Cell-responsive nanogels for anticancer drug delivery

One of the main goals in Nanomedicine is to create innovative drug delivery systems (DDS) capable of delivering drugs into a specific location with high efficiency. In the development of DDS, some essential properties are desired, such as biocompatibility and biodegradability. Furthermore, an ideal DDS should be able to deliver a drug in a controlled manner and minimize its side effects. These two objectives are still a challenge for researchers all around the world. Nanogels are an excellent vehicle to use in drug delivery and several other applications due to their biocompatibility. They are polymer-based networks, chemically or physically crosslinked, with at least 80-90% water in their composition. Their properties can be tuned, like the nanogel size, multifunctionality and degradability. Nanogels are capable of carrying in their interior bioactive molecules and deliver them into cells. The main objective of this project was to produce nanogels for the delivery of anticancer drugs with the ability of responding to existent stimuli inside cells (cellresponsiveness nanogels) and/or of controlled drug delivery. The nanogels were mainly based on alginate (AG), a natural biopolymer, and prepared using emulsion approaches. After their synthesis, they were used to encapsulate doxorubicin (Dox) which was chosen as a model drug. In the first part of the experimental work, disulfide-linked AG nanogels were prepared and, as expected, were redox-sensitive to a reducing environment like the intracellular medium. In the second part, AG nanogels crosslinked with both calcium ions and cationic poly(amidoamine) dendrimers were developed with improved sustained drug delivery. The prepared nanogels were characterized in terms of size, chemical composition, morphology, and drug delivery behavior (under redox/pH stimuli). The in vitro cytotoxicity of the nanogels was also tested against CAL-72 cells (an osteosarcoma cell line).

Análises Estruturais e atividades biológicas de exopolissacarídeo extraído do fungo comestível pleurotus Sajor-Caju e de seu derivado sulfatado quimicamente.

The exopolysaccharides are extracellular compounds produced by some species of fungi and bacteria. It is suggested that these molecules, even when in the form of complex polysaccharide-peptide, are the main bioactive molecules of many fungus. Some of the biological activities displayed by these compounds can be accentuated and others may arise when you add chemically polar or nonpolar groups to polysaccharides. The fruiting body of Pleurotus sajor-caju produces a heteropolysaccharide with antineoplastic and antimicrobial activity, but other biological activities of this polymer have not been evaluated. In this work the exopolysaccharide of Pleurotus sajor-caju was sulfated chemically and structurally characterized. We also evaluated the antiproliferative, antioxidant and anticoagulant activities from native exopolysaccharide (PN) and its sulfated derivated (PS). Polyacrylamide gel electrophoresis, infrared spectroscopy and nuclear magnetic resonance (¹³C) proved successful in sulfation of PN to obtain PS. Analysis by gas chromatography-mass spectroscopy showed that PN and PS are composed of mannose, galactose, 3-O-methyl-galactose and glucose in proportion percentage of 44,9:16,3:19,8:19 and 49, 7:14,4:17,7:18,2, respectively. The percentage of sulfate found in PS was 22.5%. Antioxidants assays revealed that the sulfation procedure affects differently the activities of exopolysaccharides, while the total antioxidant capacity, the scavenging activity of superoxide radical and ferric chelating were not affected by sulfation, on the other hand the chemical modification of PN enhanced the scavenging activity of hydroxyl radical and reducing power. PS also showed anticoagulant activity in a dose-dependent manner and clotting time was 3.0 times higher than the baseline value in APTT at 2 mg/mL. The exopolysaccharide not presented antiproliferative activity against HeLa tumor cells, but PS affects the cellular proliferation in a time-dependent manner. After 72 h, the inhibition rate of PS (2.0 mg/mL) on HeLa cells was about 60%. The results showed that PN sulfation increase some of their activities.

Avaliação das atividades antioxidantes e citotóxicas de extratos ricos em polissacarídeos extraídos das hastes de mandacaru (Cereus Jamacaru de Candolle, Cactaceae)

