Mechanisms of polymer ca2+ interaction and their effects on the characteristics of alginate microspheres and films 2

Influence of different cross-linking methods on the properties of alginate microspheres produced by emulsification.. Preparation of partially cross-linked alginate for microsphere produc

Part II Influence of different cross-linking methods on the properties of alginate microspheres produced by emulsification

A Production of the alginate microspheres by the emulsification/external linking method

cross-A1 Preparation of partially cross-linked alginate for microsphere production

A small amount of calcium chloride was incorporated into sodium alginate to produce a partially cross-linked alginate in solution The calcium chloride solution was added slowly with vigorous stirring to avoid rapid clumping of the alginates due

to cross-linking (Figure 4) Concentrations of the calcium chloride above 0.08% w/w

in calcium chloride-alginate mixture led to an almost instantaneous sol-gel tranformation in the absence of high shear stirring, making it impossible to use the mixture for microsphere formation by emulsification Therefore, 0.08 % w/w of calcium chloride was carefully incorporated as it allowed the mixture to remain in the fluid state for a sufficient period of time after stirring had stopped

During the shearing process, heat was generated which might cause degradation of the macromolecular alginate polymer into lower molecular weight fragments An exponential decrease in the viscosities of alginate solutions with decreasing molecular weight of the polymer had been reported (Konget al., 2003).

Hence a change in the viscosity of the solution could be used to indicate polymer degradation No significant difference in viscosities was observed between SA solution and S1 mixture (p>0.05), indicating that the shearing action and associated heat produced was unlikely to cause degradation of the alginate used (Figure 4 and Table 13)

Table 13 Viscosities of alginate solutions subjected to high shear with/without partial cross-linking and the properties of resultant microspheres

Properties of microspheres

viscosity

A2 Properties of microspheres produced from the partially cross-linked alginate

Different amounts of Ca2+ were added to the partially cross-link sodium alginate before it was used to produce microspheres by the emulsification/external cross-linking method The microspheres obtained were compared with those prepared from sodium alginate (i.e without partial cross-linking) This study was aimed at evaluating the effect of the cross-linking technique on the properties of the microspheres produced

The microspheres produced from the different alginate mixtures were spherical, with mean diameters ranging from 192 to 220 µm (Figure 13 and Table 13) S3 microspheres had the widest size distribution among the different batches SA and S1 microspheres were significantly smaller than S3 microspheres (p<0.05) (Table 13) The same emulsification conditions were used for all the alginate solutions/mixtures The larger S3 microspheres were attributed to less effective dispersion of the more viscous S3 mixture into globules as small as those of SA and S1 solutions A comparison of drug content and mean size of the microspheres suggested that size was not a primary factor affecting drug encapsulation efficiency (r2=0.476) The higher drug content of both SA and S1 microspheres as compared to S2 and S3 microspheres (p<0.05) showed that the use of partially cross-linked alginate in the preparation of microspheres reduced the drug encapsulation efficiency The results suggested that a more orderly arrangement of the polymeric chains resulted in less flexibility and a poor ability in entrapping drug particles Being larger in size, S2 and S3 microspheres had a smaller specific surface area than SA and S1 microspheres for drug release In spite of this, the rate of drug release from S2 and S3 microspheres was found to be comparable with that of SA and S1 microspheres The t75% values of

SA and S1 microspheres were 17.1 min and 22.9 min respectively and 14.3 min for

(a) (b)

both S2 and S3 microspheres (Table 13) This showed that the matrix of the S2 and S3 microspheres was more permeable to drug release than that of SA and S1 microspheres It could therefore be inferred that partial cross-linking of alginate had fixed the arrangement of the polymer chains with respect to one another, thereby reducing subsequent interaction which involved binary binding sites via the divalent calcium ion The more permeable matrix of S2 and S3 microspheres was expected to favour greater drug loss through diffusion during washing of the microspheres This could partially explain the lower drug content of the S2 and S3 microspheres The higher viscosity of S3 mixture as compared to S2 mixture would impede the diffusion

of the drug from the polymer phase to the organic phase during emulsification resulting in a higher drug content of S3 microspheres (p<0.05)(Table 13)

