Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

Cell Therapy by Mesenchymal Stromal Cells Versus Myoblasts in a Pig Model of Urinary Incontinence.

The leading cause of stress urinary incontinence (SUI) in women is the urethral sphincter muscle deficiency caused by mechanical stress during pregnancy and vaginal delivery. In men, prostate cancer surgery and injury of local nerves and muscles are associated with incontinence. Current treatment often fails to satisfy the patient's needs. Cell therapy may improve the situation. We therefore investigated the regeneration potential of cells in ameliorating sphincter muscle deficiency and UI in a large animal model. Urethral sphincter deficiency was induced surgically in gilts by electrocautery and balloon dilatation. Adipose tissue-derived stromal cells (ADSCs) and myoblasts from Musculus semitendinosus were isolated from male littermates, expanded, characterized in depth for expression of marker genes and in vitro differentiation, and labeled. The cells were injected into the deficient sphincter complex of the incontinent female littermates. Incontinent gilts receiving no cell therapy served as controls. Sphincter deficiency and functional regeneration were recorded by monitoring the urethral wall pressure during follow-up by two independent methods. Cells injected were detected in vivo during follow-up by transurethral fluorimetry, ex vivo by fluorescence imaging, and in cryosections of tissues targeted by immunofluorescence and by polymerase chain reaction of the sex-determining region Y (SRY) gene. Partial spontaneous regeneration of sphincter muscle function was recorded in control gilts, but the sphincter function remained significantly below levels measured before induction of incontinence (67.03% ± 14.00%, n = 6, p < 0.05). Injection of myoblasts yielded an improved sphincter regeneration within 5 weeks of follow-up but did not reach significance compared to control gilts (81.54% ± 25.40%, n = 5). A significant and full recovery of the urethral sphincter function was observed upon injection of ADSCs within 5 weeks of follow-up (100.4% ± 23.13%, n = 6, p < 0.05). Injection of stromal cells provoked slightly stronger infiltration of CD45pos leukocytes compared to myoblasts injections and controls. The data of this exploratory study indicate that ADSCs inherit a significant potential to regenerate the function of the urethral sphincter muscle.

Stratification of ovarian cancer borderline from high-grade serous carcinoma patients by quantitative serum NMR spectroscopy of metabolites, lipoproteins, and inflammatory markers.

Background: Traditional diagnosis is based on histology or clinical-stage classification which provides no information on tumor metabolism and inflammation, which, however, are both hallmarks of cancer and are directly associated with prognosis and severity. This project was an exploratory approach to profile metabolites, lipoproteins, and inflammation parameters (glycoprotein A and glycoprotein B) of borderline ovarian tumor (BOT) and high-grade serous ovarian cancer (HGSOC) for identifying additional useful serum markers and stratifying ovarian cancer patients in the future. Methods: This project included 201 serum samples of which 50 were received from BOT and 151 from high-grade serous ovarian cancer (HGSOC), respectively. All the serum samples were validated and phenotyped by 1H-NMR-based metabolomics with in vitro diagnostics research (IVDr) standard operating procedures generating quantitative data on 38 metabolites, 112 lipoprotein parameters, and 5 inflammation markers. Uni- and multivariate statistics were applied to identify NMR-based alterations. Moreover, biomarker analysis was carried out with all NMR parameters and CA-125. Results: Ketone bodies, glutamate, 2-hydroxybutyrate, glucose, glycerol, and phenylalanine levels were significantly higher in HGSOC, while the same tumors showed significantly lower levels of alanine and histidine. Furthermore, alanine and histidine and formic acid decreased and increased, respectively, over the clinical stages. Inflammatory markers glycoproteins A and B (GlycA and GlycB) increased significantly over the clinical stages and were higher in HGSOC, alongside significant changes in lipoproteins. Lipoprotein subfractions of VLDLs, IDLs, and LDLs increased significantly in HGSOC and over the clinical stages, while total plasma apolipoprotein A1 and A2 and a subfraction of HDLs decreased significantly over the clinical stages. Additionally, LDL triglycerides significantly increased in advanced ovarian cancer. In biomarker analysis, glycoprotein inflammation biomarkers behaved in the same way as the established clinical biomarker CA-125. Moreover, CA-125/GlycA, CA-125/GlycB, and CA-125/Glycs are potential biomarkers for diagnosis, prognosis, and treatment response of epithelial ovarian cancer (EOC). Last, the quantitative inflammatory parameters clearly displayed unique patterns of metabolites, lipoproteins, and CA-125 in BOT and HGSOC with clinical stages I-IV. Conclusion: 1H-NMR-based metabolomics with commercial IVDr assays could detect and identify altered metabolites and lipoproteins relevant to EOC development and progression and show that inflammation (based on glycoproteins) increased along with malignancy. As inflammation is a hallmark of cancer, glycoproteins, thereof, are promising future serum biomarkers for the diagnosis, prognosis, and treatment response of EOC. This was supported by the definition and stratification of three different inflammatory serum classes which characterize specific alternations in metabolites, lipoproteins, and CA-125, implicating that future diagnosis could be refined not only by diagnosed histology and/or clinical stages but also by glycoprotein classes.

