The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

Despite the recent advances in cancer therapy, the successful detections and treatments of cancer still remains a challenge. The existing strategies for early cancer detection are often limited due to their poor sensitivity and specificity. Also, the non-selective action of therapeutic interventions hinders treatment success. Our research was therefore directed towards the engineering of excellent bi-functionalized nanoparticles (NPs) based on carbon dots (CDs) that would improve early cancer detection and overcome the limitations of chemotherapy. With the actively targeting agent, these new NPs are expected to effectively deliver pharmacological agents directly to cancer cells. CDs are carbon-based NPs that are utilized as bioimaging agents and drug delivery systems (DDS) due to their excellent biocompatibility, non-toxicity, unique imaging, and facile surface modification. Using folic acid (FA) as targeting agent, the prepared novel CDs will carry doxorubicin (DOX) covalently and non-covalently to the cancer cells with overexpressed folate receptors. The CDs were first synthesized via the hydrothermal bottom-up approach using citric acid and ethylenediamine as precursors. The prepared CDs were then functionalized by FA via a non-cleavable peptide bond followed by complexation with DOX covalently or non-covalently to obtain the desired FA-CD-DOX NPs. All the NPs and intermediates were characterized using ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy (FL), and Fourier transform infrared spectroscopy (FTIR). Assessment of the drug loading capacity (DLC) and drug loading efficiency (DLE) with UV-vis indicated that the non-covalent NPs have low DLC but high DLE compared to the relatively low DLE and high DLC of covalent NPs. In vitro drug release studies were also carried out in phosphate buffered saline (PBS) systems with various pH. It was found that even though both non-covalent and covalent complexes released more DOX at pH 5.0 than at pH 7.0, the DOX release rate was faster in the non-covalent FA-CD-DOX NPs compared to the covalent FA-CD-DOX. Based on these results, we project increased accumulation of drugs in the more acidic (pH 4.5-5.0) microenvironment of cancer cells compared to that of normal healthy cells under physiological pH (7.4). This new FA-CD-DOX NPs could work as efficient theranostic systems to detect and treat cancer.

