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Tissue engineering scaffolds have been dedicated to regenerating damaged tissue by serving as host biomaterials for cell adhesion, growth, differentiation, and proliferation to develop new tissue. In this work, the design and fabrication of a biodegradable bilayer scaffold consisting of a ternary PLLA/PCL/CAB blend film layer and a PLGA/curcumin (CC) electrospun fiber layer were studied and discussed in terms of surface morphology, tensile mechanical properties, and molecular interactions. Three different compositions of PLLA/PCL/CAB-60/15/25 (TBF1), 75/10/15 (TBF2), and 85/5/10 (TBF3)-were fabricated using the solvent casting method. The electrospun fibers of PLGA/CC were fabricated using chloroform (CF) and dimethylformamide (DMF) co-solvents in 50:50 and 60:40 volume ratios. Spherical patterns of varying sizes were observed on the surfaces of all blend films-TBF1 (17-21 µm) > TBF2 (5-9 µm) > TBF3 (1-5 µm)-caused by heterogeneous surfaces inducing bubble nucleation. The TBF1, TBF2, and TBF3 films showed tensile elongation at break values of approximately 170%, 94%, and 43%, respectively. The PLGA/CC electrospun fibers fabricated using 50:50 CF:DMF had diameters ranging from 100 to 400 nm, which were larger than those of the PLGA fibers (50-200 nm). In contrast, the PLGA/CC electrospun fibers fabricated using 60:40 CF:DMF had diameters mostly ranging from 200 to 700 nm, which were larger than those of PLGA fibers (200-500 nm). Molecular interactions via hydrogen bonding were observed between PLGA and CC. The surface morphology of the bilayer scaffold demonstrated adhesion between these two solid surfaces resembling "thread stitches" promoted by hydrophobic interactions, hydrogen bonding, and surface roughness.
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Traditional wound dressings have not been able to satisfy the needs of the regenerative medicine biomedical area. With the aim of improving tissue regeneration, nanofiber-based wound dressings fabricated by electrospinning (ES) processes have emerged as a powerful approach. Nowadays, nanofiber-based bioactive dressings are mainly developed with a combination of natural and synthetic polymers, such as polycaprolactone (PCL) and chitosan (CHI). Accordingly, herein, PCL/CHI nanofibers have been developed with varying PCL:CHI weight ratios (9:1, 8:2 and 7:3) or CHI viscosities (20, 100 and 600 mPa·s) using a novel alternating current ES (ACES) process. Such nanofibers were thoroughly characterized by determining physicochemical and nanomechanical properties, along with wettability, absorption capacity and hydrolytic plus enzymatic stability. Furthermore, PCL/CHI nanofiber biological safety was validated in terms of cytocompatibility and hemocompatibility (hemolysis < 2%), in addition to a notable antibacterial performance (bacterial reductions of 99.90% for S. aureus and 99.91% for P. aeruginosa). Lastly, the enhanced wound healing activity of PCL/CHI nanofibers was confirmed thanks to their ability to remarkably promote cell proliferation, which make them ideal candidates for long-term applications such as wound dressings.
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A chronic nonhealing wound poses a significant risk for infection and subsequent health complications, potentially endangering the patient's well-being. Therefore, effective wound dressings must meet several crucial criteria, including: (1) eliminating bacterial pathogen growth within the wound, (2) forming a barrier against airborne microbes, (3) promoting cell proliferation, (4) facilitating tissue repair. In this study, we synthesized 8 ± 3 nm Ag NP with maleic acid and incorporated them into an electrospun polycaprolactone (PCL) matrix with 1.6 and 3.4 µm fiber sizes. The Ag NPs were anchored to the matrix via electrospraying water-soluble poly(vinyl) alcohol (PVA), reducing the average sphere size from 750 to 610 nm in the presence of Ag NPs. Increasing the electrospraying time of Ag NP-treated PVA spheres demonstrated a more pronounced antibacterial effect. The resultant silver-based material exhibited 100% inhibition of gram-negative Escherichia coli and gram-positive Staphylococcus aureus growth within 6 h while showing non-cytotoxic effects on the Vero cell line. We mainly discuss the preparation method aspects of the membrane, its antibacterial properties, and cytotoxicity, suggesting that combining these processes holds promise for various medical applications.
