Investigating the Unexpected Behavior for the Release Kinetics of Brilliant Blue Encapsulated into Calcium Alginate Beads

This work is focused on investigating the unexpected behavior for the release kinetics of brilliant blue (BB) encapsulated into calcium alginate beads. By increasing the alginate concentration from 1 - 3 % (w/v), the release of BB over time was found to follow two different behaviors. For the first two hours, the order was 1 % > 2 % > 3 %, after which it was as follow: 1 % > 3 % > 2 %! The unanticipated increase in BB release using 3 % (w/v) alginate beads after two hours over that of 2 % (w/v) alginate was examined by the swelling and bursting tests. The results were showing clear evidences by data and image the unusual behavior of 3 % (w/v) alginate beads at two hours of swelling. This unexpected behavior for the 3 % (w/v) alginate beads might be due to the higher osmotic pressure inside the beads. Overall, 2 % (w/v) calcium alginate beads were considered to be the optimum formulation showing an excellent carrier for targeting drugs to the intestine, where the swelling of the beads were 60 % in the acidic medium, it was 5000 % in the alkaline medium.


Introduction
Drug delivery systems have been extensively studied over the last years and polymers are now being studied as a method of controlling the release of drugs (1). Biodegradable polymers are one of the key materials for these devices, and have advantages over non degradable implants. The main advantage is that biodegradable devices degrade and are absorbed by the body during and/or after drug release this allows us to bypass the need for surgical removal of the device (2). One of the most known used polysaccharide in biotechnology is alginate (3). Alginate is a water-soluble linear polysaccharide extracted from brown seaweed and is composed of alternating blocks of 1-4 linked Į-L-guluronic and ȕ-D-mannuronic acid residues (4). Alginate has the ability to form hydrogel in presence of multivalent cations like Ca +2 in aqueous medium (5). Alginate shows excellent features such as immunogenecity (6), biocompatibility (7), bioadhesion (8) and non-toxicity (9), these features make it a very attractive biomaterial for use in many types of application like wound dressing (10), scaffold for tissue engineering (11) and pharmaceutical industries (12&13).
Although many authors (14&15) have studied Ca-Alg beads as a matrix for drug delivery system, optimization of the preparation parameters were not clear. Also, there are many conflicts between authors and this was the main reason for doing this work. For examples, Bajpai et al. (16) reported that using high alginate concentration 4% (w/v) gave stable beads for drug delivery system, whereas Arica et al. (17) reported that beads prepared with 1% (w/v) alginate sustained the release of 5-fluorouracil. Also the concentration of CaCl 2 as cross-linking agent showed ____________________________ *corresponding author. E-mail:magmel@gmail.com various results; Gaserod et al. (18) reported that by increasing the concentration of CaCl 2 to 3% (w/v) should increase the porosity of beads, leading to higher diffusion of entrapped drug. While Sankalia et al. (19) found that using 0.5% (w/v) of CaCl 2 gives weak gel due to insufficient cross-linking of alginate. Kim and Lee (20) reported that calcium ion content in the gel beads leveled off after 6 min of curing time in CaCl 2 solution, and there was little variation in the release of blue dextran from alginate gel beads cured for more than 6 min. Yotsuyanagi et al. (21) reported that an approximately 70 h curing was necessary to reach constant weight of alginate gels.
Thus, this work is aiming to unveil the contradiction between authors and is a concrete guide for other authors who are working with calcium alginate beads or as a comparison to other works. In this work, we comprehensively studied the influence of preparation parameters including alginate concentration, calcium chloride concentration, curing time and drying process on the encapsulation efficiency, the release rate and the beads morphology using SEM. As a second aim, calcium alginate beads has been used as carriers for targeting smart drugs such as proteins to the intestine, by studying the swelling and release of the beads in stimulated gastric and intestinal fluids using brilliant blue as a model drug.