<p>U radu je predstavljena sinteza i karakterizacija&nbsp; novog fulerenol/doksorubicin nanokompozita, sintetisanog sa ciljem dobijanja potencijalne nove nanoformulacije postojećeg antineoplastika doksorubicina, koja bi pokazala veću biolo&scaron;ku aktivnost uz smanjenje neželjenih sporednih efekata koje sam lek izaziva, na prvom mestu kardiotoksičnosti.</p><p>Nanokompozit fulerenol/doksorbicin je okarakterisan brojnim&nbsp; metodama prateći dva osnovna eksperimentalna pristupa: molekulsko-spektroskopske metode (XPS, denzitometrija i transportne osobine, NMR, UPLC, Ramanska i UV-spektroskopija, SFM) i metode&nbsp; nanokarakterizacije (DLS, AFM, TEM), kao i računske simulacije(RDF). Osnovni cilj ispitivanja je bila detekcija postojanja&nbsp;nekovalentnog nanokompozita koji ostvaruju doksorubicin i fulerenolske nanočestice u vodenom rastvoru. Rezultati karakterizacije jasno i nedvosmisleno ukazuju na postojanje nekovalentnih interakcija unutar nanokompozita, &scaron;to dalje utiče na organizaciju i udruživanje čestica, a &scaron;to uslovljava i drugačiju biolo&scaron;ku aktivnost takvog sistema u odnosu na pojedinačne komponente.&nbsp; Rezultati biolo&scaron;kih ispitivanja na&nbsp;<em>in vitro</em>&nbsp;modelu različitih tumorskih ćelijskih linija pokazuju&nbsp; značajan&nbsp;antiproliferativni efekat nanokompozita, kao i selektivnost prema tumorskim u odnosu na zdravu ćelijsku liniju. Eksperimenti na <em>in vivo</em> modelu zebrica potvrđuju smanjenje toksičnosti nanokompozita u poređenju sa lekom, primarno kardiotoksičnosti. Računske simulacije, mikroskopski i spektroskopski podaci, kao i rezultati&nbsp; <em>in vitro</em> i <em>in vivo</em> studija ukazuju na to da nekovalentne interakcije između fulerenola i doksorubicina mogu biti ključni korak u stvaranju sinergističkog sistema za dostavu leka u biolo&scaron;ki sistem.</p><p>Multipotentnost fulerenola kao nanonosača lekova i nespecifičnost strukture doksorubicina kao leka, ukazuje na to da bi fulerenol mogao biti efikasan nanonosač i drugih antineoplastika, &scaron;to daje prostora za unapređenje antitumorskih osobina lekova posredstvom istovremene administracije leka.</p> / <p>The focus of this thesis was the synthesis and characterization of a novel fullerenol/doxorubicin nanocomposite, with the aim to obtain a potential nanoformulation of antineoplastic drug doxorubicin, which would&nbsp; express greater biological activity and lower level of adverse effects than the drug itself, in the first place cardiotoxicity.</p><p>Nanocomposite fullerenol/doxorubicin was characterized by means of numerous methods&nbsp; following two main experimental approaches: molecular-spectroscopic methods (XPS, densitometry and transport properties, NMR, UPLC, Raman and UVspectroscopy, SFM) and mehods of&nbsp; anocharacterisation&nbsp; (DLS, AFM,&nbsp; TEM), as well as computer simulations (RDF). The goal of characterization was detection of&nbsp; non-covalent interactions within nanocomposite that are established between fullerenol nanoparticles and doxorubicin in aqueous solution. The results clearly indicate the existence of non- covalent interactions within nanocomposite that affect the organization and assembling of the particles, which further exhibit different biological activity of such a system in comparison to components themselves. Results of biological activity on <em>in vitro </em>model of different tumor cell lines show significant antiproliferative effect of&nbsp; nanocomposite, as well as selectivity towards tumor cell lines. Experiments conducted on <em>in vivo</em> zebrafish model confirm the lowering ofthe adverse effects of the drug, especially cardiotoxicity, in case when nanocomposite was applied. Computer simulations, microscopic and&nbsp; spectroscopic results combined with encouraging <em>in vitro </em>and <em>in vivo</em> results point out&nbsp; that non-covalent interactions between fullerenol nanoparticles and doxorubicin may present the keyrole in formation of a synergistic system for nanodrug delivery into biological system. Multipotential of fullerenol nanoparticles as a nanocarrier and non-specific structure of doxorubicin as a drug imply that fullerenol may serve as a efficient nanocarrier of numerous other antineoplastics, which further allows the improvement of antitumor properties of drugs withsimultaneous drug administration.</p>

No description available.

Chemotherapy is one of the most successful cancer treatments used today. Unfortunately, the amount of chemotherapy a patient can receive is limited by the associated negative side effects, such as cardiotoxicity, immune system suppression, and nephrotoxicity. Encapsulation of these drugs, Doxorubicin (DOX) in particular, in stabilized Pluronic micelles (Plurogel TM) shows success in limiting these harmful side effects. In previous studies, low-frequency ultrasound (US) has been shown, in vitro, to locally release DOX from these micelles. In this study, a novel drug delivery system involving the encapsulation of DOX in Plurogel and the release of the drug at the tumor site using ultrasound was studied in vivo using rats. These studies determined the effect of ultrasonically released drugs on tumor growth rate and drug delivery to the tumor tissue. Concurrently, different frequencies (20 kHz, 500 kHz) were tested for the same effects. Treatments consisted of micelle-encapsulated doxorubicin injected intravenously followed by ultrasound application to one of the two bilateral tumors. Also, in different experiments, pharmacokinetic studies of the drug in the heart, liver, leg muscle, and tumors were performed up to a period of one week after treatment. Results showed that tumors treated with ultrasound displayed, on average, slower growth rates than non-insonated tumors (P = 0.0047). Also, insonated tumors displayed a weak increased concentration of DOX than non-insonated tumors within the first eight hours after treatment (P = 0.064). However, comparison between tumors which received 20 kHz and 500 kHz ultrasound treatment showed no statistical difference (P = 0.9275) in tumor growth rate or DOX concentration. It is noteworthy that the insonated tumor has slower growth even though the amount of DOX was not that much greater in the non-insonated tumor. This suggests that US also affects the uptake and/or processing of the DOX by the tumor cells, and that the therapeutic effect may not be attributed solely to a higher concentration of drug released by insonation. Pharmacokinetic studies showed significant drug accumulation in the heart but no accumulation in the liver, skeletal leg muscle, or tumors over the course of four weeks of consecutive weekly injections of DOX-encapsulated Plurogel. After 24 hours, DOX concentration remains the greatest in the tumors, regardless of whether they received ultrasound or not.

