RESUMO
Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.
Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Adsorção , Eletrodos , Ouro/química , Cinética , Compostos de Sulfidrila/químicaRESUMO
Escherichia coli is one the most common bacteria responsible of uropathogenic diseases, which motives the search for rapid and easy methods of detection. By taking advantage of the specific interactions between mannose and type 1 fimbriae, in this work two fluorescent phenyleneethynylene (PE) trimers bearing one or two 4-aminophenyl-α-D-mannopyranoside termini groups were synthesized for the detection of E. coli. Three bacterial strains: ORN 178 (fimbriae I expression), ORN 208 (mutant serotype with no fimbriae expression) and one obtained from a local hospital (SS3) were used. Laser Scanning Confocal Microscopy (LSCM) and Surface Plasmon Resonance (SPR) were applied for the interaction studies following two different approaches: (1) mixing the oligomer solutions with the bacterial suspension, which permitted the observation of stained bacteria and by (2) biosensing as thin films, where bacteria adhered on the surface-functionalized substrate. LSCM allows one to easily visualize that two mannose groups are necessary to have a specific interaction with the fimbriae 1. The sensitivity of SPR assays to E. coli was 104 colony forming unit (CFU)/mL at 50 µL/min flow rate. The combination of PE units with two mannose groups results in a novel molecule that can be used as a specific fluorescent marker as well as a transducer for the detection of E. coli.
Assuntos
Escherichia coli , Alcinos , Aderência Bacteriana , Éteres , ManoseRESUMO
Research on the nanoscale membrane structures known as lipid rafts is relevant to the fields of cancer biology, inflammation and ischaemia. Lipid rafts recruit molecules critical to signalling and regulation of the invasion process in malignant cells, the leukocytes that provide immunity in inflammation and the endothelial cells that build blood and lymphatic vessels, as well as the patterning of neural networks. As angiogenesis is a common denominator, regulation of receptors and signalling molecules critical to angiogenesis is central to the design of new approaches aimed at reducing, promoting or normalizing the angiogenic process. The goal of this review is to highlight some of the key issues that indicate the involvement of endothelial cell lipid rafts at each step of so-called 'sprouting angiogenesis', from stimulation of the vascular endothelial growth factor to the choice of tip cells, activation of migratory and invasion pathways, recruitment of molecules that guide axons in vascular patterning and maturation of blood vessels. Finally, the review addresses opportunities for future studies to define how these lipid domains (and their constituents) may be manipulated to stimulate the so-called 'normalization' of vascular networks within tumors, and be identified as the main target, enabling the development of more efficient chemotherapeutics and cancer immunotherapies.
Assuntos
Vasos Sanguíneos/metabolismo , Microdomínios da Membrana/metabolismo , Axônios/metabolismo , Caveolinas/metabolismo , Células Endoteliais/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Transdução de Sinais , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.
Assuntos
Cavéolas/metabolismo , Células Progenitoras Endoteliais/metabolismo , Gangliosídeo G(M1)/farmacologia , Gangliosídeo G(M3)/farmacologia , Microdomínios da Membrana/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cavéolas/efeitos dos fármacos , Caveolina 1/metabolismo , Ensaio de Unidades Formadoras de Colônias , Células Progenitoras Endoteliais/efeitos dos fármacos , Humanos , Recém-Nascido , Cinética , Microdomínios da Membrana/efeitos dos fármacos , Fenótipo , Transdução de SinaisRESUMO
Phospholamban (PLN), a membrane protein present in the sarcoplasmic reticulum of cardiac myocytes, is a crucial regulator of cardiac function. It is known that PLN appears as a monomer and as a pentamer. However, the role of the PLN pentamer and its ability to generate an ion channel are a matter of debate. To address this issue we employed an experimental approach that combines electrochemical impedance spectroscopy and surface plasmon resonance measurements. In particular, we investigated the channel activity of wild-type PLN reconstituted in a tethered bilayer lipid membrane (tBLM) on a gold surface. Our results indicate that reconstituted PLN can generate ion-conducting channels in a tBLM. Experiments with a PLN monoclonal antibody support an oriented incorporation of PLN in the tBLM. We show that the binding of the antibody to the PLN cytoplasmic domain interferes with PLN channel activity.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Bicamadas Lipídicas , Citoplasma/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
THz radiation has gained great importance due to its potential applications in a wide variety of fields. For this reason, continuous efforts have been made to develop technological tools for use in this versatile band of the electromagnetic spectrum. Here, we propose a reflecting device with long focusing performances in the sub-THz band, using a bimirror device in which the relative angle is mechanically adjusted with the displacement of one of the mirrors. Despite the simplicity of the setup, the performance of this device is satisfactory down to a frequency of 0.1 THz. Theory and experience confirm that the bimirror is capable of focusing 0.1 THz radiation with a 2× magnification of the maximum input intensity while maintaining a longitudinal full width at half maximum (FWHM) of about 6 mm, which is about 12 times the depth of focus of a cylindrical optical element of the same focal length. In the absence of suitable THz equipment, the invariance property of the Fresnel diffraction integral allowed the predicted behavior to be tested in the THz range using conventional equipment operating at visible frequencies.
