Redox-Responsive Micellar Nanoparticles from Glycosaminoglycans for CD44 Targeted Drug Delivery.

Cancer progression is associated with overexpression of various receptors at the cell surface. Among these, CD44 is known to recognize and bind specifically hyaluronan (HA) and interact with less affinity to other glycosaminoglycans (GAGs), such as chondroitin sulfate (CS). In this study, we describe a simple method to obtain micellar nanoparticles with a GAG shell (HA or CS) as potential drug delivery systems that target cancer cells overexpressing CD44. Alkanethiol was conjugated at the reducing end of the respective GAG using highly efficient oxime chemistry. The alkane moiety confers amphiphilic behavior to the obtained conjugates and triggers their self-assembly into micellar nanoparticles, while the thiol group adds redox-responsiveness to the system. The properties of the particles depend on the used GAG: HA amphiphiles form more dense, smaller assemblies that are redox sensitive. Both systems allow encapsulation of either hydrophobic or hydrophilic cargos with high efficiency. We demonstrate that the GAGs exposed on the surface of the nanoparticles are with preserved bioactivity and recognized by the cellular receptors: the particles were internalized via CD44 dependent pathways.

Evaluation of the Potential of Collagen from Codfish Skin as a Biomaterial for Biomedical Applications.

Collagen is one of the most widely used biomaterials, not only due its biocompatibility, biodegradability and weak antigenic potential, but also due to its role in the structure and function of tissues. Searching for alternative collagen sources, the aim of this study was to extract collagen from the skin of codfish, previously obtained as a by-product of fish industrial plants, and characterize it regarding its use as a biomaterial for biomedical application, according to American Society for Testing and Materials (ASTM) Guidelines. Collagen type I with a high degree of purity was obtained through acid-extraction, as confirmed by colorimetric assays, SDS-PAGE and amino acid composition. Thermal analysis revealed a denaturing temperature around 16 °C. Moreover, collagen showed a concentration-dependent effect in metabolism and on cell adhesion of lung fibroblast MRC-5 cells. In conclusion, this study shows that collagen can be obtained from marine-origin sources, while preserving its bioactivity, supporting its use in biomedical applications.

Hyaluronan Receptors as Mediators and Modulators of the Tumor Microenvironment.

The tumor microenvironment (TME) is a dynamic and complex matter shaped by heterogenous cancer and cancer-associated cells present at the tumor site. Hyaluronan (HA) is a major TME component that plays pro-tumorigenic and carcinogenic functions. These functions are mediated by different hyaladherins expressed by cancer and tumor-associated cells triggering downstream signaling pathways that determine cell fate and contribute to TME progression toward a carcinogenic state. Here, the interaction of HA is reviewed with several cell-surface hyaladherins-CD44, RHAMM, TLR2 and 4, LYVE-1, HARE, and layilin. The signaling pathways activated by these interactions and the respective response of different cell populations within the TME, and the modulation of the TME, are discussed. Potential cancer therapies via targeting these interactions are also briefly discussed.

Analysis of cardamonin by square wave voltammetry.

INTRODUCTION: Several biochemical studies have already shown that cardamonin has health promoting properties, such is in agreement with typical characteristics of chalcones. Although being a very promising compound for the nutraceutical field there is a lack of studies concerning its electroanalytical properties. OBJECTIVE: To develop an electroanalytical methodology for the quantification of cardamonin in cardamom. METHODOLOGY: Cardamonin was analysed electrochemically by means of a hanging mercury drop electrode (HMDE) using square wave voltammetry (SWV). It was extracted from cardamom spice and quantified thereafter using the standard additions method to overcome matrix effects. RESULTS: A limit of detection (LOD) of 0.15 mg/L and good linearity (r² = 0.9998) were obtained. Decoction using ethanol as the extraction solvent appears to be the simplest extraction technique. Spectrophotometric analysis (maximum absorbance peak was found in ethanol at 344 nm with a value of molar extinction coefficient of (2.8 ± 0.1) × 104 L mol⁻¹ cm⁻¹) and mass spectrometry analysis by electrospray in the positive ion mode were also performed. CONCLUSION: Cardamonin was detected voltammetrically. The LOD and limit of quantification (LOQ) of the proposed voltammetric methodology are adequate for trace analysis of this compound in several phytochemical matrices.

