RESUMEN
OBJECTIVE: Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. MATERIALS AND METHODS: PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). RESULTS: Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. CONCLUSION: Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.
Asunto(s)
Imagen por Resonancia Magnética con Fluor-19/métodos , Flúor/química , Fluorocarburos/química , Nanopartículas/química , Animales , Células CHO , Colágeno/química , Medios de Contraste , Cricetulus , Combinación de Medicamentos , Emulsiones , Fluoresceínas/química , Colorantes Fluorescentes/química , Inyecciones Intravenosas , Laminina/química , Lipopolisacáridos/química , Hígado/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Proteoglicanos/química , Rodaminas/química , Bazo/diagnóstico por imagen , Absorción SubcutáneaRESUMEN
Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.
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Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimología , Membrana Celular/química , Liposomas/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMEN
The interaction of liposomal membranes composed of soybean phosphatidylcholine with the bile salts (BSs) cholate (Ch), glycocholate (GC), chenodeoxycholate (CDC), and glycochenodeoxycholate (GCDC) was studied. The BSs differed with regard to their lipophilicity, pKa values, and the size of their hydrophilic moiety. Their membrane interactions were investigated using Laurdan as a membrane-anchored fluorescent dye. The apparent membrane/water partition coefficient, D, at pH 7.4 was calculated from binding plots and compared with direct binding measurements using ultracentrifugation as a reference. The Laurdan-derived LogD values at pH 7.4 were found to be 2.10 and 2.25 for the trihydroxy BSs, i.e., Ch and GC, and 2.85 and 2.75 for the dihydroxy BSs, i.e., CDC and GCDC, respectively. For the membrane-associated glycine-conjugated GC and GCDC (pKa values of â¼3.9), no differences in the Laurdan spectra of the respective BS were found at pH 6.8, 7.4, and 8.2. Unconjugated Ch and CDC (pKa values of â¼5.0) showed pronounced differences at the three pH values. Furthermore, the kinetics of membrane adsorption and transbilayer movement differed between conjugated and unconjugated BSs as determined with Laurdan-labeled liposomes.
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2-Naftilamina/análogos & derivados , Ácidos y Sales Biliares/química , Membrana Celular/química , Colorantes Fluorescentes/química , Lauratos/química , Agua/química , 2-Naftilamina/química , Ácidos y Sales Biliares/metabolismo , Membrana Celular/metabolismo , Concentración de Iones de Hidrógeno , Absorción Intestinal , Marcaje Isotópico , Cinética , Liposomas/química , Liposomas/metabolismo , Hígado/metabolismoRESUMEN
BACKGROUND: Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. METHODS AND RESULTS: Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19F magnetic resonance imaging, together with α2-antiplasmin peptide (α2AP)-targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter<0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 1H and 19F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, α2AP-PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of α2AP-PFCs>60 minutes after thrombus induction no longer resulted in detectable 19F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2AP-PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. CONCLUSIONS: Our results demonstrate that 1H/19F magnetic resonance imaging, together with α2AP-PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19F magnetic resonance imaging.
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Colesterol/análogos & derivados , Medios de Contraste , Imagen por Resonancia Magnética con Fluor-19/métodos , Fluorocarburos , Polietilenglicoles , Embolia Pulmonar/diagnóstico , Trombosis de la Vena/diagnóstico , alfa 2-Antiplasmina/análogos & derivados , Animales , Colesterol/farmacocinética , Medios de Contraste/farmacocinética , Portadores de Fármacos , Diagnóstico Precoz , Emulsiones/farmacocinética , Factor XIIIa/metabolismo , Flúor/farmacocinética , Fluorocarburos/farmacocinética , Humanos , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología , Nanosferas , Polietilenglicoles/farmacocinética , Sensibilidad y Especificidad , Relación Señal-Ruido , Distribución Tisular , Vena Cava Inferior , alfa 2-Antiplasmina/farmacocinéticaRESUMEN
Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.
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Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Orgánulos/metabolismo , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Orgánulos/química , Orgánulos/genética , Orgánulos/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer. RESULTS: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues. CONCLUSION: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.
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Antineoplásicos/farmacología , Compuestos de Bencilideno/farmacología , Desoxicitidina/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Liposomas/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Piperidonas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Compuestos de Bencilideno/química , Compuestos de Bencilideno/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/química , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Progresión de la Enfermedad , Composición de Medicamentos , Quimioterapia Combinada , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inyecciones Subcutáneas , Liposomas/química , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Piperidonas/química , Piperidonas/farmacocinética , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.
