The aim of this study was to develop and characterize a nanoparticle-based image-contrast platform which is biocompatible, chemically stable, and accessible for radiolabeling with 201Tl. We explored whether this nanoparticle enhanced the T1 signal which might make it an MRI contrast agent as well.

(1) Background. The main goal of this work was to develop a fluorescent dye-labelling technique for our previously described nanosized platform, citrate-coated Prussian blue (PB) nanoparticles (PBNPs). In addition, characteristics and stability of the PB nanoparticles labelled with fluorescent dyes were determined. (2) Methods. We adsorbed the fluorescent dyes Eosin Y and Rhodamine B and methylene blue (MB) to PB-nanoparticle systems. The physicochemical properties of these fluorescent dye-labeled PBNPs (iron(II);iron(III);octadecacyanide) were determined using atomic force microscopy, dynamic light scattering, zeta potential measurements, scanning- and transmission electron microscopy, X-ray diffraction, and Fourier-transformation infrared spectroscopy. A methylene-blue (MB) labelled, polyethylene-glycol stabilized PBNP platform was selected for further assessment of in vivo distribution and fluorescent imaging after intravenous administration in mice. (3) Results. The MB-labelled particles emitted a strong fluorescent signal at 662 nm. We found that the fluorescent light emission and steric stabilization made this PBNP-MB particle platform applicable for in vivo optical imaging. (4) Conclusion. We successfully produced a fluorescent and stable, Prussian blue-based nanosystem. The particles can be used as a platform for imaging contrast enhancement. In vivo stability and biodistribution studies revealed new aspects of the use of PBNPs.

The in vivo biodistribution of liposomal formulations greatly influences the pharmacokinetics of these novel drugs; therefore the radioisotope labeling of liposomes and the use of nuclear imaging methods for in vivo studies are of great interest. In the present work, a new procedure for the surface labeling of liposomes is presented using the novel 99mTc-tricarbonyl complex. Liposomes mimicking the composition of two FDA approved liposomal drugs were used. In the first step of the labeling, thiol-groups were formed on the surface of the liposomes using Traut's reagent, which were subsequently used to bind 99mTc-tricarbonyl complex to the liposomal surface. The labeling efficiency determined by size exclusion chromatography was 95%, and the stability of the labeled liposomes in bovine serum was found to be 94% over 2 hours. The obtained specific activity was 50 MBq per 1 μmol lipid which falls among the highest values reported for 99mTc labeling of liposomes. Quantitative in vivo SPECT/CT biodistribution studies revealed distinct differences between the labeled liposomes and the free 99mTc-tricarbonyl, which indicates the in vivo stability of the labeling. As the studied liposomes were non-PEGylated, fast clearance from the blood vessels and high uptake in the liver and spleen were observed.

Our aim was to present a new data analysis technique for the early detection of tumorous lesions using single-photon emission computed tomography (SPECT) imaging. Beyond standardized uptake value (SUV) and standardized uptake concentration (SUC), the skewness and kurtosis parameters of whole liver activity distribution histograms were examined in SPECT images to reveal the presence of tumorous cells.

