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Preparation and properties of glutaraldehyde cross-linked whey protein-based microcapsules containing theophylline
Journal of Controlled Release 61 (1999) 123–136
Preparation and properties of glutaraldehyde cross-linked whey protein-based microcapsules containing theophylline S.J. Lee, M. Rosenberg* Department of Food Science and Technology, University of California, Davis, CA 95616, USA Received 26 February 1999; received in revised form 30 April 1999; accepted 30 April 1999
Abstract Whey protein-based microcapsules containing a model drug, theophylline, were prepared in organic phase, using glutaraldehyde-saturated toluene. In all cases, spherical microcapsules, ranging from ,400 to 1000 mm in diameter, were obtained. Results indicated that core crystals were embedded throughout the wall matrix. In all cases, retention efficiency of theophylline was higher than 74% and was not affected by cross-linking conditions. Results of theophylline release in simulated intestinal and gastric fluids at 378C indicated that the diffusion-governed core release was significantly affected by size of microcapsules, cross-linking conditions, and by type of dissolution medium. In all cases, core release in simulated intestinal fluid was faster than in simulated gastric fluid. 1999 Elsevier Science B.V. All rights reserved. Keywords: Controlled release; Microcapsules; Whey proteins; Cross-linking; Theophylline
1. Introduction Microcapsules for controlled and / or sustained core release have been developed for pharmaceutical and other applications using both synthetic and natural polymers. Natural polymers suitable for developing microcapsules for such applications include mainly proteins and polysaccharides [1–5]. Proteins have been shown to present a significant potential as wall materials for microcapsules for controlled and sustained release of different drugs and most of the reported research has been focused on different albumins [6–10]. In recent years, suitability of milk proteins as wall *Corresponding author. Tel.: 11-530-752-4682; fax: 11-530754-8145. E-mail address: [email protected] (M. Rosenberg)
materials for microcapsules and microspheres for controlled release of different drugs has been investigated. Bovine caseins were used as wall materials in developing microcapsules for sustained release of different drugs [7,11–15]. Results of these studies indicated that glutaraldehyde cross-linked caseinbased microcapsules exhibited, to a varying extent, properties desired for controlled core release. Recently, the concept of using whey proteins as microencapsulating agents has been established [16]. A series of studies has indicated that whey proteins exhibit excellent microencapsulating properties and are suitable for microencapsulation of volatile and non-volatile core materials [17–23]. It has been indicated that wall systems of spray-dried microcapsules consisting of whey proteins exhibited molecular sieve type porosity [24,25] and provided effective protection against core oxidation [26]. Most of the
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00108-X
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reported research effort pertinent to microencapsulating properties of whey proteins has been focused on developing water-soluble microcapsules. Functionality of whey proteins as wall matrices in microcapsules designed for controlled core release has been investigated only to a very limited extent. Waterinsoluble whey protein-based microcapsules, containing apolar model core, prepared by double emulsification and cross-linking with glutaraldehyde were reported by Lee and Rosenberg [27]. Core retention during microencapsulation was about 90% and microcapsules were insoluble in water at different pH and temperature conditions [27]. Heelan and Corrigan [28] reported on preparation and core release from whey protein-based microcapsules cross-linked with aqueous glutaraldehyde. Their results indicated that, in most cases, rate of core release was significantly faster than that desired in pharmaceutical applications. Although physicochemical properties of whey proteins suggest that they may be suitable for developing microcapsules for controlled and sustained core release, more research is needed in this regard. The objectives of our study were to prepare water-insoluble whey protein-based microcapsules containing a model water-soluble drug and to investigate microstructural and core release characteristics of these microcapsules.
deionized water. In all cases, the pH of wall solution was 7.2. Theophylline (1.6 g) was dispersed in 4 g of wall solution and this mixture was then suspended (at 258C) in a dispersion mixture consisting of 80 ml of dichloromethane and 50 ml of hexane containing 1% biomedical polyurethane (Thermedix Inc., Woburn, MA), in a 250-ml round-bottom flask. A stainless steel half-moon shaped paddle was used to stir the suspension at 900 rev. / min for 3 min. Protein-based wall matrices of microcapsules were cross-linked by glutaraldehyde (Sigma Chemical Co.). After stirring for 3 min, either 7.5, 15, or 30 ml of glutaraldehyde-saturated toluene (GAST), prepared according to the method reported by Longo et al. [8], was added to the suspension and cross-linking was carried out for 1 or 3 h (900 rev. / min, 258C). Wet microcapsules were separated from the dispersing solvent mixture by filtration and then washed five times, 2 min each, with 200 ml of a 1:1 mixture of dichloromethane / hexane. Microcapsules were then washed (for 2 min each) with 200 ml of 1% sodium bisulfite, with 200 ml of distilled water, and finally with 200 ml of acetone. Washed microcapsules were dried in a vacuum oven at 508C overnight. Dry microcapsule powders were separated, by sieving, into large (diameter .700 mm), medium (diameter 450–700 mm) and small (diameter ,450 mm) microcapsules and the weight proportion of capsules included in each size category was recorded. Dry capsules were kept in a desiccator pending analysis.
2. Materials and methods
2.3. Determination of total theophylline content 2.1. Wall and core materials Whey protein isolate containing 95.6% (w / w on dry basis) protein (WPI, Le Sueur Isolates, Le Sueur, MN) was used as a wall material. Theophylline (Sigma Chemical Co., St. Louis, MO) served as a model water-soluble core.
2.2. Preparation of theophylline loaded microcapsules Theophylline-containing, chemically cross-linked WPI-based microcapsules were prepared using a modification of the procedure reported by Latha et al. [7] and by Latha and Jayakrishnan [14]. Wall solution containing 20% (w / w) WPI was prepared in
Theophylline content in microcapsules included in each of the size categories was determined using a modification of the method reported by Latha and Jayakrishnan [14]. Microcapsule samples (5–10 mg) were powdered in a mortar, placed in screw-capped test tubes, and extracted with 50 ml methanol for 12 h. During extraction, test tubes were continuously rotated using a Model 7637 Roto-Troque Rotator (Cole-Parmer Instrument Company, Chicago, IL). Samples (2 ml) of supernatant were filtered through a 0.22 mm filter (Millex-GV, Durapore Membrane, Millipore, Bedford, MA), the filtrate was diluted appropriately with methanol, and absorbance at 274 nm was determined (Bausch & Lomb Spectronic 1001). The amount of extracted theophylline was
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calculated using a standard curve of theophylline in methanol. Based on the amount of core determined in this way and the known weight of the microcapsules used for analysis the core content of microcapsules was expressed as % (w / w). A preliminary study confirmed that the above methodology allowed release of 100% of the encapsulated core after 12 h extraction and was therefore suitable for determination of microcapsules core content and, subsequently, for calculating core retention. Our results agreed with previous reports that validated the suitability of methanol extraction for determination of theophylline content in milk protein-based and other microcapsules [7,14,15,30]. Core retention was expressed as the ratio (in %) of core content determined in microcapsules to a theoretical core content assuming 100% core retention during the microencapsulation process.
2.4. Release of theophylline from microcapsules Microcapsules (total weight 5.5 mg) were placed in a 200-ml double wall glass beaker containing 180 ml of either enzyme-free simulated intestinal fluid (SIF) or enzyme-free simulated gastric fluid (SGF) prepared according to the US Pharmacopeia [29]. The suspension was stirred (100 rev. / min) at 378C using a floating stir bar (Fisher Scientific, Pittsburgh, PA). Aliquots (3 ml) were withdrawn periodically, using a 5-ml syringe equipped with a 0.2-mm syringe filter (Fisher Scientific Co., Santa Clara, CA) and theophylline concentration was determined spectrophotometrically at 274 nm. In no case were microcapsules removed along with dissolution medium. In all cases, an equal volume of dissolution medium was immediately added to maintain a constant volume. Samples were withdrawn until three successive aliquots showed no increase in optical absorbance (274 nm). The amount of theophylline released from the microcapsules, at a given time, was calculated using standard curves of theophylline in SGF and SIF and expressed as percentage of total theophylline content of the investigated microcapsules.
