Targeted killing of CD133+ lung cancer stem cells using paclitaxel-loaded PLGA-PEG nanoparticles with CD133 aptamers / 南方医科大学学报

OBJECTIVE@#To construct a polylactic acid-glycolic acid-polyethylene glycol (PLGA-PEG) nanocarrier (N-Pac-CD133) coupled with a CD133 nucleic acid aptamer carrying paclitaxel for eliminating lung cancer stem cells (CSCs).@*METHODS@#Paclitaxel-loaded N-Pac-CD133 was prepared using the emulsion/solvent evaporation method and characterized. CD133+ lung CSCs were separated by magnetic bead separation and identified for their biological behaviors and gene expression profile. The efficiency of paclitaxel-loaded N-Pac-CD133 for targeted killing of lung cancer cells was assessed in vitro. SCID mice were inoculated with A549 cells and received injections of normal saline, empty nanocarrier linked with CD133 aptamer (N-CD133), paclitaxel, paclitaxel-loaded nanocarrier (N-Pac) or paclitaxel-loaded N-Pac-CD133 (n=8, 5 mg/kg paclitaxel) on days 10, 15 and 20, and the tumor weight and body weight of the mice were measured on day 40.@*RESULTS@#Paclitaxel-loaded N-Pac-CD133 showed a particle size of about 100 nm with a high encapsulation efficiency (>80%) and drug loading rate (>8%), and was capable of sustained drug release within 48 h. The CD133+ cell population in lung cancer cells showed the characteristic features of lung CSCs, including faster growth rate (30 days, P=0.001) and high expressions of tumor stem cell markers OV6(P < 0.001), CD133 (P=0.001), OCT3/4 (P=0.002), EpCAM (P=0.04), NANOG (P=0.005) and CD44 (P=0.02). Compared with N-Pac and free paclitaxel, paclitaxel-loaded N-Pac-CD133 showed significantly enhanced targeting ability and cytotoxicity against lung CSCs in vitro (P < 0.001) and significantly reduced the formation of tumor spheres (P < 0.001). In the tumor-bearing mice, paclitaxel-loaded N-Pac-CD133 showed the strongest effects in reducing the tumor mass among all the treatments (P < 0.001).@*CONCLUSION@#CD133 aptamer can promote targeted delivery of paclitaxel to allow targeted killing of CD133+ lung CSCs. N-Pac-CD133 loaded with paclitaxel may provide an effective treatment for lung cancer by targeting the lung cancer stem cells.

Synthesis, characterization, and in vitro evaluation of gamma radiationinduced PEGylated isoniazid

Background: The search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications. Results: Here, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG­INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG­INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 µM. Similar trends are observed for 0.1, 1, and 5 µM. Conclusions: In summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher antimycobacterial potency than the neat drug.

Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Effect of iodonium salt and chitosan on the physical and antibacterial properties of experimental infiltrants

Abstract Resinous infiltrants are indicated in the treatment of incipient carious lesions, and further development of these materials may contribute to greater control of these lesions. The aim of this study was to analyze the physical and antibacterial properties of experimental infiltrants containing iodonium salt and chitosan. Nine experimental infiltrants were formulated by varying the concentration of the diphenyliodonium salt (DPI) at 0, 0.5 and 1 mol%; and chitosan at 0, 0.12 and 0.25 g%. The infiltrants contained the monomeric base of triethylene glycol dimethacrylate and bisphenol-A dimethacrylate ethoxylate in a 75 and 25% proportion by weight, respectively; 0.5 mol% camphorquinone and 1 mol% ethyl 4-dimethylaminobenzoate. The degree of conversion was evaluated using Fourier transformer infrared spectroscopy, and the flexural strength and elastic modulus using the three-point bending test. Sorption and solubility in water, and antibacterial analysis (minimum inhibitory concentration and minimum bactericidal concentration) were also analyzed. Data was analyzed statistically by two-way ANOVA and Tukey's test (p<0.05), with the exception of the antibacterial test, which was evaluated by visual inspection. In general, the infiltrant group containing 0.5% DPI and 0.12% chitosan showed high values of degree of conversion, higher values of elastic modulus and flexural strength, and lower sorption values in relation to the other groups. Antibacterial activity was observed in all the groups with DPI, regardless of the concentration of chitosan. The addition of DPI and chitosan to experimental infiltrants represents a valid option for producing infiltrants with desirable physical and antibacterial characteristics.