Materials
Alginic acid sodium salt from brown algae was purchased from Fluka, brilliant blue R 250 (BB, Mw 825) was purchased from Aldrich; calcium chloride anhydrous was purchased from Gen Lab. All other reagents were of analytical grade and used as received.

Methods
As a general rule, all experiments were carried out in triplicate and data are means ± SD (n = 3).

Preparation of calcium alginate beads
Sodium alginate was dissolved in bi-distilled water at various concentrations 1, 2, 3 and 4 % (w/v), then BB 25 % (w/w) was added and suspended thoroughly by stirring. Three milliliters of this solution was dropped into a 15 ml of gelling solution through a disposable plastic syringe using a 23G needle at a dropping rate of 1 ml/min under mild agitation for various time 30 -120 min. The gelling solution contained CaCl 2 0.5 -5 % (w/v). The formed beads were collected, washed with 20 ml bidistilled water and dried at room temperature for 24 h or used in the wet state.

Encapsulation efficiency
The gelling and washing solutions remaining after removal of the beads were assayed for BB by UV/Vis spectrophotometer (Shimadzu) at 590 nm using a stander curve of known concentration in the range of 1.25 -30 mg/l with correlation coefficient R 2 = 0.9998.
The encapsulation efficiency was calculated from the difference between the initial amount of BB dissolved in alginate solution and the amount of BB measured in the gelling and washing solution (22) as shown in the following formula: Were M i is the initial amount of BB dissolved in alginate solution and M g is the amount of BB measured in the filtered solution.

Swelling study
Swelling studies were conducted using both wet and dry beads. The term wet refers to the state of the beads immediately prepared and the term dry to beads that were left to dry for 24 h in air till constant weight. Swelling studies of Ca-Alg beads were carried out in simulated intestinal fluid of 10 mM phosphate buffer (pH 7.4) and in simulated gastric fluid of 10 mM HCl buffer (pH 1.2). Accurately weighed amounts of beads were incubated in 25 ml of swelling solution at 37 o C under shaking at 100 rpm. At predetermined time intervals, the beads were separated from the medium using a stainless steel grid. Immediately, they were wiped gently with filter paper and weighed. The swelling percent of the beads was calculated according to the formula: Swelling percent % = [(Ws -W i ) / W i ] x 100 (2) Where Ws is the weight of the beads in the swollen state and W i is the initial weight of the beads.

In vitro release study
The in vitro release studies were performed in 10 mM phosphate buffer pH 7.4. Accurately weighed amounts of beads were placed in conical flasks containing 25 ml of the release medium. The samples were incubated at 37 ƕ C under shaking at 100 rpm. At predetermined time intervals, samples of 3 ml were withdraw from the release medium and were replaced with fresh phosphate buffer solution. The concentration of BB in the solution was assayed by UV/Vis spectrophotometer (Shimadzu) at 590 nm.

Morphology of the beads
The surface of the beads was examined using scanning electron microscopy (SEM, S-590, HITACHI). Prior to observation, samples were mounted on metal grids, using double-sided adhesive tape and coated by gold under vacuum before observation.

Morphology of the Beads
Scanning electron micrographs of air dried Ca-Alg beads prepared with 3% (w/v) CaCl 2 and 1, 2 and 3% (w/v) alginate concentration were illustrated in Fig. 1. At 1% (w/v) alginate, the dry beads completely lost its spherical shape (Fig. 1A) and the surface show highly roughness and large cracks ( Fig. 1B) caused by collapsing of the polymer layers during dehydration due to low mechanical strength of the gel.
By increasing the alginate concentration to 2% (w/v), beads remained its spherical shape (Fig. 1C) and the surface morphology was improved, but still showed large cracks (Fig. 1D). Further increase in alginate concentration to 3% (w/v) increases the viscosity leading to non spherical (elongated) beads ( Fig. 1E), which have smooth surface with smaller pores size due to the high gels beads mechanical strength (Fig. 1F). These results indicated that the surface morphology of Ca-Alg beads improved by increasing alginate concentration whereas increasing alginate concentration above 3% made preparation of the beads difficult because the solution become too viscous for dropping and non spherical (elongated) beads were formed.