RHAMM expression tunes the response of breast cancer cell lines to hyaluronan.

Hyaluronan (HA) synthesis and degradation are altered during carcinogenesis leading to an increased HA content in the tumor microenvironment, which correlates with poor prognosis and treatment outcomes. The main HA receptors, CD44 and RHAMM, are also overexpressed in tumors where they activate anti-apoptotic, proliferative, invasive, and migration signaling pathways. Herein, we used a unidirectional HA gradient to investigate in a high-throughput fashion the bi-directional communication between HA and breast cancer cell lines with different surface expression of CD44 and RHAMM. We found that the expression of CD44 and RHAMM depends on the HA density: the expression of these receptors is promoted at higher HA density and RHAMM is more sensitive to these changes when compared to CD44. Blocking either CD44 or RHAMM revealed different functions on binding and recognizing HA and a compensatory expression between these two receptors that maintains protumorigenic effectors such as cortactin. STATEMENT OF SIGNIFICANCE: We show that the expression of main hyaluronan (HA) receptors CD44 and RHAMM is enhanced in a HA concentration-dependent manner. Blocking activity experiments with either RHAMM or CD44 reveal the redundancy of these two receptors towards HA recognition and activation/recruitment of protumorigenic molecular effector, cortactin. These experiments also demonstrate that cells with overexpressed RHAMM are more sensitive to HA density than CD44 positive cells. The reported results are important for the development of therapies that target the hyaluronan signaling in the tumor microenvironment.

Influence of Hyaluronan Density on the Behavior of Breast Cancer Cells with Different CD44 Expression.

Molecular gradients are common in biosystems and play an essential role in physiological and pathological processes. During carcinogenesis, for example, hyaluronan (HA) homeostasis is dysregulated by cancer cells and the altered synthesis and degradation processes result in the formation of HA gradients within the tumor microenvironment. Herein, a platform is developed to study the biological role of HA gradient in breast cancer cells. Cells with different aggressiveness and expression of CD44-the main HA receptor usually overexpressed in breast cancers, are selected for this study. The developed platform is compatible with several imaging modalities and allows assessment of cell density, morphology, CD44 expression, and cell motility in a function of HA density. Using high-throughput analysis, it is shown that cells that do not express CD44 do not change along the gradient, while CD44 positive cells respond differently to the HA gradient depending on the level of CD44 expression and HA density. This different response is associated with the activation of different signaling pathways by the CD44-HA interactions.

Hyaluronan Brush-like Copolymers Promote CD44 Declustering in Breast Cancer Cells.

We report on the synthesis of hyaluronan (HA) brush-like copolymers and their application as antagonists of tumorigenic CD44-HA interactions. HA (4.8 kDa, ca. 24 saccharides) was grafted on 2-hydrohyethyl methacrylate (HEMA) by end-on oxime ligation. The obtained copolymers were compared with low and high molecular weight HA in terms of hydrolysis kinetics in the presence of hyaluronidase (isothermal titration calorimetry) and interactions with CD44 (surface plasmon resonance). The results evidenced that the high molecular weight HA and HA-g-HEMA have a much higher affinity to CD44 than low molecular weight HA. Additionally, slower enzymatic degradation was observed for the copolymer, making it an excellent candidate for active targeting of tumorigenic CD44-HA interactions. We, therefore, investigated the effect of the copolymer on cancer cell lines with different expression of CD44 and observed an efficient declustering of CD44 that is usually associated with reduction of metastasis and drug resistance.

Liposomes embedded in layer by layer constructs as simplistic extracellular vesicles transfer model.

