RESUMEN
The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.
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Glioma , Terapia de Protones , Ratas Endogámicas F344 , Sincrotrones , Animales , Ratas , Glioma/radioterapia , Glioma/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Línea Celular Tumoral , Astrocitos/efectos de la radiación , Astrocitos/metabolismo , Ácidos Nucleicos/efectos de la radiación , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismoRESUMEN
(1) Background: among all types of radiation, very heavy ions, such as Neon (Ne) or Argon (Ar), are the optimum candidates for hypoxic tumor treatments due to their reduced oxygen enhancement effect. However, their pioneering clinical use in the 1970s was halted due to severe side effects. The aim of this work was to provide a first proof that the combination of very heavy ions with minibeam radiation therapy leads to a minimization of toxicities and, thus, opening the door for a renewed use of heavy ions for therapy; (2) Methods: mouse legs were irradiated with either Ne MBRT or Ne broad beams at the same average dose. Skin toxicity was scored for a period of four weeks. Histopathology evaluations were carried out at the end of the study; (3) Results: a significant difference in toxicity was observed between the two irradiated groups. While severe da-mage, including necrosis, was observed in the broad beam group, only light to mild erythema was present in the MBRT group; (4) Conclusion: Ne MBRT is significantly better tolerated than conventional broad beam irradiations.
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The study of cell aggregation in vitro has a tremendous importance these days. In cancer biology, aggregates and spheroids serve as model systems and are considered as pseudo-tumors that are more realistic than 2D cell cultures. Recently, in the context of brain tumors (gliomas), we developed a new poly(ethylene glycol) (PEG)-based hydrogel, with adhesive properties that can be controlled by the addition of poly(L-lysine) (PLL), and a stiffness close to the brain's. This substrate allows the motion of individual cells and the formation of cell aggregates (within one day), and we showed that on a non-adhesive substrate (PEG without PLL is inert for cells), the aggregates are bigger and less numerous than on an adhesive substrate (with PLL). In this article, we present new experimental results on the follow-up of the formation of aggregates on our hydrogels, from the early stages (individual cells) to the late stages (aggregate compaction), in order to compare, for two cell lines (F98 and U87), the aggregation process on the adhesive and non-adhesive substrates. We first show that a spaceless model of perikinetic aggregation can reproduce the experimental evolution of the number of aggregates, but not of the mean area of the aggregates. We thus develop a minimal off-lattice agent-based model, with a few simple rules reproducing the main processes that are at stack during aggregation. Our spatial model can reproduce very well the experimental temporal evolution of both the number of aggregates and their mean area, on adhesive and non-adhesive soft gels and for the two different cell lines. From the fit of the experimental data, we were able to infer the quantitative values of the speed of motion of each cell line, its rate of proliferation in aggregates and its ability to organize in 3D. We also found qualitative differences between the two cell lines regarding the ability of aggregates to compact. These parameters could be inferred for any cell line, and correlated with clinical properties such as aggressiveness and invasiveness.
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Adhesión Celular , Agregación Celular , Hidrogeles/química , Modelos Biológicos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Proliferación Celular , Humanos , Cinética , Polietilenglicoles/química , Polilisina/químicaRESUMEN
The outcome of radiotherapy can be further improved by combining radiotherapy with nanoparticles. Previous biological studies showed a significant amplification of the biological damage in cells charged with nanoparticles prior to radiotherapy treatments. The rationale has been based on the physical dose enhancement. However, this subject is still a matter of controversy and there are clear indications that biochemical effects may play a key role in the radiosensitization effects of nanoparticles. Within this context, the main goal of our study was to provide new insights into the radiosensitization effects of F98 glioma cells exposed to gadolinium nanoparticles combined with clinical megavoltage beams, and compare them with respect to kilovoltage radiotherapy (commonly used in combination with nanoparticles). For this purpose, we used synchrotron-based Fourier transform infrared microspectroscopy (SR-FTIRM) to provide relevant information on the treatment-induced biochemical changes of the main cell biomolecules. Biochemical differences were evaluated after the treatments to assess cellular damage. Multivariate analysis revealed nanoparticle-dependent changes in megavoltage treated cells. The main spectral variations were related to conformational changes in the protein secondary structures, which might be induced by radiation damage and by changes or rearrangements in the nucleic acid structures due to the initiation of DNA repair mechanisms. We also observed significant changes in the phosphate I and II bands, which concerns DNA damage, while few changes were detected in the lipid region. Spectroscopic data showed that these changes increased as a function of the dose. Finally, PCA analysis did not discriminate clearly between megavoltage and kilovoltage groups treated with nanoparticles, indicating that megavoltage radiosensitization effects might not differ significantly from those in kilovoltage radiotherapy.
