Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths. However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics. These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.

Optical imaging within the shortwave infrared (SWIR, 1000-2000 nm) region of the electromagnetic spectrum has enabled high-resolution and high-contrast imaging in mice, non-invasively. Polymethine dyes, with their narrow absorption spectra and high absorption coefficients, are optimal probes for fast and multiplexed SWIR imaging. Here, we expand upon the multiplexing capabilities in SWIR imaging by obtaining brighter polymethine dyes with varied excitation wavelengths spaced throughout the near-infrared (700-1000 nm) region. Building on the flavylium polymethine dye scaffold, we explored derivatives with functional group substitution at the 2-position, deemed chromenylium polymethine dyes. The reported dyes have reduced nonradiative rates and enhanced emissive properties, enabling non-invasive imaging in mice in a single color at 300 fps and in three colors at 100 fps. Combined with polymethine dyes containing a red-shifted julolidine flavylium heterocycle and indocyanine green, distinct channels with well-separated excitation wavelengths provide non-invasive video-rate in vivo imaging in four colors.

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-020-0569-y.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

Monitoring the progression of non-alcoholic fatty liver disease is hindered by a lack of suitable non-invasive imaging methods. Here, we show that the endogenous pigment lipofuscin displays strong near-infrared and shortwave-infrared fluorescence when excited at 808 nm, enabling label-free imaging of liver injury in mice and the discrimination of pathological processes from normal liver processes with high specificity and sensitivity. We also show that the near-infrared and shortwave-infrared fluorescence of lipofuscin can be used to monitor the progression and regression of liver necroinflammation and fibrosis in mouse models of non-alcoholic fatty liver disease and advanced fibrosis, as well as to detect non-alcoholic steatohepatitis and cirrhosis in biopsied samples of human liver tissue.Label-free imaging of the endogenous pigment lipofuscin at near-infrared and shortwave-infrared wavelengths enables the longitudinal monitoring of liver injury in mice and in biopsied human livers.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-Aggregation offers a means to transform stable, near-infrared (NJR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that J-aggregates of NIR fluorophore IR-140 can be prepared inside hollow mesoporous silica nanoparticles (HMSNs) to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging in vivo with 980 nm excitation.

The first ever demonstration of temporal focusing with short wave infrared (SWIR) excitation and emission is demonstrated, achieving a penetration depth of 500 μm in brain tissue. This is substantially deeper than the highest previously-reported values for temporal focusing imaging in brain tissue, and demonstrates the value of these optimized wavelengths for neurobiological applications.

OBJECTIVE: To evaluate the feasibility of Shortwave infrared (SWIR) otoscopy in a pediatric population and establish differences with visible otoscopy. METHODS: Pediatric patients 3 years of age and older seen in the otolaryngology clinic with an audiogram and tympanogram obtained within a week of the visit were recruited for video otoscopy using visible light otoscopy and SWIR otoscopy. Videos were rated by two otolaryngologists based on ability to identify the promontory, ability to identify the ossicular chain and presence or absence of middle ear fluid. RESULTS: A total of 74 video recordings of ears were obtained in 20 patients. We obtained interpretable images in 63/74 (85.1%) ears. There was no statistical significance between ability to perform SWIR otoscopy versus white light video otoscopy as indicated by a p-value of 0.376. There was high inter-rater agreement for identification of both the promontory and the ossicular chain with Kappa values of 0.81 and 0.92 respectively. There was statistical significance between SWIR otoscopy and visible otoscopy in the ability to image the promontory (p = 0.012) and the ossicular chain (p = 0.010). Increased contrast of middle ear fluid was seen in SWIR otoscopy when compared to visible otoscopy. CONCLUSION: SWIR otoscopy is feasible in a pediatric population and could offer some advantages over visible light otoscopy such as better visualization of the middle ear structures through the tympanic membrane and increased contrast for middle ear effusions.

Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000-2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

Adipocytes possess remarkable adaptive capacity to respond to nutrient excess, fasting or cold exposure, and they are thus an important cell type for the maintenance of proper metabolic health. Although the endoplasmic reticulum (ER) is a critical organelle for cellular homeostasis, the mechanisms that mediate adaptation of the ER to metabolic challenges in adipocytes are unclear. Here we show that brown adipose tissue (BAT) thermogenic function requires an adaptive increase in proteasomal activity to secure cellular protein quality control, and we identify the ER-localized transcription factor nuclear factor erythroid 2-like 1 (Nfe2l1, also known as Nrf1) as a critical driver of this process. We show that cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity, specifically when the cells are in a state of high thermogenic activity. In mice, under thermogenic conditions, brown-adipocyte-specific deletion of Nfe2l1 (Nrf1) resulted in ER stress, tissue inflammation, markedly diminished mitochondrial function and whitening of the BAT. In mouse models of both genetic and dietary obesity, stimulation of proteasomal activity by exogenously expressing Nrf1 or by treatment with the proteasome activator PA28α in BAT resulted in improved insulin sensitivity. In conclusion, Nrf1 emerges as a novel guardian of brown adipocyte function, providing increased proteometabolic quality control for adapting to cold or to obesity.

Recent technology developments have expanded the wavelength window for biological fluorescence imaging into the shortwave infrared. We show here a mechanistic understanding of how drastic changes in fluorescence imaging contrast can arise from slight changes of imaging wavelength in the shortwave infrared. We demonstrate, in 3D tissue phantoms and in vivo in mice, that light absorption by water within biological tissue increases image contrast due to attenuation of background and highly scattered light. Wavelengths of strong tissue absorption have conventionally been avoided in fluorescence imaging to maximize photon penetration depth and photon collection, yet we demonstrate that imaging at the peak absorbance of water (near 1,450 nm) results in the highest image contrast in the shortwave infrared. Furthermore, we show, through microscopy of highly labeled ex vivo biological tissue, that the contrast improvement from water absorption enables resolution of deeper structures, resulting in a higher imaging penetration depth. We then illustrate these findings in a theoretical model. Our results suggest that the wavelength-dependent absorptivity of water is the dominant optical property contributing to image contrast, and is therefore crucial for determining the optimal imaging window in the infrared.

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 λ = 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 (∼5 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.

For imaging, the short-wavelength infrared region (SWIR; 1000-2000 nm) provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging by the biomedical research community. Here, we introduce a class of high-quality SWIR-emissive indium-arsenide-based quantum dots (QDs) that are readily modifiable for various functional imaging applications, and that exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. To demonstrate the unprecedented combination of deep penetration, high spatial resolution, multicolor imaging and fast-acquisition-speed afforded by the SWIR QDs, we quantified, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generated detailed three-dimensional quantitative flow maps of the mouse brain vasculature.

Medical imaging is routine in the diagnosis and staging of a wide range of medical conditions. In particular, magnetic resonance imaging (MRI) is critical for visualizing soft tissue and organs, with over 60 million MRI procedures performed each year worldwide. About one-third of these procedures are contrast-enhanced MRI, and gadolinium-based contrast agents (GBCAs) are the mainstream MRI contrast agents used in the clinic. GBCAs have shown efficacy and are safe to use with most patients; however, some GBCAs have a small risk of adverse effects, including nephrogenic systemic fibrosis (NSF), the untreatable condition recently linked to gadolinium (Gd) exposure during MRI with contrast. In addition, Gd deposition in the human brain has been reported following contrast, and this is now under investigation by the US Food and Drug Administration (FDA). To address a perceived need for a Gd-free contrast agent with pharmacokinetic and imaging properties comparable to GBCAs, we have designed and developed zwitterion-coated exceedingly small superparamagnetic iron oxide nanoparticles (ZES-SPIONs) consisting of ∼3-nm inorganic cores and ∼1-nm ultrathin hydrophilic shell. These ZES-SPIONs are free of Gd and show a high T contrast power. We demonstrate the potential of ZES-SPIONs in preclinical MRI and magnetic resonance angiography.

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.

