RESUMO
The photothermal properties of nanoparticles (NPs), that is, their ability to convert absorbed light into heat, have been studied since the end of the last century, mainly on gold NPs. In the new millennium, these studies have developed into a burst of research dedicated to the photothermal ablation of tumors. However, beside this strictly medical theme, research has also flourished in the connected areas of photothermal antibacterial surface coatings, gels and polymers, of photothermal surfaces for cell stimulation, as well as in purely technological areas that do not involve medical biotechnology. These include the direct conversion of solar light into heat, a more efficient sun-powered generation of steam and the use of inkjet-printed patterns of photothermal NPs for anticounterfeit printing based on temperature reading, to cite but a few. After an analysis of the photothermal effect (PTE) and its mechanism, this minireview briefly considers the antitumor-therapy theme and takes an in-depth look at all the other technological and biomedical applications of the PTE, paying particular attention to photothermal materials whose NPs have joined those based on Au.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Ouro , Temperatura Alta , Polímeros , TemperaturaRESUMO
The possibility to shape stimulus-responsive optical polymers, especially hydrogels, by means of laser 3D printing and ablation is fostering a new concept of "smart" micro-devices that can be used for imaging, thermal stimulation, energy transducing and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. In light of the increasing demand for micro-devices for nanomedicine and personalized medicine, interest is growing in the combination of composite and hybrid photo-responsive materials and digital micro-/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. Less often, the two approaches have been combined and active nanomaterials have been embedded in the microstructures. The aim of this review is to give a short overview of the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photothermal activity, chemical swelling and chemical sensing.
Assuntos
Materiais Biocompatíveis , Hidrogéis , Lasers , Polímeros , Impressão TridimensionalRESUMO
When using gold nanoparticle (AuNP) inks for writing photothermal readable secure information, it is of utmost importance to obtain a sharp and stable shape of the localized surface plasmon resonance (LSPR) absorption bands in the prints. The T increase at a given irradiation wavelength (DTl) is the retrieved information when printed patterns are interrogated with a laser source. As DTl is proportional to the absorbance at the wavelength l, any enlargement or change of the absorbance peak shape in a printed pattern would lead to wrong or unreliable reading. With the aim of preparing AuNP inks suitable for inkjet printing of patterns with stable and reliable photothermal reading, we prepared liquid solutions of spherical AuNP coated with a series of different polymers and with or without additional dispersant. The optical stability of the inks and of the printed patterns were checked by monitoring the shape changes of the sharp LSPR absorption band of AuNP in the visible (lmax 519 nm) along weeks of ageing. AuNP coated with neutral polyethylenglycol thiols (HS-PEG) of mw 2000-20,000 showed a strong tendency to rapidly agglomerate in the dry prints. The close contact between agglomerated AuNP resulted in the loss of the pristine shape of the LSPR band, that flattened and enlarged with the further appearance of a second maximum in the Near IR, due to plasmon hybridization. The tendency to agglomerate was found directly proportional to the PEG mw. Addition of the ethylcellulose (EC) dispersant to inks resulted in an even stronger and faster tendency to LSPR peak shape deformation in the prints due to EC hydrophobicity, that induced AuNP segregation and promoted agglomeration. The introduction of a charge on the AuNP coating revelead to be the correct way to avoid agglomeration and obtain printed patterns with a sharp LSPR absorption band, stable with ageing. While the use of a simple PEG thiol with a terminal negative charge, HS-PEGCOO(-) (mw 3000), was not sufficient, overcoating with the positively charged polyallylamine hydrochloride (PAH) and further overcoating with the negatively charged polystyrene sulfonate (PSS) yielded AuNP@HS-PEGCOO(-)/PAH(+) and AuNP@HS-PEGCOO(-)/PAH(+)/PSS(-), both giving stable prints. With these inks we have shown that it is possible to write photothermally readable secure information. In particular, the generation of reliable three-wavelength photothemal barcodes has been demonstrated.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Polímeros/química , Ressonância de Plasmônio de SuperfícieRESUMO
