular vesicles EVs are heterogeneous membranous vesicles 40—nm diameter that originate from different cells and are released into biofluids and the intercellular microenvironment. The smallest subpopulation 40—nm , termed exosomes, derives from inward budding of the inner endosomal membrane followed by fusion of multivesicular bodies MVBs with the plasma membrane. Exosomes are subject of intensive research and exert their biological or pathophysiological functions by delivering diverse bio-macromolecules including different types of RNAs mRNAs and noncoding regulatory RNAs , cytoplasmic proteins, membranous proteins, and lipids.

Exosomes as natural nano-sized membranous vesicles are one of the new frontiers of nanomedical research. Given their nanoscale size, potential ability to express targeting ligands and deliver bio-macromolecules, and their excellent biocompatibility, exosomes are emerging as attractive biological nanovesicle platforms for loading and carrying bio-macromolecules for nanomedicine applications. Monocytes , macrophages, and dendritic cells represent pivotal cell types in innate immunity and participate in disease pathology.

The inflammatory process is a key factor in progression and pathogenesis of different diseases including liver disease, 20 rheumatoid arthritis, 21 atherosclerosis, 22 cancer, 23 and in infections including tuberculosis, 24 human immunodeficiency virus HIV , and leishmaniasis. Due to the mechanistic role of monocytes and macrophages in inflammation, targeted drug delivery to these cells to modulate their pro-inflammatory activation has been an active line of research in recent years.

However, these cells revealed to be difficult targets, 27 , 28 particularly where intracellular delivery of an active macromolecule was necessary for gene therapy. In this study, we evaluated whether exosomes murine B cell M Our results suggest that exosomes derived from B cells can be harnessed in gene therapy to introduce exogenous miRNA inhibitor to RAW Murine B cells M Three days later, the culture media was harvested and exosomes were isolated.

RAW Afterward, samples were serially filtered through 0. After isolation, exosomes were re-suspended in PBS. Immunomagnetic beads selection method was used for characterization and optimization of the procedure. The size, concentration, morphology, and surface marker CD63 of isolated exosomes were identified by Nanoparticle Tracking Analysis NTA , transmission electron microscopy TEM and western blot as described in supplementary method section.

To standardize loading conditions of exosomes to achieve successful outcome and reproducible results, we optimized loading conditions for B cell derived exosomes. The mixtures were transferred into cold 0. After optimization of the voltage, effect of different capacitance was assessed. First, exosome were isolated using CD63 immunomagnetic beads as described previously. Then, miRNA mimic pmol was electroporated into 0.

In order to evaluate the fraction of loaded exosomes with miRNA mimic, B cell derived exosomes were isolated and loaded using the optimal conditions like voltage and concentrations , as determined in the previous section. To eliminate the presence of miRNA mimic aggregates outside of the exosomes, the exosome pellet was treated with one unit of RNase H. The fraction of miRNA mimic that was loaded into the exosomes was calculated using the following formula:.

Detailed procedures are described in supplementary method section. The exosomes were loaded with miRNA mimic or negative control 1 for miRNA mimic Ambion, Grand Island, NY and co-cultured with primary mouse hepatocytes for 6 hours followed by washing and media replacement.

The amount of miRNA was assessed after 24 hours. The same starting amount of miRNA inhibitor pmol used for loading the exosomes and chemical transfection methods. Detailed procedure of animal studies and in vivo delivery of miRNA mimic loaded exosomes to miRNA knockout mice are described in supplementary method section. Based on data distribution, Kruskal-Wallis nonparametric test or one-way analyses of variance ANOVA were performed to make comparisons between different groups.

P values less than 0. Exosomes derived from B M The identity of isolated exosomes was confirmed by the presence of exosomal marker, CD63 Figure 1B. We further examined the B cell exosome with electron microscopy and confirmed the size range of less than nm Figure 1C. As previously described by Saunderson et al. A Average size of stimulated B cell-derived exosomes was 98nm with the mode of 84nm. B Isolated exosomes from murine B cells M C TEM image of B cell exosomes showed the diameter of less than nm.

The number of exosomes was quantified using NanoSight. The results represent three independent experiments. Exosomes derived from naive non-stimulated B cells had significantly lower miRNA levels compared to the parental cells Figure 2B. These observations, first, suggested that miRNA levels can be different between the parent cells and their exosomes depending on the cell type and, second, that stimulated B cell-derived exosomes have very low levels of miRNA Since utilizing exosomes as delivery vehicles should be tailored for specific exosome, specific cargo, and cell type, 15 we optimized the loading efficiency for murine B cell M Our results showed that stimulated B cell-derived exosomes were almost devoid of endogenous miRNA and they can be used as vehicles for delivery of exogenous miRNA nucleotides.

To optimize electroporation loading, B cell-derived exosomes were isolated with CD63 immunomagnetic beads and electroporated in the presence of miRNA mimics pmol and 1mM EDTA at various voltages 0. We used EDTA in the process because EDTA prevents interactions of aluminium ions with buffer components and macromolecules in the electroporation cuvette.