Bright fluorophores in the near-infrared and shortwave infrared (SWIR) regions of the electromagnetic spectrum are essential for optical imaging in vivo. In this work, we utilized a 7-dimethylamino flavylium heterocycle to construct a panel of novel red-shifted polymethine dyes, with emission wavelengths from 680 to 1045 nm. Photophysical characterization revealed that the 1- and 3-methine dyes display enhanced photostability and the 5- and 7-methine dyes exhibit exceptional brightness for their respective spectral regions. A micelle formulation of the 7-methine facilitated SWIR imaging in mice. This report presents the first polymethine dye designed and synthesized for SWIR in vivo imaging.

With the emergence of applications based on short-wavelength infrared light, indium arsenide quantum dots are promising candidates to address existing shortcomings of other infrared-emissive nanomaterials. However, III-V quantum dots have historically struggled to match the high-quality optical properties of II-VI quantum dots. Here we present an extensive investigation of the kinetics that govern indium arsenide nanocrystal growth. Based on these insights, we design a synthesis of large indium arsenide quantum dots with narrow emission linewidths. We further synthesize indium arsenide-based core-shell-shell nanocrystals with quantum yields up to 82% and improved photo- and long-term storage stability. We then demonstrate non-invasive through-skull fluorescence imaging of the brain vasculature of murine models, and show that our probes exhibit 2-3 orders of magnitude higher quantum yields than commonly employed infrared emitters across the entire infrared camera sensitivity range. We anticipate that these probes will not only enable new biomedical imaging applications, but also improved infrared nanocrystal-LEDs and photon-upconversion technology.

Visualizing structures deep inside opaque biological tissues is one of the central challenges in biomedical imaging. Optical imaging with visible light provides high resolution and sensitivity; however, scattering and absorption of light by tissue limits the imaging depth to superficial features. Imaging with shortwave infrared light (SWIR, 1-2 μm) shares many advantages of visible imaging, but light scattering in tissue is reduced, providing sufficient optical penetration depth to noninvasively interrogate subsurface tissue features. However, the clinical potential of this approach has been largely unexplored because suitable detectors, until recently, have been either unavailable or cost prohibitive. Here, taking advantage of newly available detector technology, we demonstrate the potential of SWIR light to improve diagnostics through the development of a medical otoscope for determining middle ear pathologies. We show that SWIR otoscopy has the potential to provide valuable diagnostic information complementary to that provided by visible pneumotoscopy. We show that in healthy adult human ears, deeper tissue penetration of SWIR light allows better visualization of middle ear structures through the tympanic membrane, including the ossicular chain, promontory, round window niche, and chorda tympani. In addition, we investigate the potential for detection of middle ear fluid, which has significant implications for diagnosing otitis media, the overdiagnosis of which is a primary factor in increased antibiotic resistance. Middle ear fluid shows strong light absorption between 1,400 and 1,550 nm, enabling straightforward fluid detection in a model using the SWIR otoscope. Moreover, our device is easily translatable to the clinic, as the ergonomics, visual output, and operation are similar to a conventional otoscope.

Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.

Accurate determination of tissue concentration of ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) using T2 * MR relaxometry is still challenging. We present a reliable quantification method for local USPIO amount with the estimation of the liver specific relaxivity r2 * using monodisperse (59) Fe-core-labeled USPIO ((59) FeUSPIO). Dynamic and relaxometric in vivo characteristics of unlabeled monodisperse USPIO were determined in MRI at 3 T. The in vivo MR studies were performed for liver tissue with (59) FeUSPIO using iron dosages of 9 (n = 3), 18 (n = 2) and 27 (n = 3) µmol Fe kg(-1) body weight. The R2 * of the liver before and after USPIO injection (∆R2 *) was measured and correlated with (59) Fe activity measurements of excised organs by a whole body radioactivity counter (HAMCO) to define the dependency of ∆R2 * and (59) FeUSPIO liver concentration and calculate the r2 * of (59) FeUSPIO for the liver. Ultrastructural analysis of liver uptake was performed by histology and transmission electron microscopy. ∆R2 * of the liver revealed a dosage-dependent accumulation of (59) FeUSPIO with a percentage uptake of 70-88% of the injection dose. Hepatic ∆R2 * showed a dose-dependent linear correlation to (59) FeUSPIO activity measurements (r = 0.92) and an r2 * in the liver of 481 ± 74.9 mm(-1) s(-1) in comparison to an in vitro r2 * of 60.5 ± 3.3 mm(-1) s(-1) . Our results indicate that core-labeled (59) FeUSPIO can be used to quantify the local amount of USPIO and to estimate the liver-specific relaxivity r2 *.

