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Imaging tools are crucial for studying the vascular network and its barrier function in various physiopathological conditions. Shortwave infrared (SWIR) window optical imaging allows noninvasive, in-depth exploration. We applied SWIR imaging, combined with vessel segmentation and deep learning analyses, to study real-time dextran probe extravasation in mice experiencing intermittent hypoxia (IH)-a characteristic of obstructive sleep apnea associated with potential cardiovascular alterations due to early vascular permeability. Evidence for permeability in this context is limited, making our investigation significant. C57Bl/6 mice were exposed to normoxia or intermittent hypoxia for 14 days. Then SWIR imaging between 1,250 and 1,700 nm was performed on the saphenous artery and vein and on the surrounding tissue after intravenous injection of labeled dextrans of two different sizes (10 or 70 kDa). Postprocessing and segmentation of the SWIR images were conducted using deep learning treatment. We monitored high-resolution signals, distinguishing arteries, veins, and surrounding tissues. In the saphenous artery and vein, after 70-kD dextran injection, tissue/vessel ratio was higher after intermittent hypoxia (IH) than normoxia (N) over 500 seconds (P < 0.05). However, the ratio was similar in N and IH after 10-kD dextran injection. The SWIR imaging technique allows noninvasive, real-time monitoring of dextran extravasation in vivo. Dextran 70 extravasation is increased after exposure to IH, suggesting an increased vessel permeability in this mice model of obstructive sleep apnea.NEW & NOTEWORTHY We demonstrate that SWIR imaging technique is a useful tool to monitor real-time dextran extravasation from vessels in vivo, with a high resolution. We report for the first time an increased real-time dextran (70 kD) extravasation in mice exposed to intermittent hypoxia for 14 days compared with normoxic controls.
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Dextranos , Apneia Obstrutiva do Sono , Animais , Camundongos , Hipóxia , Artérias , Camundongos Endogâmicos C57BLRESUMO
Luminescent gold nanoclusters are rapidly gaining attention as efficient theranostic targets for imaging and therapeutics. Indeed, their ease of synthesis, their tunable optical properties and tumor targeting make them potential candidates for sensitive diagnosis and efficacious therapeutic applications. This concept highlights the key components for designing gold nanoclusters as efficient theranostics focusing on application in the field of oncology.
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Nanopartículas Metálicas , Neoplasias , Humanos , Medicina de Precisão , Ouro/uso terapêutico , Nanomedicina Teranóstica/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Nanopartículas Metálicas/uso terapêuticoRESUMO
Atomically precise gold nanoclusters are a fascinating class of nanomaterials that exhibit molecule-like properties and have outstanding photoluminescence (PL). Their ultrasmall size, molecular chemistry, and biocompatibility make them extremely appealing for selective biomolecule labeling in investigations of biological mechanisms at the cellular and anatomical levels. In this work, we report a simple route to incorporate a preformed Au25 nanocluster into a model bovine serum albumin (BSA) protein. A new approach combining small-angle X-ray scattering and molecular modeling provides a clear localization of a single Au25 within the protein to a cysteine residue on the gold nanocluster surface. Attaching Au25 to BSA strikingly modifies the PL properties with enhancement and a redshift in the second near-infrared (NIR-II) window. This study paves the way to conrol the design of selective sensitive probes in biomolecules through a ligand-based strategy to enable the optical detection of biomolecules in a cellular environment by live imaging.
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Nanopartículas Metálicas , Nanoestruturas , Ouro/química , Ligantes , Nanopartículas Metálicas/química , Soroalbumina Bovina/químicaRESUMO
A simple NIR-II emitting water-soluble system has been developed and applied in vitro and in vivo. In vitro, the fluorophore quickly accumulated in 2D and 3D cell cultures and rapidly reached the tumor in rodents, showing high NIR-II contrast for up to 1 week. This very efficient probe possesses all the qualities necessary for translation to the clinic as well as for the development of NIR-II emitting materials.
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Compostos Aza/química , Compostos de Boro/química , Corantes/química , Raios Infravermelhos , Imagem Óptica/métodos , Água/química , Linhagem Celular Tumoral , HumanosRESUMO
Gold nanoclusters (Au NCs) are an emerging class of luminescent nanomaterials but still suffer from moderate photoluminescence quantum yields. Recent efforts have focused on tailoring their emission properties. Introducing zwitterionic ligands to cap the metallic kernel is an efficient approach to enhance their one-photon excitation fluorescence intensity. In this work, we extend this concept to the nonlinear optical regime, i.e., two-photon excitation fluorescence. For a proper comparison between theory and experiment, both ligand and solvent effects should be considered. The effects of ligand shell size and of aqueous solvent on the optical properties of zwitterion functionalized gold nanoclusters have been studied by performing quantum mechanics/molecular mechanics (QM/MM) simulations.