RESUMEN
The technique of Raman tweezers microspectroscopy (RTM) for the global biomolecular content characterization of a single extracellular vesicle (EV) or a small number of EVs or other nanoscale bioparticles in an aqueous dispersion in the difficult-to-access size range of near 100 nm is described in detail. The particularities and potential of RTM are demonstrated using the examples of DOPC liposomes, exosomes from human urine and rat hepatocytes, and a mixed sample of the transfection reagent FuGENE in diluted DNA solution. The approach of biomolecular component analysis for the estimation of the main biomolecular contributions (proteins, lipids, nucleic acids, carotenoids, etc.) is proposed and discussed. Direct Raman evidence for strong intra-sample biomolecular heterogeneity of individual optically trapped EVs, due to variable contributions from nucleic acids and carotenoids in some preparations, is reported. On the basis of the results obtained, we are making an attempt to convince the scientific community that RTM is a promising method of single-EV research; to our knowledge, it is the only technique available at the moment that provides unique information about the global biomolecular composition of a single vesicle or a small number of vesicles, thus being capable of unravelling the high diversity of EV subpopulations, which is one of the most significant urgent challenges to overcome. Possible RTM applications include, among others, searching for DNA biomarkers, cancer diagnosis, and discrimination between different subpopulations of EVs, lipid bodies, protein aggregates and viruses.
Asunto(s)
Vesículas Extracelulares/química , Microscopía , Espectrometría Raman , Animales , Microscopía por Crioelectrón , Exosomas/química , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Liposomas/química , Liposomas/metabolismo , Ácidos Nucleicos/análisis , Tamaño de la Partícula , Proteínas/análisis , RatasRESUMEN
Therapeutic substances bound to nanoparticles have been shown to dissociate following excitation by various external sources of energies or chemical disturbance, resulting in controllable and efficient antitumor activity. Bioconjugation is used to produce magnetosomes associated with Rhodamine B (RhB), whose fluorescence is partially quenched by the presence of iron oxide and becomes strongly enhanced when RhB dissociates from the magnetosomes under the application of an alternating magnetic field. This novel approach enables the release of a RhB model molecule while monitoring the mechanism by fluorescence. The dissociation mechanism of RhB is highlighted by exposing a suspension of fluorescent magnetosomes to an alternating magnetic field, by magnetically isolating the supernatant of this suspension, and by showing fluorescence enhancement of the supernatant. Furthermore, to approach in vivo conditions, fluorescent magnetosomes are mixed with tissue or introduced in the mouse brain and exposed to the alternating magnetic field. Most interestingly, the percentages of RhB dissociation measured at the beginning of magnetic excitation (ΔR/δt) or 600 seconds afterwards (R600 s) are ΔR/δt â¼ 0.13% and R600 s â¼ 50% under conditions of limited temperature increases (<2.5 °C), larger values than those of ΔR/δt â¼ 0.02-0.11% and R600 s â¼ 13%, estimated for temperature increase larger than 2.5 °C. Furthermore, when magnetic excitations are repeated two to five times, the temperature increase becomes undetectable, but RhB dissociation continues to occur up to the fifth magnetic excitation. Since high heating temperatures may be damaging for tissues, this study paves the way towards the development of a safe theranostic dissociating nano-probe operating under conditions of limited temperature increase.
