PEGylated polyethyleneimine-entrapped gold nanoparticles for enhanced and targeted gene delivery applications

Gene therapy, which involves the transfer of nucleic acid into target cells in patients, has become one of the most important and widely explored strategies to treat a variety of diseases, such as cancer, infectious diseases and genetic disorders. Relative to viral vectors that have high immunogenicity, toxicity and oncogenicity, non-viral vectors have gained a lot of interest in recent years. This is largely due to their ability to mimic viral vector features including the capacity to overcome extra- and intra-cellular barriers and to enhance transfection efficiency. Polyethyleneimine (PEI) has been extensively investigated as a non-viral vector. This cationic polymer, which is able to compact nucleic acid through electrostatic interactions and to transport it across the negatively charged cell membranes, has been shown to effectively transfect nucleic acid into different cell lines. Moreover, entrapment of gold nanoparticles (Au NPs) into such an amine-terminated polymer template has been shown to significantly enhance gene transfection efficiency. In this work, a novel non-viral nucleic acid vector system for enhanced and targeted nucleic acid delivery applications was developed. The system was based on the functionalization of PEI with folic acid (FA; for targeted delivery to cancer cells overexpressing FA receptors on their surface) using polyethylene glycol (PEG) as a linker molecule. This was followed by the preparation of PEI-entrapped Au NPs (Au PENPs; for enhancement of transfection efficiency). In the synthesis process, the primary amines of PEI were first partially modified with fluorescein isothiocyanate (FI) using a molar ratio of 1:7. The formed PEI-FI conjugate was then further modified with either PEG or PEGylated FA using a molar ratio of 1:1. This process was finally followed by entrapment of Au NPs into the modified polymers. The resulting conjugates and Au PENPs were characterized by several techniques, namely Nuclear Magnetic Resonance, Dynamic Light Scattering and Ultraviolet-Visible Spectroscopy, to assess their physicochemical properties. In the cell biology studies, the synthesized conjugates and their respective Au PENPs were shown to be non-toxic towards A2780 human ovarian carcinoma cells. The role of these materials as gene delivery agents was lastly evaluated. In the gene delivery studies, the A2780 cells were successfully transfected with plasmid DNA using the different vector systems. However, FA-modification and Au NPs entrapment were not determinant factors for improved transfection efficiency. In the gene silencing studies, on the other hand, the Au PENPs were shown to effectively deliver small interfering RNA, thereby reducing the expression of the B-cell lymphoma 2 protein. Based on these results, we can say that the systems synthesized in this work show potential for enhanced and targeted gene therapy applications.

Desenvolvimento de nanosistemas farmacêuticos para terapia gênica

Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex® 355; Tween® 80; Spam® 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q®). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way

Papel da localização da carga em determinação dos parâmetros do canal iônico

To aureus α-HL channel, we used the cysteine-scanning mutagenesis technique. Twenty-four mutants were produced from the substitution of a single aminoacid of the primary structure of the α-HL pro this yzed after the incorporation of a mutant channel in planar lipid bilayer membranes. The modified proteins were studied in the absence and presence of watersoluble specific sulphydryl-specific reagents, in order to introduce a strong positive or negative harge at positions of substitution. The introduction of a negative charge in the stem region onverted the selectivity of the channel from weak anionic to more cationic. However, the troduction of a positive charge increased its selectivity to the anion. The degree of these alterations was inversely dependent on the channel radius at the position of the introduced harge (selectivity). As to the asymmetry of the conductance-voltage, the influence of the harge was more complex. The introduction of the negative charge in the stem region (the trans art of the pore) provoked a decrease. The intensity of these alterations depended on the radius, and on the type of free charge at the pore entrance. These results suggest that the free charge at surrounds the pore wall is responsible for the cation-anion selectivity of the channel. The istribution of the charges between the entrances is crucial for determining the asymmetry of e conductance-voltage curves. We hope that these results serve as a model for studies with other nanometric channels, in biological or planar lipid bilayer membranes or in iotechnological applications

