Mechanisms underlying cytotoxicity induced by engineered nanomaterials: a review of in vitro studies

Engineered nanomaterials are emerging functional materials with technologically interesting properties and a wide range of promising applications, such as drug delivery devices, medical imaging and diagnostics, and various other industrial products. However, concerns have been expressed about the risks of such materials and whether they can cause adverse effects. Studies of the potential hazards of nanomaterials have been widely performed using cell models and a range of in vitro approaches. In the present review, we provide a comprehensive and critical literature overview on current in vitro toxicity test methods that have been applied to determine the mechanisms underlying the cytotoxic effects induced by the nanostructures. The small size, surface charge, hydrophobicity and high adsorption capacity of nanomaterial allow for specific interactions within cell membrane and subcellular organelles, which in turn could lead to cytotoxicity through a range of different mechanisms. Finally, aggregating the given information on the relationships of nanomaterial cytotoxic responses with an understanding of its structure and physicochemical properties may promote the design of biologically safe nanostructures

Nanoparticles incorporating pH-responsive surfactants as a viable approach to improve the intracellular drug delivery

The pH-responsive delivery systems have brought newadvances in the field of functional nanodevices and might allow more accurate and controllable delivery of specific cargoes, which is expected to result in promising applications in different clinical therapies. Here we describe a family of chitosan TPP (tripolyphosphate) nanoparticles (NPs) for intracellular drug delivery, which were designed using two pH-sensitive amino acid-based surfactants fromthe family Nα,Nε-dioctanoyl lysine as bioactive compounds. Lowand mediummolecularweight chitosan (LMW-CS and MMW-CS, respectively) were used for NP preparation, and it was observed that the size distribution for NPs with LMW-CS were smaller (~168 nm) than that for NPs prepared with MMW-CS (~310 nm). Hemolysis assay demonstrated the pH-dependent biomembrane disruptional capability of the constructed NPs. The nanostructures incorporating the surfactants cause negligible membrane permeabilization at pH 7.4. However, at acidic pH, prevailing in endosomes, membrane-destabilizing activity in an erythrocyte lysis assay became evident. When pH decreased to 6.6 and 5.4, hemolytic capability of chitosan NPs increased along with the raise of concentration. Furthermore, studies with cell culture showed that these pH-responsive NPs displayed low cytotoxic effects against 3T3 fibroblasts. The influence of chitosan molecular weight, chitosan to TPP weight ratio, nanoparticle size and nature of the surfactant counterion on the membrane-disruptive properties of nanoparticleswas discussed in detail. Altogether, the results achieved here showed that by inserting the lysine-based amphiphiles into chitosan NPs, pH-sensitive membranolytic and potentially endosomolytic nanocarriers were developed, which, therefore, demonstrated ideal feasibility for intracellular drug delivery

Determination of Methotrexate in pH-Sensitive Chitosan Nanoparticles by Validated RP-LC and UV Spectrophotometric Methods

Nanotechnology-based drug delivery systems are in constant development and, therefore, it is of great importance to have rapid, efficient and accurate analytical methodology to quantify the encapsulated drugs. Here, simple and fast methods, by reversed-phase liquid chromatography (RP-LC) and UV spectrophotometry, were developed and validated for the determination of methotrexate (MTX) in pH-sensitive chitosan nanoparticles (CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included a surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic sodium counterion. The RP-LC method was carried out on a Waters XBridgeTM C18 column (250 mm x 4.6 mm I.D., 5μm), with mobile phase consisted of potassium phosphate buffer (0.05 M, pH 3.2): acetonitrile (86:14, v/v), and UV detection set at 303 nm. The analyses of MTX content by the UV method were also accomplished at 303 nm, using 0.1 M sodium hydroxide as diluent. The measurements were linearly correlated with concentration for both methods in the 1 - 30 μg/mL range (r > 0.9999). The specificity tests showed that there was no interference of the NP components on the quantitative analyses. Precision (repeatability and intermediate precision) was demonstrated by a relative standard deviation lower than 1.5%, whereas the accuracy was assessed by the recovery of MTX from sample matrices, given mean value of ~99%. The proposed methods were applied for the analyses of MTX in different batches of NPs, and the results showed non-significant differences (p > 0.05) between the values obtained with both methodologies. Moreover, the RP-LC method was successfully used to determine the drug entrapment efficiency, and to quantify MTX during in vitro release assays and photolytic degradation studies. In conclusion, the validated methods are suitable to assay MTX in pH-sensitive CS-NPs without any interference from the polymer or surfactant