Micelles have been employed to encapsulate the supramolecular assembly of quantum dots with palladium(II) porphyrins for the quantification of O2 levels in aqueous media and in vivo. Förster resonance energy transfer from the quantum dot (QD) to the palladium porphyrin provides a means for signal transduction under both one- and two-photon excitation. The palladium porphyrins are sensitive to O2 concentrations in the range of 0-160 Torr. The micelle-encapsulated QD-porphyrin assemblies have been employed for in vivo multiphoton imaging and lifetime-based oxygen measurements in mice with chronic dorsal skinfold chambers or cranial windows. Our results establish the utility of the QD-micelle approach for in vivo biological sensing applications.

BACKGROUND & AIMS: It is well-known that the liver can induce immune tolerance, yet this knowledge could, thus far, not be translated into effective treatments for autoimmune diseases. We have previously shown that liver sinusoidal endothelial cells (LSECs) could substantially contribute to hepatic tolerance through their ability to induce CD4+ Foxp3+ regulatory T cells (Tregs). Here, we explored whether the Treg-inducing potential of LSECs could be harnessed for the treatment of autoimmune disease. METHODS: We engineered a polymeric nanoparticle (NP) carrier for the selective delivery of autoantigen peptides to LSECs in vivo. In the well-characterized autoimmune disease model of experimental autoimmune encephalomyelitis (EAE), we investigated whether administration of LSEC-targeting autoantigen peptide-loaded NPs could protect mice from autoimmune disease. RESULTS: We demonstrate that NP-based autoantigen delivery to LSECs could completely and permanently prevent the onset of clinical EAE. More importantly, in a therapeutic approach, mice with already established EAE improved rapidly and substantially following administration of a single dose of autoantigen peptide-loaded NPs, whereas the control group deteriorated. Treatment efficacy seemed to depend on Tregs. The Treg frequencies in the spleens of mice treated with autoantigen peptide-loaded NPs were significantly higher than those in vehicle-treated mice. Moreover, NP-mediated disease control was abrogated after Treg depletion by repeated administration of Treg-depleting antibody. CONCLUSION: Our findings provide proof of principle that the selective delivery of autoantigen peptides to LSECs by NPs can induce antigen-specific Tregs and enable effective treatment of autoimmune disease. These findings highlight the importance of Treg induction by LSECs for immune tolerance.

BACKGROUND: The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging. METHODS AND RESULTS: Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDL(SPIOs) radiolabeled with (59)Fe (NP-HDL(59Fe-SPIOs)) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t-test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL(59Fe-SPIOs) uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection (P<0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis. CONCLUSIONS: In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

Semiconductor quantum dots (QD) and superparamagnetic iron oxide nanocrystals (SPIO) have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene) (PMAOD). The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr (-/-) as well as Apoe (-/-) mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα) or chemokine (C-X-C motif) ligand 10 (Cxcl10) indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications.

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle 'core', which is fully surrounded by a 'shell' of fluorescent quantum dots. A thin layer of silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.

We have recently developed compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand coated superparamagnetic iron oxide nanoparticles (SPIONs) for use in various biomedical applications. The defining characteristics of ZDS-coated SPIONs are small hydrodynamic diameters, low non-specific interactions with fetal bovine serum, the opportunity for specific labeling, and stability with respect to time, pH, and salinity. We report here on the magnetic characterization of ZDS-coated SPIONs and their in vitro and in vivo performance relative to non-specific interactions with HeLa cells and in mice, respectively. ZDS-coated SPIONs retained the superparamagnetism and saturation magnetization (M(s)) of as-synthesized hydrophobic SPIONs, with M(s) = 74 emu g(-1) [Fe]. Moreover, ZDS-coated SPIONs showed only small non-specific uptake into HeLa cancer cells in vitro and low non-specific binding to serum proteins in vivo in mice.

