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1.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
2.
DNA Cell Biol ; 38(10): 1048-1055, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433200

RESUMO

DNA condensed agents can improve the transfection efficiency of the cationic liposome delivery system. However, various condensed agents have distinct transfection efficiency and cellular cytotoxicity. The object of this study was to screen the optimal agents with the high transfection efficiency and low cytotoxicity from four polymer compressive materials, polyethylenimine (PEI), chitosan, poly-l-lysine (PLL), and spermidine. DNA was precompressed with these four agents and then combined to cationic liposomes. Subsequently, the entrapment and transfection efficiency of the obtained complexes were investigated. Finally, the particle sizes, cytotoxicity, and endocytosis fashion of these copolymers (Lipo-PEI, Lipo-chitosan, Lipo-PLL, and Lipo-spermidine) were examined. It was found that these four copolymers had significantly lower cytotoxicity and higher transfection efficiency (45.5%, 42.4%, 36.8%, and 47.4%, respectively) than those in the control groups. The transfection efficiency of Lipo-PEI and Lipo-spermidine copolymers were better than the other two copolymers. In 293T cells, nystatin significantly inhibited the transfection efficiency of Lipo-PEI-DNA and Lipo-spermidine-DNA (51.88% and 46.05%, respectively), which suggest that the endocytosis pathway of Lipo-spermidine and Lipo-PEI copolymers was probably caveolin dependent. Our study indicated that these dual-degradable copolymers especially liposome-spermidine copolymer could be used as the potential biocompatible gene delivery carriers.


Assuntos
Quitosana/química , Lipossomos/química , Polietilenoimina/química , Polilisina/química , Espermidina/química , Transfecção/métodos , Cátions , Caveolina 1/genética , Caveolina 1/metabolismo , Quitosana/metabolismo , Colesterol/química , Colesterol/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Células HEK293 , Humanos , Lipossomos/metabolismo , Nistatina/farmacologia , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/metabolismo , Polietilenoimina/metabolismo , Polilisina/metabolismo , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Espermidina/metabolismo
3.
Chem Commun (Camb) ; 55(58): 8434-8437, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31259350

RESUMO

Phosphatidylcholine is the main component of liposomes and other phospholipid-based nanocarriers in drug delivery. However, the functions and applications of these nanocarriers are extremely limited by conventional phospholipids. Here we report novel disulfide phosphatidylcholines (SS-PCs) and SS-PC based liposomes (SS-LPs) used as alternatives to traditional phospholipids and liposomes.


Assuntos
Dissulfetos/química , Portadores de Fármacos/química , Lipossomos/química , Fosfatidilcolinas/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Dissulfetos/síntese química , Dissulfetos/metabolismo , Doxorrubicina/química , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Humanos , Lipossomos/síntese química , Lipossomos/metabolismo , Camundongos , Oxirredução , Fosfatidilcolinas/síntese química , Fosfatidilcolinas/metabolismo
4.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
5.
Enzyme Microb Technol ; 128: 40-48, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186109

RESUMO

The overuse and misuse of antibiotics in treating bacterial infections cause the rapid emergence of drug-resistant bacteria, suggesting that the development of alternative strategies to control antibiotic-resistant bacteria is urgently needed. Endolysins are bacteriophage-encoded enzymes that can degrade peptidoglycan in bacterial cell walls, and they have great potential as alternative antimicrobial agents. However, exogenous application of recombinant endolysin is limited to Gram-positive bacteria because endolysins cannot penetrate the outer membrane of Gram-negative bacteria. Here, a liposome-mediated endolysin encapsulation system was developed, and its ability to penetrate the outer membrane of Gram-negative bacteria was tested. The phage-derived endolysin BSP16Lys was isolated, characterized, and used for encapsulation into a cationic liposome comprised of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and hexadecylamine. The BSP16Lys-encapsulated liposome had a high zeta potential value (over 30 mV) with an average diameter of 303 nm. The encapsulation efficiency of BSP16Lys into the liposome was 35.27%. Salmonella Typhimuriumand Escherichia coli cells treated with BSP16Lys-encapsulated liposomes showed 2.2-log CFU/mL and 1.6-log CFU/mL reductions in the viable cell numbers, respectively, without treatment of a membrane permeabilizer. These results showed potential for liposome-mediated delivery of endolysin for exogenous application against Gram-negative bacteria.