2.5. Scanning electron microscopy ( SEM) Structural features of microcapsules were investi-
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gated using SEM. For examination of outer structure, microcapsules were attached to a SEM stub using a two-sided adhesive tape (Ted Pella, Redding, CA). In order to investigate the inner structure of microcapsules, microcapsules were attached to an SEM stub, as before, and then fractured by moving a razor blade perpendicularly through the layer of microcapsules. The microstructure of microcapsules from which core has been released was also investigated. Core was released from microcapsules into SIF or SGF, using the procedure described above for the core release experiment. After reaching complete core release, microcapsules were separated from dissolution medium, dried as described above, and then were examined by SEM. Specimen preparation was as described above for core-containing microcapsules. In all cases, specimens were coated with gold using a model E-5050 Polaron Sputter Coater (Bio-Rad, San Jose, CA), and analyzed using an ISI DS-130 scanning electron microscope (International Scientific Instrument Inc., Pleasanton, CA) operated at 10 kV. Micrographs were prepared using a Type 55 Polaroid film (Polaroid Corp., Cambridge, CA).
2.6. Statistical analysis In all cases, replicate samples were analyzed at least in duplicate (n54). Significance of results was tested by ANOVA using the SigmaStat software (Jandel Scientific Software, San Rafael, CA). Significance of differences was defined at P,0.05.
3. Results and discussion
3.1. Size distribution and microstructural features In all cases, regardless of cross-linking conditions, spherical microcapsules were obtained. In all cases, microcapsule powders exhibited a relatively wide particle size distribution. The proportion of microcapsules larger than 700 mm ranged from 52 to 65%, that of microcapsules with diameter of 450– 700 mm ranged from 22 to 36%, and proportion of microcapsules smaller than 450 mm ranged from 5 to 15%. Results did not indicate effect of cross-linking conditions on microcapsule size distribution. This and the results of SEM suggested that at a given
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stirring condition, the size of microcapsules was mainly affected by number of core crystals included in wall / core droplets, prior to cross-linkage. These results agreed with those of Latha and Jayakrishnan [14] who reported an increase in microcapsule size with theophylline load. Outer topography and inner structure of microcapsules are presented in Figs. 1 and 2. It has been reported that significant shape distortions were observed when WPI-based microcapsules were crosslinked with aqueous glutaraldehyde [28]. Such difficulties were not observed in our study thus highlighting the effectiveness of cross-linking by glutaraldehyde via organic phase in allowing rapid fixation of the shape and surface morphology of the microcapsules [30]. In all cases, outer surfaces of microcapsules were not smooth and exhibited irregularities of different shapes (Fig. 1A–E). The size and shape of these structural features suggested that they represented a footprint of theophylline crystals that were originally present at the surface of the wet microcapsules or of crystals that extended beyond the microcapsule surface (Fig. 1F). The fact that these structural features were evident after the completion of crosslinking and washing suggested that theophylline crystals that were originally present at the surface were removed after the solidification of the wall matrix during cross-linking and not prior to this stage. Results thus suggested that these crystals were removed from the surface during the washing procedure. SEM of specimens prepared from microcapsules included in the aforementioned three size categories indicated that the diameter of large, medium, and small capsules was 750–1100 mm, 500–700 mm, and ,500 mm, respectively. No significant differences in outer topography of corecontaining capsules were observed between capsules included in the different size categories. In some cases, SEM results revealed the presence of some very small microspheres with smooth surfaces (Fig. 2A). In all cases, these particles had diameter smaller than 50 mm, were core-free, and exhibited no surface pores or irregularities. The presence of these particles could be attributed to the formation of corefree WPI droplets during the initial stage of the microencapsulation process. In all cases, the very small core-free microspheres were aggregated (Fig.
2A) while larger core-containing capsules exhibited no aggregation (Fig. 1A–D). This suggested that in addition to the effect of the polyurethane in preventing aggregation, surface-core crystals served as spacers that prevented contact between surfaces of wet capsules and thus prevented aggregation. The aggregation of small core-free particles indicated stickiness of the surface of wet whey-protein-based particles that led to aggregation and probably to formation of GA-cross-linked intra-particle protein bridges. Results of the SEM indicated that, in some cases, microcapsules exhibited some surface cracks (Fig. 1D and E). The presence of such cracks was especially evident when microcapsules were crosslinked for 3 h with 30 ml of GAST. Formation of these cracks could thus be attributed to high crosslinking density that rendered microcapsules fragile. Analysis of the inner structure of microcapsules (Fig. 2B–D) indicated that theophylline crystals were embedded throughout the cross-linked protein matrix. The protein matrix of the microcapsules had a very dense appearance that indicated good efficiency of the cross-linking reaction. Results (Fig. 2C and D) also indicated that core crystals were physically entrapped in the protein matrix of microcapsules and no indications for interactions between core and wall components were evident. In all cases, core load that was dispersed in wall solution exceeded the water-solubility of this drug. Thus, SEM analysis of untreated theophylline crystals (micrographs not provided here) and the presented analysis of the inner structure of microcapsules suggested that drug remained as a crystalline dispersion in the wall matrix. It has been previously reported that theophylline encapsulated in casein microcapsules, using methodology similar to that reported by us, maintained its crystalline state [14]. Results regarding the inner structure of the microcapsules agreed with earlier reports related to application of GAST in preparing casein-based microcapsules [14] and chitosan microcapsules [30]. The structural features of the wall matrix observed in this study were different and much denser that those previously reported by us for WPI-based microcapsules prepared by double emulsification and crosslinking with aqueous glutaraldehyde [27]. These findings could be related to differences between
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Fig. 1. Outer morphology of theophylline-containing WPI microcapsules. Microcapsules were cross-linked for 1 h with 7.5 or 15 ml of GAST (A and B, respectively), for 3 h with 15 ml GAST (C and F), and for 3 h with 30 ml of GAST (D and E). Arrows in D and E — surface cracks. Arrowheads in F — footprints of surface core crystals. Bar5300 mm (A–C), 30 mm (D, E), and 50 mm (F).
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Fig. 2. Outer morphology of core-free particles (A); inner structure of theophylline-containing microcapsules (B–D); outer topography and inner structure of microcapsules after complete core release into SGF at 378C (E and F, respectively). Microcapsules were cross-linked for 1 h with 30 ml GAST (A, B, C, E, and F) or for 3 h with 15 ml of GAST (D). Arrows in B, C, D — theophylline crystals. Bar550 mm (A and D), 100 mm (B), 10 mm (C and F), and 300 mm (E).
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cross-linking, by glutaraldehyde, in aqueous and organic phase [7–14]. Effect of core release on structural features of theophylline-loaded microcapsules was investigated and results are presented in Fig. 2E and F. Outer topography of microcapsules was not affected in any way during core release and was similar to that observed for freshly prepared microcapsules (Fig. 1A–C). These results thus indicated that core release was due to diffusion through the wall rather than being associated with degradation or solubilization of the protein matrix. It could also be assumed that as core release progressed, it was governed by diffusion through the wall as well as through pores left by dissolving theophylline. Examining the inner features of capsules after core has been released (Fig. 2F) revealed that the wall matrix maintained its structural features and a multitude of voids from which theophylline has been released (through diffusion) was evident. In all cases, these voids exhibited the shape of theophylline crystals that were originally embedded in them. Comparing the inner structure of capsules prior and following core release (Fig. 2B–D and F) indicated that no softening of the cross-linked wall matrix was associated with core release in water. Such softening would have significantly affected the shape and dimensions of theophylline domains (voids) within the capsules and would have led to matrix collapse. Results (Fig. 2F) indicated that no theophylline crystals could be found in capsules where 100% core release has been chemically determined. These results indicated the validity
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of the methodology used in investigating core release.
