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

Prevalência da infecção por Chlamydia Trachomatis em mulheres atendidas no Programa de rastreamento do câncer de colo do útero

Genital infection with Chlamydia trachomatis is now recognized as one of the most prevalent sexually transmitted infections (STDs). Despite major advances in laboratory diagnosis techniques, primarily the character of asymptomatic chlamydial infection in both men and in women constitutes the basis for the formation of reservoirs that perpetuate transmission and acquisition of this and other STDs. The asymptomatic in women favors the rise of infection to the upper genital tract, causing injuries that can result in infertility. An examination of population screening for early detection and treatment of asymptomatic infections is the key step in combating this major public health problem. The present study aimed to evaluate the prevalence of infection by C. trachomatis in sexually active women attended the screening program for cervical cancer of the uterus in health facilities in municipalities in different regions of the State of Rio Grande do Norte, and identify factors that may contribute to the spread of this pathogen and its relationship with the lesions of the uterine cervix. It is a cross-sectional study aimed at detecting the presence of genital tract infection by C. trachomatis either in isolated form or in association with human papilloma virus (HPV) infection in asymptomatic women. Were included in this study, a total sample of 1,134 women aged 13-76, mean 34.4 years, from March 2008 to September 2012. Specimens containing exfoliated cells of the epithelium of the uterine cervix were analyzed by examining Pap cytology for the detection of possible injuries, and the polymerase chain reaction (PCR) for detection of plasmid DNA from C. trachomatis and HPV. Infection with C. trachomatis was detected with overall prevalence rate of 8.1% in the isolated form and 2.8% in co-infection with HPV. The infection was detected in 7.4% of women with normal cytology 11.5% of those with atypical cells of undetermined significance (ASC-US) and 16.7% of those with low-grade squamous intraepithelial lesion (LSIL). We observed an association between C. trachomatis and incidence of low-grade squamous intraepithelial lesion (LSIL). The genital tract infection by C. trachomatis alone was associated with education level, ethnicity and parity, revealing that women with higher education, those of non-white ethnicity and those who had three or more pregnancies were more likely to acquire infection. Levels very close to statistical significance were observed for chronological age, age at first sexual intercourse and first pregnancy. There was no association with marital status, number of sexual partners. Co-infection with C. trachomatis and HPV was detected in 2.3% of women with normal cytology, who had 5.1% in ASC-US and 10.4% in those with LSIL. No association was found between infection C. trachomatis and increased risk of HPV infection, but women with simultaneous infection by both pathogens showed greater risk for LSIL. Co-infection was more prevalent among single women, who had in the first sexual intercourse under 18 years and those who had two or more sexual partners over a lifetime

Efeitos biológicos in vitro de agentes físicos utilizados em fisioterapia

The uses of radiobiocomplexes labeled with technetium-99m contributed to health science advances. Stannous chloride (SnCl2) has been used as a reducing agent for the labeling process. Cytotoxic and genotoxic effect of the SnCl2 have been described in several studies and with this experimental models alterations in molecular and cellular level can be evaluated. In the last years the physicals therapists acquired new devices which emits electromagnetic radiation such us Extremely Low Frequency Pulsated Electromagnetic Fields (E.L.F. P.E.M.F.), radiofrequency, Intense Pulsed Light (I.P.L.) and others which emits sonic waves such us Biorresonance. Scientific evidence of the effects and dosage is important to protect public health and to reach exposition levels that result in significant biological effects. The aim of this project is to verify the effects of these physical agents in plasmid DNA and E. coli AB1157 cultures in presence or absence of SnCl2 and the effects in blood constituents labeled with technetium-99m. Wistar rats blood was exposed to the cited sources and the labelling of blood constituents with 99mTc was carried through. Cultures of E. coli AB1157 and plasmidial samples DNA had been also exposed the physical agents. The results suggest that these agents are capable of altering neither the survival of E. coli cells or plasmid DNA electrophoresis mobility. The multidiscipline character was clearly in this study due the interaction between Nuclear Medicine department of the UERJ and the Laboratory of Physical Agents of the Maimonides University in Argentina until the union between the teacher (biomedical and physiotherapist) and student (physiotherapist), besides collaborators of the area of Physics and Biology, promoting new ideas and perspectives and also adding the knowledge of different areas and origins

Efeito de um extrato de chrysobalanus icaco na marcação de constituíntes sanguíneos com tecnécio-99m, na morfologia de hemácias, na topologia plasmidial e na ação do cloreto estanoso no dna plasmidial

