Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single-(ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles are presented, using counter-ion structure and DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Birth in Brazil: national survey into labour and birth

Background: Caesarean section rates in Brazil have been steadily increasing. In 2009, for the first time, the number of children born by this type of procedure was greater than the number of vaginal births. Caesarean section is associated with a series of adverse effects on the women and newborn, and recent evidence suggests that the increasing rates of prematurity and low birth weight in Brazil are associated to the increasing rates of Caesarean section and labour induction. Methods: Nationwide hospital-based cohort study of postnatal women and their offspring with follow-up at 45 to 60 days after birth. The sample was stratified by geographic macro-region, type of the municipality and by type of hospital governance. The number of postnatal women sampled was 23,940, distributed in 191 municipalities throughout Brazil. Two electronic questionnaires were applied to the postnatal women, one baseline face-to-face and one follow-up telephone interview. Two other questionnaires were filled with information on patients' medical records and to assess hospital facilities. The primary outcome was the percentage of Caesarean sections (total, elective and according to Robson's groups). Secondary outcomes were: post-partum pain; breastfeeding initiation; severe/near miss maternal morbidity; reasons for maternal mortality; prematurity; low birth weight; use of oxygen use after birth and mechanical ventilation; admission to neonatal ICU; stillbirths; neonatal mortality; readmission in hospital; use of surfactant; asphyxia; severe/near miss neonatal morbidity. The association between variables were investigated using bivariate, stratified and multivariate model analyses. Statistical tests were applied according to data distribution and homogeneity of variances of groups to be compared. All analyses were taken into consideration for the complex sample design. Discussion: This study, for the first time, depicts a national panorama of labour and birth outcomes in Brazil. Regardless of the socioeconomic level, demand for Caesarean section appears to be based on the belief that the quality of obstetric care is closely associated to the technology used in labour and birth. Within this context, it was justified to conduct a nationwide study to understand the reasons that lead pregnant women to submit to Caesarean sections and to verify any association between this type of birth and it's consequences on postnatal health.

Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity & Bioaccessibility

A delivery system containing polymeric (Eudragit) nanoparticles has been developed for encapsulation and controlled release of bioactive flavonoids (quercetin). Nanoparticles were fabricated using a solvent displacement method. Particle size, morphology, and charge were measured by light scattering, electron microscopy and zeta-potential. Encapsulation efficiency (EE) and release profiles were determined using electrochemical methods. Molecular interactions within the particle matrix were characterized by X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. Antioxidant properties of free and encapsulated quercetin were analyzed by TBARS and fluorescence spectroscopy. Bioaccessibility of quercetin was evaluated using an in vitro digestion model. Relatively small (d a parts per thousand aEuro parts per thousand 370 nm) anionic polymeric nanoparticles were formed containing quercetin in a non-crystalline form (EE a parts per thousand aEuro parts per thousand 67 %). The main interaction between quercetin and Eudragit was hydrogen bonding. Encapsulated quercetin remained stable during 6 months storage and maintained its antioxidant activity. Quercetin bioaccessibility within simulated small intestinal conditions was improved by encapsulation. The knowledge obtained from this study will facilitate the rational design and fabrication of polymeric nanoparticles as oral delivery systems for encapsulation, protection, and release of bioactive compounds.

Vaginal morcellation: A new strategy for large gynecological malignant tumor extraction A pilot study

Objective. Evaluate feasibility and safety of a novel technique for uterine morcellation in patients scheduled for laparoscopic treatment of gynecologic malignances. Background. The laparoscopic management of uterine malignancies is progressively gaining importance and popularity over laparotomy. Nevertheless, minimal invasive surgery is of limited use when patients have enlarged uterus or narrow vagina. In these cases, conventional uterus morcellation could be a solution but should not be recommended due to risks of tumor dissemination. Methods. Prospective pilot study of women with endometrial cancer in which uterus removal was a realistic concern due to both organ size and proportionality. Brief technique description: after completion of total laparoscopic hysterectomy and bilateral anexectomy, a nylon with polyurethane Lapsac (R) is vaginally inserted into the abdomen; the specimen is placed inside the pouch that will be closed and rotated 180 degrees toward the vaginal vault and, posteriorly, pushed into the vaginal canal; in the transvaginal phase, the surgeon pulls the edges of the bag up to vaginal introitus and all vaginal walls will be covered; inside the pouch, the operator performs a uterus bisection-morcellation. Results. In our series of 8 cases, we achieved successful completion in all patients, without conversion to laparotomy. Average operative time, blood loss and length of hospitalization were favorable. One patient presented with a vesicovaginal fistula. Conclusion. The vaginal morcellation following oncologic principles is a feasible method that permits a rapid uterine extraction and may avoid a number of unnecessary laparotomies. Further studies are needed to confirm the oncological safety of the technique. (C) 2012 Elsevier Inc. All rights reserved.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