Voltages between 0. A miRNA mimic was introduced to the exosomes using various voltages 0. B Different amount of exosomes containing 0. The exosomal protein concentrations of 0. C Schematic experimental design for evaluating effect of different isolation method in exosome recovery.

To identify the most efficient exosome-to-miRNA ratio for loading of B cell-derived exosomes, we used different exosome concentrations 0. We found that the exosomal density of 0. To assess the functional ability of exosomal delivery of miRNA mimic, we loaded B cell-derived exosomes with miRNA mimic using optimal loading conditions.

Then exosomes were co-cultured with primary mouse hepatocytes for 6 hours, followed by washing off extra exosomes and replacement of medium. The readout after 24 hours showed that exosomes were able to successfully deliver miRNA mimic to primary mouse hepatocytes, indicated by the approximately fold increase in intracellular miRNA levels Figure 4A. B cell-derived exosomes were loaded with miRNA using optimal loading conditions. A The exosomes were co-cultured with primary mouse hepatocytes for 6 hours followed by washing and media replacement.

Next, we tested ability of exosomes to mediate miRNA mimic delivery in vivo. These results suggest in vivo delivery of miRNA by exosomes to the liver. Here, using the optimized conditions of exosome loading, we investigated whether B cells-derived exosomes can act as carriers to deliver a miRNA inhibitor to RAW Thus, the miRNA inhibitor was electroporated into B cell-derived exosomes using the optimized loading conditions.

Cells were seeded 1 day before treatment and different treatment conditions and controls were applied for 24 hours. Results are representative of three independent experiments. Moreover, B cell-derived exosomes appeared to be more efficient vehicles for miRNA inhibitor delivery compared to the conventional transfection reagents. Additional advantage of the exosome-based delivery is lack of cytotoxicity and cell death compared to the transfection reagents. Regarding the prominent role of miRNA in the signaling pathways of monocytes and macrophages, 20 , 45 miRNA mimic and miRNA inhibitor were chosen as the exogenous bio-macromolecules in this study.

We optimized electroporation conditions in terms of voltage, capacitance, exosome concentration, and re-isolation method for effective miRNA loading and recovery of B cell exosomes.

The promising efficiency of exosome loading was confirmed by co-culturing miRNA mimic loaded exosomes with primary mouse hepatocytes. The level of miRNA was increased more than fold in the primary mouse hepatocytes co-cultured with miRNA loaded exosome compared to the control mimic.

Furthermore, our in vivo study using miRNA knockout mice showed that exosomes mediated successful delivery of miRNA to the liver and to primary mouse hepatocytes at 10 minutes after IV injection. Altogether, these results provide convincing evidence that the exogenous miRNA mimic was effectively introduced into the exosomes and exosomes can mediate miRNA delivery to primary mouse hepatocytes in vivo and in vitro. Recent advances in understanding biological processes have broadened the scope of therapeutic options to RNA-based therapeutics.

However, most forms of RNA are inherently unstable and prone to RNase effect, potentially immunogenic, and negatively charged. This negative charge jeopardizes their effective delivery to the hydrophobic cellular membrane even in the presence of transfection reagents.

However, the present vectors have not been completely successful in clinical trials, as they induce immunogenicity and show cytotoxicity, particularly in repeated administrations. Moreover, in contrast with chemical transfection methods, exosome-based delivery showed no cytotoxicity. Exosome-based drug delivery confers a number of advantages over other vector or liposome-based delivery methods.

First, exosomes act as natural membranous nanocarriers of bio-macromolecules between cells and they can easily transpass the plasma membrane and mediate effective RNA delivery.

Autologous exosomes, which are non-immunogenic, would be a perfect source of personalized drug vehicles. Third, non-toxicity of exosomes compared to transfection reagents is another key advantage. In summary, our results provide evidence for exosome-based delivery of RNA-based therapeutics in vitro and in vivo. Although in this study we focused on the exosome-mediated delivery of miRNA mimic to the liver and primary hepatocytes, it would be interesting to assess the bio-distribution of exosome-mediated delivery in other organs.

In vivo findings of this study mechanistically showed that exosomes can mediate successful delivery of miRNA mimic to the liver and hepatocytes as early as 10 minutes after IV injection. The in vivo study was performed at a short time point based on the reported peak of bio-distribution of exosomes, miRNAs, and RNA interfaces in pharmacokinetics studies. Our results mechanistically demonstrated that B cell-derived exosomes could be used as efficient vehicles in RNA-based therapeutic strategies and provide proof of concept for using exosomes as efficient delivery nano-vehicles with minimal cytotoxicity.

This identifies exosomes as novel frontiers in expanding nanomedicine applications. Conflict of interest: The authors have declared that no conflict of interest exists. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.

Exosome‑delivered TRPP2 siRNA inhibits the epithelial‑mesenchymal transition of FaDu cells

Matteo, Pavia Italy,. Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues.

Exosome-mimetic nanoplatforms for targeted cancer drug delivery

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