Thermal stability of extracellular hemoglobin of Glossoscolex paulistus: Determination of activation parameters by optical spectroscopic and differential scanning calorimetric studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS), optical absorption spectroscopy (UV-VIS) and differential scanning calorimetry (DSC). At pH 7.0, cyanomet-HbGp is very stable, no oligomeric dissociation is observed, while denaturation occurs at 56 degrees C, 4 degrees C higher as compared to oxy-HbGp. The oligomeric dissociation of HbGp occurs simultaneously with some protein aggregation. Kinetic studies for oxy-HbGp using UV-VIS and DES allowed to obtain activation energy (E(a)) values of 278-262 kJ/mol (DES) and 333 kJ/mol (UV-VIS). Complimentary DSC studies indicate that the denaturation is irreversible, giving endotherms strongly dependent upon the heating scan rates, suggesting a kinetically controlled process. Dependence on protein concentration suggests that the two components in the endotherms are due to oligomeric dissociation effect upon denaturation. Activation energies are in the range 200-560 kJ/mol. The mid-point transition temperatures were in the range 50-65 degrees C. Cyanomet-HbGp shows higher mid-point temperatures as well as activation energies, consistent with its higher stability. DSC data are reported for the first time for an extracellular hemoglobin. (C) 2010 Elsevier B.V. All rights reserved.

Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 M Da, It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole range. Our zeta-potential data are consistent with light scattering results. Average values apt obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5,0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.

Estrutura e dinâmica da interação de éteres de celulose com surfactantes

As técnicas de fluorimetria, condutometria, viscosimetria, turbidimetria, espalhamento de luz e espalhamento de raios-X a baixo ângulo (SAXS) foram empregadas no estudo da agregação de diferentes surfactantes aniônicos em presença de soluções aquosas diluídas de (hidroxipropil)celulose (HPC) 0,25% m/m, (hidroxipropilmetil)celulose (HPMC) 0,20% m/m e HPMC 0,10% m/m / NaCl 0,10 mol L-1. Também foram investigadas através de SAXS soluções concentradas de HPC (30, 40 e 50% m/m). Admitindo-se uma faixa geral de concentração, entre 10-4 e 10-1 mol L-1, foram utilizados neste estudo os surfactantes colato de sódio (CS), deoxicolato de sódio (DC), derivados dos sais biliares, e o alquilsintético dodecilsulafato de sódio (SDS). Observou-se que os polímeros contribuem diferentemente no processo de agregação de cada surfactante, evidenciado pela mudança dos valores da concentração de agregação crítica (CAC) em relação à concentração micelar crítica (CMC). Os resultados condutométricos confirmaram a interação éteres de celulose/sais biliares, embora a mesma tenha se mostrado mais fraca em relação a éteres de celulose/SDS. Os dados termodinâmicos demonstraram que a formação de agregados polímero/surfactante apresenta maior estabilidade do que as próprias micelas livres. Os resultados de viscosimetria e turbidimetria evidenciaram as diferenças estruturais entre HPC e HPMC, assim como entre os surfactantes. Através do espalhamento de luz dinâmico, verificou-se a existência de dois modos de correlação, rápido e lento. O primeiro é atribuído à cadeia polimérica isolada, agregados polímero/surfactante intramoleculares ou mesmo a micelas livres. Por sua vez, o modo lento relaciona-se a clusters poliméricos ou agregados polímero/surfactante intermoleculares. Adicionalmente, as curvas de distribuição dos tempos de relaxação demonstraram a influência de cada surfactante sobre a dinâmica dos polímeros. Tal influência é percebida antes mesmo da CAC, contrariando o modelo da interação polímero/surfactante proposto por Cabane. Os resultados de SAXS acusaram a formação de domínios líquido-cristalinos em xx soluções concentradas de HPC, assim como confirmaram a presença de micelas livres a altas concentrações de surfactantes nos sistemas diluídos. Em linhas gerais, os resultados indicaram a interação dos polímeros com SDS mais efetiva do que os mesmos polímeros e os sais biliares. No que tange à natureza do polímero, a HPC mostrou uma maior estabilidade na sua interação com os surfactantes do que a HPMC.