Awareness and knowledge of HPV, cervical cancer, and vaccines in young women after first delivery in São Paulo, Brazil - a cross-sectional study

Abstract Background The success of HPV vaccination programs will require awareness regarding HPV associated diseases and the benefits of HPV vaccination for the general population. The aim of this study was to assess the level of awareness and knowledge of human papillomavirus (HPV) infection, cervical cancer prevention, vaccines, and factors associated with HPV awareness among young women after birth of the first child. Methods This analysis is part of a cross-sectional study carried out at Hospital Maternidade Leonor Mendes de Barros, a large public maternity hospital in Sao Paulo. Primiparous women (15-24 years) who gave birth in that maternity hospital were included. A questionnaire that included questions concerning knowledge of HPV, cervical cancer, and vaccines was applied. To estimate the association of HPV awareness with selected factors, prevalence ratios (PR) were estimated using a generalized linear model (GLM). Results Three hundred and one primiparous women were included; 37% of them reported that they "had ever heard about HPV", but only 19% and 7%, respectively, knew that HPV is a sexually transmitted infection (STI) and that it can cause cervical cancer. Seventy-four percent of interviewees mentioned the preventive character of vaccines and all participants affirmed that they would accept HPV vaccination after delivery. In the multivariate analysis, only increasing age (P for trend = 0.021) and previous STI (P < 0.001) were factors independently associated with HPV awareness ("had ever heard about HPV"). Conclusions This survey indicated that knowledge about the association between HPV and cervical cancer among primiparous young women is low. Therefore, these young low-income primiparous women could benefit greatly from educational interventions to encourage primary and secondary cervical cancer prevention programs.

Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro

Abstract Background Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans. Results Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings. Conclusion Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

Tratamento cirúrgico e conservador da agenesia vaginal: análise de uma série de casos

OBJETIVO: Avaliar os resultados do tratamento da agenesia vaginal pela técnica cirúrgica de McIndoe-Bannister modificada e pela técnica de Frank. MÉTODOS: Este estudo retrospectivo foi conduzido com uma amostra de conveniência de 25 mulheres portadoras de agenesia vaginal em seguimento no Ambulatório de Ginecologia Infanto Puberal. Quinze mulheres foram submetidas à cirúrgica modificada de McIndoe-Bannister Grupo Cirúrgico e 10 fora tratadas com a técnica de Frank Grupo Frank. Para a análise comparativa entre essas duas amostras, foram considerados os seguintes parâmentros: vaginometria final, efeitos adversos e satisfação sexual após o tratamento. Esses dados foram obtidos por meio dos registros nos prontuários médicos. A satisfação sexual foi aferida por questão simples: como está sua vida sexual? RESULTADOS: Houve diferença em relação ao comprimento da vagina tanto naquelas submetidas à técnica de Frank (comprimento inicial 2,4±2,0 cm, após o tratamento 6,9±1,1 cm, p<0,0001), quanto naquelas submetidas à técnica cirúrgica (comprimento inicial 0,9±1,4 cm, após o tratamento 8,0±0,8 cm, p<0,0001). A vaginometria foi maior no Grupo Cirúrgico (Grupo Frank=7,0±0,9 cm versus Grupo Cirúrgico=8,0±0,8 cm, p=0,0005). Quarenta por cento do Grupo Cirúrgico tiveram complicações cirúrgicas. Não foram registradas complicações pela técnica de Frank. A satisfação sexual foi referida pela totalidade das pacientes. CONCLUSÃO: Os dados do presente estudo indicam que ambas as técnicas, cirúrgica e conservadora são eficientes para o tratamento da agenesia vaginal, resultando na construção da vagina favorável à realização do coito e com satisfação sexual. Os aspectos favoráveis da técnica de Frank estão relacionados com o baixo custo e baixos índices de complicações.

Nurses and care delivery to elderly women: a social phenomenological approach

OBJECTIVE: To understand how nurses see care delivery to elderly women. METHODS: In this phenomenological study, ten nurses working at Primary Health Care Units were interviewed between September 2010 and January 2011. RESULTS: In care delivery, nurses consider the elderly women's knowledge background and biographical situation, and also value the family's participation as a care mediator. These professionals have the acuity to capture these women's specific demands, but face difficulties to deliver care to these clients. Nurses expect to deliver qualified care to these women. CONCLUSION: The theoretical and methodological approach of social phenomenology permitted revealing that the nurse designs qualified care to elderly women, considering the possibilities in the context. This includes the participation of different social actors and health sectors, assuming collective efforts in action strategies and professional training, in line with the particularities and care needs of elderly women nurses identify.

Antifungal activity of lectins against yeast of vaginal secretion

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.