J . MICROENCAPSULATION,
1991, VOL. 8,
NO,
4, 537-545
Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: (11). Effect of temperature and pH on the stability and release behaviour of microspheres
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S. Y. LINTS, Y. L. TZANg, C. N. WENGB and C. J. LEE§ ?Department of Medical Research, Veterans General Hospital-Taipei, Taipei, Republic of China $Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan, Republic of China YDepartment of Pathology, Pig Research Institute, Chu-Nan, Taiwan, Republic of China (Received 10 December 1990; accepted 20 February 1991)
T h e in vitro stability (temperature and pH) and dissolution study (pH7.4 phosphate buffer solution and pH changed medium) of the enteric-coated microspheres containing Mycoplasma hyopneumoniae vaccine (MHV) were examined. The MHV microspheres were thermally more stable than the unencapsulated MHV. More than 90% of antigenicity was retained in the M H V microspheres for 3 weeks when stored at 4°C. T h e MHV microspheres in p H 1.2 and pH 3.0 medium were more stable than the unencapsulated MHV. T h e M H V enteric-coated microspheres exhibited an excellent enteric function to prevent pH-related inactivation. T h e influence of particle size, CAP concentration and span 80 concentration on the MHV released from microspheres was also determined. T h e smaller the particle size, the higher the dissolution rate due to the larger surface area of the smaller particle. T h e higher the concentration of span 80 used, the more the greater the amount of MHV released. This was attributed to the more porous structure of microspheres prepared by the higher concentration of span 80. By increasing the CAP concentration, however, the release rate of MHV was decreased due to the larger amount of CAP and the more compact structure of microspheres.
Introduction Mycoplasma hyopneumoniae may induce mycoplasmal pneumonia of swine, and directly causes considerable pig fatality and economic loss, which is one of the important considerations in animal husbandry. Weng has indicated that pigs show a protective effect after intramuscular inoculation with formalin-inactivated M H V in incomplete Freund’s adjuvant, and boosted intra-intestinally with M H V alone via Peyer’s patches (Weng 1986). We have found that M H V seems to be thermally stable and can preserve 90% of antigenicity at 37°C for 96h, but it is very sensitive to low pH values. At pH value below 3, MHV may lose its activity very quickly; but in p H 5 environment it is quite stable and can preserve at least 90% of its activity after 120 min of exposure (Tzan et al. 1989a). In order to protect vaccine from destruction by enzymes, or by the low p H environment of the stomach, we have used the solvent evaporation method, to prepare MHV enteric-coated microspheres (Lin et 01. 1991). T h e operating tTo whom correspondence should be addressed. 0265-2048/91 83.00 0 1991 Taylor & Francis Ltd
538
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Journal of Microencapsulation Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
conditions are optimized to obtain MHV microspheres with uniform content. In this study the stability of MHV enteric-coated microspheres in different p H conditions and temperatures was studied. T h e in oitro release behaviour of these enteric-coated microspheres was determined in p H 7-4 phosphate buffer solution and in the different pH media to evaluate their enteric action.