Effect of alginate concentration
The effect of alginate concentration on the encapsulation efficiency was studied using 1 -4 % (w/v) alginate beads hardened with 3% (w/v) CaCl 2 and cured for 30 min. Figure 2 shows a gradual increase in the EE from 86 -93 % when alginate concentration was increased from 1 -4 % (w/v). However, the EE % of beads using 4 % alginate was close to that of 3 % alginate, thus for further optimization, 1 -3 % (w/v) alginate were used.
This increase in the EE can be explained as follow, increasing the alginate concentration provided more binding sites of alginate for Ca 2+ ions resulting in the A D B E F C formation of a more compact gel membrane, with smaller pores size so leaching of BB to the curring medium during preparation decreased (19, 22 & 23).

Effect of CaCl 2 concentration
Keeping the alginate concentration and curing time fixed at 2 % and 30 min, respectively, and increasing CaCl 2 concentration from 0.5 -5 % (w/v) significantly increased the EE % from 62 -90 % reaching a plateau after 3% (w/v) CaCl 2 as shown in Fig. 3. This can be explained as follow, at low concentration of CaCl 2 0.5 & 1 % (w/v) the amount of Ca +2 ions were insufficient for fast cross-linking of alginate and the formed gels have low mechanical strength and large pores size so leakage of BB to the external medium took place leading to decrease of the EE. While at high concentration of CaCl 2 , 2 & 3 % (w/v) the cross-linking of alginate was more efficient and quick due to a great quantity of Ca +2 ions were available to cross-linking alginate, so strong gels with smaller pores size were formed, which reduced leakage of BB during preparation. Further increasing in CaCl 2 concentration more than 3 % (w/v) showed no effect on encapsulation efficiency due to saturation of carboxylate group of alginate with Ca 2+ ions (24 -26) thus for further optimization, 0.5 -3 % (w/v) CaCl 2 were used.

Effect of curing time
Increasing the gelation time from 30 -120 min during preparation of Ca-Alg beads using 2 % (w/v) sodium alginate and 3 % (w/v) CaCl 2 solution decreased EE from 90 -84 % as shown in Fig. 4. The ionic interaction between carboxylate group in alginate and the small bivalent calcium ions was so quick. Further soaking of the alginate beads in the CaCl 2 solution lead to more leaching of BB. This may be due to the high solubility of BB in aqueous medium (20). Thus, a curing time of 30 min, which gives the maximum EE has been chosen for further experiments.

In vitro release study
As described previously (2) the release of low molecular weight drugs depends on diffusion through pores while drugs with high molecular weight as protein releases through swelling and disintegration of polymeric matrix. In our case, BB was used as model drug for low molecular weight drugs so its release was expected to be controlled by diffusion mechanism but we noticed that the release profile obeyed another mechanism according to the preparation condition.

Effect of CaCl 2
Increasing CaCl 2 concentration from 2 to 3 % (w/v) and keeping the alginate concentration and curing time at 2 % (w/v) and 30 min, respectively decreased the release rate of BB in phosphate buffer, pH 7.4 (stimulated intestinal fluid), which is in agreement with previous studies (25 & 26). For example, the release percent after 120 min was 66 and 84 % using 3 and 2 % (w/v) CaCl 2 , respectively as shown in This can be explained by the fact that at higher concentration of CaCl 2 cross-linking of alginate was more efficient, so strong gel with smaller pores size was formed, which retarded penetration of dissolution medium into the beads, which in turn decreased the release rate (24). Also the efficient cross linking of alginate beads at higher CaCl 2 retarded disintegration of beads which take place due to the presence of phosphate ions in the buffer (phosphate buffer) which have a high affinity for Ca +2 ions. Dainty et al. (27) reported that the disruption of Ca-Alg beads occurred faster in phosphate buffer above pH 5.5 by chelating action of phosphate ions, at these higher pH values, the affinity of phosphate ions toward calcium ions is higher than that of alginate, and solubility of calcium phosphate complex is high.