RESUMO
A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca(2+) and Mg(2+), the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Membranas Artificiais , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Adsorção , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Eletroquímica , Magnésio/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
THz radiation has assumed great importance thanks to the efforts in the development of technological tools used in this versatile band of the electromagnetic spectrum. Here, we propose a reflective biprism device with wavelength-independent long-focusing performances in the THz band by exploiting the high thermo-mechanical deformation of the elastomer polydimethylsiloxane (PDMS). This deformation allows for achieving significant optical path modulations in the THz band and effective focusing. The surface of a PDMS layer is covered with a gold thin film acting as a heater thanks to its absorption of wavelengths below ~500 nm. An invariance property of the Fresnel integral has been exploited to experimentally verify the THz performances of the device with an ordinary visible laser source, finding excellent agreement with the theoretical predictions at 1 and 3 THz. The same property also allowed us to experimentally verify that the reflective biprism focus has a longitudinal extension much greater than that exhibited by a benchmark convex cylindrical mirror with the same optical power. The device is thermo-mechanically stable up to a heating power of 270 mW, although it might be potentially exploited at higher powers with minor degradation of the optical performances.
RESUMO
Submicron long focusing range beams are gaining attention due to their potential applications, such as in optical manipulation, high-resolution lithography and microscopy. Here, we report on the theoretical and experimental characterization of an elastomeric polydimethylsiloxane/single layer graphene (PDMS/SLG) axicon-like tunable device, able to generate diffraction-resistant submicrometric spots in a pump and probe configuration. The working principle is based on the phase change of an input Gaussian beam induced in the elastomer via the thermo-optical effect, while the heating power is produced by the optical absorption of the SLG. The phase-modified beam is transformed by an objective into a long focus with submicron diameter. Our foci reach an experimental full width at half maximum (FWHM) spot diameter of 0.59 µm at the wavelength of 405 nm, with the FWHM length of the focal line greater than 90 µm. Moreover, the length of the focal line and the diameter of the focus can be easily tuned by varying the pump power. The proposed thermo-optical device can thus be useful for the simple and cheap improvement of the spatial resolution on long focus lines.
RESUMO
We investigated the chemisorption of self-assembled monolayers of sulfur-functionalized 4-amino-7-nitrobenzofurazan on gold and silver nanoisland films (NIFs) by means of surface-enhanced fluorescence (SEF) and surface-enhanced Raman scattering (SERS). The ligand is a push-pull molecule, where an intramolecular charge transfer occurs between an electron-donor and an electron-acceptor group, thus exhibiting nonlinear optical properties that are related to both SERS and SEF effects. The presence of different heteroatoms in the molecule ensures the possibility of chemical interaction with both silver and gold substrates. The SERS spectra suggest that furazan is bound to silver via lone pairs of the nitrogen atoms, whereas the ligand is linked to gold via a sulfur atom. Silver NIFs provide more efficient enhancement of both fluorescence and Raman scattering in comparison with gold NIFs. The present SEF and SERS investigation could provide useful information for foreseeing changes in the nonlinear responses of this push-pull molecule.
RESUMO
Imidacloprid is the most widely used insecticide in agriculture and its intensive use over the last 30 years has caused a global concern due to its potentially toxic effects on the ecosystem. Considering the recent scientific interest in novel simple methods for imidacloprid analysis, we propose a label-free sensitive and specific localised surface plasmon resonance system for the detection of the insecticide based on 2D nanostructured metasurfaces with highly performing plasmonic properties. The specificity of the sensor proposed was achieved by covalent bio-functionalization of the metasurface using a smart and easy one-step procedure mediated by carbon disulphide. The biosensor produced was tested using a set of imidacloprid standard solutions showing a competitive limit of detection, lower than 1 ng mL-1. Our novel nanosensing configuration represents a valid and reliable solution to realize low-cost portable POC tests as an alternative to the laborious and expensive methods traditionally used for insecticide detection.
Assuntos
Inseticidas/análise , Neonicotinoides/análise , Nitrocompostos/análise , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Ouro/química , Inseticidas/imunologia , Limite de Detecção , Nanopartículas Metálicas/química , Neonicotinoides/imunologia , Nitrocompostos/imunologiaRESUMO
Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.
RESUMO
We observed Förster resonance energy transfer (FRET) with a covalently linked donor-acceptor pair D-A consisting of two naphthalene groups acting as the donors and a benzofurazan group acting as the acceptor and adsorbed onto Ag or Au nanoisland films. The use of metal nanoisland films, which caused a strong enhancement of the Raman signal, permitted description of the adsorption mechanism onto the two metals. The intense fluorescence response of molecular adsorbates and the different behavior of the antenna on Ag and Au nanoislands are partly explained in terms of the radiating plasmon model.
