RESUMEN
Microsphere size is a primary determinant of solute release velocity. We present here a rational way for producing PLGA microspheres with different and controlled sizes. The following process variables were studied: Stirring velocity during the second emulsion step, dispersed and continuous phases volume ratio, and poly(vinyl alcohol) concentration in the continuous phase. A full factorial experimental design 2(3) with triplicate at the central point was used to determine the influence of variables on PLGA microsphere mean size. The stirring velocity and poly(vinyl alcohol) concentration were the main variables at 0.95 significance level. An influence of PVA and stirring velocity on microspheres size is observed, there is no correlation for DP/CP volume ratio on size of microspheres. By combining the two variables--the stirring velocity and poly(vinyl alcohol) concentration, the surface response was analyzed. The increase of poly(vinyl alcohol) concentration with concomitant increase on stirring velocity produced microspheres with the lower sized. In contrast the lower poly(vinyl alcohol) concentration and the lower stirring velocity used produced the higher microspheres sized. Uniformly spherical and smooth microspheres (4-15 microm of diameter) were obtained. No significant difference was observed on Ponca S loading within the experimental region. Our results open the possibility of formulating PLGA microspheres with custom sizes performing a minimum of experiments as required for specific applications.
Asunto(s)
Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Nanoestructuras/química , Nanotecnología/métodos , Poliglactina 910/química , Propiedades de Superficie , Antígenos/química , Materiales Biocompatibles/química , Microesferas , Tamaño de la Partícula , Alcohol Polivinílico/químicaRESUMEN
Liposomes have been used since the 1970's to encapsulate drugs envisaging enhancement in efficacy and therapeutic index, avoidance of side effects and increase in the encapsulated agent stability. The major problem when encapsulating snake venoms is the liposomal membrane instability caused by venom phospholipases. Here the results obtained encapsulating Crotalus durissimus terrificus and a pool of Bothropic venoms within liposomes (LC and LB, respectively) used to produce anti-venom sera are presented. The strategy was to modify the immunization protocol to enhance antibody production and to minimize toxic effects by encapsulating inactivated venoms within stabilized liposomes. Chemically modified venoms were solubilized in a buffer containing an inhibitor and a chelating agent. The structures of the venoms were analyzed by UV, CD spectroscopy and ELISA. In spite of the differences in the helical content between natural and modified venoms, they were recognized by horse anti-sera. To maintain long-term stability, mannitol was used as a cryoprotectant. The encapsulation efficiencies were 59 % (LB) and 99 % (LC), as followed by filtration on Sephacryl S1000. Light scattering measurements led us to conclude that both, LB (119 ±47 nm) and LC (147±56 nm) were stable for 22 days at 4 °C, even after lyophilization. Genetically selected mice and mixed breed horses were immunized with these formulations. The animals did not show clinical symptoms of venom toxicity. Both, LB and LC enhanced by at least 30 % the antibody titers 25 days after injection and total IgG titers remained high 91 days after immunization. The liposomal formulation clearly exhibited adjuvant properties.
Asunto(s)
Antivenenos/inmunología , Venenos de Crotálidos/química , Venenos de Crotálidos/inmunología , Animales , Bothrops , Venenos de Crotálidos/administración & dosificación , Crotalus , Caballos , Inmunización , Liposomas , Ratones , Fosfolípidos/químicaRESUMEN
Chitosan, alpha-(1-4)-amino-2-deoxy-beta-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide present in crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid linear molecular structure make this macromolecule ideal as drug carrier. The association between chitosan and liposomes was carefully described, where REVs (reverse phase evaporation vesicles) were sandwiched by chitosan. The usage of these particles in vaccine formulation is here proposed for the first time in the literature. The Chitosan-REVs now stabilized by polyvinilic alcohol were the vehicle for Diphtheria toxoid (Dtxd). Round chitosan-sandwiched REVs (REVs-Chi) particles of 373 +/- 17 nm containing 65% Dtxd were obtained. After 200 min of incubation in a simulated gastric fluid, 70% of the Dtxd was liberated from REVs-Chi in comparison to 100% of Dtxd liberated from pure REVs. In PBS, the Dtxd liberation from REVS-Chi was about 60%. Mice were immunized with Dtxd encapsulated within REVs-Chi and with other REVs/Dtxd formulations adsorbed onto Freund adjuvant or alumen [AIF and Al(OH)(3)]. The response patterns and the immune maturity were measured by IgG(1) and IgG(2a) titrations. REVs-Chi containing Dtxd elicited both antibodies production giving the animals higher immune response and selectivity. It was interesting that the memory of those mice immunized with REVs-Chi containing Dtxd enhanced, after booster, antibody production by 47% in contrast with 17 and 7% in mice immunized with the antigen vehiculated in REVs-AIF or REVs-Al(OH)(3), respectively.