Although discrete and spherical calcium alginate microspheres were successfully produced from the partially cross-linked alginate solution using an emulsification method, this technique of cross-linking was not recommened for the development of drug carriers Partial cross-linking of the alginate reduced the subsequent interaction with Ca2+, resulting in lower drug encapsulation efficiency and relatively rapid drug release

B Production of alginate microspheres by the emulsification/internal linking method

cross-B1 Determination of the amount of CaCO 3 and volume of glacial acetic acid needed

Calcium carbonate was added to a beaker of water (50 g) and dispersed with a magnetic stirrer (Figure 14) Glacial acetic acid was added slowly and the pH was monitored The minimum volume of glacial acetic acid needed to fully react with the

Figure 14 Setup for the determination of amount of CaCO3 and volume of glacial acetic acid needed

CaCO3 was determined.The end point was marked by the first sign of complete disappearance of white CaCO3 particles The reaction between H+ and CaCO3 was rapid and was completed within 15 min Thus, it could be inferred that all the Ca2+had been released from the CaCO3 during emulsification after 15 min The pH of the solutions formed was observed to be above pH 4 and would not affect the stability of the alginate

B2 Treatment of alginate microspheres produced

The alginate microspheres were produced and subjected to different treatments

as shown in Figure 5 The amount of CaCO3 was varied in order to evaluate the influence of different amounts of the cross-linker on the alginate microspheres M1

microspheres were discrete and spherical prior to in vacuo filtration They appeared

distorted in shape after filtration and formed massive clumps after oven-drying This was attributed to the deformation of the alginate matrices when subjected to strong

compression pressure during in vacuo filtration and adhesion of microspheres in close

contact with one another during drying in the oven Similar observations were made for M2 microspheres despite crosslinking with more Ca2+than the M1 microspheres (Figure 5)

In an attempt to increase the matrix strength and remove as much moisture as possible from the wet microspheres, glutaraldehyde and isopropyl alcohol were added Glutaraldehyde had been used as a crosslinking agent in the preparation of calcium alginate beads(Kulkarni et al., 2000) as well as a hardening agent for beads

made from pectin (Sriamornsak and Nunthanid, 1998) Similarly, isopropyl alcohol

had been used to harden and dehydrate alginate microspheres (Wan et al., 1992)

The treated microspheres were found to be discrete and spherical after in vacuo filtration However, they still exhibited a distorted shape and marked clumping

after drying in an oven Both glutaraldehyde and isopropyl alcohol were unable to produce adequate hardening of the microspheres Although the amounts of CaCO3, glutaraldehyde and isopropyl alcohol used were more than sufficient for the intended function, they failed to produce the desired discrete microspheres Carbon dioxide was liberated from CaCO3 in the presence of an acid It was probable that the above observations were due to the rapid liberation of carbon dioxide This resulted in the formation of porous microsphere matrices which were weaker and easily succumbed

to the pressure of filtration

Freeze-drying was attempted to dry the product As freeze-drying is also suitable for drying thermolabile materials and causes minimal structural changes to

the product, it was used to eliminate the adverse effects of in vacuo filtration and oven

drying on the structural integrity of the alginate microspheres The M5 and M6 microspheres produced with 0.5 g and 0.8 g of CaCO3 were found to be discrete but

‘millet’ in shape (Figure 15)