RESUMO
HYPOTHESIS: Bimetallic nanoparticles made of iron oxide and Ag could be fabricated by pulsed laser ablation of iron and silver targets in pure water by a two-step route. These nanoparticles could exhibit both magnetic and plasmonic properties. EXPERIMENTS: Bimetallic nanoparticles were fabricated by using a focused Nd:YAG nanosecond laser source emitting a 1064nm wavelength radiation and characterized with ζ-potential, Dynamic Light Scattering (DLS), UV-vis absorption, Transmission Electron Microscopy (TEM), High Resolution TEM (HRTEM), Energy Dispersive X-ray Spectrometry (EDX), and Selected Area Electron Diffraction (SAED). The magnetic character of the nanoparticles was ascertained by observing attraction by a magnet and complete removing from the water environment, while their SERS (surface-enhanced Raman scattering) response was checked by decorating them with 2,2'-bipyridine as molecular reporter and performing Raman tests with green (514.5nm) and far-red (785nm) excitation wavelengths. FINDINGS: The observed magnetic attraction was due to magnetite (Fe3O4), the only ferromagnetic iron oxide form evidenced by the characterization tests in the aqueous colloidal system, where silver nanoparticles were also embedded. UV-vis and SERS spectra confirmed the presence of nanostructured silver as plasmonic constituent of the fabricated metal nanoparticles.
RESUMO
Cell therapies are treatments in which stem or progenitor cells are stimulated to differentiate into specialized cells able to home to and repair damaged tissues. After their discovery, endothelial progenitor cells (EPCs) stimulated worldwide interest as possible vehicles to perform autologous cell therapy of tumors. Taking into account the tumor-homing properties of EPCs, two different approaches to control cancer progression have been pursued by combining cell-based therapy with gene therapy or with nanomedicine. The first approach is based on the possibility of engineering EPCs to express different transgenes, and the second is based on the capacity of EPCs to take up nanomaterials. Here we review the most important progress covering the following issues: the characterization of bona fide endothelial progenitor cells, their role in tumor vascularization and metastasis, and preclinical data about their use in cell-based tumor therapy, considering antiangiogenic, suicide, immune-stimulating, and oncolytic virus gene therapy. The mixed approach of EPC cell therapy and nanomedicine is discussed in terms of plasmonic-dependent thermoablation and molecular imaging.
Assuntos
Portadores de Fármacos/uso terapêutico , Células Progenitoras Endoteliais/transplante , Neoplasias/tratamento farmacológico , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Portadores de Fármacos/química , Células Progenitoras Endoteliais/química , Terapia Genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Vírus Oncolíticos/genéticaRESUMO
In the photothermal treatments (PTs) of tumor, the localization of a high number of near-infrared (NIR) absorbing gold nanoparticles in the tumor mass is still a challenging issue. Here, we propose a promising strategy to deliver therapeutic chitosan-coated gold nanoparticles to tumor cells as hidden cargo of Endothelial Colony Forming Cells (ECFCs) endowed with an innate tumor-tropism. Remarkably, ECFC gold enrichement doesn't affect cell viability and preserves the endothelial lineage characteristics such as capillary morphogenesis and cell migration. We demonstrate that heavily Au-doped ECFCs are able to efficiently warm up the tumor environment, and kill the cancer cells via hyperthermic heating both in vitro as well as in vivo. Thus, we show an excellent thermotransductive property of gold enriched ECFCs and their capability to kill melanoma cells at moderate NIR light intensities.
Assuntos
Células Endoteliais/citologia , Ouro/química , Melanoma/terapia , Nanopartículas Metálicas/química , Neoplasias Cutâneas/terapia , Animais , Movimento Celular , Sobrevivência Celular , Quitosana/química , Coloides/química , Células Endoteliais/metabolismo , Feminino , Humanos , Íons , Luz , Melanoma/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neovascularização Fisiológica , Fotoquímica , Receptores CXCR4/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente TumoralRESUMO
A procedure based on surface plasmon resonance (SPR) is proposed to monitor the lateral mobility of lipid molecules in solid-supported bilayer lipid membranes (ssBLMs), an essential prerequisite for the formation of important microdomains called lipid rafts (LRs). The procedure relies on the marked tendency of the ganglioside GM1 to be recruited by LRs and to act as a specific receptor of the beta-subunit of the cholera toxin (ChTB). In the presence of both GM1 and ChTB, spontaneous formation of lipid rafts domains in mobile ssBLMs is accompanied by an appreciable increase in the amount of adsorbed ChTB, as monitored by SPR.
RESUMO
UV polymerization of self-assembled monolayers of a novel carbazolyl-diacetylene (CDS9) chemisorbed on silver films was demonstrated by surface plasmon resonance (SPR) and surface enhanced Raman scattering (SERS) experiments. SPR tests performed during UV exposure permitted one to observe the growth of the absorption coefficient, associated with the formation of the polymeric backbone. The Raman spectra of polymerized monolayers exhibited the bands associated with the C=C stretching modes of the conjugated backbone, typical of the blue and red polymeric phases usually present in polydiacetylenes, with a clear predominance of the red form. Moreover, the strong surface enhancement of the Raman band corresponding to the aromatic C=C stretching modes suggested that carbazolyl groups arrange nearly perpendicularly to the metal surface. In contrast, the absence of a SERS signal in the region of conjugated C[triple bond]C bond stretchings confirmed a polymerization scheme with conjugated triple bonds nearly parallel to the plane of the metal.