Materials and methods Materials Cellulose acetate phthalate (CAP), glucose and span 80 were purchased from Wako Pure Chem. Ind. Ltd (Osaka, Japan), Sigma (St Louis, MO, USA) and Hayashi Pure Chem. Ind. Ltd (Osaka, Japan), respectively. All other materials were of analytical reagent grade. Mycoplasma hyopneumoniae vaccine ( M H V ) Mycoplasma hyopneumoniae isolated by the Pig Research Institute, Taiwan, ROC, was cultured in Friis medium and treated as in our previous study (Tzan et al. 1989b). The concentrated antigen was freeze-dried in phosphate buffer solution to form a powder of Mycoplasma hyopneumoniae vaccine (MHV) and stored at - 18°C for further use. T h e ELISA analytical method was used to determine the antigenicity of MHV (Tzan et al. 1989a, b, Weng et al. 1991). Microencapsulation of M H V enteric-coated microspheres T h e solvent evaporation method was employed to prepare the MHV entericcoated microspheres, as described previously (Lin et al. 1991). Operating conditions were controlled at 20°C, 400rpm, 0.4% of span 80 and 32.4% of CAP. T h e MHV microspheres were screened by using U S P standard sieves. T h e surface topography of microspheres was examined with a scanning electron microscope (Hitachi 520, Tokyo, Japan). T h e content of MHV in microspheres was determined by the ELISA method. T h e particle size of MHV microspheres were fractioned by sieving. Stability test Temperature. T h e thermal effect on the unencapsulated and encapsulated MHV was determined under various temperature conditions, i.e. 25"C, 37"C, 50°C and 60°C. Vials were filled with a specified amount of samples. T h e vials were all kept at different temperatures. After the prescribed intervals one of the vials was sampled to assay the remaining antigenicity of MHV by the ELISA method. T h e long-term stability of the MHV enteric-coated microcapsules was also observed at 4"C, 20"C, 27°C and 37°C for 3 weeks. p H effect. The same amount of unencapsulated and encapsulated MHV was added to vials containing media at different pH values, i.e. p H 1.2, 3.0 or 5.0. At prescribed intervals the sample was filtered to assay the remaining antigenicity of MHV. T h e different size fractions of MHV microspheres were also determined for their remaining antigenicity in pH 1.2 medium at 25°C. All tests were performed in duplicate. In vitro dissolution study T h e dissolution behaviour of MHV enteric-coated microspheres was determined at 37°C by the pH changing method. T h e 250mg samples of MHV microspheres were put into five jacketed beakers containing 200ml of p H 1.2 solution. T h e rotation
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speed of the paddle was controlled at 50 rpm. During the first 2 h of dissolution in pH 1.2 solution, one beaker was taken out at 30 min intervals; the microspheres were analysed for residual antigenicity. Then the microspheres contained in the remaining beakers were filtered and the pH 1.2 solution was discarded. T h e microspheres were immediately rinsed with distilled water and then dispersed in 200 ml of pH 7.4 phosphate buffer solution at 37°C for further dissolution study. At the prescribed intervals 1 ml of solution was sampled, and the amount of MHV released from the microspheres was assayed by the ELISA method. One millilitre of MHV freedissolution medium was added to the dissolution vessel to maintain the volume of the dissolution vessel. T h e test was performed in duplicate to obtain the mean. T h e dissolution study was also performed in p H 7.4 phosphate buffer solution at 37°C using the paddle method (50 rpm).
Results and discussion Stabilization of peptides or proteins means either a decrease of rate constants of inactivation or an increase of half-lives of inactivation under denaturing conditions at certain temperature or pH values (Klibanov 1979). Immobilization is a general and universal method of stabilizing proteins or peptides; microencapsulation is one method of immobilization for protecting them from pH-related inactivation and mitigating thermal effects (Moss and Lim 1983).
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Figurel. Effect of temperature on the stability of MHV microspheres (A)and unencapsulated MHV (B).Key: 0,25"C; A , 37°C; 0,SO"C; 0,60"C. (C) Long-term test: 0 , 4°C; A , 20°C; W , 27°C; 37°C.
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Figure 1 shows the effect of temperature on the stability of unencapsulated and encapsulated MHV. It is apparent that the encapsulated MHV was more stable than the unencapsulated one. T h e higher the temperature used the lower the antigenicity retained. At the initial stage of the higher temperature, the degraded amount of inactivation of the unencapsulated MHV was higher than that of the encapsulated MHV, leading to lower remaining antigenicity. In long-term storage, more than 90% of antigenicity was retained in the MHV microspheres for 3 weeks when it was stored at 4°C (figure 1C). However, the remaining antigenicity was 75%, 67%or 35% when stored at 2 0 T , 27°C or 37"C, respectively. This suggests that 4°C might be the most appropriate storage temperature for MHV microspheres. T h e effect of p H on the stability of unencapsulated and encapsulated MHV is shown in figure 2. T h e result indicates that the MHV enteric-coated microspheres were more stable than the unencapsulated MHV at p H 1.2 and p H 3.0, since the remaining antigenicity of MHV microspheres after 2 h of incubation was about 80% (pH 3-0) and 70% (pH 1-2) when compared with that of the unencapsulated MHV [61% (pH 3.0) and 35% (pH 1.2)]. This obviously illustrates that MHV entericcoated microspheres exhibited an excellent enteric function to prevent pH-related inactivation. In pH 5 medium, both unencapsulated and encapsulated MHV were quite stable, since they still preserved about 90% antigenicity after 2 h incubation in pH 5.0 medium. Figure 2C shows the stability of MHV microspheres with different