3.2. Core retention during microencapsulation Theophylline content in microcapsules ranged from 49.5 to 52.5% (w / w) and was not significantly affected (P.0.05) by cross-linking variables (Table 1). Results indicated significant differences in theophylline content between microcapsules of different size (Table 1). Core content in large- and mediumsize microcapsules cross-linked for 3 h with 30 ml GAST was similar (P.0.05) however, in all other cases, core content in microcapsules (from a given batch) was proportionally related to microcapsule size (P,0.05). Some significant differences were observed between microcapsules from a given size category prepared at different cross-linking conditions (Table 1). However, no specific trend relating cross-linking conditions to ultimate core content could be identified. Theophylline content ranged from 51.5 to 55%, from 44.5 to 50.5%, and from 35 to 42% for large, medium, and small microcapsules, respectively (Table 1). The highest (P,0.05) core content among large microcapsules was found in the system cross-linked for 3 h with 15 ml of GAST; in all other systems, no effect of cross-linking conditions on core content was detected (P.0.05). Among the medium-size microcapsules, the highest and lowest core content was found in systems crosslinked for 3 h with 30 ml GAST and for 3 h with 7.5
Table 1 Theophylline content and core retention efficiency in microcapsules prepared at different cross-linking conditions Glutaraldehyde (ml)a
Reaction time (h)
Large b
Medium
Small
Overall c
Retention (%)
37.5 42.0* 35.0* 38.5 39.6 38.4
49.5 50.3 52.5 50.5 49.5 51.0
74.0 75.0 78.0 76.5 73.5 75.5
Theophylline content (% w / w) 7.5 7.5 15 15 30 30 a
3 1 3 1 3 1
51.5 53.5 55.0* 53.5 52.5 52.0
44.5* 49.0 47.5 47.5 50.5* 48.0
Volume of glutaraldehyde-saturated toluene used. Large, medium, and small microcapsules (see text). c Calculated overall core content. * Means in a column followed by an asterisk are significantly different (P,0.05). b
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ml GAST, respectively (P,0.05); core content in all other medium-size capsules was similar (P.0.05). The highest and lowest core content in small microcapsules was found in systems cross-linked for 7.5 ml GAST and for 3 h with 15 ml GAST, respectively (P,0.05). The overall core content in microcapsules (Table 1) was not affected by crosslinking conditions (P.0.05). Core retention ranged from 73.5 to 78% and was not significantly affected by cross-linking conditions (Table 1). Our results thus indicated that the microencapsulation process conditions allowed, in all cases, high core retention. Core losses during the process could be mainly attributed to effects of the washing stage. Theophylline is soluble, to different extents, in the washing agents that were used. Core losses from the wet capsules could thus be attributed to removal of core crystals from the outer surface of microcapsules as well as to some losses from inner parts of the microcapsules, by a diffusion-driven leaching process. Indications for removal of surface core crystals during washing stages were also provided by the aforementioned results of structure analysis. Differences in core content between microcapsules of different size could be explained in light of the effect of number of core crystals initially included in the microcapsule and that of surface-to-volume ratio on core loss. It is clear that small microcapsules, that contain fewer core crystals and have an overall larger surface, would exhibit higher core loss than that obtained with larger microcapsules with overall smaller surface area and more core crystals. It could thus be concluded that core retention in microcapsules was mainly affected by core content and size of wet microcapsules at the washing stage. Our results were similar to those reported by Thanoo et al. [30] who reported core retention of about 80% in preparing chitosan microcapsules containing about 50% theophylline. Theophylline content of large microcapsules and overall core retention obtained in our study were similar to results of Latha and Jayakrishnan [14] and those of Latha et al. [7] regarding theophylline-containing casein microcapsules. Core retention level obtained in our study was higher than that reported by Heelan and Corrigan [28] for microencapsulation of different water-soluble core materials in WPI-based microcapsules.
3.3. Theophylline release from microcapsules Release of theophylline from microcapsules into SIF and SGF at 378C is depicted in Figs. 3–5. Comparing data regarding water-solubility of theophylline [31] with that regarding the ratio of theophylline to volume of fluid in our release experiments indicated that sink conditions existed in all cases. In a preliminary study, de novo microcapsules were prepared, using the same procedure used in preparing theophylline-containing capsules, and their solubility in SGF and SIF at 378C was investigated. Results indicated that the GAST-cross-linked WPI particles were insoluble in both fluids. This and the aforementioned results of structure analysis indicated that core release from theophylline-loaded microcapsules could be attributed to diffusion-driven mechanism rather than solubilization of the protein matrix. Results presented in Figs. 3–5 indicated that core release was affected by type of simulated digestive fluid, cross-linking conditions, and by microcapsule size. In all cases, the rate of core release from given microcapsules into SIF was significantly higher than that into SGF. For example, results obtained with large microcapsules prepared with 30 ml GAST and cross-linking time of 3 h indicated that complete core release was obtained after 75 and 150 min in SIF and SGF, respectively (Fig. 5A). Although not directly tested in our research, differences in release obtained in SIF and SGF could be attributed to differences in extent of swelling of WPI-based microcapsules in the two fluids [28]. It has been established that proteinbased microcapsules swelled more in SIF than in SGF and that core release was proportional to extent of swelling [14]. Effects of the nature of simulated digestive fluid on core release observed in our study were similar to those earlier reported for other protein-based microcapsules. Among these were results of Thanoo et al. [30] for release of theophylline from chitosan microcapsules, results of Latha and Jayakrishnan [14] for release of theophylline from casein-based capsules, and results of Heelan and Corrigan [28] regarding core release from WPI-based capsules. Results obtained with microcapsules from the three size categories indicated that in all cases, core release was time-dependent and was affected, to
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Fig. 3. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 7.5 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
varying extent, by cross-linking conditions (Figs. 3–5). Core release from small microcapsules (,450 mm) was significantly faster (P,0.05) than that from larger capsules. Complete core release from these capsules was observed after 20 min with SIF, regardless of cross-linking conditions, and after 30– 50 min with SGF (Figs. 3C, 4C, and 5C). Results thus indicated only a limited effect of cross-linking conditions on core release from small microcapsules and, in light of the overall fast core release, sug-
gested that small capsules obtained in our study had only limited potential when practicality of controlled and / or sustained core release applications were considered. However, results obtained with large and medium size microcapsules indicated suitability of the systems for controlled core release applications. Results obtained with large microcapsules (.700 mm) indicated that complete core release into SIF was obtained after 50–75 min and was affected, to a certain extent, by cross-linking conditions (Figs. 3A,
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Fig. 4. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 15 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
4A, and 5A). The time needed for complete core release from these microcapsules into SGF ranged from 90 to 150 min and was proportional to both amount of GAST and cross-linking reaction time. Results obtained with SGF indicated that core release from capsules cross linked with 7.5 ml GAST for 3 h or with 15 or 30 ml GAST for 1 h was 30% slower than that obtained with microcapsules cross linked for 1 h with 7.5 ml GAST. Results also indicated that regardless of amount of GAST used, core release from microcapsules cross linked for 3 h was 25–30%
slower than that from microcapsules cross-linked for 1 h. Results obtained with medium size microcapsules (500–700 mm) indicated trends similar to those observed for large microcapsules however, core release was faster (P,0.05) than that obtained with large capsules (Figs. 3B, 4B, and 5B). Complete core release from these microcapsules into SIF and SGF was obtained after 30 to 50 min and after 50 to 110 min, respectively and indicated a significant (P,0.05) effect of dissolution medium. Core release
