Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles

International audience

Creation of protein loaded biodegradable microparticles via ultrasonic atomization suitable for nasal delivery

Improved biopharmaceutical delivery may be achieved via the use of biodegradable microspheres as delivery vehicles. Biodegradable microspheres offer the advantages of maintaining sustained protein release over time whilst simultaneously protecting the biopharmaceutical from degradation. Particle samples produced by ultrasonic atomization were studied in order to determine a feed stock capable of producing protein loaded poly-ε-caprolactone (PCL) particles suitable for nasal delivery (i.e., less than 20 μm). A 40 kHz atomization system was used with a 6 mm full wave atomization probe. The effect of solids percent, feed flow rate, volumetric ratio of the polymer stock to the protein stock, and protein concentration in the protein stock on particle size characteristics were determined. It was shown that feed stocks containing 100 parts of 0.5 or 1.0% w/v PCL in acetone with one part 100 mg ml -1 BSA and 15 mg ml -1 PVA produced particles with a mass moment diameter (D[4,3]) of 13.17 μm and 9.10 μm, respectively in addition to displaying high protein encapsulation efficiencies of 93 and 95%, respectively. The biodegradable PCL particles were shown to be able to deliver encapsulated protein in vitro under physiological conditions.

Chitosan nanoparticle as protein delivery carrier - Systematic examination of fabrication conditions for efficient loading and release

Chitosan nanoparticles fabricated via different preparation protocols have been in recent years widely studied as carriers for therapeutic proteins and genes with varying degree of effectiveness and drawbacks. This work seeks to further explore the polyionic coacervation fabrication process, and associated processing conditions under which protein encapsulation and subsequent release can be systematically and predictably manipulated so as to obtain desired effectiveness. BSA was used as a model protein which was encapsulated by either incorporation or incubation method, using the polyanion tripolyphosphate (TPP) as the coacervation crosslink agent to form chitosan-BSA-TPP nanoparticles. The BSA-loaded chitosan-TPP nanoparticles were characterized for particle size, morphology, zeta potential, BSA encapsulation efficiency, and subsequent release kinetics, which were found predominantly dependent on the factors of chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The BSA loaded nanoparticles prepared under varying conditions were in the size range of 200-580 nm, and exhibit a high positive zeta potential. Detailed sequential time frame TEM imaging of morphological change of the BSA loaded particles showed a swelling and particle degradation process. Initial burst released due to surface protein desorption and diffusion from sublayers did not relate directly to change of particle size and shape, which was eminently apparent only after 6 h. It is also notable that later stage particle degradation and disintegration did not yield a substantial follow-on release, as the remaining protein molecules, with adaptable 3-D conformation, could be tightly bound and entangled with the cationic chitosan chains. In general, this study demonstrated that the polyionic coacervation process for fabricating protein loaded chitosan nanoparticles offers simple preparation conditions and a clear processing window for manipulation of physiochemical properties of the nanoparticles (e.g., size and surface charge), which can be conditioned to exert control over protein encapsulation efficiency and subsequent release profile. The weakness of the chitosan nanoparticle system lies typically with difficulties in controlling initial burst effect in releasing large quantities of protein molecules. (C) 2007 Elsevier B.V. All rights reserved.

Controlling microencapsulation and release of micronized proteins using poly(ethylene glycol) and electrospraying

The fabrication of tailored microparticles for delivery of therapeutics is a challenge relying upon a complex interplay between processing parameters and materials properties. The emerging use of electrospraying allows better tailoring of particle morphologies and sizes than current techniques, critical to reproducible release profiles. While dry encapsulation of proteins is essential for the release of active therapeutics from microparticles, it is currently uncharacterized in electrospraying. To this end, poly(ethylene glycol) (PEG) was assessed as a micronizing and solubilizing agent for dry protein encapsulation and release from electrosprayed particles made from polycaprolactone (PCL). The physical effect of PEG in protein-loaded poly(lactic-co-glycolic acid) (PLGA) particles was also studied, for comparison. The addition of 5–15 wt% PEG 6 kDa or 35 kDa resulted in reduced PCL particle sizes and broadened distributions, which could be improved by tailoring the electrospraying processing parameters, namely by reducing polymer concentration and increasing flow rate. Upon micronization, protein particle size was reduced to the micrometer domain, resulting in homogenous encapsulation in electrosprayed PCL microparticles. Microparticle size distributions were shown to be the most determinant factor for protein release by diffusion and allowed specific control of release patterns.