RESUMEN
We report a method of fabrication of fluorescent magnetosomes, designated as MCR400, in which 400 µM of rhodamine B are introduced in the growth medium of AMB-1 magnetotactic bacteria and fluorescent magnetosomes are then extracted from these bacteria. These fluorescent magnetosomes behave differently from most fluorescent nanoprobes, which often lead to fluorescence losses over time due to photobleaching. Indeed, when MCR400 are heated to 30-90 °C, brought to an acidic pH, or exposed to radiations, we observed that their fluorescence intensity increased. We attributed this behavior to the dissociation of rhodamine B from the magnetosomes. Interestingly, enhanced fluorescence was also observed in vitro when MCR400 were mixed with either primary macrophages or tumor cells (TC1-GFP or RG2-Cells) or in vivo when MCR400 were introduced in rat glioblastoma. We showed that MCR400 internalize in tumor and immune cells (macrophages) leading to enhanced fluorescence, suggesting that fluorescent magnetosomes could be used during cancer treatments such as magnetic hyperthermia to image cells of interest such as immune or tumor cells.
RESUMEN
BACKGROUND: Biologics magnetics nanoparticles, magnetosomes, attract attention because of their magnetic characteristics and potential applications. The aim of the present study was to develop and characterize novel magnetosomes, which were extracted from magnetotactic bacteria, purified to produce apyrogen magnetosome minerals, and then coated with Chitosan, Neridronate, or Polyethyleneimine. It yielded stable magnetosomes designated as M-Chi, M-Neri, and M-PEI, respectively. Nanoparticle biocompatibility was evaluated on mouse fibroblast cells (3T3), mouse glioblastoma cells (GL-261) and rat glioblastoma cells (RG-2). We also tested these nanoparticles for magnetic hyperthermia treatment of tumor in vitro on two tumor cell lines GL-261 and RG-2 under the application of an alternating magnetic field. Heating, efficacy and internalization properties were then evaluated. RESULTS: Nanoparticles coated with chitosan, polyethyleneimine and neridronate are apyrogen, biocompatible and stable in aqueous suspension. The presence of a thin coating in M-Chi and M-PEI favors an arrangement in chains of the magnetosomes, similar to that observed in magnetosomes directly extracted from magnetotactic bacteria, while the thick matrix embedding M-Neri leads to structures with an average thickness of 3.5 µm2 per magnetosome mineral. In the presence of GL-261 cells and upon the application of an alternating magnetic field, M-PEI and M-Chi lead to the highest specific absorption rates of 120-125 W/gFe. Furthermore, while M-Chi lead to rather low rates of cellular internalization, M-PEI strongly associate to cells, a property modulated by the application of an alternating magnetic field. CONCLUSIONS: Coating of purified magnetosome minerals can therefore be chosen to control the interactions of nanoparticles with cells, organization of the minerals, as well as heating and cytotoxicity properties, which are important parameters to be considered in the design of a magnetic hyperthermia treatment of tumor.
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Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/uso terapéutico , Glioma/terapia , Magnetosomas/química , Nanopartículas/química , Nanopartículas/uso terapéutico , Células 3T3 , Animales , Línea Celular Tumoral , Quitosano/química , Quitosano/uso terapéutico , Difosfonatos/química , Difosfonatos/uso terapéutico , Hipertermia Inducida , Campos Magnéticos , Magnetospirillum/química , Ratones , Polietileneimina/química , Polietileneimina/uso terapéutico , RatasRESUMEN
One strategy to improve the clinical outcome of radiotherapy is to use nanoparticles as radiosensitizers. Along this line, numerous studies have shown the enhanced effectiveness of tumour cell killing when nanoparticles are exposed to irradiation. However, the mechanisms of action are not clear yet. In addition to the damage due to a possible local radiation dose enhancement, the interaction of nanoparticles with essential biological macromolecules could lead to changes in the cells, such as cell arrest at radiosensitive phases. Within this framework, vibrational spectroscopy was used to investigate the biochemical changes in F98 glioma cells induced by X-ray irradiations combined with gadolinium nanoparticles. Fourier transform infrared (FTIR) microspectroscopy experiments were performed at the Emira laboratory of the SESAME synchrotron (Jordan), allowing the characterisation of spectral signatures of nanoparticle-induced effects in glioma cells. Multivariate analysis of the spectra recorded using principal component analysis reveals clear differences in the DNA, protein and lipid regions in the presence of nanoparticles. Prior to irradiation, results show that nanoparticles induce biochemical modifications in the cells, probably due to changes in the cellular function. Biochemical alterations are amplified in the presence of radiation. In particular, variations in the intensity and in the position of the PO2(-) symmetric and asymmetric modes are observed due to radiation damage to the DNA, which is increased in nanoparticle-treated cells. At 24 hours post-irradiation, biochemical changes related to the hallmark characteristics of cell death are detected. This includes a shift towards low wavenumbers in the amide I and II bands, relative amplitude changes in the CH2 and CH3 stretching modes, along with DNA chromatin condensation indications. Results were confirmed by two complementary cell viability assays.
