MR and fluorescence imaging of gadobutrol-induced optical clearing of red fluorescent protein signal in an in vivo cancer model.

Multimodality registration of optical and MR images in the same tissue volume in vivo may be enabled by MR contrast agents with an optical clearing (OC) effect. The goals of this study were to (a) investigate the effects of clinical MR contrast agent gadobutrol (GB) and its combinations as a potential OC agent assisting in fluorescence intensity (FI) imaging in vivo and (b) evaluate MRI as a tool for imaging of topical or systemic application of GB for the purpose of OC. Subcutaneous tumor xenografts expressing red fluorescent marker protein were used as disease models. MRI was performed at 1 T 1 H MRI using T1 -weighted 3D gradient-echo (T1w-3D GRE) sequences to measure time-dependent MR signal intensity changes by region of interest analysis after image segmentation. Topical application of 1.0 M or 0.7 M GB-containing OC mixture with water and dimethyl sulfoxide showed similar 30-40% increases of tumor FI during the initial 15 min. Afterwards, the OC effect of GB on FI and tumor/background FI ratio showed a decrease over time in the case of 1.0 M GB, unlike the 0.7 M GB mixture, which resulted in a steady increase of FI and tumor/background ratio for 15-60 min. The use of T1w-3D GRE MR pulse sequences showed that concentrated 1.0 M GB resulted in MR signal loss of the skin due to high magnetic susceptibility and that signal loss coincided with the OC effect. Intravenous injection of 0.3 mmol GB/kg resulted in a rapid but transient 40% increase of FI of the tumors. Overall, 1 T MRI enabled tracking of GB-containing OC compositions on the skin surface and tumor tissue, supporting the observation of a time-dependent FI increase in vivo.

Imaging NF-κB activity in a murine model of early stage diabetes.

Early pro-inflammatory signaling in the endocrine pancreas involves activation of NF-κB, which is believed to be important for determining the ultimate fate of ß-cells and hence progression of type 1 diabetes (T1D). Thus, early non-invasive detection of NF-κB in pancreatic islets may serve as a potential strategy for monitoring early changes in pancreatic endocrine cells eventually leading to T1D. We investigated the feasibility of optical imaging of NF-κB transcription factor activation induced by low-dose streptozocin (LD-STZ) treatment in the immunocompetent SKH1 mouse model of early stage diabetes. In this model, we showed that the levels of NF-κB may be visualized and measured by fluorescence intensity of specific near-infrared (NIR) fluorophore-labeled oligodeoxyribonucleotide duplex (ODND) probes. In addition, NF-κB activation following LD-STZ treatment was validated using immunofluorescence and transgenic animals expressing NF-κB inducible imaging reporter. We showed that LD-STZ-treated SKH1 mice had significantly higher (2-3 times, P < .01) specific NIR FI in the nuclei and cytoplasm of islets cells than in non-treated control mice and this finding was corroborated by immunoblotting and electrophoretic mobility shift assays. Finally, using semi-quantitative confocal analysis of non-fixed pancreatic islet microscopy we demonstrated that ODND probes may be used to distinguish between the islets with high levels of NF-κB transcription factor and control islet cells.

19F MRI of Polymer Nanogels Aided by Improved Segmental Mobility of Embedded Fluorine Moieties.

Using fluorinated probes for 19F MRI imaging is an emerging field with potential utility in cellular imaging and cell tracking in vivo, which complements conventional 1H MRI. An attractive feature of 19F-based imaging is that this is a bio-orthogonal nucleus and the naturally abundant isotope is NMR active. A significant hurdle however in the 19F MRI arises from the tendency of organic macromolecules, with multiple fluorocarbon substitutions, to aggregate in the aqueous phase. This aggregation results in significant loss of sensitivity, because the T2 relaxation times of these aggregated 19F species tend to be significantly lower. In this report, we have developed a strategy to covalently trap nanoscopic states with an optimal degree of 19F substitutions, followed by significant enhancement in T2 relaxation times through increased segmental mobility of the side chain substituents facilitated by the stimulus-responsive elements in the polymeric nanogel. In addition to NMR relaxation time based evaluations, the ability to obtain such signals are also evaluated in mouse models. The propensity of these nanoscale assemblies to encapsulate hydrophobic drug molecules and the availability of surfaces for convenient introduction of fluorescent labels suggest the potential of these nanoscale architectures for use in multimodal imaging and therapeutic applications.