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particle sizes in pH 1.2 solution. It is obvious that larger MHV microspheres had a better protective action than smaller ones, possibly because the larger particles might have smaller surface area and therefore make pH-related inactivation less probable. The antigenicity of MHV microspheres after 4 h incubation in p H 1.2 solution remained about 82%, 77% or 65% for 1300pm, 1015 pm or 595 pm particle sizes, respectively. However, the unencapsulated MHV had only 15% retained after 4 h incubation in pH 1.2 medium. T h e scanning electron micrographs of MHV enteric-coated microspheres of different CAP concentration are shown in figure 3. T h e porous topography appearing on the surface of microspheres prepared by lower CAP concentration is apparent. T h e higher the concentration of CAP used, the more compact the microspheres, leading to a rigid particle. In order to prevent aggregation of coacervates in the liquid paraffin during microencapsulation, a hydrophobic surfactant, span 80, was added (Lin et al. 1991). T h e effect of span 80 concentration on the surface topography of microspheres is indicated in figure 4. When span 80 concentration was 0.05% the surface of the microspheres was smooth with few pores. As concentration increased, the surface appearance gradually became porous and shrunken (figure 4C). If the concentration was >2%, the microspheres became brittle. This might be due to the higher concentration of span 80 deposited in microspheres being removed when the microspheres was soaked in chloroform to wash the residual liquid paraffin, resulting in the porous structure and the internal structure on the surface of microspheres.
Figure 3. Scanning electron micrographs of MHV microspheres made by different CAP concentration. Key: A: 242%, B: 39-0%,C: 545%.
Journal of Microencapsulation Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
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Figure 4. Scanning electron micrographs of MHV microspheres prepared by different concentrations of span 80. Key: A: OOS%, B: O4%, C: 2%. In our previous study we found that MHV was uniformly distributed in each particle size (Lin et al. 1991). T h e influence of particle size of the microspheres on the release of MHV in p H 7.4phosphate buffer solution at 37°C is shown in figure 5 . It can be seen that the smaller the particle size, the more rapid the release. Moreover, the microspheres gradually sank into the p H 7-4phosphate buffer solution due to the penetration of medium. T h e larger surface area of the smaller particle size might be responsible for this rapid release behaviour (Pongpaibul et al. 1988). Figure 6 shows the effect of the concentration of span 80 on the release of MHV microspheres. It is apparent that the release of MHV from microspheres was dependent on concentration of span 80. T h e higher the concentration of span 80 used, the greater the release amount obtained, since the microspheres made by the higher concentration of span 80 were more porous than those made by the lower concentration of span 80, resulting in more rapid release behaviour. This was proved by the surface topography of microspheres prepared by the different concentrations of span 80 (figure 4). In order to simulate the p H change of the gastrointestinal tract of swine, the dissolution with p H changed method was performed as modified from drug release of enteric-coated articles, specified in USP XXI. T h e release was carried out in pH 1.2 solution at the initial 2 h, then the dissolution was studied in p H 7.4 phosphate buffer solution. Figure 7 indicates the release patterns of MHV enteric-coated microspheres prepared by various CAP concentrations. MHV release rate, whether
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Figure 5. Release profiles of different particle sizes of MHV microspheres in pH7.4 phosphate buffer solution at 37°C. Key: 0, 1300prn; A , 1015prn; A , 670prn; 0 , 595 pm; 0, 3235 prn.
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Figure 6. Effect of span 80 concentration of the release amount of MHV microspheres in 0.6%; A , 1%; 0 , 2%. pH 7.4 phosphate buffer solution, Key: M , 005%; A , 0.1%; 0,
in pH 1.2 solution or in p H 7.4 phosphate buffer solution, was dependent on CAP concentration. By increasing CAP concentration the release rate of M H V from microspheres was decreased; this phenomenon was proved by the compact surface topography of MHV microspheres made from the higher CAP concentration, as shown in figure 3. It was also shown that these enteric-coated microspheres exhibited slower release rate in p H 1.2 solution than in the p H 7.4 phosphate buffer solution. Figure 8A shows the p H dependency of M H V released from microspheres. It is clear that the M H V microspheres showed a slower release rate in the p H S
S. Y.Lin e t al.
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Figure 7. Effect of CAP concentration on the release amount of MHV microspheres in pH 39% A , 47.4%; 0 , 54.5%. changed medium. Key: 0, 16.7%; 0 , 242%; A, 32.4%; 0 ,
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Figure 8. Effect of pH of medium on the release properties of MHV microspheres (A)and the relationship betweeh t , , and CAP concentration or pH medium (B).Key: 0 , pH 5.0; A , pH 6.0; 0 , pH 7.4; 0 , pH 8.0.