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With the objective to evaluate the potential of ultra-small gold (Au) nanoclusters (NCs) for optical image-guided surgery, we synthesized and characterized AuNCs shelled by zwitterionic or pegylated ligands. The toxicity of the different AuNCs was evaluated on the Head and Neck Squamous Cell Carcinoma (HNSCC) CAL-33 and SQ20B cell lines in vitro. The safer AuNCs were administrated intravenously to mice for the determination of the pharmacokinetic properties. Biodistributions were performed on orthotopic CAL-33 HNSCC-bearing mice. Finally, the AuNCs were used for image-guided surgery, allowing the increase of the survival time vs. control animals, and the number of animals without any local recurrence.
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Meios de Contraste/química , Ouro/química , Neoplasias de Cabeça e Pescoço/cirurgia , Nanopartículas Metálicas/química , Cirurgia Assistida por Computador , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Contraste/farmacocinética , Endocitose , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Distribuição TecidualRESUMO
The use of visible/NIR-emitting gold nanoclusters (Au NCs), previously proposed for in vivo imaging, has been limited to some extent by low quantum yields (QYs) and the limited penetration of visible light in tissue. Here we report short wavelength infrared (SWIR, λ = 1-2 µm) emitting Au NCs with a good photoluminescence QY for this wavelength range (0.6% to 3.8% for λem = 1000 to 900 nm) and excellent stability under physiological conditions. We show that surface ligand chemistry is critical to achieving these properties. We demonstrate the potential of these SWIR-emitting Au NCs for in vivo imaging in mice. The Au NCs have a hydrodynamic diameter that is small (â¼5 nm) enough that they exhibit a rapid renal clearance, and images taken in the SWIR region show better resolution of the blood vessels than in the NIR region.
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Betaína/análogos & derivados , Ouro/química , Substâncias Luminescentes/química , Nanopartículas Metálicas/química , Imagem Óptica/métodos , Animais , Betaína/análise , Betaína/química , Ouro/análise , Raios Infravermelhos , Luz , Substâncias Luminescentes/análise , Medições Luminescentes/métodos , Nanopartículas Metálicas/análise , Camundongos , Ondas de RádioRESUMO
Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.
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Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.
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Nanopartículas Metálicas , Nanopartículas , Ouro/química , Nanopartículas/química , Polímeros , Espécies Reativas de Oxigênio/metabolismo , Nanopartículas Metálicas/químicaRESUMO
Using fluorescence-guided surgery (FGS) to cytoreductive surgery helps achieving complete resection of microscopic ovarian tumors. The use of visible and NIR-I fluorophores has led to beneficial results in clinical trials; however, involving NIR-II dyes seems to outperform those benefits due to the deeper tissue imaging and higher signal/noise ratio attained within the NIR-II optical window. In this context, we developed NIR-II emitting dyes targeting human epidermal growth factor receptor 2 (HER2)-positive ovarian tumors by coupling water-soluble NIR-II aza-BODIPY dyes to the FDA-approved anti-HER2 antibody, namely, trastuzumab. These bioconjugated NIR-II-emitting dyes displayed a prolonged stability in serum and a maintained affinity toward HER2 in vitro. We obtained selective targeting of HER2 positive tumors (SKOV-3) in vivo, with a favorable tumor accumulation. We demonstrated the fluorescence properties and the specific HER2 binding of the bioconjugated dyes in vivo and thus their potential for NIR-II FGS in the cancer setting.
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Anticorpos Monoclonais , Neoplasias Ovarianas , Feminino , Humanos , Trastuzumab/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Corantes FluorescentesRESUMO
Lateral diffusion of nano-objects on lipid membranes is a crucial process in cell biology. Recent studies indicate that nanoparticle lateral diffusion is affected by the presence of membrane proteins and deviates from Brownian motion. Gold nanoparticles (Au NPs) stabilized by short thiol ligands were dispersed near a free-standing bilayer formed in a 3D microfluidic chip. Using dark-field microscopy, the position of single NPs at the bilayer surface was tracked over time. Numerical analysis of the NP trajectories shows that NP diffusion on the bilayer surface corresponds to Brownian motion. The addition of bovine serum albumin (BSA) protein to the solution led to the formation of a protein corona on the NP surface. We found that protein-coated NPs show anomalous superdiffusion and that the distribution of their relative displacement obeys Lévy flight statistics. This superdiffusive motion is attributed to a drastic reduction in adhesive energies between the NPs and the bilayer in the presence of the protein corona. This hypothesis was confirmed by numerical simulations mimicking the random walk of a single particle near a weakly adhesive surface. These results may be generalized to other classes of nano-objects that experience adsorption-desorption behaviour with a weakly adhesive surface.