Assuntos
Antibacterianos , Materiais Biocompatíveis , Escherichia coli , Poliésteres , Álcool de Polivinil , Prata , Staphylococcus aureus , Álcool de Polivinil/química , Álcool de Polivinil/farmacologia , Prata/química , Prata/farmacologia , Poliésteres/química , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Células Vero , Animais , Chlorocebus aethiops , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Nanopartículas Metálicas/química , Alicerces Teciduais/química , Testes de Sensibilidade MicrobianaRESUMO
Orally dispersible films (ODFs) prepared by an electrospinning are a novel type of pharmaceutical formulation. This dosage form has the potential to be beneficial for small children and the elderly, who can have problems with administration of classical tablets due to the increased risk of choking and difficulty with swallowing. Due to the highly porous nanofiber morphology, the ODFs examined in this study achieve rapid disintegration into drug microparticles when in contact with saliva. The suspension is then easier to swallow. In this study, we focus on the impact of film composition (polymer matrix composition) on the properties of electrospun membranes. In particular, we prepared ODFs composed of a mixture of PEG 100 000 with HPMC E5 and PVP k90 with HPMC E5. We found significant differences in the structure of electrospinned membranes, where samples containing PEG 100 000 and HPMC E5 exhibited much narrower distribution of fibers. Furthermore, nanofibers containing PVP k90 exhibit a faster disintegration rate, while dissolution of the drug was faster in the case of PEG 100 000 containing ODFs. The improvement was caused by both the structure and composition of the membranes.
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Sistemas de Liberação de Medicamentos , Polímeros , Criança , Humanos , Idoso , Polímeros/química , Solubilidade , Composição de Medicamentos , ComprimidosRESUMO
The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no vasa vasorum were found to be present. Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.
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The purpose of this work is to explore the preparation of nanofibrous orally dispersible films (ODFs) by needleless electrospinning from the active pharmaceutical ingredient (API) Tadalafil using particles suspended in a solution of polymers and other excipients. The prepared films were characterized by a combination of scanning electron microscopy, mechanical tests, measurements of the disintegration time and dissolution characteristic, X-ray diffraction, and differential scanning calorimetry. Furthermore, we investigated the impact of lamination pressures in the range of 0 to 5 bars combined with films at various relative humidity values on the mechanical properties of the ODF. An increase in lamination pressure resulted in higher Young's modulus values, with the maximum value observed for a sample laminated at a pressure of 5 bar and the maximum stress and strain of the prepared ODF at a lamination pressure of 1.2 bar. Moreover, there was a significant increase in the disintegration time with increase in lamination pressure. The disintegration time ranged from 0.35 s for non-laminated samples to 12 s for samples laminated at a pressure of 5 bar. On the contrary, the lamination pressure did not reveal to have any impact on the dissolution kinetics. These results confirmed that the lamination pressure can improve the processability of ODFs without affecting the API dissolution kinetics.
Assuntos
Química Farmacêutica , Nanofibras , Varredura Diferencial de Calorimetria , Química Farmacêutica/métodos , Excipientes/química , SolubilidadeRESUMO
This paper aims at an in-depth and comprehensive analysis of mechanical and microstructural properties of AISI 316L austenitic stainless steel (W. Nr. 1.4404, CL20ES) produced by laser powder bed fusion (LPBF) additive manufacturing (AM) technology. The experiment in its first part includes an extensive study of the anisotropy of mechanical and microstructural properties in relation to the built orientation and the direction of loading, which showed significant differences in tensile properties among samples. The second part of the experiment is devoted to the influence of the process parameter focus level (FL) on mechanical properties, where a 48% increase in notched toughness was recorded when the level of laser focus was identical to the level of melting. The FL parameter is not normally considered a process parameter; however, it can be intentionally changed in the service settings of the machine or by incorrect machine repair and maintenance. Evaluation of mechanical and microstructural properties was performed using the tensile test, Charpy impact test, Brinell hardness measurement, microhardness matrix measurement, porosity analysis, scanning electron microscopy (SEM), and optical microscopy. Across the whole spectrum of samples, performed analysis confirmed the high quality of LPBF additive manufactured material, which can be compared with conventionally produced material. A very low level of porosity in the range of 0.036 to 0.103% was found. Microstructural investigation of solution annealed (1070 °C) tensile test samples showed an outstanding tendency to recrystallization, grain polygonization, annealing twins formation, and even distribution of carbides in solid solution.
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Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.
