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The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and ß-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25 °C), ionic strength (150 mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was â0.1 mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50-60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43 nM chlorpyrifos and 0.28 nM penicillin G. They even permitted the quantification of 14.3 µM chlorpyrifos and 2.8 µM penicillin G. Also, immobilization conferred higher stability to AChE (â94% activity after 20 days storage at 4 °C) and allowed to reuse the BL up to 12 cycles.
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Técnicas Biosensibles , Cloropirifos , Contaminantes Ambientales , Nanopartículas de Magnetita , Acetilcolinesterasa/química , Nanopartículas de Magnetita/química , Agua , beta-Lactamasas , Enzimas Inmovilizadas/química , Temperatura , Técnicas Biosensibles/métodos , Concentración de Iones de HidrógenoRESUMEN
Magnetite nanorods (MNRs) are synthesized based on the use of hematite nanoparticles of the desired geometry and dimensions as templates. The nanorods are shown to be highly monodisperse, with a 5:1 axial ratio, and with a 275 nm long semiaxis. The MNRs are intended to be employed as magnetic hyperthermia and photothermia agents, and as drug vehicles. To achieve a better control of their photothermia response, the particles are coated with a layer of gold, after applying a branched polyethyleneimine (PEI, 2 kDa molecular weight) shell. Magnetic hyperthermia is performed by application of alternating magnetic fields with frequencies in the range 118-210 kHz and amplitudes up to 22 kA/m. Photothermia is carried out by subjecting the particles to a near-infrared (850 nm) laser, and three monochromatic lasers in the visible spectrum with wavelengths 480 nm, 505 nm, and 638 nm. Best results are obtained with the 505 nm laser, because of the proximity between this wavelength and that of the plasmon resonance. A so-called dual therapy is also tested, and the heating of the samples is found to be faster than with either method separately, so the strengths of the individual fields can be reduced. Due to toxicity concerns with PEI coatings, viability of human hepatoblastoma HepG2 cells was tested after contact with nanorod suspensions up to 500 µg/mL in concentration. It was found that the cell viability was indistinguishable from control systems, so the particles can be considered non-cytotoxic in vitro. Finally, the release of the antitumor drug doxorubicin is investigated for the first time in the presence of the two external fields, and of their combination, with a clear improvement in the rate of drug release in the latter case.
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A large number of different types of cancer have been shown to be associated with an abnormal metabolism of phosphatidylcholine (PC), the main component of eukaryotic cell membranes. Indeed, the overexpression of choline kinase α1 (ChoKα1), the enzyme that catalyses the bioconversion of choline to phosphocholine (PCho), has been found to associate with cell proliferation, oncogenic transformation and carcinogenesis. Hence, ChoKα1 has been described as a possible cancer therapeutic target. Moreover, the choline transporter CTL1 has been shown to be highly expressed in several tumour cell lines. In the present work, we evaluate the antiproliferative effect of PL48, a rationally designed inhibitor of ChoKα1, in MCF7 and HepG2 cell lines. In addition, we illustrate that the predominant mechanism of cellular choline uptake in these cells is mediated by the CTL1 choline transporter. A possible correlation between the inhibition of both choline uptake and ChoKα1 activity and cell proliferation in cancer cell lines is also highlighted. We conclude that the efficacy of this inhibitor on cell proliferation in both cell lines is closely correlated with its capability to block choline uptake and ChoKα1 activity, making both proteins potential targets in cancer therapy.
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The synergy between directed chemotherapy and thermal therapy (both magnetic hyperthermia and photothermia) mediated by a nanoassembly composed of functionalized biomimetic magnetic nanoparticles (BMNPs) with the chemotherapeutic drug doxorubicin (DOXO) covered by the polymer poly(lactic-co-glycolic acid) (PLGA), decorated with TAT peptide (here referred to as TAT-PLGA(DOXO-BMNPs)) is explored in the present study. The rationale behind this nanoassembly lies in an optimization of the nanoformulation DOXO-BMNPs, already demonstrated to be more efficient against tumor cells, both in vitro and in vivo, than systemic traditional therapies. By embedding DOXO-BMNPs into PLGA, which is further functionalized with the cell-penetrating TAT peptide, the resulting nanoassembly is able to mediate drug transport (using DOXO as a drug model) and behaves as a hyperthermic agent (induced by an alternating magnetic field (AMF) or by laser irradiation with a laser power density of 2 W/cm2). Our results obtained using the HepG2 cell line show that there is a synergy between chemotherapy and thermal therapy that results in a stronger cytotoxic effect when compared to that caused by the soluble DOXO. This is probably due to the enhanced DOXO release occurring upon the application of the thermal therapy, as well as the induced local temperature rise mediated by BMNPs in the nanoassembly following exposition to AMF or to near-infrared (NIR) laser irradiation. These results represent a proof of concept demonstrating that TAT-PLGA(DOXO-BMNPs) can be used to efficiently combine therapies against tumor cells, which is a step forward in the transition from systemic to local treatments.
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The formation of fibrillar aggregates of the amyloid beta peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aß40 and Aß42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aß40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aß aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aß40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aß40 and is, therefore, a property acquired during the aggregation process. In contrast to Aß40, we did not observe the same self-catalytic fibrillation in Aß42 spheroidal oligomers, which could neither be induced to fibrillate by the Aß40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.
