Angela Maria Moraes

Kanamycin incorporation in lipid vesicles prepared by ethanol injection designed for tuberculosis treatment

The primary goal of this study was the production of liposomes encapsulating kanamycin for drug administration by inhalation. The selected drug is indicated for multiresistant tuberculosis, and administration through inhalation allows both local delivery of the drug to the lungs and systemic therapy. The ethanol injection method used for the liposome production is easily scaled up and is characterized by simplicity and low cost. Vesicles were prepared using different lipid compositions, including hydrogenated soybean phosphatidylcholine and cholesterol (SPC/Chol), egg phosphatidylcholine and cholesterol (EPC/Chol), distearoyl phosphatidylcholine and cholesterol (DSPC/Chol), distearoyl phosphatidylcholine, dimyristoyl phosphatidylethanolamine and cholesterol (DSPC/DMPE/Chol), dipalmitoyl phosphatidylcholine and cholesterol (DPPC/Chol) and dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol and cholesterol (DPPC/DPPG/Chol). The effects of different operational conditions for vesicle production and drug encapsulation were evaluated, aiming at a compromise between final process cost and suitable vesicle characteristics. The best performance concerning drug incorporation was achieved with the DSPC/Chol system, although its production cost was considerably larger than that of the natural lipids formulations. Encapsulation efficiencies up to 63% and final drug to lipid molar ratios up to 0.1 were obtained for SPC/Chol vesicles presenting mean diameters of 132 nm incubated at 60°C with the drug for 60 min at an initial drug‐to‐lipid molar ratio of 0.16.

Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring.

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.

Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture

Insect cell culture has become increasingly useful for the production of heterologous proteins as well as of baculovirus polyhedra, and several different culture media formulations can be employed for this purpose. The goal of this work was to assess the potential of lyophylized milk whey ultra filtration retentate (MWR) when associated to yeast extract (YE), glucose and Pluronic F68 (PF68) to partially replace fetal calf serum (FBS) in Spodoptera frugiperda Sf9 cells culture in Graces medium, aiming AgMNPV baculovirus production. Batch cultivation results showed that the yeast extract and the milk whey concentrate effectively increased cell concentration to about half the level commonly verified for the Sf900II serum-free medium. A 4-fold increase in viable cell concentration was achieved when employing 5% (w/v) MWR, 8 g/L YE, 1% (v/v) of FBS and 2.7 g.l -1 of glucose, resulting in 1.6 x 10 7 polyhedra.ml -1 after infection with baculovirus.

Characterization of Liposomal Systems Entrapping Boron-Containing Compounds in Response to pH Gradients

Boron neutron capture therapy (BNCT) is based on the nuclear reaction that occurs when a stable isotope, Boron-10, is irradiated with low-energy thermal neutrons to yield ionizing Helium and Lithium ions that are highly damaging and usually lethal to cells. The successful treatment of cancer by BNCT requires the selective concentration of Boron-10 within malignant tumors. Liposomes have been used as drug delivery vehicles for in vivo application, including several anticancer agents. The ability of two Boron-containing compounds, 1-p-borono-phenylalanine (BPA) HCl and o-carboranylpropylamine chloride (CPA) to accumulate within unilamellar liposomes passively and in response to a transmembrane pH gradient are compared. Characterization of the obtained systems is performed for conventional and polyethylene glycol (PEG)-modified (stealth) liposomes, in terms of lipid and drug contents, vesicle size and stability. The results indicate that BPA can be successfully encapsulated in conventional liposomes by passive loading, while the active loading approach is more suitable for the entrapment of CPA.

Comparison of the Cultivation of Wild and Transfected Drosophila Melanogaster S2 Cells in Different Media

Insect cells have been increasingly employed for the production of recombinant proteins. One of the most widely used dipteran cells in transfection studies is the Schneider 2 (S2) cell line, established from Drosophila melanogaster embryonic tissue. In this work, the growth and proliferation of wild and transfected S2 cells expressing the G glycoprotein from rabies virus (GPV) were compared employing different culture media. For the transfected (S2AcGPV2) cell contruction, the plasmid pGPV encoding the sequence of interest and the vectors pAc 5.1/V5-His A under the control of the Drosophila actin promoter were utilized. The selection vector pCoHygro carrying genes coding for hygromicin-inactivating enzymes was also employed and the cells were transfected using lipofectin. Due to wide utilization of TNM-FH and TC100 media (both requiring supplementation with fetal bovine serum) for insect cells, their low cost and the low protein content of SF900II medium, these three media and the mixture TNM-FH- SF900II (1:1) were evaluated for the choice of a suitable media for S2 cells. The results indicated that the wild and the transfected cells presented different growth characteristics in the distinct media. In SF900II medium, larger accumulation and consumption of lactate were observed for the wild cell culture. In TC100, however, S2AcGPV2 cells did not produce lactate. In TNM-FH, the S2 cells presented lower growth rate (P max=0.0078 h -1 ) when compared to the other media (P max=0.0375 h -1 and P max=0.0112 h -1 for SF900II and TC100, respectively), with an accentuated viability drop during the first days of culture. The mixture of TNM-FH and SF900II at a 1:1 volume ratio resulted in cell growth (P max=0.0377 h -1 ) similar to that observed in SF900II medium only, allowing significant culture media cost reduction. Transfected

Metabolic Modeling of Drosophila melanogaster Cells Under the Balanced Growth Condition

Although insect cells have quite an analogous performance to mammalian cells, in general, they show a higher cell growth, ammonium (NH4+) release and alanine (ALA) formation, besides lower lactate (LAC) accumulation rates, even in glucose excess conditions. In the present work, the metabolic pathway of Drosophila melanogaster Schneider 2 cells (S2AcGPV2) transfected to express a rabies virus glycoprotein (RVGP) cultivated in a serum free media was proposed and utilized for flux analysis by employing measurements of biomass and extracellular species during the exponential phase of batch cultivation. The resulted elementary fluxes were translated into a set of overall reactions and a dynamical mass model was then established. Such methodology allowed establishing an unstructured model to be oriented by the main metabolic reactions.

Preparation and Characterization of Inclusion Complexes of Cyclodextrins and Tuberculosis Primary Treatment Drugs

Tuberculosis is an infectuous disease of slow recovery that is the second cause of death according to the World Health Organization. Tuberculosis has affected about one-third of the world’s population, killing about three million patients each year and beeing the single most important infectious cause of death on earth (Bloom, 1992). It is common to observe in the conventional treatment side effects, systemic toxicity, resistance of the strains to various antibiotics, and short time in between medicine intake. Hard-to-follow long-term treatments have shown poor adhesion by patients and this limits effectiveness and cure. On the other hand, when followed correctly, conventional therapies are successful but not free from toxicity. Nowadays, some different approaches have been proposed for the therapy of tuberculosis, using encapsulated drugs or delivery systems. Cyclodextrins (CDs) are products of high added value that have found applications as carriers for therapeutic drugs. Inclusion complexes of drugs with cyclodextrins may modify drug properties such as physical and chemical stability and bioavailability (Szejtli, 1988). Otherwise inclusion complexes in aqueous solutions can be associated to other drug delivery systems such as liposomes, increasing the efficacy of healing and decreasing unwanted side effects of drugs (Loukas et al., 1995). These properties make cyclodextrins attractive candidates for improving the treatment regimes for serious diseases characterized by slow healing such as tuberculosis.