BACKGROUND & AIMS: The liver can mitigate the inflammatory activity of infiltrating T cells by mechanisms that are not entirely clear. Here we investigated the role of liver sinusoidal endothelial cells (LSECs) in regulating the activity of inflammatory CD4 T cells. METHODS: Interactions between T helper (Th) 1 or Th17 cells and LSEC were studied by intravital microscopy and by in vitro stimulation assays. RESULTS: Circulating CD4 T cells established lasting and repeated interactions with liver endothelium in vivo. Stimulation of Th1 and Th17 cells by LSEC greatly inhibited their capacity to secrete interferon-γ or interleukin-17 in vitro; in contrast, stimulation by dendritic cells (DCs) resulted in considerable secretion of both cytokines. Cytokine release by Th1 or Th17 cells seemed to be actively suppressed by LSEC, as indicated by the inhibition of cytokine secretion even in the presence of Th1- and Th17-promoting DC. This inhibition of CD4 T cell effector function seemed to depend on the dominance of inhibitory over activating co-stimulatory signals on LSEC, since (1) cytokine secretion could be restored by increased CD28 co-activation; (2) LSEC from interleukin-10(-/-) mice, which manifest increased activating signals, such as MHC II, and decreased inhibitory signals, such as PD-L1, failed to suppress cytokine secretion; and (3) cytokine secretion by Th1 or Th17 cells that lacked PD-1, the ligand for inhibitory PD-L1, could not be suppressed by LSEC. CONCLUSIONS: LSEC inhibit inflammatory cytokine secretion of Th1 and Th17 effector CD4 T cells in dependence of interleukin-10 and PD-1.

Nanotechnology deals with structures with a maximum size of 100 nanometers and is applied in various scientific disciplines. The basis for this is its potential to create many new materials such as nanoparticles which are suitable for a vast range of applications in electronics or energy production but also in biomedicine. Nanoparticles have exceptional physical properties useful for different applications ranging from material sciences to biomedical imaging. In life sciences nanoparticles provide a novel tool to study metabolic processes such as the metabolism of lipoproteins or to noninvasively detect diseases in a very early stage. Major hallmarks of early atherosclerotic lesion formation are endothelial dysfunction and accumulation of large amounts of lipoprotein-derived cholesterol esters in macrophages within the vessel wall. Since conventional methods such as plasma marker analyses are not specific and sensitive enough to reliably assess the risk of cardiovascular events at an early stage, nanoparticles-based imaging technologies might provide a valuable tool for the non-invasive assessment of atherosclerotic lesions in the future. In this review, we will give an overview on the characteristics of modern nanoparticles and will emphasize the current studies utilizing nanoparticles for the visualization of both lipoprotein metabolism and atherosclerosis.

A simple, fast, efficient, and widely applicable method to radiolabel the cores of monodisperse superparamagnetic iron oxide nanoparticles (SPIOs) with (59)Fe was developed. These cores can be used as precursors for a variety of functionalized nanodevices. A quality control using filtration techniques, size-exclusion chromatography, chemical degradation methods, transmission electron microscopy, and magnetic resonance imaging showed that the nanoparticles were stably labeled with (59)Fe. Furthermore, the particle structure and the magnetic properties of the SPIOs were unchanged. In a second approach, monodisperse SPIOs stabilized with (14)C-oleic acid were synthesized, and the stability of this shell labeling was studied. In proof of principle experiments, the (59)Fe-SPIOs coated with different shells to make them water-soluble were used to evaluate and compare in vivo pharmacokinetic parameters such as blood half-life. It could also be shown that our radiolabeled SPIOs embedded in recombinant lipoproteins can be used to quantify physiological processes in closer detail than hitherto possible. In vitro and in vivo experiments showed that the (59)Fe label is stable enough to be applied in vivo, whereas the (14)C label is rapidly removed from the iron core and is not adequate for in vivo studies. To obtain meaningful results in in vivo experiments, only (59)Fe-labeled SPIOs should be used.

