IV. Results and Discussion Part I. Influence of viscosity and uronic acid composition on the properties of alginate films and microspheres produced by emulsification. A. Film study Calcium alginate films were produced by a solvent evaporation technique using different grades of sodium alginate (Table 6). The films were used to assess the influence of alginate composition on the tensile properties of alginate microsphere matrix. At least 0.15 M CaCl2 was required to produce smooth films of sufficient strength which could be handled without any breakage. Lower CaCl2 concentrations gave rise to small protuberances in LH and FL films. In addition, there was little change in film weight (LH, MP (Table 10). There was no significant difference (p>0.05) in drug content between LH and MP microspheres despite their difference in mean size and extent of cross-linking. The same trend was observed between NI and FL microspheres with respect to mean size. MA, FL and NI had viscosities in the range of 308-390 mPa.s and produced comparable extent of microsphere aggregation. The difference in the degree of agglomeration of the yield among between NI, FL and MA microspheres is less than % (Table 10). Among these, MA microspheres had markedly larger mean size (6-13 %) but only slightly higher drug content (4-5 %). Overall, it was clearly seen that the drug content was not predominantly affected by microsphere size and extent of cross-linking. The viscosity of sodium alginate played a more important role as indicated by their linear relationship with drug content (r2 =0.977). The viscous nature of sodium alginate acted as a barrier to the diffusion of drug out of the alginate globules during emulsification with less drug lost to the continuous phase during the process than if alginate was not present. 89 100 µm (a) (b) Figure 10. (a) Blank alginate microspheres. (b) Alginate microspheres containing sulphaguanidine (indicated by bright drug crystals present in the microspheres). 90 Microspheres produced from the different types of sodium alginate showed relatively rapid rate of release with more than 50 % of sulphaguanidine released at the end of 60 (or 3600s). Drug release from LH microspheres was the fastest, followed by MP, NI, MA and FL microspheres (Table 11). As sodium alginate was cross-linked to form insoluble calcium alginate microspheres, the influence of viscosity was expected to be manifested primarily through its effect on microsphere morphology. LH and MP microspheres produced from alginates of lower viscosity were largely discrete. The smaller LH microspheres exhibited a faster drug release than the larger MP microspheres. For alginates of higher viscosities (NI, FL and MA), extents of aggregation and cross-linking played much more important roles (Figure 11). Drug release trends in distilled water and in 0.1 N HCl (pH ≈ 1.2) were similar but release rates were much higher in the acidic medium (Table 11). The latter was attributed to the greater solubility of sulphaguanidine in acidic medium and conversion of calcium alginate to alginic acid, which is more permeable to drug diffusion. Similar findings in beads were observed by other researchers (Østberg et al., 1994). Further investigations were carried out to examine the relative influence of the afore-mentioned factors. Alginate microsphere batches were separated into different size fractions and the effect of microsphere size on the encapsulation properties and drug release characteristics was studied. This would provide more conclusive evidence on the effects of alginate composition and the extent of calcium crosslinking on the drug encapsulation efficiency and release properties of the microspheres with defined size range of microspheres. Microsphere batches of 54-75 µm were obtained for each type of alginate microspheres by sieving. Sieved microspheres in the size range of 54-75 µm were denoted as S microspheres to 91 Table 11. Release parameters of alginate microspheres in different media. Release in deionised water Alginate code S microspheres* Release in 0.1 N HCl Calcium alginate microspheres Calcium alginate microspheres t25% (s) t50% (s) t25% (s) t50% (s) t25% (s) t50% (s) LH 408.75 ± 12.74 2220.63 ± 139.69 41.50 ± 4.66 104.50 ± 10.24 47.60 ± 5.70 69.00 ± 6.04 MP 127.75 ± 6.36 626.25 ± 72.78 67.75 ± 7.69 201.50 ± 9.56 41.75 ± 2.50 69.50 ± 6.76 NI 173.67 ± 11.23 946.67 ± 90.08 155.00 ± 16.96 1421.00 ± 207.98 64.83 ± 4.94 