medium. When p H was beyond 5.0 a rapid release rate of MHV was found, since CAP dissolved at approximately p H 5.4 (Delporte 1979). T h i s result agreed with the release profiles of the enteric-coated CAP microcapsules prepared by the spraydrying technique or wet granulation (Takenaka et al. 1980, Lin et al. 1987). Figure 8B shows the relationship between t 5 0 (time for 50% released) and CAP concentration or p H of dissolution medium. T h e higher the concentration of CAP the higher the value of t S O , for which the compact surface and wall thickness of microspheres might be responsible (Wagner 1971, Takenaka et al. 1979). T h e lower solubility of CAP in the dissolution medium of lower p H might result in the large t50 value. From the above results of an in w h o stability and release study, the protective and enteric function of MHV microspheres is assured. Field studies on animals are currently in progress.
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Acknowledgement This project was supported by t h e Foundation of VGH-TH, Taipei, ROC ( V G H - T H 78-018-02).
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References DEI-PORTE, J. P., 1979, Effects of aging on physico-chemical properties of free cellulose acetate phthalate films. Pharmaceutical Industry, 41, 984-990. M., K I ~ I B A NA. OV , 1979, Enzyme stabilization by immobilization. Analytical Biochemistry, 93, 1-25. LIN,S. Y., and KAWASHIMA, Y., 1987, Drug release from tablets containing cellulose acetate phthalate as an additive or enteric-coating material. Pharmaceutical Research, 4,70-74. Y. L., LEE,C. J., and WENG,C. N., 1991, Preparation of enteric-coated LIN,S. Y., TZAN, microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: I. Formation condition and micromeritic properties. Journal of Microencapsulation, 8, 3 17-325. Moss,-R. D., and LIM,F., 1983, Biomedical Applications of Microencapsulation, edited by F. Lim. (CRC Press, Florida), pp. 119-135. PONGPAIBUL, Y., MARYAMA, K., and IWATSURU, M., 1988, Formation and in-vivro evaluation of theophylline-loaded poly(methy1 methacrylate) microspheres. Journal of Pharmacy and Pharmacology, 40, 530-533. H., KAWASHIMA, Y., and LIN, S. Y., 1979. T h e effects of wall thickness and TAKENAKA, amount of hardening agent on the release characteristics of sulfarnethoxazole microcapsules prepared by gelatin-acacia complex coacervation. Chemical Pharmaceutical Bulletin, 27, 3054-3060. TAKENAKA, H., KAWASHIMA, Y., and LIN, S. Y., 1980. Preparation of enteric-coated microcapsules of tableting by a spray drying technique and in vitro simulation of drug release from the tablet in GI tract. Journal of Pharmaceutical Sciences, 70, 1256-1260. TZAN,Y. L., WENG,C. N., LEE,C. J., and LIN, S. Y., 1989a, Determination of antigenic characteristics of Mycoplasma hyopneumoniae by enzyme-linked immunosorbent assay. I I. The stabilities of Mycoplasma hyopneumoniae antigen. Journal of the Chinese Society of Veterinary Science, 15, 199-206. TZAN, Y. L., WENG,C. N., LEE,C. J., and LIN, S. Y., 1989b, Determination of antegenic characteristics of Mycoplasma hyopneumoniae by enzyme-linked immunosorbent assay. I. The establishment and performance of ELISA. Journal of the Chinese Society of Veterinary Science, 15, 193-198. J. G., 1971. Riopharmaceuticals and Releoant Pharmacokinetics, (Drug Intelligence WAGNER, Publications, Hamilton). p. 123. WENG,C. N., 1986, Annual Report of Pig Research Znstitute (Taiwan, ROC), 3 2 4 1 . WENG,C. N., LIN, S. Y., TZAN, Y. L., and LEE,C. J., 1991, Determination of antigenic characteristics & stability of Mycoplasma hyopneumoniae. Biotechnology Progress, 7 , 69-7 1 .