Assuntos
Antibacterianos/metabolismo , Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Escherichia coli/efeitos dos fármacos , Lipossomos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Antibacterianos/síntese química , Parede Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Lipossomos/síntese química , Peptidoglicano/metabolismo
6.
Nat Commun ; 10(1): 2370, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147549

RESUMO

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Forma das Organelas/genética , Autofagia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico/genética
7.
Nat Commun ; 10(1): 2334, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133642

RESUMO

Pseudomonas aeruginosa, a significant opportunistic pathogen, can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals leads to altered biofilm formation and increased tolerance to various antibiotics, and requires the histidine kinase PA1396. Here, we show that the membrane-associated sensory input domain of PA1396 has five transmembrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds to develop novel adjuvants improving the efficacy of existing antibiotics.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ácidos Graxos Insaturados/metabolismo , Histidina Quinase/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Animais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/imunologia , Histidina Quinase/genética , Humanos , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Mutagênese , Fosforilação , Polimixinas/farmacologia , Polimixinas/uso terapêutico , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991632

RESUMO

The development of effective nanosystems for drug delivery represents a key challenge for the improvement of most current anticancer therapies. Recent progress in the understanding of structure and function of extracellular vesicles (EVs)-specialized membrane-bound nanocarriers for intercellular communication-suggests that they might also serve as optimal delivery systems of therapeutics. In addition to carrying proteins, lipids, DNA and different forms of RNAs, EVs can be engineered to deliver specific bioactive molecules to target cells. Exploitation of their molecular composition and physical properties, together with improvement in bio-techniques to modify their content are critical issues to target them to specific cells/tissues/organs. Here, we will discuss the current developments in the field of animal and plant-derived EVs toward their potential use for delivery of therapeutic agents in different pathological conditions, with a special focus on cancer.


Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/química , Preparações Farmacêuticas/administração & dosagem , Animais , Portadores de Fármacos/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Lipossomos/química , Lipossomos/metabolismo , Plantas/química , Plantas/metabolismo , RNA Interferente Pequeno/administração & dosagem
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 217: 278-287, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30952094

RESUMO

By using first-principles approaches based on Density Functional Theory, we explore the possibility of using dendritic macromolecular structures as carriers of the doxorubicin anticancer drug. In particular, we consider macromolecular cavities of different sizes composed of phenylene-, thiophene-, phenyl-cored thiophen- and thioazole-based dendrimers. The comparison between the optimized molecular geometries of the monomers and of the host-guest complexes reveals that only slight structural changes are observed in doxorubicin upon complexation. Also, the encapsulation energies for the host-guest complexes suggest that these systems are of potential use for pharmacology applications in vivo. The interaction of the guest doxorubicin with the macromolecular cavities exploits different types of weak intermolecular forces including σ, π and hydrogen bond interactions. The electronic structure of these complexes is discussed, with particular emphasis placed on the role of the charge distribution and the nature of the frontier molecular orbitals in the encapsulation process. Spectroscopic properties of these complexes are derived to facilitate their detection in laboratory and in vivo. These include IR vibrational frequencies, absorption wavelengths and relative oscillator strengths for the main transitions in the UV-Vis spectrum.


