Amaia Esquisabel

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres.

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freunds complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.

Influence of formulation variables on the in-vitro release of albumin from biodegradable microparticulate systems

Poly(D,L-lactide-co-glycolide) microspheres containing BSA were prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface absorbed protein, encapsulation efficiency and release kinetics. The influence of two formulation variables (the procedure to obtain the first emulsions and the lyophilization of the microspheres once obtained) on the physical characteristics and release behaviour of the microspheres was also investigated. Sonicated microspheres were smooth and spherical, with a mean particle size of 20 microns and an encapsulation efficiency of 81%. When the first emulsion was prepared by vortex mixing the particles were irregular and porous, with a mean size of 31 microns and a lower encapsulation efficiency (56%). The sonication allows a more homogeneous emulsion as well as a lower percentage of albumin adsorbed on the surface. The in vitro release profile was described as a biexponential process with an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a second sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release of BSA was dependent on the preparation method. The greatest burst release was found for microspheres formulated using the vortex mixer, 58% of the encapsulated protein was released during the first 24 h, whereas sonicated microspheres released 32.2%. This burst effect could be reduced by lyophilizing the microspheres following their preparation. The amount of protein released decreased to 28.3% and 51.6% in sonicated and non-sonicated microspheres respectively, when they were lyophilized.

Stability of BSA encapsulated into PLGA microspheres using PAGE and capillary electrophoresis

Bovine serum albumin (BSA, Mw 66 200 Da) has been encapsulated as a model protein drug within poly(d,l-lactide-co-glycolide) (PLGA 50:50) microspheres using a w/o/w double emulsion method. The microspheres prepared were smooth and spherical with a mean particle size of 1.32 μm. The total protein loading and surface-associated protein were 8.61 and 16.60%, respectively. The microspheres showed a triphasic in vitro release profile with an initial burst effect due to the release of the protein adsorbed on the microsphere surface, a second sustained release phase due to protein diffusion through the pores or channels formed in the polymer matrix, and a third phase due to polymer bioerosion. The purpose of this paper was to evaluate the effect of the microencapsulation process on the integrity of the entrapped protein using polyacrylamide gel electrophoresis and capillary electrophoresis. The stability of the protein released during in vitro assays was also assessed. The results obtained showed that there was no apparent effect of the drastic encapsulation conditions (contact with dichloromethane (DCM), probe sonication, and vigorous shaking) on the structural integrity of the protein. On the other hand, it was found that after 1 week of incubation the protein released from the microspheres starts to hydrolyze to smaller fragments, probably due to a significant decrease in the medium pH as a result of the accumulation of the polymer degradation products.

Production of BCG alginate-PLL microcapsules by emulsification/internal gelation

A biocompatible emulsification method for microencapsulation of live cells and enzymes within a calcium alginate matrix applied to Bacillus Calmette-Guérin (BCG) has been developed. Small-diameter alginate beads (microcapsules) were formed via internal gelation of an alginate solution emulsified within vegetable oil. Five different oils (sesame, sweet almond, perhydrosqualene, camomile and jojoba) were used. The rheological analysis of the oils showed a Newtonian behaviour, with viscosities = 30.0, 37.7, 51.2, 59.3 and 67.1 mPa.s for perhydrosqualene, jojoba, camomile, sesame and sweet almond oil respectively. The particle size of the microcapsules obtained ranged from 30.3 microns for the microcapsules prepared with sweet almond oil to 57.0 microns for those made with perhydrosqualene. The mean particle diameter obtained was found to be dependent on the viscosity of the oil employed, according to the equation: phi (micron) = 76.6-0.628 eta (mPa.s) (r2 = 0.943). The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Freeze-drying of the microcapsules was carried out to ensure their stability during storage. Two batches of microcapsules (those prepared with sesame and jojoba oil) and four types of cryoprotectors (glucose, trehalose, mannitol and sorbitol), at three concentration levels (5, 10 and 20% w/v) were studied. The parameters evaluated were particle size, physical appearance, reconstitution of lyophilizates and microscopical evaluation. For both batches of microcapsules the best results were obtained with trehalose 5%, showing particle sizes of 42.1 microns in the case of the microcapsules prepared with sesame oil, and of 45.3 microns for those prepared with jojoba.

A new oral vaccine candidate based on the microencapsulation by spray-drying of inactivated Vibrio cholerae

The aim of this work was to evaluate the microencapsulation by spray-drying of inactivated Vibrio cholerae, using methacrylic copolymers Eudragit® L30D-55 and FS30D. The microparticles obtained presented a particle size around 3.0 μm. The preparation temperature affected the morphology and the antigenicity of microparticles, but it did not affect the V. cholerae content. In vitro release studies showed that in acid medium less than 5% of bacteria was released, and in neutral medium, Eudragit® L30D-55 microparticles released 86% after 24 h, whereas FS30D released less than 30%. Rats inoculated with microparticles exhibited vibriocidal antibody titres. Microencapsulation by spray-drying of inactivated V. cholerae could be proposed as a method to obtain an oral vaccine which provides controlled release of the bacteria.