Asunto(s)
Comunicación Celular , Extensiones de la Superficie Celular/metabolismo , Enfermedad de Hodgkin/metabolismo , Antígeno Ki-1/metabolismo , Células de Reed-Sternberg/metabolismo , Vesículas Secretoras/metabolismo , Transducción de Señal , Microambiente Tumoral , Biomarcadores de Tumor/metabolismo , Ligando CD30/metabolismo , Línea Celular Tumoral , Extensiones de la Superficie Celular/inmunología , Extensiones de la Superficie Celular/ultraestructura , Microscopía por Crioelectrón , Eosinófilos/inmunología , Eosinófilos/metabolismo , Citometría de Flujo , Granulocitos/inmunología , Granulocitos/metabolismo , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/patología , Humanos , Interleucina-8/metabolismo , Espectrometría de Masas , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Tamaño de los Orgánulos , Células de Reed-Sternberg/inmunología , Células de Reed-Sternberg/ultraestructura , Vesículas Secretoras/inmunología , Vesículas Secretoras/ultraestructuraRESUMEN
Inflammatory processes can reliably be assessed by (19)F MRI using perfluorocarbons (PFCs), which is primarily based on the efficient uptake of emulsified PFCs by circulating cells of the monocyte-macrophage system and subsequent infiltration of the (19)F-labeled cells into affected tissue. An ideal candidate for the sensitive detection of fluorine-loaded cells is the biochemically inert perfluoro-15-crown-5 ether (PFCE), as it contains 20 magnetically equivalent (19)F atoms. However, the biological half-life of PFCE in the liver and spleen is extremely long, and so this substance is not suitable for future clinical applications. In the present study, we investigated alternative, nontoxic PFCs with predicted short biological half-lives and high fluorine content: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD) and trans-bis-perfluorobutyl ethylene (F-44E). Despite the complex spectra of these compounds, we obtained artifact-free images using sine-squared acquisition-weighted three-dimensional chemical shift imaging and dedicated reconstruction accomplished with in-house-developed software. The signal-to-noise ratio of the images was maximized using a Nutall window with only moderate localization error. Using this approach, the retention times of the different PFCs in murine liver and spleen were determined at 9.4 T. The biological half-lives were estimated to be 9 days (PFD), 12 days (PFOB) and 28 days (F-44E), compared with more than 250 days for PFCE. In vivo sensitivity for inflammation imaging was assessed using an ear clip injury model. The alternative PFCs PFOB and F-44E provided 37% and 43%, respectively, of the PFCE intensities, whereas PFD did not show any signal in the ear model. Thus, for in vivo monitoring of inflammatory processes, PFOB emerges as the most promising candidate for possible future translation of (19)F MR inflammation imaging to human applications.
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Flúor , Fluorocarburos , Procesamiento de Imagen Asistido por Computador , Inflamación/diagnóstico , Imagen por Resonancia Magnética , Animales , Emulsiones , Semivida , Humanos , Hidrocarburos Bromados , Cinética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Relación Señal-Ruido , Bazo/metabolismo , Factores de Tiempo , Presión de VaporRESUMEN
Poly(n-butyl-cyanoacrylate)-nanocapsules filled by perfluorodecalin (PFD) are proposed as potential oxygen carriers for blood substitute. The capsule dispersion is prepared via interfacial polymerisation from a PFD emulsion in water which in turn is generated by spontaneous phase separation. The resulting dispersion is capable of carrying approximately 10% of its own volume of gaseous oxygen, which is approximately half of the capacity of human blood. The volumes of the organic solvents and water are varied within a wide range, connected to a change of the capsule radius between 200 and 400 nm. The principal suitability of the capsule dispersion for intravenous application is proven in first physiological experiments. A total amount of 10 ml/kg body weight has been infused into rats, with the dispersion supernatant and a normal saline solution as controls. After the infusion of nanocapsules, the blood pressure as well as the heart rate remains constant on a normal level.
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Sustitutos Sanguíneos , Cianoacrilatos , Fluorocarburos , Nanocápsulas/química , Oxígeno , Animales , Sustitutos Sanguíneos/química , Sustitutos Sanguíneos/farmacología , Cianoacrilatos/química , Cianoacrilatos/farmacología , Evaluación Preclínica de Medicamentos , Enbucrilato , Fluorocarburos/química , Fluorocarburos/farmacología , Humanos , Masculino , Nanocápsulas/ultraestructura , Tamaño de la Partícula , Ratas , Ratas WistarRESUMEN
The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRß chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRß chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.