Effects of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) release from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.

Model resin composites incorporating ZnO-NP: activity against S. mutans and physicochemical properties characterization

Abstract Although resin composites are widely used in the clinical practice, the development of recurrent caries at composite-tooth interface still remains as one of the principal shortcomings to be overcome in this field. Objectives To evaluate the activity against S. mutans biofilm of model resin composites incorporating different concentrations of ZnO-nanoparticles (ZnO-NP) and characterize their physicochemical properties. Materials and Methods Different concentrations of ZnO-NP (wt.%): E1=0, E2=0.5, E3=1, E4=2, E5=5 and E6=10 were incorporated into a model resin composite consisting of Bis-GMA-TEGDMA and barium borosilicate particles. The activity against S. mutans biofilm was evaluated by metabolic activity and lactic acid production. The following physicochemical properties were characterized: degree of conversion (DC%), flexural strength (FS), elastic modulus (EM), hardness (KHN), water sorption (Wsp), water solubility (Wsl) and translucency (TP). Results E3, E4, E5 and E6 decreased the biofilm metabolic activity and E5 and E6 decreased the lactic acid production (p<0.05). E6 presented the lowest DC% (p<0.05). No significant difference in FS and EM was found for all resin composites (p>0.05). E5 and E6 presented the lowest values of KHN (p<0.05). E6 presented a higher Wsp than E1 (p<0.05) and the highest Wsl (p<0.05). The translucency significantly decreased as the ZnO- NP concentration increased (p<0.05). Conclusions The incorporation of 2 - 5 wt.% of ZnO-NP could endow antibacterial activity to resin composites, without jeopardizing their physicochemical properties.

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells

Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

Influence of addition of [2-(methacryloyloxy)ethyl]trimethylammonium chloride to an experimental adhesive

Abstract The aim of this study was to develop an experimental adhesive with addition of [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC) and to evaluate its mechanical and biological properties and its in vitro antibacterial activity. An experimental adhesive resin was formulated with Bis-GMA, TEGDMA, and HEMA. The antibacterial monomer was added at concentrations of 1%, 2.5%, and 5% (METAC groups). A group without METAC addition was used as control. The experimental adhesives were evaluated as to their antibacterial potential against Streptococcus mutans, degree of conversion, and softening in ethanol for 2 hours. The data were analyzed by one-way ANOVA, Tukey’s post-hoc test, and the paired Student’s t-test (significance level of 0.05). METAC showed antibacterial activity against S. mutans at all concentrations (p < 0.05). There was no statistical difference across METAC groups (p > 0.05). The 1%, 2.5%, and 5% groups yielded the highest mean values for degree of conversion (p < 0.05). The 1% group did not differ from the control group (p > 0.05). There was no statistical difference in baseline microhardness values (p > 0.05) and microhardness values after immersion in ethanol were lower than at baseline for all groups (p < 0.05). There was no statistical difference in the reduction of Knoop hardness number (KHN) after immersion in ethanol for any of the groups (p > 0.05). The results of the present study indicate that METAC is a promising antibacterial agent when added to an adhesive system.

Comparative evaluation of propofol in nanoemulsion with solutol and soy lecithin for general anesthesia / Avaliação comparativa do propofol em nanoemulsão com solutol e com lecitina de soja para anestesia geral