Extracellular vesicles (EVs) are particles originating from the exfoliation of the cellular membrane. They are involved in cell-to-cell and cell-to-matrix signaling, exchange of bioactive molecules, tumorigenesis and metastasis, among others. To mitigate the limited understanding of EVs transfer phenomena, we developed a simplistic model that mimics EVs and their interactions with cells and the extracellular matrix. The proposed model is a layer by layer (LbL) film built from the polycationic poly-l-lysine (PLL) and the glycosaminoglycan hyaluronic acid (HA) to provide ECM mimicry. Positively charged 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N1,N1,N14,N14-tetramethyl-N1,N14-ditetradecyltetradecane-1,14-diaminium dibromide (GS14) liposomes were embedded in this construct to act as EVs analogs. To simulate EVs carrying substances, Nile Red was loaded as a model of lipophilic cargo molecules. The integration of each component was followed by quartz crystal microbalance measurements, which confirmed the immobilization of intact liposomes on the underlying (PLL/HA)3 soft film. The release of Nile Red from liposomes either embedded in the LbL construct or exposed at its surface revealed a fast first order release. This system was validated as a model for EV/cell interactions by incubation with breast cancer cells MDA-MB-231. We observed higher internalization for embedded liposomes when compared with surface-exposed ones, showcasing that the ECM mimic layers do not constitute a barrier to liposome/cell interactions but favor them.

Development and characterisation of cytocompatible polyester substrates with tunable mechanical properties and degradation rate.

Although it has been repeatedly indicated the importance to develop implantable devices and cell culture substrates with tissue-specific rigidity, current commercially available products, in particular cell culture substrates, have rigidity values well above most tissues in the body. Herein, six resorbable polyester films were fabricated using compression moulding with a thermal presser into films with tailored stiffness by appropriately selecting the ratio of their building up monomers (e.g. lactide, glycolide, trimethylene carbonate, dioxanone, ε-caprolactone). Typical NMR and FTIR spectra were obtained, suggesting that the fabrication process did not have a negative effect on the conformation of the polymers. Surface roughness analysis revealed no apparent differences between the films as a function of polymer composition. Subject to polymer composition, polymeric films were obtained with glass transition temperatures from -52 °C to 61 °C; contact angles in water from 81 ° to 94 °; storage modulus from 108 MPa to 2,756 MPa and loss modulus from 8 MPa to 507 MPa (both in wet state, at 1 Hz frequency and at 37 °C); ultimate tensile strength from 8 MPa to 62 MPa, toughness from 23 MJ/m3 to 287 MJ/m3, strain at break from 3 % to 278 %, macro-scale Young's modulus from 110 MPa to 2,184 MPa (all in wet state); and nano-scale Young's modulus from 6 kPa to 15,019 kPa (in wet state). With respect to in vitro degradation in phosphate buffered saline at 37 °C, some polymeric films [e.g. poly(glycolide-lactide) 30 / 70] started degrading from day 7 (shortest timepoint assessed), whilst others [e.g. poly(glycolide-co-ε-caprolactone) 10 / 90] were more resilient to degradation up to day 21 (longest timepoint assessed). In vitro biological analysis using human dermal fibroblasts and a human monocyte cell line (THP-1) showed the potential of the polymeric films to support cell growth and controlled immune response. Evidently, the selected polymers exhibited properties suitable for a range of clinical indications.

Co-localization and crosstalk between CD44 and RHAMM depend on hyaluronan presentation.

CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM) are the main hyaluronan (HA) receptors. They are commonly overexpressed in different cancers activating signaling pathways related to tumor progression, metastasis and chemoresistance. Besides their involvement in signal transduction via interaction with HA, currently, there is a little information about the possible crosstalk between CD44 and RHAMM and the role of HA in this process. In the present work, we used immunocytochemistry combined with Förster resonance energy transfer (FRET) microscopy and co-immunoprecipitation to elucidate the involvement of HA in CD44 and RHAMM expression, co-localization and crosstalk. We studied breast cancer cells lines with different degrees of invasiveness and expression of these receptors in the absence of exogenous HA and compared the data with the results obtained for cultures supplemented with either soluble HA or seeded on substrates with end-on immobilized HA. Our results demonstrated that cells response depends on the HA presentation: CD44/RHAMM complexation was upregulated in all cell lines upon interaction with immobilized HA, but not with its soluble form. Moreover, the results showed that the expression of both CD44 and RHAMM is regulated via interactions with HA indicating cell-specific feedback loop(s) in the signaling cascade.