A facile pathway to prepare enzymatically degradable microcapsules with tunable capsule shell properties

Dextran sulfate (DS)/poly-L-lysine (PLL) microcapsules are fabricated by an in situ coacervation method using DS-doped CaCO3 microparticles as templates. Twinned superstructures or spherical CaCO3 microparticles are produced depending on DS concentration in the starting Solution. DS/PLL microcapsules with ellipsoidal or spherical outline are obtained after removal of templates in disodium ethylene diamine tetraacetate dehydrate (EDTA) without PLL. Their shell thickness and negative surface charges increase with the DS weight percentage in the templates. The surface potential of DS/PLL microcapsules.

Microencapsulation of casein hydrolysate by complex coacervation with SPI/pectin

The aim of this work was to encapsulate casein hydrolysate by complex coacervation with soybean protein isolate (SPI)/pectin. Three treatments were studied with wall material to core ratio of 1:1, 1:2 and 1:3. The samples were evaluated for morphological characteristics, moisture, hygroscopicity, solubility, hydrophobicity, surface tension, encapsulation efficiency and bitter taste with a trained sensory panel using a paired comparison test. The samples were very stable in cold water. The hydrophobicity decreased inversely with the hydrolysate content in the microcapsule. Encapsulated samples had lower hygroscopicity values than free hydrolysate. The encapsulation efficiency varied from 91.62% to 78.8%. Encapsulated samples had similar surface tension, higher values than free hydrolysate. The results of the sensory panel test considering the encapsulated samples less bitter (P < 0.05) than the free hydroly-state, showed that complex coacervation with SPI/pectin as wall material was an efficient method for microencapsulation and attenuation of the bitter taste of the hydrolysate. (C) 2009 Elsevier Ltd. All rights reserved.

Diphtheria toxoid conformation in the context of its nanoencapsulation within liposomal particles sandwiched by chitosan

Chitosan (alpha alpha-(1-4)-amino-2-deoxy-beta beta-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd alpha alpha-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, POencapsulation within ideal conditions. This was a technological breakthrough, because a simple solution, such as salt, addition avoided heterologous protein use.

Investigation of properties affecting controlled release of macromolecules from PLGA microspheres

