Natural or Synthetic RNA Delivery: A Stoichiometric Comparison of Extracellular Vesicles and Synthetic Nanoparticles.
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ABSTRACT:
SUBMITTER: Murphy DE
PROVIDER: S-EPMC8023702 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundOral delivery remains unattainable for nucleic acid therapies. Many nanoparticle-based drug delivery systems have been investigated for this, but most suffer from poor gut stability, poor mucus diffusion and/or inefficient epithelial uptake. Extracellular vesicles from bovine milk (mEVs) possess desirable characteristics for oral delivery of nucleic acid therapies since they both survive digestion and traverse the intestinal mucosa.ResultsUsing novel tools, we comprehensively examine the intestinal delivery of mEVs, probing whether they could be used as, or inform the design of, nanoparticles for oral nucleic acid therapies. We show that mEVs efficiently translocate across the Caco-2 intestinal model, which is not compromised by treatment with simulated intestinal fluids. For the first time, we also demonstrate transport of mEVs in novel 3D 'apical-out' and monolayer-based human intestinal epithelial organoids (IEOs). Importantly, mEVs loaded with small interfering RNA (siRNA) induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in macrophages. Using inflammatory bowel disease (IBD) as an example application, we show that administration of anti-tumour necrosis factor alpha (TNFα) siRNA-loaded mEVs reduced inflammation in a IBD rat model.ConclusionsTogether, this work demonstrates that mEVs could either act as natural and safe systems for oral delivery or nucleic acid therapies, or inform the design of synthetic systems for such application.
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Project description:Alzheimer's disease is a progressive neurodegenerative disorder, with the strongest disease-associated changes observed at clinical or end-stage disease. Transcriptomic deregulation of miRNA expression can spread via "horizontal" RNA transfer through extracellular vesicles (EVs) to act in conjunction with proteins, leading to changes in mRNA, which can provide early signals to indicate forthcoming neuropathological changes in the brain. Here, we analysed the small RNA content, in particular, miRNA, contained in brain-derived EVs isolated from the frontal cortex of Alzheimer's subjects (n = 8) and neurological control subjects (n = 9). Brain-derived EVs were found to contain an upregulation of disease-associated miRNA. RNA species from brain-derived EVs were correlated with miRNA profiles obtained from matching total brain homogenate. These results provide a blueprint into the biological pathways potentially effected during disease that may be assisted by brain-derived EV RNA horizontal transfer.We also correlated the miRNA changes in the brain with those detected in peripheral EVs collected from serum of Alzheimer's disease patients (n = 23, and healthy controls, n = 23) and revealed a panel of miRNA that could be used as a liquid brain biopsy. Overall, our study provides the first interrogation of the small RNA contents in brain-derived EVs and how they could be used to understand the early pathological changes in Alzheimer's disease which will benefit the development of an early diagnostic blood test.
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Project description:Extracellular vesicles (EVs) are carriers of biological signals through export and delivery of RNAs and proteins. Of increasing interest is the use of EVs as a platform for delivery of biomolecules. Preclinical studies have effectively used EVs to treat a number of diseases. Uniquely, endogenous machinery within cells can be manipulated in order to produce desirable loading of cargo within secreted EVs. In order to inform the development of such approaches, an understanding of the cellular mechanisms by which cargo is sorted to EVs is required. Here, the current knowledge of cargo sorting within EVs is reviewed. Here is given an overview of recent bioengineering approaches that leverage these advances. Methods of externally manipulating EV cargo are also discussed. Finally, a perspective on the current challenges of EVs as a drug delivery platform is offered. It is proposed that standardized bioengineering methods for therapeutic EV preparation will be required to create a well-defined clinical product.
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Project description:We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.
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Project description:The rapid and non-invasive pulmonary drug delivery (PDD) has attracted great attention compared to the other routes. However, nanoparticle platforms, like liposomes (LPs) and extracellular vesicles (EVs), require extensive reformulation to suit the requirements of PDD. LPs are artificial vesicles composed of lipid bilayers capable of encapsulating hydrophilic and hydrophobic substances, whereas EVs are natural vesicles secreted by cells. Additionally, novel LPs-EVs hybrid vesicles may confer the best of both. The preparation methods of EVs are distinguished from LPs since they rely mainly on extraction and purification, whereas the LPs are synthesized from their basic ingredients. Similarly, drug loading methods into/onto EVs are distinguished whereby they are cell- or non-cell-based, whereas LPs are loaded via passive or active approaches. This review discusses the progress in LPs and EVs as well as hybrid vesicles with a special focus on PDD. It also provides a perspective comparison between LPs and EVs from various aspects (composition, preparation/extraction, drug loading, and large-scale manufacturing) as well as the future prospects for inhaled therapeutics. In addition, it discusses the challenges that may be encountered in scaling up the production and presents our view regarding the clinical translation of the laboratory findings into commercial products.
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