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.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Encapsulation of Local Anesthetic Bupivacaine in Biodegradable Poly(DL-lactide-co-glycolide) Nanospheres: Factorial Design, Characterization and Cytotoxicity Studies

Local anesthetic agents cause temporary blockade of nerve impulses productiong insensitivity to painful stimuli in the area supplied by that nerve. Bupivacaine (BVC) is an amide-type local anesthetic widely used in surgery and obstetrics for sustained peripheral and central nerve blockade. in this study, we prepared and characterized nanosphere formulations containing BVC. To achieve these goals, BVC loaded poly(DL-lactide-co-glycolide) (PLGA) nanospheres (NS) were prepared by nanopreciptation and characterized with regard to size distribution, drug loading and cytotoxicity assays. The 2(3-1) factorial experimental design was used to study the influence of three different independent variables on nanoparticle drug loading. BVC was assayed by HPLC, the particle size and zeta potential were determined by dynamic light scattering. BVC was determined using a combined ultrafiltration-centrifugation technique. The results of optimized formulations showed a narrow size distribution with a polydispersivity of 0.05%, an average diameter of 236.7 +/- 2.6 nm and the zeta potential -2.93 +/- 1,10 mV. In toxicity studies with fibroblast 3T3 cells, BVC loaded-PLGA-NS increased cell viability, in comparison with the effect produced by free BVC. In this way, BVC-loaded PLGA-NS decreased BVC toxicity. The development of BVC formulations in carriers such as nanospheres could offer the possibility of controlling drug delivery in biological systems, prolonging the anesthetic effect and reducing toxicity.

Identificação e avaliação de propriedades de polissacarídeos sulfatados de diferentes fontes naturais que possibilitem sua aplicabilidade biotecnológica

Sulfated polysaccharides (SP) are widely distributed in animals and seaweeds tissues. These polymers have been studied in light of their important pharmacological activities, such as anticoagulant, antioxidant, antitumoral, anti-inflammatory, and antiviral properties. On other hand, SP potential to synthesize biomaterials like as nanoparticules has not yet been explored. In addition, to date, SP have only been found in six plants and all inhabit saline environments. However, the SP pharmacological plant activities have not been carrying out. Furthermore, there are no reports of SP in freshwater plants. Thus, do SP from marine plants show pharmacological activity? Do freshwater plants actually synthesize SP? Is it possible to synthesize nanoparticles using SP from seaweed? In order to understand this question, this Thesis was divided into tree chapters. In the first chapter a sulfated polysaccharide (SPSG) was successfully isolated from marine plant Halodule wrightii. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan contains glucose and xylose. Several assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test; in the next chapter using different tools such as chemical and histological analyses, energy-dispersive X-ray analysis (EDXA), gel electrophoresis and infra-red spectroscopy we confirm the presence of sulfated polysaccharides in freshwater plants for the first time. Moreover, we also demonstrate that SP extracted from E. crassipes root has potential as an anticoagulant compound; and in last chapter a fucan, a sulfated polysaccharide, extracted from the brown seaweed was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution for hydrophobic chains of 1H NMR was approximately 93%. SNFfuc-TBa125 in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, measured bydynamic light scattering. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0 43.7% at SNFuc concentrations of 0.05 0.5 mg/ mL and RAEC non-tumor cell line proliferation displayed inhibition of 8.0 22.0%. On the other hand, nanogel improved CHO and RAW non-tumor cell line proliferation in the same concentration range. Flow cytometric analysis revealed that this fucan nanogel inhibited 786 cell proliferation through caspase and caspaseindependent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle

(-)-Hinokinin-loaded poly(d,l-lactide-co-glycolide) microparticles for Chagas disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Síntese e caracterização de tensoativos e estudo de equilíbrio de fase dos sistemas CnH2n+2/H2O/CnH2n+1COONa/But-OH

The growing utilization of surfactants in several different areas of industry has led to an increase on the studies involving solutions containing this type of molecules. Due to its amphiphilic nature, its molecule presents one polar part and one nonpolar end, which easily interacts with other molecules, being able to modify the media properties. When the concentration in which its monomers are saturated, the airliquid system interface is reached, causing a decrease in interfacial tension. The surfactants from pure fatty acids containing C8, C12 and C16 carbonic chains were synthesized in an alcoholic media using sodium hydroxide. They were characterized via thermal analysis (DTA and DTG) and via infrared spectroscopy, with the intention of observing their purity. Physical and chemical properties such as superficial tension, critical micelle concentration (c.m.c), surfactant excess on surface and Gibbs free energy of micellization were determined in order to understand the behaviour of these molecules with an aqueous media. Pseudo-ternary phase diagrams were obtained aiming to limit the Windsor equilibria conditions so it could be possible to understand how the surfactants carbonic chain size contributes to the microemulsion region. Solutions with known concentrations were prepared to study how the surfactants can influence the dynamic light scattering spectroscopy (DLS) and how the diffusion coefficient is influenced when the media concentration is altered. The results showed the variation on the chain size of the studied surfactant lipophilic part allows the conception of surfactants with similar interfacial properties, but dependent on the size of the lipophilic part of the surfactant. This variation causes the surfactant to have less tendency of microemulsionate oil in water. Another observed result is that the n-alcanes molecule size promoted a decrease on the microemulsion region on the obtained phase diagrams

Studies on the encapsulation of diclofenac in small unilamellar liposomes of soya phosphatidylcholine