The viability of a drug delivery system which encapsulates chemotherapeutic drugs (Doxorubicin) in the hydrophobic core of polymeric micelles and triggers release by ultrasound application was investigated at an applied frequency of 500 kHz. The investigation also included elucidating the mechanism of drug release at 70 kHz, a frequency which had previously been shown to induce drug release. A fluorescence detection chamber was used to measure in vitro drug release from both Pluronic and stabilized micelles and a hydrophone was used to monitor bubble activity during the experiments. A threshold for release between 0.35 and 0.40 in mechanical index was found at 70 kHz and shown to correspond with the appearance of the subharmonic signal in the acoustic spectrum. Additionally, drug release was found to correlate with increase in subharmonic emission. No evidence of drug release or of the subharmonic signal was detected at 500 kHz. These findings confirmed the role of cavitation in ultrasonic drug release from micelles. A mathematical model of a bubble oscillator was solved to explore the differences in the behavior of a single 10 um bubble under 70 and 500 kHz ultrasound. The dynamics were found to be fundamentally different; the bubble follows a period-doubling route to chaos at 500 kHz and an intermittent route to chaos at 70 kHz. It was concluded that this type of "intermittent subharmonic" oscillation is associated with the apparent drug release. This research confirmed the central role of cavitation in ultrasonically-triggered drug delivery from micelles, established the importance of subharmonic bubble oscillations as an indicator, and expounded the key dynamic differences between 70 and 500 kHz ultrasonic cavitation.

Solid tumors possess biological features that are different from those in healthy tissues, which provides opportunities of anticancer treatment by nanomedicines. Due to the presence of the fenestrated tumor vasculatures, nanomedicines can selectively accumulate in tumor tissues by the enhanced permeability and retention (EPR) effect. The acidic pH in tumor interstitium (pH 6.0-7.0) also provides a promising mechanism to trigger the nanomedicines to promote the cellular uptake of cargo drugs. The previously reported stealth liposomes coated with PEG are known to accumulate in tumors owing to their prolonged circulation time. The PEG coating on liposomes can hinder serum protein adsorption and thus prevent rapid elimination by the reticuloendothelial system, thus increasing the liposome circulation time. However, liposomal interaction with cancer cells can also be hindered by the PEG coating. In order to improve the anticancer activity of stealth liposomes, novel synthetic imidazole-based lipids were introduced to the composition of stealth liposomes to develop the pH-sensitive imidazole-based convertible liposomes (ICL). At acidic pH, the imidazole-based lipids would protonate to acquire positive charges, thus clustering with the negatively charged PEGylated lipids. Such lipid-lipid electrostatic interaction would induce phase separation of the bilayer to generate a PEG-free domain that displays excess positive charges. Such newly converted, cationic liposomes at acidic pH in tumor interstitium would have better interaction with negatively charged cancer cells and/or enhanced drug release, therefore overcoming the drawback of traditional stealth liposomes. After synthesizing the imidazole-based lipids DHI, DHMI and DHDMI, we constructed doxorubicin (DOX)-loaded ICL formulations. The physicochemical properties of ICL were characterized, and factors influencing such properties were explored. The pH-triggered acquisition of positive charges of ICL was confirmed by the elevation of ζ- potentials and aggregation with negatively charged model liposomes that mimic bio-membranes at acidic pH 6.0-7.0. Acidic pH-triggered release of ICL was confirmed by drug release assays. It was also found that although the incorporation of cholesterol can remarkably reduce the size and increase the encapsulation efficiency (EE) of ICL, it also hinders the pH-sensitivity of ICL. The morphology of ICL at both pH 7.4 and pH 6.0 was characterized under transmission electron microscopy (TEM), which showed morphological changes in response to acidic pH 6.0, which further supported the proposed pH-sensitivity of ICL. Cytotoxicity assays on 3D MCS of HeLa, A549, MDA-MB-231 and MDA-MB-468 cell lines were conducted to evaluate the anticancer activity of ICL formulations. ICL formulations without cholesterol showed considerably enhanced anticancer activities against MCS compared with the non-sensitive stealth liposomes (NSL). However, incorporation of cholesterol decreased such activities. The IC50 values of cholesterol-free ICL and ICL with cholesterol against MCS strongly suggested that the pH-sensitivity introduced by the imidazole-based lipids would enhance the anticancer activity of stealth liposomes, while the hindrance of the pH-sensitivity by cholesterol would reduce such activities. Taken together, ICL’s pH-sensitivity is correlated with their enhanced anticancer activity than non-sensitive stealth liposomes.