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Colesterol/metabolismo , Nanopartículas/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Esfingomielinas/metabolismo , Animales , Membrana Celular/metabolismo , Supervivencia Celular , Análisis por Conglomerados , Reactivos de Enlaces Cruzados/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Humanos , Ligandos , Ratones , Péptidos/metabolismo , Fosfatidilcolinas/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Transferrina , Liposomas Unilamelares/metabolismoRESUMEN
Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Membrana Celular/microbiología , Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/microbiología , Proteínas Citotóxicas Formadoras de Poros/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Línea Celular , Membrana Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , Enterocolitis Seudomembranosa/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG2000 was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG1100 was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG2000 or sterol-PEG1100 into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG1100 in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.
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Liposomas/química , Fusión de Membrana , Polietilenglicoles/química , Ésteres del Colesterol/química , Concentración de Iones de Hidrógeno , Cinética , Liposomas/síntesis química , Fosfatidiletanolaminas/químicaRESUMEN
We applied protein-mediated lipid transfer using recombinant His-tagged pro-sterol carrier protein 2 (pro-SCP2) to prepare asymmetrical membrane vesicles (AMV) featuring an unequal transmembrane distribution of the negative phospholipid egg-phosphatidylglycerol (EPG). Pure egg-phosphatidylcholine (EPC) vesicles were used as the acceptor and EPC:EPG 90:10 mol % vesicles as the donor populations. The changes in surface charge during EPG transfer were used to quantify the degree of asymmetry by free-flow electrophoresis (FFE). The relative deflection in FFE correlated with EPG content in the outer monolayer (x(EPG)). The initial transfer rates and first order rate constants for the transfer process were determined. The addition of pro-SCP2 at a molar protein-to-lipid ratio R(P/L) of (15-20) x 10(-5) accelerated the EPG transfer to half-times of between 2 and 3 h. Thus, the transmembrane redistribution of EPG by flip-flop, which reduces the degree of asymmetry and occurs at half-times of tens of hours, was minimized during the transfer process. We investigated the influence of membrane curvature on the transfer rate using 50 and 100 nm vesicles with very low size distribution widths (RSD of 13-17%). Transfer occurred with a 55.7% higher initial rate between the smaller vesicles. The use of equally sized acceptor and donor populations of such narrow size distributions was shown to be important for the preparation of AMV with a uniform degree of asymmetry. Under these conditions, AMV were obtained after less than 3 h by preparative FFE separation. In the case of the acceptor vesicles, EPG transfer increased x(EPG) to 3 mol %, whereas it was reduced to 6 mol % in the donor vesicles.
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Proteínas Portadoras/química , Electroforesis/métodos , Lípidos/química , Membranas Artificiales , Liposomas , Proteínas Recombinantes/químicaRESUMEN
Poly(ethersulfone) membranes were surface modified in a one-step procedure. For this purpose, the membranes were soaked with aqueous solutions of different low-molecular weight molecules bearing diverse hydrophilic functionalities and subject to electron beam treatment. No catalysts, photoinitiators, organic solvents or other toxic reagents were used, and no additional synthetic or purification steps were required.
RESUMEN
The aim of this study was to investigate the performance of a newly devised high-performance thin-layer chromatography (HPTLC) method in quantifying common liposome membrane components, including the five phospholipids (PLs), phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, as well as cholesterol, cholesteryl hemisuccinate, and linoleic acid. Besides strictly keeping to a standardized procedure, three parameters were particularly critical for proper quantification. First, a relative humidity of higher than 60% caused migration distances to increase and reduced the resolution of the PLs on a silica-gel 60 HPTLC plate. Second, PLs underwent oxidative combustion during storage for 2 or 24 hours on an HPTLC plate, with peak losses of up to 25-44%. These losses could be prevented by storage under nitrogen and, to some extent, by the addition of the antioxidant, DL-alpha-tocopherol. Third, even with automated sample application, the accuracy and consistency of the application volume proved to be an important cause of error and needs routine verification. Considering these parameters, the method was found to accurately and precisely determine the composition of three different liposome preparations. The recovery was 97.2-101.8%, compared to secondary methods, and consistent over different days and with different operators (mean RSD of the recovery: 2.03 +/- 1.16%, n = 9). The working range was determined to be 100-300 ng in the case of the PLs (individual limit of determination between 40 and 80 ng) and 20-60 ng in the case of cholesterol (limit of determination: 16 ng).