ABSTRACT INTRODUCTION: The vehicle for propofol in 1 and 2% solutions is soybean oil emulsion 10%, which may cause pain on injection, instability of the solution and bacterial contamination. Formulations have been proposed aiming to change the vehicle and reduce these adverse reactions. OBJECTIVES: To compare the incidence of pain caused by the injection of propofol, with a hypothesis of reduction associated with nanoemulsion and the occurrence of local and systemic adverse effects with both formulations. METHOD: After approval by the CEP, patients undergoing gynecological procedures were included in this prospective study: control (n = 25) and nanoemulsion (n = 25) groups. Heart rate, noninvasive blood pressure and peripheral oxygen saturation were monitored. Demographics and physical condition were analyzed; surgical time and total volume used of propofol; local or systemic adverse effects; changes in variables monitored. A value of p < 0.05 was considered significant. RESULTS: There was no difference between groups regarding demographic data, surgical times, total volume of propofol used, arm withdrawal, pain during injection and variables monitored. There was a statistically significant difference in pain intensity at the time of induction of anesthesia, with less pain intensity in the nanoemulsion group. CONCLUSIONS: Both lipid and nanoemulsion formulations of propofol elicited pain on intravenous injection; however, the nanoemulsion solution elicited a less intense pain. Lipid and nanoemulsion propofol formulations showed neither hemodynamic changes nor adverse effects of clinical relevance.

RESUMO INTRODUÇÃO: O veículo do propofol em soluções a 1 e 2% é a emulsão de óleo de soja a 10%, que pode provocar dor à injeção, instabilidade da solução e contaminação bacteriana. Formulações foram propostas com o objetivo de alterar o veículo e reduzir essas reações adversas. OBJETIVOS: Comparar a incidência de dor à injeção do propofol com a hipótese de redução associada à nanoemulsão e a ocorrência de efeitos adversos locais e sistêmicos com as duas formulações. MÉTODO: Após aprovação pelo Conselho de Ética em Pesquisa, foram incluídos neste estudo prospectivo pacientes submetidas a procedimentos cirúrgicos ginecológicos: grupos controle (n = 25) e nanoemulsão (n = 25). Foram monitorados frequência cardíaca, pressão arterial não invasiva e saturação periférica de oxigênio. Foram analisados dados demográficos e estado físico; tempo cirúrgico e volume total usado de propofol; efeitos adversos locais ou sistêmicos; alterações nas variáveis de monitoramento. Considerou-se significativo valor de p < 0,05. RESULTADOS: Não houve diferença entre os grupos em relação a: dados demográficos, tempos cirúrgicos, volume total usado de propofol, retirada do braço, presença de dor durante a injeção e variáveis de monitoramento. Verificou-se diferença estatística significativa na intensidade da dor no momento da indução da anestesia, com menor intensidade no grupo nanoemulsão. CONCLUSÕES: Ambas as formulações de propofol, lipídica e em nanoemulsão, elicitaram dor à injeção venosa, porém a solução de nanoemulsão promoveu dor em menor intensidade. O propofol lipídico e o propofol em nanoemulsão não apresentaram alterações hemodinâmicas e efeitos adversos de relevância clínica.

Síntese e caracterização de derivados de hemoglobina para aplicação terapêutica / Synthesis and characterization of hemoglobin based oxygen carriers for therapeutic application

A transfusão de sangue é uma intervenção terapêutica capaz de salvar muitas vidas. Entretanto, transfusões também apresentam uma alta gama de possíveis eventos adversos, questões logísticas, econômicas e sociais. Dentre as principais preocupações terapêuticas estão a incompatibilidade (principalmente do sistema ABO), a transmissão de microrganismos patogênicos, os distúrbios imunomodulatórios, as reações hemolíticas, o aumento estatístico do risco de morte proporcional ao volume de sangue infundido, dentre outros. Diversas alternativas às transfusões sanguíneas são propostas na literatura científica, dentre elas o desenvolvimento de transportadores de oxigênio que utilizam a hemoglobina, comumente intitulados substitutos sanguíneos. Neste âmbito, o presente estudo teve como objetivo o desenvolvimento de uma rota de síntese e a síntese de partículas de gelatina contendo hemoglobina polimerizada. Para tanto, realizou-se a síntese do polietileno glicol bis-[succinimidil succinato], extraiu-se e polimerizou-se com glutaraldeído ou polietileno glicol bis-[succinimidil succinato] hemoglobina de sangue bovino e, partículas de gelatina coriácea ou óssea contendo hemoglobina polimerizada foram sintetizadas e caracterizadas. A síntese do polietileno glicol bis-[succinimidil succinato] (SSPEG) foi caracterizada por espectroscopia RAMAN, análise diferencial de calorimetria (DSC) e os resultados obtidos indicaram o sucesso das reações. O produto da reação de polimerização da hemoglobina e albumina com o SSPEG foi verificado por SDS-PAGE e os resultados obtidos indicaram a formação com sucesso de polímeros de alta massa molecular. As partículas contendo hemoglobina polimerizada geradas com gelatina coriácea apresentaram diâmetro hidrodinâmico de 1370 nm, dispersividade de 0,029 e potencial zeta de -36,1 mV. As partículas contendo hemoglobina polimerizada geradas com gelatina óssea apresentaram diâmetro hidrodinâmico de 438 nm, dispersividade de 0,563 e potencial zeta de -24,5 mV. Os resultados obtidos sugerem a aplicabilidade da gelatina coriácea para a produção de partículas contendo hemoglobina polimerizada com possível aplicação como transportador de oxigênio