1991, VOL. 8,
NO,
4, 537-545
Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: (11). Effect of temperature and pH on the stability and release behaviour of microspheres
Journal of Microencapsulation Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
S. Y. LINTS, Y. L. TZANg, C. N. WENGB and C. J. LEE§ ?Department of Medical Research, Veterans General Hospital-Taipei, Taipei, Republic of China $Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan, Republic of China YDepartment of Pathology, Pig Research Institute, Chu-Nan, Taiwan, Republic of China (Received 10 December 1990; accepted 20 February 1991)
T h e in vitro stability (temperature and pH) and dissolution study (pH7.4 phosphate buffer solution and pH changed medium) of the enteric-coated microspheres containing Mycoplasma hyopneumoniae vaccine (MHV) were examined. The MHV microspheres were thermally more stable than the unencapsulated MHV. More than 90% of antigenicity was retained in the M H V microspheres for 3 weeks when stored at 4°C. T h e MHV microspheres in p H 1.2 and pH 3.0 medium were more stable than the unencapsulated MHV. T h e M H V enteric-coated microspheres exhibited an excellent enteric function to prevent pH-related inactivation. T h e influence of particle size, CAP concentration and span 80 concentration on the MHV released from microspheres was also determined. T h e smaller the particle size, the higher the dissolution rate due to the larger surface area of the smaller particle. T h e higher the concentration of span 80 used, the more the greater the amount of MHV released. This was attributed to the more porous structure of microspheres prepared by the higher concentration of span 80. By increasing the CAP concentration, however, the release rate of MHV was decreased due to the larger amount of CAP and the more compact structure of microspheres.
Introduction Mycoplasma hyopneumoniae may induce mycoplasmal pneumonia of swine, and directly causes considerable pig fatality and economic loss, which is one of the important considerations in animal husbandry. Weng has indicated that pigs show a protective effect after intramuscular inoculation with formalin-inactivated M H V in incomplete Freund’s adjuvant, and boosted intra-intestinally with M H V alone via Peyer’s patches (Weng 1986). We have found that M H V seems to be thermally stable and can preserve 90% of antigenicity at 37°C for 96h, but it is very sensitive to low pH values. At pH value below 3, MHV may lose its activity very quickly; but in p H 5 environment it is quite stable and can preserve at least 90% of its activity after 120 min of exposure (Tzan et al. 1989a). In order to protect vaccine from destruction by enzymes, or by the low p H environment of the stomach, we have used the solvent evaporation method, to prepare MHV enteric-coated microspheres (Lin et 01. 1991). T h e operating tTo whom correspondence should be addressed. 0265-2048/91 83.00 0 1991 Taylor & Francis Ltd
538
S. Y . Lin et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
conditions are optimized to obtain MHV microspheres with uniform content. In this study the stability of MHV enteric-coated microspheres in different p H conditions and temperatures was studied. T h e in oitro release behaviour of these enteric-coated microspheres was determined in p H 7-4 phosphate buffer solution and in the different pH media to evaluate their enteric action.