Assuntos
Antibióticos Antineoplásicos/química , Dendrímeros/química , Doxorrubicina/química , Lipossomos/química , Substâncias Macromoleculares/química , Modelos Teóricos , Antibióticos Antineoplásicos/metabolismo , Benchmarking , Cápsulas , Dendrímeros/metabolismo , Doxorrubicina/metabolismo , Lipossomos/metabolismo , Substâncias Macromoleculares/metabolismo , Teoria Quântica , Termodinâmica
10.
Contrast Media Mol Imaging ; 2019: 2645928, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30956626

RESUMO

In solid tumors, rapid local intravascular release of anticancer agents, e.g., doxorubicin (DOX), from thermosensitive liposomes (TSLs) can be an option to overcome poor extravasation of drug nanocarriers. The driving force of DOX penetration is the drug concentration gradient between the vascular compartment and the tumor interstitium. In this feasibility study, we used fibered confocal fluorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumor after its intravascular release from TSLs, Thermodox®. Cell uptake kinetics of the released DOX was quantified, along with an in-depth assessment of released-DOX penetration using an evolution model. A subcutaneous rat R1 rhabdomyosarcoma xenograft was used. The rodent was positioned in a setup including a water bath, and FCFM identification of functional vessels in the tumor tissue was applied based on AngioSense. The tumor-bearing leg was immersed in the 43°C water for preheating, and TSLs were injected intravenously. Real-time monitoring of intratumoral (i.t.) DOX penetration could be performed, and it showed the progressing DOX wave front via its native fluorescence, labeling successively all cell nuclei. Cell uptake rates (1/k) of 3 minutes were found (n=241 cells), and a released-DOX penetration in the range of 2500 µm2·s-1 was found in the tumor extravascular space. This study also showed that not all vessels, identified as functional based on AngioSense, gave rise to local DOX penetration.


Assuntos
Doxorrubicina/farmacocinética , Hipertermia Induzida , Lipossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Cinética , Microscopia Confocal , Ratos , Rabdomiossarcoma/metabolismo
11.
ACS Appl Mater Interfaces ; 11(17): 15389-15400, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951282

RESUMO

The antimicrobial effects of Laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ potential measurements, and confocal microscopy. As a result of the net negative charge of Laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the Laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite their net positive charge, peptide-loaded Laponite nanoparticles did not kill Gram-negative Escherichia coli bacteria or disrupt anionic model liposomes. They did however cause bacteria flocculation, originating from the interaction of Laponite and bacterial lipopolysaccharide (LPS). Free LL-37, in contrast, is potently antimicrobial through membrane disruption but does not induce bacterial aggregation in the concentration range investigated. Through LL-37 loading of Laponite nanoparticles, the combined effects of bacterial flocculation and membrane lysis are observed. However, bacteria aggregation seems to be limited to Gram-negative bacteria as Laponite did not cause flocculation of Gram-positive Bacillus subtilis bacteria nor did it bind to lipoteichoic acid from bacterial envelopes. Taken together, the present investigation reports several novel phenomena by demonstrating that nanoparticle charge does not invariably control membrane destabilization and by identifying the ability of anionic Laponite nanoparticles to effectively flocculate Gram-negative bacteria through LPS binding. As demonstrated in cell experiments, such aggregation results in diminished LPS-induced cell activation, thus outlining a promising approach for confinement of infection and inflammation caused by such pathogens.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Nanopartículas/química , Silicatos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Nanopartículas/metabolismo , Tamanho da Partícula , Silicatos/metabolismo
12.
J Agric Food Chem ; 67(19): 5544-5551, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31007021

RESUMO

The purpose of the study was to investigate the transepithelial transport route of Arg-Leu-Ser-Phe-Asn-Pro (RLSFNP), a milk-derived angiotensin-converting enzyme (ACE) inhibitory peptide, and to encapsulate RLSFNP in a liposome to improve its intestinal bioavailability. The transport route was investigated using transport inhibitors in a human intestinal Caco-2 cell monolayer model. Sodium azide and wortmannin significantly reduced the permeability of RLSFNP ( P < 0.01), indicating that energy-dependent transcytosis is involved in the transport of RLSFNP across Caco-2 cells. The hexapeptide RLSFNP was then embedded in liposomes, and the RLSFNP liposome was characterized. Afterward, the cellular uptake and transepithelial transport ability of the RLSFNP liposome across Caco-2 cell monolayers was observed. The results demonstrated that the RLSFNP liposome was successfully prepared, having a significant sustained release and storage capability. The RLSFNP liposome can be absorbed by Caco-2 cells, with an increased intestinal absorption of RLSFNP compared to RLSFNP alone. The results showed a new way to improve RLSFNP intestinal bioavailability.