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Gadolinio/química , Gadolinio/farmacología , Glioma/patología , Nanopartículas del Metal , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Sincrotrones , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Ratas , Rayos XRESUMEN
Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria are shown to be highly efficient for cancer therapy when they are exposed to an alternative magnetic field. When a suspension containing MDA-MB-231 breast cancer cells was incubated in the presence of various amounts of extracted chains of magnetosomes, the viability of these cells remained high in the absence of an alternative magnetic field. By contrast, when this suspension was exposed to an alternative magnetic field of frequency 183 kHz and field strengths of 20, 40, or 60 mT, up to 100% of these cells were destroyed. The antitumoral activity of the extracted chains of magnetosomes is demonstrated further by showing that they can be used to fully eradicate a tumor xenografted under the skin of a mouse. For that, a suspension containing â¼1 mg of extracted chains of magnetosomes was administered within the tumor and the mouse was exposed to three heat cycles of 20 min, during which the tumor temperature was raised to â¼43 °C. We also demonstrate the higher efficiency of the extracted chains of magnetosomes compared with various other materials, i.e., whole inactive magnetotactic bacteria, individual magnetosomes not organized in chains, and two different types of chemically synthesized superparamagnetic iron oxide nanoparticles currently tested for alternative magnetic field cancer therapy. The higher efficiency of the extracted chains of magnetosomes compared with that of the other nanoparticles is attributed to three factors: (i) a specific absorption rate higher for the magnetosomes than for the chemically synthesized superparamagnetic iron oxide nanoparticles, (ii) a more uniform heating for the chains of magnetosomes than for the individual magnetosomes and (iii) the ability of the chains of magnetosomes to penetrate within the cancer cells or bind at the cell membrane following the application of the alternative magnetic field, which enables efficient cell destruction. Biodistribution studies revealed that extracted chains of magnetosomes administered directly within xenografted breast tumors progressively left the tumors during the 14 days following their administration and were then eliminated in large proportion in the feces.
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Bacterias/citología , Neoplasias de la Mama/terapia , Magnetoterapia/métodos , Magnetosomas , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ácido Cítrico/química , Ácido Edético/química , Femenino , Calor , Humanos , Magnetosomas/química , Ratones , Nanopartículas/química , Nanopartículas/uso terapéutico , Polietilenglicoles/química , Dodecil Sulfato de Sodio/farmacología , Factores de TiempoRESUMEN
The coordination complexes (DIP)(2)Ru(CH(3)bpyCOOH) and (DIP)(2)Ru(COOHbpyCOOH), where DIP and bpy are diphenylphenanthroline and bispyridine, have been recently proposed as fluorescent markers of nuclear DNA (Musatkina et al., J. Inorg. Biochem. 101:1086-1089, 2007), but no DNA binding investigation and no quantitative fluorescence evaluations had been done. Both complexes, as well as the smaller ones with bpy's in place of DIP's, have been investigated here by spectroscopic DNA titrations (UV-vis absorption, fluorescence, circular dichroism) and by in vitro cellular studies (flow cytometry and fluorescence imaging). Contrary to previous reports, neither the carboxylic function nor the more extended DIP ligand ensures any appreciable binding to DNA. This is clearly illustrated by the appearance of an isosbestic point of a second kind and by the proportionality of the fluorescence maximum intensity to the absorbance at the excitation wavelength. Above all, the lack of enhanced fluorescence in the presence of DNA definitively rules out the use of such complexes as DNA markers. Moreover, there is no detectable nuclear uptake. However, the fluorescent complexes with the DIP ligands, especially (DIP)(2)Ru(CH(3)bpyCOOH), are massively incorporated into the cytoplasm while preserving cell integrity, which could suggest other types of biological application.