On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção e avaliação de nanoemulsão catiônica como possível vetor não-viral para pIRES2-EGFP

Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355®, 1.2 % of Tween 80®, 0.8 % of Span 80®, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 μS/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ºC. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/μL); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier

Cobre e zinco no material percolado e no solo com a aplicação de água residuaria de suinocultura em solo cultivado com soja

O objetivo deste trabalho foi avaliar possíveis efeitos da aplicação de taxas de água residuária de suinocultura (ARS), quanto às concentrações de cobre e zinco, tanto no solo quanto na água percolada de lisímetros de drenagem, durante o ciclo da cultura da soja. O experimento foi conduzido em ambiente protegido, onde havia 24 lisímetros de drenagem. Semeou-se soja cultivar CD 202, e foram aplicadas ARS nas seguintes dosagens: 0; 100; 200 e 300 m³ ha-1 no ciclo, combinadas com duas adubações na semeadura (com e sem adição da adubação recomendada na semeadura), em três repetições por tratamento. Foram feitas seis coletas do material percolado durante o ciclo da cultura e realizadas determinações de cobre e zinco. No solo, foram feitas três coletas em cada parcela experimental, para a determinação do pH, da matéria orgânica, da capacidade de troca catiônica, do cobre e do zinco. O delineamento foi em blocos casualizados, num esquema fatorial de 4 x 2. Observou-se que o cobre no solo não apresentou diferenças significativas com a aplicação de ARS e adubação, enquanto as concentrações de zinco no solo foram influenciadas pelas taxas aplicadas de ARS. As transferências de cobre e de zinco por percolação foram pouco expressivas.

Relações entre ocorrência do Mal-de-Panama em bananeira da cv. Nanicão e nutrientes no solo e nas folhas

O presente trabalho teve como objetivo verificar se os sintomas incitados na bananeira cv. Nanicão, do subgrupo Cavendish, na região do Vale do Ribeira, estão relacionados com níveis de nutrientes no solo e nas folhas. Foram separadas 16 áreas na região, sendo a metade com plantas sintomáticas e a outra com plantas sadias. Nessas áreas, coletou-se a terceira folha de cinco plantas e o solo junto a essas mesmas plantas, nas profundidades de 0 a 20 cm e de 20 a 40 cm. em ambas as profundidades do solo amostrado, níveis de Ca, Mg, PO(-3)4, S e da capacidade de trocas catiônicas (CTC) foram significativamente diferentes entre as áreas, sendo que os valores baixos destes elementos estavam presentes nas áreas contendo plantas sintomáticas. em ambas as profundidades, o Mg, o Al e o H em relação à CTC foram significativamente diferentes entre as áreas, sendo que o valor baixo de Mg e alto de Al e H estava presente nas áreas com plantas sintomáticas. O N, K e S nas folhas foram significativamente diferentes entre as áreas. Estes elementos apresentaram valores baixos nas áreas contendo plantas sintomáticas. Apesar de algumas quantidades de macronutrientes do solo e das folhas estarem presentes somente nas áreas contendo plantas de Nanicão com sintomas semelhantes aos de fusariose, deve-se buscar comprovação de uma possível ocorrência da raça 4 do patógeno no Vale do Ribeira.

Atributos químicos do solo decorrentes da aplicação em superfície de calcário e gesso em sistema plantio direto recém-implantado

