Effect of the composition, pH and type of drying on enzyme release from dried and wet polyelectrolyte capsules

SANTAGAPITA, PATRICIO R.; MAZZOBRE, M. FLORENCIA; BUERA, M. PILAR

Trieste, Italia

Conferencia; Frontiers in Water Biophysics Conference; 2010

University of Trieste

The encapsulation procedure takes advantages of the immobilization process, which facilitates a wide variety of chemical and biological process. In search of suitable matrices for biomolecule stabilization and delivery, ionically crosslinked hydrogels, such as alginate capsules have been thoroughly investigated. On the other hand, the conservation of labile biomolecules is generally performed in dehydrated or frozen media, being saccharides (especially trehalose) among of the most used excipient. Alginate and pectin are of the most popular anionic polyelectrolytes used for capsule preparation and form gel structures in contact with CaCl2 solution; chitosan, a cationic polyelectrolyte, is frecuently used for coating beads produced after alginate or pectin gelation. As an alternative to the conventional composition for the preparation of capsules, trehalose was added to alginate, alginate chitosan-coated and alginate/ pectin beads in the external calcium gelation media in order to evaluate the release of an enzyme (invertase) on dried and wet capsules. Drying was performed by two different methods: freeze-drying and vacuum-drying. Invertase (β-fructofuranosidase) is an enzyme of high technological impact in the production of invert sugar. The purpose of this work was to evaluate the effect of capsule composition, pH and drying methods on the release of invertase from dried and wet capsules. Methodology Capsules were obtained by dropping alginate (1% w/v) or alginate/pectin (1% w/v, 3:1 mass ratio) solution containing invertase (0,06 mg/mL) on a 2.5 % w/v CaCl2 solution with or without trehalose (20% w/v) or with or without chitosan (0.25% w/v) with a syringe, under stirring. Both solutions were adjusted to pH value of 3.8. A peristaltic pump was employed for regulating the flux of the dropping solution. Wet beads were dried by freeze-drying (12 h) and vacuum-drying (4 h at 25°C). Release studies were performed in wet beads in 50 mM acetate buffer. Three different pH values around the isoelectric point of invertase were studied: pH 3.8, 4.5 and 5.5. The remaining enzymatic activity was determined spectrophotometrically by employing di-nitro salicylic acid method. FTIR and DSC determinations, and optical and FEG-SEM observations were conducted in order to complete the characterization. A semi-empirical model for determining the transport mechanism of the enzyme from the beads was applied (1): Where At is the enzyme activity at time t; A is the enzyme maximum activity; k is a constant related to geometrical, structural and macromolecular network of the beads; t is time of release; n is a parameter indicating the type of transport mechanism. Results and discussion Spherical capsules were obtained with an average diameter of 1.5 mm. Alginate, pectin and chitosan interaction was confirmed by FTIR and DSC analysis. The rate of invertase release from wet beads was mainly dependent on pH: the slowest rate occurred at pH 3.8, at which the enzyme and alginate had global opposite charges. The evaluation of the release kinetics through the semi-empirical model allowed to relate the obtained data with the transport mechanism. The parameter n provided adequate information on the transport mechanism. At pH 3.8 and 4.5 the wet beads showed a fickean diffusional transport (n = 0.43), while at pH 5.5 an anomalous behavior was observed involving both diffusion and the relaxation of polymer chains (0.43 < n < 0.85). Dried beads showed a departure from fickean mechanism, but not through the anomalous behavior since n < 0.43, which was attributable to the porous nature of the beads. Enzyme release was slower from freeze-dried capsules than from vacuum-dried capsules. Conclusions The pH value does not only influence the relative charges of polymers and enzyme but also influences the relaxation of polymers chains which affects enzyme release. The transport mechanism form wet and dried capsules was not affected by their composition but it was mainly affected by pH and drying treatment. Freeze-dried beads showed to be an excellent system for enzyme conservation and also presented release kinetics slower than wet beads. The presence of trehalose in alginate beads improved enzyme conservation and slower the enzyme release. pH and drying methods should be regarded as important factors in order to control the release of enzyme from alginate beads. A second polymer in the beads coating formulation also may change the transport mechanism and the rate of release. References 1. P. L. Ritger and N.A. Peppas, 1987. J. Controlled Release, 5, 23-36.