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Antibacterianos/administração & dosagem , Bandagens , Nanofibras , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Cicatrização/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , CamundongosRESUMO
We have prepared a candidate biocompatible construct for skin wound healing based on electrospun polycaprolactone (PCL) nanofibrous membranes. The membrane material was loaded either with L-arginine or with alaptide, or with a mixture of both bioactive components. Alaptide is a spirocyclic synthetic dipeptide, an analogue of melanocyte-stimulating hormone release-inhibiting factor. L-arginine is an amino acid with a basic guanidine side chain. It is a direct precursor of nitric oxide, which plays a pivotal role in skin repair. The presence and the distribution of the additives were proved with high-performance liquid chromatography, Fourier-transform infrared spectroscopy and Raman spectroscopy. The influence of L-arginine and alaptide on the morphology of the membrane was characterized using scanning electron microscopy. No statistically significant correlation between fiber diameter and drug concentration was observed. The membranes were then tested in vitro for their cytotoxicity, using primary human dermal fibroblasts, in order to obtain the optimal concentrations of the additives for in vivo tests in a rat model. The membranes with the highest concentration of L-arginine (10 wt. %) proved to be cytotoxic. The membranes with alaptide in concentrations from 0.1 to 2.5 wt.%, and with the other L-arginine concentrations (1 and 5 wt.%), did not show high toxicity. In addition, there was no observed improvement in cell proliferation on the membranes. The in vivo experiments revealed that membranes with 1.5 wt.% of alaptide or with 1.5 wt.% of alaptide in combination with 5 wt.% of L-arginine markedly accelerated the healing of skin incisions, and particularly the healing of skin burns, i.e. wounds of relatively large extent. These results indicate that our newly-developed nanofibrous membranes are promising for treating wounds with large damaged areas, where a supporting material is needed.
Assuntos
Arginina/química , Materiais Biocompatíveis/química , Nanofibras/química , Peptídeos/química , Cicatrização/efeitos dos fármacos , Animais , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Sistemas de Liberação de Medicamentos , Eletroquímica , Eletrodos , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia de Fluorescência , Neuropeptídeos/química , Peptídeos Cíclicos/química , Ratos , Ratos Wistar , Pele/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Polycaprolactone (PCL) was electrospun with the addition of arginine (Arg), an α-amino acid that accelerates the healing process. The efficient needleless electrospinning technique was used for the fabrication of the nanofibrous layers. The materials produced consisted mainly of fibers with diameters of between 200 and 400 nm. Moreover, both microfibers and beads were present within the layers. Higher bead sizes were observed with the increased addition of arginine. The arginine content within the layers as well as the weight of the resultant electrospun materials were enhanced with the increased addition of arginine to the electrospinning solution (1, 5 and 10 wt%). The PCL + 1% Arg nanofibrous layer contained 5.67 ± 0.04% of arginine, the PCL + 5% Arg layer 22.66 ± 0.24% of arginine and the PCL + 10% Arg layer 37.33 ± 0.39% of arginine according to the results of the elemental analysis. A high burst release within 5 h of soaking was recorded for the PCL + 5% and PCL + 10% nanofibrous layers. However, the release rate of arginine from the PCL + 1% Arg was significantly slower, reaching a maximum level after 72 h of soaking. The resulting materials were hydrophobic. Hemocompatibility testing under static conditions revealed no effect on hemolysis following the addition of arginine and the prolongation of the prothrombin time with the increased addition of arginine, thus exerting an influence on the extrinsic and common pathway of coagulation activation. The results of the dynamic hemocompatibility assessment revealed that the numbers of blood cells and platelets were not affected significantly by the various electrospun samples during incubation. The TAT, ß-thromboglobulin and SC5-b9 concentrations were characterized by a moderate increase in the PCL group compared to those of the control group. The presence of arginine resulted in a decrease in the investigated hemocompatibility markers. The PMN elastase levels were comparable with respect to all the groups.