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Nanopartículas Metálicas , Nanopartículas , Coroa de Proteína , Ouro , Bicamadas Lipídicas , Proteínas de Membrana , Nanopartículas/metabolismo , Soroalbumina Bovina , Compostos de SulfidrilaRESUMO
The influence of solvent polarity and surface ligand rigidification on the SWIR emission profile of gold nanoclusters with an anistropic surface was investigated. A strong enhancement of the SWIR emission band at 1200 nm was observed when measuring in different local environments: in solution, in polymer composites, and in solids. SWIR in vivo imaging of mice assisted by deep learning after intravenous administration of these gold nanoclusters provides high definition pseudo-3D views of vascular blood vessels.
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Ouro/química , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Animais , Ligantes , Camundongos , Espectrofotometria Infravermelho/métodos , Propriedades de SuperfícieRESUMO
Following intravenous administration, the interaction of fluorescent exogenous molecules with circulating endogenous transporters can influence their photophysical properties as well as their fate and distribution, and possibly their recognition by different cell types. This type of interaction can be used to optimize the drug delivery but also the imaging properties of a compound of interest. In this study, we investigated the behavior of SWIR-WAZABY-01 fluorophore, a water-soluble aza-BODIPY dye emitting in the NIR-II region, both in vitro and in vivo. While the fluorescence emission of SWIR-WAZABY-01 was weak in aqueous solutions, it was intensely magnified in plasma (â¼ ×30). Further analyses using lipoprotein gel electrophoresis and ultracentrifugation revealed interactions between SWIR-WAZABY-01 and plasma lipoproteins in vitro and ex vivo, in particular with LDL. The tumor uptake mechanism of SWIR-WAZABY-01 was investigated based on the presence of low-density lipoprotein (LDL) receptors and passive tumor uptake. Overall, we found that SWIR-WAZABY-01 interacts with lipoproteins enhancing their NIR-II fluorescence emission, and driving the tumor accumulation with regards to the expression of lipoprotein receptors (LDLR, SR-BI). Moreover, SWIR-WAZABY-01, by exploiting endogenous lipoproteins, arises as a new, potent and relevant tool to efficiently label LDL involved in pathologies.
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Neoplasias , Receptores de Lipoproteínas , Humanos , Fluorescência , Corantes Fluorescentes , Lipoproteínas LDL/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológicoRESUMO
Ultrasmall metal nanoclusters (NCs) are employed in an array of diagnostic and therapeutic applications due to their tunable photoluminescence, high biocompatibility, polyvalent effect, ease of modification, and photothermal stability. However, gold nanoclusters' (AuNCs') intrinsically antimicrobial properties remain poorly explored and are not well understood. Here, we share an insight into the antimicrobial action of atomically precise AuNCs based on their ability to passively translocate across the bacterial membrane. Functionalized by a hydrophilic modified-bidentate sulfobetaine zwitterionic molecule (AuNC-ZwBuEt) or a more hydrophobic monodentate-thiolate, mercaptohexanoic acid (AuNC-MHA) molecule, 2 nm AuNCs were lethal to both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. The bactericidal efficiency was found to be bacterial strain-, time-, and concentration-dependent. The direct visualizations of the translocation of AuNCs and AuNC-cell and subcellular interactions were investigated using cryo-soft X-ray nano-tomography, transmission electron microscopy (TEM), and scanning TEM energy-dispersive spectroscopy analyses. AuNC-MHA were identified in the bacterial cytoplasm within 30 min, without evidence of the loss of membrane integrity. It is proposed that the bactericidal effect of AuNCs is attributed to their size, which allows for efficient energy-independent translocation across the cell membrane. The internalization of both AuNCs caused massive internal damage to the cells, including collapsed subcellular structures and altered cell morphology, leading to the eventual loss of cellular integrity.