Antiretroviral Hydrophobic Core Graft-Copolymer Nanoparticles: The Effectiveness against Mutant HIV-1 Strains and in Vivo Distribution after Topical Application.

PURPOSE: Developing and testing of microbicides for pre-exposure prophylaxis and post-exposure protection from HIV are on the list of major HIV/AIDS research priorities. To improve solubility and bioavailability of highly potent anti-retroviral drugs, we explored the use of a nanoparticle (NP) for formulating a combination of two water-insoluble HIV inhibitors. METHODS: The combination of a non-nucleoside HIV reverse transcriptase inhibitor (NNRTI), Efavirenz (EFV), and an inhibitor of HIV integrase, Elvitegravir (ELV) was stabilized with a graft copolymer of methoxypolyethylene glycol-polylysine with a hydrophobic core (HC) composed of fatty acids (HC-PGC). Formulations were tested in TZM-bl cells infected either with wild-type HIV-1IIIB, or drug-resistant HIV-1 strains. In vivo testing of double-labeled NP formulations was performed in female rats after a topical intravaginal administration using SPECT/CT imaging and fluorescence microscopy. RESULTS: We observed a formation of stable 23-30 nm NP with very low cytotoxicity when EFV and ELV were combined with HC-PGC at a 1:10 weight ratio. For NP containing ELV and EFV (at 1:1 by weight) we observed a remarkable improvement of EC50 of EFV by 20 times in the case of A17 strain. In vivo imaging and biodistribution showed in vivo presence of NP components at 24 and 48 h after administration, respectively. CONCLUSIONS: insoluble orthogonal inhibitors of HIV-1 life cycle may be formulated into the non-aggregating ultrasmall NP which are highly efficient against NNRTI-resistant HIV-1 variant.

Proceedings of the third international molecular pathological epidemiology (MPE) meeting.

Molecular pathological epidemiology (MPE) is a transdisciplinary and relatively new scientific discipline that integrates theory, methods, and resources from epidemiology, pathology, biostatistics, bioinformatics, and computational biology. The underlying objective of MPE research is to better understand the etiology and progression of complex and heterogeneous human diseases with the goal of informing prevention and treatment efforts in population health and clinical medicine. Although MPE research has been commonly applied to investigating breast, lung, and colorectal cancers, its methodology can be used to study most diseases. Recent successes in MPE studies include: (1) the development of new statistical methods to address etiologic heterogeneity; (2) the enhancement of causal inference; (3) the identification of previously unknown exposure-subtype disease associations; and (4) better understanding of the role of lifestyle/behavioral factors on modifying prognosis according to disease subtype. Central challenges to MPE include the relative lack of transdisciplinary experts, educational programs, and forums to discuss issues related to the advancement of the field. To address these challenges, highlight recent successes in the field, and identify new opportunities, a series of MPE meetings have been held at the Dana-Farber Cancer Institute in Boston, MA. Herein, we share the proceedings of the Third International MPE Meeting, held in May 2016 and attended by 150 scientists from 17 countries. Special topics included integration of MPE with immunology and health disparity research. This meeting series will continue to provide an impetus to foster further transdisciplinary integration of divergent scientific fields.

Synthesis and Testing of Modular Dual-Modality Nanoparticles for Magnetic Resonance and Multispectral Photoacoustic Imaging.