Effect of alginate concentration
Increasing the alginate concentration from 1 -3 % (w/v) at constant CaCl 2 concentration and curing time of 3 % (w/v) and 30 min, respectively showed a significant effect on the release of BB in phosphate buffer at pH 7.4. The rate of release could be divided into two sections, a) before two hours: The release of BB was found to follow this order: 1 % > 2 % > 3 % and b) after two hours: the release of BB was as follow: 1 % > 3 % > 2 % as shown in Fig.6. For the rate of release before two hours, i.e. 1 % > 2 % > 3 %, it could be explained by the fact that increasing the alginate concentration increases the viscosity and provided more number of binding sites of alginate for Ca 2+ ions resulting in the formation of a more stable and compact gel membrane with smaller pore size so penetration of dissolution medium into beads retarded and the release rate decreased (22). This was also supported by studying the release kinetics as shown in Fig. 7 where the release of drug from simple swellable polymeric matrix followed power law expression (28) as shown in equation 3.

Log [Mt/M ] = log K+ n log t (3)
Where [Mt/M ] is the drug released fraction at time t, K is a constant and n is the release exponent.
There are three scenarios for n values: 1) If n 0.5, the mechanism is called Fickian and the release is diffusionally controlled.
2) If n 0.85, the mechanism is called case II Transport and the release is depending only on the relaxation/swelling of the polymer.
3) If 0.5 > n > 0.85, the mechanism is called non Fickian (anomalous) and the release is depending on both the diffusion and relaxation of the polymer. The increase in alginate concentration from 1 -3 % (w/v) tended to increase the n values. Alginate concentration of 1% (w/v) showed n = 0.55 being close to Fickian mechanism due to large pore size and low mechanical strength of beads, which disintegrated rapidly so the release depended only on diffusion mechanism. Further increase of alginate concentration to 2 % increased n value to 0.64 following anomalous transport (non Fickian) due to increase in the gel mechanical strength so the release undergoes through diffusion and swelling (relaxation) of the polymer network. While using 3 % alginate, the n value increased to 0.97 shifting the release mechanism from anomalous transport to case II transport, which depended mainly on swelling/relaxation of the polymer network.
For the rate of release after two hours, the results were unexpected, i.e. the rate of release using 3 % (w/v) alginate started to be faster than that of 2 % (w/v) alginate (1 % > 3 % > 2 %) as shown in Fig. 6. For example, at 210 min, where all alginate formulations reached the plateau, the release of BB using 2 % (w/v) alginate was 86 % compared to 91 % using 3 % (w/v) alginate. Theoretically, it was expected for the 3 % (w/v) alginate beads to release about 70 % of the BB! To understand this unexpected behavior, the swelling study for Ca-Alg beads of 2 % and 3 % (w/v) alginate concentration was carried out in phosphate buffer pH 7.4 as shown in Fig. 8.  Two observations were noticed from the swelling study. The first, that beads prepared with 3 % (w/v) alginate showed lower swelling percent than that of 2 % (w/v) alginate, which was in harmony with the release results and release kinetics. The second was bursting and disruption of beads prepared with 3 % (w/v) alginate after 2 h while no bursting in case of 2 % (w/v) alginate, which showed a decline in swelling percent after 2 h for 3 % (w/v) alginate beads. The later was supported by the optical images of the beads after incubation in the release medium as shown in Fig. 9. This may be due to the higher osmotic pressure inside beads prepared with 3 % (w/v) alginate compared to that prepared with 2 % (w/v) alginate (29). From above results and discussion, we discerned that increasing alginate concentration from 2 % to 3 % (w/v) increased the mechanical strength

A B
of the beads, increased the EE and delayed the drug release. However, the higher alginate concentration caused inability to use hypodermic syringe to prepare smaller beads where the size of dried beads was increased from 544 to 638 μm compared to 2 % (w/v) alginate, respectively (Fig. 1). In addition, the use of highly viscous alginate solution caused non homogenous cross-linking of beads, which retarded the diffusion of Ca +2 ions into the interior of beads, causing improper mixing of drug with alginate solution and leading to bursting of beads due to the high osmotic pressure (16).