267.50 ± 48.90 FL 357.50 ± 28.47 1628.75 ± 139.69 593.33 ± 47.38 3441.67 ± 174.02 96.25 ± 5.33 758.13 ± 101.85 MA 230.00 ± 19.69 1468.75 ± 82.37 255.00 ± 19.24 2364.00 ± 186.67 79.00 ± 6.54 455.00 ± 31.83 *Sieved microspheres in the size range of 54-75 µm. 92 92 800 4500 4000 700 3500 600 3000 2500 400 2000 t50% (s) t25% (s) 500 300 1500 200 1000 100 500 50 55 60 Ca 65 2+ 70 75 80 85 90 95 content of microspheres (mg) t25% t50% Figure 11. Changes in t25% and t50% with increased Ca2+ content of alginate microspheres 93 differentiate from the unsieved products. Highly discrete MP microspheres were separated into different size fractions. The larger size fractions showed comparable drug contents, which were significantly lower than that of the smallest fraction (p0.94). Increase in drug release rate with decrease in microsphere size was attributed to the larger surface area to volume ratio of smaller microspheres which promoted faster drug release (Table 12). Significant differences between the t25% and the t50% values of each size fraction of microspheres further emphasized the pronounced effect of the microsphere size on drug release from alginate microspheres. For microspheres within the size range of 54-75 µm, MA microspheres had the highest drug content followed by NI, FL, LH and MP (Table 10). A general increase in drug content was observed with higher alginate viscosity and greater extent of cross-linking (Tables and 10). The results suggested that the viscosity of the alginate and the amount of Ca2+ cross-linked with alginate played more dominant roles than microsphere size in controlling encapsulation efficiency of the microspheres. Regression analysis between viscosity and drug content of S microspheres (r2>0.8, p value= 0.031) as well as between Ca2+ contents and drug content of the S microspheres (r2< 0.6, p value= 0.130) revealed that the viscosity of alginates might have a greater influence on the encapsulation efficiency of the microspheres. The high viscosity grade alginate could impede drug loss by diffusion during the formation of the microspheres. In addition, the higher drug contents observed in S 94 Table 12. Drug content and drug release parameters of MP microspheres of different size fractions. Size range of microspheres (µm) Drug content (%) t25% (s) t50% (s) 32-53 27.77 ± 0.038 76.83 ± 4.39 184.17 ± 14.225 54-75 23.83 ± 0.200 127.50 ± 5.95 626.25 ± 72.783 76-100 23.86 ± 0.109 305.00 ± 26.61 2217.50 ± 39.449 101-125 22.78 ± 0.106 459.00 ± 18.33 2720.00 ± 171.464 95 microspheres produced from alginates of high viscosity grades (NI, FL and MA) unequivocally reflected the stronger influence of viscosity over the amount of crosslinking calcium on the drug content of these S microspheres (Tables and 10). For S microspheres, it was generally observed that MP exhibited the fastest rate of drug release followed by NI, MA, FL and LH microspheres (Table 11). Figure 12 suggests that the alginate matrix must be sufficiently cross-linked with at least a minimum amount of Ca2+ to be effective as a barrier to drug diffusion. It was also noted that the decrease in the rate of drug release with increased Ca2+ content was only observed in microspheres produced with alginates of higher viscosity (Tables and 10 and Figure 12). Among these alginates, MA and NI had low G while FL had high G contents. Thus, this further supported the earlier suggestion that the viscosity of the alginate would influence the interaction between calcium and alginate to a greater extent than the monomeric composition of the alginate as a consequence of cross-linking by emulsification. In fact, the S microspheres that were produced from alginates with lower viscosities (LH and MP) also had lower calcium contents (Tables and 10). These microspheres were less well cross-linked and swelled to a great extent during dissolution studies, forming a gelatinous barrier which slowed down the rate of drug release. 96 500 1250 1000 750 300 t50% (s) t25% (s) 400 500 200 250 100 60 65 70 75 80 85 90 95 Ca2+ content of alginate microspheres (mg) t25% t50% Figure 12. Influence of Ca2+ content on the rate of drug release of S microspheres. 97 [...]... increased Ca2+ content was only observed in microspheres produced with alginates of higher viscosity (Tables 8 and 10 and Figure 12 ) Among these alginates, MA and NI had low G while FL had high G contents Thus, this further supported the earlier suggestion that the viscosity of the alginate would influence the interaction between calcium and alginate to a greater extent than the monomeric composition of the. .. ± 4.39 18 4 .17 ± 14 .225 54-75 23.83 ± 0.200 12 7.50 ± 5.95 626.25 ± 72.783 76 -10 0 23.86 ± 0 .10 9 305.00 ± 26. 