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Fig. 5. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 30 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
into SGF from microcapsules cross-linked for 3 h was 80, 40 and 46% slower than that obtained with capsules cross-linked for 1 h for 7.5, 15 and 30 ml GAST, respectively. Effect of cross-linking conditions on release into SIF was only to a very limited extent (P.0.05). As stated above for large and medium size microcapsules, results suggested that generally, core release was inversely proportional to cross-linking density. However, core release from large and medium size capsules cross-linked for 3 h with 15 ml
GAST was slower than that from the same size capsules cross-linked for 3 h with 30 ml GAST (Figs. 4 and 5). These results could be attributed to the presence of cracks at the surface of microcapsules cross-linked for 3 h with 30 ml GA (Fig. 1D and E). The presence of such cracks allowed direct contact between dissolution medium and inner parts of microcapsules and thus compromised, to a certain extent, the role of the cross-linked wall matrix in governing the diffusion-controlled core release. These results suggest that although adjusting cross-
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linking conditions and hence cross-linking density can modulate rate of core release from WPI-based microcapsules, effects on structural and mechanical properties of microcapsules should be carefully assessed. Results obtained with large and medium size microcapsules indicated that effects of cross-linking conditions on core release were manifested to a greater extent with SGF than with SIF (Figs. 3–5). These results could be explained in light of the profound effect of swelling in governing diffusiondriven core release from the microcapsules. Overall, effects of microcapsule size on core release observed in our study were similar to those reported by Thanoo et al. [30] and those reported by Latha et al. [7] and could be attributed to effect of surface-tovolume ratio on the diffusion-governed core release. Heelan and Corrigan [28] reported that release of different water-soluble core materials from WPIbased microcapsules cross-linked with aqueous glutaraldehyde was very fast and was not affected by cross-linking conditions. The differences between the results of our study and those of Heelan and Corrigan [28] could be attributed to effects of the GAST used in our study. It has been suggested that crosslinking with GAST provided significantly better uniformity and efficiency of cross-linking at the surface of dispersed protein-based droplets than that accomplished with aqueous glutaraldehyde [28]. It has been also indicated [7,14] that cross-linking with GAST led to the formation of a surface net, with high cross-linking density, effective in modulating core release. It has been reported [14] that theophylline release from casein microcapsules cross-linked by GAST could be described by the Higuchi model [32]: Q 5 kt 1 / 2 where Q is the cumulative amount of drug released at time t and k depends on the surface areas and the diffusion coefficient. For systems that meet the above conditions and assuming constant surface area and diffusion coefficient, plotting Q vs. t 1 / 2 should yield a straight line. Theophylline release data presented in Figs. 3–5 were processed according to the Higuchi model and
representative results are depicted in Fig. 6. Results indicated that in all cases, the release profile was characterized by two linear segments that differed in their slope, followed by a non-linear segment, where the Higuchi model was no longer valid. Results indicated that the linear function of the Higuchi model was true only up to about 80–85% of the time needed for complete release, similar to what has been indicated by Alvarez et al. [33]. The identified changes in release rate suggested changes in the release mechanism [34]. This could probably be attributed to combined effect of diffusion through the capsule wall system and, at later stages, effects of liquid-filled pores and empty core domains. Results indicated that slopes of the linear segments and the time at which rate of release changed were mainly affected by the type of dissolution medium. This could be attributed to differences in matrix swelling, as discussed above. Overall, results presented in Fig. 6 could be explained in light of the aforementioned discussion pertaining to effects of dissolution medium and cross-linking conditions on core release.
4. Conclusion Results of our study indicated that WPI could be used in preparing water-insoluble microcapsules containing about 50% of water-soluble model drug. In all cases, theophylline retention during microencapsulation was higher than 70%, indicating the high efficiency of the microencapsulation process. Results indicated that cross-linking of the protein-based wall matrices of microcapsules by glutaraldehyde-saturated toluene via organic phase was effective in influencing rate of core release. Results obtained with crack-free capsules indicated that core release was controlled by a diffusion-driven mechanism and that the rate of core release was inversely related to cross-linking time and to the amount of GAST used. Core release was significantly affected by microcapsule size and dispersing medium type, and could be described by the Higuchi model. Results of this study indicated that whey proteins might be used as microencapsulating agents in developing microcapsules for controlled and / or sustained core release.
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Fig. 6. Theophylline release vs. square root of time in simulated gastric or intestinal fluids (SGF and SIF, respectively) at 378C from large and medium size microcapsules. Microcapsules were cross-linked for 1 h or 3 h with 30 ml or for 3 h with 15 ml of glutaraldehyde-saturated toluene (1h30, 3h30, and 3h15, respectively). In all cases, mean values (n$4) are presented.
Acknowledgements This research was supported by a grant from Dairy Management Inc. (DMI).
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[5] M.C. Levy, M.C. Andry, Mixed-walled microcapsules made of cross-linked proteins and polysaccharides: preparation and properties, J. Microencaps. 8 (1991) 335–347. [6] A. Arshady, Albumin microspheres and microcapsules: methodology of manufacturing techniques, J. Control. Release 14 (1990) 111–131. [7] M.S. Latha, K. Rathinam, P.V. Mohanan, A. Jayakrishnan, Bioavailability of theophylline from glutaraldehyde crosslinked microspheres in rabbits following oral administration, J. Control. Release 34 (1995) 1–7. [8] E.W. Longo, H. Iwata, A. Lindheimer, E.P. Goldberg, Preparation of hydrophilic albumin microspheres using polymeric dispersing agents, J. Pharm. Sci. 71 (1982) 1323– 1328. [9] S.S. Davis, L. Illum, J.G. McVie, E. Tomlinson, Microspheres and Drug Therapy, Pharmaceutical, Immunological and Medical Aspects, Elsevier, Amsterdam, 1984. [10] A.M. Saleh, L.K. El-Kordagui, D.A. Robb, A.T. Florence, Effect of some drugs and additives on the cross-linking of bovine serum albumin by glutaraldehyde, Int. J. Pharm. 57 (1989) 205–210. [11] Y. Chen, N. Willmott, J. Anderson, A.T. Florence, Comparison of albumin and casein microspheres as carrier for doxorubicin, J. Pharm. Pharmacol. 39 (1987) 978–985. [12] Y. Willmott, G.A. Magee, J. Cummings, G.W. Halbert, J.F. Smith, Doxorubicin-loaded casein microspheres: protean nature of drug incorporation, J. Pharm. Pharmacol. 44 (1992) 472–475. [13] W.A. Knepp, A. Jayakrishnan, J.M. Quigg, H.S. Sitren, J.J. Bagnall, E.P. Goldberg, Synthesis, properties and intratumoral evaluation of mitoxantrone-loaded casein microspheres in Lewis lung carcinoma, J. Pharm. Pharmacol. 45 (1993) 887–891. [14] M.S. Latha, A. Jayakrishnan, Glutaraldehyde cross-linked bovine casein microspheres as a matrix for the controlled release of theophylline: in-vitro studies, J. Pharm. Pharmacol. 46 (1994) 8–13. [15] M.S. Latha, A. Jayakrishnan, K. Rathiman, M. Mohanty, Casein as a carrier matrix for 5-fluorouracil: drug release from microspheres, drug-protein conjugates and in-vivo degradation of microspheres in rat muscle, J. Pharm. Pharmacol. 46 (1994) 858–862. [16] M. Rosenberg, Milk derived whey protein-based microencapsulating agents and a method of use, US Patent 5,601,760, February 11, 1997. [17] D.L. Moreau, M. Rosenberg, Microstructure and fat extractability in microcapsules based on whey proteins or mixtures of whey proteins and lactose, Food Struct. 12 (1993) 457– 468. [18] M. Rosenberg, S.Y. Lee, Microstructure of anhydrous milkfat / whey proteins emulsions, Food Struct. 12 (1993) 267– 274.