Determination of salbutamol enantiomers by high-performance capillary electrophoresis and its application to dissolution assays

Capillary zone electrophoresis was successfully applied to the chiral separation of salbutamol after addition of a suitable cyclodextrin chiral selector to the electrophoresis buffer. Parameters important in achieving enantiomeric separation are cyclodextrin type, mobile phase pH and applied field strength. In our study, salbutamol enantiomeric separation was obtained with the following conditions: heptakis (2,6-di-O-methyl)-beta-cyclodextrin in 40 mM Tris (pH 2.5) and at 15 kV, obtaining a 3.09 resolution with migration times of 13.74 min for (R)-salbutamol and 13.98 min for (S)-salbutamol. Linearity, limit of quantitation, precision and accuracy were established using this method. The calibration curve was linear in a range of 1-40 micrograms ml-1 of racemic salbutamol (0.5-20 micrograms ml-1 of each enantiomer). This method was applied to evaluate the enantioselective release of salbutamol and taking into account the hypothesis that one enantiomer of a chiral drug would be released faster than the other from a pharmaceutical dosage form containing a racemic drug and a chiral excipient. For this purpose, matrix tablets formed by chiral excipients such as hydroxypropylmethylcellulose (HPMC) were considered. The release of the enantiomers of salbutamol from the formulations containing HPMC was found to be equivalent, with constant dissolution values (K) of 1.187 +/- 0.223% min-n for (R)-salbutamol and 1.076 +/- 0.268% min-n for (S)-salbutamol.

The state-of-the-art of approved and under-development cholera vaccines.

Cholera remains a huge public health problem. Although in 1894, the first cholera vaccination was reported, an ideal vaccine that meets all the requirements of the WHO has not yet been produced. Among the different approaches used for cholera vaccination, attenuated vaccines represent a major category; these vaccines are beneficial in being able to induce a strong protective response after a single administration. However, they have possible negative effects on immunocompromised patient populations. Both the licensed CVD103-HgR and other vaccine approaches under development are detailed in this article, such as the Vibrio cholerae 638 vaccine candidate, Peru-15 or CholeraGarde(®) and the VA1.3, VA1.4, IEM 108 VCUSM2 and CVD 112 vaccine candidates. In another strategy, killed V. cholerae vaccines have been developed, including Dukoral(®), mORCAX(®) and Sanchol™. The killed vaccines are already sold, and they have successfully demonstrated their potential to protect populations in endemic areas or after natural disasters. However, these vaccines do not fulfill all the requirements of the WHO because they fail to confer long-term protection, are not suitable for children under two years, require more than a single dose and require a distribution chain with cold storage. Lastly, other vaccine strategies under development are summarized in this review. Among these strategies, vaccine candidates based on alternative drug delivery systems that have been reported lately in the literature are discussed, such as microparticles, proteoliposomes, LPS subunits, DNA vaccines and rice seeds containing toxin subunits. Preliminary results reported by many groups working on alternative delivery systems for cholera vaccines demonstrate the importance of new technologies in addressing old problems such as cholera. Although a fully ideal vaccine has not yet been designed, promising steps have been reported in the literature resulting in hope for the fight against cholera.

Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis

Lung impairment is the most life-threatening factor for cystic fibrosis patients. Indeed, Pseudomonas aeruginosa is the main pathogen in the pulmonary infection of these patients. In this work, we developed sodium colistimethate loaded lipid nanoparticles, namely, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), as a strategy to enhance the antimicrobial therapy against P. aeruginosa in cystic fibrosis patients. The nanoparticles obtained displayed a 200-400 nm size, high drug entrapment (79-94%) and a sustained drug release profile. Moreover, both SLN and NLC presented antimicrobial activity against clinically isolated P. aeruginosa. The integrity of the nanoparticles was not affected by nebulization through a mesh vibrating nebulizer. Moreover, lipid nanoparticles appeared to be less toxic than free sodium colistimethate in cell culture. Finally, an in vivo distribution experiment showed that nanoparticles spread homogenously through the lung and there was no migration of lipid nanoparticles to other organs, such as liver, spleen or kidneys.

Effect of lecithins on BCG-alginate-PLL microcapsule particle size and stability upon storage.

Alginate-PLL microcapsules containing BCG were prepared by emulsification/ internal gelation of an alginate solution dispersed within a vegetable oil containing lecithins as emulsifiers. The lecithins studied were soy bean lecithin at 0.1, 0.5, 1 and 2%concentration; and dried egg yolk lecithin at 0.1, 0.25, 0.5 and 1%. The microcapsule particle size showed a dependence upon the amount and type of lecithin added to the oil. Increasing the emulsifying agent concentration was found to reduce particle size, from 50.9mum obtained when lecithins were not used in the emulsification step, to 13.9mum obtained when 1% dried egg yolk lecithin was employed. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-fried using 5%trehalose as cryoprotectant in order to preserve their stability upon storage. The stability of the microcapsules was assayed over 12 months at room temperature, finding that alginate-PLL microcapsules were stable up to 6 months. Moreover, in the case of microcapsules prepared with lecithins, a significant increase in particle size was observed, from 16.9mum at the beginning of the study to 25.2mum at 12 months storage.Alginate-PLL microcapsules containing BCG were prepared by emulsification/internal gelation of an alginate solution dispersed within a vegetable oil containing lecithins as emulsifiers. The lecithins studied were soy bean lecithin at 0.1, 0.5, 1 and 2% concentration; and dried egg yolk lecithin at 0.1, 0.25, 0.5 and 1%. The microcapsule particle size showed a dependence upon the amount and type of lecithin added to the oil. Increasing the emulsifying agent concentration was found to reduce particle size, from 50.9 microm obtained when lecithins were not used in the emulsification step, to 13.9 microm obtained when 1% dried egg yolk lecithin was employed. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-dried using 5% trehalose as cryoprotectant in order to preserve their stability upon storage. The stability of the microcapsules was assayed over 12 months at room temperature, finding that alginate-PLL microcapsules were stable up to 6 months. Moreover, in the case of microcapsules prepared with lecithins, a significant increase in particle size was observed, from 16.9 microm at the beginning of the study to 25.2 microm at 12 months storage.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.