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Abstract
Floating gellan gum beads were prepared using Calcium chloride or Zinc sulphate as crosslinking agent and
CaCO3 as buoyancy imparting agent. Chemical and morphological change of prepared gellan beads were
studied by FT-IR, DSC & SEM. Both FTIR and DSC studies revealed the electrostatic interaction of COOgroups of gellan with Ca++ or Zn++, however, affinity seemed to be different. SEM studies revealed that
Zn++ cross-linked gellan gum beads were significantly denser than Ca++ cross-linked beads. The %
entrapment efficiency; buoyancy and drug release from the beads were investigated using metronidazole as
a model drug. Our results showed that Zn++ cross-linked floating beads exhibited significantly high drug
entrapment efficiency (p<0.05) compared to Ca++ cross-linked beads. The difference in observed behavior
may be attributed to differences in ionic radius, hard or soft character and selectivity or non-selectivity of
Ca++ and Zn++ to the gellan gum gelation.. The findings of this study could provide potential benefits in the
development of more scientific approach towards designing sustained/controlled release drug delivery
systems based on gellan gum.
Key words: Gellan gum, ionotropic gelation, ionic radius, floating beads.
1. Introduction
Gellan gum is an extracellular polysaccharide that has capacity to forms gels with both mono and divalent
cations. In case of divalent cations, the gelation and aggregation of gellan gum occurs via a chemical
bonding between divalent cations and carboxylic groups in the gellan chains, in addition to the screening
effect (Kanesaka et al., 2004). Apart from alkaline earth metals (Ca++, Mg++or Ba++), transition elements
such as Cu++, Zn++ etc have also been reported as complexing agent to form gel gellan gum solutions
(Singh & Kim, 2005, Aminabhavi at al., 2006). Gellan is markedly different from other uronic acid
containing polysaccharides like alginate and pectin because it exhibit lack of specificity among the alkaline
earth cations and selectivity among the transition elements. For all the divalent ions the affinity series for
increasing gel strength has been reported to be: Mg++, Ca++, Sr++, Ba++ < Zn++ < Cu++ < Pb++ (Grasdalen &
Smidsroed, 1987). Although both Ca++ and Zn++ crosslinked gellan gum beads has been studied previously
for oral sustained or stomach specific delivery of drugs but there is paucity of literature on the effect of
these complexing agents on the crosslinking of gellan gum. In the present investigation the effect of
calcium chloride or zinc sulphate as crosslinking agent on the properties of floating gellan gum beads were
investigated. The findings of this study could provide potential benefits in the development of more
scientific approach towards designing sustained release systems based on gellan gum as drug release;
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encapsulation efficiency, surface morphology etc are largely dependent upon concentration and nature of
divalent cations.
1.1 Material and methods
1.1.1 Materials
Deacetylated Gellan gum (GG) (GelzanTM CM) was procured from Sigma Lifesciences. De-ionized water
(HPLC grade) was purchased from Quilagens, India. All other chemicals used were of analytical grade (AR
Grade).
1.1.2 Methods
Preparation of Ca++ and Zn++ crosslinked floating gellan gum beads
The beads were prepared (Table 1) by ionotropic gelation technique (Choi et al., 2002). Gellan (GG)
solutions were prepared by dissolving the gellan gum in deionized water by heating at 90 C. The resultant
homogeneous bubble free slurry dispersion was dropped through a 20G syringe needle into coagulation
medium containing different concentration of either Ca++ or Zn++ as CaCl2 or ZnSO4 dissolved in deionized
water containing 10% v/v acetic acid, which was kept under mild stirring to improve the mechanical
strength of the beads and also to prevent aggregation of the formed beads. Immediate formation of gelled
beads took place; after 3 minutes of curing time; the formed beads separated by filtration, washed thrice
with deionized water and dried at 40 C in a hot air oven (DR 101, Universal, India) overnight.
Table 1: Formulation composition of various batches of floating beads.
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predetermined intervals, a 1ml aliquot was withdrawn and replenished with an equal volume of fresh
dissolution medium .The withdrawn samples were analyzed UV spectrophotometrically at 277nm.
1.1.3.8 Statistical analysis
All the data were analyzed by Students t test and one-way ANOVA to determine statistical difference in
the results. A probability value p < 0.05 was considered statistically significant. The software used was
SigmaPlot 11 (Systat Software Inc).
1.4 Results and discussion
The interaction of Ca++ or Zn++ with COO- group on gellan network may be assumed to occur in three
possible ways; as an intramolecule hydrated salt-type formed within the single chain (I); as an
intermolecule hydrated salt-type through the cross-linking structure which connects two adjacent COOgroups between the chains (II); and an alternative formation, may be interpreted as a pendent half-salt (III).
Formations (I) and (11) contribute significantly to enhancement of the chain stiffness, lower the degree of
free rotation of the chains, and increase the chain entanglements, thereby the mechanical strength of the
crosslinked beads.
1.4.1 Characterization of beads
1.4.1.1 Thermal characterization
The DSC thermogram of GG (Fig.1) showed a broad endothermic peak at 159 0C could be attributed to
evaporation of bound water.