Antibacterial treatment is an essential issue in many diverse fields, from medical device treatments (for example prostheses coating) to food preservation. However, there is a need of novel and light-weight materials with high antibacterial efficiency (preferably due to the physical activation). Utilization of photo-thermally active nanoparticles can lead to novel and re-usable materials that can be remotely activated on-demand to thermally eradicate bacteria and mitigate biofilm formation, therefore meeting the above challenge. In this study polyvinyl alcohol (PVA) hydrogel films containing non-toxic and highly photo-thermally active Prussian blue (PB) nanoparticles were fabricated. The confocal microscopy studies indicated a uniform nanoparticle distribution and a low degree of aggregation. Upon near-infrared (NIR; 700 and 800 nm) light irradiation of PVA-PB films, the local temperature increases rapidly and reaches a plateau (up to ΔT â 78 °C), within ≈6-10 s under relatively low laser intensities, I â 0.3 W cm-2. The high and localized increase of temperature on the fabricated films resulted in an efficient antibacterial effect on Pseudomonas aeruginosa (P. aeruginosa) bacteria. In addition, the localized photo-thermal effect was also sufficient to substantially mitigate biofilms growth.
Assuntos
Antibacterianos/síntese química , Biofilmes/efeitos dos fármacos , Ferrocianetos/química , Nanopartículas/química , Fototerapia/métodos , Álcool de Polivinil/química , Ferrocianetos/farmacologia , Temperatura Alta , Lasers , Terapia com Luz de Baixa Intensidade/métodos , Álcool de Polivinil/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologiaRESUMO
The study of the dynamics of biological systems requires one to follow relaxation processes in time with micron-size spatial resolution. This need has led to the development of different fluorescence correlation techniques with high spatial resolution and a tremendous (from nanoseconds to seconds) temporal dynamic range. Spatiotemporal information can be obtained even on complex dynamic processes whose time evolution is not forecast by simple Brownian diffusion. Our discussion of the most recent applications of image correlation spectroscopy to the study of anomalous sub- or superdiffusion suggests that this field still requires the development of multidimensional image analyses based on analytical models or numerical simulations. We focus in particular on the framework of spatiotemporal image correlation spectroscopy and examine the critical steps in getting information on anomalous diffusive processes from the correlation maps. We point out how a dual space-time correlative analysis, in both the direct and the Fourier space, can provide quantitative information on superdiffusional processes when these are analyzed through an empirical model based on intermittent active dynamics. We believe that this dual space-time analysis, potentially amenable to mathematical treatment and to the exact fit of experimental data, could be extended to include the rich phenomenology of subdiffusive processes, thereby quantifying relevant parameters for the various motivating biological problems of interest.
Assuntos
Difusão , Modelos Biológicos , Imagem Molecular , Espectrometria de Fluorescência , Processos EstocásticosRESUMO
Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.
Assuntos
Cromatina/metabolismo , Lamina Tipo A/metabolismo , Movimento , Animais , Difusão , Fibroblastos/citologia , Fibroblastos/metabolismo , CamundongosRESUMO
Microfluidic devices reproducing 3D networks are particularly valuable for nanomedicine applications such as tissue engineering and active cell sorting. There is however a gap in the possibility to measure how the flow evolves in such 3D structures. We show here that it is possible to map 3D flows in complex microchannel networks by combining wide field illumination to image correlation approaches. For this purpose, we have derived the spatiotemporal image correlation analysis of time stacks of single-plane illumination microscopy images. From the detailed analytical and numerical analysis of the resulting model, we developed a fitting method that allows us to measure, besides the in-plane velocity, the out-of-plane velocity component down to vz â 65 µm/s. We have applied this method successfully to the 3D reconstruction of flows in microchannel networks with planar and 3D ramifications. These different network architectures have been realized by exploiting the great prototyping ability of a 3D printer, whose precision can reach few tens of micrometers, coupled to poly dimethyl-siloxane soft-printing lithography.