Há interesse em implantar o sistema plantio direto em áreas anteriormente cultivadas no sistema convencional ou sob pastagem, corrigindo a acidez com calagem superficial após a implantação do sistema. Essa prática pode ser possível desde que não haja impedimento físico do solo ao crescimento radicular. Nesse sentido, o gesso é uma alternativa para diminuição da atividade do Al3+ e aumento da saturação por bases (V), principalmente Ca2+, nas camadas do subsolo, podendo ser utilizado como produto complementar ao calcário. Este trabalho objetivou avaliar as alterações dos atributos químicos do solo (pH CaCl2, H + Al, Al3+, Ca2+, Mg2+, S-SO4(2-), V e teor de micronutrientes catiônicos) decorrentes da aplicação de calcário e de gesso agrícola em superfície em sistema plantio direto recém-implantado em região de inverno seco. O experimento foi conduzido em um Latossolo Vermelho distroférrico de Botucatu (SP). O delineamento experimental foi em blocos casualizados com parcelas subdivididas e quatro repetições. As parcelas foram constituídas por quatro doses de calcário dolomítico com PRNT de 71,2 % (0, 1.100, 2.700 e 4.300 kg ha-1), visando elevar a saturação por bases a 50, 70 e 90 %, respectivamente. As subparcelas foram constituídas pelas doses de gesso agrícola de 0 e 2.100 kg ha-1. Amostras de terra foram coletadas nas profundidades de 0-0,05, 0,05-0,10, 0,10-0,20, 0,20-0,40, 0,40-0,60 e 0-0,20 m, aos três, seis, 12 e 18 meses da aplicação dos produtos. A aplicação superficial de calcário diminuiu a acidez e elevou os teores de Ca e Mg trocável, principalmente nas camadas superficiais do solo. A aplicação de gesso agrícola aumentou os teores de Ca trocável e S-SO4(2-), e diminuiu os teores de Al no solo, contribuindo para que os efeitos da calagem superficial nas características químicas do solo alcançassem, de forma mais rápida, as camadas do subsolo. Os valores de saturação por bases obtidos na profundidade de 0-0,20 m com a calagem foram menores do que os estimados pelo método da elevação da saturação por bases, principalmente nas maiores doses, mesmo com a aplicação de gesso agrícola. A calagem em superfície não alterou os teores de micronutrientes na camada de 0-0,20 m de profundidade.

Purificação da glicerina bruta obtida a partir da transesterificação do óleo de algodão

The crude glycerine is a raw material that can be used in a wide variety of products. Even with all the impurities inherent in the process of being obtained, the crude glycerin is already in a marketable product. However, the market is much more favorable to the commercialization of purified glycerine. The glycerin is a byproduct gotten from the process of transesterification of waste oils and fats in the production of biodiesel. More recently, the deployment of the new Federal Law of Brazil, related to the implementation of energy resources, forces, from 2008, the increase of 2% biodiesel in diesel common with prospects for 5% (B5). Therefore, it is indispensable that new routes of purification as well as new markets are developed. The objective of this work was to purify, through ion exchange, the crude glycerin, obtained from the reaction of transesterification of cottonseed oil. The cottonseed oil was characterized as the fatty acid composition and physical-chemical properties. The process of ion exchange was conducted in batch. In this process were used strong cation, low anion resins and a mixed resin used to de-ionize water. The purified glycerin was characterized as the content of metals. Tests were performed with activated charcoal adsorption, and for this, it was made tests of time contact with coal as well as quantity of coal used. The time of activation, the amount of the activation solution, the contact time of the glycerol solution in resins, the amount and type of resin applied were evaluated. Considering the analysis made with activated charcoal, when the glycerin solution was treated using the resins individually it was observed that in the conditions for treatment with 10 g of resin, 5 hours of contact with each resin and 50 mL of glycerin solution, its conductivity decreased to a cationic resin, increased to the anionic resin and had a variable value with respect to resin mixed. In the treatment in series, there was a constant decrease in the conductivity of the solution of glycerin. Considering two types of treatment, in series and individually, the content of glycerol in glycerin pre-purified solution with the different resins varied from 12,46 to 29.51% (diluted solution). In analysis performed without the use of activated charcoal, the behavior of the conductivity of the solution of glycerin were similar to results for treatment with activated charcoal, both in series as individually. The solution of glycerin pre-purified had a glycerol content varying from 8.3 to 25.7% (diluted solution). In relation to pH, it had a behavior in accordance with the expected: acid for the glycerin solution treated with cationic resin, basic when the glycerin solution was treated with the anionic resin and neutral when treated with the mixed resin, independent of the kind of procedure used (with or without coal, resins individually or in series). In relation to the color of the glycerin pre-purified solution, the resin that showed the best result was the anionic (colorless), however this does not mean that the solution is more in pure glycerol. The chromatographic analysis of the solutions obtained after the passage through the resins indicated that the treatment was effective by the presence of only one component (glycerol), not considering the solvent of the analysis

