Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems.

Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP-siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP-siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP-siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a "currant bun" morphology as visualized by cryo-TEM. This structure is fully consistent with LNP-siRNA structure predicted by molecular modeling.

Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. In the present study, we used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy between fluorescently labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly before endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only present in docked vesicles; unexpected residual "orphan SNARE complexes" also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis-conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatiotemporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid intermolecular interactions between small proportions of protein partners in cellular subcompartments.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.

Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005 mg kg(-1) in mice and less than 0.03 mg kg(-1) in non-human primates.

Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells.

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

Rational design of cationic lipids for siRNA delivery.

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.