RESUMO
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour).
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/análise , Saccharomyces cerevisiae/metabolismo , Análise de Célula Única/métodos , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/análise , Corantes Fluorescentes/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
In vivo studies of blood circulation pathologies have great medical relevance and need methods for the characterization of time varying flows at high spatial and time resolution in small animal models. We test here the efficacy of the combination of image correlation techniques and single plane illumination microscopy (SPIM) in characterizing time varying flows in vitro and in vivo. As indicated by numerical simulations and by in vitro experiments on straight capillaries, the complex analytical form of the cross-correlation function for SPIM detection can be simplified, in conditions of interest for hemodynamics, to a superposition of Gaussian components, easily amenable to the analysis of variable flows. The possibility to select a wide field of view with a good spatial resolution along the collection optical axis and to compute the cross-correlation between regions of interest at varying distances on a single time stack of images allows one to single out periodic flow components from spurious peaks on the cross-correlation functions and to infer the duration of each flow component. We apply this cross-correlation analysis to the blood flow in Zebrafish embryos at 4 days after fertilization, measuring the average speed and the duration of the systolic and diastolic phases.
Assuntos
Hemodinâmica , Peixe-Zebra/fisiologia , Animais , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Microesferas , Pulso Arterial , Rodaminas/química , Tempo , Lipossomas Unilamelares/químicaRESUMO
Gold nanocages (AuNCs) have been shown to be a useful tool for harnessing imaging and hyperthermia therapy of cancer, thanks to their unique optical properties, low toxicity, and facile surface functionalization. Herein, we use AuNCs for selective targeting of prostate cancer cells (PC3) via specific interaction between neuropeptide Y (NPY) receptor and three different NPY analogs conjugated to AuNCs. Localized surface plasmon resonance band of the nanoconjugates was set around 800 nm, which is appropriate for in vivo applications. Long-term stability of nanoconjugates in different media was confirmed by UV-vis and DLS studies. Active NPY receptor targeting was observed by confocal microscopy showing time-dependent AuNCs cellular uptake. Activation of ERK1/2 pathway was evaluated by Western blot to confirm the receptor-mediated specific interaction with PC3. Cellular uptake kinetics were compared as a function of peptide structure. Cytotoxicity of nanoconjugates was evaluated by MTS and Annexin V assays, confirming their safety within the concentration range explored. Hyperthermia studies were carried out irradiating the cells, previously incubated with AuNCs, with a pulsed laser at 800 nm wavelength, showing a heating enhancement ranging from 6 to 35 °C above the culture temperature dependent on the irradiation power (between 1.6 and 12.7 W/cm2). Only cells treated with AuNCs underwent morphological alterations in the cytoskeleton structure upon laser irradiation, leading to membrane blebbing and loss of microvilli associated with cell migration. This effect is promising in view of possible inhibition of proliferation and invasion of cancer cells. In summary, our Au-peptide NCs proved to be an efficient theranostic nanosystem for targeted detection and activatable killing of prostate cancer cells.
Assuntos
Terapia de Alvo Molecular/métodos , Nanopartículas , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/terapia , Nanomedicina Teranóstica/métodos , Linhagem Celular Tumoral , Desenho de Fármacos , Ouro , Humanos , Lasers , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Peptídeos/síntese química , Peptídeos/química , Neoplasias da Próstata/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Termografia/métodosRESUMO
Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.