Prediction of paclitaxel pharmacokinetic based on in vitro studies: Interaction with membrane models and human serum albumin.

Interactions of paclitaxel (PTX) with models mimicking biological interfaces (lipid membranes and serum albumin, HSA) were investigated to test the hypothesis that the set of in vitro assays proposed can be used to predict some aspects of drug pharmacokinetics (PK). PTX membrane partitioning was studied by derivative spectrophotometry; PTX effect on membrane biophysics was evaluated by dynamic light scattering, fluorescence anisotropy, atomic force microscopy and synchrotron small/wide-angle X-ray scattering; PTX distribution/molecular orientation in membranes was assessed by steady-state/time-resolved fluorescence and computer simulations. PTX binding to HSA was studied by fluorescence quenching, derivative spectrophotometry and dynamic/electrophoretic light scattering. PTX high membrane partitioning is consistent with its efficacy crossing cellular membranes and its off-target distribution. PTX is closely located in the membrane phospholipids headgroups, also interacting with the hydrophobic chains, and causes a major distortion of the alignment of the membrane phospholipids, which, together with its fluidizing effect, justifies some of its cellular toxic effects. PTX binds strongly to HSA, which is consistent with its reduced distribution in target tissues and toxicity by bioaccumulation. In conclusion, the described set of biomimetic models and techniques has the potential for early prediction of PK issues, alerting for the required drug optimizations, potentially minimizing the number of animal tests used in the drug development process.

Probing the efficacy of a heterologous Leishmania/L. Viannia braziliensis recombinant enolase as a candidate vaccine to restrict the development of L. infantum in BALB/c mice.

In the present study, the Leishmania braziliensis enolase protein was evaluated as a vaccine candidate against visceral leishmaniasis (VL). The DNA sequence was cloned and the recombinant protein (rEnolase) was evaluated as a vaccine, associated with saponin, as an immune adjuvant. The protective efficacy of the rEnolase plus saponin combination was investigated in BALB/c mice against Leishmania infantum infection. The results revealed that the vaccine induced higher levels of IFN-γ, IL-12, and GM-CSF when a capture ELISA and flow cytometry were performed, as well as an antileishmanial nitrite production after using in vitro stimulation with rEnolase and an antigenic Leishmania preparation. The vaccinated animals, when compared to the control groups, showed a lower parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes when both a limiting dilution technique and RT-PCR assay were performed. In addition, these mice showed low levels of antileishmanial IL-4, IL-10, and anti-Leishmania IgG1 isotype antibodies. Partial protection was associated with IFN-γ production, which was mainly mediated by CD4+ T cells. In conclusion, the present study's data showed that the L. braziliensis enolase protein could be considered a vaccine candidate that offers heterologous protection against VL.

Novel application of square-wave adsorptive-stripping voltammetry for the determination of xanthohumol in spent hops.

This paper reports the development of a novel electrochemical assay for xanthohumol (XN) by square-wave adsorptive-stripping voltammetry (SWAdSV) with a hanging mercury drop electrode. The method showed good repeatability (CV < 2%) and linearity (between 10 and 250 µg L(-1)), as well as suitable limits of detection (2.6 µg L(-1)) and quantification (8.8 µg L(-1)). The method was applied for the quantification of this compound in spent hops, and the results obtained were compared with the HPLC-UV method. XN contents determined by the SWAdSV method were 16 ± 1 and 100 ± 4 µg L(-1) for aqueous and methanolic extracts, respectively. The developed new methodology considerably reduces the analysis time, approximately from 25 min (HPLC-UV method) to 7 min, enabling a high sample throughput. In addition, the detection and quantification limits were approximately 5-fold lower than those obtained with the chromatographic method.