Increased blood-brain barrier (BBB) permeability and extensive neuronal changes have been described earlier in both healthy and pathological aging like apolipoprotein B-100 (APOB-100) and amyloid precursor protein (APP)-presenilin-1 (PSEN1) transgenic mouse models. APOB-100 hypertriglyceridemic model is a useful tool to study the link between cerebrovascular pathology and neurodegeneration, while APP-PSEN1 humanized mouse is a model of Alzheimer's disease. The aim of the current study was to characterize the inflammatory changes in the brain with healthy aging and in neurodegeneration. Also, the cerebro-morphological and cognitive alterations have been investigated. The nose-to-brain delivery of a P-glycoprotein substrate model drug (quinidine) was monitored in the disease models and compared with the age-matched controls. Our results revealed an inflammatory balance shift in both the healthy aged and neurodegenerative models. In normal aging monocyte chemoattractant protein-1, stem cell factor and Rantes were highly upregulated indicating a stimulated leukocyte status. In APOB-100 mice, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), and interleukin-17A (IL-17A) were induced (vascular reaction), while in APP-PSEN1 mice resistin, IL-17A and GM-CSF were mostly upregulated. The nasal drug absorption was similar in the brain and blood indicating the molecular bypass of the BBB. The learning and memory tests showed no difference in the cognitive performance of healthy aged and young animals. Based on these results, it can be concluded that various markers of chronic inflammation are present in healthy aged and diseased animals. In APOB-100 mice, a cerebro-ventricular dilation can also be observed. For development of proper anti-aging and neuroprotective compounds, further studies focusing on the above inflammatory targets are suggested.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Radiotherapy is one of the most frequently applied treatments in oncology. Tissue-absorbed ionizing radiation damages not only targeted cells but the surrounding cells too. The consequent long-term induced oxidative stress, irreversible tissue damage, or second malignancies draw attention to the urgent need of a follow-up medical method by which personalized treatment could be attained and the actually dose-limiting organ could be monitored in the clinical practice. We worked out a special hemisphere irradiation technique for mice which mimics the radiation exposure during radiotherapy. We followed up the changes of possible brain imaging biomarkers of side effects, such as cerebral blood flow, vascular endothelial function, and cellular metabolic processes for 60 days. BALB/c mice were divided into two groups (n=6 per group) based on the irradiation doses (5 and 20 Gy). After the irradiation procedure arterial spin labeling (ASL), diffusion-weighted imaging (DWI) in magnetic resonance modality and [18F]fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) scans of the brain were obtained at several time points (3, 7, 30, and 60 days after the irradiation). Significant physiological changes were registered in the brain of animals following the irradiation by both applied doses. Elevated standard uptake values were detected all over the brain by FDG-PET studies 2 months after the irradiation. The apparent diffusion coefficients from DWI scans significantly decreased one month after the irradiation procedure, while ASL studies did not show any significant perfusion changes in the brain. Altogether, our sensitive multimodal imaging protocol seems to be an appropriate method for follow-up of the health status after radiation therapy. The presented approach makes possible parallel screening of healthy tissues and the effectiveness of tumor therapy without any additional radiation exposure.

The "back-translation" of clinically available protocols to measure myocardial perfusion to preclinical imaging in mouse models of human disease is attractive for basic biomedical research. With respect to single-photon emission computed tomography (SPECT) approaches, clinical myocardial perfusion imaging protocols are established with different 99mTc-labeled perfusion tracers; however, studies evaluating and optimizing protocols for these tracers in high-resolution pinhole SPECT in mice are lacking. This study aims at evaluating two clinically available 99mTc-labeled myocardial perfusion tracers (99mTc-sestamibi vs. 99mTc-Tetrofosmin) in mice using four different imaging protocols.

Bc-DLFL.1 is a novel spontaneous, high-grade transplantable mouse B-cell lymphoma model for selective serosal propagation. These cells attach to the omentum and mesentery and show dissemination in mesenteric lymph nodes. We aimed to investigate its early stage spread at one day post-intraperitoneal inoculation of lymphoma cells (n = 18 mice), and its advanced stage at seven days post-inoculation with in vivo [18F]FDG-PET and [18F]PET/MRI, and ex vivo by autoradiography and Cherenkov luminescence imaging (CLI). Of the early stage group, nine animals received intraperitoneal injections, and nine received intravenous [18F]FDG injections. The advanced stage group (n = 3) received intravenous FDG injections. In the early stage, using autoradiography we observed a marked accumulation in the mesentery after intraperitoneal FDG injection. Using other imaging methods and autoradiography, following the intravenous injection of FDG no accumulations were detected. At the advanced stage, tracer accumulation was clearly detected in mesenteric lymph nodes and in the peritoneum after intravenous administration using PET. We confirmed the results with immunohistochemistry. Our results in this model highlight the importance of local FDG administration during diagnostic imaging to precisely assess early peritoneal manifestations of other malignancies (colon, stomach, ovary). These findings also support the importance of applying topical therapies, in addition to systemic treatments in peritoneal cancer spread.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