[19] M. Rosenberg, S.L. Young, Whey proteins as microencapsulating agents. Microencapsulation of anhydrous milkfat — structure evaluation, Food Struct. 12 (1993) 31–41. [20] S.L. Young, X. Sarda, M. Rosenberg, Microencapsulating properties of whey proteins. 1. Microencapsulation of anhydrous milk fat, J. Dairy Sci. 76 (1993) 2868–2877. [21] S.L. Young, X. Sarda, M. Rosenberg, Microencapsulating properties of whey proteins. 2. Combination of whey proteins with carbohydrates, J. Dairy Sci. 76 (1993) 2878–2885. [22] T.Y. Sheu, M. Rosenberg, Microencapsulation by spraydrying ethyl caprylate in whey protein and carbohydrate wall systems, J. Food Sci. 60 (1995) 98–103. [23] M. Rosenberg, T.Y. Sheu, Microencapsulation of volatiles by spray-drying in whey protein-based wall systems, Int. Dairy J. 6 (1996) 273–284. [24] D.L. Moreau, M. Rosenberg, Porosity of whey protein-based microcapsules containing anhydrous milkfat measured by gas displacement pycnometry, J. Food Sci. 63 (1998) 819–823. [25] D.L. Moreau, M. Rosenberg, Porosity of microcapsules with wall systems consisting of whey proteins and lactose measured by gas displacement pycnometry, J. Food Sci. 63 (1999) in press. [26] D.L. Moreau, M. Rosenberg, Oxidative stability of anhydrous milkfat microencapsulated in whey proteins, J. Food Sci. 61 (1996) 39–43. [27] S.J. Lee, M. Rosenberg, Preparation and some properties of water-insoluble, whey protein-based microcapsules, J. Microencaps. (1999) in press. [28] B.A. Heelan, O.I. Corrigan, Preparation and evaluation of microspheres prepared from whey protein isolate, J. Microencaps. 15 (1) (1998) 93–105. [29] US Pharmacopeia, US Pharmacopeia National Formulary Supplement, USP 23-NF 18, Rockville, MD, 1995. [30] B.C. Thanoo, M.C. Sunny, A. Jayakrishnan, Cross-linked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals, J. Pharm. Pharmacol. 44 (1992) 283–286. [31] E.A. Swinyard, in: Respiratory Drugs, Remington’s Pharmaceutical Sciences, Vol. XVI, Mack Publishing Company, Easton, PA, 1980, pp. 811–812. [32] T. Higutchi, Mechanism of sustained action medication, J. Pharm. Sci. 52 (1963) 1145–1149. [33] V. Pillay, C.M. Dangor, T. Govender, K.R. Moopanar, N. Hurbans, Drug release modulation from cross-linked calcium alginate microdiscs, 2: compression and stability of the hydrodynamically-sensitive calcium alginate matrix and the associated drug release mechanism, Drug Del. 5 (1) (1998) 35–46. [34] A.M.R. Alvarez, A.M.H. Villafuerte, M. Fernandez-Arevalo, J.M.J. Dorado, Inert matrix tablets as a controlled release dosage form for carteolol hydrochloride, Eur. J. Pharm. Biopharm. 37 (1991) 147–153.
Preparation and properties of glutaraldehyde cross-linked whey protein-based microcapsules containing theophylline S.J. Lee, M. Rosenberg* Department of Food Science and Technology, University of California, Davis, CA 95616, USA Received 26 February 1999; received in revised form 30 April 1999; accepted 30 April 1999
Abstract Whey protein-based microcapsules containing a model drug, theophylline, were prepared in organic phase, using glutaraldehyde-saturated toluene. In all cases, spherical microcapsules, ranging from ,400 to 1000 mm in diameter, were obtained. Results indicated that core crystals were embedded throughout the wall matrix. In all cases, retention efficiency of theophylline was higher than 74% and was not affected by cross-linking conditions. Results of theophylline release in simulated intestinal and gastric fluids at 378C indicated that the diffusion-governed core release was significantly affected by size of microcapsules, cross-linking conditions, and by type of dissolution medium. In all cases, core release in simulated intestinal fluid was faster than in simulated gastric fluid. 1999 Elsevier Science B.V. All rights reserved. Keywords: Controlled release; Microcapsules; Whey proteins; Cross-linking; Theophylline
1. Introduction Microcapsules for controlled and / or sustained core release have been developed for pharmaceutical and other applications using both synthetic and natural polymers. Natural polymers suitable for developing microcapsules for such applications include mainly proteins and polysaccharides [1–5]. Proteins have been shown to present a significant potential as wall materials for microcapsules for controlled and sustained release of different drugs and most of the reported research has been focused on different albumins [6–10]. In recent years, suitability of milk proteins as wall *Corresponding author. Tel.: 11-530-752-4682; fax: 11-530754-8145. E-mail address: [email protected] (M. Rosenberg)
materials for microcapsules and microspheres for controlled release of different drugs has been investigated. Bovine caseins were used as wall materials in developing microcapsules for sustained release of different drugs [7,11–15]. Results of these studies indicated that glutaraldehyde cross-linked caseinbased microcapsules exhibited, to a varying extent, properties desired for controlled core release. Recently, the concept of using whey proteins as microencapsulating agents has been established [16]. A series of studies has indicated that whey proteins exhibit excellent microencapsulating properties and are suitable for microencapsulation of volatile and non-volatile core materials [17–23]. It has been indicated that wall systems of spray-dried microcapsules consisting of whey proteins exhibited molecular sieve type porosity [24,25] and provided effective protection against core oxidation [26]. Most of the
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00108-X
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reported research effort pertinent to microencapsulating properties of whey proteins has been focused on developing water-soluble microcapsules. Functionality of whey proteins as wall matrices in microcapsules designed for controlled core release has been investigated only to a very limited extent. Waterinsoluble whey protein-based microcapsules, containing apolar model core, prepared by double emulsification and cross-linking with glutaraldehyde were reported by Lee and Rosenberg [27]. Core retention during microencapsulation was about 90% and microcapsules were insoluble in water at different pH and temperature conditions [27]. Heelan and Corrigan [28] reported on preparation and core release from whey protein-based microcapsules cross-linked with aqueous glutaraldehyde. Their results indicated that, in most cases, rate of core release was significantly faster than that desired in pharmaceutical applications. Although physicochemical properties of whey proteins suggest that they may be suitable for developing microcapsules for controlled and sustained core release, more research is needed in this regard. The objectives of our study were to prepare water-insoluble whey protein-based microcapsules containing a model water-soluble drug and to investigate microstructural and core release characteristics of these microcapsules.
deionized water. In all cases, the pH of wall solution was 7.2. Theophylline (1.6 g) was dispersed in 4 g of wall solution and this mixture was then suspended (at 258C) in a dispersion mixture consisting of 80 ml of dichloromethane and 50 ml of hexane containing 1% biomedical polyurethane (Thermedix Inc., Woburn, MA), in a 250-ml round-bottom flask. A stainless steel half-moon shaped paddle was used to stir the suspension at 900 rev. / min for 3 min. Protein-based wall matrices of microcapsules were cross-linked by glutaraldehyde (Sigma Chemical Co.). After stirring for 3 min, either 7.5, 15, or 30 ml of glutaraldehyde-saturated toluene (GAST), prepared according to the method reported by Longo et al. [8], was added to the suspension and cross-linking was carried out for 1 or 3 h (900 rev. / min, 258C). Wet microcapsules were separated from the dispersing solvent mixture by filtration and then washed five times, 2 min each, with 200 ml of a 1:1 mixture of dichloromethane / hexane. Microcapsules were then washed (for 2 min each) with 200 ml of 1% sodium bisulfite, with 200 ml of distilled water, and finally with 200 ml of acetone. Washed microcapsules were dried in a vacuum oven at 508C overnight. Dry microcapsule powders were separated, by sieving, into large (diameter .700 mm), medium (diameter 450–700 mm) and small (diameter ,450 mm) microcapsules and the weight proportion of capsules included in each size category was recorded. Dry capsules were kept in a desiccator pending analysis.