Crianças que cuidam de irmãos com necessidades especiais / Children caretaking for siblings with special needs

Este estudo analisou situações em que um irmão/ã ajuda a cuidar de uma criança com necessidades especiais. Foram entrevistadas as mães e as crianças cuidadoras de 10 famílias de baixa renda, em Salvador-Bahia, com filhos portadores de paralisia cerebral. São detalhados os comportamentos de cuidado indicados pelas mães e pelas crianças cuidadoras, a participação das crianças em tarefas domésticas e suas atividades de lazer. Descrevem-se dificuldades e satisfação/insatisfação dos cuidadores com a tarefa, e expectativas de mães e crianças em relação ao futuro. Discute-se o grau de responsabilidade atribuído às crianças. Apontam-se limitações do estudo e algumas direções de intervenção para profissionais de saúde que atendem essas famílias.

This study analyzed situations in which a child shares part of the caretaking tasks required by a brother or sister with special needs. Mothers and child caretakers of 10 low-income families, in Salvador-Bahia, with a cerebral palsy child, were interviewed. Caretaking behaviors indicated by mothers and child caretakers, children's participation in domestic tasks, and their leisure activities are detailed. The caretakers' satisfaction/dissatisfaction and difficulties with their role, and their mother's and their own expectations for the future are described. The degree of responsibility attributed to the children is discussed. Limitations of the study and some possible directions of intervention for health professionals attending families in similar situations are pointed out.

A influência do estigma na rede social do portador de Aids / The stigma influence in the Aids hearer acquaintance net

Introdução: a síndrome da imunodeficiência adquirida carrega consigo forte estigma social, que rotula seu portador de impróprio, imoral e inaceitável socialmente. Além do portador, pessoas da rede de relações próximas ao portador também podem tornar-se alvo de estigma e rejeição por serem vistas com o portador. Objetivo: identificar como o estigma aparece no discurso de pessoas que pertencem ao grupo de relações do portador e que comparecem ao horário de visita de um hospital geral. Métodos: coleta de dados através de entrevistas que posteriormente foram analisadas. Resultados: os conteúdos derivam das disposições adquiridas junto ao grupo social a que pertença, durante o tempo longo, o tempo vivido e o tempo presente. O visitante além dos conteúdos do tempo histórico é influenciado pelo tempo vivido, ou seja, pela convivência anterior com o doente. Essa experiência fará parte do tempo vivido do visitante e do visitado. Conclusão: hoje, apesar do aumento do conhecimento sobre a aids, o paciente e sua rede de relações ainda são submetidos a práticas de isolamento.

Introduction: aids carries a strong social stigma, which labels its bearer as unsuitable, immoral and socially unacceptable. Besides the bearer, theacquaintances of the Aids bearer may also suffer from stigma and rejection once they are seen as the bearer. Objective: the present work has as a goal identify how the stigma comes up in the speech of people who belong to the acquaintance net of the bearer and who frequent the general hospital in the visit time. Methods: the data collection happened through interviews. Results: the contents derive from the dispositions acquired together with the belonging social group during the long time, lived time and present time. The visitor, besides the contents of the historic time, is influenced by the lived time, i.e, by the previous living time with the ill person. This experience will make part of the visitor?s and visited person?s lived time. Conclusion: nowadays, despite the increased knowledge on Aids, the patient and his acquaitance net are still subject to isolation practices.

Vínculos e redes sociais em contextos familiares e institucionais: uma reflexão conceitual / Bonds and social networks in family and institutional contexts: a conceptual reflection

Propõe-se uma reflexão conceitual sobre os conceitos de vínculo e rede social. São resumidos quatro trabalhos que utilizam esses conceitos e discutidas suas convergências e divergências. Carvalho sintetiza estudos sobre vínculo na interação criança-criança. Sampaio problematiza a vinculação entre crianças de rua e educadores. Rabinovich relata observações de famílias em uma comunidade quilombola. Bastos reflete sobre redes sociais com base no discurso de mães em grupos de encontro. A comparação dessas pesquisas em termos dos conceitos de vínculo e rede social indica a conveniência de explicitação dos seus sentidos em cada caso, para permitir diálogo entre enfoques diversificados sobre as relações humanas.

