comparative evaluation of ca++ and zn++ cross-linked gellan

Archives Des Sciences Vol 65, No. 2;Feb 2012

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Comparative evaluation of Ca++ and Zn++ cross-linked gellan gum based floating beads

Anurag Verma*1, Jayant K. Pandit2 1Department of Pharmaceutics, School of Pharmacy, IFTM University, Moradabad, 244001, India (Corresponding Author). Tel: +919412581046, E-mail: [email protected] 2 Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, India.

* Corresponding author. Tel: +919451570863, E-mail: [email protected]

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 COO- groups 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


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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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1.1.3 Characterization of floating beads

1.1.3.1 FTIR Spectroscopic characterization

FTIR spectra were recorded for pure polymer, blank Ca++ and Zn++ crosslinked beads using a FTIR facility (Shimadzu, model 8400S). The samples were prepared in KBr disks (2 mg sample in 200 mg KBr). The scanning range was 400-4000 cm-1.

1.1.3.2 Differential scanning calorimetric characterization

DSC thermograms of pure polymer, blank Ca++ and Zn++ crosslinked beads were recorded. Samples were heated in a hermetically sealed aluminum pan and heat runs from 30 to 350 0C at a heating rate of 5 0C/min, using a temperature modulated DSC (TA Instruments, USA, Model: SDT 2960). The characteristic peaks and heat of melting endotherm were recorded.

1.1.3.3 Microscopic and scanning electron microscopic (SEM) characterization The size of beads was determined with an optical microscope (Model BH-2, Olympus, Japan) fitted with a stage and an ocular micrometer. Twenty dried beads were measured determine the mean diameter of the beads. All measurements were in triplicate. The shape, surface morphology and internal structure of the dried beads were assessed with a scanning electron microscope (Leo 435VP, variable pressure, Oxford, U.K.) at various magnifications. 1.1.3.4 Measurement of density of floating beads Density of the beads was measured by immersing the beads in 0.02% w/v Tween 80 solution for 3 days. Beads sunken after this process, were used for density measurement carried out by the displacement method using n-hexane as a nonsolvent (Soppimath, 2001). 1.1.3.5 Assessment of in vitro buoyancy of the floating beads In-vitro study of bead buoyancy was performed using a USP XXVII dissolution apparatus type II (paddle type, Electrolab, Mumbai, India). The beads were dispersed in 500ml of 0.1M HCl (pH 1.2) at 3710C with continuous agitation at 50 rpm. The floating beads were separated from submerged beads and their proportion (%) was determined (USP 27/ National Formulary 22). 1.1.3.6 Determination of drug entrapment efficiency The metronidazole entrapment efficiency of each formulation was determined by extracting the crushed beads with 0.1M HCl (pH 1.2) for 45 min at 37 0C and then centrifuged at 5000 rpm. The supernatant layer was taken and suitably diluted with 0.1M HCl, quantifying the amount of drug UV spectrophotometrically at 277 nm. The entrapment efficiency (EE) was calculated according to the relationship:

1.1.3.7 In vitro drug release studies In-vitro release of metronidazole from the beads was evaluated with a USP XXVII dissolution apparatus type II (paddle type, Electrolab, Mumbai, India) at 50rpm in 500ml 0.1M HCl (pH 1.2) at 370.50C. At


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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 COO- groups 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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Figure 2: FTIR spectra of GG, Ca++ and Zn++ crosslinked GG beads. 1.4.1.3 Microscopic and Scanning Electron Microscopic (SEM) characterization of beads The scanning electron micrographs (SEM) of various gellan beads are shown in Fig. 3. Ca++ cross-linked floating beads were spherical with rough outer surface as evident by cracks on the surface of bead, whereas, Zn++ cross-linked beads were irregular in shape with very rough outer surfaces. Further, Ca++ cross-linked floating beads appeared to be more porous with larger pores than Zn++ cross-linked beads. Internal porous structure of the beads directly related to the presence of gas generating agents. Violent generation of CO2 occurred upon its contact with acidic gelation medium and this has resulted in rough surface with appearance of cracks because of the bursting effect of the larger amounts of CO2 before the walls were sufficiently hardened. In case of Zn++ crosslinked beads, the reason for relatively smaller internal pores could be due to the incomplete reaction between gas generating agent in the beads and acidic coagulation medium. The above observation may be explained by considering two facts, firstly, that transition metals are more potent gel former than alkaline earth metals and there is complete lack of selectivity among the alkaline earth metals and selectivity for transition elements, which distinguished gellan gum from other uronic acid containing polysaccharides like alginates and pectin (Grasdalen & Smidsroed, 1987). Secondly, the ability of metals to form complexes usually increases with the increase of positive charge of the metal ion and with the decrease of its ionic radius.