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ADN/química , ADN/metabolismo , Absorción , Dicroismo Circular , Complejos de Coordinación , Fluorescencia , Ligandos , Salicilatos , Análisis EspectralRESUMEN
Bisphosphonates have been used for decades in the standard therapy of bone-related diseases, including bone metastasis of various malignancies, and they might as well be toxic on early cancer cells themselves. In order to allow a better delivery of neridronate (a N-containing bisphosphonate with relatively poor activity), liposomes were evaluated in vitro on cancer cell lines (MDA-MB-231, U87-MG and Caco2). After chemical synthesis, this water-soluble molecule was encapsulated into liposomes containing DOPC:DOPG:Chol (72:27:1 molar ratio). The influence of neridronate (free or liposomal) on cell viability or proliferation after treatment was evaluated using the MTT method, as well as cell migration and invasion assays; these techniques showed a drastic improvement of the action of neridronate on MDA-MB-231 cells with an EC(50) 50 times lower when neridronate was encapsulated. Internalization of liposomes was followed by flow cytometry and fluorescence microscopy, demonstrating internalization via the endocytic pathway. Furthermore, since overexpression of matrix metalloproteinases (particularly MMP-2 and MMP-9) has been correlated to poor prognosis in many cancer types, detection of MMP expression is a satisfactory indication of the therapy efficiency and was then performed on treated cells. On MDA-MB-231 cells, MPPs expression was also significantly reduced by neridronate while entrapped in liposomes.
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Neoplasias de la Mama , Difosfonatos/metabolismo , Difosfonatos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Células CACO-2 , Cápsulas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Difosfonatos/síntesis química , Femenino , Humanos , Liposomas , Metaloproteinasas de la MatrizRESUMEN
Time-resolved confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin-ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin-ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.
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Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Oligonucleótidos/química , Porfirinas/química , Células 3T3 , Animales , Cationes , Núcleo Celular/metabolismo , Sistemas de Liberación de Medicamentos , Fluorescencia , Melanoma Experimental , Ratones , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Oligonucleótidos Antisentido/química , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Flow cytometry light scattering was used to monitor size increase of Candida albicans (isolate ATCC 10231) cells in the presence or absence of the antifungal drug amphotericin B (AmB). This non-invasive and descriptive method allowed for the differentiation of dead and dormant sub-populations of cells. When inoculated into a growth medium without AmB, a progressive increase in light scattering was observed over a period of approximately 4 h, but without proliferation of the yeast. After this period, the light scattering distribution regressed to baseline level, whereas cell proliferation started. In the presence of AmB, all the cells shrank in size within approximately 4 h and proliferation was temporarily halted. However, in the presence of 0.4 microM AmB, a progressive increase of light scattering occurred after 21 h which was similar to that observed within the first 4 h in the absence of the antifungal. After approximately 24 h of incubation at this concentration of AmB, proliferation resumed. These observations indicate that this renewed cell proliferation was due to the reawakening of dormant cells in the presence of AmB (45% in the presence of 0.4 microM AmB) rather than the result of the development of viable cells that had escaped detection. This simple descriptive approach could be extended to other fungal strains or species, to other antifungal drugs and possibly to bacteria.
