RESUMO
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.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Hipocampo/citologia , Neurônios/citologia , Proteínas SNARE/metabolismo , Sinapses/fisiologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cerulenina/metabolismo , Estimulação Elétrica , Embrião de Mamíferos , Exocitose/efeitos dos fármacos , Exocitose/genética , Feminino , Proteínas de Fluorescência Verde/genética , Processamento de Imagem Assistida por Computador , Masculino , Modelos Biológicos , Mutação/genética , Dinâmica não Linear , Técnicas de Patch-Clamp , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos , Proteínas SNARE/genética , Sinapses/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Fatores de Tempo , Transfecção , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species. In particular, the low potency of LNP containing DLinDAP can be attributed to hydrolysis by endogenous lipases following uptake. LNP containing DLinKC2-DMA, DLinKDMA, or DLinDMA, which lack ester linkages, are not vulnerable to lipase digestion and facilitate much more potent gene silencing. The superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to higher uptake and improved ability to stimulate siRNA release from endosomes subsequent to uptake. FROM THE CLINICAL EDITOR: This study reports on the in vivo gene silencing potency of lipid nanoparticle-siRNA systems containing ionizable cationic lipids. It is concluded that the superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to their higher uptake thus improved ability to stimulate siRNA release from endosome.
Assuntos
Lipídeos/química , Macrófagos/metabolismo , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Cátions/química , Cátions/metabolismo , Linhagem Celular , Clatrina/metabolismo , Endocitose , Lipase/metabolismo , Metabolismo dos Lipídeos , Camundongos , Pinocitose , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Ribonucleases/metabolismoRESUMO
Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.
Assuntos
Lipídeos/química , Microfluídica/métodos , Nanopartículas/química , Tamanho da Partícula , Triglicerídeos , ÁguaRESUMO
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.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Cátions/química , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Lipídeos/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Western Blotting , Medula Óssea , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Endocitose , Citometria de Fluxo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Lipossomos , Fígado/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Here we report that highly concentrated cationic lipid/helper lipid-nucleic acid complexes (lipoplexes) can facilitate reproducible delivery of a variety of oligonucleotides and plasmids to chicken embryos or to mouse embryonic mesenchyme. Specifically, liposomes composed of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)/1,2 dioleoyl glycero-3-phosphorylethanolamine (DOPE) prepared at 18-mM concentrations produced high levels of transfection of exogenous genes in vivo and in vitro. Furthermore, we report sufficient uptake of plasmids expressing interference RNA to decrease expression of both exogenous and endogenous genes. The simplicity of preparation, implementation, and relatively low toxicity of this transfection reagent make it an attractive alternative for developmental studies in post-gastrulation vertebrate embryos.
Assuntos
Lipossomos/química , Transfecção/métodos , Animais , Embrião de Galinha , Embrião de Mamíferos , Lipossomos/administração & dosagem , Mesoderma/metabolismo , Camundongos , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/químicaRESUMO
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.
Assuntos
Inativação Gênica , Lipídeos/administração & dosagem , Fígado/fisiologia , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Aminas/química , Animais , Feminino , Terapia Genética/métodos , Humanos , Cinética , Lipídeos/química , Lipossomos/administração & dosagem , Lipossomos/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , RNA Interferente Pequeno/químicaRESUMO
We have developed a minimally-perturbing strategy that enables labeling and subcellular visualization of endogenous dendritic receptors on live, wild-type neurons. Specifically, calcium-permeable non-NMDA glutamate receptors expressed in hippocampal neurons can be targeted with this novel synthetic tri-functional molecule. This ligand-directed probe was targeted towards AMPA receptors and bears an electrophilic group for covalent bond formation with an amino acid side chain on the extracellular side of the ion channel. This molecule was designed in such a way that the use-dependent, polyamine-based ligand accumulates the chemically-reactive group at the extracellular side of these polyamine-sensitive receptors, thereby allowing covalent bond formation between an electrophilic moiety on the nanoprobe and a nucleophilic amino acid sidechain on the receptor. Bioconjugation of this molecule results in a stable covalent bond between the nanoprobe and the target receptor. Subsequent photolysis of a portion of the nanoprobe may then be employed to effect ligand release allowing the receptor to re-enter the non-liganded state, all the while retaining the fluorescent beacon for visualization. This technology allows for rapid fluorescent labeling of native polyamine-sensitive receptors and further advances the field of fluorescent labeling of native biological molecules.