61 2 217 .50 ± 39.449 10 1 -12 5 22.78 ± 0 .10 6 459.00 ± 18 .33 2720.00 ± 17 1.464 95 microspheres produced from alginates of high viscosity grades (NI, FL and MA) unequivocally reflected the stronger influence of viscosity over the amount of crosslinking calcium on the drug content of these S microspheres. .. that the viscosity of the alginate and the amount of Ca2+ cross-linked with alginate played more dominant roles than microsphere size in controlling encapsulation efficiency of the microspheres Regression analysis between viscosity and drug content of S microspheres (r2>0.8, p value= 0.0 31) as well as between Ca2+ contents and drug content of the S microspheres (r2< 0.6, p value= 0 .13 0) revealed that the. .. viscosity of alginates might have a greater influence on the encapsulation efficiency of the microspheres The high viscosity grade alginate could impede drug loss by diffusion during the formation of the microspheres In addition, the higher drug contents observed in S 94 Table 12 Drug content and drug release parameters of MP microspheres of different size fractions Size range of microspheres (µm) Drug content... indicating a strong influence of the polymer viscosity on the extent of cross-linking in the microspheres In film formation, the sodium alginate was in direct contact with the cross-linking solution while in microsphere formation, the interaction between dispersed globules of sodium 88 alginate and cross-linking agent was dependent on the random collision between the globules (Heng et al., 2003) Alginates... the encapsulation properties and drug release characteristics was studied This would provide more conclusive evidence on the effects of alginate composition and the extent of calcium crosslinking on the drug encapsulation efficiency and release properties of the microspheres with defined size range of microspheres Microsphere batches of 54-75 µm were obtained for each type of alginate microspheres by... fraction of microspheres further emphasized the pronounced effect of the microsphere size on drug release from alginate microspheres For microspheres within the size range of 54-75 µm, MA microspheres had the highest drug content followed by NI, FL, LH and MP (Table 10 ) A general increase in drug content was observed with higher alginate viscosity and greater extent of cross-linking (Tables 8 and 10 ) The. .. by the emulsification process In comparison with films, the microspheres showed a different trend in crosslinking Despite having lower M/G ratios, LH and FL microspheres were cross-linked to a lower extent as indicated by their relatively low Ca2+ content (Tables 6 and 8) On the other hand, a fairly linear relationship (r2 =0.86) was observed between the viscosity of sodium alginate and the Ca2+ content,... trends in the properties of the alginate matrix Microscopic examination showed that the microspheres were filled with drug particles (Figure 10 ) The drug content of the microspheres increased in the following order: MA>NI, FL>LH, MP (Table 10 ) There was no significant difference (p>0.05) in drug content between LH and MP microspheres despite their difference in mean size and extent of cross-linking The same... between NI and FL microspheres with respect to mean size MA, FL and NI had viscosities in the range of 308-390 mPa.s and produced comparable extent of microsphere aggregation The difference in the degree of agglomeration of the yield among between NI, FL and MA microspheres is less than 5 % (Table 10 ) Among these, MA microspheres had markedly larger mean size (6 -13 %) but only slightly higher drug content . difference LH 81. 73 ± 2.58 92.43 ± 1. 89 +13 .09 2337.26 ± 72.52 3236 .16 ± 11 3.36 +38.46 18 .64 ± 1. 10 10 .15 ± 1. 16 -8.49 MP 87. 91 ± 2.48 10 2.08 ± 2.50 +16 .12 2302.57 ± 74 .10 3346.87. +45.35 25 .18 ± 1. 48 8.44 ± 0.36 -16 .74 NI 57.50 ± 1. 91 10 0.69 ± 2.69 +75 .11 19 55. 71 ± 56.47 3409 .17 ± 86.02 +74.32 19 .68 ± 1. 53 11 .68 ± 0.77 -8.00 FL 55.27 ± 1. 90 90. 91 ± 0.85. between the viscosity of sodium alginate and the Ca 2+ content, indicating a strong influence of the polymer viscosity on the extent of cross-linking in the microspheres. In film formation, the