Ionically cross-linked carrageenan-alginate hydrogel beads

J. Biomater. Sci. Polymer Edn, Vol. 19, No. 1, pp. 47–59 (2008) VSP 2008.Also available online - www.brill.nl/jbs

Ionically cross-linked carrageenan-alginate hydrogel beads

Z. MOHAMADNIA, M. J. ZOHURIAAN-MEHR ∗, K. KABIRI,A. JAMSHIDI and H. MOBEDIIran Polymer and Petrochemical Institute (IPPI), P.O. Box 14965-115, Tehran, Iran

Received 4 December 2006; accepted 17 July 2007

Abstract—Hydrogel beads based on the carbohydrate biopolymers kappa-carrageenan and sodiumalginate were newly prepared. Both classical and experimental design (Taguchi) methods were usedto obtain the optimum conditions for the full-polysaccharide hydrogel preparation. The carrageenan-alginate (Caralgi) beads exhibited a surface morphology smoother than that of the one-polysaccharidenetwork beads. Infrared spectroscopy and DSC/TGA thermal methods were used to study thechemical structure and thermal properties of the beads. The carrageenan parts appreciably enhancedthermostability of the networks. The fully carbohydrate-based hydrogel beads are expected to bebiologically compatible and degradable. They are being considered as new carriers for drug loadingand controlled delivery systems.

Key words: Hydrogel; carrageenan; sodium alginate; ionic cross-linking; bead.

INTRODUCTION

Carbohydrate polymers from marine sources have been studied and utilized inpharmaceutical and biotechnological industries for many years. Alginates are afamily of linear polysaccharides, produced by brown algae which contain varyingamounts of 1,4-linked β-D-mannuronic acid and α-L-guluronic acid residues [1].The structure of these units is shown in Fig. 1. Besides its desired mucoadhesiveproperties, which enabled it to be used as a matrix for the entrapment and deliveryof variety of proteins cell, biotechnological applications are based either on specificbiological effects of the alginate molecule itself or on its unique, gentle and almosttemperature-independent sol/gel transition in the presence of multivalent cations,e.g., Ca2+ [1, 2]. Alginate gelation takes place when divalent (usually Ca2+) ortrivalent cations interact ionically with blocks of guluronic acid residues, resulting

∗To whom correspondence should be addressed.E-mail: [email protected] or [email protected]

48 Z. Mohamadnia et al.

Figure 1. Structure of alginate repeating units.

Figure 2. Repeating saccharide units of kappa-carrgeenan (κC).

in formation of three-dimensional network which is usually described by the ‘egg-box’ model [1, 3]. Alginate-Ca2+ has been widely investigated in oral and nasaldrug-release studies [3].

Carrageenans are commercially important hydrophilic polysaccharides that occurin numerous species of seaweeds. These linear sulfated biopolymers are composedof D-galactose and 3,6-anhydro-D-galactose units [1, 2]. Carrageenans are classifiedas kappa, iota, alpha, beta, lambda and theta, according to the number and positionof sulfate groups in the repeating units [1]. Schematic diagram of the most importanttype of the carragreenan family, i.e., kappa-carrageenan (κC), is presented in Fig. 2.Carrageenan is used in pharmaceutical formulations, industrial and commercialapplications as gelling, thickening and stabilizing agent, especially in food products[1, 2].

Ionically cross-linked carrageenan-alginate hydrogel beads 49

The κC beads are prepared by gelling with monovalent ions (often K+) andsometimes divalent ions. Preparation of carrageenan beads and loading/controlledrelease studies for some drugs, like verapamil HCl and ibuprofen, have beenreported [4].

It has been found that the sulfate groups in a hydrogel improve its swelling in saltsolutions, as it has been shown for the hydrolyzed starch sulfate-g-polyacrylonitrilehydrogels [5]. Pourjavadi et al. [6, 7] reported that the hydrogel networks composeda sulfate-containing polysaccharide, κC, swelled highly in ionic media when theyhad been grafted with desired acrylic monomers. In the present work, this inherentcharacteristic of κC was an initial reason to choose this biopolymer for preparationof a drug delivery system as hydrogel beads. Therefore, the main motivation of thiswork has been the preparation of novel fully natural hydrogel beads for oral deliveryof drugs.

Therefore, as an initial step, the hydrogel beads themselves were prepared andthe preparation conditions were optimized empirically. In this preliminary report,we have investigated the preparation and characterization of carrageenan-alginatecomplex beads. A literature review revealed that such ionically cross-linkedpolymer network beads have not been reported so far. We have called the novelhydrogels “Caralgi” to (i) abbreviate the new material name properly and (ii) implytheir structure–property relationship that might be a combination of those of bothbiopolymers.