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Liposomas/química , Fosfolípidos/análisis , Cromatografía en Capa DelgadaRESUMEN
BACKGROUND: In this study, we developed and validated a new approach for in vivo visualization of inflammatory processes by magnetic resonance imaging using biochemically inert nanoemulsions of perfluorocarbons (PFCs). METHODS AND RESULTS: Local inflammation was provoked in 2 separate murine models of acute cardiac and cerebral ischemia, followed by intravenous injection of PFCs. Simultaneous acquisition of morphologically matching proton ((1)H) and fluorine ((19)F) images enabled an exact anatomic localization of PFCs after application. Repetitive (1)H/(19)F magnetic resonance imaging at 9.4 T revealed a time-dependent infiltration of injected PFCs into the border zone of infarcted areas in both injury models, and histology demonstrated a colocalization of PFCs with cells of the monocyte/macrophage system. We regularly found the accumulation of PFCs in lymph nodes. Using rhodamine-labeled PFCs, we identified circulating monocytes/macrophages as the main cell fraction taking up injected nanoparticles. CONCLUSIONS: PFCs can serve as a "positive" contrast agent for the detection of inflammation by magnetic resonance imaging, permitting a spatial resolution close to the anatomic (1)H image and an excellent degree of specificity resulting from the lack of any (19)F background. Because PFCs are nontoxic, this approach may have a broad application in the imaging and diagnosis of numerous inflammatory disease states.
Asunto(s)
Isquemia Encefálica/patología , Inflamación/diagnóstico , Imagen por Resonancia Magnética/métodos , Isquemia Miocárdica/patología , Animales , Emulsiones , Fluorocarburos , Inflamación/etiología , Ganglios Linfáticos , Macrófagos , Ratones , MonocitosRESUMEN
We have prepared nine new dodecaborate cluster lipids with potential use in boron neutron capture therapy of tumors. This new generation of boron lipids is only singly negatively charged and consists of a pyridinium core with C(12), C(14), and C(16) chains as lipid backbone, connected through the nitrogen atom via a butylene, pentylene, or ethyleneoxyethylene linker to the oxygen atom on the dodecaborate cluster as headgroup. The lipids were obtained by nucleophilic attack of 4-(bisalkylmethyl)pyridine on the tetrahydrofurane, the dioxane, and a newly prepared tetrahydropyrane derivative, respectively, of closo-dodecaborate. All of these boron lipids are able to form closed vesicles in addition to some bilayers in the pure state and in the presence of helper lipids. The thermotropic behavior was found to be increasingly complex and polymorphic with increasing alkyl chain length. Except for two lipids, all lipids have low in vitro toxicity, and longer alkyl chains lead to a significant decrease in toxicity. The choice of the linker plays no major role with respect to their ability to form liposomes and their thermotropic properties, but the toxicity is influenced by the linkers in the case of short alkyl chains.
Asunto(s)
Compuestos de Boro/química , Terapia por Captura de Neutrón de Boro/métodos , Lípidos/síntesis química , Piridinas/química , Animales , Compuestos de Boro/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Lípidos/química , Lípidos/uso terapéutico , Lípidos/toxicidad , Liposomas/química , Estructura Molecular , Piridinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 x 10(6) RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the zeta potential of 7.17 +/- 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 x 10(6) RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , Liposomas/química , Plásmidos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Cationes , Línea Celular , Colesterol/genética , Electroforesis en Gel de Agar , Células HeLa , Humanos , Luciferasas/genética , Tamaño de la Partícula , Compuestos de Amonio Cuaternario/química , SonicaciónRESUMEN
The exchange reaction between sulfopropyl-dextran (Sephadex C-25) and propranolol hydrochloride (prop-HCl) as a model drug was studied. Binding experiments were carried out using a dissolution tester and photometric quantification of the amount of drug bound. The thermodynamic parameters of the binding reaction were derived by isothermal titration calorimetry (ITC) and equilibrium studies. Equilibrium was reached within approximately 12 min and was nicely described by the Langmuir equation for low degree of occupation of binding sites (concentrations of prop-HCl in equilibrium up to 3.6 mmol/L) with respect to the exchange capacity (2.0 mmol/g ion exchanger). However, the heat effects measured in the ITC experiment for step-wise addition of drug solution surprisingly increased for the first injections up to a maximum and only thereafter decreased. The heat effects do not exclusively represent the exchange reaction, but rather include a more complex pattern of reactions: shrinking of SP Sephadex C-25 upon addition of prop-HCl solution squeezes considerable amounts of the drug molecules out of the complex. Although the Langmuir fit is excellent, it cannot reveal information about the equilibrium.
Asunto(s)
Dextranos/química , Resinas de Intercambio Iónico/química , Propranolol/química , Sitios de Unión , Calorimetría/métodos , Química Farmacéutica/métodos , Intercambio Iónico , Cinética , Fotometría , TermodinámicaRESUMEN
Nanoparticle brushes: Complex nanostructures can be formed by self assembly of amphiphilic CdSe/CdS core-shell nanoparticles that bear a brushlike layer of poly(ethylene oxide) chains. This route is based on controlling the volume fractions of hydrophilic and hydrophobic moieties within the particles and allows the formation of micellar, cylindrical, or vesicular nanoobjects (see picture).