György Trencsényi

99mTc-labelled nanosystem as tumour imaging agent for SPECT and SPECT/CT modalities

We report the synthesis, in vitro and in vivo investigation of folate-targeted, biocompatible, biodegradable self-assembled nanoparticles radiolabelled with (99m)Tc, as potential new SPECT or SPECT/CT imaging agent. Nanoparticles with hydrodynamic size in the range of 75-200 nm were prepared by self-assembly of chitosan and folated poly-γ-glutamic acid, and then radiolabelled with (99m)Tc. The nanoparticles target tumour cells overexpressing folate receptors and internalize specifically into them to realize early tumour diagnosis detected by SPECT and SPECT/CT modalities. Rat hepatocellular carcinoma cells were used as model system. Cell specificity and tumour targeting efficacy of these nanosystems were investigated in vitro, and in vivo using SPECT and fusion nanoSPECT/CT imaging. In vitro results showed that the radiolabeled nanosystem was efficiently internalized by tumour cells. Whole-body biodistribution of the new radiolabelled, folate-targeted nanoparticles revealed higher uptake in the tumorous kidney compared to the non-tumorous contralateral side. Uptake by the lungs and thyroids was negligible, which confirmed the stability of the nanoparticles in vivo. In vivo SPECT and SPECT/CT imaging visually reinforced the uptake results and were in accordance with the biodistribution data: the new nanoparticles as a targeted contrast agent improve tumour targeting and are able to detect folate-receptor-overexpressing tumours in animal models with enhanced contrast.

Time-lapse analysis of cell death in mammalian and fungal cells.

Time-lapse video microscopy was designed to follow the movement of single cells for an unlimited period of time under physiological conditions. The system is based on two inverted microscopes located in a CO(2) incubator and equipped with charge-coupled device cameras connected to the computer. Frames were recorded every minute and the subsequent video sequence was converted to database form. The system was applied to describe the movements of normal HaCaT cells and Pb-treated cells causing the so-called apoptotic dance during cell death. The apoptotic movement was also followed in high-osmolarity glycerol-type mitogen-activated protein kinase (MAPK) null mutant of Fusarium proliferatum, a filamentous fungus, during osmotic stress. The shortest (20 min) and most vigorous death movements were observed in apoptotic fungal cells subjected to salt stress. The necrotic process at higher Pb concentration (50 microM) took 2-3 h, whereas the apoptotic process at lower Pb concentrations lasted from minutes to days.

In vivo imaging of Aminopeptidase N (CD13) receptors in experimental renal tumors using the novel radiotracer 68Ga-NOTA-c(NGR)

PURPOSE Aminopeptidase N (APN/CD13) plays an important role in tumor neoangiogenic process and the development of metastases. Furthermore, it may serve as a potential target for cancer diagnosis and therapy. Previous studies have already shown that asparagine-glycine-arginine (NGR) peptides specifically bind to APN/CD13. The aim of the study was to synthesize and investigate the APN/CD13 specificity of a novel (68)Ga-labeled NOTA-c(NGR) molecule in vivo using miniPET. METHODS c[KNGRE]-NH2 peptide was conjugated with p-SCN-Bn-NOTA and was labeled with Ga-68 ((68)Ga-NOTA-c(NGR)). Orthotopic and heterotopic transplanted mesoblastic nephroma (NeDe) bearing Fischer-344 rats were prepared, on which biodistribution studies and miniPET scans were performed for both (68)Ga-NOTA-c(NGR) and ανβ3 integrin selective (68)Ga-NODAGA-[c(RGD)]2 tracers. APN/CD13 receptor expression of NeDe tumors and metastases was analyzed by western blot. RESULTS (68)Ga-NOTA-c(NGR) was produced with high specific activity (5.13-5.92GBq/μmol) and with excellent radiochemical purity (95%<), at all cases. Biodistribution studies in normal rats showed that uptake of the (68)Ga-NOTA-c(NGR) was significantly (p⩽0.05) lower in abdominal organs in comparison with (68)Ga-NODAGA-[c(RGD)]2. Both radiotracers were mainly excreted from the kidney. In NeDe tumor bearing rats higher (68)Ga-NOTA-c(NGR) accumulation was found in the tumors than that of the (68)Ga-NODAGA-[c(RGD)]2. Using orthotopic transplantation, metastases were developed which showed specific (68)Ga-NOTA-c(NGR) uptake. Western blot analysis confirmed the presence of APN/CD13 expression in NeDe tumors and metastases. CONCLUSION Our novel radiotracer (68)Ga-NOTA-c(NGR) showed specific binding to the APN/CD13 expressed ortho- and heterotopic transplanted NeDe tumors. Therefore, (68)Ga-NOTA-c(NGR) is a suitable tracer for the detection of APN/CD13 positive tumors and metastases in vivo.