The influences of various parameters on the bead formation are investigated andthe preparative conditions are optimized by both classical and an experimentaldesign methods. The structure, morphology and thermal properties of the beadswere also characterized by infrared spectroscopy, scanning electron microscopy andthermal analyses, respectively.

MATERIALS AND METHODS

Materials

Sodium alginate (Alg) and κ-carrageenan (κC) were purchased from Fluka(Switzerland) and Condinson (Denmark), respectively. Calcium chloride and potas-sium chloride were of analytical grade and used as received.

Preparation of hydogel bead

The alginate-carrageenan complex beads were prepared by dropping aqueoussolution of alginate and carrageenan into calcium chloride and potassium chloridesolutions [4, 8].

General procedure. Alg and κC solutions were prepared separately by dissolv-ing the biopolymers in distilled water, and heating each at 70◦C (for Alg) and 80◦C


(for κC), for 30 min, while stirring constantly [9]. The ionotropic method was usedfor the preparation of beads [10]. The polysaccharide solutions were thoroughlymixed and, through a plastic syringe, added drop-wise (20 µl) into a stirred salt solu-tion. The bath temperature was between 65–70◦C. Experimental conditions such asdistance between the syringe and gelation media (12 cm), average number of dropssolution falling into gelation media per minute (30 drops/min), type of magnet (rodtype, 3 cm), rate of agitation (50 rpm) and the temperature were fixed. To completegelation, bead-like particles were maintained in the solution for 30 min then filtered,followed by washing with distilled water and then allowed to dry overnight at 37◦C.

Caralgi bead preparation optimized via Taguchi method. The experimentaldesign using Taguchi approach was used to optimize the Caralgi bead preparationthrough decreased number of the experiments.

Four parameters, solution concentration of Alg, κC, KCl and CaCl2, were selectedas essential factors for the Caralgi bead preparation. Three levels for each factorwere selected as given in Table 1. According to the method, four mentioned factorsat three levels were designed by Minitab 12 software, as shown in Table 2. Each ofthe nine experiments was conducted according to the general procedure.

Caralgi bead preparation optimized via classical method. An alginate solution(3.5%, w/v) and carrgeenan solution (3.5%, w/v) were prepared similar to thegeneral procedure. Then different ratios of these solutions, given in Table 3, were

Table 1.The experimental control factors and their levels (%, w/v)

Control factor Level 1 Level 2 Level 3Alginate (F1) 2.5 3.5 4.5KCl (F2) 2.0 3.0 4.0Carrageenan (F3) 1.5 2.5 3.5CaCl2 (F4) 2.0 3.0 4.0

Table 2.The orthogonal array for the experiments T1–T9 and the product yield as thediscrimination index (α)

Trial No. F1 F2 F3 F4 Discrimination index (α)T1 1 1 1 1 1.01T2 1 2 2 2 1.53T3 1 3 3 3 0.82T4 2 1 2 3 1.28T5 2 2 3 1 1.19T6 2 3 1 2 1.62T7 3 1 3 2 1.09T8 3 2 1 3 1.64T9 3 3 2 1 0.80


Table 3.The classical design for preparing Caralgi beads including various Carrageenan/Algweight ratios and the product yield as the discrimination index (α)

Exp. Code Carrageenan/Alg weight ratio Discrimination index (α)

C1 10/0 1.31C2 9/1 1.21C3 8/2 1.29C4 7/3 1.24C5 6/4 1.27C6 5/5 1.24C7 4/6 1.27C8 3/7 1.30C9 2/8 1.37C10 1/9 1.28C11 0/10 1.44

added into 200 ml of the salt solution mixture of (3% (w/v) CaCl2 and 3% (w/v)KCl). All the experiments were run in triplicate.

The discrimination index (α). To find the optimized formulation of Caralgibeads, we defined a dimensionless quantity named “discrimination index”, α. Thisfactor was calculated from the weight of dried beads divided by the total weightof the initial polysaccharide(s). The index for all of the formulations of beadformation, both classic and Taguchi, was calculated and given in Tables 2 and 3.

It should be pointed that quantitative determination of the bead composition wasnot our aim in this step. Certainly, excess amounts of the ionic cross-linkers (i.e.,Ca2+ and K+) were required to ensure sufficiency of cross-linkers. Thus, accordingto the empirical values of α, we can be sure that approximately no non-reactedpolysaccharide remained in the bead-forming media.