Materials and methods Materials Cellulose acetate phthalate (CAP), glucose and span 80 were purchased from Wako Pure Chem. Ind. Ltd (Osaka, Japan), Sigma (St Louis, MO, USA) and Hayashi Pure Chem. Ind. Ltd (Osaka, Japan), respectively. All other materials were of analytical reagent grade. Mycoplasma hyopneumoniae vaccine ( M H V ) Mycoplasma hyopneumoniae isolated by the Pig Research Institute, Taiwan, ROC, was cultured in Friis medium and treated as in our previous study (Tzan et al. 1989b). The concentrated antigen was freeze-dried in phosphate buffer solution to form a powder of Mycoplasma hyopneumoniae vaccine (MHV) and stored at - 18°C for further use. T h e ELISA analytical method was used to determine the antigenicity of MHV (Tzan et al. 1989a, b, Weng et al. 1991). Microencapsulation of M H V enteric-coated microspheres T h e solvent evaporation method was employed to prepare the MHV entericcoated microspheres, as described previously (Lin et al. 1991). Operating conditions were controlled at 20°C, 400rpm, 0.4% of span 80 and 32.4% of CAP. T h e MHV microspheres were screened by using U S P standard sieves. T h e surface topography of microspheres was examined with a scanning electron microscope (Hitachi 520, Tokyo, Japan). T h e content of MHV in microspheres was determined by the ELISA method. T h e particle size of MHV microspheres were fractioned by sieving. Stability test Temperature. T h e thermal effect on the unencapsulated and encapsulated MHV was determined under various temperature conditions, i.e. 25"C, 37"C, 50°C and 60°C. Vials were filled with a specified amount of samples. T h e vials were all kept at different temperatures. After the prescribed intervals one of the vials was sampled to assay the remaining antigenicity of MHV by the ELISA method. T h e long-term stability of the MHV enteric-coated microcapsules was also observed at 4"C, 20"C, 27°C and 37°C for 3 weeks. p H effect. The same amount of unencapsulated and encapsulated MHV was added to vials containing media at different pH values, i.e. p H 1.2, 3.0 or 5.0. At prescribed intervals the sample was filtered to assay the remaining antigenicity of MHV. T h e different size fractions of MHV microspheres were also determined for their remaining antigenicity in pH 1.2 medium at 25°C. All tests were performed in duplicate. In vitro dissolution study T h e dissolution behaviour of MHV enteric-coated microspheres was determined at 37°C by the pH changing method. T h e 250mg samples of MHV microspheres were put into five jacketed beakers containing 200ml of p H 1.2 solution. T h e rotation
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speed of the paddle was controlled at 50 rpm. During the first 2 h of dissolution in pH 1.2 solution, one beaker was taken out at 30 min intervals; the microspheres were analysed for residual antigenicity. Then the microspheres contained in the remaining beakers were filtered and the pH 1.2 solution was discarded. T h e microspheres were immediately rinsed with distilled water and then dispersed in 200 ml of pH 7.4 phosphate buffer solution at 37°C for further dissolution study. At the prescribed intervals 1 ml of solution was sampled, and the amount of MHV released from the microspheres was assayed by the ELISA method. One millilitre of MHV freedissolution medium was added to the dissolution vessel to maintain the volume of the dissolution vessel. T h e test was performed in duplicate to obtain the mean. T h e dissolution study was also performed in p H 7.4 phosphate buffer solution at 37°C using the paddle method (50 rpm).
Results and discussion Stabilization of peptides or proteins means either a decrease of rate constants of inactivation or an increase of half-lives of inactivation under denaturing conditions at certain temperature or pH values (Klibanov 1979). Immobilization is a general and universal method of stabilizing proteins or peptides; microencapsulation is one method of immobilization for protecting them from pH-related inactivation and mitigating thermal effects (Moss and Lim 1983).
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Figurel. Effect of temperature on the stability of MHV microspheres (A)and unencapsulated MHV (B).Key: 0,25"C; A , 37°C; 0,SO"C; 0,60"C. (C) Long-term test: 0 , 4°C; A , 20°C; W , 27°C; 37°C.
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Journal of Microencapsulation Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
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Figure 1 shows the effect of temperature on the stability of unencapsulated and encapsulated MHV. It is apparent that the encapsulated MHV was more stable than the unencapsulated one. T h e higher the temperature used the lower the antigenicity retained. At the initial stage of the higher temperature, the degraded amount of inactivation of the unencapsulated MHV was higher than that of the encapsulated MHV, leading to lower remaining antigenicity. In long-term storage, more than 90% of antigenicity was retained in the MHV microspheres for 3 weeks when it was stored at 4°C (figure 1C). However, the remaining antigenicity was 75%, 67%or 35% when stored at 2 0 T , 27°C or 37"C, respectively. This suggests that 4°C might be the most appropriate storage temperature for MHV microspheres. T h e effect of p H on the stability of unencapsulated and encapsulated MHV is shown in figure 2. T h e result indicates that the MHV enteric-coated microspheres were more stable than the unencapsulated MHV at p H 1.2 and p H 3.0, since the remaining antigenicity of MHV microspheres after 2 h of incubation was about 80% (pH 3-0) and 70% (pH 1-2) when compared with that of the unencapsulated MHV [61% (pH 3.0) and 35% (pH 1.2)]. This obviously illustrates that MHV entericcoated microspheres exhibited an excellent enteric function to prevent pH-related inactivation. In pH 5 medium, both unencapsulated and encapsulated MHV were quite stable, since they still preserved about 90% antigenicity after 2 h incubation in pH 5.0 medium. Figure 2C shows the stability of MHV microspheres with different