Blood transfusion is a therapeutic intervention that can save many lives. However, transfusion is also related to several possible adverse therapeutic events and logistic, economic and social concerns. Among the major therapeutic concerns are incompatibility (mainly of the ABO group system), pathogenic microorganisms' transmission, immunomodulatory disturbances, hemolytic reactions, death risk increase that is proportional to the infused volume, among others. Several alternatives to blood transfusion are proposed in the scientific literature. Among them is the development of hemoglobin based oxygen carriers, commonly entitle blood substitutes. To this extent, the present work aimed to develop a synthetic route and to synthesize gelatin particles containing polymerized hemoglobin. To this purpose PEG bis(succinimidyl succinate) was synthesized, bovine hemoglobin was extracted and polymerized with glutaraldehyde or PEG and polyhemoglobin contained particles of gelatin from leather or bones were synthesized and characterized. PEG bis(succinimidyl succinate) synthesis was characterized by RAMAN spectroscopy and by differential scanning calorimetry (DSC) and the obtained results indicated the successful synthesis. The reaction product of the polymerization of hemoglobin or albumin with PEG was verified by SDS-PAGE and the results indicated the successful formation of high molecular mass polymers. The particles generated with leather gelatin and polyhemoglobin had a hydrodynamic diameter of 1370 nm, dispersity of 0.029 and zeta potential of -36.1 mV. Particles generated with bone gelatin and polyhemoglobin had hydrodynamic diameter of 438 nm, dispersity of 0.563 and zeta potential of -24.5 mV. The obtained results suggest the applicability of leather gelatin for the production of polyhemoglobin containing particles aiming to the development of a hemoglobin based oxygen carrier

Formulation and in vitro release studies of pegylated mucin based matrix tablets

The effects of polymer concentration on the flow properties of granules and in-vitro release profiles from matrix tablets of three model drugs formulated from pegylated mucin base was investigated. Mucin was extracted from the African giant snail and in combination with PEG was used to produce a copolymer matrix base, which was mixed with the model drugs using wet granulation method. The granules and tablets were evaluated according to official and unofficial requirements. Results showed best flow with Acetylsalicylic acid [ASA] and Chloroquine Phosphate [CQ] granules with Hausner ratio of 1.04-1.2, Carr's index of 4.2-17.5% and angle of repose between 19°-26°. The tablets met B.P specifications with respect to tablet weights, friability and drug content. The release profiles showed faster release of the drug with high content of PEG and a slower release with high concentration of mucin. Pegylated mucin base will find useful application in the development of a wide range of formulations

Obturation Over an S1 ProTaper Instrument Fragment in a Mandibular Molar with Three Years of Follow-up

This case report describes root canal filling performed over a large S1 ProTaper file fragment in a second mandibular molar with irreversible pulpitis. An S1 ProTaper file was fractured during the instrumentation of the mesiobuccal canal. Approximately 10 mm of file fragment remained in the apical and middle thirds of the canal. The obturation was performed over this fragment using thermomechanically compacted gutta-percha and sealer. Radiographic findings and the absence of clinical signs and symptoms at 3-year follow up indicated successful treatment. Cone-beam computed tomography images revealed absence of periapical lesion and details of intracanal file fragment related to root fillings and apex morphology. In this case, the presence of a large intracanal fractured instrument did not have a negative impact on the endodontic prognosis during the follow up evaluation period.

