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

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

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

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

Obtenção de emulsões asfálticas modificadas utilizando resíduos industriais

The main objective of this research was the development and characterization of conventional and modified cationic asphalt emulsions. The asphalt emulsions were developed by using the Petroleum Asphalt Cement (CAP 50-70) from Fazenda Belém (Petrobras -Aracati-Ce). The first step in this research was the development of the oil phase (asphalt + solvent) and the aqueous phase (water + emulsifying agent + acid + additives), separately. During the experiments for the obtaining of the conventional asphalt emulsion, the concentration of each constituent was evaluated. For the obtaining of the oil phase, kerosene was used as solvent at 15 and 20 wt.%. For the development of the aqueous phase, the emulsifying agent was used at 0.3 and 3.0 wt.%, whereas the acid and the additive were set at 0.3 wt.%. The percentage of asphalt in the asphalt emulsion was varied in 50, 55, and 60 wt.% and the heating temperature was set at 120 °C. The aqueous phase in the asphalt emulsion was varied from 16.4 to 34.1 wt.% and the heating temperature was set at 60 °C. After the obtaining of the oil and the aqueous phases, they were added at a colloidal mill, remaining under constant stirring and heating during 15 minutes. Each asphalt emulsion was evaluated considering: sieve analysis, Saybolt Furol viscosity, pH determination, settlement and storage stability, residue by evaporation, and penetration of residue. After the characterization of conventional emulsions, it was chosen the one that presented all properties in accordance with Brazilian specifications (DNER-EM 369/97). This emulsion was used for the development of all modified asphalt emulsions. Three polymeric industrial residues were used as modifier agents: one from a clothing button industry (cutouts of clothing buttons) and two from a footwear industry (cutouts of sandals and tennis shoes soles), all industries located at Rio Grande do Norte State (Brazil).The polymeric residues were added into the asphalt emulsion (1 to 6 wt.%) and the same characterization rehearsals were accomplished. After characterization, it were developed the cold-mix asphalts. It was used the Marshall mix design. For cold-mix asphalt using the conventional emulsion, it was used 5, 6 and 7 wt.% asphalt emulsion. The conventional mixtures presented stability values according Brazilian specification (DNER-369/97). For mixtures containing asphalt modified emulsions, it was observed that the best results were obtained with emulsions modified by button residue

Sistemas microemulsionados aplicados à remoção da cor de efluente têxtil

Effluent color resulting from textile dyeing processes has been one of the biggest environmental problems faced by the textile industry. In particular, reactive dyes are highly resistant to conventional wastewater treatment methods. New technologies have been contemplated, some of which have been applied in industrial treatment plants, but color removal has not been efficiently attained. Since microemulsion systems provide good results in heavy metals and proteins extraction processes, their use in dyes extraction has been suggested and investigated. In this work, a real textile wastewater from an exhaustion dyebath has been treated, which contains the following reactive dyes: Procion Yellow H-E4R (CI Reactive Yellow 84), Procion Blue H-ERD (CI Reactive Blue 160) and Procion Red H-E3B (CI Reactive Red 120), in addition to auxiliary compounds normally found in dyeing processes with reactive dyes. The dyes Remazol Blue RR and Remazol Turquoise Blue G (Reactive Blue 21) have also been examined in view of the presence of heavy metals in these molecules. The microemulsion system comprised dodecyl ammonium chloride (as a cationic surfactant), water or wastewater as aqueous phase, kerosene as oil phase, and one of the following alcohols as cosurfactant: isoamyl alcohol, n-butyl alcohol and n-octyl alcohol. The pseudo-ternary diagrams were constructed in order to define Winsor s equilibrium regions. The influence of parameters such as pH, C/S (cosurfactant/surfactant) ratio, distribution coefficient, initial dye concentration, salinity, temperature, phases relative amounts, loading capacity of the microemulsion phase and dye reextraction rate has also been investigated. An experimental planning (Scheffé Net) was used to optimize the extraction process. The removal of color and metals reached levels as high as 99%

Desenvolvimento de sistemas farmacêuticos emulsionados para veiculação gênica

Broadly speaking, the concept of gene therapy involves the transfer of a genetic material into a cell, tissue, or organ in order to cure a disease or at least improve the clinical status of a patient. Making it simple, gene therapy consists in the insertion of functional genes into cells containing defective genes by substituting, complementing or inhibiting them. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, it is a key issue to create systems able to transfer and protect the DNA until it reaches the target, the vectors. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easily produced, present controllable stability and enable transfection. The aim of this work was to develop an emulsion for gene therapy and evaluate its ability to compact nucleic acids by the development of a complex with the plasmid pIRES2-EGFP. The first step was to determine the Hydrophilic Lipophilic Balance (HLB) of the Captex® 355 (oily internal phase of the emulsion) through long and short term stability assays. Based on the results, emulsions composed of Captex® 355, Tween 20® and Span 60® with 10.7 HLB were produced by three different methods: phase inversion, spontaneous emulsification and sonication. The results showed that the lowest diameter and best stability of the emulsions were achieved by the sonication method. The cationic emulsions were made by adding DOTAP to the basic emulsion. Its association with pIRES2-EGFP was evaluated by electrophoresis. Several rates of emulsion and DNA were evaluated and the results showed that 100% of the complex was formed when the rate DOTAP/DNA(nmol/µg) was 130. In conclusion, the overall results show the ability of the proposed emulsion to compact pIRES2-EGFP, which is a requirement to a successful transfection. Therefore, such formulation may be considered a promising candidate for gene therapy