The use of medicinal plants to cure and treat various diseases is a common practice in the world and in Brazil. In several regions of the Brazil´s Northeast, the cactus Cereus jamacaru, known as mandacaru, is used popularly as a treatment to many diseases, including those related to heart respiratory diseases, gastric ulcers, scurvy, and kidney diseases. However, there is a scarcity in the scientific literature that proves scientifically the popular application of this cactus. Like other plants, Cereus jamacaru synthesizes several potentially bioactive molecules, like as polysaccharides. In this work, three polysaccharides-rich aqueous extracts, MCA80, MPM and MCP60, were obtained from this plant and analyzed chemically, as well as their cytotoxic and antioxidant potential. The data showed that all extracts consist mainly of polysaccharides (89.42 to 95.76%), but also protein (> 2%) and phenolic (3 to 8.87%) contaminants were detected. All extracts are rich in galactose, glucose and mannose. In addition, glucuronic acid was found in MCA80 and MCP60. The extracts showed total antioxidant capacity ranged from 55.21 to 68.13 of ascorbic acid equivalents (AAE). Besides, they exhibited reducer power and cupric chelation in a dose-dependent manner. None of the extracts inhibited the MTT reduction in the presence of prostate tumor cells (PC-3). However, MCP60 was the most effective extract by preventing the reduction of MTT by about 80% in the presence of cells 786. Nuclear fragmentation tests showed that this extract induces cell death. The data indicated that mandacaru synthesizes bioactive polysaccharides with potential as antioxidant and antitumor agents. For future studies, it is intended to purify and characterize these polysaccharides and its antioxidant and antitumor mechanisms

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.

Immunomodulatory effects of tick saliva

Ticks are bloodsucking ectoparasites that cause great damage to host organisms, so these ectoparasites are of great importance in medicine and veterinary medicine. All the biological success achieved by ticks is due to the action of bioactive components present in their saliva, which are synthesized by the salivary glands. These components have great diversity of functions such as enabling feeding and the permanence of ectoparasites on hosts, since they modulate immune system acting as complement inhibitors, immunosuppressors, cytokine expression modulator and chemokine binders of hosts. In addition, these components are an important source of protective antigens. In this sense, salivary glands/saliva are considered a potential source of multifunctional molecules. In this context, many studies have been conducted aiming at searching to establish a better understanding on the biology and morphophysiology of some organs such as salivary glands, as well as elucidate the complex relationship of these ectoparasites with their hosts. Such studies are conducted with the main objective of developing new immunobiological products aimed at the alternative control of ticks, as well as for the identification and isolation of bioactive molecules with pharmacological properties and with great therapeutic potential in the search for treatments for some diseases.

Geranylation of benzoic acid derivatives by enzymatic extracts from Piper crassinervium (Piperaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-Candida properties of urauchimycins from actinobacteria associated with Trachymyrmex ants

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation of new molecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexplored environments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated from workers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed that seven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition of Candida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated the rare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medically important Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited the growth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Our results concur with the predictions that the attine ant-microbe symbiosis may be a source of bioactive metabolites for biotechnology and medical applications. © 2013 Thais D. Mendes et al.

Utilização de células de linhagem odontoblástica (MDPC-23) em estudos de citotoxicidade e na avaliação do efeito de fatores de crescimento sobre a expressão de genes específicos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Síntese e funcionalização de superfícies de óxido de ferro superparamagnéticos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo do mecanismo do nitrofeniletano na prostaglandina-endoperóxido sintase e relação estrutura-propriedade de nitroderivados

Estudos anteriores sugerem que o óleo essencial da Aniba canelilla contendo altos níveis de 1-nitro-2-feniletano apresenta atividade antinociceptiva, antiinflamatória e hipotensora. Um estudo teórico de correlação entre estrutura química versus atividade biológica do 1-nitro-2-feniletano e a prostaglandinaendoperóxido sintase foi realizado. Além disso, foi feita uma análise conformacional do nitrofeniletano, bem como modificações em sua estrutura química, obtendo-se derivados nitroestirenos e cinamatos, para a posterior avaliação da relação estrutura-propriedade. A análise conformacional do nitrofeniletano foi realizada usando o método DFT/B3LYP com o conjunto de base 6-31G(d) através da variação da torção do angulo dihedro entre as ligações dos carbonos C₁-C₂ ligadas aos grupos fenila e nitro, com variação de intervalos de 5°, na faixa de 0° a 180°, visando a busca dos confôrmeros de menor energia. O confôrmero de 180º do dihedro O₂N-C₁-C₂-C₆H₅ foi significativamente mais estável e isto pode ser explicado em termos do equilíbrio pela presença de sete geometrias de mais baixas energias. A análise de HOMO e LUMO mostra que os grupamentos fenila e metileno contribuem para a formação deste orbital. O cálculo de energia e determinação de propriedades eletrônicas relacionadas com o mecanismo de interação com a ciclooxigenase demonstrou boa correlação entre os grupos nitro e carboxílico, os quais podem ser classificados como bioisósteros. Os resultados das propriedades físico-químicas calculadas indicam uma maior correlação do nitrofeniletano com o ácido acetilsalicílico, podendo ter seu mecanismo de ação relacionado através de interações com a Arg-120 e Ser-530.Cálculos realizados com a guanidina protonada pelo DFT/B3LYP/6-31G(d,p) parao complexo contendo o grupo carboxilato exibem uma energia de interação mais favorável, com valor de -136,34 kcal/mol, quando comparado com a molécula contendo o grupo nitro, com valor de -12,84 kcal/mol, o que pode explicar a maior efetividade da indometacina, semelhante aos resultados biológicos. Os derivados do nitrofeniletano apresentaram uma relação estrutural com derivados de cinamatos, diferindo nas propriedades redox, o que pode favorecer o desenvolvimento de estruturas protótipo como moléculas bioativas a partir destes esqueletos moleculares.

