Antimicrobial Particles from Cationic Lipid and Polyelectrolytes

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CM C, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta= 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of p. aeruginosa viability (10(7) CFU/mL) at 1 or 2 mu g/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 mu g/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.

Supramolecular assemblies of a new class of nonplanar cationic metalloporphyrins and anionic metallophthalocyanines

The heteroaggregation behavior between a new class of nonplanar cationic beta-octabrominated meso-alkylpyridinium zinc(II)-porphyrins (beta-Br(8)(ZnP)) and anionic tetrasulfonated metallophthalocyanines (MTSPc, M = Ni(II) and Cu(II)) has been studied by UV-Vis electronic spectroscopy, in dimethylsulfoxide (DMSO) solution. The heteroaggregate stoichiometry and the association constants were determined by means of Job plots. Dimers and unexpected trimers, taking into account the existence of axially coordinated DMSO molecules to the central metal in both beta-Br(8)(ZnP) and MTSPc complexes, are formed in solution. The spectroscopic properties of the heteroaggregates are markedly different from those observed in the correspondent planar cationic derivatives, the heteroaggregates showing major changes predominantly in the beta-Br(8)(ZnP) Soret band region and minor effects in the MTSPc Q bands. The observed changes in the Soret band region (red/blue shifts, decrease in the absorption intensities) depend on the nature of the alkyl substituent attached to the meso-pyridinium group. The greater versatility of the nonplanar porphyrins accommodating the meso-substituents in out-of-plane and in-plane conformations is proposed to explain the observed stoichiometries and the differences on the heteroaggregates spectroscopic properties for each beta-Br(8)(ZnP) compound. The likely conformations assumed by the meso-substituents in these beta-Br(8)(ZnP) compounds and its spectroscopic characteristics are in accordance with the participation of the substituents as the main factor on the extent of the observed red-shifted spectra in nonplanar porphyrins. The obtained association constants (K(IP)) for the dimers and trimers are lower than those previously found for the similar planar cationic porphyrin systems, due to the lack of extensive pi-pi interactions and to the less effective approximation between the ionic groups, resulting in loosened heteroaggregates, particularly for the trimeric systems. Furthermore, the experimental results suggest that the NiTSPc is more distorted in DMSO solution than the CuTSPc derivative, favoring the interaction with the nonplanar beta-Br(8)(ZnP) compounds. (C) 2007 Elsevier B.V. All rights reserved.

Stereoselective Nucleophilic Addition of Potassium Alkyltrifluoroborates to Cyclic N-Acyliminium Ions: a Simple and Mild Approach to Chiral 5-Alkyl-pyrrolidin-2-ones

The stereoselective nucleophilic addition of potassium alkyltrifluoroborates to cyclic N-acyliminium ions derived from N-benzyl-3,4,5-triacetoxy-pyrrolidin-2-one, which affords 5-substituted-pyrrolidin-2-ones, is described. The products are obtained in moderate to good yields and are produced predominantly as the anti diastereomer.

Micellar properties of surface active ionic liquids: A comparison of 1-hexadecyl-3-methylimidazolium chloride with structurally related cationic surfactants

Ionic liquids, ILs, carrying long-chain alkyl groups are surface active, SAIIs. We investigated the micellar properties of the SAIL 1-hexadecyl-3-methylimidazolium chloride, C(16)MeImCl, and compared the data with 1-hexadecylpyridinium chloride, C(16)PYCl, and benzyl (3-hexadecanoylaminoethyl)dimethylammonium chloride, C(15)AEtBzMe(2)Cl. The properties compared include critical micelle concentration, cmc; thermodynamic parameters of micellization; empirical polarity and water concentrations in the interfacial regions. In the temperature range from 15 to 75 degrees C, the order of cmc in H(2)O and in D(2)O is C(16)PYCl > C(16)MeImCl > C(15)AEtBzMe(2)Cl. The enthalpies of micellization, Delta H(mic)(degrees), were calculated indirectly from by use of the van`t Hoff treatment; directly by isothermal titration calorimetry, ITC. Calculation of the degree of counter-ion dissociation, alpha(mic), from conductivity measurements, by use of Evans equation requires knowledge of the aggregation numbers, N(agg), at different temperatures. We have introduced a reliable method for carrying out this calculation, based on the volume and length of the monomer, and the dependence of N(agg) on temperature. The N(agg) calculated for C(16)PyCl and C(16)MeImCl were corroborated by light scattering measurements. Conductivity- and ITC-based Delta H(mic)(degrees) do not agree; reasons for this discrepancy are discussed. Micelle formation is entropy driven: at all studied temperatures for C(16)MeImCl; only up to 65 degrees C for C(16)PyCl; and up to 55 degrees C for C(15)AEtBzMe(2)Cl. All these data can be rationalized by considering hydrogen-bonding between the head-ions of the monomers in the micellar aggregate. The empirical polarities and concentrations of interfacial water were found to be independent of the nature of the head-group. (C) 2010 Elsevier Inc. All rights reserved.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [

