Protein Encapsulation via Porous CaCO₃ Microparticles Templating

Abstract

Porous microparticles of calcium carbonate with an average diameter of 4.75 microm were prepared and used for protein encapsulation in polymer-filled microcapsules by means of electrostatic layer-by-layer assembly (ELbL). Loading of macromolecules in porous CaCO3 particles is affected by their molecular weight due to diffusion-limited permeation inside the particles and also by the affinity to the carbonate surface. Adsorption of various proteins and dextran was examined as a function of pH and was found to be dependent both on the charge of the microparticles and macromolecules. The electrostatic effect was shown to govern this interaction. This paper discusses the factors which can influence the adsorption capacity of proteins. A new way of protein encapsulation in polyelectrolyte microcapsules is proposed exploiting the porous, biocompatible, and decomposable microparticles from CaCO3. It consists of protein adsorption in the pores of the microparticles followed by ELbL of oppositely charged polyelectrolytes and further core dissolution. This resulted in formation of polyelectrolyte-filled capsules with protein incorporated in interpenetrating polyelectrolyte network. The properties of CaCO3 microparticles and capsules prepared were characterized by scanning electron microscopy, microelectrophoresis, and confocal laser scanning microscopy. Lactalbumin was encapsulated by means of the proposed technique yielding a content of 0.6 pg protein per microcapsule. Horseradish peroxidase saves 37% of activity after encapsulation. However, the thermostability of the enzyme was improved by encapsulation. The results demonstrate that porous CaCO3 microparticles can be applied as microtemplates for encapsulation of proteins into polyelectrolyte capsules at neutral pH as an optimal medium for a variety of bioactive material, which can also be encapsulated by the proposed method. Microcapsules filled with encapsulated material may find applications in the field of biotechnology, biochemistry, and medicine.

Preserving Catalytic Activity and Enhancing Biochemical Stability of the Therapeutic Enzyme Asparaginase by Biocompatible Multilayered Polyelectrolyte Microcapsules

“…Enzyme-loaded microparticles formed by rapid mixing of Na 2 CO 3 and CaCl 2 in the presence of ScASNaseI exhibited an average size of 1−2 μm. We observed that microparticles of more homogeneous size and spherical morphology ( Figure S5) were obtained when lower than suggested 28,30 concentrations of Na 2 CO 3 and CaCl 2 were used. Therefore, the initial concentrations of Na 2 CO 3 and CaCl 2 were 0.15 M, rather than 1 M or 0.33 M, as reported before.…”

“…At this point, it is worth mentioning that ScASNaseI has a calculated isoelectric point (pI) of ∼5.3, indicating that, at the working pH of 7.5, the enzyme is negatively charged, while on the other hand, CaCO 3 particles are positively charged at pH values below 9.0. 30 Therefore, one may assume that high loading efficiency can be attributed to favorable electrostatic interactions between the protein and the calcium carbonate template, in addition to the high degree of adsorption of the enzyme to interior cavity surfaces of the template during the formation of the cores. When loading the enzyme following the same procedure at pH 6 (closer to the enzyme's pI), we observed a 5-fold higher protein loss in the coprecipitation as well as in the washing steps ( Figure S6).…”

Polymeric multilayer capsules delivering biotherapeutics

“…For this purpose, emulsions [10,11] are used to encapsulate hydrophobic compounds -these are dissolved into an organic phase which is subsequently coated with a multilayer film -while porous inorganic templates can adsorb both hydrophilic as well as hydrophobic molecules in their pores. Mesoporous silica (SiO 2 ) [12][13][14] and calcium carbonate (CaCO 3 ) [15][16][17][18] microparticles have been used for this purpose. Mesoporous silica (Figure 2) benefits from the advantage that such templates can be obtained in a monodisperse state and that they are stable over the whole pH range which allows the use of polyelectrolyte solutions with acidic pH.…”

“…So far the following biotherapeutics have been encapsulated in CaCO 3 templated PMLC: dextran, a-lactalbumin, lysosyme, horseradish peroxidise, glucose oxidase, catalase, ovalbumin, bovie serum albumin, achymotrypsin, insulin DNA and pronase. [15,17,18,[24][25][26][27][28] A third category of template particles that have gathered considerable attention in literature are hydrogel beads. Hydrogels are strongly hydrated 3D networks which, due to their aqueous environment offer good preservation of the biotherapeutics' bioactivity upon encapsulation.…”

Deposition Mechanisms in Layer-by-Layer or Step-by-Step Deposition Methods: From Elastic and Impermeable Films to Soft Membranes with Ion Exchange Properties

“…The success of this coating technology relies on its simplicity and versatility as it can be easily automated ( Figure 1) and, since that the LBL coatings can be deposited not only on planar substrates but also on colloids, nanoparticles [5] and in the pores [6] of all these materials.…”