Treatment with stem cells is in an exceptional ascent in various fields of medicine, including cardiology, neurology and oncology. The evaluation of the success of the treatment is still difficult due to the limited possibilities of the in vivo monitoring of transplanted cells, despite the multitude of imaging, diagnostic and laboratory methods nowadays available. The radiollabeling of stem cells with various isotopes bound in complexes (radiopharmaceuticals) represents an appropriate method that enables in vivo cell tracking after administration. Scintigram (scan), which is a visual representation of the spatial distribution of labelled cells in the body after the transplantation, can be used for quantitative estimation of the absolute or relative number of cells in the region of interest, e.g. an organ like heart, as well. In the field of nuclear medicine imaging, radiopharmaceuticals labelled with indium-111 (oxine, tropolone) and technetium-99m (HMPAO) are routinely used to label cells (leukocytes, platelets). Both isotopes are gamma emitters, with appropriate energy and half-life for use in medicine. Both isotopes form highly lipophilic complexes with ligands, by which all the cells that come into contact with, can be non-selectively radiolabelled. 111In-labelled oxine (8-hydroxyquinoline) was the first ligand to be used in the radiolabelling of leukocytes in the field of nuclear medicine imaging. Both, oxine and the tropolone, form a 3:1 complex with indium-111 that are neutral and highly lipophilic, which allows the diffusion of the complex across the cell membrane. In the cell, partial or complete dissociation of the complexes and the binding of indium-111 to intracellular proteins occur. Technetium-99m labelled hexamethylpropyleneamine oxime (HMPAO) or exametazim is a radiopharmaceutical that was developed to monitor changes in regional cerebral blood flow. The [99mTc][Tc(HMPAO)] complex exhibits high lipophilicity, has no charge, and has a molecular weight low enough to pass through the blood brain barrier. The described mechanism is being exploited in routine nuclear medicine imaging practice for cell (leukocytes) radiolabelling. After the passive diffusion of the primary complex across the cell membrane, the cell enzymes transform the complex into a secondary complex with lower lipophilicity and thus remain in the cell. Galium-68, copper-64 and zirconium-89 are positron emitters, used in PET (Positron Emission Tomography) nuclear imaging diagnostics. Compared to SPECT (Single Photon Emission Computed Tomography), PET is characterized by higher sensitivity (10-times higher), better resolution and easier quantification, allowing the detection of smaller lesions and more accurate tracking of a small number of radiolabelled cells. The 68Ge/68Ga radionuclide generator allows the gallium-68 to be produced at the site of use. Use of the automated synthesis modules enables the production of tracers labelled with gallium-68 of appropriate quality for routine clinical use, despite its short half-life (68 min). Copper-64 and zirconium-89 have longer half-lives (12.7 h, 78.4 h) that could allow longer tracking of radiolabelled cells, and represent excellent candidates for the synthesis of radiopharmaceuticals for radiolabelling cells. All three isotopes, like indium-111, belong to a group of radiometals. They have similar chemistry as indium-111, and form similar complexes, enabling the formation of complexes with oxine (8-hydroxyquinoline) and tropolone (2-hydroxy-2,4,6-cycloheptatrienone) that can be further used for non-selective radiolabelling of cells. The doctoral thesis is comprised of three content-related sections. In the first section, covering the main goal of the doctoral thesis, development and evaluation of radiolabelling of haematopoietic stem cells (CD34+) were conducted. We radiolabeled haematopoietic stem cells (CD34+) with [99mTc][Tc(HMPAO)], a technique that today represents the gold standard for radiolabelling of WBC in daily clinical practice. The treatment with haematopoietic stem cells (CD34+) is clinically proven to be useful in the treatment of ischemic conditions, e.g. ischemic heart disease, in patients who are not responding to standard pharmacological treatment or treatment with revascularization. CD34+ cells obtained by bone marrow stimulation were labelled with [99mTc][Tc(HMPAO)] and the effect of radiolabelling on the physiological state (effect on cell viability, cytotoxicity, efflux of radiopharmaceuticals from cells) was evaluated. No influence of cell labelling on the physiological status of the cells was observed. Radiolabelling of CD34+ cells with [99mTc][Tc(HMPAO)] enables short term cell tracking of radiolabelled cells an engraftment evaluation using SPECT imaging. With the obtained data, we can significantly contribute to the assessment of the effectiveness of stem cell therapy and possible changes in treatment. Up to now the preparation of cell radiolabelling tracers of appropriate quality and small enough volumes for PET, with its superiority over SPECT (better sensitivity, quantification and resolution) is still a major limitation for wider use. Reasons are generally connected with possibilities of automation of the tracer preparation and depending on the radionuclide, properties of PET tracer itself, enabling cell radiolabelling. Our goal of preparing PET radiopharmaceuticals for cell radiolabelling is explained in two sections. In the second section of the thesis, we studied and evaluated the use of a self-shielded radiosynthesis module for fully automated preparation of radiotracers labelled with gallium-68 and/or other PET and beta emitters. The use of such a module is necessary in order to reduce the radiation load of the personnel preparing the radiopharmaceuticals, while at the same time allowing the preparation of radiopharmaceuticals of appropriate quality and in suitable volumes. In the third section we developed and optimized the method for the concentration of PET radioisotopes (gallium-68, copper-64, zirconium-89), subsequent radiolabelling of oxine and tropolone on the anion exchange column and the elution of the complexes formed on the column in the physiological medium in volumes small enough for radiolabelling of cells and potentially suitable for the automation. As a proof of concept, we radiolabelled erythrocytes with prepared radiopharmaceuticals and evaluated efflux of the radioactivity from the cells, followed by radiolabelling of leukocytes with the most promising compounds (64Cu-labelled tropolone and oxine). The suitability for potential use in clinical practice was confirmed in an animal model (BALB/c mice) of inflammation and infection.
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