Graphene oxide and central nervous system : evaluation of effects on blood brain barrier and nanotoxicological profile

Orientador: Maria Alice da Cruz HoflingTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A barreira hematoencefálica (BHE), localizada na interface sangue-cérebro, é uma estrutura dinâmica destinada a manter a homeostasia do sistema nervoso central (SNC) através da regulação altamente restritiva propiciada por barreiras físicas e moleculares, tanto paracelulares quanto transcelulares. Entretanto, a BHE também restringe a entrada de agentes terapêuticos importantes para o tratamento de desordens do SNC. Dentre as abordagens utilizadas para superar o problema, a utilização de nanomateriais é particularmente promissora. Os nanomateriais de carbono despertam grande interesse devido às extraordinárias propriedades físico-químicas que apresentam e ao fato de que a (sub)organização celular do SNC e dos nanomateriais a base de carbono compartilharem semelhanças morfológicas e funcionais. Na primeira fase deste projeto, analisamos (i) a capacidade do óxido de grafeno reduzido (rGO) modular a permeabilidade da BHE de ratos Wistar adultos, (ii) os possíveis mecanismos envolvidos neste processo, (iii) a toxicidade deste nanomaterial 15 minutos, 1 hora, 3 horas e 7 dias após administração de uma dose intravenosa única (7 mg/kg). rGO concentrou-se principalmente no tálamo e hipocampo, com pico de distribuição ocorrendo às 3 horas, como detectado por espectroscopia de massas por imagem. A presença sistêmica de rGO induziu abertura transitória da BHE no hipocampo dos animais como demonstrado em nível anatômico (infusão de azul de Evans), subcelular (microscopia eletrônica de transmissão) e molecular (expressão de proteínas da via paracelular e transcelular). Os ratos tratados com rGO mostraram-se clinicamente indistinguíveis dos animais controle injetados com veículo (água destilada). Análises hematológicas, histopatológicas (marcadores de neurônios e astrócitos), bioquímicas (avaliação nefrotoxicidade e hepatotoxicidade) e testes genotóxicos mostraram que a injeção de rGO produziu efeitos toxicológicos mínimos nos tempos avaliados. Comparando com o grupo controle, os animais tratados com rGO apresentaram diminuição nos níveis de ureia 3 horas pós-tratamento e aumento na atividade de superóxido-dismutase 1 hora e 7 dias pós-tratamento. Alterações intra-rGO-grupos foram leucocitose (rGO-1 hora vs. rGO-3 horas) e leucopenia (rGO-3 horas vs. rGO-7 dias), no entanto, nenhuma resposta inflamatória foi observada no soro e no hipocampo, região onde a BHE foi permeabilizada. Na segunda fase do projeto, foram realizados testes in vivo e in vitro com rGO funcionalizado com o polímero polietilenoglicol (PEG 6.000). A peguilação tem sido usada para melhorar as características físico-químicas dos nanomateriais. Com a concentração mais elevada (100 ug/ml), rGO não funcionalizado apresentou baixa toxicidade, enquanto rGO-PEG induziu efeitos deletérios e morte em cultura primárias de astrócitos e células endoteliais de cérebro de ratos. Corroborando os dados in vitro, houve progressiva diminuição na expressão de marcadores astrocitários (GFAP e conexina-43) e marcadores da junção de oclusão, adesão e lâmina basal (ocludina, ?-catenina e laminina) após injeção de rGO-PEG in vivo, dados estes indicativos de ruptura da BHE. A formação intracelular de espécies reativas de oxigênio in vitro e o aumento no sistema antioxidante enzimático in vivo induzido por rGO-PEG sugerem danos mediados por estresse oxidativo. Concluímos que dentro das condições experimentais utilizadas rGO, mas não rGO-PEG, pode ser uma ferramenta promissora a ser testada como veículo para entrega de fármacos que possuem difícil acesso ao SNCAbstract: The blood brain barrier (BBB), located on the blood-brain interface, is a dynamic structure destined to maintain the homeostasis of central nervous system (CNS) through the highly restrictive control afforded by physical and molecular barriers from paracellular and transcellular pathway. However, BBB also restricts the entry of important therapeutic agents for the treatment of CNS disorders. Among the approaches used to overcome this problem, the use of nanomaterials is particularly promising. Carbon-based nanomaterials arouse great interest due to their extraordinary physicochemical properties and morphological and functional similarities shared between the (sub)cellular organisation of the CNS and carbon-based nanomaterials. In the first part of the project, we analyze (i) the ability of reduced graphene oxide (rGO) to modulate the permeability of BBB in adults Wistar rats, (ii) the possible mechanisms involved in this process, (iii) the toxicity of this nanomaterial 15 minutes, 1 hour, 3 hours and 7 days after a single intravenous administration (7 mg/kg). rGO were mainly concentrated in thalamus and hippocampus, with peak of distribution occurring at 3 hours, as detected by mass spectroscopy imaging. Systemic presence of rGO induced transient opening of the BBB in the hippocampus of animals as shown at anatomical (Evans blue dye infusion), subcellular (transmission electron microscopy) and molecular (expression of paracellular ans transcellular pathway proteins) levels. The rGO-treated rats were clinically indistinguishable from controls animals injected with vehicle (distilled water). Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO injection seemed to produce minimal toxicological effects at the time points assessed. Regarding the control group, rGO-treated animals exhibited reduction in blood urea nitrogen level 3 hours post-treatment and increases in superoxide dismutase activity 1 hour and 7 days post-treatment. Intra-treated groups alterations involved leukocytosis (rGO-1 hour vs. rGO-3 hours) and leukopenia (rGO-3 hours vs. rGO-7 days), nevertheless, no inflammatory response was induced in the serum and hippocampus, the permeabilized region of BBB. In the second part of the project, in vivo and in vitro tests were carried out using rGO functionalized with polyethylene glycol (PEG 6,000). The PEGylation has been used to improve the physicochemical characteristics of nanomaterials. Using the highest concentration (100 µg/ml), non-functionalized rGO showed low toxicity whereas rGO-PEG induced deleterious effects and death in primary culture of astrocytes and rat brain endothelial cells. Corroborating the in vitro data, there was a progressive decrease in the expression of astrocytic markers (GFAP and connexin-43), tight and adherens junctions and basal lamina (occludin, ?-catenin and laminin) markers after rGO-PEG injection in vivo, indicating BBB disruption. The formation of intracellular ROS in vitro and the increase in the enzymatic antioxidant system in vivo induced by PEGylated rGO indicated oxidative stress-mediated damage. We concluded that within the experimental conditions used rGO, but not rGO-PEG, could be a promising tool to be tested as a carrier for delivery of drugs that have difficult access to the CNSDoutoradoFarmacologiaDoutora em Farmacologia2012/24782-5, 2015/03254-9FAPES