Este relato de caso descreve a obturação do canal radicular realizada sobre um grande fragmento da lima ProTaper S1 em um segundo molar inferior com pulpite irreversível. Uma lima ProTaper S1 fraturou durante a instrumentação do canal mésio-vestibular. Aproximadamente 10 mm de remanescente do fragmento da lima permaneceu nos terços apical e médio do canal. A obturação foi realizada sobre este fragmento usando guta-percha compactada termomecanicamente e cimento endodôntico. Achados radiográficos e ausência de sinais e sintomas clínicos após 3 anos de acompanhamento indicaram o sucesso do tratamento. Imagens de tomografia computadorizada de feixes cônicos revelaram a ausência de lesão periapical e detalhes do fragmento da lima intracanal relacionados à obturação do canal radicular e à morfologia do ápice. Neste caso, a presença de grande instrumento fraturado intracanal não teve impacto negativo no prognóstico endodôntico durante o período de acompanhamento.

The impact of pegylated interferon and ribavirin combination treatment on lipid metabolism and insulin resistance in chronic hepatitis C patients

BACKGROUND/AIMS: Lipid profile and insulin resistance (IR) are associated with hepatitis C virus (HCV) and may predict the chronic hepatitis C (CHC) treatment response. The aim of this study was to determine the association between CHC treatment response and lipid profile and IR change during treatment. METHODS: In total, 203 CHC patients were reviewed retrospectively between January 2005 and December 2011 at Soon Chun Hyang University Hospital. The lipid profile, homeostasis model for assessment (HOMA) of IR (HOMA-IR), and HOMA of beta cells (HOMA-beta) were evaluated before interferon plus ribavirin therapy (BTx), at the end of treatment (DTx), and 24 weeks after the end of treatment (ATx). RESULTS: A sustained virologic response (SVR) was achieved by 81% of all patients (49/60), 60% (n=36) of whom possessed genotype 1, with the remainder being non-genotype-1 (40%, n=24). Apart from age, which was significantly higher in the non-SVR group (SVR, 48.0+/-11.2 years, mean+/-SD; non-SVR, 56.6+/-9.9 years; P2.5), HOMA-IR was significantly changed at DTx in the SVR group. CONCLUSIONS: LDL-C appears to be associated with HCV treatment in SVR patients. Furthermore, eradication of HCV may improve whole-body IR and insulin hypersecretion, as well as high baseline insulin resistance (HOMA-IR >2.5).

Utilização de sistemas poliméricos de duas fases aquosas (SPDFA) compostos por polietileno glicol/ácido poliacrílico (PEG/APA) para extração de ácido clavulânico / Utilization of aqueous two phase systems (ATPS) composed of polyethylene glycol/polyacrylic acid (PEG/APA) in the extraction of clavulanic acid