Assuntos
Arginina/química , Hemólise , Teste de Materiais/métodos , Poliésteres/química , Alicerces Teciduais/química , Cicatrização , Materiais Biocompatíveis/química , Eletricidade , Humanos , Nanofibras/química , Tempo de Protrombina , Engenharia TecidualRESUMO
Polymer-metal based material with unique 3D structure is an attractive substrate for the development of biomedical applications. A novel preparation of the composite from polymer fibres and silver nanoparticles has been designed through: (1) preparation of silver nanoparticles by phytosynthesis and (2) incorporation of these nanoparticles in a fibrous membrane prepared by electrospinning. The nanoparticle biosynthesis was performed in a pure environmental-friendly, easy, static, bottom-up in vitro regime using Tilia sp. leachate. TEM and XRD depict the formation, stabilisation and encapsulation of crystalline silver (14 ± 9 nm) nanoparticles (NPs) in one simple step with low tendency to aggregate. We achieved successful incorporation in the uniform electrospun 221 ± 24 nm poly(vinylalcohol) fibres, and this confirms the possibility of its use in the biomedical field. Both SEM with EDX and TEM analysis determined fibre uniformity with the presence of silver NPs, and ICP-AES confirmed the relatively similar metal concentration throughout the triplicate measurement of fibre structures on the 2 × 2 cm area in the following manner: 0.303 ± 0.018 wt. %, 0.282 ± 0.017 wt. %, and 0.281 ± 0.017 wt. %. Our hypothesis is based on previously verified preparation of active silver NPs and the easily prepared PVA electrospun fibres which act as a water soluble matrix. The simple methodology of incorporating biosynthetically prepared NPs in the PVA fibers highlights the effectiveness of this material, with simple release from water-soluble PVA and final activation of the prepared NPs.
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Nanotechnologies allow the production of yarns containing nanofibres for use in composites, membranes and biomedical materials. Composite yarns with a conventional thread core for mechanical strength and a nanofibrous envelope for functionality, e.g. biological, catalytic, have many advantages. Until now, the production of such yarns has been technologically difficult. Here, we show an approach to composite yarn production whereby a plume of nanofibers generated by high throughput AC needleless and collectorless electrospinning is wound around a classic thread. In the resulting yarn, nanofibres can form up to 80% of its weight. Our yarn production speed was 10 m/min; testing showed this can be increased to 60 m/min. After the yarn was embedded into knitwear, scanning electron microscope images revealed an intact nanofibrous envelope of the composite yarn. Our results indicate that this production method could lead to the widespread production and use of composite nanofibrous yarns on an industrial scale.
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The study involved the electrospinning of the copolymer poly(L-lactide-co-ε-caprolactone) (PLCL) into tubular grafts. The subsequent material characterization, including micro-computed tomography analysis, revealed a level of porosity of around 70%, with pore sizes of 9.34 ± 0.19 µm and fiber diameters of 5.58 ± 0.10 µm. Unlike fibrous polycaprolactone, the electrospun PLCL copolymer promoted fibroblast and endothelial cell adhesion and proliferation in vitro. Moreover, the regeneration of the vessel wall was detected following implantation and, after six months, the endothelialization of the lumen and the infiltration of arranged smooth muscle cells producing collagen was observed. However, the degradation rate was found to be accelerated in the rabbit animal model. The study was conducted under conditions that reflected the clinical requirements-the prostheses were sutured in the end-to-side fashion and the long-term end point of prosthesis healing was assessed. The regeneration of the vessel wall in terms of endothelialization, smooth cell infiltration and the presence of collagen fibers was observed after six months in vivo. A part of the grafts failed due to the rapid degradation rate of the PLCL copolymer.
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Prótese Vascular , Artérias Carótidas/patologia , Poliésteres/química , Enxerto Vascular , Células 3T3 , Animais , Aorta/patologia , Adesão Celular , Colágeno/metabolismo , Cães , Células Endoteliais , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento Tridimensional , Camundongos , Miócitos de Músculo Liso/citologia , Polímeros/química , Porosidade , Coelhos , Ratos , Regeneração , Suínos , Engenharia Tecidual/métodos , Alicerces Teciduais , Microtomografia por Raio-XRESUMO
Biodegradable polyesters, namely polycaprolactone (PCL) and copolymer of polylactide and polycaprolactone (PLCL) were electrospun into various fibrous structures and their hemocompatibility was evaluated in vitro. Firstly, hemolytic effect was evaluated upon incubation with diluted whole blood. The results showed that the degree of hemolysis depended on chemical composition and fibrous morphology. Electrospun polycaprolactone induced slight degree of hemolysis depending on its molecular weight and fibrous morphology; copolymer PLCL did not cause detectable hemolysis. The influence of coagulation pathways was examined by measurement of coagulation times. It was showed that intrinsic coagulation pathway assessed by activated partial thromboplastin time (APTT) was moderately accelerated after incubation with PCL and prolonged after incubation with copolymer PLCL. Extrinsic activation of coagulation tested by prothrombin time (PT) was slightly accelerated after incubation with all tested electrospun samples. Thrombogenicity assessment of fibrous samples revealed high thrombogenic properties of fibrous materials that was comparable to high degree of collagen thrombogenicity. The level of platelet activation was dependent on chemical composition and surface morphology of tested materials.