Significantly less clumping was observed when the microspheres were dried, as compared to those that were oven-dried Additional cross-linking for 30 min with calcium chloride solution produced less clumping of the microspheres The microspheres produced with 0.8 g of CaCO3 showed less clumping than those produced with 0.5 g of calcium carbonate Interestingly, 1.0 g of CaCO3 produced distorted microspheres with a higher degree of clumping This might be attributed to the excessive production of carbon dioxide when a large amount of CaCO3 was used, resulting in adverse structural effects, producing more porous and weaker microsphere matrices

freeze-Figure 15 Photographs of freeze-dried microspheres produced with (a) 0.5 g and (b) 0.8 g of CaCO3 obtained using a light microscope under 400X magnification

(a)

(b)

The properties of microspheres produced by different cross-linking methods clearly showed a strong influence of cross-linking techniques on the integrity of the calcium alginate matrix formed Partially cross-linked alginate microspheres and externally cross-linked alginate microspheres could be successfully produced by emulsification The calcium alginate microspheres were discrete, spherical, sufficiently strong to be harvested and could be handled with ease However, calcium alginate microspheres produced by an internal cross-linking method were ‘millet in shape’ and appeared weak (Figure 15) The results obtained seemed to suggest that the different techniques of producing microspheres involved different cross-linking mechanisms that affected the matrix structure differently and the properties of the matrix to different extents Unravelling the cross-linking mechanisms and their effects

on matrix integrity would assist in the control of the interaction between Ca2+ and alginate with the use of more appropriate cross-linking techniques

C Mechanisms of external and internal cross-linking of the alginate matrix

C1 Alginate film as a matrix model

Films were used as matrix models as standard test samples could be easily prepared to evaluate the properties of the matrix and the influence of size and shape could be avoided The properties that affected the quality of the alginate matrix such

as thickness, extent of cross-linking, tensile strength, elastic modulus, solubility, degree of hydration and drug permeability were evaluated In addition, the surface topography of the matrix was determined as this parameter was reported to affect the

success rate of encapsulated cells in immuno-isolation devices (Lanza et al., 1991)

C1.1 Process parameters for cross-linking

C1.1.1 Type of cross-linking agent used

When insoluble calcium salts are used, the Ca2+ must be released for linking action at pH values above the pKa of alginates Immersion of alginates in media with a pH lower than the pKa value would result in the conversion of the alginates to alginic acid Hence, some insoluble calcium salts such as calcium oxalate and calcium tartrate are not suitable as the Ca2+ are not released within a suitable pH range

cross-CaCO3 was chosen as the cross-linking agent as the Ca2+ could easily be released from the insoluble carbonate by lowering the pH of the medium to pH 6.5

(Poncelet et al., 1995) Only a small volume of glacial acetic acid was required to

bring about the required pH change CaCO3 was used in both external and internal cross-linking studies to maintain similarity in the source of Ca2+ used and minimize experimental variability

C1.1.2 Amount of cross-linking agent used

Films when externally cross-linked using less than 0.15 g of CaCO3 were fragile and broke into small pieces when handled Therefore, the films were cross-linked using at least 0.15 g of CaCO3 Different amounts of CaCO3 were used in the study to evaluate the influence of various amounts of cross-linking agent on the gelation mechanisms

C1.1.3 Medium used for cross-linking of alginate matrices

A review of the literature showed that the media used for internal and external cross-linking methods were not standardized and often, the pH values of the media used were was not reported Thus, comparisons between studies and results are not

meaningful (Choi et al., 2002; Liu et al., 2002; Quong et al., 1997; Vanderberg and

De La Noüe, 2001) In this present study, an aqueous medium was used within a small

pH range so that the effects of internal and external cross-linking methods on the properties of the cross-linked matrix could be compared A number of studies had reported the formation of less dense internally cross-linked alginate matrices with

larger pore sizes when compared to externally cross-linked products (Choi et al., 2002; Liu et al., 2002; Quong et al., 1997; Vanderberg and De La Noüe, 2001) Liu et

al (2002)attributed this to the displacement of Ca2+ by H+ from the acid added While the acid liberated Ca2+ from the insoluble salt, it also competed with Ca2+ for interaction with the alginate Therefore, the pH range of the cross-linking media had