Multifunctional PLGA nanoparticles combining transferrin-targetability and pH-stimuli sensitivity enhanced doxorubicin intracellular delivery and in vitro antineoplastic activity in MDR tumor cells

Targeted delivery aims to enhance cellular uptake and improve therapeutic outcome with higher disease specificity. The expression of transferrin receptor (TfR) is upregulated on tumor cells, which make the protein Tf and its receptor vastly relevant when applied to targeting strategies. Here, we proposed Tf-decorated pH-sensitive PLGA nanoparticles containing the chemosensitizer poloxamer as a carrier for doxorubicin delivery to tumor cells (Tf-DOX-PLGA-NPs), aiming at alleviating multidrug resistance (MDR). We performed a range of in vitro studies to assess whether targeted NPs have the ability to improve DOX antitumor potential on resistant NCI/ADR-RES cells. All evaluations of the Tf-decorated NPs were performed comparatively to the nontargeted counterparts, aiming to evidence the real role of NP surface functionalization, along with the benefits of pH-sensitivity and poloxamer, in the improvement of antiproliferative activity and reversal of MDR. Tf-DOX-PLGA-NPs induced higher number of apoptotic events and ROS generation, along with cell cycle arrest. Moreover, they were efficiently internalized by NCI/ADR-RES cells, increasing DOX intracellular accumulation, which supports the greater cell killing ability of these targeted NPs with respect to MDR cells. Altogether, these findings supported the effectiveness of the Tf-surface modification of DOX-PLGA-NPs for an improved antiproliferative activity. Therefore, our pH-responsive Tf-inspired NPs are a promising smart drug delivery system to overcome MDR effect at some extent, enhancing the efficacy of DOX antitumor therapy

Inclusion of a pH‑responsive amino acid‑based amphiphile in methotrexate‑loaded chitosan nanoparticles as a delivery strategy in cancer therapy

The encapsulation of antitumor drugs in nanosized systems with pH-sensitive behavior is a promising approach that may enhance the success of chemotherapy in many cancers. The nanocarrier dependence on pH might trigger an efficient delivery of the encapsulated drug both in the acidic extracellular environment of tumors and, especially, in the intracellular compartments through disruption of endosomal membrane. In this context, here we reported the preparation of chitosan-based nanoparticles encapsulating methotrexate as a model drug (MTX-CS-NPs), which comprises the incorporation of an amino acid-based amphiphile with pH-responsive properties (77KS) on the ionotropic complexation process. The presence of 77KS clearly gives a pH-sensitive behavior to NPs, which allowed accelerated release of MTX with decreasing pH as well as pH-dependent membrane-lytic activity. This latter performance demonstrates the potential of these NPs to facilitate cytosolic delivery of endocytosed materials. Outstandingly the cytotoxicity of MTX-loaded CS-NPs was higher than free drug to MCF-7 tumor cells and, to a lesser extent, to HeLa cells. Based on the overall results, MTX-CS-NPs modified with the pH-sensitive surfactant 77KS could be potentially useful as a carrier system for intracellular drug delivery and, thus, a promising targeting anticancer chemotherapeutic agent

Teste de Morisky-Green e Brief Medication Questionnaire para avaliar adesão a medicamentos