2. Materials and methods
2.3. Determination of total theophylline content 2.1. Wall and core materials Whey protein isolate containing 95.6% (w / w on dry basis) protein (WPI, Le Sueur Isolates, Le Sueur, MN) was used as a wall material. Theophylline (Sigma Chemical Co., St. Louis, MO) served as a model water-soluble core.
2.2. Preparation of theophylline loaded microcapsules Theophylline-containing, chemically cross-linked WPI-based microcapsules were prepared using a modification of the procedure reported by Latha et al. [7] and by Latha and Jayakrishnan [14]. Wall solution containing 20% (w / w) WPI was prepared in
Theophylline content in microcapsules included in each of the size categories was determined using a modification of the method reported by Latha and Jayakrishnan [14]. Microcapsule samples (5–10 mg) were powdered in a mortar, placed in screw-capped test tubes, and extracted with 50 ml methanol for 12 h. During extraction, test tubes were continuously rotated using a Model 7637 Roto-Troque Rotator (Cole-Parmer Instrument Company, Chicago, IL). Samples (2 ml) of supernatant were filtered through a 0.22 mm filter (Millex-GV, Durapore Membrane, Millipore, Bedford, MA), the filtrate was diluted appropriately with methanol, and absorbance at 274 nm was determined (Bausch & Lomb Spectronic 1001). The amount of extracted theophylline was
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calculated using a standard curve of theophylline in methanol. Based on the amount of core determined in this way and the known weight of the microcapsules used for analysis the core content of microcapsules was expressed as % (w / w). A preliminary study confirmed that the above methodology allowed release of 100% of the encapsulated core after 12 h extraction and was therefore suitable for determination of microcapsules core content and, subsequently, for calculating core retention. Our results agreed with previous reports that validated the suitability of methanol extraction for determination of theophylline content in milk protein-based and other microcapsules [7,14,15,30]. Core retention was expressed as the ratio (in %) of core content determined in microcapsules to a theoretical core content assuming 100% core retention during the microencapsulation process.
2.4. Release of theophylline from microcapsules Microcapsules (total weight 5.5 mg) were placed in a 200-ml double wall glass beaker containing 180 ml of either enzyme-free simulated intestinal fluid (SIF) or enzyme-free simulated gastric fluid (SGF) prepared according to the US Pharmacopeia [29]. The suspension was stirred (100 rev. / min) at 378C using a floating stir bar (Fisher Scientific, Pittsburgh, PA). Aliquots (3 ml) were withdrawn periodically, using a 5-ml syringe equipped with a 0.2-mm syringe filter (Fisher Scientific Co., Santa Clara, CA) and theophylline concentration was determined spectrophotometrically at 274 nm. In no case were microcapsules removed along with dissolution medium. In all cases, an equal volume of dissolution medium was immediately added to maintain a constant volume. Samples were withdrawn until three successive aliquots showed no increase in optical absorbance (274 nm). The amount of theophylline released from the microcapsules, at a given time, was calculated using standard curves of theophylline in SGF and SIF and expressed as percentage of total theophylline content of the investigated microcapsules.
2.5. Scanning electron microscopy ( SEM) Structural features of microcapsules were investi-
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gated using SEM. For examination of outer structure, microcapsules were attached to a SEM stub using a two-sided adhesive tape (Ted Pella, Redding, CA). In order to investigate the inner structure of microcapsules, microcapsules were attached to an SEM stub, as before, and then fractured by moving a razor blade perpendicularly through the layer of microcapsules. The microstructure of microcapsules from which core has been released was also investigated. Core was released from microcapsules into SIF or SGF, using the procedure described above for the core release experiment. After reaching complete core release, microcapsules were separated from dissolution medium, dried as described above, and then were examined by SEM. Specimen preparation was as described above for core-containing microcapsules. In all cases, specimens were coated with gold using a model E-5050 Polaron Sputter Coater (Bio-Rad, San Jose, CA), and analyzed using an ISI DS-130 scanning electron microscope (International Scientific Instrument Inc., Pleasanton, CA) operated at 10 kV. Micrographs were prepared using a Type 55 Polaroid film (Polaroid Corp., Cambridge, CA).
2.6. Statistical analysis In all cases, replicate samples were analyzed at least in duplicate (n54). Significance of results was tested by ANOVA using the SigmaStat software (Jandel Scientific Software, San Rafael, CA). Significance of differences was defined at P,0.05.
3. Results and discussion
3.1. Size distribution and microstructural features In all cases, regardless of cross-linking conditions, spherical microcapsules were obtained. In all cases, microcapsule powders exhibited a relatively wide particle size distribution. The proportion of microcapsules larger than 700 mm ranged from 52 to 65%, that of microcapsules with diameter of 450– 700 mm ranged from 22 to 36%, and proportion of microcapsules smaller than 450 mm ranged from 5 to 15%. Results did not indicate effect of cross-linking conditions on microcapsule size distribution. This and the results of SEM suggested that at a given
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stirring condition, the size of microcapsules was mainly affected by number of core crystals included in wall / core droplets, prior to cross-linkage. These results agreed with those of Latha and Jayakrishnan [14] who reported an increase in microcapsule size with theophylline load. Outer topography and inner structure of microcapsules are presented in Figs. 1 and 2. It has been reported that significant shape distortions were observed when WPI-based microcapsules were crosslinked with aqueous glutaraldehyde [28]. Such difficulties were not observed in our study thus highlighting the effectiveness of cross-linking by glutaraldehyde via organic phase in allowing rapid fixation of the shape and surface morphology of the microcapsules [30]. In all cases, outer surfaces of microcapsules were not smooth and exhibited irregularities of different shapes (Fig. 1A–E). The size and shape of these structural features suggested that they represented a footprint of theophylline crystals that were originally present at the surface of the wet microcapsules or of crystals that extended beyond the microcapsule surface (Fig. 1F). The fact that these structural features were evident after the completion of crosslinking and washing suggested that theophylline crystals that were originally present at the surface were removed after the solidification of the wall matrix during cross-linking and not prior to this stage. Results thus suggested that these crystals were removed from the surface during the washing procedure. SEM of specimens prepared from microcapsules included in the aforementioned three size categories indicated that the diameter of large, medium, and small capsules was 750–1100 mm, 500–700 mm, and ,500 mm, respectively. No significant differences in outer topography of corecontaining capsules were observed between capsules included in the different size categories. In some cases, SEM results revealed the presence of some very small microspheres with smooth surfaces (Fig. 2A). In all cases, these particles had diameter smaller than 50 mm, were core-free, and exhibited no surface pores or irregularities. The presence of these particles could be attributed to the formation of corefree WPI droplets during the initial stage of the microencapsulation process. In all cases, the very small core-free microspheres were aggregated (Fig.
