Alexei Bogdanov

X-ray attenuating contrast agents for imaging thrombi directly during endovascular thrombectomy (EVT) are urgently needed for shortening the wait time for treatment and for reducing the chances of blood clot fragmentation. Neutrophil extracellular traps (NETs) are a product of an innate immune system response by which neutrophils release decondensed chromatin strands decorated with granule and cytosolic proteins, including neutrophil elastase and citrullinated histone H3 (CitH3). NETs are frequently found within fibrous thrombi in pathology and represent a promising target for thrombi-specific imaging agents due to their common occurrence in human cerebrovascular thrombi. We designed and tested 200 nm lipid vesicles (LV) formulated in the presence of a combination of hydrophilic and hydrophobic computed tomography (CT) contrast agents with resultant efficacy of X-ray attenuation corresponding to 312 ± 54 mg/mL iodine. The LV incorporated trans-cyclooctene-terminated pegylated distearoylphosphatidylethanolamine (TCO-PEG-DSPE) for rapid conjugation of methyltetrazine(mTz)-modified monoclonal immunoglobulin G (IgG) with anti-citH3 binding specificity. By using differential fluorescent labeling of the antibody and lipid components, we determined that 80 ± 3% of mTz-linked IgG coprecipitated with the LV after conjugation in contrast to 0.1-0.2% of control IgG. The engineered NET-specific LV were tested in vitro using differentiated human HL60 promyeloblasts (dHL60) as a standard model of NETing neutrophils. Using fibrin meshwork-incorporated dHL60 as well as monolayer cell cultures, we determined that anti-citH3 LV showed specific and high-affinity binding to dHL60 cells, which were stimulated to undergo NETosis. This work suggests the high promise of NET-specific agents in providing thrombus-specific imaging contrast during EVT.

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 (r ₁) 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 r ₁ at 3 T. Relaxometry in the presence of blood plasma showed no more than a 10% increase of r ₁, 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/mm², 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.

We performed synthesis and testing of Fab’-based NIR fluorescent probes with the goal of achieving sufficiently high tumor/background (muscle) ratios at the earlier time points after intravenous injection in comparison to the full-size IgG or larger fragments. Conjugation of NIRDye 800CW to Fab’ resulted in partially quenched imaging probes. Fluorescence intensity imaging of orthotopic xenografts in both EGFR-attenuated and wild-type triple negative breast cancer tumors using Fab’-800CW allowed earlier detection of EGFR expression in vivo.

The catalytically inactive mutant of Cas9 (dCas9) endonuclease has multiple biomedical applications, with the most useful being the activation/repression of transcription. dCas9 family members are also emerging as potential experimental tools for gene mapping at the level of individual live cells and intact tissue. We performed initial testing on a set of tools for Cas9-mediated visualization of nuclear compartments. We investigated doxycycline (Dox)-inducible (Tet-On) intracellular distribution of constructs encoding dCas9 orthologs from St. thermophilus (St) and N. meningitides (Nm) fused with EGFP and mCherry fluorescent proteins (FP) in human A549 cells. We also studied time-dependent expression of these chimeric fluorescent constructs (dCas9-FP) after Tet-On induction in live cells and compared it with the time course of dCas9-FP expression in experimental dCas9-FP-expressing tumor xenografts using a combination of fluorescence imaging and in vivo contrast-assisted magnetic resonance imaging for assessing the extent of tumor perfusion. In vivo Dox-induction of mCherry-chimera expression occurred in tumor xenografts as early as 24 h post-induction and was visualized by using optical clearing (OC) of the skin. OC via topical application of gadobutrol enabled high-contrast imaging of FP expression in tumor xenografts due to a 1.1–1.2-fold increase in FI in both the red and green channels.

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 ¹H 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.