Magnetic resonance (MR) and photoacoustic (PA) imaging are currently being investigated as complementing strategies for applications requiring sensitive detection of cells in vivo. While combined MR/PAI detection of cells requires biocompatible cell labeling probes, water-based synthesis of dual-modality MR/PAI probes presents significant technical challenges. Here we describe facile synthesis and characterization of hybrid modular dextran-stabilized gold/iron oxide (Au-IO) multimetallic nanoparticles (NP) enabling multimodal imaging of cells. The stable association between the IO and gold NP was achieved by priming the surface of dextran-coated IO with silver NP resulting from silver(I) reduction by aldehyde groups, which are naturally present within the dextran coating of IO at the level of 19-23 groups/particle. The Au-IO NP formed in the presence of silver-primed Au-IO were stabilized by using partially thiolated MPEG5-gPLL graft copolymer carrying residual amino groups. This stabilizer served as a carrier of near-infrared fluorophores (e.g., IRDye 800RS) for multispectral PA imaging. Dual modality imaging experiments performed in capillary phantoms of purified Au-IO-800RS NPs showed that these NPs were detectible using 3T MRI at a concentration of 25 µM iron. PA imaging achieved approximately 2.5-times higher detection sensitivity due to strong PA signal emissions at 530 and 770 nm, corresponding to gold plasmons and IRDye integrated into the coating of the hybrid NPs, respectively, with no "bleaching" of PA signal. MDA-MB-231 cells prelabeled with Au-IO-800RS retained plasma membrane integrity and were detectable by using both MR and dual-wavelength PA at 49 ± 3 cells/imaging voxel. We believe that modular assembly of multimetallic NPs shows promise for imaging analysis of engineered cells and tissues with high resolution and sensitivity.

Hydrophobic-core PEGylated graft copolymer-stabilized nanoparticles composed of insoluble non-nucleoside reverse transcriptase inhibitors exhibit strong anti-HIV activity.

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150-250 nm stable nanoparticles (NP). The NP and HC-PGC were non-toxic in experiments with TZM-bl reporter cells. Nanoparticles containing selected efficient candidate Z107 NNRTI preserved the ability to inhibit HIV-1 reverse transcriptase polymerase activities with no appreciable change of EC50. The formulation with HC-PGC bearing residues of oleic acid resulted in nanoparticles that were nearly identical in anti-HIV-1 potency when compared to Z107 solutions in DMSO (EC50=7.5±3.8 vs. 8.2±5.1 nM). Therefore, hydrophobic core macromolecular stabilizers form nanoparticles with insoluble NNRTI while preserving the antiviral activity of the drug cargo.

Imaging Inflammation in Cerebrovascular Disease.

Imaging inflammation in large intracranial artery pathology may play an important role in the diagnosis of and risk stratification for a variety of cerebrovascular diseases. Looking beyond the lumen has already generated widespread excitement in the stroke community, and the potential to unveil molecular processes in the vessel wall is a natural evolution to develop a more comprehensive understanding of the pathogenesis of diseases, such as ICAD and brain aneurysms.

Gold nanoparticles stabilized with MPEG-grafted poly(l-lysine): in vitro and in vivo evaluation of a potential theranostic agent.

As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with (99m)Tc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers "stealth" properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy.

Myeloperoxidase in human intracranial aneurysms: preliminary evidence.

BACKGROUND AND PURPOSE: Noninvasive imaging identifying a predictive biomarker of the bleeding risk of unruptured intracranial aneurysms (UIAs) is needed. We investigated a potential biomarker of UIA instability, myeloperoxidase, in human aneurysm tissue. METHODS: Human brain aneurysms were harvested after clipping and were histologically and biochemically evaluated for the presence of myeloperoxidase. Of the tissue collected, 3 were from ruptured aneurysms and 20 were from UIAs. For each UIA, its 5-year aneurysm rupture risk was determined using the Population, Hypertension, Age, Size of Aneurysm, Earlier Subarachnoid Hemorrhage From Another Aneurysm and Site of Aneurysm (PHASES) model. RESULTS: All ruptured aneurysms were myeloperoxidase positive. Of the UIAs, half were myeloperoxidase positive. The median 5-year aneurysm rupture risk was higher for myeloperoxidase-positive UIA (2.28%) than myeloperoxidase-negative UIA (0.69%), and the distributions were statistically different (P<0.005, Wilcoxon-Mann-Whitney test). The likelihood for myeloperoxidase-positive UIA was significantly associated (P=0.031) with aneurysm rupture risk (odds ratio, 4.79; 95% confidence limits, 1.15-19.96). CONCLUSIONS: Myeloperoxidase is associated with PHASES estimated risk of aneurysm rupture and may potentially be used as an imaging biomarker of aneurysm instability.