Effect of drying
The release was carried out for wet beads after immediately prepared and for beads dried in air for 24 h till constant weight. The release profile shows that the drying of beads accelerates the release rate as shown in Fig. 10. This may be due to the dehydration of alginate gel leading to formation of cracks and large gaps on the surface of beads which accelerate the release of BB through it (30).

Swelling study
Swelling studies were carried out to investigate the behavior of Ca-Alg beads in the release medium (gastro intestinal track) by monitoring the swelling percent and disintegration of the wet and dry beads in acidic medium pH 1.2 (stimulate gastric medium) and in alkaline medium pH 7.4 (SIF). The swelling of wet Ca-Alg beads in alkaline medium as shown in Fig.11 exhibited a swelling of 236 % at 60 min and then began to disintegrate. This was due to the presence of Na + ions in the phosphate buffer which undergo ionexchange with the Ca 2+ ions present within the alginate chains. So the repulsion force between the negatively charge carboxylate group (i.e. -COOgroups) of alginate increased and the degree of crosslinking decreased due to loss of Ca 2+ ions. This ultimately results in a rather loose structure and hence the beads take up more water until bursting of the beads take place and the beads start to disintegrate (31).  The same beads tend to shrink when exposed to acidic environment, pH 1.2. Ouwerx et al. (32) have shown that at low pH values < 4, the carboxylate groups of alginate are protonized and hence the electrostatic repulsion among these groups decreased and shrinkage is favored so the interior water rejected out side the bead and a decrease in its weigh take place.
On the other hand the swelling of the dried beads show highly degree of swelling compared to wet beads as shown in Fig. 12. Calcium alginate beads exhibited nearly 5000 % swelling percent in phosphate buffer, pH 7.4 at 120 min then beads started to disintegrate. This was due to the hydration of the dried beads and the ion exchange mechanism between Na + and Ca 2+ ions, which was explained previously (31). The dried beads showed a very small swelling of 60 % in HCl buffer without disintegration. This was due to hydration of the hydrophilic groups of alginate (16). These results indicated that Ca-Alg beads have a resistance towards acidic medium while it showed highly swelling percent and disintegration in alkaline medium. This phenomenon can be exploited in targeting the release of drugs to the intestinal region.

Conclusion
The optimum conditions for preparing alginate beads based on calcium chloride were studied inclusively using brilliant blue as a model of drugs. Results showed that the use of high alginate concentration, 2% (w/v) alginate could i) increase the mechanical strength of beads, ii) increase the EE, iii) delayed the drug release. But use of highly viscous alginate solution, 2% (w/v) alginate, may create some other complication such as i) non homogenous crosslinking of beads as highly viscous medium of droplet could retard the diffusion of Ca +2 ions into the interior of beads, ii) inability to use hypodermic syringe to prepare smaller beads where the size of dried beads were increased from 544 to 638 μm with increasing alginate concentration from 2 to 3 % (w/v), respectively , iii) improper mixing of drug with alginate solution, iv) bursting of beads due to the high osmotic pressure. Consequently, we concluded that Ca-Alg beads prepared using 2 % (w/v) alginate and hardened with 3% (w/v) CaCl 2 and cured for 30 min are showing the most suitable conditions for controlled Brilliant Blue (BB) release. We discerned that Also, Ca-Alg beads were found to be an excellent carrier for targeting BB to the intestine, where the swelling of the beads were 5000 % in alkaline medium compared to 60 % in acidic medium.
Overall, the carrier could be used for targeting smart drugs such as proteins for gastric passage and controlled intestinal release.