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particle sizes in pH 1.2 solution. It is obvious that larger MHV microspheres had a better protective action than smaller ones, possibly because the larger particles might have smaller surface area and therefore make pH-related inactivation less probable. The antigenicity of MHV microspheres after 4 h incubation in p H 1.2 solution remained about 82%, 77% or 65% for 1300pm, 1015 pm or 595 pm particle sizes, respectively. However, the unencapsulated MHV had only 15% retained after 4 h incubation in pH 1.2 medium. T h e scanning electron micrographs of MHV enteric-coated microspheres of different CAP concentration are shown in figure 3. T h e porous topography appearing on the surface of microspheres prepared by lower CAP concentration is apparent. T h e higher the concentration of CAP used, the more compact the microspheres, leading to a rigid particle. In order to prevent aggregation of coacervates in the liquid paraffin during microencapsulation, a hydrophobic surfactant, span 80, was added (Lin et al. 1991). T h e effect of span 80 concentration on the surface topography of microspheres is indicated in figure 4. When span 80 concentration was 0.05% the surface of the microspheres was smooth with few pores. As concentration increased, the surface appearance gradually became porous and shrunken (figure 4C). If the concentration was >2%, the microspheres became brittle. This might be due to the higher concentration of span 80 deposited in microspheres being removed when the microspheres was soaked in chloroform to wash the residual liquid paraffin, resulting in the porous structure and the internal structure on the surface of microspheres.
Figure 3. Scanning electron micrographs of MHV microspheres made by different CAP concentration. Key: A: 242%, B: 39-0%,C: 545%.
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Figure 4. Scanning electron micrographs of MHV microspheres prepared by different concentrations of span 80. Key: A: OOS%, B: O4%, C: 2%. In our previous study we found that MHV was uniformly distributed in each particle size (Lin et al. 1991). T h e influence of particle size of the microspheres on the release of MHV in p H 7.4phosphate buffer solution at 37°C is shown in figure 5 . It can be seen that the smaller the particle size, the more rapid the release. Moreover, the microspheres gradually sank into the p H 7-4phosphate buffer solution due to the penetration of medium. T h e larger surface area of the smaller particle size might be responsible for this rapid release behaviour (Pongpaibul et al. 1988). Figure 6 shows the effect of the concentration of span 80 on the release of MHV microspheres. It is apparent that the release of MHV from microspheres was dependent on concentration of span 80. T h e higher the concentration of span 80 used, the greater the release amount obtained, since the microspheres made by the higher concentration of span 80 were more porous than those made by the lower concentration of span 80, resulting in more rapid release behaviour. This was proved by the surface topography of microspheres prepared by the different concentrations of span 80 (figure 4). In order to simulate the p H change of the gastrointestinal tract of swine, the dissolution with p H changed method was performed as modified from drug release of enteric-coated articles, specified in USP XXI. T h e release was carried out in pH 1.2 solution at the initial 2 h, then the dissolution was studied in p H 7.4 phosphate buffer solution. Figure 7 indicates the release patterns of MHV enteric-coated microspheres prepared by various CAP concentrations. MHV release rate, whether
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Figure 5. Release profiles of different particle sizes of MHV microspheres in pH7.4 phosphate buffer solution at 37°C. Key: 0, 1300prn; A , 1015prn; A , 670prn; 0 , 595 pm; 0, 3235 prn.
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Figure 6. Effect of span 80 concentration of the release amount of MHV microspheres in 0.6%; A , 1%; 0 , 2%. pH 7.4 phosphate buffer solution, Key: M , 005%; A , 0.1%; 0,
in pH 1.2 solution or in p H 7.4 phosphate buffer solution, was dependent on CAP concentration. By increasing CAP concentration the release rate of M H V from microspheres was decreased; this phenomenon was proved by the compact surface topography of MHV microspheres made from the higher CAP concentration, as shown in figure 3. It was also shown that these enteric-coated microspheres exhibited slower release rate in p H 1.2 solution than in the p H 7.4 phosphate buffer solution. Figure 8A shows the p H dependency of M H V released from microspheres. It is clear that the M H V microspheres showed a slower release rate in the p H S
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Figure 7. Effect of CAP concentration on the release amount of MHV microspheres in pH 39% A , 47.4%; 0 , 54.5%. changed medium. Key: 0, 16.7%; 0 , 242%; A, 32.4%; 0 ,
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Figure 8. Effect of pH of medium on the release properties of MHV microspheres (A)and the relationship betweeh t , , and CAP concentration or pH medium (B).Key: 0 , pH 5.0; A , pH 6.0; 0 , pH 7.4; 0 , pH 8.0.