The encapsulation of acid (AD) and sodium diclofenac (SD) in small unilamellar liposomes (SUV) as well as the interactions of the drug with the bilayer was studied. SUV was prepared by sonication from multilamellar liposomes containing soya phosphatidylcholine and diclofenac at various proportions. The size distribution obtained from dynamic light scattering showed that the incorporation of SD decreases significantly the size of the liposomes suggesting that the drug interacts with the bilayer of the liposomes. This size decrease is related with the phase transition of liposomes to mixed micelar solution. The encapsulation of the hydrophilic dye indocyanine green in the aqueous compartment of liposomes showed that the rate of captured dye decreases with SD concentration suggesting the transition of liposomes to mixed micelles. The P-31 NMR analysis indicates that SD interacts with the phosphate of phosphatidylcholine head groups. A schematic model for interaction of SD with phosphatidylcholine of the liposomes in which the diclofenac anion interacts with the ammonium group of the phospholipid and the dichloropheryl ring occupies a more internal site of bilayer near phosphate group was proposed. (C) 2004 Elsevier B.V. All rights reserved.

Potential tuberculostatic agent: Micelle-forming pyrazinamide prodrug

Pyrazinamide was condensed with the poly(ethylene glycol)-poly(aspartic acid) copolymer (PEG-PASP), a micelle-forming derivative was obtained that was characterized in terms of its critical micelle concentration (CMC) and micelle diameter. The CMC was found by observing the solubility of Sudan III in Poly(ethylene glycol)-poly(pyrazinamidomethyl aspartate) copolymer (PEG-PASP-PZA) solutions. The mean diameter of PEG-PASP-PZA micelles, obtained by analyzing the dynamic light-scattering data, was 78.2 nm. The PEG-PASP-PZA derivative, when assayed for anti-Mycobacterium activity, exhibited stronger activity than the simple drug.

On the incorporation of the non-steroidal anti-inflammatory naproxen into cationic O/W microemulsions

Microemulsions (ME) containing hexadecyltrimethylammonium bromide (HTAB)/ethanol as surfactant, isopropylmyristate (IM) or butylstearate (BS) as oil phase and aqueous buffer were studied. Pseudo-ternary phase diagrams of the investigated systems were obtained at constant surfactant/cosurfactant molar ratio (1:5) by titration in order to characterize the proportions between the components to obtain clear systems. Oil in water microemulsions were prepared in a wide range of phase volume (phi). UV-vis absorption spectra of naproxen at pH 5.5 showed that the solubility of Np increases significantly in the presence of O/W ME in high phase volumes. For both, IM and BS microemulsions, the dynamic light scattering experiments showed that the size of the oil droplets remains constant in low values of phi, increasing abruptly in high phi values. Phase solubility study revealed that for both IM and BS microemulsions, the drug incorporation followed a straight-line profile in all range of phi. The data could be analyzed through the phase-separation model and the association constants (K) calculated varied from 27 to 90 M-1, depending on the pH and on the microemulsion oil phase. (c) 2005 Elsevier B.V. All rights reserved.

Preparation of polymeric micelles for use as carriers of tuberculostatic drugs

Purpose: This paper focuses on the characterization of polymeric micelle-forming tuberculostatic prodrugs and the antimycobacterial activity of these prodrugs.Method: By the condensation of hydroxymethylpyrazinamide, isoniazid and rifampin with free carboxyl groups on the copolymer poly(ethyleneglycol)-poly(aspartic acid), micelle-forming carrier-drug conjugates were obtained. These micelles were characterized by dynamic light scattering, to measure the micelle diameter; by acid-base titration, to determine the percentage of carboxylic groups occupied by the tuberculostatic; by Sudan III solubility tests, to estimate the critical micelle concentration (CMC); and visual control and spectrophotometric measurement, to determine the stability of micelles. These micelles were tested in vitro against several Mycobacterium strains.Results: As expected, the size and distribution of the micelle-forming tuberculostatic prodrugs found to be small (78.2nm, 84.2nm and 98.9 nm) while the level of the drug conjugated was high (65.02-85.7%). Furthermore, the micelles were stable in vitro, exhibiting a low level of CMC and stronger antimycobacterial activity than the original drugs.Conclusion: the results demonstrate that polymeric micelles can be used as efficient carriers for drugs, which alone, exhibit undesired pharmacokinetics, poor solubility, and low stability. The synthesized micelle-forming tuberculostatic prodrugs opens a perspective of alternative prodrugs that prolong action and decrease the toxicity of the tuberculostatic drugs of choice.

Study of the diclofenac/phospholipid interactions with liposomes and monolayers

The interaction of diclofenae sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting In[1 - (Gamma/Gamma(infinity))] versus t(1/2) (C) 2004 Elsevier B.V. All rights reserved.