Dual-Modality Surface-Enhanced Resonance Raman Scattering and Multispectral Optoacoustic Tomography Nanoparticle Approach for Brain Tumor Delineation.

Difficulty in visualizing glioma margins intraoperatively remains a major issue in the achievement of gross total tumor resection and, thus, better clinical outcome of glioblastoma (GBM) patients. Here, the potential of a new combined optical + optoacoustic imaging method for intraoperative brain tumor delineation is investigated. A strategy using a newly developed gold nanostar synthesis method, Raman reporter chemistry, and silication method to produce dual-modality contrast agents for combined surface-enhanced resonance Raman scattering (SERRS) and multispectral optoacoustic tomography (MSOT) imaging is devised. Following intravenous injection of the SERRS-MSOT-nanostars in brain tumor bearing mice, sequential MSOT imaging is performed in vivo and followed by Raman imaging. MSOT is able to accurately depict GBMs three-dimensionally with high specificity. The MSOT signal is found to correlate well with the SERRS images. Because SERRS enables uniquely sensitive high-resolution surface detection, it could represent an ideal complementary imaging modality to MSOT, which enables real-time, deep tissue imaging in 3D. This dual-modality SERRS-MSOT-nanostar contrast agent reported here is shown to enable high precision depiction of the extent of infiltrating GBMs by Raman- and MSOT imaging in a clinically relevant murine GBM model and could pave new ways for improved image-guided resection of brain tumors.

DNA-Nanostructure-Gold-Nanorod Hybrids for Enhanced In Vivo Optoacoustic Imaging and Photothermal Therapy.

A functional cancer theranostic nanoplatform is developed, specifically tailored toward the optoacoustic modality by combining gold nanorods with DNA nanostructures (D-AuNR). DNA origami is used as an efficient delivery vehicle owing to its prominent tumor-targeting property. The D-AuNR hybrids display an enhanced tumor diagnostic sensitivity by improved optoacoustic imaging and excellent photothermal therapeutic properties in vivo.

High-Resolution Multispectral Optoacoustic Tomography of the Vascularization and Constitutive Hypoxemia of Cancerous Tumors.

Diversity of the design and alignment of illumination and ultrasonic transducers empower the fine scalability and versatility of optoacoustic imaging. In this study, we implement an innovative high-resolution optoacoustic mesoscopy for imaging the vasculature and tissue oxygenation within subcutaneous and orthotopic cancerous implants of mice in vivo through acquisition of tomographic projections over 180° at a central frequency of 24 MHz. High-resolution volumetric imaging was combined with multispectral functional measurements to resolve the exquisite inner structure and vascularization of the entire tumor mass using endogenous and exogenous optoacoustic contrast. Evidence is presented for constitutive hypoxemia within the carcinogenic tissue through analysis of the hemoglobin absorption spectra and distribution. Morphometric readouts obtained with optoacoustic mesoscopy have been verified with high-resolution ultramicroscopic studies. The findings described herein greatly extend the applications of optoacoustic mesoscopy toward structural and multispectral functional measurements of the vascularization and hemodynamics within solid tumors in vivo and are of major relevance to basic and preclinical oncological studies in small animal models.

Synthesis and evaluation of condensed magnetic nanocrystal clusters with in vivo multispectral optoacoustic tomography for tumour targeting.