Flotação por ar dissolvido para os minerais quartzo e feldspato utilizando coletores catiônicos

The pegmatite rocks in Rio Grande do Norte are responsible for much of the production of industrial minerals like quartz and feldspar. Quartz and feldspar are minerals from pegmatite which may occur in pockets with metric to centimetric dimensions or as millimetric to sub millimetric intergrowths. The correct physical liberation of the mineral of interest, in case of intergrowths, requires an appropriate particle size, acquired by size reduction operations. The method for treating mineral which has a high efficiency fines particles recovery is flotation. The main purpose of the present study is to evaluate the recovery of quartz and potassium feldspar using cationic diamine and quaternary ammonium salt as collectors by means of dissolved air flotation DAF. The tests were performed based on a central composite design 24, by which the influence of process variables was statistically verified: concentration of the quaternary ammonium salt and diamine collectors, pH and conditioning time. The efficiency of flotation was calculated from the removal of turbidity of the solution. Results of maximum flotation efficiency (60%) were found in the level curves, plotted in conditions of low concentrations of collectors (1,0 x 10-5 mol.L-1). These high flotation efficiencies were obtained when operating at pH 4 to 8 with conditioning time ranging from 3 to 5 minutes. Thus, the results showed that the process variables have played important roles in the dissolved air flotation process concerning the flotability of the minerals.

Aplicação de microemulsão na recuperação de petróleo de reservatórios carbonáticos

The large investment in exploration activities offshore Brazil has generated new findings, generally in carbonate reservoirs, with different wettability conditions usually considered in the sandstone, strongly water-wet. In general, the carbonates reservoirs tend to be oil-wet, it difficult to mobilize of oil these reservoirs. These oils can be mobilized by different methods, or it may reverse the wettability of the surface of the reservoir and facilitate the flow of oil, improving production rates. Thus, the objective of this work was to study the influence of inversion on the wettability of the rock in the production and recovery of petroleum from carbonate reservoirs, using microemulsions. Three systems were chosen with different classes of surfactants: a cationic (C16TAB), an anionic (SDS) and nonionic (Unitol L90). Studies of the influence of salinity on the formation of the microemulsion as well as the characterization of fluids using density and viscosity measurements were also performed. To verify the potential of microemulsion systems in changing the wettability state of the chalk oil-wet to water-wet, contact angle measurements were performed using chalk of neutral-wet as surface material. Overall, with respect to the ionic character of the surfactants tested, the cationic surfactant (C16TAB) had a greater potential for reversal in wettability able to transform the rock wettability neutral to strongly water-wet, when compared with the anionic surfactant (SDS) and nonionic (Unitol L90), which showed similar behavior, improving the wettability of the rock to water. The microemulsions of all surfactants studied were effective in oil recovery, resulting in 76.92% for the system with C16TAB, 67.42% for the SDS and 66.30% for Unitol L90 of residual oil

Variações no balanço catiônico do solo induzidas pela adubação potássica e efeitos na cultura do alho vernalizado