Antigen-specific redirection of immune effector cells with chimeric antigen receptors (CARs) demonstrated high therapeutic potential for targeting cancers of different origins. Beside CAR-T cells, natural killer (NK) cells represent promising alternative effectors that can be combined with CAR technology. Unlike T cells, primary NK cells and the NK cell line NK-92 can be applied as allogeneic off-the-shelf products with a reduced risk of toxicities. We previously established a modular universal CAR (UniCAR) platform which consists of UniCAR-expressing immune cells that cannot recognize target antigens directly but are redirected by a tumour-specific target module (TM). The TM contains an antigen-binding moiety fused to a peptide epitope which is recognized by the UniCAR molecule, thereby allowing an on/off switch of CAR activity, and facilitating flexible targeting of various tumour antigens depending on the presence and specificity of the TM. Here, we provide proof of concept that it is feasible to generate a universal off-the-shelf cellular therapeutic based on UniCAR NK-92 cells targeted to tumours expressing the disialoganglioside GD2 by GD2-specific TMs that are either based on an antibody-derived single-chain fragment variable (scFv) or an IgG4 backbone. Redirected UniCAR NK-92 cells induced specific killing of GD2-expressing cells in vitro and in vivo, associated with enhanced production of interferon-γ. Analysis of radiolabelled proteins demonstrated that the IgG4-based format increased the in vivo half-life of the TM markedly in comparison to the scFv-based molecule. In summary, UniCAR NK-92 cells represent a universal off-the-shelf platform that is highly effective and flexible, allowing the use of different TM formats for specific tumour targeting.

Nutritional interventions may highly contribute to the maintenance or restoration of human health. Grapes (Vitis vinifera) are one of the oldest known beneficial nutritional components of the human diet. Their high polyphenol content has been proven to enhance human health beyond doubt in statistics-based public health studies, especially in the prevention of cardiovascular disease and cancer. The current review concentrates on presenting and classifying polyphenol bioactive molecules (resveratrol, quercetin, catechin/epicatechin, etc.) available in high quantities in Vitis vinifera grapes or their byproducts. The molecular pathways and cellular signaling cascades involved in the effects of these polyphenol molecules are also presented in this review, which summarizes currently available in vitro and in vivo experimental literature data on their biological activities mostly in easily accessible tabular form. New molecules for different therapeutic purposes can also be synthesized based on existing polyphenol compound classes available in high quantities in grape, wine, and grape marc. Therefore an overview of these molecular structures is provided. Novel possibilities as dendrimer nanobioconjugates are reviewed, too. Currently available in vitro and in vivo experimental literature data on polyphenol biological activities are presented in easily accessible tabular form. The scope of the review details the antidiabetic, anticarcinogenic, antiviral, vasoprotective, and neuroprotective roles of grape-origin flavonoids. The novelty of the study lies in the description of the processing of agricultural by-products (grape seeds and skins) of industrial relevance, and the detailed description of the molecular mechanisms of action. In addition, the review of the clinical therapeutic applications of polyphenols is unique as no summary study has yet been done.

Ivermectin, an antiparasitic drug, has been repurposed for COVID-19 treatment during the SARS-CoV-2 pandemic. Although its antiviral efficacy was confirmed early in vitro and in preclinical studies, its clinical efficacy remained ambiguous. Our purpose was to assess the efficacy of ivermectin in terms of time to viral clearance based on the meta-analysis of available clinical trials at the closing date of the data search period, one year after the start of the pandemic. This meta-analysis was reported by following the PRISMA guidelines and by using the PICO format for formulating the question. The study protocol was registered on PROSPERO. Embase, MEDLINE (via PubMed), Cochrane Central Register of Controlled Trials (CENTRAL), bioRvix, and medRvix were searched for human studies of patients receiving ivermectin therapy with control groups. No language or publication status restrictions were applied. The search ended on 1/31/2021 exactly one year after WHO declared the public health emergency on novel coronavirus. The meta-analysis of three trials involving 382 patients revealed that the mean time to viral clearance was 5.74 days shorter in case of ivermectin treatment compared to the control groups [WMD = -5.74, 95% CI (-11.1, -0.39), p = 0.036]. Ivermectin has significantly reduced the time to viral clearance in mild to moderate COVID-19 diseases compared to control groups. However, more eligible studies are needed for analysis to increase the quality of evidence of ivermectin use in COVID-19.