2A) while larger core-containing capsules exhibited no aggregation (Fig. 1A–D). This suggested that in addition to the effect of the polyurethane in preventing aggregation, surface-core crystals served as spacers that prevented contact between surfaces of wet capsules and thus prevented aggregation. The aggregation of small core-free particles indicated stickiness of the surface of wet whey-protein-based particles that led to aggregation and probably to formation of GA-cross-linked intra-particle protein bridges. Results of the SEM indicated that, in some cases, microcapsules exhibited some surface cracks (Fig. 1D and E). The presence of such cracks was especially evident when microcapsules were crosslinked for 3 h with 30 ml of GAST. Formation of these cracks could thus be attributed to high crosslinking density that rendered microcapsules fragile. Analysis of the inner structure of microcapsules (Fig. 2B–D) indicated that theophylline crystals were embedded throughout the cross-linked protein matrix. The protein matrix of the microcapsules had a very dense appearance that indicated good efficiency of the cross-linking reaction. Results (Fig. 2C and D) also indicated that core crystals were physically entrapped in the protein matrix of microcapsules and no indications for interactions between core and wall components were evident. In all cases, core load that was dispersed in wall solution exceeded the water-solubility of this drug. Thus, SEM analysis of untreated theophylline crystals (micrographs not provided here) and the presented analysis of the inner structure of microcapsules suggested that drug remained as a crystalline dispersion in the wall matrix. It has been previously reported that theophylline encapsulated in casein microcapsules, using methodology similar to that reported by us, maintained its crystalline state [14]. Results regarding the inner structure of the microcapsules agreed with earlier reports related to application of GAST in preparing casein-based microcapsules [14] and chitosan microcapsules [30]. The structural features of the wall matrix observed in this study were different and much denser that those previously reported by us for WPI-based microcapsules prepared by double emulsification and crosslinking with aqueous glutaraldehyde [27]. These findings could be related to differences between
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Fig. 1. Outer morphology of theophylline-containing WPI microcapsules. Microcapsules were cross-linked for 1 h with 7.5 or 15 ml of GAST (A and B, respectively), for 3 h with 15 ml GAST (C and F), and for 3 h with 30 ml of GAST (D and E). Arrows in D and E — surface cracks. Arrowheads in F — footprints of surface core crystals. Bar5300 mm (A–C), 30 mm (D, E), and 50 mm (F).
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Fig. 2. Outer morphology of core-free particles (A); inner structure of theophylline-containing microcapsules (B–D); outer topography and inner structure of microcapsules after complete core release into SGF at 378C (E and F, respectively). Microcapsules were cross-linked for 1 h with 30 ml GAST (A, B, C, E, and F) or for 3 h with 15 ml of GAST (D). Arrows in B, C, D — theophylline crystals. Bar550 mm (A and D), 100 mm (B), 10 mm (C and F), and 300 mm (E).
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cross-linking, by glutaraldehyde, in aqueous and organic phase [7–14]. Effect of core release on structural features of theophylline-loaded microcapsules was investigated and results are presented in Fig. 2E and F. Outer topography of microcapsules was not affected in any way during core release and was similar to that observed for freshly prepared microcapsules (Fig. 1A–C). These results thus indicated that core release was due to diffusion through the wall rather than being associated with degradation or solubilization of the protein matrix. It could also be assumed that as core release progressed, it was governed by diffusion through the wall as well as through pores left by dissolving theophylline. Examining the inner features of capsules after core has been released (Fig. 2F) revealed that the wall matrix maintained its structural features and a multitude of voids from which theophylline has been released (through diffusion) was evident. In all cases, these voids exhibited the shape of theophylline crystals that were originally embedded in them. Comparing the inner structure of capsules prior and following core release (Fig. 2B–D and F) indicated that no softening of the cross-linked wall matrix was associated with core release in water. Such softening would have significantly affected the shape and dimensions of theophylline domains (voids) within the capsules and would have led to matrix collapse. Results (Fig. 2F) indicated that no theophylline crystals could be found in capsules where 100% core release has been chemically determined. These results indicated the validity
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of the methodology used in investigating core release.
3.2. Core retention during microencapsulation Theophylline content in microcapsules ranged from 49.5 to 52.5% (w / w) and was not significantly affected (P.0.05) by cross-linking variables (Table 1). Results indicated significant differences in theophylline content between microcapsules of different size (Table 1). Core content in large- and mediumsize microcapsules cross-linked for 3 h with 30 ml GAST was similar (P.0.05) however, in all other cases, core content in microcapsules (from a given batch) was proportionally related to microcapsule size (P,0.05). Some significant differences were observed between microcapsules from a given size category prepared at different cross-linking conditions (Table 1). However, no specific trend relating cross-linking conditions to ultimate core content could be identified. Theophylline content ranged from 51.5 to 55%, from 44.5 to 50.5%, and from 35 to 42% for large, medium, and small microcapsules, respectively (Table 1). The highest (P,0.05) core content among large microcapsules was found in the system cross-linked for 3 h with 15 ml of GAST; in all other systems, no effect of cross-linking conditions on core content was detected (P.0.05). Among the medium-size microcapsules, the highest and lowest core content was found in systems crosslinked for 3 h with 30 ml GAST and for 3 h with 7.5
Table 1 Theophylline content and core retention efficiency in microcapsules prepared at different cross-linking conditions Glutaraldehyde (ml)a
Reaction time (h)
Large b
Medium
Small
Overall c
Retention (%)
37.5 42.0* 35.0* 38.5 39.6 38.4
49.5 50.3 52.5 50.5 49.5 51.0
74.0 75.0 78.0 76.5 73.5 75.5
Theophylline content (% w / w) 7.5 7.5 15 15 30 30 a
3 1 3 1 3 1
51.5 53.5 55.0* 53.5 52.5 52.0
44.5* 49.0 47.5 47.5 50.5* 48.0
Volume of glutaraldehyde-saturated toluene used. Large, medium, and small microcapsules (see text). c Calculated overall core content. * Means in a column followed by an asterisk are significantly different (P,0.05). b
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ml GAST, respectively (P,0.05); core content in all other medium-size capsules was similar (P.0.05). The highest and lowest core content in small microcapsules was found in systems cross-linked for 7.5 ml GAST and for 3 h with 15 ml GAST, respectively (P,0.05). The overall core content in microcapsules (Table 1) was not affected by crosslinking conditions (P.0.05). Core retention ranged from 73.5 to 78% and was not significantly affected by cross-linking conditions (Table 1). Our results thus indicated that the microencapsulation process conditions allowed, in all cases, high core retention. Core losses during the process could be mainly attributed to effects of the washing stage. Theophylline is soluble, to different extents, in the washing agents that were used. Core losses from the wet capsules could thus be attributed to removal of core crystals from the outer surface of microcapsules as well as to some losses from inner parts of the microcapsules, by a diffusion-driven leaching process. Indications for removal of surface core crystals during washing stages were also provided by the aforementioned results of structure analysis. Differences in core content between microcapsules of different size could be explained in light of the effect of number of core crystals initially included in the microcapsule and that of surface-to-volume ratio on core loss. It is clear that small microcapsules, that contain fewer core crystals and have an overall larger surface, would exhibit higher core loss than that obtained with larger microcapsules with overall smaller surface area and more core crystals. It could thus be concluded that core retention in microcapsules was mainly affected by core content and size of wet microcapsules at the washing stage. Our results were similar to those reported by Thanoo et al. [30] who reported core retention of about 80% in preparing chitosan microcapsules containing about 50% theophylline. Theophylline content of large microcapsules and overall core retention obtained in our study were similar to results of Latha and Jayakrishnan [14] and those of Latha et al. [7] regarding theophylline-containing casein microcapsules. Core retention level obtained in our study was higher than that reported by Heelan and Corrigan [28] for microencapsulation of different water-soluble core materials in WPI-based microcapsules.