Avaliaram-se os efeitos das relações (Ca+Mg)/K do solo sobre o desenvolvimento, absorção de nutrientes e produção de bulbos na cultura do alho cv. Roxo Pérola de Caçador conduzindo experimento no período de maio a outubro de 1990 em vasos de cimento amianto contendo 50 kg de terra, em casa de vegetação telada. Os tratamentos consistiram de aplicações de doses de potássio visando atingir relações (Ca+Mg)/K no complexo de troca do solo próximas a 170; 50; 35; 20; 12,5; 7,5; 5,0; 3,5 e 2,0. Verificaram-se aumentos na concentração de K, Ca e Mg na solução do solo proporcionais à elevação do teor de K trocável. As concentrações foliares de K e Mn diminuiram proporcionalmente às relações (Ca+Mg)/K do solo, ocorrendo o inverso com as concentrações de Ca e Mg. A absorção dos demais nutrientes não foi influenciada pelos tratamentos. A relação (Ca+Mg)/K influenciou a produção de bulbos e a altura das plantas durante todo o ciclo, sendo que os menores valores desta relação, caracterizados por excesso de K, inibiram mais o desenvolvimento e a produção de bulbos. A porcentagem de K no complexo de troca, seguida da relação K/(Ca+Mg)1/2 e do K trocável do solo foram os índices que melhor correlacionaram com a produção de bulbos.

Uso da tecnologia de troca iônica no tratamento de águas contaminadas com Nitrato do Aqüífero Dunas-Barreiras, Natal/RN-Brasil

The city of Natal comprises an area of about 170 km² (65,63 squares miles). The Dunas-Barreiras Aquifer is the most important reservoir of the coastal basin of RN. It is being responsible for the water supplying of about 70% of the population, however, due to the sewage disposal system by cesspools and drains, it is presently affected in a great extent by nitrates contamination. Thus, the present work proposes to research the utilization of contaminated water by nitrates of this fountainhead and find cost of the potable water through the ionic exchange technology. This technology consists in the removal of mineral salts by the exchange of cations for one ion of hydrogen (H+), through the passage of water by cationic resin bed and, secondly, by the exchange of the anions for hydroxyl ions (OH-) through a anionic resin bed. The obtained results have showed the waters derived from fountains, big water holes and shallow wells were microbiologically contaminated, while the waters derived from deep wells (above 70 m 76,58 yards) were free of contamination. Thus, only these ones are suitable to the use of ionic technology. The experiments were conducted with the resin IMAC-HP-555 such as kinetic, thermodynamic, and adsorption by fixed bed studies, being obtained several project variables for the experimental column, as follow: work temperature of 25oC; resin maximum capacity maximum e mean of adsorption ==0,01692 g NO3-1/g R e 0,0110 g NO3-1/g R, respectively. On the experimental column were performed breakthrough tests which pointed for an average ideal average speed of work of 13.2 m / h, with an average efficiency of 45% of adsorption, an optimal concentration of NaCl desorption of 8%, and an ideal desorption time of 80 minutes for the equilibrium conditions of water from the Dunas-Barreiras aquifer. Scale projection for ion-exchange column for denitrification, for these variables, using a computer modeling programme, to project the column of ion exchange ROREX-420/2000, obtained a cost for the drinking water denitrified by this system of R$ 0,16 / m3

Estado nutricional e componentes da produção de plantas de pimentão conduzidas em sistema de fertirrigação durante indução de estresse salino em cultivo protegido

O uso das técnicas de fertirrigação em ambiente protegido vem aumentando no Brasil devido à praticidade de condução e redução dos custos de produção. Apesar dessas vantagens, salienta-se que esta prática também pode trazer riscos, principalmente pelo acúmulo de sais na superfície do solo e na região periférica do bulbo radicular. em caso de salinização do solo pelo manejo incorreto do sistema podem ocorrer alterações na disponibilidade de nutrientes, com conseqüências fisiológicas no desenvolvimento das plantas. Neste trabalho, utilizou-se o pimentão (Capsicum annuum L.) como planta-modelo para estudos da interferência da salinidade na nutrição das plantas, pela alteração da condutividade elétrica (CE) na solução do solo. Os resultados revelaram indícios de antagonismo entre nutrientes, notadamente entre as formas catiônicas em razão do aumento da concentração de KCl nos tratamentos com alta CE. A suplementação do solo com adubo orgânico não atenuou os efeitos do aumento da concentração salina e os valores extremos de CE interferiram drasticamente na produção.