Dynamic monitoring of blood-brain barrier integrity using water exchange index (WEI) during mannitol and CO2 challenges in mouse brain.

The integrity of the blood-brain barrier (BBB) is critical to normal brain function. Traditional techniques for the assessment of BBB disruption rely heavily on the spatiotemporal analysis of extravasating contrast agents. However, such methods based on the leakage of relatively large molecules are not suitable for the detection of subtle BBB impairment or for the performance of repeated measurements in a short time frame. Quantification of the water exchange rate constant (WER) across the BBB using strictly intravascular contrast agents could provide a much more sensitive method for the quantification of the BBB integrity. To estimate WER, we have recently devised a powerful new method using a water exchange index (WEI) biomarker and demonstrated BBB disruption in an acute stroke model. Here, we confirm that WEI is sensitive to even very subtle changes in the integrity of the BBB caused by: (i) systemic hypercapnia and (ii) low doses of a hyperosmolar solution. In addition, we have examined the sensitivity and accuracy of WEI as a biomarker of WER using computer simulation. In particular, the dependence of the WEI-WER relation on changes in vascular blood volume, T1 relaxation of cellular magnetization and transcytolemmal water exchange was explored. Simulated WEI was found to vary linearly with WER for typically encountered exchange rate constants (1-4 Hz), regardless of the blood volume. However, for very high WER (>5 Hz), WEI became progressively more insensitive to increasing WER. The incorporation of transcytolemmal water exchange, using a three-compartment tissue model, helped to extend the linear WEI regime to slightly higher WER, but had no significant effect for most physiologically important WERs (WER < 4 Hz). Variation in cellular T1 had no effect on WEI. Using both theoretical and experimental approaches, our study validates the utility of the WEI biomarker for the monitoring of BBB integrity.

Characterization of optical clearing mechanisms in muscle during treatment with glycerol and gadobutrol solutions.

The recent increasing interest in the application of radiology contrasting agents to create transparency in biological tissues implies that the diffusion properties of those agents need evaluation. The comparison of those properties with the ones obtained for other optical clearing agents allows to perform an optimized agent selection to create optimized transparency in clinical applications. In this study, the evaluation and comparison of the diffusion properties of gadobutrol and glycerol in skeletal muscle was made, showing that although gadobutrol has a higher molar mass than glycerol, its low viscosity allows for a faster diffusion in the muscle. The characterization of the tissue dehydration and refractive index matching mechanisms of optical clearing was made in skeletal muscle, namely by the estimation of the diffusion coefficients for water, glycerol and gadobutrol. The estimated tortuosity values of glycerol (2.2) and of gadobutrol (1.7) showed a longer path-length for glycerol in the muscle.

Surface modification of neurovascular stents: from bench to patient.

Flow-diverting stents (FDs) for the treatment of cerebrovascular aneurysms are revolutionary. However, these devices require systemic dual antiplatelet therapy (DAPT) to reduce thromboembolic complications. Given the risk of ischemic complications as well as morbidity and contraindications associated with DAPT, demonstrating safety and efficacy for FDs either without DAPT or reducing the duration of DAPT is a priority. The former may be achieved by surface modifications that decrease device thrombogenicity, and the latter by using coatings that expedite endothelial growth. Biomimetics, commonly achieved by grafting hydrophilic and non-interacting polymers to surfaces, can mask the device surface with nature-derived coatings from circulating factors that normally activate coagulation and inflammation. One strategy is to mimic the surfaces of innocuous circulatory system components. Phosphorylcholine and glycan coatings are naturally inspired and present on the surface of all eukaryotic cell membranes. Another strategy involves linking synthetic biocompatible polymer brushes to the surface of a device that disrupts normal interaction with circulating proteins and cells. Finally, drug immobilization can also impart antithrombotic effects that counteract normal foreign body reactions in the circulatory system without systemic effects. Heparin coatings have been explored since the 1960s and used on a variety of blood contacting surfaces. This concept is now being explored for neurovascular devices. Coatings that improve endothelialization are not as clinically mature as anti-thrombogenic coatings. Coronary stents have used an anti-CD34 antibody coating to capture circulating endothelial progenitor cells on the surface, potentially accelerating endothelial integration. Similarly, coatings with CD31 analogs are being explored for neurovascular implants.