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Anti-Infecciosos , Nanopartículas Metálicas , Antibacterianos/farmacologia , Bactérias , Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Pseudomonas aeruginosa , Staphylococcus aureusRESUMO
Human transferrin has been biolabelled with gold nanoclusters (Au NCs) using a simple, fast and non-toxic method. These nanocrystals (<2 nm) are stabilized in the protein via sulfur groups and have a high fluorescence emission in the near infrared region (QY=4.3%; λem=695 nm). Structural investigation and photophysical measurements show a high population of clusters formed of 22-33 gold atoms covalently bound to the transferrin. In solutions with pH ranging from 5 to 10 and in buffer solutions (PBS, HEPES), those biolabelled proteins exhibit a good stability. No significant quenching effect of the fluorescent transferrin has been detected after iron loading of iron-free transferrin (apoTf) and in the presence of a specific polyclonal antibody. Additionally, antibody-induced agglomeration demonstrates no alteration in the protein activity and the receptor target ability. MTT and Vialight® Plus tests show no cytotoxicity of these labelled proteins in cells (1 µg ml(-1)-1 mg ml(-1)). Cell line experiments (A549) indicate also an uptake of the iron loaded fluorescent proteins inside cells. These remarkable data highlight the potential of a new type of non-toxic fluorescent transferrin for imaging and targeting.
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Ouro/química , Nanopartículas Metálicas/química , Transferrina/metabolismo , Apoproteínas/metabolismo , Linhagem Celular Tumoral , Fluorescência , Humanos , Ferro/metabolismo , Tamanho da Partícula , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de TempoRESUMO
Shortwave infrared window (SWIR: 1000-1700 nm) represents a major improvement compared to the NIR-I region (700-900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIR-IIb region (1500-1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images.
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Aprendizado Profundo , Animais , Raios Infravermelhos , Redes Neurais de Computação , Ondas de RádioRESUMO
We evaluated the impact of light-scattering effects on spatial resolution in different shortwave infrared (SWIR) sub-regions by analyzing two SWIR emissive phantoms made of polydimethylsiloxane (PDMS)-gold nanoclusters (Au NCs) composite covered with mice skin, or capillary tubes filled with Au NCs or IRDye 800CW at different depth in intralipids and finally, after administration of the Au NCs intravenously in mice. Our findings highlighted the benefit of working at the highest tested spectral range of the SWIR region with a 50% enhancement of spatial resolution measured in artificial model when moving from NIR-II (1000-1300 nm) to NIR-IIa (1300-1450 nm) region, and a 25% reduction of the scattering from the skin determined by point spread function analysis from the NIR-II to NIR-IIb region (1500-1700 nm). We also confirmed that a series of Monte Carlo restoration of images significantly improved the spatial resolution in vivo in mice in deep tissues both in the NIR-II and NIR-IIa spectral windows.
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Ouro , Ondas de Rádio , Animais , Raios Infravermelhos , Camundongos , Imagem Óptica , Imagens de FantasmasRESUMO
In parallel with the rapidly growing and widespread use of nanomedicine in the clinic, we are also witnessing the development of so-called theranostic agents that combine diagnostic and therapeutic properties. Among them, ultra-small gold nanoclusters (Au NCs) show promising potential due to their optical properties and activatable therapeutic activities under irradiation. Furthermore, due to their hydrodynamic diameter of smaller than 6 nm and unique biophysical properties, they also present intriguing behaviors in biological and physio-pathological environments. In this review, we aim to present the latest research studies published on such nanoparticles in animals. We also propose guidelines to identify the main physico-chemical parameters that govern the behaviour of Au NCs after administration in small animals, notably concerning their renal elimination and their ability to accumulate in tumors. Then, we present recent advances in their use as theranostic agents putting them in parallel with other contrast agents.
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Antineoplásicos/química , Meios de Contraste/química , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Animais , Antineoplásicos/farmacocinética , Meios de Contraste/farmacocinética , Tratamento Farmacológico , Humanos , Rim , Imageamento por Ressonância Magnética , Nanopartículas Metálicas/administração & dosagem , Imagem Óptica , Tamanho da Partícula , Técnicas Fotoacústicas , Fototerapia , Tomografia por Emissão de Pósitrons , Radioterapia , Propriedades de Superfície , Nanomedicina Teranóstica , Tomografia Computadorizada por Raios XRESUMO
A library of ultra-small red photoluminescent gold nanoclusters (Au NCs) were synthesized with an increasing amount of positive charges provided by the addition of mono-, di- or trivalent-glutathione modified arginine peptides. We then studied how the arginine content impacted on the interaction of Au NCs with negatively charged artificial lipid bilayers and cell membranes. Results indicated that increasing the arginine content enhanced Au NCs' adsorption on lipid bilayers and on cell membranes followed by an increased cellular uptake in melanoma cells (COLO 829). Surprisingly, the presence of up to 40% serum for highly positively charged Au NCs did not hinder their interaction with lipid bilayers that contain glycolipids, suggesting a reduced opsonization of these Au NCs. In addition, these Au NCs are usually not toxic, except those with the highest arginine contents. Thus, controlled grafting of arginine peptides onto Au NCs is an elegant strategy to improve their binding and internalization by tumor cells while still keeping their anti-fouling properties.