A viabilidade da produção em escala industrial de produtos biotecnológicos de interesse comercial e terapêutico, como os fármacos, depende significativamente das técnicas de separação e purificação utilizadas. A aplicação do sistema de duas fases aquosas (SDFA) é proposta como alternativa para a purificação, pois permite a separação e análise de biomoléculas, de modo que estas não percam sua atividade ou propriedades desejadas. Esta técnica é interessante para a purificação em larga escala, pois permite partição seletiva, com potencial de obtenção de altos rendimentos, além de apresentar boa relação custo-benefício. O presente trabalho estudou a purificação por extração líquido-líquido do ácido clavulânico em SDFA utilizando um novo sistema polimérico aquoso, formado pelos polímeros polietileno glicol (PEG) e ácido poliacrílico (APA). Foram estudadas diferentes composições do sistema polimérico aquoso PEG/APA, empregando diferentes massas molares e concentrações para o PEG e utilizando a massa molar 8000g/mol para o APA. Com base nas informações obtidas o melhor ponto de extração para o ácido clavulânico na presença de Na2SO4 foi definido como MPEG=400 g/mol, CPEG=17,5% (m/m) e CNaPA=22,5% (m/m) com K= 19,14, ηT=91,21%, BM=101,69 e R=0,45. Enquanto que na presença de NaCl, o melhor ponto encontrado foi: MPEG=400 g/mol, CPEG=35% (m/m) e CNaPA=10% (m/m) com K=11,96 ηT=80,04%, BM=90,18 e R=0,66. No trabalho será avaliada, também, a influência da temperatura, pH e força iônica nesse sistema. Estabeleceram-se os melhores parâmetros de separação do ácido clavulânico presente em meio fermentado produzido por Streptomyces clavuligerus utilizando a metodologia de fermentação extrativa com SDFA PEG/APA. O efeito do ácido clavulânico no diagrama de fases do sistema PEG-APA, bem como sua partição na forma pura e na presença de homogeneizado celular, foi estudado principalmente através da determinação do coeficiente de partição e recuperação do respectivo fármaco

The viability of industrial scale production of commercial and therapeutical biotechnological products, such as drugs, is significantly dependent on the separation and purification techniques applied. The use of two-aqueous phase systems (ATPS) is proposed as an alternative to purification because it allows the separation and analysis of biomolecules, so that they do not lose their activities or desired properties. This technique is interesting for large scale purification because it allows selective partition with high potential yield and good cost/benefit ratio. The present work studied the purification of clavulanic acid (CA) by liquid-liquid extraction in ATPS applying a new aqueous polymeric system composed of two polymers, namely polyethylene-glicol (PEG) and sodium polyacrylate (NaPA). Different compositions of the aqueous polymeric system (PEG/PAA) were utilized, employing different PEG molar masses (MPEG) and concentrations (CPEG) and a molar mass of PAA of 8000 g/mol. In the light of the results obtained, the best conditions for clavulanic acid extraction, in the presence of Na2SO4, were MPEG = 400 g/mol, CPEG = 17.5% (m/m) and CNaPA = 22.5% (m/m), which allowed obtaining a partition coefficient (K) of 19.14, a yield in the top phase (ηT) of 91.21%, a mass balance (MB) of 101.69 and a volume ratio (R) of 0.45. On the other hand, in the presence of NaCl, the best results (K = 11.96, ηT = 80.04%, MB = 90.18 and R = 0.66) were found at: MPEG = 400 g/mol, CPEG = 35% m/m and CNaPA = 10% m/m. The effect of clavulanic acid in the PEG-PAA system phase diagram and its partition either in its pure form or in the cell homogenate were studied mainly through both the determination of the partition coefficient and the recovery of the drug selected for this study

A reduced dose of ribavirin does not influence the virologic response during pegylated interferon alpha-2b and ribavirin combination therapy in patients with genotype 1 chronic hepatitis C

BACKGROUND/AIMS: When combined with pegylated interferon alpha-2b (Peg-IFN alpha-2b) for the treatment of genotype 1 chronic hepatitis C (CHC) in Korea, the current guideline for the initial ribavirin (RBV) dose is based on body weight. However, since the mean body weight is lower for Korean patients than for patients in Western countries, current guidelines might result in Korean patients being overdosed with RBV. METHODS: We retrospectively reviewed the medical records of patients with genotype 1 CHC who were treated with Peg-IFN alpha-2b and RBV combination therapy. We divided the patients into groups A (> or =15 mg/kg/day, n=23) and B (<15 mg/kg/day, n=26), given that the standard dose is 15 mg/kg/day. The clinical course in terms of the virologic response, adverse events, and dose modification rate was compared between the two groups after therapy completion. RESULTS: The early response rates (92.0% vs. 83.3%, P=0.634) and sustained virologic response rates (82.6% vs. 73.1%, P=0.506) did not differ significantly between the two groups. During the treatment period, the RBV dose reduction rate was significantly higher in group A than in group B (60.9% vs. 23.1%, P=0.01). CONCLUSIONS: RBV dose reduction is performed frequently when patients are treated according to the current Korean guidelines. Given that lowering the RBV dose did not appear to decrease the virologic response during therapy, reducing RBV doses below the current Korean guideline may be effective for treatment, especially in low-weight patients.