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Péptidos beta-Amiloides/química , Benzotiazoles/química , Fenómenos Biofísicos , Catálisis , Línea Celular Tumoral , Supervivencia Celular , Humanos , Cinética , Peso Molecular , Conformación ProteicaRESUMEN
MamC-mediated biomimetic magnetic nanoparticles (BMNPs) have emerged as one of the most promising nanomaterials due to their magnetic features (superparamagnetic character and large magnetic moment per particle), their novel surface properties determined by MamC, their biocompatibility and their ability as magnetic hyperthermia agents. However, the current clinical application of magnetic hyperthermia is limited due to the fact that, in order to be able to reach an effective temperature at the target site, relatively high nanoparticle concentration, as well as high magnetic field strength and/or AC frequency are needed. In the present study, the potential of BMNPs to increase the temperature upon irradiation of a laser beam in the near infrared, at a wavelength at which tissues become partially transparent, is explored. Moreover, our results also demonstrate the synergy between photothermia and chemotherapy in terms of drug release and cytotoxicity, by using BMNPs functionalized with doxorubicin, and the effectiveness of this combination therapy against tumor cells in in vitro experiments. Therefore, the findings of the present study open the possibility of a novel, alternative approach to fight localized tumors.
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Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nanoparticles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lactic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Internalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanoformulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here.
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The design of novel nanomaterials that can be used as multifunctional platforms allowing the combination of therapies is gaining increased interest. Moreover, if this nanomaterial is intended for a targeted drug delivery, the use of several guidance methods to increase guidance efficiency is also crucial. Magnetic nanoparticles (MNPs) allow this combination of therapies and guidance strategies. In fact, MNPs can be used simultaneously as drug nanocarriers and magnetic hyperthermia agents and, moreover, they can be guided toward the target by an external magnetic field and by their functionalization with a specific probe. However, it is difficult to find a system based on MNPs that exhibits optimal conditions as a drug nanocarrier and as a magnetic hyperthermia agent. In this work, a novel nanoformulation is proposed to be used as a multifunctional platform that also allows dual complementary guidance. This nanoformulation is based on mixtures of inorganic magnetic nanoparticles (M) that have been shown to be optimal hyperthermia agents, and biomimetic magnetic nanoparticles (BM), that have been shown to be highly efficient drug nanocarriers. The presence of the magnetosome protein MamC at the surface of BM confers novel surface properties that allow for the efficient and stable functionalization of these nanoparticles without the need of further coating, with the release of the relevant molecule being pH-dependent, improved by magnetic hyperthermia. The BM are functionalized with Doxorubicin (DOXO) as a model drug and with an antibody that allows for dual guidance based on a magnetic field and on an antibody. The present study represents a proof of concept to optimize the nanoformulation composition in order to provide the best performance in terms of the magnetic hyperthermia agent and drug nanocarrier.
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Recent studies have shown the potential of magnetic hyperthermia in cancer treatments. However, the underlying mechanisms involved have not been yet fully described. In particular, the cell death related to magnetic hyperthermia observed in cultures incubated with low concentration of magnetic nanoparticles and under a low intensity alternating magnetic field, in which a macroscopic temperature rise is not observed, is still not understood. In the present study, we investigate the production of intracellular Reactive Oxygen Species (ROS) as a mechanism to induce cell death under these conditions. In this study, the production and influence of ROS on the viability of HepG2 human hepatoma cells (used as a model cell line) are analyzed under the application of variable magnetic fields using hyperthermia agents, such as biomimetic magnetic nanoparticles (BMNPs) mediated by magnetosome MamC protein from Magnetococcus marinus MC-1. The results show that intracellular ROS production increases up to â¼90% following upon the exposure of AMF to HepG2 cells containing BMNPs, which could determine the loss of cell viability (up to â¼40% reduction) without a significant rise in temperature. Such ROS production is linked to mitochondrial dysfunction caused by the application of AMF to cells containing BMNPs.
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Campos Magnéticos , Especies Reactivas de Oxígeno/metabolismo , Materiales Biomiméticos/farmacología , Supervivencia Celular , Células Hep G2 , HumanosRESUMEN
Choline kinase α1 (ChoKα1) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoKα1 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoKα1. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia.
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A full understanding of the molecular mechanism of action of choline kinase α (ChoKα) inhibitors at the cell level is essential for developing therapeutic and preventive approaches for cancer. The aim of the present study was to evaluate the effects of the ChoKα inhibitors EB-3D and EB-3P on lipid metabolism in HepG2 cells. We used [methyl-14C]choline, [1,2-14C]acetic acid and [2-3H]glycerol as exogenous precursors of the corresponding phospholipids and neutral lipids. [Methyl-14C]choline was also used to determine choline uptake. Protein levels were determined by Western blot. Ultrastructural alterations were investigated by transmission electron microscopy. In this work, we demonstrate that EB-3D and EB-3P interfere with phosphatidylcholine biosynthesis via both CDP-choline pathway and choline uptake by the cell. Moreover, the synthesis of both diacylglycerols and triacylglycerols was affected by cell exposure to both inhibitors. These effects were accompanied by a substantial decrease in cholesterol biosynthesis, as well as alterations in the expression of proteins related to cholesterol homeostasis. We also found that EB-3D and EB-3P lowered ChoKα protein levels. All these effects could be explained by the modulation of the AMP-activated protein kinase signalling pathway. We show that both inhibitors cause mitochondrial alteration and an endoplasmic reticulum stress response. EB-3D and EB-3P exert effects on ChoKα expression, AMPK activation, apoptosis, endoplasmic reticulum stress and lipid metabolism. Taken together, results show that EB-3D and EB-3P have potential anti-cancer activity through the deregulation of lipid metabolism.