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DNA methylation is a crucial epigenetic hallmark of cancer development but the experimental methods able to prove nanoscale modifications are very scarce. Over time, Raman and its counterpart, surface-enhanced Raman scattering (SERS), became one of the most promising techniques capable to investigate nanoscale modifications of DNA bases. In our study, we employed Raman/SERS to highlight the differences between normal and leukemia DNA samples and to evaluate the effects of a 5-azacytidine treatment on leukemia cells. To obtain spectral information related to DNA base modifications, a DNA incubation step of 4 min at 94 °C, similar to the one performed in the case of RT-PCR experiments, was conducted prior to any measurements. In this way, reproducible Raman/SERS spectra were collected for all genomic DNA samples. Our Raman results allowed discrimination between normal and cancer DNAs based on their different aggregation behavior induced by the distinct methylation landscape present in the DNA samples. On the other hand, the SERS spectra collected on the same DNA samples show a very intense vibrational band located at 1008 cm-1 assigned to a rocking vibration of 5-methyl-cytosine. The intensity of this band strongly decreases in cancer DNA due to the modification of the methylation landscape occurring in cancers. We believe that under controlled experimental conditions, this vibrational band could be used as a powerful marker for demonstrating epigenetic reprogramming in cancer by means of SERS.
Asunto(s)
Leucemia , Vibración , Humanos , Desmetilación del ADN , Espectrometría Raman/métodos , ADN/química , Leucemia/genéticaRESUMEN
We report a very simple, rapid and reproducible method for the fabrication of anisotropic silver nanostars (AgNS) that can be successfully used as highly efficient SERS substrates for different bioanalytes, even in the case of a near-infra-red (NIR) excitation laser. The nanostars have been synthesized using the chemical reduction of Ag+ ions by trisodium citrate. This is the first research reporting the synthesis of AgNS using only trisodium citrate as a reducing and stabilizing agent. The key elements of this original synthesis procedure are rapid hydrothermal synthesis of silver nanostars followed by a cooling down procedure by immersion in a water bath. The synthesis was performed in a sealed bottom flask homogenously heated and brought to a boil in a microwave oven. After 60 s, the colloidal solution was cooled down to room temperature by immersion in a water bath at 35 °C. The as-synthesized AgNS were washed by centrifugation and used for SERS analysis of test molecules (methylene blue) as well as biological analytes: pharmaceutical compounds with various Raman cross sections (doxorubicin, atenolol & metoprolol), cell lysates and amino acids (methionine & cysteine). UV-Vis absorption spectroscopy, (Scanning) Transmission Electron Microscopy ((S)TEM) and Atomic Force Microscopy (AFM) have been employed for investigating nanostars' physical properties.
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Plata , Espectrometría Raman , Microscopía de Fuerza Atómica , Microondas , Plata/química , Espectrometría Raman/métodos , AguaRESUMEN
In this label-free surface-enhanced Raman scattering (SERS) study of genomic DNA, we demonstrate that the cancer-specific DNA methylation pattern translates into specific spectral differences. Thus, DNA extracted from an acute myeloid leukemia (AML) cell line presented a decreased intensity of the 1005 cm-1 band of 5-methylcytosine compared to normal DNA, in line with the well-described hypomethylation of cancer DNA. The unique methylation pattern of cancer DNA also influences the DNA adsorption geometry, resulting in higher adenine SERS intensities for cancer DNA. The possibility of detecting cancer DNA based on its SERS spectrum was validated on peripheral blood genomic DNA samples from n = 17 AML patients and n = 17 control samples, yielding an overall classification of 82% based on the 1005 cm-1 band of 5-methylcytosine. By demonstrating the potential of SERS in assessing the methylation status in the case of real-life DNA samples, the study paves the way for novel methods of diagnosing cancer. Graphical abstract.
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Metilación de ADN , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Espectrometría Raman/métodos , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/patología , MasculinoRESUMEN
Even if considered a cumulative and not a proliferative CD5+ B-cell neoplasm, chronic lymphocytic leukemia (CLL) has a proliferation rate higher than that recognized earlier, especially in the lymphoid tissues. Some patients with CLL develop a clinical syndrome entitled Richter syndrome (RS). Understanding CLL genetics and epigenetics may help to elucidate the molecular basics of the clinical heterogeneity of this type of malignancy. In the present project we aimed to identify a microRNA species that can predict the evolution of therapy-resistant CLL towards RS. In the first phase of our study, microRNA-19b was identified as a possible target, and in the second phase, we transfected three different CLL cell lines with microRNA-19b mimic and inhibitor and assessed the potential role on leukemia cells in vitro. The mechanism by which miR-19b acts were identified as the upregulation of Ki67 and downregulation of p53. This was further supported through RT-PCR and western blotting on CLL cell lines, as well as by next generation sequencing on two patients diagnosed with CLL that evolved into RS.