Molecular Magnetic Resonance Imaging of Aneurysmal Inflammation Using a Redox Active Iron Complex.

OBJECTIVES: Inflammation plays a key role in driving brain aneurysmal instability and rupture, but clinical tools to noninvasively differentiate between inflamed and stable aneurysms are lacking. We hypothesize that imaging oxidative changes in the aneurysmal microenvironment driven by myeloid inflammatory cells may represent a noninvasive biomarker to evaluate rupture risk. In this study, we performed initial evaluation of the oxidatively activated probe Fe-PyC3A as a tool for magnetic resonance imaging (MRI) of inflammation in a rabbit model of saccular aneurysm. MATERIALS AND METHODS: The difference in longitudinal relaxivity ( r1 ) in reduced and oxidized states of Fe-PyC3A was measured in water and blood plasma phantoms at 3 T. A rabbit saccular aneurysm model was created by endovascular intervention/elastinolysis with subsequent decellularization in situ. Rabbits were imaged at 4 weeks (n = 4) or 12 weeks (n = 4) after aneurysmal induction, when luminal levels of inflammation reflected by the presence of myeloperoxidase positive cells are relatively high and low, respectively, using a 3 T clinical scanner. Both groups were imaged dynamically using a 2-dimensional T1-weighted fast field echo pulse MRI sequence before and up to 4 minutes postinjection of Fe-PyC3A. Dynamic imaging was then repeated after an injection of gadobutrol (0.1 mmol/kg) as negative control probe. Rabbits from the 12-week aneurysm group were also imaged before and 20 minutes and 3 hours after injection of Fe-PyC3A using an axial respiratory gated turbo-spin echo (TSE) pulse sequence with motion-sensitized driven equilibrium (MSDE) preparation. The MSDE/TSE imaging was repeated before, immediately after dynamic acquisition (20 minutes postinjection), and 3 hours after injection of gadobutrol. Aneurysmal enhancement ratios (ERs) were calculated by dividing the postinjection aneurysm versus skeletal muscle contrast ratio by the preinjection contrast ratio. After imaging, the aneurysms were excised and inflammatory infiltrate was characterized by fluorometric detection of myeloperoxidase activity and calprotectin immunostaining, respectively. RESULTS: In vitro relaxometry showed that oxidation of Fe-PyC3A by hydrogen peroxide resulted in a 15-fold increase of r1 at 3 T. Relaxometry in the presence of blood plasma showed no more than a 10% increase of r1 , indicating the absence of strong interaction of Fe-PyC3A with plasma proteins. Dynamic imaging with Fe-PyC3A generated little signal enhancement within the blood pool or adjacent muscle but did generate a transient increase in aneurysmal ER that was significantly greater 4 weeks versus 12 weeks after aneurysm induction (1.6 ± 0.30 vs 1.2 ± 0.03, P < 0.05). Dynamic imaging with gadobutrol generated strong aneurysmal enhancement, but also strong enhancement of the blood and muscle resulting in smaller relative ER change. In the 12-week group of rabbits, MSDE/TSE imaging showed that ER values measured immediately after dynamic MRI (20 minutes postinjection) were significantly higher ( P < 0.05) in the case of Fe-PyC3A (1.25 ± 0.06) than for gadobutrol injection (1.03 ± 0.03). Immunohistochemical corroboration using anticalprotectin antibody showed that leukocyte infiltration into the vessel walls and luminal thrombi was significantly higher in the 4-week group versus 12-week aneurysms (123 ± 37 vs 18 ± 7 cells/mm 2 , P < 0.05). CONCLUSIONS: Magnetic resonance imaging using Fe-PyC3A injection in dynamic or delayed acquisition modes was shown to generate a higher magnetic resonance signal enhancement in aneurysms that exhibit higher degree of inflammation. The results of our pilot experiments support further evaluation of MRI using Fe-PyC3A as a noninvasive marker of aneurysmal inflammation.