Figure 1: DSC thermograms of gellan gum, Ca++ and Zn++ crosslinked beads.
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The endothermic peak at 233 0C suggested melting of the polymer, whereas, exothermic peak at 2540C
indicated decomposition of polymer. The DSC thermogram of Ca++ crosslinked blank GG beads showed
two endothermic peaks. A broad endothermic peak appeared at 138 0C may be attributed to the dehydration
(gel water as well as water coordinated with Ca++) and the second endothermic peak at 217 0C may be due
to change in rotational energy of the molecular chains due to the formation of salt complexes. The DSC
thermogram also indicated disappearance of exothermal peak of GG; this may attributed to enhanced chain
stiffness due to crosslinking with Ca++. On the other hand DSC thermogram of Zn++ crosslinked blank GG
beads showed a weak endothermic peak at 208 0C. This may imply that the chain rotation energy was
reduced by the formation of [(Zn++)-COO -].
1.4.1.2 FTIR Spectroscopic characterization
In FTIR spectrum of pure GG (Fig. 2), the band at 3421.49cm-1 is due to the presence of OH- group of
glucopyranose ring. The band at 2924.41cm-1 is due to the stretching vibrations of CH2 group, while those
appearing at 1614.25 and 1416.02cm-1 due to asymmetric and symmetric stretching of COO- group. The
FTIR spectrum of blank Ca++ crosslinked floating beads showed shifting of O-H stretching vibration to
3434.88 cm-1 indicating involvement of hydroxyl group with Ca++. The band at 1748.47 indicates that some
COO- group of the GG transformed into carboxylic group due to interaction with Ca++. The band due to
asymmetric and symmetric vibrations of COO- group shifted to higher wavenumber, that is, 1620.23 and
1454.69 cm-1 and there is a new band at 1550.78 cm-1 indicating involvement of COO- group in
coordination process with Ca++. Further, the band at 1030 cm-1 in the GG spectra is shifted to 1037.09 cm-1
indicating involvement of OH group in coordination with Ca++. The difference value [ =as COO- --s COO-] between the asymmetric and symmetric stretching frequencies is found to be 130 for Ca++
crosslinked GG beads. The difference value can reflect the coordination modes of COO- group. Values of
> 200 cm-1 indicate a single bond between the metal and carboxylate group, whereas, < 150 cm-1
indicate a twice bonded structure (Scot & Owen, 2001). On the other hand, the FTIR spectra of blank Zn++
crosslinked GG beads showed shifting of O-H stretching to relatively low wave number, that is, 3430.42
cm-1, whereas, band in the region of 1740 cm-1 is missing compared to spectra of Ca++ crosslinked GG
beads. Further, no change is observed in the frequency of asymmetric vibrations of COO-, whereas,
symmetric vibrations of COO-group were shifted to 1443.14 cm-1 compared to 1454.69 cm-1 in case of Ca++
crosslinked beads. The difference value for Zn++ crosslinked system is found to be 156 corresponds to the
chelating mode.
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When Ca++ [coordination number in water range from seven to nine] (Eugene, 2009) or Zn++ [coordination
number in water range from 4 to 6] (Siegbahn et al., 1998, Jalilehvand et al., 2001) goes into aqueous
solution, they became hydrated with water molecules and, therefore, formed hydration shells, according to
their hydration number. Water molecules that surrounds the cations suppress the shielding ability of
cationic species and thus, during the process of gelation together with their specificity, there is an easy
ingress of Zn++ between pairs of double helices compared to Ca++, which resulted in deeper, tenacious and
rapid crosslinking of gellan network due to its interaction with COO- groups on gellan network, and, hence
little time for the entrapped gas generating agent to completely react with acidic coagulation medium.
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Figure 3: Scanning electron microscopy images of various formulations of floating beads: (a), (b) and (c)
showing shape, surface and transverse section of Ca++ crosslinked beads. Scanning electron microscopy
images of various formulations of floating beads: (d), (e) and (f) showing shape, surface and transverse
section of Zn++ cross-linked beads.
1.4.1.4 Assessment of in vitro buoyancy of the beads
The propensity of beads to exhibit buoyancy depends on the true density of the fabricated beads. The
density of beads was in the range of 0.7073 to 0.8889 gm/cm3 i.e., less than the density of the gastric juice
(approx. 1.004 g/cm3). The density of Zn++ crosslinked beads ranged from 0.8873 to 0.8889 gm/cm3,
whereas, density of Ca++ crosslinked beads ranged from 0.7073 to 0.721 g/cm3. There was no lag time as
the beads floated immediately when placed in 0.1M HCl (pH 1.2). Ca++ crosslinked beads floated for up to
14 hours whereas Zn++ cross-linked beads floated for 7-8 hours. Besides from explanation given in the
morphological evaluation section the observed difference may also be attributed to the difference in
ionization potentials of Ca++ and Zn++. The high values of Zn++ ionization potentials are reflected in its
stronger tendency to form covalent bonds (Eugene, 2009, Siegbahn et al., 1998) this could have resulted in
the formation of strong and rigid covalent bonded structures, which could not float in the gastric fluid for
long period.
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