medium. When p H was beyond 5.0 a rapid release rate of MHV was found, since CAP dissolved at approximately p H 5.4 (Delporte 1979). T h i s result agreed with the release profiles of the enteric-coated CAP microcapsules prepared by the spraydrying technique or wet granulation (Takenaka et al. 1980, Lin et al. 1987). Figure 8B shows the relationship between t 5 0 (time for 50% released) and CAP concentration or p H of dissolution medium. T h e higher the concentration of CAP the higher the value of t S O , for which the compact surface and wall thickness of microspheres might be responsible (Wagner 1971, Takenaka et al. 1979). T h e lower solubility of CAP in the dissolution medium of lower p H might result in the large t50 value. From the above results of an in w h o stability and release study, the protective and enteric function of MHV microspheres is assured. Field studies on animals are currently in progress.
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Acknowledgement This project was supported by t h e Foundation of VGH-TH, Taipei, ROC ( V G H - T H 78-018-02).
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References DEI-PORTE, J. P., 1979, Effects of aging on physico-chemical properties of free cellulose acetate phthalate films. Pharmaceutical Industry, 41, 984-990. M., K I ~ I B A NA. OV , 1979, Enzyme stabilization by immobilization. Analytical Biochemistry, 93, 1-25. LIN,S. Y., and KAWASHIMA, Y., 1987, Drug release from tablets containing cellulose acetate phthalate as an additive or enteric-coating material. Pharmaceutical Research, 4,70-74. Y. L., LEE,C. J., and WENG,C. N., 1991, Preparation of enteric-coated LIN,S. Y., TZAN, microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: I. Formation condition and micromeritic properties. Journal of Microencapsulation, 8, 3 17-325. Moss,-R. D., and LIM,F., 1983, Biomedical Applications of Microencapsulation, edited by F. Lim. (CRC Press, Florida), pp. 119-135. PONGPAIBUL, Y., MARYAMA, K., and IWATSURU, M., 1988, Formation and in-vivro evaluation of theophylline-loaded poly(methy1 methacrylate) microspheres. Journal of Pharmacy and Pharmacology, 40, 530-533. H., KAWASHIMA, Y., and LIN, S. Y., 1979. T h e effects of wall thickness and TAKENAKA, amount of hardening agent on the release characteristics of sulfarnethoxazole microcapsules prepared by gelatin-acacia complex coacervation. Chemical Pharmaceutical Bulletin, 27, 3054-3060. TAKENAKA, H., KAWASHIMA, Y., and LIN, S. Y., 1980. Preparation of enteric-coated microcapsules of tableting by a spray drying technique and in vitro simulation of drug release from the tablet in GI tract. Journal of Pharmaceutical Sciences, 70, 1256-1260. TZAN,Y. L., WENG,C. N., LEE,C. J., and LIN, S. Y., 1989a, Determination of antigenic characteristics of Mycoplasma hyopneumoniae by enzyme-linked immunosorbent assay. I I. The stabilities of Mycoplasma hyopneumoniae antigen. Journal of the Chinese Society of Veterinary Science, 15, 199-206. TZAN, Y. L., WENG,C. N., LEE,C. J., and LIN, S. Y., 1989b, Determination of antegenic characteristics of Mycoplasma hyopneumoniae by enzyme-linked immunosorbent assay. I. The establishment and performance of ELISA. Journal of the Chinese Society of Veterinary Science, 15, 193-198. J. G., 1971. Riopharmaceuticals and Releoant Pharmacokinetics, (Drug Intelligence WAGNER, Publications, Hamilton). p. 123. WENG,C. N., 1986, Annual Report of Pig Research Znstitute (Taiwan, ROC), 3 2 4 1 . WENG,C. N., LIN, S. Y., TZAN, Y. L., and LEE,C. J., 1991, Determination of antigenic characteristics & stability of Mycoplasma hyopneumoniae. Biotechnology Progress, 7 , 69-7 1 .