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Transformación Celular Neoplásica , Exosomas , Leucemia Linfocítica Crónica de Células B , MicroARNs , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Exosomas/química , Exosomas/metabolismo , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , MicroARNs/sangre , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Pronóstico , Síndrome , Células Tumorales CultivadasRESUMEN
Efficient use of magnetic hyperthermia in clinical cancer treatment requires biocompatible magnetic nanoparticles (MNPs), with improved heating capabilities. Small (~34 nm) and large (~270 nm) Fe3O4-MNPs were synthesized by means of a polyol method in polyethylene-glycol (PEG) and ethylene-glycol (EG), respectively. They were systematically investigated by means of X-ray diffraction, transmission electron microscopy and vibration sample magnetometry. Hyperthermia measurements showed that Specific Absorption Rate (SAR) dependence on the external alternating magnetic field amplitude (up to 65 kA/m, 355 kHz) presented a sigmoidal shape, with remarkable SAR saturation values of ~1400 W/gMNP for the small monocrystalline MNPs and only 400 W/gMNP for the large polycrystalline MNPs, in water. SAR values were slightly reduced in cell culture media, but decreased one order of magnitude in highly viscous PEG1000. Toxicity assays performed on four cell lines revealed almost no toxicity for the small MNPs and a very small level of toxicity for the large MNPs, up to a concentration of 0.2 mg/mL. Cellular uptake experiments revealed that both MNPs penetrated the cells through endocytosis, in a time dependent manner and escaped the endosomes with a faster kinetics for large MNPs. Biodegradation of large MNPs inside cells involved an all-or-nothing mechanism.
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Compuestos Férricos/farmacocinética , Hipertermia Inducida/métodos , Nanopartículas de Magnetita/química , Tamaño de la Partícula , Adsorción , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Glicol de Etileno/química , Compuestos Férricos/química , Humanos , Campos Magnéticos , Ratones , Microscopía Electrónica de Transmisión , Polietilenglicoles/química , Difracción de Rayos XRESUMEN
The magnetic properties of nanocrystalline MxFe3-xO4 ferrites with M=Fe, Co, and Zn were investigated. The data support a core-shell model, where the core is ferrimagnetically ordered, and the shell shows a spin glass type behavior. The reduced magnetizations of spin glass components follow an mg = (1 - b/H-1/2) field dependence. The b values are strongly correlated with the intensities of exchange interactions. The field dependences of the magnetoresistances of Fe3O4 and ZnxFe3-xO4 nanoparticles pellets, experimentally determined, are well described if instead of the core reduced magnetization, commonly used, that of the shell is taken into account. For similar compositions of the nanoparticles, identical b values are obtained both from magnetization isotherms and magnetoresistances studies. The half-metallic behavior of spinel Fe3O4 based nanoparticles is discussed comparatively with those of double perovskites.
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Co1−xZnxFe2O4 nanoparticles (0 ≤ x ≤ 1) have been synthesized via a green sol−gel combustion method. The prepared samples were studied using X-ray diffraction measurements (XRD), transmission electron microscopy (TEM), Raman, and magnetic measurements. All samples were found to be single phases and have a cubic Fd-3m structure. EDS analysis confirmed the presence of cobalt, zinc, iron, and oxygen in all studied samples. Raman spectra clearly show that Zn ions are preferentially located in T sites for low Zn concentrations. Due to their high crystallinity, the nanoparticles show high values of the magnetization, which increases with the Zn content for x < 0.5. The magnetic properties are discussed based on Raman results. Co ferrite doped with 30% of Zn produced the largest SAR values, which increase linearly from 148 to 840 W/gMNPs as the H is increased from 20 to 60 kA/m.
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Our study addresses the problematics of magnetic skyrmions, nanometer-size vortex-like swirling topological defects, broadly studied today for applications in classic, neuromorphic and quantum information technologies. We tackle some challenging issues of material properties versus skyrmion stability and manipulation within a multiple-scale modeling framework, involving complementary ab-initio and micromagnetic frameworks. Ab-initio calculations provide insight into the anatomy of the magnetic anisotropy, the Dzyaloshinskii-Moriya asymmetric exchange interaction (DMI) and their response to a gating electric field. Various multi-layered heterostructures were specially designed to provide electric field tunable perpendicular magnetization and sizeable DMI, which are required for skyrmion occurrence. Landau-Lifshitz-Gilbert micromagnetic calculations in nanometric disks allowed the extraction of material parameter phase diagrams in which magnetic textures were classified according to their topological charge. We identified suitable ranges of magnetic anisotropy, DMI and saturation magnetization for stabilizing skyrmionic ground states or writing/manipulating them using either a spin-transfer torque of a perpendicular current or the electric field. From analyzing the different contributions to the total magnetic free energy, we point out some critical properties influencing the skyrmions' stability. Finally, we discuss some experimental issues related to the choice of materials or the design of novel magnetic materials compatible with skyrmionic applications.
