Karsa / Stephenson Encapsulation and Controlled Release

1. Auflage 1993
ISBN: 978-1-84569-821-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 178 Seiten

Reihe: Woodhead Publishing Series in Food Science, Technology and Nutrition

Encapsulation and controlled release combines basic information on the subject with details of the latest research, making it suitable for both newcomers to the field and those with experience of encapsulation technology. It will also be of great interest to those working on water-soluble or dispersible polymers, as well as application chemists and biochemists in diverse areas.

Autoren/Hrsg.

Karsa, D R

Stephenson, R A

Leseproben

The Use of Gelatin and Alginate for the Immobilization of Bioactive Agents
E. Schacht1*, J.C. Vandichel1, A. Lemahieu2, N. De Rooze1 and S. Vansteenkiste1,     1DEPARTMENT OF ORGANIC CHEMISTRY, BIOMATERIALS RESEARCH GROUP, UNIVERSITY OF GHENT, GHENT, B-9000, BELGIUMz; 2SANOFI BIOINDUSTRIES BENELUX, BRUSSELS, B-1400, BELGIUM 1 Introduction
For many bioactive agents (pharmaceuticals, pesticides, enzymes) there is a need to prolong the duration of activity, reduce the side effects and have a more efficient utilization of the active agent. In the particular case of biocatalysts (enzymes, microorganisms) there may be a need for an increased stability, a more practical processibility or a better recovery of the biocatalyst. One approach to achieve these objectives is by optimizing the dosage form in which the bioactive agent is presented to the biological environment. For that purpose natural and synthetic polymers can serve as depot systems in which the agent can be incorporated and from which it is subsequently released over an extended and ideally controllable period of time. For preparing such reservoir systems, natural polymers like alginate and gelatin are attractive candidate matrices because of their accessibility and biodegradability. In the present paper we like to discuss some techniques for the immobilization of pesticides, respectively enzymes and microorganisms, in alginate, respectively in gelatin. 2 Immobilization of a microorganism and enzymes in gelatin
Study of the crosslinkage of gelatin by dextran dialdehydes
Gelatin is a protein material derived from collagen by alkaline or acidic treatment. The most frequent sequence identified in the protein backbone is -gly-pro-X-1. Gelatin is composed of a-chains (monomeric), ß-chains (dimers) and ?-chains (trimers). A typical molecular weight for the a-chains is about 80,000 daltons2. The pendant -amino groups in the lysine and hydroxylysine residues are suitable sites for the crosslinkage or hardening of gelatine. A variety of hardening procedures have been described in the literature3,4. Among the most important organic hardeners are formaldehyde and glutaraldehyde. Polyaldehydes such as polyacrolein5 or periodate oxidized starch8 and plant gums7 were also reported as suitable crosslinking agents. Polymeric crosslinkers have, in comparison with their low molecular weight analogues, a limited diffusivity in a gelatin gel matrix. This may be advantageous for certain applications, e.g. the immobilization of microorganisms, as will be further demonstrated in this paper. Recently, we have used periodate oxidized dextrans as crosslinking agents for gelatin. This system was applied for immobilizing biocatalysts in gelatin beads. Periodate oxidation of polysaccharides is a convenient method for preparing polyaldehyde derivatives. Since in dextran the repeat unit contains 3 vicinal hydroxyl groups, partial oxidation leads to different types of dialdehydes8 (Fig.1):
Figure 1 Dialdehyde structures in partial oxidized dextran The ratio of the different dialdehyde structures depends on the initial dextran/periodate ratio and the reaction conditions8. The characterization of the dextran dialdehydes used in this study is summarized in Table 1. Table 1 Characterization of dextran dialdehydes Type of dextran Degree of oxidation (%) meq aldehydes/g T-40 5 0.7 T-40 10 1.4 T-40 20 2.3 T-40 50 6.0 T-40 70 7.9 T-70 20 2.1 T-200 20 2.2 T-500 20 2.2 The degree of oxidation is defried as the percentage of anhydroglumsides oxidized. The polyaldehydes can easily react in aqueous medium with amines or polyamines like gelatin with the formation of Schiff’s base conjugates. In the latter case a crosslinked hydrogel is formed. The (simplified) structure of a gelatin/dextran polyaldehyde network is schematically represented in Fig. 2.