Assuntos
Ouro/química , Ouro/metabolismo , Espaço Intracelular/metabolismo , Microscopia Confocal , Modelos Biológicos , Movimento , Nanoestruturas , Sobrevivência Celular , Difusão , Endocitose , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Análise Espaço-TemporalRESUMO
The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose. A maximum 15% dye release in aqueous solution was found when the bulk temperature of gold nanostars solutions was increased to T = 42 °C, the maximum physiological temperature. This fraction reduces 3-5% for temperatures lower than 40 °C. Similar results were found when the temperature increase was obtained by laser excitation of the near-infrared (NIR) localized surface plasmon resonance of the GNSs, which are photothermally responsive. Besides the direct impact of temperature, an increased BDP-SH release was observed upon changing the chemical composition of the solvent from pure water to phosphate-buffered saline and culture media solutions. Moreover, also a significant fraction of PEG-SH was released from the GNS surface due to the increase in temperature. We monitored it with a different approach, that is, by using a coating of α-mercapto-ω-amino PEG labeled with tetramethylrhodamine isothiocyanate on the amino group, that after heating was separated from GNS by ultracentrifugation and the released PEG was determined by spectrofluorimetric techniques on the supernatant solution. These results suggest some specific limitations in the use of the gold-thiolate bond for coating of nanomaterials with organic compounds in biological environments. These limitations come from the duration and the intensity of the thermal treatment and from the medium composition and could also be exploited in biological media to modulate the in vivo release of drugs.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Fatores de Transcrição NFATC/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipase C gama/metabolismo , Quinases da Família src/metabolismoRESUMO
The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.
Assuntos
Proteínas de Fluorescência Verde/química , Histidina/química , Dobramento de Proteína , Estabilidade Proteica , Cinética , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Análise Espectral , Relação Estrutura-Atividade , TermodinâmicaRESUMO
We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within =/~20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are =/~3 and =/~100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia.
Assuntos
Ouro/química , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Temperatura , Fenômenos Ópticos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.
RESUMO
Novel amphiphilic copolymers have been synthesized based on a biocompatible poly(hydroxyethylaspartamide) (PHEA) backbone, bearing both anchoring groups for gold nanoparticles, such as thiols and disulfide, and conjugable moieties, such as amino groups, the latter as points suitable for appending further functional agents. The strategy was to functionalize α,ß-poly[(N-2-hydroxyethyl)-D,L-aspartamide] (PHEA) with PEG2000-NH2 and with ethylenediamine (EDA) obtaining a partially pegylated copolymer with a large number of pendant primary amino groups. A fraction of the latter was conjugated with molecules bearing terminal thiol moieties such as 12-mercaptododecanoic acid (MDA) and disulfide groups such as lipoic acid (LA), obtaining the two amphiphilic derivatives PHEA-PEG2000-EDA-MDA (PPE-MDA) and PHEA-PEG2000-EDA-LA (PPE-LA), which also proved intrinsically able to self-assemble in polymeric micelles. The two copolymers efficiently coated gold nanostars (GNSs, size ≈ 40 nm), wrapping around the surface increasing only slightly the hydrodynamic diameter (reaching ≈ 45 nm), imparting them stability and a pH-switchable surface charge, due to the unreacted amino groups. Remarkably, the poor solvation and the huge steric hindrance experienced by the amino groups lowers the observed logarithmic protonation constants to 5.6-5.7. In vitro experiments demonstrated that PPE-MDA and PPE-LA copolymers have an intrinsic excellent biocompatibility in both the human brain neuroblastoma (SH-SY5Y) and human bronchial epithelial (16-HBE) cell lines. Interaction of the same cell lines with "nude" GNS and GNS coated with PPE-LA was also studied, disclosing a completely satisfactory biocompatibility of the latter.