The use of radionuclides has contributed for advances in Health Sciences, to research or to the diagnosis and/or treatment of diseases. These advances have been possible with the utilization of radiopharmaceuticals labeled with technetium-99m (99mTc). Stannous chloride (SnCl2) has the main reducing agent utilized to obtain radiopharmaceuticals labeled with technetium-99m. It has been reported that several natural or synthetic drugs are capable to alter the labeling of blood constituents with 99mTc, as well as the red blood cells morphology. The aim of this study was to evaluate possible alterations of Chrysobalanus icaco extract on the labeling of blood constituents with 99mTc, on the morphology of RBC of blood of Wistar rats, on the breakage of plasmid DNA and on the effects of stannous chloride on plasmid DNA. The results showed significant (P<0.05) alteration of the labeling of blood constituents with 99mTc, as well as, modification of the morphology and morphometry (perimeter/area ratio) of the RBC in presence of the extract. These data suggest that this abajeru extract could alter the labeling of blood constituents with 99mTc by its chelating/antioxidant action and/or effects on membrane structures. Moreover C. icaco extract altered the electrophoretic profile and decreased significantly (p<0.05) the effect of SnCl2 on plasmid DNA. The results obtained in this work could indicate a dose-dependent protective action against the SnCl2 and a genotoxic effect of C. icaco extract on plasmid DNA

Avaliação do potencial de mutagenicidade e toxidade da lectina hipoglicemiante de folha de Bauhinia monandra (pata-de-vaca)

Medicinal plants have been used since antiquity to treat various human diseases. The leaves of Bauhinia monandra are widely used in Brazil as herbal remedies in the treatment of Diabetes Mellitus. From the leaves of B. monandra was purified a galactose-specific lectin, called BmoLL, which also showed a significant hypoglycemic capacity. Following the proposed rules by decree No 116 of 1996/08/08 of the Ministry of Health of Brazil, the study aimed to evaluate the potential for toxicity and mutagenicity of BmoLL from the use of tests with Escherichia coli strain CC104 (Forward mutagenesis assay) with Salmonella typhimurium strain TA (Kado test), with plasmid pBCKS (Break occurrences in plasmid DNA) and enzyme exonuclease III (Search of abasic sites). The results demonstrated that the lectin was unable to increase the frequency of reverse mutation of strains of S. typhimurium, with and without metabolic activity. However, a significant decrease in the frequency of spontaneous mutation was observed in strains of E. coli, especially in poor repair (CC104mutMmutY), indicating an antioxidant potential of the lectin. BmoLL is unable to generate genotoxic and cytotoxic damage, based on the concentrations and the tests performed

Avaliação de efeitos DW extratos de sambucus australis E sambucus nigra na marcação de constituintes sanguineos com tecnécio-99M, na morfologia de Hemácias de ratos wistar, na topologia plasmidial e na ação do cloreto estanoso no DNA Plasmidial

Radiobiocomplexes are used in nuclear medicine to obtain images and to treat diseases. Blood constituents have been used as radiobiocomplexes. Natural or synthetic products can influence on the labeling of blood constituents with technetium-99m (99mTc), the morphology of red blood cells and on the stannous chloride (SnCl2) action on plasmid DNA. Sambucus australis and Sambucus nigra are used in popular culture for treating diseases. The aim of this work was to evaluate the effects of the extracts of Sambucus australis and Sambucus nigra on the labeling of blood constituents with 99mTc, on morphology of red blood cells of Wistar rats, on the topology of plasmids DNA and the action against the SnCl2 effects on the DNA of plasmids pBSK. On the labeling of blood constituents with 99mTc it was verified that both extracts were capable to decrease significantly the radioactivity in the cellular compartment and in the insoluble fraction of plasma. Sambucus australis also decreased the labeling of insoluble fraction of blood cells with 99mTc. Both extracts did not alter the morphology of red blood cells. Moreover, it was verified that Sambucus nigra did not alter the electrophoretic profile of plasmid DNA, but decreased the effect of SnCl2 on plasmid DNA. These last results sugest a genotoxic effect and a protective action of Sambucus nigra extract against the SnCl2 action on plasmid DNA. This work was developed with the contribution of several Departments of biomedical area of the Hospital Universitário Pedro Ernesto, of the UERJ, characterizing a multidisciplinary experimental research

Avaliação de efeitos biológicos de antiinflamatórios derivados do ácido propiônico através de modelos experimentais em nível molecular e celular

Derivatives of propionic acid NSAIDs are irreversible inhibitors of cyclooxygenase enzyme widely used. The aim of this study was to evaluate, through different experimental models, biological effects of derivatives of propionic acid (fenoprofen, naproxen, ibuprofen and ketoprofen) in cellular and molecular level. The labeling of blood constituents with technetium-99m (99mTc) and morphological analysis of erythrocytes of blood of rats, as well as growth, survival of cultures of Escherichia coli (E. coli) and the assessment of bacterial plasmid electrophoretic profiles were models used for experimental evaluation of possible biological effects of antiinflammatory drugs. The results show that, in general, anti-inflammatory drugs evaluated were not able to alter the labeling of blood constituents with 99mTc, the morphology of red blood cells from blood of rats, as well as the growth of cultures of E. coli and the electrophoretic profile of plasmid DNA. However, naproxen appears to cytotoxic effect on bacterial cultures, plasmids and genotoxic effects in reducing the action of stannous chloride in cultures of E. coli. The use of experimental fast performance and low cost was important for assessment of biological effects, contributing to a better understanding of the properties of propionic acid derivatives studied. anti-inflammatory, blood constituents, technetium-99m, stannous chloride, Escherichia coli; DNA

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

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