Colloidal clusters of magnetic iron oxide nanocrystals (MIONs), particularly in the condensed pattern (co-CNCs), have emerged as new superstructures to improve further the performance of MIONs in applications pertaining to magnetic manipulation (drug delivery) and magnetic resonance imaging (MRI). Exploitation of the advantages they represent and their establishment in the area of nanomedicine demands a particular set of assets. The present work describes the development and evaluation of MION-based co-CNCs featuring for the first time such assets: High magnetization, as well as magnetic content and moment, high relaxivities (r2 = 400 and r2* = 905 s(-1) mMFe(-1)) and intrinsic loss power (2.3 nH m(2) kgFe(-1)) are combined with unprecedented colloidal stability and structural integrity, stealth and drug-loading properties. The reported nanoconstructs are endowed with additional important features such as cost-effective synthesis and storage, prolonged self-life and biocompatibility. It is finally showcased with in vivo multispectral optoacoustic tomography how these properties culminate in a system suitable for targeting breast cancer and for forceful in vivo manipulation with low magnetic field gradients.

Optoacoustic imaging enabled biodistribution study of cationic polymeric biodegradable nanoparticles.

Nanosized contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. However, many solid nanoparticles exhibit a great potential to induce toxicity, hindering their use for clinical applications. On the other hand, near-infrared (NIR) dyes have also been used for extensive biological applications, but show some limitations due to their poor aqueous stability, tendency to aggregation and rapid elimination from the body. An alternative proposed in this work to overcome these limitations is the use of NIR dye-loaded nanoparticles. Here we introduce nanoparticles constructed with poly(D,L-lactide-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer widely used for biomedical applications, attached to the polycation polyethyleneimine (PEI) to obtain positively charged nanoparticles. The in vivo biodistribution of the cationic PEI-PLGA nanoparticles was investigated after administration through three different routes (intravenous, intraperitoneal and subcutaneous) using multispectral optoacoustic tomography (MSOT). The prepared nanoparticles exhibited good colloidal stability and adequate optical properties for optoacoustic imaging. The in vivo biodistribution assays indicated a strong accumulation of the particles in the liver and spleen, and retention in these organs for at least 24 h. Therefore, these nanoparticles could find promising applications in MSOT due to a sharp and characteristic optoacoustic spectrum and high optoacoustic signal generation, and become a promising building block for theranostic strategies.

Optoacoustic imaging of naphthalocyanine: potential for contrast enhancement and therapy monitoring.

UNLABELLED: We investigated in vitro and in vivo the optoacoustic responses of silicon 2,3-naphthalocyanine bi(trihexylsilyloxide) (SiNc), considered herein as a reporter molecule for optoacoustic imaging, elucidating its efficiency for optoacoustic (photoacoustic) signal generation and examining the in vivo performance achieved. METHODS: SiNc solutions were prepared using Cremophor EL in water and evaluated for light-absorbing and optoacoustic contrast-generating properties. Photostability and singlet oxygen generation were investigated under pulsed laser illumination and validated using photoabsorbance. HT-29 mouse tumor models were used to assess the biodistribution of the compound and its performance as an optoacoustic contrast agent in vivo. RESULTS: SiNc was found to generate superior optoacoustic signals compared with the commonly used indocyanine green. Multispectral optoacoustic tomography of mouse tumors efficiently resolved the biodistribution of SiNc and the underlying perfusion parameters in vivo. In addition, we demonstrate how light-triggered SiNc reactions with molecular oxygen can be potentially sensed and discuss the relation of these measurements to the biochemical process involved in photothermal treatment. CONCLUSION: SiNc appears to be a promising family of contrast agent for optoacoustic imaging. Further development possibilities promise to expand its use in purely contrast generation settings, as well as its photodynamic therapy application.

Dynamic imaging of PEGylated indocyanine green (ICG) liposomes within the tumor microenvironment using multi-spectral optoacoustic tomography (MSOT).

Multispectral optoacoustic tomography (MSOT) is a powerful modality that allows high-resolution imaging of photo-absorbers deep within tissue, beyond the classical depth and resolution limitations of conventional optical imaging. Imaging of intrinsic tissue contrast can be complemented by extrinsically administered gold nanoparticles or fluorescent molecular probes. Instead, we investigated herein generation of re-engineered clinically-used PEGylated liposomes incorporating indocyanine green (LipoICG) as a contrast strategy that combines materials already approved for clinical use, with strong photo-absorbing signal generation available today only from some metallic nanoparticles (e.g. gold nanorods). Using MSOT we confirmed LipoICG as a highly potent optoacoustic agent and resolved tissue accumulation in tumor-bearing animals over time with high-sensitivity and resolution using two tumor models of different vascularisation. We further showcase a paradigm shift in pharmacology studies and nanoparticle investigation, by enabling detailed volumetric optical imaging in vivo through the entire tumor tissue non-invasively, elucidating never before seen spatiotemporal features of optical agent distribution. These results point to LipoICG as a particle with significant advantageous characteristics over gold nanoparticles and organic dyes.