Assuntos
Materiais Revestidos Biocompatíveis/síntese química , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Polietilenoglicóis/química , Tensoativos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/toxicidade , Etilenodiaminas/química , Humanos , Teste de Materiais , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Tamanho da PartículaRESUMO
It is well known that copper oxide nanoparticles (CuO NPs) are heavily toxic on in vitro systems. In human alveolar epithelial cells, the mechanism of toxicity is mostly related to oxidative insults, coming from intracellularly dissolved copper ions, finally leading to apoptotic or autophagic cell death. Our hypothesis is based on possible early oxidative events coming from specific NP surface reactivity able to undermine the cell integrity and to drive cell to death, independently from Lysosomal-Enhanced Trojan Horse mechanism. Two types of CuO NPs, with different oxidative potential, were selected and tested on A549 cells for 1 h and 3 h at 10, 25, 50 and 100 µg/ml. Cells were then analyzed for viability and oxidative change of the proteome. Oxidative by-products were localized by immunocytochemistry and cell-NP interactions characterized by confocal and electron microscopy techniques. The results show that CuO NPs induced oxidative changes soon after 1 h exposure as revealed by the increase in protein carbonylation and reduced-protein-thiol oxidation. In parallel, cell viability significantly decreased, as shown by MTT assay. Such effects were higher for CuO NPs with more crystalline defects and with higher ROS production than for fully crystalline NPs. At these exposure times, although NPs efficiently interacted with cell surface and were taken up by small endocytic vesicles, no ion dissolution was visible inside the lysosomal compartment and no effects were produced by extracellularly dissolved copper ions. In conclusion, a specific NP surface-dependent oxidative cell injury was demonstrated. More detailed studies are required to understand which targets precociously react with CuO NPs, but these results introduce new paradigms for the toxicity of the metal-based NPs, beyond the Lysosomal-Enhanced Trojan horse-related mechanism, and open-up new opportunities to investigate the interactions and effects at the bio-interface for designing safer as well as more effective CuO-based biocides.
Assuntos
Cobre , Nanopartículas Metálicas , Humanos , Cobre/química , Espécies Reativas de Oxigênio/metabolismo , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/química , Estresse Oxidativo , Carbonilação ProteicaRESUMO
Prussian blue (PB) is a coordination polymer based on the Fe2+ CN Fe3+ sequence. It is an FDA-approved drug, intended for oral use at the acidic pH of the stomach and of most of the intestine track. However, based on FDA approval, a huge number of papers proposed the use of PB nanoparticles (PBnp) under "physiological conditions", meaning pH buffered at 7.4 and high saline concentration. While most of these papers report that PBnp are stable at this pH, a small number of papers describes instead PBnp degradation at the same or similar pH values, i.e. in the 7-8 range. Here we give a definitively clear picture: PBnp are intrinsically unstable at pH ≥ 7, degrading with the fast disappearance of their 700 nm absorption band, due to the formation of OH- complexes from the labile Fe3+ centers. However, we show also that the presence of a polymeric coating (PVP) can protect PBnp at pH 7.4 for over 24 h. Moreover, we demonstrate that when "physiological conditions" include serum, a protein corona is rapidly formed on PBnp, efficiently avoiding degradation. We also show that the viability of PBnp-treated EA.hy926, NCI-H1299, and A549 cells is not affected in a wide range of conditions that either prevent or promote PBnp degradation.
Assuntos
Nanopartículas , Nanopartículas/química , Ferrocianetos/química , Concentração de Íons de HidrogênioRESUMO
Heating has recently been applied as an alternative to electrical stimulation to modulate excitability and to induce neuritogenesis and the expression of neuronal markers; however, a long-term functional differentiation has not been described so far. Here, we present the results obtained by a new approach for scalable thermal stimulation on the behavior of a model of dorsal root ganglion neurons, the F-11 cell line. Initially, we performed experiments of bulk stimulation in an incubator for different time intervals and temperatures, and significant differences in neurite elongation and in electrophysiological properties were observed in cultures exposed at 41.5 °C for 30 min. Thus, we exposed the cultures to the same temperature increase using a near-infrared laser to irradiate a disc of Prussian blue nanoparticles and poly-vinyl alcohol that we had adhered to the outer surface of the petri dish. In irradiated cells, neurites were significantly longer, and the electrophysiological properties (action potential firing frequency and spontaneous activity) were significantly increased compared to the control. These results show for the first time that a targeted thermal stimulation could induce morphological and functional neuronal differentiation and support the future application of this method as a strategy to modify neuronal behavior in vivo.