Physicochemical characterization of the beads

Fourier transform infrared (FT-IR) spectroscopy (Bruker IFS 48 spectrophotometer)was taken in KBr pellets to confirm the structure of the gums and beads. Surfacemorphology of the particles was studied by scanning electron microscopy (SEM,Cambridge S-360). A simultaneous thermal analysis system (STA 625, PolymerLaboratories) was used to record the scanning differential calorimetry (DSC) andthermogravimetric analysis (TGA) with a heating rate of 20◦C/min under nitrogenatmosphere. Elemental analysis was performed by a CHNOS analyzer system(Elementar, model Vario EL III).


RESULTS AND DISCUSSION

Hydrogel bead preparation

Known alginate-Ca2+ and kappa-carrageenan-K+ cross-linked networks are formedwhen each polysaccharide is treated individually with the corresponding saltsolution [1–3]. Alginate is cross-linked with Ca2+ via ionic inter-chain bondingaccording to the egg-box model. In κ-carrageenan (κC), however, a differentgelation mechanism has been proposed. Potassium acts as intramolecular glue,forming electrostatic attraction with the sulfate esters and anhydro-oxygen atomsof κC [1]. The basic hypothesis of the present work is based on the simultaneousformation of alginate-Ca2+ and κC-K+ networks, resulting in polymer networkhydrogel. This intimate mixture or alloy of homo-cross-linked polymers will behold together by permanent entanglements, while no chemical bonds or graftsbetween them are formed.

In preliminary experiments based on the literature [4, 8], different possiblepriorities of addition of reagents (aqueous solutions of Alg, κC, CaCl2 and KCl)for bead preparation were examined to achieve the best procedure for resultingin isolated beads. Meanwhile, it was found that no bead formation was observedby dropping κC into CaCl2 (and also dropping Alg into KCl). Conversely, stablegel beads were clearly formed when the polysaccharides were dropped into theircorresponding salt solutions. Therefore, it was almost impossible to retain non-cross-linked polysaccharide when excess amounts of the ionic cross-linkers wereused in the bead formation media. Empirically, Alg-Ca beads were formed fasterthan κC-K beads. In addition, κC basically formed softer beads with less sphericalthan the alginate-based beads. Each polysaccharide is ionically cross-linked withthe corresponding cation. With another cation, the yield of the bead formation ordiscrimination index (α) was decreased. Thus, alginate and carrageenan prefer toform a stable gel with CaCl2 and KCl, respectively.

Overall, the best way to the hydrogel bead preparation was concluded as thedropwise addition of the Alg-κC mixture to solution containing both CaCl2 andKCl. The optimum concentration of the reactants was then determined using ofeither Taguchi or classical methods discussed below.

The Taguchi method for Caralgi bead preparation optimization

The aim of this study was to minimize the optimizing experiments for findingthe best bead formulation leading to highest discrimination index (α) and desiredmorphology. Four parameters containing solution concentration of Alg, κC, KCland CaCl2 were selected as essential factors for the Caralgi bead preparation. Threelevels for each factor were selected (Table 1). Based on the preliminary experiments,the lowest and highest levels were chosen to achieve a complete formation of gelbeads with reasonable size and dimensional stability during the cation diffusion andionic cross-linking leading to hardening the beads without diffusion. In addition,the polysaccharide mixture concentration was limited to applicable levels so that


the thickened mixtures could be injected through a syringe to yield droplets withdesired sizes. Meanwhile, excessive amounts of polysaccharide had to be used toensure the complete gel with sufficient cations became available.

The four mentioned factors at three levels were designed by the software asshown in Table 2. After conducting the nine experiments according to the generalprocedure, α values were calculated and loaded into the software. Nine experimentswere designed for optimizing the variable reactants. According to the softwareoutput, κC in level 1 (i.e., 1.5%), Alg in level 2 (i.e., 3.5%), KCl in level 2 (i.e.,3.0%) and CaCl2 in level 2 (i.e., 3.0%) were the optimum levels derived. Figure3c and 3g shows the photomicrographs of the optimized formulation of the Caralgibeads in different magnifications.

The classical method for Caralgi bead preparation optimization

It was realized from the above investigations that 3.5% (w/v) for each polysaccha-ride is suitable and 3% (w/v) for each salt solution is an excessive concentrationwith which the polysaccharide can form a gel with sufficient cation concentration.Table 3 shows the various κC/Alg weight ratios and the product yield given as thediscrimination index (α). The highest α value for C11 practically confirms a verygood gel formation. In fact, the amount of α for C1 is also very high; however,experiments C1 and C11 are outside the scope of this study because no cross-linkednetwork is formed in these two cases.