Monoclonal antibody-targeted PEGylated liposome-ICG encapsulating doxorubicin as a potential theranostic agent.

Indocyanine green (ICG) is an FDA-approved, strongly photo-absorbent/fluorescent probe that has been incorporated into a clinically-relevant PEGylated liposome as a flexible optoacoustic contrast agent platform. This study describes the engineering of targeted PEGylated liposome-ICG using the anti-MUC-1 "humanized" monoclonal antibody (MoAb) hCTM01 as a tumour-specific theranostic system. We aimed to visualise non-invasively the tumour accumulation of these MoAb-targeted liposomes over time in tumour-bearing mice using multispectral optoacoustic tomography (MSOT). Preferential accumulation of targeted PEGylated liposome-ICG was studied after intravenous administration in comparison to non-targeted PEGylated liposome-ICG using both fast growing (4T1) and slow growing (HT-29) MUC-1 positive tumour models. Monitoring liposomal ICG in the tumour showed that both targeted and non-targeted liposome-ICG formulations preferentially accumulated into the tumour models studied. Rapid accumulation was observed for targeted liposomes at early time points mainly in the periphery of the tumour volume suggesting binding to available MUC-1 receptors. In contrast, non-targeted PEGylated liposomes showed accumulation at the centre of the tumour at later time points. In an attempt to take this a step further, we successfully encapsulated the anticancer drug, doxorubicin (DOX) into both targeted and non-targeted PEGylated liposome-ICG. The engineering of DOX-loaded targeted ICG liposome systems present a novel platform for combined tumour-specific therapy and diagnosis. This can open new possibilities in the design of advanced image-guided cancer therapeutics.

siRNA liposome-gold nanorod vectors for multispectral optoacoustic tomography theranostics.

Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained.

Polyglycerolsulfate functionalized gold nanorods as optoacoustic signal nanoamplifiers for in vivo bioimaging of rheumatoid arthritis.

We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied.

Gold nanoprisms as optoacoustic signal nanoamplifiers for in vivo bioimaging of gastrointestinal cancers.

Early detection of cancer greatly increases the chances of a simpler and more effective treatment. Traditional imaging techniques are often limited by shallow penetration, low sensitivity, low specificity, poor spatial resolution or the use of ionizing radiation. Hybrid modalities, like optoacoustic imaging, an emerging molecular imaging modality, contribute to improving most of these limitations. However, this imaging method is hindered by relatively low signal contrast. Here, gold nanoprisms (AuNPrs) are used as signal amplifiers in multispectral optoacoustic tomography (MSOT) to visualize gastrointestinal cancer. PEGylated AuNPrs are successfully internalized by HT-29 gastrointestinal cancer cells in vitro. Moreover, the particles show good biocompatibility and exhibit a surface plasmon band centered at 830 nm, a suitable wavelength for optoacoustic imaging purposes. These findings extend well to an in vivo setting, in which mice are injected with PEGylated AuNPrs in order to visualize tumor angiogenesis in gastrointestinal cancer cells. Overall, both our in vitro and in vivo results show that PEGylated AuNPrs have the capacity to penetrate tumors and provide a high-resolution signal amplifier for optoacoustic imaging. The combination of PEGylated AuNPrs and MSOT represents a significant advance for the in vivo imaging of cancers.

Multispectral opto-acoustic tomography (MSOT) of the brain and glioblastoma characterization.