Reduced Graphene Oxide Modified the Interdigitated Chain Electrode for an Insulin Sensor

Insulin is a key regulator in glucose homeostasis and its deficiency or alternations in the human body causes various types of diabetic disorders. In this paper, we present the development of a reduced graphene oxide (rGO) modified interdigitated chain electrode (ICE) for direct capacitive detection of insulin. The impedance properties of rGO-ICE were characterized by equivalent circuit modeling. After an electrochemical deposition of rGO on ICE, the electrode was modified with self-assembled monolayers and insulin antibodies in order to achieve insulin binding reactions. The impedance spectra and capacitances were measured with respect to the concentrations of insulin and the capacitance change (ΔC) was analyzed to quantify insulin concentration. The antibody immobilized electrode showed an increment of ΔC according to the insulin concentration in human serum ranging from 1 ng/mL to 10 µg/mL. The proposed sensor is feasible for label-free and real-time measuring of the biomarker and for point-of-care diagnosis

Reduced Graphene Oxide Modified the Interdigitated Chain Electrode for an Insulin Sensor

Insulin is a key regulator in glucose homeostasis and its deficiency or alternations in the human body causes various types of diabetic disorders. In this paper, we present the development of a reduced graphene oxide (rGO) modified interdigitated chain electrode (ICE) for direct capacitive detection of insulin. The impedance properties of rGO-ICE were characterized by equivalent circuit modeling. After an electrochemical deposition of rGO on ICE, the electrode was modified with self-assembled monolayers and insulin antibodies in order to achieve insulin binding reactions. The impedance spectra and capacitances were measured with respect to the concentrations of insulin and the capacitance change (ΔC) was analyzed to quantify insulin concentration. The antibody immobilized electrode showed an increment of ΔC according to the insulin concentration in human serum ranging from 1 ng/mL to 10 µg/mL. The proposed sensor is feasible for label-free and real-time measuring of the biomarker and for point-of-care diagnosis

Development of a low-cost graphene-based impedance biosensor

PhD ThesisThe current applicability and accuracy of point-of-care devices is limited, with the need of future technologies to simultaneously target multiple analytes in complex human samples. Graphene’s discovery has provided a valuable opportunity towards the development of high performance biosensors. The quality and surface properties of graphene devices are critical for biosensing applications with a preferred low contact resistance interface between metal and graphene. However, each graphene production method currently results in inconsistent properties, quality and defects thus limiting its application towards mass production. Also, post-production processing, patterning and conventional lithography-based contact deposition negatively impact graphene properties due to chemical contamination. The work of this thesis focuses on the development of fully-functional, label-free graphene-based biosensors and a proof-of-concept was established for the detection of prostate specific antigen (PSA) in aqueous solution using graphene platforms. Extensive work was carried out to characterize different graphene family nanomaterials in order to understand their potential for biosensing applications. Two graphene materials, obtained via a laser reduction process, were selected for further investigations: reduced graphene oxide (rGO) and laser induced graphene from polyimide (LIG). Electrically conductive, porous and chemically active to an extent, these materials offer the advantage of simultaneous production and patterning as capacitive biosensing structures, i.e. interdigitated electrode arrays (IDE). Aiming to enhance the sensitivity of these biosensors, a novel, radio-frequency (RF) detection method was investigated and compared with conventional electrochemical impedance spectroscopy (EIS) on a well-known biocompatible material: gold (standard). It was shown that the RF detection methods require careful design and testing setup, with conventional EIS performing better in the given conditions. The method was further used on rGO and LIG IDE devices for the electrochemical impedance detection of PSA to assess the feasibility of the graphene based materials as biosensors. The graphene-based materials were successfully functionalized via the available carboxylic groups, using the EDC-NHS chemistry. Despite the difficulty of producing reproducible graphene-based electrodes, highly required for biosensor development, extensive testing was carried out to understand their feasibility. The calibration curves obtained via successive PSA addition showed a moderate-to-high ii sensitivity of both rGO and LIG IDE. However, further adsorption and drift testing underlined some major limitations in the case of LIG, due to its complex morphology and large porosity. To enable low contact resistance to these biosensors, the electroless nickel coating process is shown to be compatible with various graphene-based materials. This was demonstrated by tuning the chemical nickel bath and method conditions for pristine graphene and rGO for nickel contacts deposition