This paper suggests a conceptual reflection on bonds and social network concepts. Four studies that use such concepts are synthesized and their convergences and divergences are discussed. Carvalho synthesizes studies on bonds in child-child interaction. Sampaio is concerned with bonds concerning homeless children and educators. Rabinovich reports observations of families in a Quilombola community. Bastos makes reflections on social network, based on the talks of some mothers during group meetings. The comparison between the studies mentioned, in terms of both concepts, that is, bonds or linkings and social network, shows the convenience of a clear explanation in relation to the meanings in each of the cases, so that the dialogue among different approaches on human relationships can be allowed.

Se propone una reflexión conceptual sobre los conceptos de vínculo y red social. Son resumidos cuatro trabajos que utilizan esos conceptos y discutidas sus convergencias y divergencias. Carvalho sintetiza estudios sobre vínculo en la interacción niño-niño. Sampaio problematiza la vinculación entre "niños de la calle" y educadores. Rabinovich relata observaciones de familias en una comunidad quilombola. Bastos refleja sobre redes sociales basado en el discurso de madres en grupos de encuentro. La comparación de esas pesquisas en términos de los conceptos de vínculo y red social indica la conveniencia de explicitación de sus sentidos en cada caso, para permitir diálogo entre enfoques diversificados sobre las relaciones humanas.

O uso de entrevistas em estudos com crianças / Using interviews with children

Este artigo relata algumas experiências de utilização de entrevistas com crianças e reflete sobre sua utilidade e limitações. A entrevista com crianças é uma técnica ainda relativamente pouco explorada na literatura. Em nosso próprio trabalho - baseado principalmente em observação direta - entrevistas individuais ou em grupo foram utilizadas esporadicamente, a título de complementar dados observacionais, oferecer material qualitativo para exploração posterior ou responder a certos tipos de pergunta. Com base nessa experiência, estruturamos este texto em termos de situações- exemplo ou casos, dos quais alguns ressaltam aspectos problemáticos ou limitadores, e outros ilustram empregos dessa técnica que consideramos bem-sucedidos.

A família em situações disruptivas provocadas por hospitalização / The family in disruptive situations caused by hospitalization

Este trabalho focaliza a situação disruptiva provocada pela hospitalização de um familiar, vítima de doença ou acidente súbitos, requerendo uma reação de enfrentamento por parte da família. São apresentados resultados relativos a seis famílias que viveram essa situação, obtidos por meio de entrevista em profundidade com um dos familiares. Inicialmente, as famílias e as situações são caracterizadas, e são descritas as estratégias de enfrentamento identificadas em cada caso. A seguir, aprofunda-se a análise das estratégias recorrendo a semelhanças e diferenças entre os casos. Em todos os casos ocorre uma reorganização da família em resposta ao evento e às demandas decorrentes; estratégias de enfrentamento variaram, sendo a mais presente o suporte social. Sugerem-se algumas questões para estudos futuros e para a preparação de profissionais de saúde.

Vínculos afetivos na vida adulta: o visitante do paciente com AIDS / Affective bonds in adulthood: the visitor of the patient with AIDS

Compreender a experiência de visitar o paciente adulto internado com AIDS. Utilizou-se o método de representação social formulada por Spink, desenvolvida numa unidade de internação para soropositivos num hospital geral de Ribeirão Preto - SP

Carta ao professor de uma criança com câncer / Letter to the teacher of a child with cancer

Por ser o professor uma das pessoas ,mais significativas para a criança, o Grupo de Apoio à criança com câncer de Ribeiräo Preto considera extremamente importante seu envolvimento no processo terapêutico e de reabilitaçäo do aluno portador de câncer. Para isso elaborou a Carta do Professor