Deposition of selenium thin layers on gold surfaces from sulphuric acid media: Studies using electrochemical quartz crystal microbalance, cyclic voltammetry and AFM

In this paper we report here new considerations about the relationship between the mass and charge variations (m/z relationship) in underpotential deposition (UPD), bulk deposition and also in the H(2)Se formation reaction. Nanogravimetric experiments were able to show the adsorption of H(2)SeO(3) on the AuO surface prior to the voltammetric sweep and that, after the AuO reduction, 0.40 monolayer of H(2)SeO(3) remains adsorbed on the newly reduced Au surface, which was enough to gives rise to the UPD layer. The UPD results indicate that the maximum coverage with Se(ads) on polycrystalline gold surface corresponds to approximately 0.40 monolayer, in good agreement with charge density results. The cyclic voltammetry experiments demonstrated that the amount of bulk Se obtained during the potential scan to approximately 2 Se monolayers, which was further confirmed by electrochemical quartz crystal microbalance (EQCM) measurements that pointed out a mass variation corresponding of 3 monolayers of Se. In addition, the Se thin films were obtained by chronoamperometric experiments, where the Au electrode was polarized at +0.10V during different times in 1.0 M H(2)SO(4) + 1.0 mM SeO(2). The topologic aspects of the electrodeposits were observed in Atomic Force Microscope (AFM) measurements. Finally, in highly negative potential polarizations, the H(2)Se formation was analyzed by voltammetric and nanogravimetric measurements. These finding brings a new light on the selenium electrodeposition and point up to a proposed electrochemical model for molecule controlled surface engineering. (c) 2009 Elsevier Ltd. All rights reserved.

Cationic and neutral phenylmercury(II) complexes with heterocyclic thione ligands. X-ray structures of [HgPh(dmpymtH)][BF(4)] center dot H(2)O and [{HgPh}(2)(mu-dtu)]

The neutral complex [HgPh(dmpymt)] 1 (dmpymtH = 4,6-dimethylpyrimidine-2(1H)-thione) reacts with HBF(4) to give the cationic complex [HgPh(dmpymtH)][BF(4)] 2. The X-ray molecular structure of the later revealed a [2+1] coordination sphere about the mercury(II) atom (C-Hg-S and Hg center dot center dot center dot N). In the dinuclear complex [(HgPh)(2)(mu-dtu)] 3 [dtuH(2) = 2,4(1H,3H)-pyrimidinedithione or dithiouracil] the coordination spheres are also [2+1] although dissimilar regarding the Hg center dot center dot center dot N secondary bonds. NMR spectroscopy ((1)H, (13)C and (199)Hg) studies were undertaken in solution and the results discussed in the light of the X-ray structures. (C) 2008 Elsevier B. V. All rights reserved.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Taking Advantage of Electrostatic Interactions To Grow Langmuir-Blodgett Films Containing Multilayers of the Phospholipid Dipalmitoylphosphatidylglycerol

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

Determination of Analgesics (Dipyrone and Acetaminophen) in Pharmaceutical Preparations by Cyclic Voltammetry at a Copper(II) Hexacyanoferrate(III) Modified Carbon Paste Electrode

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Selectivity of the cyclic carbonate formation by fixation of carbon dioxide into epoxides catalyzed by Lewis bases

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

Inclusion complex of piroxicam with beta-cyclodextrin and incorporation in cationic microemulsion. In vitro drug release and in vivo topical anti-inflammatory effect

Topical formulations of piroxicam were evaluated by determination of their in vitro release and in vivo anti-inflammatory effect. The in vitro release assay demonstrated that the microemulsion (ME) systems provided a reservoir effect for piroxicam release. However, the incorporation of the ME into carboxyvinilic gel provoked a greater reduction in the release of piroxicam than the ME system alone. Anti-inflammatory activity was carried out by the cotton pellet granuloma inhibition bioassay. Topical anti-inflammatory effect of the piroxicam inclusion complex/ME contained in carboxyvinilic gel showed significant inhibition of the inflammation process (36.9%, P < 0.05). Subcutaneous administration of the drug formulations showed a significant effect on the inhibition of inflammation, 68.8 and 70.5%, P <0.05, when the piroxicam was incorporated in ME and in the combined system beta -cyclodextrin (B-CD)/ME, respectively, relative to the buffered piroxicam (42.2%). These results demonstrated that the ME induced prolonged effects, providing inhibition of the inflammation for 9 days after a single dose administration. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide-cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the alpha(1,2,3)-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO-cyclic GMP pathway. (C) 2003 Elsevier Ltd. All rights reserved.