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We report the successful synthesis and a complete magnetic characterization of CoFe2O4@SiO2@Au magnetoplasmonic nanoparticles. The CoFe2O4 magnetic nanoparticles were prepared using the hydrothermal method. A subsequent SiO2 shell followed by a plasmonic Au shell were deposited on the magnetic core creating magnetoplasmonic nanoparticles with a core-shell architecture. A spin-glass-type magnetism was shown at the surface of the CoFe2O4 nanograins. Depending on the external magnetic field, two types of spin-glass were identified and analyzed in correlation with the exchange field acting on octahedral and tetrahedral iron sites. The magnetization per formula unit of the CoFe2O4 core is not changed in the case of CoFe2O4@SiO2@Au nanocomposites. The gold nanoparticles creating the plasmonic shell show a giant diamagnetic susceptibility, dependent on their crystallite sizes.
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Increasing the biocompatibility, cellular uptake, and magnetic heating performance of ferromagnetic iron-oxide magnetic nanoparticles (F-MNPs) is clearly required to efficiently induce apoptosis of cancer cells by magnetic hyperthermia (MH). Thus, F-MNPs were coated with silica layers of different thicknesses via a reverse microemulsion method, and their morphological, structural, and magnetic properties were evaluated by multiple techniques. The presence of a SiO2 layer significantly increased the colloidal stability of F-MNPs, which also enhanced their heating performance in water with almost 1000 W/gFe as compared to bare F-MNPs. The silica-coated F-MNPs exhibited biocompatibility of up to 250 µg/cm2 as assessed by Alamar Blues and Neutral Red assays on two cancer cell lines and one normal cell line. The cancer cells were found to internalize a higher quantity of silica-coated F-MNPs, in large endosomes, dispersed in the cytoplasm or inside lysosomes, and hence were more sensitive to in vitro MH treatment compared to the normal ones. Cellular death of more than 50% of the malignant cells was reached starting at a dose of 31.25 µg/cm2 and an amplitude of alternating magnetic field of 30 kA/m at 355 kHz.
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We report the synthesis of magnetite nanoparticles (IOMNPs) using the polyol method performed at elevated temperature (300 °C) and high pressure. The ferromagnetic polyhedral IOMNPs exhibited high saturation magnetizations at room temperature (83 emu/g) and a maximum specific absorption rate (SAR) of 2400 W/gFe in water. The uniform dispersion of IOMNPs in solid matrix led to a monotonous increase of SAR maximum (3600 W/gFe) as the concentration decreased. Cytotoxicity studies on two cell lines (cancer and normal) using Alamar Blues and Neutral Red assays revealed insignificant toxicity of the IOMNPs on the cells up to a concentration of 1000 µg/mL. The cells internalized the IOMNPs inside lysosomes in a dose-dependent manner, with higher amounts of IOMNPs in cancer cells. Intracellular hyperthermia experiments revealed a significant increase in the macroscopic temperatures of the IOMNPs loaded cell suspensions, which depend on the amount of internalized IOMNPs and the alternating magnetic field amplitude. The cancer cells were found to be more sensitive to the intracellular hyperthermia compared to the normal ones. For both cell lines, cells heated at the same macroscopic temperature presented lower viability at higher amplitudes of the alternating magnetic field, indicating the occurrence of mechanical or nanoscale heating effects.
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Surface enhanced Raman spectroscopy (SERS) represents a promising technique in providing specific molecular information that could have a major impact in biomedical applications, such as early cancer detection. SERS requires the presence of a suitable plasmonic substrate that can generate enhanced and reproducible diagnostic relevant spectra. In this paper, we propose a new approach for the synthesis of such a substrate, by using concentrated silver nanoparticles purified using the Tangential Flow Filtration method. The capacity of our substrates to generate reproducible and enhanced Raman signals, in a manner that can allow cancer detection by means of Multivariate Analysis (MVA) of Surface Enhanced Raman (SER) spectra, has been tested on blood plasma samples collected from 35 healthy donors and 29 breast cancer patients. All the spectra were analyzed by a combined Principal Component-Linear Discriminant Analysis. Our results facilitated the discrimination between healthy donors and breast cancer patients with 90% sensitivity, 89% specificity and 89% accuracy. This is a direct consequence of substrates' ability to generate diagnostic relevant spectral information by performing SERS measurements on pristine blood plasma samples. Our results suggest that this type of solid substrate could be employed for the detection of other types of cancer or other diseases by means of MVA-SERS procedure.