Brain research depends strongly on imaging for assessing function and disease in vivo. We examine herein multispectral opto-acoustic tomography (MSOT), a novel technology for high-resolution molecular imaging deep inside tissues. MSOT illuminates tissue with light pulses at multiple wavelengths and detects the acoustic waves generated by the thermoelastic expansion of the environment surrounding absorbing molecules. Using spectral unmixing analysis of the data collected, MSOT can then differentiate the spectral signatures of oxygenated and deoxygenated hemoglobin and of photo-absorbing agents and quantify their concentration. By being able to detect absorbing molecules up to centimeters deep in the tissue it represents an ideal modality for small animal brain imaging, simultaneously providing anatomical, hemodynamic, functional, and molecular information. In this work we examine the capacity of MSOT in cross-sectional brain imaging of mice. We find unprecedented optical imaging performance in cross-sectional visualization of anatomical and physiological parameters of the mouse brain. For example, the potential of MSOT to characterize ischemic brain areas was demonstrated through the use of a carbon dioxide challenge. In addition, indocyanine green (ICG) was injected intravenously, and the kinetics of uptake and clearance in the vasculature of the brain was visualized in real-time. We further found that multiparameter, multispectral imaging of the growth of U87 tumor cells injected into the brain could be visualized through the intact mouse head, for example through visualization of deoxygenated hemoglobin in the growing tumor. We also demonstrate how MSOT offers several compelling features for brain research and allows time-dependent detection and quantification of brain parameters that are not available using other imaging methods without invasive procedures.

Optical imaging of cancer heterogeneity with multispectral optoacoustic tomography.

PURPOSE: To investigate whether multispectral optoacoustic tomography (MSOT) can reveal the heterogeneous distributions of exogenous agents of interest and vascular characteristics through tumors of several millimeters in diameter in vivo. MATERIALS AND METHODS: Procedures involving animals were approved by the government of Upper Bavaria. Imaging of subcutaneous tumors in mice was performed by using an experimental MSOT setup that produces transverse images at 10 frames per second with an in-plane resolution of approximately 150 µm. To study dynamic contrast enhancement, three mice with 4T1 tumors were imaged before and immediately, 20 minutes, 4 hours, and 24 hours after systemic injection of indocyanine green (ICG). Epifluorescence imaging was used for comparison. MSOT of a targeted fluorescent agent (6 hours after injection) and hemoglobin oxygenation was performed simultaneously (4T1 tumors: n = 3). Epifluorescence of cryosections served as validation. The accumulation owing to enhanced permeability and retention in tumors (4T1 tumors: n = 4, HT29 tumors: n = 3, A2780 tumors: n = 2) was evaluated with use of long-circulating gold nanorods (before and immediately, 1 hour, 5 hours, and 24 hours after injection). Dark-field microscopy was used for validation. RESULTS: Dynamic contrast enhancement with ICG was possible. MSOT, in contrast to epifluorescence imaging, showed a heterogeneous intratumoral agent distribution. Simultaneous imaging of a targeted fluorescent agent and oxy- and deoxyhemoglobin gave functional information about tumor vasculature in addition to the related agent uptake. The accumulation of gold nanorods in tumors seen at MSOT over time also showed heterogeneous uptake. CONCLUSION: MSOT enables live high-spatial-resolution observations through tumors, producing images of distributions of fluorochromes and nanoparticles as well as tumor vasculature.

Optical and opto-acoustic interventional imaging.

Many clinical interventional procedures, such as surgery or endoscopy, are today still guided by human vision and perception. Human vision however is not sensitive or accurate in detecting a large range of disease biomarkers, for example cellular or molecular processes characteristic of disease. For this reason advanced optical and opto-acoustic (photo-acoustic) methods are considered for enabling a more versatile, sensitive and accurate detection of disease biomarkers and complement human vision in clinical decision making during interventions. Herein, we outline developments in emerging fluorescence and opto-acoustic sensing and imaging techniques that can lead to practical implementations toward improving interventional vision.

Assembly/disassembly of drug conjugates using imide ligation.

A strategy is described that allows the easy assembly and controlled disassembly of drug conjugates. Imide ligation, that is, the reaction of a peptide thioacid with an azidoformate, is used for conjugate assembly. The imide bond participates also with an endopeptidase-triggered cyclization-based disassembly mechanism.

New NSAIDs-NO hybrid molecules with antiproliferative properties on human prostatic cancer cell lines.

The design of profen hybrids containing a NO donor moiety connected to an aliphatic spacer led to compounds with a similar cyclooxygenase inhibition compared to their parent profen and with significant antiproliferative activities on PC3 cells. However, inhibition of COX-2 pathway alone did not seem sufficient to inhibit cancer cell proliferation, and NO-release in a time-dependent manner strongly contributes to this activity.