Seminal Vesicle Secretion 2 Acts as a Protectant of Sperm Sterols and Prevents Ectopic Sperm Capacitation in Mice

ABSTRACT Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET‐MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration.

AAZTA: An Ideal Chelating Agent for the Development of 44Sc PET Imaging Agents

Unprecedented fast and efficient complexation of ScIII was demonstrated with the chelating agent AAZTA (AAZTA=1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) under mild experimental conditions. The robustness of the 44 Sc(AAZTA)- chelate and conjugated biomolecules thereof is further shown by in vivo PET imaging in healthy and tumor mice models. The new results pave the way towards development of efficient Sc-based radiopharmaceuticals using the AAZTA chelator.

Cancer cell targeting and imaging with biopolymer-based nanodevices

We report here the synthesis, in vitro and in vivo investigation of magnetic resonance imaging (MRI) active nanoparticles, which target folate receptor overexpressing tumor cells. Self-assembled nanoparticles with a hydrodynamic size of 50-200 nm were prepared from poly-γ-glutamic acid and chitosan biopolymers with Gd-ions. The nanoparticles are biocompatible, non-toxic and stable for several months in aqueous media. In vitro assays using confocal microscopy, flow cytometry and MR imaging on HeLa human cervix carcinoma tumor cells showed that folic acid targeted nanoparticles were internalized specifically in a folate receptor dependent manner. In vivo study confirmed, that, considerable accumulation of nanosystems was found compared with the control animal represented by the MR images. Relaxometry measurements demonstrated that the nanoparticle-Gd complexes drastically change the signal intensity of the tumor cells. Because of the contrast enhancement, they are attractive candidates as potential contrast agents for a variety of diagnostic applications including early diagnosis of tumors.

Incomplete chromatin condensation in enlarged rat myelocytic leukemia cells.

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.

18FDG, [18F]FLT, [18F]FAZA, and 11C-Methionine Are Suitable Tracers for the Diagnosis and In Vivo Follow-Up of the Efficacy of Chemotherapy by miniPET in Both Multidrug Resistant and Sensitive Human Gynecologic Tumor Xenografts

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[18F]fluoro-2-deoxy-D-glucose (18FDG), 11C-methionine, 3′-deoxy-3′-[18F]fluorothymidine (18F-FLT), and [18F]fluoroazomycin-arabinofuranoside (18FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive (Pgp+) and negative (Pgp−) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp+ and Pgp− A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp+ tumors. Our results demonstrate that 18FDG, 18F-FLT, 18FAZA, and 11C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacy of tumor chemotherapy in both Pgp+ and Pgp− human tumor xenografts by miniPET.

Multiparametric labeling optimization and synthesis of 68Ga-labeled compounds applying a continuous-flow microfluidic methodology

AbstractThe synthesis and functional evaluation of a wide variety of radiolabeled chelator—biomolecule conjugates with high specific activity and radiochemical purity are crucial to development of personalized nuclear medicine. An excellent platform technology for achieving this objective involves use of generator-produced positron emission tomography (PET)-radionuclide 68Ga. Currently, applied manual methodology for optimization and development for new labeling techniques offers only slow screening with relatively high precursor consumption. A capillary-based microfluidic synthesis module with online high-performance liquid chromatography (HPLC) was constructed for the optimization of reaction parameters of 68Ga-PET tracers. This approach enables performance of 68Ga-labeling reactions in 10 μL volumes, followed by sample analysis. The high-throughput capacity of the system allows very rapid optimization. The optimal pH and ligand concentration from the experiments were utilized directly to the production of 68Ga-NODAGA-(RGD)2 and 68Ga-NOPO-RGD. Applying optimal parameters to production of these aforementioned radiopharmaceuticals allowed their synthesis with high radiochemical purity (over 95%) and with surprisingly negligible retention of residual activity in the system.