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Manganese and zinc ferrite magnetic nanoparticles (MNPs) were successfully synthesizedusing the polyol method in ethylene glycol and were found to have high saturation magnetizationvalues (90-95 emu/g at 4 K) when formed by ~30-nm crystallites assembled in an ~80-nm multicorestructure. Hyperthermia data revealed a sigmoidal dependence of the specific absorption rate (SAR)on the alternating magnetic field (AMF) amplitude, with remarkable saturation SAR values in waterof ~1200 W/gFe+Mn and ~800 W/gFe+Zn for the Mn and Zn ferrites, respectively. The immobilizationof the MNPs in a solid matrix reduced the maximum SAR values by ~300 W/gFe+Mn, Zn for bothferrites. The alignment of the MNPs in a uniform static magnetic field, before their immobilizationin a solid matrix, significantly increased their heating performance. Toxicity assays performed infour cell lines revealed a lower toxicity for the Mn ferrites, while in the case of the Zn ferrites, only~50% of cells were viable upon their incubation for 24 h with 0.2 mg/mL of MNPs. Cellular uptakeexperiments revealed that both MNPs entered the cells in a time-dependent manner, as they werefound initially in endosomes and later in the cytosol. All of the studied cell lines were more sensitiveto the ZnFe2O4 MNPs.
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By carefully controlling the electrostatic interactions between cationic liposomes, which already incorporate magnetic nanoparticles in the bilayers, and anionic gold nanoparticles, a new class of versatile multifunctional nanohybrids (plasmonic magneto-liposomes) that could have a major impact in drug delivery and controlled release applications has been synthesized. The experimental results confirmed the successful synthesis of hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and polyethylene glycol functionalized (PEGylated) gold nanoparticles (AuNPs). The SPIONs were incorporated in the liposomal lipidic bilayers, thus promoting the formation of cationic magnetoliposomes. Different concentrations of SPIONs were loaded in the membrane. The cationic magnetoliposomes were decorated with anionic PEGylated gold nanoparticles using electrostatic interactions. The successful incorporation of SPIONs together with the modifications they generate in the bilayer were analyzed using Raman spectroscopy. The plasmonic properties of the multifunctional nanohybrids were investigated using UV-Vis absorption and (surface-enhanced) Raman spectroscopy. Their hyperthermic properties were recorded at different frequencies and magnetic field intensities. After the synthesis, the nanosystems were extensively characterized in order to properly evaluate their potential use in drug delivery applications and controlled release as a result of the interaction with an external stimulus, such as an NIR laser or alternating magnetic field.
RESUMEN
Iron oxide magnetic nanoparticles (IOMNPs) have been successfully synthesized by means of solvothermal reduction method employing polyethylene glycol (PEG200) as a solvent. The as-synthesized IOMNPs are poly-dispersed, highly crystalline, and exhibit a cubic shape. The size of IOMNPs is strongly dependent on the reaction time and the ration between the amount of magnetic precursor and PEG200 used in the synthesis method. At low magnetic precursor/PEG200 ratio, the cubic IOMNPs coexist with polyhedral IOMNPs. The structure and morphology of the IOMNPs were thoroughly investigated by using a wide range of techniques: TEM, XRD, XPS, FTIR, and RAMAN. XPS analysis showed that the IOMNPs comprise a crystalline magnetite core bearing on the outer surface functional groups from PEG200 and acetate. The presence of physisorbed PEG200 on the IOMNP surface is faintly detected through FT-IR spectroscopy. The surface of IOMNPs undergoes oxidation into maghemite as proven by RAMAN spectroscopy and the occurrence of satellite peaks in the Fe2p XP spectra. The magnetic studies performed on powder show that the blocking temperature (TB) of IOMNPs is around 300 K displaying a coercive field in between 160 and 170 Oe. Below the TB, the field-cooled (FC) curves turn concave and describe a plateau indicating that strong magnetic dipole-dipole interactions are manifested in between IOMNPs. The specific absorption rate (SAR) values increase with decreasing nanoparticle concentrations for the IOMNPs dispersed in water. The SAR dependence on the applied magnetic field, studied up to magnetic field amplitude of 60 kA/m, presents a sigmoid shape with saturation values up to 1700 W/g. By dispersing the IOMNPs in PEG600 (liquid) and PEG1000 (solid), it was found that the SAR values decrease by 50 or 75 %, indicating that the Brownian friction within the solvent was the main contributor to the heating power of IOMNPs.