RESUMO
Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.
Assuntos
Membrana Celular/química , Força Compressiva , Proteínas de Membrana/química , Estresse Mecânico , Humanos , Conformação ProteicaRESUMO
The present study was designed to test the hypothesis that programmed cell death-1 (PD-1) siRNA can downregulate PD-1 expression in macrophages in culture and in tumor tissues in mice and inhibit tumor growth in a mouse model. PD-1 siRNA was encapsulated in solid lipid nanoparticles (SLNs), and the physical properties of the resultant SLNs were characterized. The ability of the PD-1 siRNA-SLNs to downregulate PD-1 expression was confirmed in J774A.1 macrophages in culture and in tumor tissues in mice. Moreover, the antitumor activity of the PD-1 siRNA-SLNs was evaluated in a mouse model. The PD-1 siRNA-SLNs were roughly spherical, and their particle size, polydispersity index, and zeta potential were 141 ± 5 nm, 0.17 ± 0.02, and 20.7 ± 4.7 mV, respectively, with an siRNA entrapment efficiency of 98.9%. The burst release of the PD-1 siRNA from the SLNs was minimal. The PD-1 siRNA-SLNs downregulated PD-1 expression on J774A.1 macrophage cell surface as well as in macrophages in B16-F10 tumors pre-established in mice. In mice with pre-established B16-F10 tumors, the PD-1 siRNA-SLNs significantly inhibited the tumor growth, as compared with siRNA-SLNs prepared with non-functional, negative control siRNA. In conclusion, the PD-1 siRNA-SLNs inhibited tumor growth, likely related to their ability to downregulate PD-1 expression by tumor-associated macrophage (TAMs).
Assuntos
Lipídeos/administração & dosagem , Macrófagos/metabolismo , Nanopartículas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Animais , Regulação para Baixo , Camundongos , Neoplasias Experimentais/patologia , Receptor de Morte Celular Programada 1/genéticaRESUMO
Most small molecule chemotherapeutics must cross one or more cellular membrane barriers to reach their biochemical targets. Owing to the relatively low solubility of chemotherapeutics in the lipid membrane environment, high doses are often required to achieve a therapeutic effect. The resulting systemic toxicity has motivated efforts to improve the efficiency of chemotherapeutic delivery to the cellular interior. Toward this end, liposomes containing lipids with cationic head groups have been shown to permeabilize cellular membranes, resulting in the more efficient release of encapsulated drugs into the cytoplasm. However, the high concentrations of cationic lipids required to achieve efficient delivery remain a key limitation, frequently resulting in toxicity. Toward overcoming this limitation, here, we investigate the ability of ternary lipid mixtures to enhance liposomal delivery. Specifically, we investigate the delivery of the chemotherapeutic, doxorubicin, using ternary liposomes that are homogeneous at physiological temperature but have the potential to undergo membrane phase separation upon contact with the cell surface. This approach, which relies upon the ability of membrane phase boundaries to promote drug release, provides a novel method for reducing the overall concentration of cationic lipids required for efficient delivery. Our results show that this approach improves the performance of doxorubicin by up to 5-fold in comparison to the delivery of the same drug by conventional liposomes. These data demonstrate that ternary lipid compositions and cationic lipids can be combined synergistically to substantially improve the efficiency of chemotherapeutic delivery in vitro.
Assuntos
Doxorrubicina/química , Lipossomos/química , Propano/química , Propriedades de SuperfícieRESUMO
Cellular delivery of therapeutic macromolecules such as proteins, peptides, and nucleic acids remains limited due to inefficient transport across the cellular plasma membrane. Gap junction channels, composed of connexin proteins, provide a mechanism for direct transfer of small molecules across membranes, and recent evidence suggests that the transfer of larger, polymer-like molecules such as microRNAs may be possible. Here, we report direct evidence of gap junction-mediated transfer of polymeric macromolecules. Specifically, we examined the transport of dextran chains with molecular weights ranging from 10 to 70 kDa. We found that dextran chains of up to 40 kDa can diffuse through at least five cell layers in a gap junction-dependent manner within a 30 min time frame. Further, we evaluated the ability of connectosomes, cell-derived vesicles containing functional connexin proteins, to be loaded with dextran chains. By opening connexon hemichannel pores within the membranes of connectosomes, we found that 10 kDa dextran was loaded into more than 90% of vesicles, with reduced levels of loading for dextran chains of larger molecular weight. Upon delivering 10 kDa dextran-loaded connectosomes to cells, we further found that connectosomes transferred these membrane-impermeable molecules to the cellular cytosol with dramatically improved efficiency in comparison to the delivery of free, unencapsulated dextran. Collectively, these results reveal that polymeric macromolecules can be delivered to cells via gap junctions, suggesting that the gap junction route may be useful for the delivery of polymeric therapeutic molecules, such as nucleic acids and peptides.
Assuntos
Dextranos , Ácidos Nucleicos , Conexinas/química , Conexinas/metabolismo , Dextranos/metabolismo , Junções Comunicantes/metabolismo , Ácidos Nucleicos/metabolismo , Polímeros/metabolismoRESUMO
Chemotherapeutic delivery is limited by inefficient transport across cellular membranes. Here, we harness the cellular gap junction network to release therapeutic cargos directly into the cytosol. Specifically, cell-derived vesicles, termed connectosomes, contain gap junction transmembrane proteins that open a direct passageway to the cellular interior. Connectosomes were previously shown to substantially improve chemotherapeutic delivery in vitro. Here, we test connectosomes in vivo, using a murine breast tumor model. We demonstrate that connectosomes improve chemotherapeutic delivery to cellular targets within tumors by up to 16-fold, compared to conventional drug-loaded liposomes, suggesting an efficient alternative pathway for intracellular delivery.
Assuntos
Lipossomos , Neoplasias , Animais , Membrana Celular , Conexinas , Junções Comunicantes , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
Optimizing powder flow and compaction properties are critical for ensuring a robust tablet manufacturing process. The impact of flow and compaction properties of the active pharmaceutical ingredient (API) becomes progressively significant for higher drug load formulations, and for scaling up manufacturing processes. This study demonstrated that flow properties of a powder blend can be improved through API particle engineering, without critically impacting blend tabletability at elevated drug loadings. In studying a jet milled API (D50=24µm) and particle engineered wet milled API (D50=70µm and 90µm), flow functions of all API lots were similarly poor despite the vast difference in average particle size (ffc<4). This finding strays from the common notion that powder flow properties are directly correlated to particle size distribution. Upon adding excipients, however, clear trends in flow functions based on API particle size were observed. Wet milled API blends had a much improved flow function (ffc>10) compared with the jet milled API blends. Investigation of the compaction properties of both wet and jet milled powder blends also revealed that both jet and wet milled material produced robust tablets at the drug loadings used. The ability to practically demonstrate this uncommon observation that similarly poor flowing APIs can lead to a marked difference upon blending is important for pharmaceutical development. It is especially important in early phase development during API selection, and is advantageous particularly when material-sparing techniques are utilized.