OBJETIVO: Analisar a confiabilidade e o desempenho da versão em português de instrumentos de avaliação da adesão ao tratamento anti-hipertensivo. MÉTODOS: Pacientes hipertensos atendidos de janeiro a setembro de 2010 em uma unidade de atenção primária em Porto Alegre, RS, foram selecionados aleatoriamente (n = 206). Na avaliação da adesão foram utilizadas versões em português do Teste de Morisky-Green (TMG) e do Brief Medication Questionnaire (BMQ). Foram analisados consistência interna, estabilidade temporal e desempenho com relação a três padrões-ouro: controle inadequado da pressão arterial (&gt; 140/90 mmHg); taxa insuficiente de retirada de medicação na farmácia da Unidade Básica de Saúde (< 80%); e a combinação de ambos. RESULTADOS: Dos pacientes avaliados, 97 utilizavam medicamentos dispensados somente pela farmácia da Unidade Básica de Saúde. Os testes apresentaram boa consistência interna: BMQ &#945; de Cronbach de 0,66 (IC95% 0,60;0,73) e o TMG 0,73 (IC95% 0,67;0,79). O desempenho do BMQ no domínio regime apresentou sensibilidade de 77%, especificidade de 58% e área sob a curva ROC de 0,70 (IC95% 0,55;0,86), e o TMG sensibilidade de 61%, especificidade de 36% e área sob a curva ROC de 0,46 (IC95% 0,30;0,62). A correlação entre o BMQ e o TMG foi de r = 0,28, p &gt; 0,001. A baixa adesão ao BMQ está associada a maiores níveis tensionais quando comparada com pacientes aderentes (148,4 [dp 20,1] vs 128,8 [dp 17,8], p < 0,001), mas não para o TMG. CONCLUSÕES: O BMQ apresentou melhor desempenho que o TMG, com maiores sensibilidade e especificidade. A avaliação da adesão pode auxiliar o clinico na discriminação entre uso inadequado da medicação e esquema terapêutico insuficiente.OBJETIVO: Analizar la confiabilidad y el desempeño de la versión en portugués de instrumentos de evaluación de la adherencia al tratamiento antihipertensivo. MÉTODOS: Pacientes hipertensos atendidos de enero a septiembre de 2010 en una unidad de atención primaria en Porto Alegre, Sur de Brasil, fueron seleccionados aleatoriamente (n=206). En la evaluación de la adherencia fueron utilizadas versiones en portugués de la Prueba de Morisky-Green (TMG) y del Brief Medication Questionnarie (BMQ). Se analizaron consistencia interna, estabilidad temporal y desempeño con relación a tres patrones-oro: control inadecuado de la presión arterial (&gt; 140/90 mmHg); tasa insuficiente de retirada de medicación en la farmacia de la Unidad Básica de Salud (< 80%) y la combinación de ambos. RESULTADOS: De los pacientes evaluados, 97 utilizaban medicamentos dispensados solamente por la farmacia de la Unidad Básica de Salud. Las pruebas presentaron buena consistencia interna: BMQ &#945; de Cronbach de 0,66 (IC95% 0,60;0,73) y el TMG 0,73 (IC95% 0,67;0,79). El desempeño del BMQ en el dominio régimen presentó sensibilidad de 77%, especificidad de 58%, y área bajo la curva ROC de 0,70 (IC95% 0,55;0,86) y el TMG sensibilidad de 61%, especificidad de 36% y área bajo la curva ROC de 0,46 (IC95% 0,30;0,62). La correlación entre el BMQ y el TMG fue de r=0,28, p&gt;0,001. La baja adherencia al BMQ está asociada a mayores niveles tensionales al compararlo con pacientes adherentes 148,4 [de 0,1] vs 128,8 [de 17,8], pOBJECTIVE: To analyze the reliability and performance of the Portuguese version of questionnaires used to evaluate adherence to hypertensive treatment. METHODS: Hypertensive patients attending a primary healthcare unit in Porto Alegre, Southern Brazil, from January to September 2010, were randomly selected (n = 206). To evaluate adherence, Portuguese versions of the Morisky-Green test (MGT) and the Brief Medication Questionnaire (BMQ) were used. The analysis considered internal consistency, temporal stability and performance compared to three gold standards, which are: inadequate control of blood pressure (BP &gt; 140/90 mmHg); insufficient rate of medication acquisition at the institution's pharmacy