Avaliação antifungica, farmacognóstica e toxicológica sazonal de Petiveria alliacea L. (Phytolaccaceae)

O estudo das plantas medicinais desperta grande interesse científico, principalmente devido às mesmas serem consideradas fontes potenciais de moléculas bioativas com estrutura diferenciada e mecanismo de ação inovador. A importância de pesquisas voltadas para a descoberta e produção de novos fitoterápicos deve-se a grande contribuição que estes vêm apresentando diante de diversas patologias. A espécie Petiveria alliacea é uma planta medicinal utilizada amplamente pela população da região amazônica e destaca-se por apresentar diversas alegações de uso e ainda algumas classes de metabólitos com comprovadas ações terapêuticas. O presente trabalho teve o objetivo de avaliar os parâmetros farmacognósticos sazonais da espécie, o potencial antifúngico dos extratos produzidos em diferentes períodos de coleta sobre espécies de Aspergillus e a toxicidade dos mesmos in vitro e in vivo. Na avaliação farmacognóstica sazonal de P. alliacea, utilizando métodos descritos na Farmacopéia Brasileira, os resultados demonstraram parâmetros reprodutíveis para o controle de qualidade da droga vegetal, não havendo diferença na presença dos constituintes químicos do pó e do extrato hidroalcoólico, sendo observada a presença de saponinas, acúcares e alcaloides em toda a planta e nos extratos da raiz apenas sesquiterpenolactonas e depsídeos/depsidonas. Os resultados do método da microdiluição realizadas com extratos das raízes de dois períodos, evidenciaram fraca atividade antifúngica in vitro, porém não foi observado nenhum efeito dos extratos das partes aéreas. A atividade citotóxica, avaliada pelo método colorimétrico MTT, demonstrou que o extrato hidroalcóolico da raiz dos dois períodos não reduz a viabilidade celular em nenhuma das concentrações testadas. Também não foram detectados sinais de toxicidade aguda do extrato na dose de 5000mg/kg em camundongos. Estes dados são considerados relevantes e o estudo em questão evidenciou que P. alliacea é uma espécie medicinal promissora, porém investigações mais detalhadas são necessárias para que sejam confirmadas suas várias alegações de uso e para que a planta seja utilizada no desenvolvimento de um novo agente fitoterápico.

Caracterização e avaliação da citocompatibilidade de nanocompositos baseados em celulose bacteriana e fibroína para aplicação em regenração tecidual

Pós-graduação em Ciências Odontológicas - FOAR

Tissue-engineering-based Strategies for Regenerative Endodontics

Stemming from in vitro and in vivo pre-clinical and human models, tissue-engineering-based strategies continue to demonstrate great potential for the regeneration of the pulp-dentin complex, particularly in necrotic, immature permanent teeth. Nanofibrous scaffolds, which closely resemble the native extracellular matrix, have been successfully synthesized by various techniques, including but not limited to electrospinning. A common goal in scaffold synthesis has been the notion of promoting cell guidance through the careful design and use of a collection of biochemical and physical cues capable of governing and stimulating specific events at the cellular and tissue levels. The latest advances in processing technologies allow for the fabrication of scaffolds where selected bioactive molecules can be delivered locally, thus increasing the possibilities for clinical success. Though electrospun scaffolds have not yet been tested in vivo in either human or animal pulpless models in immature permanent teeth, recent studies have highlighted their regenerative potential both from an in vitro and in vivo (i.e., subcutaneous model) standpoint. Possible applications for these bioactive scaffolds continue to evolve, with significant prospects related to the regeneration of both dentin and pulp tissue and, more recently, to root canal disinfection. Nonetheless, no single implantable scaffold can consistently guide the coordinated growth and development of the multiple tissue types involved in the functional regeneration of the pulp-dentin complex. The purpose of this review is to provide a comprehensive perspective on the latest discoveries related to the use of scaffolds and/or stem cells in regenerative endodontics. The authors focused this review on bioactive nanofibrous scaffolds, injectable scaffolds and stem cells, and pre-clinical findings using stem-cell-based strategies. These topics are discussed in detail in an attempt to provide future direction and to shed light on their potential translation to clinical settings.