Herein, exploration of potential synthesis routes for developing key components for efficient, implantable, and bio-absorbable triboelectric nanogenerators was performed. Further, their triboelectric charging, thermal, infrared spectroscopic properties along with biocompatibility were investigated. To obtain polymers with improved triboelectric charging efficiency, copolymers of sorbitol and sebacic acid (PSS) and hexanediol and citric acid (PHC) were synthesized using polycondensation mechanism. Whereas citric acid, glutaric acid, 1,2-propanediol and 1,6-hexanediol copolymer (CHGP) was synthesized via sequential ring-opening, and polycondensation mechanisms. Synthesis pathways of PSS and PHC were used as described earlier, while a novel synthesis route was applied to obtain CHGP copolymer. Triboelectric measurements of the synthesized copolymers demonstrated higher triboelectric surface charge – an order of magnitude higher than commercially available acid- and ester-terminated poly(D,L-lactide-co-glycolides) (PLGA) elastomers, as well as up to two orders of magnitude higher than well-known triboelectric materials such as polydimethylsiloxane and polytetrafluorethylene. Thermal analysis of polymers performed through thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) displayed a typical degradation and phase change behavior, associated with copolymers. Moreover, revealing the effect of crosslinking in CHGP polymer on glass transition temperature (T g ), melting temperature (T m ) was analyzed. Finally, cell-culture cytotoxicity tests have shown that CHGP copolymers with the highest cross-linking displayed lowest cytotoxicity and low cytoproliferative effect. Developed materials showed improved triboelectric properties with noticeable biocompatibility. These materials own distinctive collective properties for utilisation in fabrication of an alternative energy source, powering next generation of implantable biomedical and healthcare devices.

Multimodality imaging of triple-negative mammary adenocarcinoma metastases by using luminescence enabled differentiation between osteolytic and nonosteolytic phenotypes, whereas specific assessment... Purpose To develop multimodality imaging techniques for measuring epidermal growth factor receptor (EGFR) as a therapy-relevant and metastasis-associated molecular marker in triple-negative mammary...

Introduction Animal models of saccular aneurysms are stable following creation, and do not actively remodel or rupture. Recently, rodent models of aneurysm wall remodeling and rupture have been described by surgical grafting of decellularized arterial tissue.¹ We sought to 1) develop and apply a modified in situ decellularization approach, not require complex grafting procedures, in a large animal model of saccular aneurysms and 2) investigate modified aneurysmal expansion and inflammation using in vivo imaging. Methods 18 New Zealand White rabbits (3.0–3.5 kg) underwent standard elastinolysis aneurysm creation of the right common carotid artery. The animals were randomized 2:1 to receive isolated vessel SDS treatment (perfusion with 1% solution for 45 min) or nothing. Every two weeks until week 8, three of the rabbits (2 SDS+elastase, 1 elastase), were subjected to MR imaging,² followed by contrast injection with stable macrocyclic gadolinium(III)-based contrast agent that has been shown to selectively enhance the areas of vessel wall myeloperoxidase (MPO) accumulation.^{3 4} The MRI was repeated at 3 hours post contrast injection and the enhancement ratio (ER) was calculated. All aneurysms were measured for size by digital subtraction angiography. Results Of the 17 rabbits included in the study, 11 were treated in situ with SDS following elastase incubation and 6 served as controls. One SDS-treated rabbit was excluded due to rupture of the carotid artery during recovery. During follow-up MR imaging the ER was greater than 1 for all SDS-treated animals, whereas the controls had an ER of greater than 1 only at the two-week timepoint (consistent with the expected healing process). Average ER for SDS-treated animals was 1.63±0.20, while control animals had an ER of 1.02±0.06 (pConclusions During the 12 weeks post aneurysm creation, MPO-mediated MR signal enhancement was continuously detected within the wall of the aneurysm, and other than at 2 weeks, only in the SDS-treated animals. This novel modified large animal aneurysm model is expected to be useful for testing of new therapies that rely on not only the mechanical properties of the aneurysm but also the biological processes that make aneurysms vulnerable to rupture. References Stroke 2014;45:248–254. AJNR2015;36:146–52. Radiology 2009;252:696–703. Sci Rep 2018;8:7687. Disclosures R. King: None. J. Caroff: 1; C; FullBright. A. Leopardi: None. A. Bogdanov: None. M. Gounis: None.

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.