Drag reduction by polyethylene glycol in the tail arterial bed of normotensive and hypertensive rats

This study was designed to evaluate the effect of drag reducer polymers (DRP) on arteries from normotensive (Wistar) and spontaneously hypertensive rats (SHR). Polyethylene glycol (PEG 4000 at 5000 ppm) was perfused in the tail arterial bed with (E+) and without endothelium (E-) from male, adult Wistar (N = 14) and SHR (N = 13) animals under basal conditions (constant flow at 2.5 mL/min). In these preparations, flow-pressure curves (1.5 to 10 mL/min) were constructed before and 1 h after PEG 4000 perfusion. Afterwards, the tail arterial bed was fixed and the internal diameters of the arteries were then measured by microscopy and drag reduction was assessed based on the values of wall shear stress (WSS) by computational simulation. In Wistar and SHR groups, perfusion of PEG 4000 significantly reduced pulsatile pressure (Wistar/E+: 17.5 ± 2.8; SHR/E+: 16.3 ± 2.7 percent), WSS (Wistar/E+: 36; SHR/E+: 40 percent) and the flow-pressure response. The E- reduced the effects of PEG 4000 on arteries from both groups, suggesting that endothelial damage decreased the effect of PEG 4000 as a DRP. Moreover, the effects of PEG 4000 were more pronounced in the tail arterial bed from SHR compared to Wistar rats. In conclusion, these data demonstrated for the first time that PEG 4000 was more effective in reducing the pressure-flow response as well as WSS in the tail arterial bed of hypertensive than of normotensive rats and these effects were amplified by, but not dependent on, endothelial integrity. Thus, these results show an additional mechanism of action of this polymer besides its mechanical effect through the release and/or bioavailability of endothelial factors.

Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion

Normal bone remodeling is maintained by a balance between osteoclast and osteoblast activity, whereas defects in osteoclast activity affecting such balance result in metabolic bone disease. Macrophage-macrophage fusion leading to multinucleated osteoclasts being formed is still not well understood. Here we present PEG-induced fusion of macrophages from both U937/A and J774 cell lines and the induced differentiation and activation of osteoclast-like cells according to the expression of osteoclast markers such as tartrate resistant acid phosphatase (TRAP) and bone resorptive activity. PEG-induced macrophage fusion, during the non-confuent stage, signifcantly increased the osteoclastogenic activity of macrophages from cell lines compared to that of spontaneous cell fusion in the absence of PEG (polyethylene glycol). The results shown in this work provide evidence that cell fusion per se induces osteoclast-like activity. PEG-fused macrophage differential response to pretreatment with osteoclastogenic factors was also examined in terms of its ability to form TRAP positive multinucleated cells (TPMNC) and its resorptive activity on bovine cortical bone slices. Our work has also led to a relatively simple method regarding those previously reported involving cell co-cultures. Multinucleated osteoclast-like cells obtained by PEG-induced fusion of macrophages from cell lines could represent a suitable system for conducting biochemical studies related to basic macrophage fusion mechanisms, bone-resorption activity and the experimental search for bone disease therapeutic alternatives.

Hypolipidemic activity of Hibiscus rosa sinensis root in rats.

The hypolipidemic activity of Hibiscus rosa sinensis (family Malvaceae) root extract was studied on triton and cholesterol-rich high fat diet (HFD) induced models of hyperlipidemia in rats. In triton WR-1339-induced hyperlipidemia, feeding with root extract (500 mg/kg body wt/day p.o.) exerted lipid-lowering effect, as assessed by reversal of plasma levels of total cholesterol (TC), phospholipids (PL) and triglycerides (TG) and reactivation of post-heparin lipolytic activity (PHLA) of plasma. The other model was fed with cholesterol-rich HFD and root extract (500 mg/kg body wt/ day p.o.) simultaneously for 30 days. This also caused lowering of lipid levels in plasma and liver homogenate and reactivation of plasma PHLA and hepatic total lipoprotein lipase activity. The hypolipidemic activity of Hibiscus rosa sinensis root was compared with a standard drug guggulipid (200 mg/kg body wt/day p.o.), a known lipid- lowering agent in both models. Histopathological findings in rat liver supported the protective role of H. rosa sinensis root extract in preventing cholesterol-rich HFD-induced hepatic steatosis.

