RESUMEN
Polystyrene sulfate (PSS) particles (301 nm mean diameter) were covered with single cationic dioctadecyldimethylammonium bromide (DDA) bilayers and used for antigen adsorption and presentation. The antigen was a mixture of purified 18/14 Taenia crassiceps proteins (18/14-Tcra). Firstly, the DDA/PSS assembly was characterized at 1mM NaCl and 5 x 10(9) PSS particles/mL over a range of DDA concentrations (0.001-1mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.01 mM DDA is enough to produce homodisperse and cationic bilayer-covered particles. Secondly, under these experimental conditions, 18/14-Tcra adsorption isotherms onto biomimetic particles or aluminium hydroxide (Al(OH)3) yield limiting adsorption of 0.36 and 1.32 mg protein/mg biomimetic particles or Al(OH)3, respectively. Finally, in mice, superior humoral and cellular immunoresponse from serum IgG and footpad swelling was obtained for antigen/biomimetic particles in comparison to conventional Al(OH)3. Cationic bilayer-covered particles are a novel, highly organized and, possibly, general immunoadjuvant for antigen presentation and subunit vaccine design.
Asunto(s)
Antígenos Helmínticos/inmunología , Portadores de Fármacos , Membrana Dobles de Lípidos , Lípidos/química , Poliestirenos/química , Compuestos de Amonio Cuaternario/química , Taenia/inmunología , Vacunas/inmunología , Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Hidróxido de Aluminio/inmunología , Animales , Formación de Anticuerpos , Antígenos Helmínticos/química , Cationes , Química Farmacéutica , Composición de Medicamentos , Femenino , Hipersensibilidad Tardía/inmunología , Luz , Ratones , Tamaño de la Partícula , Dispersión de Radiación , Cloruro de Sodio/química , Vacunas/químicaRESUMEN
The synthesis of hybrid particles was carried out by emulsion polymerization of styrene in complexes formed by carboxymethyl cellulose (CMC), a polyanion, and a cationic surfactant, cetyltrimethylammonium bromide (CTAB). CMC chains with variable molecular weights and degrees of substitution were tested. The polymerization condition chosen was that corresponding to CMC chains fully saturated with CTAB and to the onset of pure surfactant micelle formation, namely, at the critical aggregation concentration. The hybrid particles were characterized by zeta potential and light scattering measurements. The period of colloidal stability in the ionic strength of 2.0 mol L(-)(1) NaCl was observed visually. Upon increasing the CMC chain length, the particle characteristics remained practically unchanged, but the colloid stability was increased. The increase in the CMC degree of substitution led to particles with more negative zeta potential values. The adsorption of copper ions (Cu(2+)) on the surface of hybrid particles could be described by the Langmuir model, as determined by potentiometric measurements. The increase in the mean zeta potential values and X-ray absorption near-edge spectra evidenced the immobilization of Cu(2+) ions on the hybrid particles.
Asunto(s)
Carboximetilcelulosa de Sodio/química , Cobre/química , Poliestirenos/química , Cationes/química , Tamaño de la PartículaRESUMEN
Multiple uses for synthetic cationic liposomes composed of dioctadecyldimethylammonium bromide (DODAB) bilayer vesicles are presented. Drugs or biomolecules can be solubilized or incorporated in the cationic bilayers. The cationic liposomes themselves can act as antimicrobial agents causing death of bacteria and fungi at concentrations that barely affect mammalian cells in culture. Silica particles or polystyrene microspheres can be functionalized by coverage with DODAB bilayers or phospholipid monolayers. Negatively charged antigenic proteins can be carried by the cationic liposomes which generate a remarkable immunoadjuvant action. Nucleotides or DNA can be physically adsorbed to the cationic liposomes to be transferred to mammalian cells for gene therapy. An overview of the interactions between DODAB vesicles and some biomolecules or drugs clearly points out their versatility for useful applications in a near future.
Asunto(s)
Antibacterianos/química , Liposomas/química , Compuestos de Amonio Cuaternario/química , Tensoactivos/química , Portadores de Fármacos , Interacciones Farmacológicas , Escherichia coli/efectos de los fármacosRESUMEN
The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.
Asunto(s)
Antibacterianos/farmacología , Liposomas/farmacología , Compuestos de Amonio Cuaternario/farmacología , Tensoactivos/farmacología , Cationes , Electricidad , Floculación , Bacilos y Cocos Aerobios Gramnegativos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Especificidad de la Especie , Staphylococcus aureus/efectos de los fármacos , Propiedades de SuperficieRESUMEN
The 18 kDa antigenic protein from Mycobacterium leprae (P) or its N-acyl derivative (AP) was incorporated in dioctadecyldimethylammonium bromide (DODAB) liposomes in water or in phosphate-buffered saline (PBS). In water, 100% P incorporation in liposomes contrasts with 65% in PBS. There is 75-80% AP incorporation to liposomes in water against 55-65% in PBS, showing that attachment of hydrophobic residues to the protein, instead of increasing, further decreases incorporation to the liposomes. From protein adsorption on latex, P affinity is larger than AP affinity for the latex surface whereas limiting adsorption for AP is much larger than that obtained for P, possibly due to AP aggregation in solution. P-induced rupture of liposomes containing [14C]sucrose was evaluated from dialysis of protein/liposomes mixtures. In water, P incorporation to the liposomes causes leakage of radioactive contents contrasting with the absence of leakage for P incorporation in PBS. Immunization tests for delayed type hypersensitivity indicate a enhancement of cell-mediated immunological response towards P/DODAB complexes that is not obtained for the isolated protein. Absence of leakage for P in PBS is associated with a P "lying-over" on the liposome and optimization of protein presentation to the immunological system.
Asunto(s)
Adyuvantes Inmunológicos/química , Proteínas Bacterianas/química , Proteínas de Choque Térmico/química , Hipersensibilidad Tardía , Liposomas/química , Mycobacterium leprae/inmunología , Adsorción , Animales , Proteínas Bacterianas/inmunología , Cationes/metabolismo , Fenómenos Químicos , Química Física , Femenino , Proteínas de Choque Térmico/inmunología , Ratones , Ratones Endogámicos BALB C , Microesferas , Compuestos de Amonio Cuaternario/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Sacarosa/química , AguaRESUMEN
Small dioctadecyldimethylammonium chloride (DODAC) vesicles prepared by sonication fuse upon addition of NaCl as detected by several methods (electron microscopy, trapped volume determinations, temperature-dependent phase transition curves, and osmometer behavior. In contrast, small sodium dihexadecyl phosphate (DHP) vesicles mainly aggregate upon NaCl addition as shown by electron microscopy and the lack of osmometer behavior. Scatter-derived absorbance changes of small and large DODAC or DHP vesicles as a function of time after salt addition were obtained for a range of NaCl or amphiphile concentration. These changes were interpreted in accordance with a phenomenological model based upon fundamental light-scattering laws and simple geometrical considerations. Short-range hydration repulsion between DODAC (or DHP) vesicles is possibly the main energy barrier for the fusion process.