A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo.

Bis-phenylamides and bis-hydroxyindolamides of diethylenetriaminepentaacetic acid-gadolinium (DTPA(Gd)) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, gadolinium chelates of bis-5-hydroxytryptamide-DTPA (bis-5HT-DTPA(Gd)) have been used to image localized inflammation in animal models by detecting neutrophil-derived myeloperoxidase (MPO) activity at the inflammation site. However, in other preclinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here we report a novel MPO-sensing probe obtained by replacing the reducing substrate serotonin (5-HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using nuclear magnetic resonance spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd) (1) improves solubility in water; (2) acts as a substrate for both horseradish peroxidase and MPO enzymes; (3) induces cross-linking of proteins in the presence of MPO; (4) produces oxidation products, which bind to plasma proteins; and (5) unlike bis-5HT-DTPA(Gd), does not follow first-order reaction kinetics. In vivo magnetic resonance imaging (MRI) in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to 5 days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. Bis-HTrp-DTPA(Gd) should offer improvements for MRI of MPO-mediated inflammation in vivo, especially in high-field MRI, which requires a higher dose of contrast agent.

In situ decellularization of a large animal saccular aneurysm model: sustained inflammation and active aneurysm wall remodeling.

OBJECTIVE: To investigate in situ decellularization of a large animal model of saccular aneurysm as a strategy for achieving aneurysmal growth and lasting inflammation. METHODS: 18 New Zealand White rabbits were randomized 2:1 to receive endoluminal sodium dodecyl sulfate infusion (SDS, 1% solution, 45 min) following elastase or elastase-only treatment (control). All aneurysms were measured by digital subtraction angiography every 2 weeks. Every 2 weeks, three of the rabbits (two elastase + SDS, one control) underwent MRI, followed by contrast injection with myeloperoxidase (MPO)-sensing contrast agent. MRI was repeated 3 hours after contrast injection and the enhancement ratio (ER) was calculated. Following MRI, aneurysms were explanted and subjected to immunohistopathology. RESULTS: During follow-up MRI, the average ER for SDS-treated animals was 1.63±0.20, compared with 1.01±0.06 for controls (p<0.001). The width of SDS-treated aneurysms increased significantly in comparison with the elastase aneurysms (47% vs 20%, p<0.001). Image analysis of thin sections showed infiltration of MPO-positive cells in decellularized aneurysms and surroundings through the 12-week observation period while control tissue had 5-6 times fewer cells present 2 weeks after aneurysm creation. Immunohistochemistry demonstrated the presence of MPO-positive cells surrounding decellularized lesions at early time points. MPO-positive cells were found in the adventitia and in the thrombi adherent to the aneurysm wall at later time points. CONCLUSIONS: In situ decellularization of a large animal model of saccular aneurysms reproduces features of unstable aneurysms, such as chronic inflammation (up to 12 weeks) and active aneurysm wall remodeling, leading to continued growth over 8 weeks.

Synthesis and applications of theranostic oligonucleotides carrying multiple fluorine atoms.

The use of various oligonucleotide (ON) syntheses and post-synthetic strategies for targeted chemical modification enables improving their efficacy as potent modulators of gene expression levels in eukaryotic cells. However, the search still continues for new approaches designed for increasing internalization, lysosomal escape, and tissue specific delivery of ON. In this review we emphasized all aspects related to the synthesis and properties of ON derivatives carrying multifluorinated (MF) groups. These MF groups have unique physico-chemical properties because of their simultaneous hydrophobicity and lipophobicity. Such unusual combination of properties results in the overall modification of ON mode of interaction with the cells and making multi-fluorination highly relevant to the goal of improving potency of ON as components of new therapies. The accumulated evidence so far is pointing to high potential of ON probes, RNAi components and ON imaging beacons carrying single or multiple MF groups for improving the stability, specificity of interaction with biological targets and delivery of ONs in vitro and potentially in vivo.

Magnetic resonance contrast agents in optical clearing: Prospects for multimodal tissue imaging.