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Anfotericina B/farmacología , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Polienos/farmacología , Candida albicans/crecimiento & desarrollo , Recuento de Colonia Microbiana , Medios de Cultivo , Citometría de Flujo/métodosRESUMEN
Solution secondary structures of three synthetic cationic peptides, currently used in antisense oligonucleotide delivery into living cells, have been analyzed by means of circular dichroism (CD) and Raman scattering in different buffers as a function of concentration and time. All three peptides are of minimalist conception, i.e., formed by only two types of amino acids (leucine: L and lysine: K). Two of these peptides contain 15 aminoacids: N(ter)- KLLKLLLKLLLKLLK (L(10)K(5)), N(ter)-KLKLKLKLKLKLKLK (L(7)K(8)), and the third one has only 9 residues: N(ter)-KLKLKLKLK (L(4)K(5)). The conformational behavior of the 15-mers in pure water differs considerably one from another. Although both of them are initially disordered in the 50-350 microM range, L(10)K(5) gradually undergoes a disordered to alpha-helix transition for molecular concentrations above 100 microM. In all other solvents used, L(10)K(5) adopts a stable alpha-helical conformation. In methanol and methanol/Tris mixture, nonnative alpha-helices can be induced in both KL-alternating peptides, i.e., L(7)K(8) and L(4)K(5). However, in major cases and with a time delay depending on peptide concentration, beta-like structures can be gradually formed in both solutions. In PBS and methanol/PBS mixture, the tendency for L(7)K(8) and L(4)K(5) is to form structures belonging to beta-family. A discussion has been undertaken on the effect of counterions as well as their nature in the stabilization of ordered structures in both KL-alternating peptides.
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Leucina/química , Lisina/química , Péptidos/química , Solventes/química , Análisis Espectral , Secuencia de Aminoácidos , Dicroismo Circular , Cinética , Metanol/química , Datos de Secuencia Molecular , Oligopéptidos/análisis , Oligopéptidos/química , Péptidos/análisis , Conformación Proteica , Estructura Secundaria de Proteína , Espectrometría Raman , Trometamina/químicaRESUMEN
The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.
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Acridinas/farmacología , Antineoplásicos/farmacología , Pirazoles/farmacología , Animales , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células K562 , Cinética , Leucemia/patología , Ratones , Células 3T3 NIH , Células Tumorales CultivadasRESUMEN
Interaction, i.e., cellular uptake and intracellular distribution, of synthetic modified antisense oligonucleotide with the B16 melanoma cell line was studied using cationic polyene antibiotic, amphotericin B 3-dimethylaminopropyl amide, as a carrier vector. The antisense oligonucleotide--dT(15) oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3'-O-P-CH(2)-O-5'--was labeled with fluorescent tetramethylrhodamine marker. The oligonucleotide itinerancy across the cell membrane and its distribution inside the cell was visualized using fluorescence microimaging. During the first several hours a strong preference staining of the cell nucleus was found. Fluorescence lifetime measurements from the intracellular environment (confocal laser microspectrofluorimeter, frequency domain phase/modulation technique in 1 to 200 MHz frequency region) yielded two spectral components of 4.9 and 1.4 ns lifetime, respectively. While the former component correlates with the previously characterized effect of the fluorophore binding to biomolecular targets in membranes and/or cytoplasm, the latter component is newly observed and its possible origin is discussed.
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Oligonucleótidos/farmacocinética , Espectrometría de Fluorescencia/métodos , Anfotericina B/farmacología , Animales , Cationes , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Rayos Láser , Melanoma Experimental , Ratones , Microscopía Confocal , Oligonucleótidos Antisentido/farmacocinéticaRESUMEN
Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C.
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Lisina/química , Oligonucleótidos/química , Oligopéptidos/química , Tionucleótidos/química , Transporte Biológico , Dicroismo Circular , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Leucina/química , Microscopía Fluorescente , Oligonucleótidos/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Organofosfatos/química , Organofosfatos/metabolismo , Espectrometría Raman , Tionucleótidos/metabolismo , Células Tumorales CultivadasRESUMEN
This chapter provides a basic overview of most of the oligonucleotide delivery systems available for an in vitro use. Two major classes are described: systems that act through an endocytosis process (e.g., lipid-based vectors, nanoparticles, and polycations) and systems that by-pass this endocytosis process (e.g., peptides and pore-forming agents). Each technique is briefly described to allow a critical choice of the best delivery systems suitable for specific purposes in cultured cells.