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Richard Bach

mendel

Výzkum

Study of Fluorescence of Doxorubicin in Muscle Tissue using Highly Sensitive Fluorescence Sensing

Malignant diseases represent 25 % of the cause of death in developed countries. Colorectal cancer, breast cancer, prostate cancer and lung cancer are the most common types of cancer. On the other hand, mortality caused by these diseases decreases and survival time increases due to early diagnosis and effective treatment [1]. Magnetic resonance imaging (MRI), ultrasound (US), positron emission tomography (PET), computed tomography (CT), single-photon emission computed tomography (SPECT) and optical imaging methods belong to the contemporary used imaging methods in diagnostics and monitoring of the therapy [2,3]. Optical methods are relatively low cost non-ionizing methods based on the specific optical properties. They represent an important tool for non-invasive diagnosis with still developing properties as resolution [4,5]. It is advantageous to use inherent fluorescent properties of substances and labelled molecules [6].

Fluorescence reflectance imaging (FRI) enables imaging of fluorescent probes in tissues. In this case, the source of radiation and detector are at the same side of the object. Connection of laser and sensitive CCD device together with advanced mathematical models allows sensitive detection and evaluation of fluorescence intensity. Fluorescence-mediated molecular tomography enables reconstruction of three dimensional images of fluorescent probes in a tissue [7-9]. Organic fluorophores (fluorescein, rhodamine), biological fluorophores (green fluorescence protein), or quantum dots can be sources of fluorescence for these purposes. In addition, inherent fluorescence of some drugs (doxorubicin, ellipticine) can be used [6,10-12]. In the area of basic research, detection of fluorescence of therapeutics is beneficial particularly in the development of targeted therapy and control of drugs targeting into place affected by a tumour. Doxorubicin is a highly effective and widely used ant hracycline antibiotic, important antineoplastic drug intercalating DNA and causing oxidation stress that is used to treat leukaemia and solid tumours [13-19]. However, its application is limited by high cardiotoxicity, therefore it is necessary to monitor the applied dose [11,17]. Stationary techniques, such as spectrophotometric methods [20] including fluorimetry [21-23], but also separation methods, such as high performance liquid chromatography [24-28] and capillary electrophoresis [29-32], can be used for in vitro characterization and pharmacological evaluation of doxorubicin. In vivo studying of interactions and distribution of doxorubicin can be performed in a microscale using microscopic techniques in different arrangements as laser scanning microscopy [33-35], fluorescence life time microscopy [14] and scanning electron microscopy [36]. At the macroscale level, high-frequency ultrasound imaging [37], PET imaging [38] and fluorescence imaging using quantum dots [39] have been used for the monitoring doxorubicin or doxorubicin-modified nanoparticles in tissues. The aim of this work was to study the fluorescent properties of doxorubicin and to study the behaviour of doxorubicin diluted to different concentrations in water or methanol. In addition, doxorubicin was injected into muscle tissue to monitor its behaviour and to detect its fluorescence (emission) at different depths because of the fact that a major problem with the clinical use of doxorubicin in addition to adverse side effects common to all cytostatics like myelosupression, nausea and vomiting, mouth ulcers, local agressivity and alopecia, is their cardiotoxicity. Cardiotoxicity limits administration of doxorubicin exceeding an accumulated dose of approximately 450-550 mg/m2 [40]. The toxic effects of anthracyclines to cardiomyocytes are not the result of inhibition of DNA synthesis, because these cells do not replicate [41]. The mechanisms of the anthracyclines cardiotoxicity are not fully understood, but cardiac tissue is vulnerable to free radical because of the low activity of antioxidant enzyme systems in cardiomyocytes altered by the effect of doxorubicin [17].From the point of view of cadiotoxicity, doxorubicin and endogenous formaldehyde form a conjugate of two anthracycline molecules with three methylene groups, two forming oxazolidine rings and one binding the oxazolidines together at their 3´-amino nitrogens. This conjugate may hydrolyze to produce an active monomeric metabolite in which the carbon of formaldehyde is recovered in the form of a Schiff’s base at the aminogroup of daunosamine [42]. Anthracycline-formaldehyde conjugates intercalates into DNA by covalent bonding of the Schiff’s base with the 2-amino group of a G-base in the minor groove of DNA. If the interaction with DNA occurs at the trinucleotide 5´-NGC-3´, then the drug intercalates between N and G and covalently bonds to the G-base on one strand using formaldehyde, and to the G-base on the opposing strand using hydrogen bonds. This combination of intercalation, covalent bonding, and hydrogen bonding is referred to as the virtual cross-linking of DNA by anthracyclines [43].

Podpořeno projekty: NANOLABSYS CZ.1.07/2.3.00/20.0148

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