Assuntos
Corantes Fluorescentes/química , Hipocampo/química , Receptores de AMPA/análise , Animais , Células Cultivadas , Humanos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/químicaRESUMO
Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Delta9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Delta9 displayed smaller amperometric "foot-current" currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Delta9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.
Assuntos
Fusão de Membrana , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Sinalização do Cálcio , Bovinos , Células Cromafins/citologia , Células Cromafins/metabolismo , Exocitose , CinéticaRESUMO
The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.
Assuntos
Células Cromafins/ultraestrutura , Grânulos Cromafim/química , Grânulos Cromafim/ultraestrutura , Vesículas Secretórias/química , Vesículas Secretórias/ultraestrutura , Animais , Catecolaminas/análise , Bovinos , Células Cultivadas , Células Cromafins/química , Células Cromafins/fisiologia , Grânulos Cromafim/efeitos dos fármacos , Exocitose , Membranas Intracelulares/ultraestrutura , Levodopa/farmacologia , Neurotransmissores/análise , Técnicas de Patch-Clamp , Reserpina/farmacologia , Vesículas Secretórias/efeitos dos fármacosRESUMO
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.
Assuntos
Nanopartículas Metálicas , Microfluídica , Fosfatidilcolinas/química , Microscopia Crioeletrônica , Ouro/química , Microscopia Eletrônica de Transmissão , RNA Interferente Pequeno/químicaRESUMO
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.
RESUMO
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.
Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , Desenho de Fármacos , Lipídeos/química , RNA Interferente Pequeno/química , Transfecção/métodos , Cátions , RNA Interferente Pequeno/administração & dosagemRESUMO
Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of catecholamines near the cell surface and not due to slow dissociation from the granular matrix.
Assuntos
Células Cromafins/fisiologia , Exocitose , Microeletrodos , Platina , Análise Serial de Tecidos/métodos , Laranja de Acridina/análise , Laranja de Acridina/metabolismo , Animais , Transporte Biológico , Catecolaminas/análise , Catecolaminas/metabolismo , Bovinos , EletroquímicaRESUMO
Focal release of cytotoxic proteins by eosinophils onto the target surface plays an important role in parasite killing. Degranulation was stimulated by intracellular application of calcium and guanosine 5'-3-O-(thio)triphosphate via the recording patch pipette or via streptolysin-O permeabilization. Exocytotic fusion was monitored by capacitance measurements, whereas release of fluorescent weak bases, which accumulate selectively within eosinophil granules, was followed by fluorescence imaging. Several distinct types of granule fusion events were directly observed by simultaneous capacitance and fluorescence measurements. These are fusion of a single granule with the plasma membrane, intracellular granule-granule fusion, fusion of large compounds of pre-fused granules with the plasma membrane (compound exocytosis), and sequential fusion of granules to granules previously fused to the plasma membrane. Extensive granule-granule fusion was also observed by electron microscopy of permeabilized cells. All these fusion mechanisms contribute to focal release. The coexistence of distinct modes of exocytosis suggests that their regulation may modulate effector functions of eosinophils during helminth infection and allergic response.
Assuntos
Eosinófilos/fisiologia , Eosinófilos/ultraestrutura , Exocitose , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Grânulos Citoplasmáticos/fisiologia , Grânulos Citoplasmáticos/ultraestrutura , Capacitância Elétrica , Condutividade Elétrica , Corantes Fluorescentes , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cavalos/sangue , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Microscopia Eletrônica , Microscopia de Fluorescência , Técnicas de Patch-ClampRESUMO
Dynamics of degranulation was studied in horse eosinophils by patch clamp capacitance measurements. Degranulation was stimulated by intracellular application of calcium, and GTPgammaS or guanosine 5'-(beta,gamma-imido)triphosphate at different concentrations via the patch pipette. Degranulation was quantified by measuring the delay time between the beginning of intracellular perfusion and the first exocytotic event, determining the distribution of time intervals between fusion events and the capacitance step size distributions under the different conditions. The degranulation dynamics could be well reproduced using a computer model assuming three independent rate constants for granule-plasma membrane fusion, granule fusion with already exocytosed granules, and intracellular granule-granule fusion. The rate of granule-plasma membrane fusion is sensitive to both, the GTP analog and [Ca2+]i. The rate of granule-exocytosed granule fusion is sensitive to [Ca2+]i but insensitive to the GTP analogs, and the rate of granule-to-granule fusion is sensitive to the GTP analog but insensitive to [Ca2+]i. Granule fusions with the three different target compartments thus involve different regulatory mechanisms.