Among the hydrogel beads, C9 showed the maximum amount of α, near to thatof C11. Figure 3d and 3h shows the morphology of the optimally prepared Caralgibead, i.e., C9.

The elemental analysis of C9 showed that it is composed of 21.34% C, 4.4% Hand 0.988% S. According to the repeating unit of the polysaccharides (Figs 1 and 2),alginate and carrageenan have 24 and 18 carbon atoms, respectively. Carrageenanhas also two sulfur atoms while alginate does not. This is the basis of a calculationresulted in the relative composition of the Caralgi hydrogel. Thus, the alginate tocarrageenan repeating unit ratio is found to be 2.05. It means the hydrogel formationprocess has produced beads having both of the polysaccharides without a majorpreference of one of them. A minor carrageenan loss is inevitable in this ionicallycross-linking system. It can be attributed to a relatively weaker carrageenan–K+interaction.

Morphology of the hydrogel beads

Typical SEM photomicrographs of the hydrogel particles are shown in Fig. 3.According to Fig. 3a, κC-K exhibits non-spherical cylinder-like bead-like particles.The as-prepared beads are very soft and geometrically unstable; therefore, theylead to non-spherical beads. In contrast, Alg-Ca bead particles are spherical anduniformly shaped. As observed in higher magnification, these one-polysaccharide


Figure 3. SEM micrographs of (a) carrageenan (κC)-K beads, (b) sodium alginate (Alg)-Ca beads,(c) Caralgi beads optimized by the Taguchi method and (d) Caralgi beads (C9) optimized bythe classical method (magnification ×80). Pictures (e)–(h) are close-ups of the pictures (a)–(d),respectively (magnification ×500).


hydrogels comprise special regular textures (Fig. 3e and 3f). Such morphology in asimilar sample has been reported elsewhere [8].

Based upon the SEM pictures shown in Fig. 3g and 3h, in comparison to Fig. 3eand 3f, formation of the hydrogel composed of both polysaccharides resulted in asmoother surface having bead-like morphology. We have no exact reasoning forthe morphological difference at this stage. However, in the case of these two-polysaccharide beads, the lower mechanically stabilized beads generally possessedsmoother morphologies. Related to this fact, the classically optimized bead withcoarser morphology (i.e., sample C9; Table 3, Fig. 3) was chosen as an optimalproduct.

Spectral and thermal characterization

Infra-red spectroscopy was employed to characterize the structure and the possi-ble interactions in carrageenan and alginate networks comparing with the intactpolysaccharides. Figure 4 shows the FT-IR spectra of alginate, carrageenan andtheir mixture cast film. In addition to the carbohydrate C–C and C–O vibrationsat the finger print area, a very wide band appeared at 2600–3600 cm−1 that is re-lated to the –COO− groups of Alg (Fig. 4a). The bands observed at 915, 1024 and1227 cm−1 can be attributed to 3,6-anhydro-D-galactose, glycosidic linkage andester sulfate stretching of carrageenan backbone, respectively [7] (Fig. 4b). Thebroad band at 3200–3600 cm−1 is due to the stretching of O–H groups of the car-bohydrates. The spectrum of the physical mixture of the polysaccharides (Fig. 4c)comprises the major peaks of both the components. The wavenumbers near 1600and 1400 cm−1 were attributed to the symmetrical vibration of carboxylate ions onalginate and film blend.

DSC and TGA analyses were applied to thermally characterize the Caralgi beadsin comparison with the one-polysaccharide beads and the intact gums (Fig. 5). Theintense endothermic wide peaks in all of the thermograms (70–150◦C) are due toremoving the moisture (5–14%) absorbed by the polysaccharide. This value isminimized for κC-K beads, which may be attributed to lower relative numbers ofhydrophilic OH groups in κC (anhydride bridge) compared with Alg. The DSCcurve of intact κC shows two additional endothermic peaks that may be attributedto a melting point and a kind of thermal decomposition. No intensive exothermaltransition is observed in DSC traces of κC, κC-K and Caralgi hydrogel.