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well.In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD(+) serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261-279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival.

In this study we systematically developed a potential MR T(1) contrast agent based on very small PEGylated iron oxide nanoparticles. We adjusted the size of the crystalline core providing suitable relaxometric properties. In addition, a dense and optimized PEG coating provides high stability under physiological conditions together with low cytotoxicity and low nonspecific phagocytosis into macrophage cells as a part of the reticulo endothelial system at biologically relevant concentrations. The as developed contrast agent has the lowest r(2)/r(1) ratio (2.4) at 1.41 T reported so far for PEGylated iron oxide nanoparticles as well as a r(1) relaxivity (7.3 mM(-1) s(-1)) that is two times higher compared to that of Magnevist as a typical T(1) contrast agent based on gadolinium as a clinical standard.

Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals have physical properties that are well suited for biomedical imaging. Previously, we have shown that iron oxide nanocrystals embedded within the lipid core of micelles show optimized characteristics for quantitative imaging. Here, we embed quantum dots and superparamagnetic iron oxide nanocrystals in the core of lipoproteins--micelles that transport lipids and other hydrophobic substances in the blood--and show that it is possible to image and quantify the kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic magnetic resonance imaging. The lipoproteins were taken up by liver cells in wild-type mice and displayed defective clearance in knock-out mice lacking a lipoprotein receptor or its ligand, indicating that the nanocrystals did not influence the specificity of the metabolic process. Using this strategy it is possible to study the clearance of lipoproteins in metabolic disorders and to improve the contrast in clinical imaging.

Uptake of colloidal polyelectrolyte-coated particles and polyelectrolyte multilayer capsules has been demonstrated by living cells. The capsules for this study were prepared by poly(diallyldimethylammoniumchloride) (PDMAC) and poly(styrene sulfonate) (PSS) on SiO2. SiO2 template cores were used because of their negative charge. It was observed during the study that SiO2 templates dissolved in hydrofluoric acid after the formation of capsule-shell. The study also found that heat treatment can be used for stable encapsulation of materials inside the microcapsuels made on SiO 2. It was also found that calcium carbonate microparticles can provide a loading capacity for biological materials. The study used a confocal laser scanning microscopy (CLSM) to examine the capsule incorporation by the cells.

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.

Superparamagnetic MnFe2O4 nanocrystals of different sizes were synthesized in high-boiling ether solvent and transferred into water using three different approaches. First, we applied a ligand exchange in order to form a water soluble polymer shell. Second, the particles were embedded into an amphiphilic polymer shell. Third, the nanoparticles were embedded into large micelles formed by lipids. Although all approaches lead to effective negative contrast enhancement, we observed significant differences concerning the magnitude of this effect. The transverse relaxivity, in particular r2*, is greatly higher for the micellar system compared to the polymer-coated particles using same-sized nanoparticles. We also observed an increase in transverse relaxivities with increasing particle size for the polymer-coated nanocrystals. The results are qualitatively compared with theoretical models describing the dependence of relaxivity on the size of magnetic spheres.

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.

Due to their greater photostability compared to established organic fluorescence markers, semiconductor quantum dots provide an attractive alternative for the biolabeling of living cells. On the basis of a comparative investigation using differently sized thiol-stabilized CdTe nanocrystals in a variety of commonly used biological buffers, a method is developed to quantify the stability of such a multicomponent system. Above a certain critical size, the intensity of the photoluminescence of the nanocrystals is found to diminish with pseudo-zero-order kinetics, whereas for specific combinations of particle size, ligand, and buffer there appears to be no decay below this critical particle size, pointing out the necessity for thorough investigations of this kind in the view of prospect applications of semiconductor nanocrystals in the area of biolabeling.