The traditional anticancer drugs are distributed in vivo through systemic blood circulation with a very small portion reaching the tumor site. Targeted drug delivery systems are developed in efforts to concentrate the drug molecules in the tissue of interest while reducing the drug distribution to healthy tissues to reduce the side effects. Liposomes are colloidal systems composed of amphiphilic molecules that assemble into vesicle structures in aqueous media. They are common carriers for targeted drug delivery with the advantages of low toxicity, low immunogenicity and the ability of encapsulating both lipophilic and hydrophilic drugs. Prior research indicated the advantages of triggered release in drug delivery systems. As a specific example, a series of trans-2-aminocyclohexanol based lipids (flipids) have been reported to illustrate a promising strategy to render pH-triggered drug delivery systems: pH-triggered conformational switch. Based on the foregoing, we hypothesize that incorporation of lipids with a pH-sensitive conformational switch and a long-saturated lipid tail can improve the anticancer activities of stealth liposomes. In this study, six new flipids with C-16 saturated hydrocarbon tails were designed. Such lipids were synthesized with high yields by introducing a catalyst (Copper (II) tetrafluoroborate) at a key step of the synthetic scheme. pH-sensitive liposomes (fliposomes) composed of flipids were prepared and loaded with the anticancer drug doxorubicin with high encapsulation efficiency. The physicochemical properties of doxorubicin-loaded fliposomes were characterized and their pH-dependent leakage were investigated. The results showed that among all groups fliposomes containing the C-16 trans-2-morpholylcyclohexanol-based flipid (Mor-C16) exhibited the largest increase of release as the pH dropped form pH 7.4 to 6.0, indicating its good potential of serving as a component in pH-triggered drug delivery systems. Three-dimensional multicellular spheroids (3D MCS) are self-assembled microscale tissue analogs in vitro. They better mimic the native and complex tumor microenvironment than the conventional two-dimensional cell culture systems. In this dissertation study, 3D MCS of six different human cancer cells were successfully cultured and their growing conditions were optimized to obtain 3D MCS of tight structure and reproducible size. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells as well as acidic inside pH based on confocal microscopic imaging studies. The penetration of doxorubicin-loaded Mor-C16 fliposomes into 3D MCS was imaged by confocal microscopy in comparison to doxorubicin-loaded non pH-sensitive liposomes and free doxorubicin. The anticancer activities of doxorubicin-loaded Mor-C16 fliposomes against 3D MCS of three different cell lines was also evaluated by cell viability. Both the fliposome and the non pH-sensitive liposome formulations more efficiently penetrated into two of the three types of 3D MCS compared to free doxorubicin after 4h drug exposure. However, doxorubicin-loaded Mor-C16 fliposome imposed higher cytotoxicity to all three types of 3D MCS compared to doxorubicin-loaded non pH-sensitive liposome over 72 h drug exposure. Taken together, we propose that fliposomes achieved superior activity against 3D MCS by efficient penetration into 3D MCS, followed by enhanced release of the anticancer drug doxorubicin.

This work focused on engineering bi-functionalized nanoparticles (NPs) based on carbon dots (CDs) to improve early cancer detection and treatment. Therefore, using folic acid (FA) as a targeting agent, the CDs were prepared to deliver high concentrations (HC) of doxorubicin (DOX) and gemcitabine (GEM) covalently and non-covalently to cancer cells. The prepared FA-CDs-DOX/GEM-HC NPs were characterized using ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. Assessment of the drug loading capacity (DLC) and drug loading efficiency (DLE) indicated that the non-covalent NPs have low DLC but high DLE compared to the relatively low DLE and high DLC of covalent NPs. In vitro drug release studies showed that the DOX/GEM release rate was faster at pH 5.0 in the non-covalent FA-CDs-DOX/GEM-HC NPs than covalent. Also, the non-covalent FA-CDs-DOX-HC NPs showed greater percentage cumulative drug release and lower cell viability in the MDA-MB-231 breast cancer cell line compared to covalent.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.