To observe other possible endothermic transition overlapped by the wide peak ofthe absorbed moisture, the Caralgi C9 sample was completely dried and analyzedagain. According to Fig. 6, the moisture-free Caralgi sample exhibited a glasstransition around 66◦C and several endothermic transitions from 180 to 210◦C.The peaks at 189–197◦C may be attributed to cation–alginate interactions withtwo different uronic acid units of alginate [11, 12]. The rest peaks located at181–184◦C can be attributed to carrageenan–cation interactions. As shown inFig. 6, the endothermic transitions are multiple and relatively large. It impliesthe ionic/macromolecular interactions involving in the polysaccharide network are


Figure 4. FT-IR spectra of (a) sodium alginate (Alg), (b) carrageenan, (c) Alg-carrageenan mixture asa cast film, (d) Alg-Ca2+ bead sample, (e) carrageenan-K+ bead sample and (f) Caralgi bead sample.

various and very complicated. This intricacy has been mentioned by severalresearchers [13]. For example, Morris et al. [14] proposed that the carrageenangel formation occurred in the presence of K+, Rb+, Cs+ and high concentrationof Na+ is due to a double-helix-based aggregated-domain structure as the gelledstructure. Structural characterization of the mixed polysaccharide gels, Caralginetworks, meets a more critical challenge due to different ionotropic gelationmechanisms for the individual polysaccharides. Meanwhile, an interpenetratedpolymer network structure formation is probable, but not certain, under the ioniccross-linking conditions. Deeper investigations have yet to be conducted toelucidate the nature of interactions involved in the ionically cross-linked Caralginetworks.


Figure 5. DSC/TGA thermograms of intact carrageenan (κC), intact sodium alginate (Alg), κC-Kbeads, Alg-Ca beads and Caralgi beads. Heating rate 20◦C/min under pure N2.

A remarkable thermostability improvement is observed in the ionically cross-linked networks, according to TGA traces of Caralgi, κC-K and Alg-Ca beads.Comparing the intact gums and one-polysaccharide networks shows that the car-rageenan component improves the thermal stability. Most likely the presence ofsulfate groups in the carrageenan structure is the main factor inducing the ther-mostability. This possibility is supported by the fact that the char yield (at 600◦C)of carrageenan-K beads is recorded to be higher than that of Caralgi beads (69.9%vs. 52.4%).


Figure 6. DSC thermogram of Caralgi bead C9 after complete drying. Heating rate 20◦C/min underpure N2.

CONCLUSION

Alginate-Ca2+ is a well-known easily produced hydrogel network with goodmechanical integrity. However, in the present work, carrageenan-K+ was addedto it for a few reasons.

First, as mentioned in the Introduction, the presence of “sulfate groups” inhydrogel networks favors a higher swelling capacity in saline solutions. Since weplanned to prepare hydrogel beads for drug delivery controlled via the “swelling”mechanism, we intended to obtain beads with the least swelling-loss in appropriateionic media such as intestine.

Second, carrageenan is an edible, cheap and available biopolymer. Particularly, itcan be simply cross-linked ionically with a biological species, i.e., potassium (K+).Third, based on the present experience, carrageenan-K+ and alginate-Ca2+ can beformed simultaneously without any interference leading to main loss of physicalproperties. Meanwhile, the carrageenan parts enhance the thermostability of thegels. Forth, the produced Caralgi is a fully-natural hydrogel network; therefore, itdoes not suffer from synthetic regions or residual reactants/reagents. Finally, thenovelty of the work has been guaranteed because, to the best of our knowledge, thepreparation of such hydrogels has not been reported in the literature.

Thus, both classical and experimental design (Taguchi) methods were used toachieve the optimum conditions for the hydrogel bead-like particle preparation.Overall, the optimized preparative conditions may be as follows (formulation C9):8.0 g 3.5% Alg solution, 2.0 g 3.5% carrageenan solution, 100 ml 3.0% CaCl2


solution and 100 ml 3.0% KCl solution (temperature 65–70◦C, time 30 min). Thecarrageenan-alginate (Caralgi) beads exhibited a surface morphology smoother thanthat of the one-polysaccharide network beads. It can be seen that the optimizedformulation is richer in alginate to carrageenan. The reason may be attributed to astronger interaction of alginate-Ca2+ than carrageenan-K+.

The chemical structure and thermal properties of the beads were also studied byinfrared spectroscopy and DSC/TGA methods, respectively. The multiplicity andlargeness of the thermal transitions before degradation (Fig. 6) imply the intricacyof the ionic interactions of the polysaccharide network. Meanwhile, under theionic cross-linking conditions, the formation of an interpenetrated polymer networkstructure is probable. However, the exact structure of the hydrogel network remainsas a subject of debate.

Improved thermal stability was observed for the formulations with higher car-rageenan content. The fully carbohydrate-based hydrogel beads are expected tobe biologically compatible and degradable. They have been prepared under highlypractical conditions, so the preparative method can be easily scaled up. The Car-algi bead formation system was applied in consequent drug loading and controlled-release studies that are in progress in this group.

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Ionically cross-linked carrageenan-alginate hydrogel beads

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