Rationale: Human induced pluripotent stem cell-derived endothelial cells can be candidates for engineering therapeutic vascular grafts. Methods: Here, we studied the role of three-dimensional culture on their characteristics and function both in vitro and in vivo. Results: We found that differentiated hPSC-EC can re-populate decellularized biomatrices; they remain viable, undergo maturation and arterial/venous specification. Human PSC-EC develop antifibrotic, vasoactive and anti-inflammatory properties during recellularization. In vivo, a robust increase in perfusion was detected at the engraftment sites after subcutaneous implantation of an hPSC-EC-laden hydrogel in rats. Histology confirmed survival and formation of capillary-like structures, suggesting the incorporation of hPSC-EC into host microvasculature. In a canine model, hiPSC-EC-seeded onto decellularised vascular segments were functional as aortic grafts. Similarly, we showed the retention and maturation of hiPSC-EC and dynamic remodelling of the vessel wall with good maintenance of vascular patency. Conclusions: A combination of hPSC-EC and biomatrices may be a promising approach to repair ischemic tissues.

Selective portal vein ligation (PVL) allows the two-stage surgical resection of primarily unresectable liver tumours by generating the atrophy and hypertrophy of portally ligated (LL) and non-ligated lobes (NLL), respectively. To evaluate critically important underlying functional alterations, present study characterised in vitro and vivo liver function in male Wistar rats (n = 106; 210-250 g) before, and 24/48/72/168/336 h after PVL. Lobe weights and volumes by magnetic resonance imaging confirmed the atrophy-hypertrophy complex. Proper expression and localization of key liver transporters (Ntcp, Bsep) and tight junction protein ZO-1 in isolated hepatocytes demonstrated constantly present viable and well-polarised cells in both lobes. In vitro taurocholate and bilirubin transport, as well as in vivo immunohistochemical Ntcp and Mrp2 expressions were bilaterally temporarily diminished, whereas LL and NLL structural acinar changes were divergent. In vivo bile and bilirubin-glucuronide excretion mirrored macroscopic changes, whereas serum bilirubin levels remained unaffected. In vivo functional imaging (indocyanine-green clearance test; 99mTc-mebrofenin hepatobiliary scintigraphy; confocal laser endomicroscopy) indicated transitionally reduced global liver uptake and -excretion. While LL functional involution was permanent, NLL uptake and excretory functions recovered excessively. Following PVL, functioning cells remain even in LL. Despite extensive bilateral morpho-functional changes, NLL functional increment restores temporary declined transport functions, emphasising liver functional assessment.

Although an extensive research is being undertaken, the ideal bone graft and evaluation method of the bone formation draw still a warranted attention. The purpose of this study was to develop a novel multimodal radiomics evaluation method, utilizing X-ray computed tomography (CT) and single photon emission computed tomography (SPECT) with Tc-99m-Methyl diphosphonate (Tc-99m-MDP) tracer. These modalities are intended to provide quantitative data concerning the mineral bone density (after evaluation it is referred to as opacity) and the osteoblast activity, at the same time. The properties of bone formation process within poly (methyl methacrylate)-based bone cement graft (PMMA) was compared to that of albumin coated, sterilized, antigen-extracted freeze-dried human bone grafts (HLBC), in caudal vertebrae (C5) of rats. The animals were scanned at 3 and 8 weeks after surgery. In both groups, the mean opacity increased, while the mean Tc-99m-MDP activity decreased. The later parameter was significant (n = 4, p = 0.002) only in HLBC group. The linear regression analysis of PMMA-treated group variables (mean opacity increase; mean Tc-99m-MDP activity decrease), revealed a negative correlation with the medium strength (r = 0.395, p = 0.605). Whereas, it showed strong positive correlation when HLBC group variables were analyzed (r = 0.772, p = 0.012). These results indicate that using HLBC grafts is advantageous in terms of the osteoblast activity and bone vascularization over PMMA cement. Using this regression analysis method, we were able to distinguish characteristics that otherwise could not be distinguished by a regular data analysis. Hence, we propose utilizing this novel method in preclinical tests, and in clinical monitoring of bone healing, in order to improve diagnosis of bone-related diseases.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.

Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries. Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation. Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry. Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.