to be carefully controlled Addition of glacial acetic acid enabled liberation of Ca2+ from the insoluble calcium salt was attempted, but it also reduced the pH of the medium drastically Hence, sodium acetate was also added in the medium to keep the

pH relatively constant, at about 4

C1.2 FTIR of alginate films

The presence of a very strong peak at a wavenumber of around 1737cm-1 for

alginic acid has been reported by Dupuy et al (1994) The occurance of the peak was

suggested to be due to the carbonyl (C=O) stretch, attributed to the free carboxyl COOH) group present in alginic acid In this study, strong peaks around 1737 cm-1were absent in the FTIR spectra of the calcium cross-linked alginate films, indicating potential interactions involving C=O groups (Figure 16) It could therefore be inferred that the pH of the medium used promoted interactions between Ca2+ and alginate The displacement of Ca2+ by H+ could be controlled by careful manipulation of the pH of the medium The FTIR spectra of the cross-linked alginate films

Figure 16 FTIR spectra of alginic acid and alginate matrices

1737 cm -1

appeared similar, without significant detectable changes in the matrix due to different

cross-linking mechanisms

C1.3 Morphology, surface topography and calcium content of alginate matrices

Films were produced by both external and internal cross-linking methods using various amounts of CaCO3 (Table 5) The dried cross-linked alginate films were

relatively transparent Examination of internally cross-linked films under 40X

magnification showed the presence of abundant minute cavities which were not seen

in the externally cross-linked films Cross-linking by either method increased the surface roughness of the films (Table 14 and Figure 17) It was postulated that Ca2+ in the external cross-linking method interacted extensively with the film surface first as contact began at the surface This initial cross-linking action drew the polymer chains closer together to form a less permeable surface to the diffusion of Ca2+ into the interior Consequently, a less homogeneous alginate matrix formed, due to a relatively poor cross-linked interior and a highly cross-linked film surface A homogeneous cross-linked matrix is essential for uniform contraction of the matrix during drying, which, in turn, is critical to the formation of a smooth surface The surface roughness and Ca2+ content of externally cross-linked alginatefilms increased significantly (p<0.05) when the amount of CaCO3 used was increased from 0.15 g to 0.25 g (Table 14) Further increase in the amount of CaCO3 to 0.35 g did not have a significant impact on surface roughness thereby indicating that 0.25 g was sufficient to produce a highly cross-linked surface that was able to impede diffusion of Ca2+ into the interior However, in earlier studies, alginate microspheres were found to release drug rapidly

(Chan et al., 1997; Wan et al., 1994) Since some of the drug molecules studied were

Table 14 Surface roughness, mechanical properties and calcium content of dried calcium alginate films

Film Code Ra Value

(nm)

Tensile Strength (N/mm 2 )

Elastic Modulus (N/mm 2 )

Ca 2+ content (mg/g of film)

SA 44.68 ± 3.00 78.78 ± 2.13 1674.66 ± 81.41 -

CAE 0.15 45.09 ± 0.77 89.28 ± 3.55 3141.97 ± 91.28 67.01 ± 0.89 CAE 0.25 53.53 ± 1.16 95.89 ± 2.78 3017.95 ± 127.73 76.42 ± 1.72 CAE 0.35 52.62 ± 1.26 99.86 ± 4.92 2533.22 ± 163.79 79.48 ± 1.84 CAI 0.15 51.03 ± 1.23 88.23 ± 3.16 2974.40 ± 112.15 65.71 ± 0.39 CAI 0.25 59.00 ± 1.62 86.56 ± 4.67 2646.34 ± 79.91 73.55 ± 0.48 CAI 0.35 66.22 ± 2.21 87.23 ± 2.13 2210.71 ± 220.17 76.24 ± 0.72 CAI 0.25S - 84.51 ± 3.26 3230.33 ± 199.01 -

CAI 0.25N - 90.22 ± 1.61 3270.21 ± 287.63 -