Assuntos
Materiais Biocompatíveis/química , Polímeros/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Células Sanguíneas/citologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Colágeno/química , Hemólise/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Tempo de Tromboplastina Parcial , Poliésteres/química , Polímeros/síntese química , Tempo de ProtrombinaRESUMO
We optimized and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of six metabolites of homocysteine metabolism: homocysteine, methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine and betaine. The detection limits for these metabolites were in the nanomolar range, and the intra- and inter-day precisions were lower than 20% of the relative standard deviations. The method was specifically designed for the determination of the intracellular concentrations of the metabolites in cultured cells. To study the role of betaine-homocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT ((BHMT) HepG2) were treated with homocysteine or with a specific inhibitor of BHMT, and metabolite levels were subsequently measured. Severely compromised methyl group metabolism in the HepG2 cells, which is typical of cancer-derived cells, prevented clear evaluation of the changes caused by the external manipulations of homocysteine metabolism. However, the ease of handling these cells and the almost unlimited source of experimental material supplied by cells in permanent culture allowed us to develop a reliable methodology. The precautions concerning intracellular metabolite determinations using LC-MS/MS in cultured cells that are expressed in this work will have global validity for future metabolomics studies.
Assuntos
Betaína-Homocisteína S-Metiltransferase/metabolismo , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Calibragem , Cromatografia Líquida , Células Hep G2 , Homocisteína/análise , Homocisteína/química , Humanos , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
Metabolism of benzene, an important environmental and industrial carcinogen, produces three electrophilic intermediates, namely, benzene oxide and 1,2- and 1,4-benzoquinone, capable of reacting with the DNA. Numerous DNA adducts formed by these metabolites in vitro have been reported in the literature, but only one of them was hitherto identified in vivo. In a search for urinary DNA adducts, specific LC-ESI-MS methods have been developed for the determination in urine of six nucleobase adducts, namely, 7-phenylguanine, 3-phenyladenine, 3-hydroxy-3,N(4) -benzethenocytosine, N(2) -(4-hydroxyphenyl)guanine, 7-(3,4-dihydroxyphenyl)guanine and 3-(3,4-dihydroxyphenyl)-adenine (DHPA), with detection limits of 200, 10, 260, 50, 400 and 200 pg ml(-1) , respectively. Mice were exposed to benzene vapors at concentrations of 900 and 1800 mg m(-3) , 6 h per day for 15 consecutive days. The only adduct detected in their urine was DHPA. It was found in eight out of 30 urine samples from the high-exposure group at concentrations of 352 ± 146 pg ml(-1) (mean ± SD; n = 8), whereas urines from the low-exposure group were negative. Assuming the DHPA concentration in the negative samples to be half of the detection limit, conversion of benzene to DHPA was estimated to 2.2 × 10(-6) % of the absorbed dose. Thus, despite the known high mutagenic and carcinogenic potential of benzene, only traces of a single DNA adduct in urine were detected. In conclusion, DHPA is an easily depurinating adduct, thus allowing indication of only high recent exposure to benzene, but not long-term damage to DNA in tissues.
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Adenina/análogos & derivados , Benzeno/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/urina , Adenina/urina , Administração por Inalação , Animais , Biomarcadores , Cromatografia Líquida de Alta Pressão , Masculino , Espectrometria de Massas , Camundongos , Padrões de Referência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We describe a method of fabrication of nanoporous flexible probes which work as artificial proboscises. The challenge of making probes with fast absorption rates and good retention capacity was addressed theoretically and experimentally. This work shows that the probe should possess two levels of pore hierarchy: nanopores are needed to enhance the capillary action and micrometer pores are required to speed up fluid transport. The model of controlled fluid absorption was verified in experiments. We also demonstrated that the artificial proboscises can be remotely controlled by electric or magnetic fields. Using an artificial proboscis, one can approach a drop of hazardous liquid, absorb it and safely deliver it to an analytical device. With these materials, the paradigm of a stationary microfluidic platform can be shifted to the flexible structures that would allow one to pack multiple microfluidic sensors into a single fiber.