Figure 2 Schematic presentation of the structure of a dextran dialdehyde crosslinked gelatin. The rate of crosslinkage of gelatin depends on a number of factors, including structural parameters of gelatin (isoelectric point, molecular weight, gel strength), the molecular weight and degree of oxidation of the dextran dialdehyde and the reaction conditions (pH, ion strength and temperature). The effect of some of these parameters will now be discussed in more detail. A typical crosslinking experiment is as follows: gelatin (0.5 g) and dextran dialdehyde (0.5 g), prepared as described by Ruys9, are dissolved separately in a buffer solution (5 ml). Both solutions are then mixed during 30 seconds and a 1 ml sample is transferred into the recipient of a low shear rotation viscometer (Haake, RV 100-CV-100 with Mooney-Erwart coni-cylindrical measuring system). The viscosity is then recorded as a function of time at a shear of 120 rpm. The effect of the degree of oxidation of the dextran on the onset of gelation is illustrated in Figure 3. It is clear that the gelation proceeds more rapidly with increasing aldehyde content in the dextran derivative.
Figure 3 Influence of the degree of oxidation of dextran on the rate of gelation of gelatin (I.P. 7.0, pH=8, 40°C), — 5%, *—* 10%, ?—? 20%, — 50% and — 70%. It was further observed that the gelation occurs faster as the molecular weight of the dextran increases (Fig. 4). For a given degree of oxidation (number of dialdehydes per hundred anhydroglucose units) the functionality per molecule increases as the molecular weight increases. Hence, the critical conversion at which gelation takes off will decrease with increasing molecular weight.
Figure 4 Influence of the molecular weight of the dextran dialdehyde on the rate of gelation (gelatin I.P.=7.0, pH=8, 40°C, degree of oxidation = 20%), — T10, ?—? T40, — T70, *—* T200 and — T500. For a given type of gelatin and dextran dialdehyde, the rate of gelation is strongly dependent on the pH of the buffer, the ionic strength and the type of the buffer used. Figure 5 and 6 illustrate the effect of buffer concentration and the nature of the buffer on the time to onset of gelation for a series of reactions in different buffers of varying ionic strength. For phosphate and maleate buffers, the gelation proceeds faster with increasing buffer strength. On the other hand, in acetate buffer the influence is minimal whereas in citrate buffer gelation occurs slower with increasing ionic strenght of the medium. A plausible explanation for this influence of the nature of the buffer on the gelation time may be a difference in salting-in capacity. It has been reported before that phosphates have a salting-in effect on hydrogels10. It was proposed that the phosphate may complex with the hydrophobic parts of the gel matrix. An analogous complexation of phosphate and maleate with the gelatin matrix would result in a coil expansion caused by charge repulsions and consequently an increased availability of the lysine amine groups for reaction with the aldehydes.
Figure 5 Crosslinkage of gelatin (I.P.=6.4, pH=6, 40°C) in different buffers of ion strength I=0.05 mol l-1, *—* phosphate, — citrate, x—x acetate and — maleate buffer.
Figure 6 Crosslinkage of gelatin (I.P.=6.4, pH=6, 40°C) in different buffers of ion strength I=0.2 mol l-1, ?—? phosphate, *—* citrate, — acetate and — maleate buffer. The above data clearly demonstrate that the rate of crosslinkage of gelatin by dextran dialdehyde can be widely varied by the choice of the reagents and the reaction conditions. This will be exploited for the immobilization of biocatalysts in crosslinked gelatin beads as will be discussed below. Immobilization of a microorganism and enzymes in crosslinked gelatin beads
Immobilization procedure. For the immobilization of microorganisms or enzymes, a gelatin of isoelectric point 7.25 and gel strength 200 Bloom was selected. The dextran dialdehyde is derived from dextran T-40 (MW 36,000), the degree of oxidation is 20%. The procedure followed for the bead preparation is illustrated in Fig....