M. Igartua

Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres

BSA was entrapped in particles of different sizes (200, 500 and 1000 nm) prepared from poly(D,L-lactic-co-glycolic) acid by a double emulsion method. The particles were given, either intranasally, orally or subcutaneously, to Balb/c mice and the serum IgG, IgG1 and IgG2a response elicited was compared to that obtained by the subcutaneous administration of either free antigen, free antigen emulsified 1:1 with Freunds Complete Adjuvant (FCA), or free antigen administered with Al(OH)(3). The administration of 1000 nm particles generally elicited a higher serum IgG response than that obtained with the administration of 500 or 200 nm sized nanospheres, the immune response for 500 nm particles being similar than that obtained with 200 nm by the subcutaneous and the oral route, and higher by the intranasal route. PLGA nanoparticles can elicit serum IgG2a responses by the three routes studied. No significant differences on the serum IgG2a/IgG1 ratios were found after the subcutaneous, the oral and the intranasal administration of the different spheres but those were in general higher compared to the administration of either free antigen or free antigen adsorbed to alum. The route of administration influences the serum IgG2a/IgG1 ratio after the administration of free antigen, but not after the administration of the particles. Therefore, differences on the total serum IgG response induced by particles of different sizes do not result in differences on the IgG1 or IgG2a-type immune responses, suggesting that the antigen processing and presentation is similar in all cases tested for PLGA particles.

Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates

The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

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.

Survival of different cell lines in alginate-agarose microcapsules

Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

Development and optimisation of alginate-PMCG-alginate microcapsules for cell immobilisation

Mechanical stability, uniformity of size, complete encapsulation of cells and optimal microenvironment are major challenges in the design and development of microcapsules for cell immobilisation purposes. In this work, a novel microcapsule chemistry based on polyelectrolyte complexation between alginate and poly(methylene-co-guanidine) (PMCG) is presented. We have characterised the effect of PMCG concentration and time of exposure on microcapsule diameter and membrane thickness, selecting a PMCG concentration of 0.5% (v/v) and an exposure time of 1 min as optimal parameters for a correct coating. Afterwards, the mechanically most resistant alginate-PMCG-alginate (A-PMCG-A) microcapsule type was chosen according to two different stability studies. Beads with a solid core and an inhomogeneous internal configuration resulted in stronger microcapsules. Further, the selected A-PMCG-A beads presented both an increased stability compared to classical Ca(2+)/alginate and alginate-poly-L-lysine-alginate (APA) microcapsules, and had an adequate microenvironment for cell viability. This new chemistry allows the controlled adjustment of microcapsule size and wall thickness, offering new alternatives for cell transplantation.

Biodegradable PLGA microspheres as a delivery system for malaria synthetic peptide SPf66

SPf66 is the first chemically synthesised vaccine to elicit a partial protective immune response against malaria. The aluminium hydroxide (alum)-adsorbed SPf66 vaccine is weakly immunogenic and of poor to moderate efficacy in humans. To investigate the possibility of improving SPf66 vaccine immunogenicity, a delivery system based on poly-D,L-lactide-co-glycolide (PLGA) microspheres was developed and the immune response induced after its subcutaneous administration into mice was evaluated. Microspheres were prepared by a solvent extraction/double emulsion (w/o/w) method and characterised for morphology, size, peptide loading, release profile and peptide integrity. The in vitro and in vivo results obtained showed that there was no apparent effect of the encapsulation procedure on SPf66 integrity and immunogenicity. The subcutaneous administration of microspheres showed a significantly higher immune response (serum IgG levels) than that obtained with alum adsorbed SPf66 and it was comparable to that of SPf66 emulsified with Freunds adjuvant (FA). These observations illustrate the potential of PLGA microspheres as a delivery system for chemically synthesised antigens.

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.

Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres.

Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigens microencapsulation in PLGA microspheres by double emulsion method. Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.

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.