Assuntos
Células Epiteliais/metabolismo , Lipossomos/química , Peptídeos/metabolismo , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Transporte Biológico , Células CACO-2 , Composição de Medicamentos , Humanos , Mucosa Intestinal/metabolismo , Lipossomos/metabolismo , Modelos Biológicos , Peptídeos/química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo
13.
Nat Commun ; 10(1): 1952, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028251

RESUMO

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.


Assuntos
Toxinas Biológicas/metabolismo , Yersinia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Lipossomos/metabolismo , Toxinas Biológicas/genética , Yersinia/genética
14.
Nat Commun ; 10(1): 1800, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996302

RESUMO

Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (ß-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.


Assuntos
Células Artificiais/metabolismo , Permeabilidade da Membrana Celular , Lipossomos/metabolismo , Microfluídica/métodos , Células Artificiais/química , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Dispositivos Lab-On-A-Chip , Lipossomos/química , Microfluídica/instrumentação , Transição de Fase
15.
Nat Commun ; 10(1): 1608, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962439

RESUMO

Membrane traffic operates by vesicles that bud from precursor organelles and are transported to their target compartment where they dock and fuse. Targeting requires tethering factors recruited by small GTPases and phosphoinositides whereas fusion is carried out by SNARE proteins. Here we report that vesicles containing the Q-SNAREs syntaxin 13 (Stx13) and syntaxin 6 (Stx6) together are targeted to a different endosomal compartment than vesicles containing only Stx6 using injection of artificial vesicles. Targeting by Stx6 requires Vps51, a component of the GARP/EARP tethering complexes. In contrast, targeting by both Stx6 and Stx13 is governed by Vps13B identified here as tethering factor functioning in transport from early endosomes to recycling endosomes. Vps13B specifically binds to Stx13/Stx6 as well as to Rab14, Rab6, and PtdIns(3)P. We conclude that SNAREs use a combinatorial code for recruiting tethering factors, revealing a key function in targeting that is independent of SNARE pairing during fusion.


Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Lipossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas Qa-SNARE/metabolismo , Animais , Cercopithecus aethiops , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Microscopia Intravital , Microscopia Confocal , Ligação Proteica/fisiologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem com Lapso de Tempo , Células Vero
16.
Analyst ; 144(9): 3030-3037, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30901016

RESUMO

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.


Assuntos
Transporte Biológico/fisiologia , Corantes Fluorescentes/química , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Rodaminas/química , Animais , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Células COS , Bovinos , Linhagem Celular Tumoral , Cercopithecus aethiops , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal/métodos , Fragmentos de Peptídeos/metabolismo , Fosfatidiletanolaminas/síntese química , Fosfatidiletanolaminas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rodaminas/síntese química , Rodaminas/metabolismo , Soroalbumina Bovina/química
17.
Biosystems ; 179: 48-54, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851346

RESUMO

We adopt a model in which the time evolution of a solution-charged liposome with selective permeability is seen as a sequence of two-stage cycles. In the first stage the lipid vesicle with a certain input concentration of the active substance is swelling up in an osmotic process until it reaches a size that one pore is formed on its surface. The second stage follows, where the vesicle content is partially eliminated through the pore to induce a decrease of the vesicle volume up to its original size. A new cycle is then initiated with the remaining concentration of the active substance for input, and so on. The process is described mathematically by an ordinary differential equation for the first stage and by a system of three such equations for the second but the numerical treatment is quite demanding for the system because this is stiff. We use a recently proposed Runge-Kutta method of a special form which, in contrast to the standard versions, shares the salient advantages of being both explicit and A-stable. The results on a case with realistic biophysical parameters are given for the swelling time in each cycle, total number of cycles, and the active substance amount delivered during each cycle. The model can be successfully used in a large variety of situations of direct practical interest, in particular when the active substance is pharmacological.