Poly(lactide-co-glycolide), or PLGA, microspheres offer a widely-studied biodegradable option for controlled release of therapeutics. An array of fabrication methodologies have been developed to produce these microspheres with the capacity to encapsulate therapeutics of various types; and produce microspheres of a wide range of sizes for different methods of delivery. The encapsulation, stability, and release profiles of therapeutic release based on physical and thermodynamic properties has also been studied and modeled to an extent. Much research has been devoted to tailoring formulations for improved therapeutic encapsulation and stability as well as selective release profiles. Despite the breadth of available research on PLGA microspheres, further analysis of fundamental principles regarding the microsphere degradation, formation, and therapeutic encapsulation is necessary. This work aims to examine additional fundamental principles related to PLGA microsphere formation and degradation from solvent-evaporation of preformed polymer. In particular, mapping the development of the acidic microenvironment inside the microsphere during degradation and erosion is discussed. Also, the effect of macromolecule size and conformation is examined with respect to microsphere diameter and PLGA molecular weight. Lastly, the effects of mechanical shearing and protein exposure to aqueous media during microsphere formation are examined. In an effort to better understand the acidic microenvironment development across the microsphere diameter, pH sensitive dye conjugated to protein that undergoes conformational change at different acidic pH values was encapsulated in PLGA microspheres of diameters ranging from 40 µm to 80 µm, and used in conjunction with fluorescence resonance energy transfer to measure the radial pH change in the microspheres. Qualitative analysis of confocal micrographs was used to correlate fluorescence intensity with pH value, and obtain the radial pH across the center of the microsphere. Therapeutic encapsulation and release from polymeric microspheres is governed by an interconnected variety of factors, including the therapeutic itself. The globular protein bovine serum albumin, and the elongated and significantly smaller enzyme, lysozyme, were encapsulated in PLGA microspheres ranging from 40 µm to 80 µm in diameter. The initial surface morphology upon microsphere formation, release profiles, and microsphere erosion characteristics were explored in an effort to better understand the effect of protein size, conformation, and known PLGA interaction on the formation and degradation of PLGA microspheres and macromolecule release, with respect to PLGA molecular weight and microsphere diameter. In addition to PLGA behavior and macromolecule behavior, the effect of mechanical stresses during fabrication was examined. Two similar solvent extraction techniques were compared for the fabrication of albumin loaded microspheres. In particular, the homogeneity of the microspheres as well as capacity to retain encapsulated albumin were compared. This preliminary study paves the way for a more rigorous treatment of the effect of mechanical forces present in popular microsphere fabrication. Several factors affecting protein release from PLGA microspheres are examined herein. The technique explored for spatial resolution of the pH inside the microsphere proved mildly effective in producing a reliable method of mapping microsphere pH changes. However, notable trends with respect to microsphere size, PLGA molecular weight, and microsphere porosity were observed. Proposed methods of improving spatial resolution of the acidic microenvironment are also provided. With respect to microsphere formation, studies showed that albumin and lysozyme had little effect on the internal homogeneity of the microsphere. Rather, ionic interactions with PLGA played a more significant role in the encapsulation and release of each macromolecule. Studies also showed that higher instances of mechanical stress led to less homogeneous microspheres with lower protein encapsulation. This suggests that perhaps instead of or in addition to modifying the microsphere formation formulation, the fabrication technique itself should be more closely considered in achieving homogeneous microspheres with desired loading.

Scaffolds for controlled release of cartilage growth factors

International audience

Sustained release of TGF-β1 from biodegradable microparticles prepared by a new green process in CO2 medium

International audience

Co-encapsulation and characterisation of omega-3 fatty acids and probiotic bacteria in whey protein isolate-gum Arabic complex coacervates

Omega-3 fatty acids and probiotic bacteria were co-encapsulated in a single whey protein isolate (WPI)-gum Arabic (GA) complex coacervate microcapsule. Tuna oil (O) and Lactobacillus casei 431 (P) were used as models of omega-3 and probiotic bacteria, respectively. The co-microcapsules (WPI-P-O-GA) and L.casei containing microcapsules (WPI-P-GA) were converted into powder by using spray and freeze drying. The viability of L.casei was significantly higher in WPI-P-O-GA co-microcapsules than in WPI-P-GA. The oxidative stability of tuna oil was significantly higher in spray dried co-capsules than in freeze dried ones.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

Encapsulation of Hydrocortisone and Mesalazine in Zein Microparticles

Zein was investigated for use as an oral-drug delivery system by loading prednisolone into zein microparticles using coacervation. To investigate the adaptability of this method to other drugs, zein microparticles were loaded with hydrocortisone, which is structurally related to prednisolone; or mesalazine, which is structurally different having a smaller LogP and ionizable functional groups. Investigations into the in vitro digestibility, and the electrophoretic profile of zein, and zein microparticles were conducted to shed further insight on using this protein as a drug delivery system. Hydrocortisone loading into zein microparticles was comparable with that reported for prednisolone, but mesalazine loading was highly variable. Depending on the starting quantities of hydrocortisone and zein, the average amount of microparticles equivalent to 4 mg hydrocortisone, (a clinically used dose), ranged from 60-115 mg, which is realistic and practical for oral dosing. Comparatively, an average of 2.5 g of microparticles was required to deliver 250 mg of mesalazine (a clinically used dose), so alternate encapsulation methods that can produce higher and more precise mesalazine loading are required. In vitro protein digestibility revealed that zein microparticles were more resistant to digestion compared to the zein raw material, and that individual zein peptides are not preferentially coacervated into the microparticles. In combination, these results suggest that there is potential to formulate a delivery system based on zein microparticles made using specific subunits of zein that is more resistant to digestion as starting material, to deliver drugs to the lower gastrointestinal tract.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.