Conspectus The ability of gene or RNA interference (RNAi) delivery to increase or decrease virtually any protein in a cell opens the path for cures to most diseases that afflict humans. However, their high molecular weight, anionic nature, and instability in the presence of enzymes, pose major obstacles to nucleic acid delivery and frustrates their use as human therapies. This Account describes current ideas on the mechanisms in non-viral nucleic acid delivery and how lipidic and polymeric carriers overcome some of the critical barriers to delivery. A multitude of polymeric and lipidic vectors have been developed over the last 20 years, only a small fraction of them have progressed into clinical trials. Given that none of these vectors has received FDA approval, indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. Despite this, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery. A smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane are required. The barriers to siRNA delivery are fewer: siRNA is more readily released from the carrier, siRNA is more resistant to enzymatic degradation and the target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RISC formation and activity is unknown. This makes specific targeting of the RISC for more efficient siRNA delivery difficult. Furthermore, identifying the factors favoring the binding of siRNA to Ago-2 and understanding how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity could increase siRNA delivery efficiency. In this manuscript we concisely review the current synthetic vectors and for a few of these, propose alternative strategies. We suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we raise the question if some carriers are delivering the siRNA to cells capable of repackaging the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells where the siRNA effect is manifest. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.

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Mesenchymal stem cell (MSC)-derived exosomes mediate tissue regeneration in a variety of diseases including ischemic heart injury, liver fibrosis, and cerebrovascular disease. Despite an increasing number of studies reporting the therapeutic effects of MSC exosomes, the underlying molecular mechanisms and their miRNA complement are poorly characterized. Here we microRNA (miRNA)-profiled MSC exosomes and conducted a network analysis to identify the dominant biological processes and pathways modulated by exosomal miRNAs. At a system level, miRNA-targeted genes were enriched for (cardio)vascular and angiogenesis processes in line with observed cardiovascular regenerative effects. Targeted pathways were related to Wnt signaling, pro-fibrotic signaling via TGF-β and PDGF, proliferation, and apoptosis. When tested, MSC exosomes reduced collagen production by cardiac fibroblasts, protected cardiomyocytes from apoptosis, and increased angiogenesis in HUVECs. The intrinsic beneficial effects were further improved by virus-free enrichment of MSC exosomes with network-informed regenerative miRNAs capable of promoting angiogenesis and cardiomyocyte proliferation. The data presented here help define the miRNA landscape of MSC exosomes, establish their biological functions through network analyses at a system level, and provide a platform for modulating the overall phenotypic effects of exosomes.

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A potential siRNA carrier for pulmonary gene delivery was assessed by encapsulating siRNA into biodegradable polyester nanoparticles consisting of tertiary-amine-modified polyvinyl alcohol (PVA) backbones grafted to poly(d,l-lactide-co-glycolide) (PLGA). The resulting siRNA nanoparticles were prepared using a solvent displacement method that offers the advantage of forming small nanoparticles without using shear forces. The nanoparticles were characterized with regard to particle size, zeta-potential, and degradation at pH 7.4 using dynamic and static light scattering. SiRNA release studies were performed and correlated to the nanoparticle degradation. In vitro knockdown of firefly luciferase reporter gene was used to assess the potential of the nanoparticles as siRNA carriers in a human lung epithelial cell line, H1299 luc. The amine-modified-PVA-PLGA/siRNA nanoparticles form 150-200 nm particles with zeta-potentials of +15-+20 mV in phosphate buffered saline (PBS). Break down of the nanoparticles was seen within 4 h in PBS with sustained release of siRNA. These nanoparticles have achieved 80-90% knockdown of a luciferase reporter gene with only 5 pmol anti-luc siRNA, even after nebulization. Hence we conclude that amine-modified-PVA-PLGA/siRNA nanoparticles could be a promising siRNA carrier for pulmonary gene delivery due to their fast degradation and potent gene knockdown profile.

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A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs.

We report the synthesis and biophysical characterization of a novel class of zwitterionic lipids (ZL) with head groups containing a 3° or 4° amine and carboxylate. ZL form stable liposomes that exhibit head group dependent, pH-responsive biophysical characteristics. These lipids may be suitable components for drug delivery applications due to their ease of synthesis and unique pH-dependent properties.