Assuntos
Simulação por Computador , Lipossomos/química , Lipossomos/metabolismo , Matemática , Fosfatidilcolinas/química , Humanos , Permeabilidade
18.
Chemistry ; 25(33): 7798-7814, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30889296

RESUMO

Synthesizing proteins inside liposomes and other microcompartments is a well-established practice. However, the origin of this research is not from the distant past, dating back to 1999-2004, when the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in bottom-up approaches), and, in particular, it strongly contributes to the construction of artificial cell-like systems. These systems, often called "synthetic cells", can be used to model cellular processes, including membrane-centered ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular, it does not include all kinds of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is to note the importance of mastering liposome technology together with cell-free systems for the successful realization of this specific type of synthetic cell. With this aim, four currently employed protocols are compared and discussed, with a major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency.


Assuntos
Células Artificiais/química , Lipossomos/química , Proteínas/síntese química , Expressão Gênica , Bicamadas Lipídicas/química , Lipossomos/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Biologia Sintética
19.
Methods Mol Biol ; 1960: 101-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798525

RESUMO

Macrophages are innate immune cells, which have important roles in the inflammatory response to infections or tissue injury, and have an equally important role in the resolution of inflammation. Macrophages play a key part in directing the innate immune response and subsequent adaptive immune response. They can acquire a variety of distinct but also overlapping activation states, depending on the local microenvironment, in order to perform these functions. Stimuli, such as IFNγ and LPS, can promote an inflammatory activation state, which is associated with the production of reactive oxygen species, and pro-inflammatory cytokines and chemokines. Immune complexes and LPS can promote an anti-inflammatory activation state to prevent damage to the host, which is associated with the production of high levels of the anti-inflammatory cytokine IL-10 and low levels of pro-inflammatory cytokines. Wound-healing macrophages can be activated by IL-4 or IL-13 and have roles in tissue remodeling and the resolution of inflammation. Macrophages are present in nearly every tissue of the body and are important for maintaining homeostasis, but their dysfunction can also lead to diseases, such as inflammatory bowel disease. To study the role macrophages play in a complex in vivo environment, depletion and reconstitution experiments can be utilized. Clodronate liposomes are an effective and versatile way to deplete macrophages in vivo; they can allow selective depletion from tissues of interest and can be used on transgenic mice. However, clodronate liposomes deplete all types of macrophages as well as dendritic cells, so other strategies are required in parallel to determine whether macrophages or macrophages of a particular activation state are required. Reconstitution of macrophages by adoptive transfer can be performed, with or without prior depletion, to further suggest that the observed effect is macrophage dependent. Macrophages activated ex vivo or macrophages from transgenic mice can be adoptively transferred during disease models to determine whether a specific protein or activation state affects disease outcome. Macrophage contribution to health and disease can be effectively studied using depletion with clodronate liposomes and by macrophage reconstitution, as demonstrated in this chapter.


Assuntos
Macrófagos/metabolismo , Animais , Ácido Clodrônico/metabolismo , Inflamação/metabolismo , Interferon gama/farmacologia , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Lipossomos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos
20.
Zebrafish ; 16(2): 171-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30724716

RESUMO

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.


Assuntos
Sistemas de Liberação de Medicamentos/veterinária , Compostos de Epóxi/química , Lipossomos/metabolismo , Macrófagos/metabolismo , Compostos Organofosforados/química , Piperidinas/química , Peixe-Zebra , Animais , Antioxidantes/química , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/química , Modelos Animais
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