Table 2: Physiochemical characteristics of floating beads.

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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1.4.1.5 Drug entrapment efficiency of floating beads Metronidazole entrapment ranged from 53% to 81% (Table 2). It was observed that Zn++ crosslinked systems showed significant improvement in entrapment efficiency compared to Ca++ crosslinked systems (Z5 compared to Z6; p


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References 1. Kanesaka, S. et al. (2004). Binding effect of Cu++ as a trigger on the sol-to-gel and the coil-to-

helix transition processes of polysaccharide, gellan gum. Biomacromolecules, 5, 863868. doi:10.1021/bm030072t, http://dx.doi.org/10.1021/bm030072t 2. Singh, B.N. & Kim, K.H. (2005). Effects of divalent cations on drug encapsulation efficiency of

deacylated gellan gum. Journal of Microencapsulation, 22, 761771. doi:10.1080/02652040500273704, http://dx.doi.org/10.1080/02652040500273704

3. Aminabhavi, T.M. at al. (2006).Controlled release of cephalexin through gellan gum beads: effect of formulation parameters on entrapment efficiency, size, and drug release. European Journal Pharmaceutics and Biopharmaceutics, 63, 249-61. doi:10.1016/j.ejpb.2005.12.008, http://dx.doi.org/10.1016/j.ejpb.2005.12.008,

4. Grasdalen, H & Smidsroed, O. Gelation of gellan gum. (1987). Carbohydrate Polymers, 7, 371393. doi:10.1016/0144-8617(87)90004-X, http://dx.doi.org/10.1016/0144-8617(87)90004-X

5. Choi, B.Y. et al. (2002). Preparation of alginate beads for floating drug delivery: effects of CO2 gas forming agents. International Journal of Pharmaceutics, 239, 81-91. doi:10.1016/S0378-5173(02)00054-6, http://dx.doi.org/10.1016/S0378-5173(02)00054-6

6. Soppimath K.S. et al. (2001). Microspheres as floating drug delivery system to increase the gastric residence of drugs. Drug Metabolism Reviewa. 33, 149-160. doi:10.1081/DMR-100104401, http://dx.doi.org/10.1081/DMR-100104401

7. United States Pharmacopoeia 27/National Formulary 22, USP Pharmacopoeial Convention, Rockville (MD) 2000.

8. Scot, T.M. & Owen W. D. (2001). Surface complexation and dissolution of hematite by C1-C6 dicarboxylic acids at pH 5.0. Geochimica et Cosmochimica Acta, 65, 4289 4301. doi:10.1016/S0016-7037(01)00696-2, http://dx.doi.org/10.1016/S0016-7037(01)00696-2

9. Eugene AP. (2009). Metalloproteomics. Wiley InterScience, USA, pp-37-58 and 150-172. 10. Siegbahn, P.E.M. et al. (1998). Hydration of Beryllium, Magnesium, Calcium, and Zinc Ions

Using Density Functional Theory. Journal of Physical Chemistry A, 102, 219-228. doi:10.1021/jp972072r

11. Jalilehvand, F., et al. (2001). Hydration of the calcium ion. An EXAFS, large-angle x-ray scattering, and molecular dynamics simulation study. Journal of American Chemical Society, 123, 431-41. doi:10.1021/ja001533a

12. Dipankar, D. (1992). On Pearson's HSAB principle. Inorganic Chemistry. 31, 27972800. doi: 10.1021/ic00039a025

13. Fernando, M. et al. (2004). Introduction of external field effects in the frontier molecular orbital theory of chemical reactivity. International Journal of Quantam Chemistry, 44(S26), 751 760. doi:10.1002/qua.560440866

14. Engberts, J.B.F.N. et al. (1996). Lewis-acid catalysis of carbon- carbon bond forming reactions in water. Recueil des Travaux Chimiques des Pays-Bas, 115, 457-464. doi:10.1002/recl.19961151103, http://dx.doi.org/10.1002/recl.19961151103

15. Aiedeh, K. et al. (2007). Effect of ionic crosslinking on the drug release properties of chitosan diacetate matrices. Journal of Pharmaceutical Science, 96, 3843. doi:10.1002/jps.20764, http://dx.doi.org/10.1002/jps.20764

comparative evaluation of ca++ and zn++ cross-linked gellan

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