Skin optical clearing effect ex vivo and in vivo was achieved by topical application of low molecular weight paramagnetic magnetic resonance contrast agents. This novel feature has not been explored before. By using collimated transmittance the diffusion coefficients of three clinically used magnetic resonance contrast agents, that is Gadovist, Magnevist and Dotarem as well as X-ray contrast agent Visipaque in mouse skin were determined ex vivo as (4.29 ± 0.39) × 10-7 cm2 /s, (5.00 ± 0.72) × 10-7 cm2 /s, (3.72 ± 0.67) × 10-7 cm2 /s and (1.64 ± 0.18) × 10-7 cm2 /s, respectively. The application of gadobutrol (Gadovist) resulted in efficient optical clearing that in general, was superior to other contrast agents tested and allowed to achieve: (a) more than 12-fold increase of transmittance over 10 minutes after application ex vivo; (b) markedly improved images of skin architecture obtained with optical coherence tomography; (c) an increase of the fluorescence intensity/background ratio in TagRFP-red fluorescent marker protein expressing tumor by five times after 15 minutes application into the skin in vivo. The obtained results have immediate implications for multimodality imaging because many contrast agents are capable of simultaneously enhancing the contrast of multiple imaging modalities.

Carotid artery brain aneurysm model: in vivo molecular enzyme-specific MR imaging of active inflammation in a pilot study.

PURPOSE: To demonstrate the feasibility of using a myeloperoxidase (MPO)-specific paramagnetic magnetic resonance (MR) contrast agent to identify active inflammation in an animal model of common carotid artery (CCA) aneurysm. MATERIALS AND METHODS: All animal experiments were approved by the institutional animal care and use committee. Elastase-induced saccular aneurysms were created at the root of the right CCA in 16 New Zealand white rabbits. Intramural and perivascular injection of Escherichia coli lipopolysaccharide (LPS) was performed with an endovascular approach to induce aneurysm inflammation. After intraarterial injection of an MPO-specific (di-5-hydroxytryptamide of gadopentetate dimeglumine, 0.1 mmol per kilogram of bodyweight) or a non-MPO-specific (di-tyrosine of gadopentetate dimeglumine, 0.1 mmol/kg) contrast agent, animals underwent 3-T MR imaging. Intramural presence of MPO in aneurysms in which LPS had been injected was confirmed at immunohistologic analysis. Active MPO activity was verified by measuring the spectrophotometric oxidation of guaiacol. RESULTS: Endovascular injection of LPS resulted in inflammatory cell infiltration into the aneurysm wall, and there was a difference in active MPO expression between aneurysms in which LPS had been injected and control aneurysms (20.3 ng of MPO per milligram of tissue vs 0.12 ng of MPO per milligram of tissue, respectively; P < .002). MR imaging with di-5-hydroxytryptamide of gadopentetate dimeglumine revealed a difference in enhancement ratio between inflamed aneurysms in which LPS had been injected and control aneurysms (1.55 +/- 0.05 vs 1.16 +/- 0.10, respectively; P < .02). In inflamed aneurysms, di-5-hydroxytryptamide of gadopentetate dimeglumine exhibited delayed washout kinetics compared with the kinetics of di-tyrosine of gadopentetate dimeglumine. This finding enabled the verification of MPO specificity. CONCLUSION: The findings of this pilot study established the feasibility of an animal model of saccular aneurysm inflammation that can be seen with clinical-field-strength MR imaging and use of the enzyme-sensitive MR contrast agent di-5-hydroxytryptamide of gadopentetate dimeglumine, which is a paramagnetic MPO substrate that specifically enhances MR signal.

Merging molecular imaging and RNA interference: early experience in live animals.

The rapid development of non-invasive imaging techniques and imaging reporters coincided with the enthusiastic response that the introduction of RNA interference (RNAi) techniques created in the research community. Imaging in experimental animals provides quantitative or semi-quantitative information regarding the biodistribution of small interfering RNAs and the levels of gene interference (i.e., knockdown of the target mRNA) in living animals. In this review we give a brief summary of the first imaging findings that have potential for accelerating the development and testing of new approaches that explore RNAi as a method for achieving loss-of-function effects in vivo and as a promising therapeutic tool.