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Microquímica/instrumentação , Micromanipulação/instrumentação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/instrumentação , Soluções/isolamento & purificação , Manejo de Espécimes/instrumentação , Absorção , Desenho de Equipamento , Análise de Falha de Equipamento , Micromanipulação/métodos , Tamanho da Partícula , Manejo de Espécimes/métodosRESUMO
Among numerous adducts formed by reaction of DNA with p-benzoquinone (p-BQ), an electrophilic metabolite of benzene, only N2-(4-hydroxyphenyl)guanine (N2HPG) has been confirmed in vivo. If excreted in urine N2HPG would be a candidate non-invasive biomarker of the DNA damage caused by benzene. To test this hypothesis, biotransformation of N2HPG was studied in rats. Unchanged N2HPG in urine amounted to 8.0 ± 2.2% and 9.1 ± 1.7% (mean ± SE) at the dose of 2 mg/kg excreted within 1 and 2 days after ip dosage, respectively. After acidic hydrolysis of the urine a slight but consistent increase in urinary N2HPG to 9.5 ± 3.2% and 11 ± 2.6% of dose was found within 1 and 2 days, respectively, indicating formation of hydrolysable conjugates. An oxidised metabolite was detected by LC-ESI-MS and identified by comparison with authentic standard as N2-(4-hydroxyphenyl)-8-oxoguanine (N2HPOG). Its excretion amounted to 2.7 ± 0.2% of dose and increased to 12.0 ± 2.7% of dose when N2HPOG was released from its conjugates by acidic hydrolysis. Glucuronides and sulphates of both N2HPG and N2HPOG were confirmed in urine by LC-ESI-MS and by enzymatic treatment with glucuronidase/sulphatase. These results indicate an extensive metabolism of N2HPG in vivo, which must be taken into account when considering N2HPG as a possible biomarker of exposure to benzene.
Assuntos
Benzoquinonas/metabolismo , Carcinógenos/metabolismo , Adutos de DNA/farmacocinética , Guanina/análogos & derivados , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Biomarcadores/urina , Biotransformação , Cromatografia Líquida de Alta Pressão , Adutos de DNA/química , Adutos de DNA/metabolismo , Adutos de DNA/urina , Glucuronidase/metabolismo , Glucuronídeos/química , Glucuronídeos/metabolismo , Glucuronídeos/urina , Guanina/química , Guanina/metabolismo , Guanina/farmacocinética , Guanina/urina , Concentração de Íons de Hidrogênio , Hidrólise , Masculino , Oxirredução , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Sulfatases/metabolismo , Sulfatos/química , Sulfatos/metabolismo , Sulfatos/urina , Espectrometria de Massas em TandemRESUMO
Benzetheno adducts derived from p-benzoquinone (p-BQ), a reactive metabolite of benzene, were reported to be formed by the reaction of p-BQ with DNA in vitro but have never been detected either in vivo or in experiments with living cells. Two of them, 3-hydroxy-3,N(4)-benzetheno-2'-deoxycytidine (DCBQ) and 7-hydroxy-1,N(2)-benzetheno-2'-deoxyguanosine (DGBQ), were administered to rats by single ip injections at the doses of 2 mg/kg each. The excretion of unchanged compounds DCBQ and DGBQ within 2 days amounted to 8.2 ± 1.9 and 4.5 ± 1.2% (mean ± SE) of the dose, respectively. Additionally, deribosylated metabolites of DCBQ and DGBQ, 3-hydroxy-3,N(4)-benzethenocytosine (CBQ) and 7-hydroxy-1,N(2)-benzethenoguanine (GBQ), were found amounting to 45.7 ± 10.2 and 2.9 ± 2.1% of the dose, respectively. An additional portion of CBQ and GBQ was liberated from their corresponding conjugates by acidic hydrolysis. Therefore, total recoveries of CBQ and GBQ in urine were 82.1 ± 13.5 and 11.6 ± 5.1% of the dose. To identify conjugated metabolites, DCBQ and DGBQ were administered intraperitoneally at the doses 10.5 and 11.0 mg/kg, respectively, to one animal each. Glucuronides of DCBQ, DGBQ, and GBQ as well as sulfates of DGBQ, CBQ, and GBQ were identified by ESI-LC-MS according to (M - H)(-) ions and their fragmentation. In addition, two oxygenated metabolites and their corresponding conjugates were detected for DGBQ and GBQ. One of these metabolites was identified as 2,7-dihydroxy-1,N(2)-benzethenoguanine OGBQ1. It coeluted with the product obtained by the reaction of HQ and p-BQ mixture with 8-hydroxy-2'-deoxyguanosine followed by acid hydrolysis. These findings suggest that both DCBQ and DGBQ undergo extensive biotransformation in vivo. CBQ appears to be the only p-BQ derived DNA adduct, which can be efficiently recovered from its conjugates and might be therefore useful in molecular dosimetry of benzene.