17 research outputs found

    Profiling and exploiting lipid-based nanoparticles in vitro and in vivo

    Get PDF
    One of the major hurdles for therapeutic applications is the efficient delivery of bioactive molecules to the site of action. The high flexibility and biosafety of lipid-based nanoparticles has greatly enhanced their employment as delivery systems not only for synthetic but also for natural molecules such as proteins and nucleic acids. This thesis was brought about to investigate the nucleic acid delivery potential of synthetic lipid-based nanoparticles as well as to look into the composition and delivery patterns of their natural counterparts, extracellular vesicles (EVs), in order to set ground for future lipid-based therapeutic interventions. Firstly, in Paper I we explored the potency of a lipid-based delivery agent, Lipofectamine 2000 which after being frozen and thawed showed orders of magnitude higher nucleic acid delivery efficiency in vitro and in vivo than the non-frozen counterpart. This effect was consistent across different cryo-manipulations, cell lines and also various types of nucleic acid. Further analysis with different methodologies revealed that the underlying potency plausibly relies on the elevated sedimentation and spreading of the complexes and/or relates to the specific structure or composition of the carrier. These findings illustrate that a simple freeze-thawing procedure allows to drastically reduce the amount of transfection reagent for cellular nucleic acid delivery, whilst not losing the desired activity. Secondly, we shifted our focus to natural lipid-based carriers, EVs in order to shed light on the vesicular and non-vesicular (non-EV) small RNA patterns and their relation to the EV proteome (Paper II and III). Though the studies exploited different EV enrichment methods the relative depletion of vesicular small RNAs was confirmed in both instances. A detailed analysis of the secretory repertoire of small RNAs showed a significant depletion of microRNA (miRNA) sequences, matching well with the depletion of “miRNA related” proteins in EVs. The relative expression level of cellular, EV and non-EV miRNAs correlated well and though some differentially expressed (DE) miRNAs were detected, these had a relatively low expression in both the source cells as well as in the secretory fractions. We also quantified the total level of selected miRNAs in EVs and non-EV fraction investigating both the basal as well as overexpressed levels and could verify that the vast majority of mature miRNA is secreted to the non-EV portion of the secretome. Paper IV was brought about to gain a comprehensive overview of the biodistribution of exogenous EVs. This study confirmed that fluorescent lipophilic dyes are suitable for membrane labelling and in vivo tracking of EVs. The general biodistribution pattern of EVs was seen to follow a common mononuclear phagocytic system (MPS) uptake pattern with the majority of EVs accumulating in the liver, spleen and lungs. Nevertheless, depending on the cell source, administration route, dose and the presence of targeting moieties this distribution could be altered. The present findings are important to gain a thorough understanding of the nucleic acid delivery capacity of lipid-based nanoparticles, especially EVs and thereby progress their employment as therapeutic nucleic acid carriers

    Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

    Get PDF
    Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.Peer reviewe

    Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

    Get PDF
    Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

    Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

    Get PDF
    While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

    mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells

    Get PDF
    mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy

    Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion

    No full text
    The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2 = 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future

    Proteome profiling of whole plasma and plasma-derived extracellular vesicles facilitates the detection of tissue biomarkers in the non-obese diabetic mouse Authors

    Get PDF
    The mechanism by which pancreatic beta cells are destroyed in type 1 diabetes (T1D) remains to be fully understood. Recent observations indicate that the disease may arise because of different pathobiological mechanisms (endotypes). The discovery of one or several protein biomarkers measurable in readily available liquid biopsies (e.g. blood plasma) during the pre-diabetic period may enable personalized disease interventions. Recent studies have shown that extracellular vesicles (EVs) are a source of tissue proteins in liquid biopsies. Using plasma samples collected from pre-diabetic non-obese diabetic (NOD) mice (an experimental model of T1D) we addressed if combined analysis of whole plasma samples and plasma-derived EV fractions increases the number of unique proteins identified by mass spectrometry (MS) compared to the analysis of whole plasma samples alone. LC-MS/MS analysis of plasma samples depleted of abundant proteins and subjected to peptide fractionation identified more than 2300 proteins, while the analysis of EV-enriched plasma samples identified more than 600 proteins. Of the proteins detected in EV-enriched samples, more than a third were not identified in whole plasma samples and many were classified as either tissue-enriched or of tissue-specific origin. In conclusion, parallel profiling of EV-enriched plasma fractions and whole plasma samples increases the overall proteome depth and facilitates the discovery of tissue-enriched proteins in plasma. If applied to plasma samples collected longitudinally from the NOD mouse or from models with other pathobiological mechanisms, the integrated proteome profiling scheme described herein may be useful for the discovery of new and potentially endotype specific biomarkers in T1D.Medicin
    corecore