3.3. Theophylline release from microcapsules Release of theophylline from microcapsules into SIF and SGF at 378C is depicted in Figs. 3–5. Comparing data regarding water-solubility of theophylline [31] with that regarding the ratio of theophylline to volume of fluid in our release experiments indicated that sink conditions existed in all cases. In a preliminary study, de novo microcapsules were prepared, using the same procedure used in preparing theophylline-containing capsules, and their solubility in SGF and SIF at 378C was investigated. Results indicated that the GAST-cross-linked WPI particles were insoluble in both fluids. This and the aforementioned results of structure analysis indicated that core release from theophylline-loaded microcapsules could be attributed to diffusion-driven mechanism rather than solubilization of the protein matrix. Results presented in Figs. 3–5 indicated that core release was affected by type of simulated digestive fluid, cross-linking conditions, and by microcapsule size. In all cases, the rate of core release from given microcapsules into SIF was significantly higher than that into SGF. For example, results obtained with large microcapsules prepared with 30 ml GAST and cross-linking time of 3 h indicated that complete core release was obtained after 75 and 150 min in SIF and SGF, respectively (Fig. 5A). Although not directly tested in our research, differences in release obtained in SIF and SGF could be attributed to differences in extent of swelling of WPI-based microcapsules in the two fluids [28]. It has been established that proteinbased microcapsules swelled more in SIF than in SGF and that core release was proportional to extent of swelling [14]. Effects of the nature of simulated digestive fluid on core release observed in our study were similar to those earlier reported for other protein-based microcapsules. Among these were results of Thanoo et al. [30] for release of theophylline from chitosan microcapsules, results of Latha and Jayakrishnan [14] for release of theophylline from casein-based capsules, and results of Heelan and Corrigan [28] regarding core release from WPI-based capsules. Results obtained with microcapsules from the three size categories indicated that in all cases, core release was time-dependent and was affected, to
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Fig. 3. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 7.5 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
varying extent, by cross-linking conditions (Figs. 3–5). Core release from small microcapsules (,450 mm) was significantly faster (P,0.05) than that from larger capsules. Complete core release from these capsules was observed after 20 min with SIF, regardless of cross-linking conditions, and after 30– 50 min with SGF (Figs. 3C, 4C, and 5C). Results thus indicated only a limited effect of cross-linking conditions on core release from small microcapsules and, in light of the overall fast core release, sug-
gested that small capsules obtained in our study had only limited potential when practicality of controlled and / or sustained core release applications were considered. However, results obtained with large and medium size microcapsules indicated suitability of the systems for controlled core release applications. Results obtained with large microcapsules (.700 mm) indicated that complete core release into SIF was obtained after 50–75 min and was affected, to a certain extent, by cross-linking conditions (Figs. 3A,
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Fig. 4. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 15 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
4A, and 5A). The time needed for complete core release from these microcapsules into SGF ranged from 90 to 150 min and was proportional to both amount of GAST and cross-linking reaction time. Results obtained with SGF indicated that core release from capsules cross linked with 7.5 ml GAST for 3 h or with 15 or 30 ml GAST for 1 h was 30% slower than that obtained with microcapsules cross linked for 1 h with 7.5 ml GAST. Results also indicated that regardless of amount of GAST used, core release from microcapsules cross linked for 3 h was 25–30%
slower than that from microcapsules cross-linked for 1 h. Results obtained with medium size microcapsules (500–700 mm) indicated trends similar to those observed for large microcapsules however, core release was faster (P,0.05) than that obtained with large capsules (Figs. 3B, 4B, and 5B). Complete core release from these microcapsules into SIF and SGF was obtained after 30 to 50 min and after 50 to 110 min, respectively and indicated a significant (P,0.05) effect of dissolution medium. Core release
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Fig. 5. In vitro release profile of large (A), medium size (B) and small (C) theophylline-containing WPI microcapsules in simulated gastric and intestinal fluids (SGF and SIF, respectively) at 378C. Microcapsules were cross-linked for 1 or 3 h with 30 ml of glutaraldehydesaturated toluene. In all cases, mean values (n$4) are presented.
into SGF from microcapsules cross-linked for 3 h was 80, 40 and 46% slower than that obtained with capsules cross-linked for 1 h for 7.5, 15 and 30 ml GAST, respectively. Effect of cross-linking conditions on release into SIF was only to a very limited extent (P.0.05). As stated above for large and medium size microcapsules, results suggested that generally, core release was inversely proportional to cross-linking density. However, core release from large and medium size capsules cross-linked for 3 h with 15 ml
GAST was slower than that from the same size capsules cross-linked for 3 h with 30 ml GAST (Figs. 4 and 5). These results could be attributed to the presence of cracks at the surface of microcapsules cross-linked for 3 h with 30 ml GA (Fig. 1D and E). The presence of such cracks allowed direct contact between dissolution medium and inner parts of microcapsules and thus compromised, to a certain extent, the role of the cross-linked wall matrix in governing the diffusion-controlled core release. These results suggest that although adjusting cross-
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linking conditions and hence cross-linking density can modulate rate of core release from WPI-based microcapsules, effects on structural and mechanical properties of microcapsules should be carefully assessed. Results obtained with large and medium size microcapsules indicated that effects of cross-linking conditions on core release were manifested to a greater extent with SGF than with SIF (Figs. 3–5). These results could be explained in light of the profound effect of swelling in governing diffusiondriven core release from the microcapsules. Overall, effects of microcapsule size on core release observed in our study were similar to those reported by Thanoo et al. [30] and those reported by Latha et al. [7] and could be attributed to effect of surface-tovolume ratio on the diffusion-governed core release. Heelan and Corrigan [28] reported that release of different water-soluble core materials from WPIbased microcapsules cross-linked with aqueous glutaraldehyde was very fast and was not affected by cross-linking conditions. The differences between the results of our study and those of Heelan and Corrigan [28] could be attributed to effects of the GAST used in our study. It has been suggested that crosslinking with GAST provided significantly better uniformity and efficiency of cross-linking at the surface of dispersed protein-based droplets than that accomplished with aqueous glutaraldehyde [28]. It has been also indicated [7,14] that cross-linking with GAST led to the formation of a surface net, with high cross-linking density, effective in modulating core release. It has been reported [14] that theophylline release from casein microcapsules cross-linked by GAST could be described by the Higuchi model [32]: Q 5 kt 1 / 2 where Q is the cumulative amount of drug released at time t and k depends on the surface areas and the diffusion coefficient. For systems that meet the above conditions and assuming constant surface area and diffusion coefficient, plotting Q vs. t 1 / 2 should yield a straight line. Theophylline release data presented in Figs. 3–5 were processed according to the Higuchi model and
representative results are depicted in Fig. 6. Results indicated that in all cases, the release profile was characterized by two linear segments that differed in their slope, followed by a non-linear segment, where the Higuchi model was no longer valid. Results indicated that the linear function of the Higuchi model was true only up to about 80–85% of the time needed for complete release, similar to what has been indicated by Alvarez et al. [33]. The identified changes in release rate suggested changes in the release mechanism [34]. This could probably be attributed to combined effect of diffusion through the capsule wall system and, at later stages, effects of liquid-filled pores and empty core domains. Results indicated that slopes of the linear segments and the time at which rate of release changed were mainly affected by the type of dissolution medium. This could be attributed to differences in matrix swelling, as discussed above. Overall, results presented in Fig. 6 could be explained in light of the aforementioned discussion pertaining to effects of dissolution medium and cross-linking conditions on core release.
4. Conclusion Results of our study indicated that WPI could be used in preparing water-insoluble microcapsules containing about 50% of water-soluble model drug. In all cases, theophylline retention during microencapsulation was higher than 70%, indicating the high efficiency of the microencapsulation process. Results indicated that cross-linking of the protein-based wall matrices of microcapsules by glutaraldehyde-saturated toluene via organic phase was effective in influencing rate of core release. Results obtained with crack-free capsules indicated that core release was controlled by a diffusion-driven mechanism and that the rate of core release was inversely related to cross-linking time and to the amount of GAST used. Core release was significantly affected by microcapsule size and dispersing medium type, and could be described by the Higuchi model. Results of this study indicated that whey proteins might be used as microencapsulating agents in developing microcapsules for controlled and / or sustained core release.
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Fig. 6. Theophylline release vs. square root of time in simulated gastric or intestinal fluids (SGF and SIF, respectively) at 378C from large and medium size microcapsules. Microcapsules were cross-linked for 1 h or 3 h with 30 ml or for 3 h with 15 ml of glutaraldehyde-saturated toluene (1h30, 3h30, and 3h15, respectively). In all cases, mean values (n$4) are presented.
Acknowledgements This research was supported by a grant from Dairy Management Inc. (DMI).
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