O polietilenoglicol aumenta a penetração do vírus da diarréia viral bovina, do vírus da estomatite vesicular e do vírus sincicial respiratório bovino em células de cultivo / Polyethylene glycol increases the penetration of bovine viral diarrhea virus, vesicular stomatitis virus and bovine respiratory syncytial virus in cultured cells

A baixa eficiência de penetração de alguns vírus em células de cultivo pode representar uma dificuldade para o isolamento e a multiplicação viral in vitro. No presente estudo investigou-se o efeito do polietilenoglicol (PEG) na replicação de sete vírus bovinos em células de linhagem de rim bovino (MDBK). A eficiência de penetração e replicação foi mensurada pela contagem do número de placas virais produzidas em tapetes celulares, após adsorção do inóculo viral (100 DICC50 mL-1) com ou sem a adição de PEG a 5 por cento (peso molecular 6.000). A adição de PEG ao inóculo resultou em aumentos significativos do número de placas para o vírus da diarréia viral bovina (BVDV) (aumento de 3,4 vezes), vírus da estomatite vesicular (VSV) (2,2 vezes) e vírus respiratório sincicial bovino (BRSV) (1,5 vezes). A adição de PEG não produziu aumento significativo no número de placas dos herpesvírus bovinos 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5). Por outro lado, o PEG produziu uma redução do número de placas (1,4 vezes) produzidas pelo vírus da parainfluenza bovina (bPI-3V). A adição de PEG a 5 por cento também aumentou a sensibilidade de detecção (entre 10 e 100 vezes) do BVDV no soro de três bezerros persistentemente infectados. Para o BRSV, a adição de PEG aumentou em duas vezes a sensibilidade do isolamento viral de secreções nasais de duas ovelhas infectadas experimentalmente. Esses resultados demonstram que o PEG aumenta a eficiência de infecção do BVDV, VSV e BRSV em células de cultivo, podendo ser utilizado para aumentar a sensibilidade de detecção desses vírus em amostras clínicas (isolamento viral) e/ou, para aumentar os títulos de vírus produzidos em cultivo celular.

The low efficiency of penetration of some viruses in cultured cells may represent an obstacle for viral isolation and/or viral multiplication in tissue culture. This study investigated the effect of polyethylene glycol (PEG) on the penetration and replication of seven bovine enveloped viruses in culture cells. Penetration efficiency was measured by counting the number of viral plaques produced in bovine kidney cells (MDBK). The addition of 5 percent PEG (molecular weight 6.000) to the viral inoculum containing 100 TCID50 mL-1 (tissue culture median infectious dosis) of each virus, during adsorption for 2h at 37ºC, resulted in a significant increase in the number of plaques for bovine viral diarrhea virus (BVDV) (increase of 3.4-fold), vesicular stomatitis virus (VSV) (2.2-fold) and bovine respiratory syncytial virus (BRSV) (1.5-fold). The addition of 5 percent PEG to the inoculum of bovine herpesviruses 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) did not increase the number of viral plaques. On the other hand, PEG produced a reduction in the number of plaques by bovine parainfluenza virus (bPI-3V) (1.4-fold). Furthermore, the addition of 5 percent PEG produced a 10- to 1000-fold increase in the sensitivity of BVDV detection in the serum of three persistently infected calves; and doubled the sensitivity of detection of BRSV in nasal secretions of two experimentally infected sheep. These results demonstrate that PEG enhances the efficiency of infection by BVDV, VSV and BRSV in cultured bovine cells and therefore may be used to increase the sensitivity of virus detection in clinical samples (viral isolation), and/or to increase virus titers in cell cultures.