Improving the performance of transglutaminase-crosslinked microparticles for enteric delivery

a b s t r a c t
Various agents for cross-linking have been investigated for stabilizing and controlling the barrier properties of microparticles for enteric applications. Transglutaminase, in addition to being commercially available for human consumption, presents inferior cross-linking action compared to glutaraldehyde. In this study, the inten- sity of this enzymatic cross-linking was investigated in microparticles obtained by complex coacervation between gelatin and gum Arabic. The effectiveness of cross-linking in these microparticles was evaluated based on swelling, release of a model substance (parika oleoresin: colored and hydrophobic) and gastrointestinal assays. The cross-linked microparticles remained intact under gastric conditions, whereas the uncross-linked microparticles have been dissolved. However, all of the microparticles have been dissolved under intestinal conditions. The amount of oily core that was released decreased as the amount of transglutaminase increased. For the most efficient microparticles (50 U/g of protein), the performance was improved by increasing the pH of cross-linking from 4.0 to 6.0, resulting in a release of 17.1% rather than 32.3% of the core material. These results were considerably closer to the 10.3% of core material released by glutaraldehyde-cross-linked microparticles (1 mM/g of protein).

Orally administered polymeric microparticles that encapsulate drugs must resist degradation in gastric juices while ensuring complete drug release in the intestinal tract (Song, Zhang, Yang, & Yan, 2009; Zhang, Pan, & Chung, 2011). In most cases, this ideal objective can be achieved through structural modifications of the polymeric wall, which can delay degradation of the microparticles until a specific time or location.These polymeric carrier systems can be produced using various encapsulation techniques, including spray chilling, spray drying, ionic gelation, fluidized-bed coating and liposome entrapment (Desai & Park, 2005). Another such technique, complex coacervation, presents various advantages, including high encapsulation efficiency without the use of extreme production conditions; therefore, this technique can be used in the preparation of pharmaceuticals and foods. However, the nature of the ionic interactions between the polymers leads to low mechanical and thermal resistances of the resulting polymeric wall. Hence, it is necessary to strengthen the wall via cross-linking (Burgess & Ponsart, 1998; Sanchez & Renard, 2002) such that the rate of release of the active component can be precisely controlled.
For many years, aldehydes have been used as cross-linking agents (Thies, 2006). Aldehydes, such as glutaraldehyde (GLU), produce irreversible bonds and thus enhance the resistance of the wall to extreme pH and temperature conditions (Babin & Dickinson, 2001; Hernández-Muñoz, Villalobos, & Chiralt, 2004). These cross-linking agents covalently bind to the free unprotonated amino groups of a pro- tein. During the reaction, aldehydes are also inserted into the chemical binding; however, they are toxic, which limits their use for human consumption (Gallieta, Di Giola, Guilbert, & Cuq, 1998).

Transglutaminase (TG) is a non-toxic enzyme that catalyzes intra- and intermolecular cross-linking (ε-(λ-glutamyl)-lysyl) between pro- tein molecules (Motoki & Seguro, 1998). TG has also been studied as a cross-linking agent, particularly for materials that are produced for human consumption (Kuraishi, Yamazaki, & Susa, 2001; Mariniello, Di Pierro, Giosafatto, Sorrentino, & Porta, 2008; Tang, Chen, Li, & Yang, 2006). The conditions typically used for cross-linking with transglu- taminase include a temperature of 20 °C, pH of 5.0 and long reaction times, such as 15 to 24 h.In the present study, the intensity of transglutaminase cross-linking in microparticles produced by complex coacervation between gum Arabic and gelatin was investigated. The effects of adding different levels of TG (0, 10, 30 and 50 U/g of protein) on the mean size, morphology, release of oily core material and resistance to gastrointestinal conditions were compared to those of the typical concentration of glutaraldehyde (1 mM/g of protein). Because the activity of enzymes such as TG is highly sensitive to pH, the pH of cross-linking was also evaluated, as well as its effects on the release of core material under simulated gastric conditions.

2.Materials and methods
Gum Arabic (IRX49345, Colloides Naturels, SP, Brazil) and type A gelatin (244 bloom, Gelita South America, SP, Brazil) were used as wall materials. Commercial soy oil (Liza®) containing 10:1 paprika oleoresin (Citromax, SP, Brazil) was used as the core material. Microbial transglutaminase (Ca2+ independent, 100 U/g of nominal enzymatic ac- tivity, Activa TG-S®, Ajinomoto, SP, Brazil) and glutaraldehyde (Sigma, St. Louis, USA) were used as cross-linking agents. Pepsin (3180 U/mg of protein) and pancreatin (3 × USP unit of enzyme activity) enzymes, which were used to simulate gastric conditions, were purchased from Sigma-Aldrich (St. Louis, USA). All solvents and other reagents were of analytical grade, and deionized water was used in all the experiments.Microparticles were produced by emulsifying 2.5 g of the core material in 100 mL of gum Arabic solution (2.5 wt.%) for 1 min at 14,000 rpm at 50 °C using an Ultra-Turrax homogenizer (T-18 basic, IKA, Staufen, Germany) followed by incorporating the emulsion into 100 mL of gelatin solution (2.5 wt.%), also at 50 °C, and adding 400 mL of deionized water at 50 °C. The pH was reduced to 4.0 using 0.1 M HCl, and the system was gradually cooled to 10 °C. After 12 h in a refrig- erator (5 °C), the particles settled and the excess liquid was drained. These moist microparticles were freeze-dried in an Edwards Pirani 501 Freeze Dryer (West Sussex, UK) and characterized.To determine the efficiency of encapsulation, the freeze-dried micro- particles (100 mg) were dissolved in a tube with 1 mL of pancreatin solution (0.30 mg/mL in NaHCO3, 0.1 M, pH 7.0). Pancreatin, a protease, was used to hydrolyze the wall of particles and allow the release of the oily core. After incubating for 30 min at 37 °C, 20 mL of sunflower oil, a hydrophobic medium, was added to the tube to extract the hydrophobic core material released from the aqueous system. The system was con- tinuously stirred at 25 rpm for 1 h at 37 ± 0.5 °C. The content of the tubes was filtered through glass wool to separate the particulate mate- rials and centrifuged (10 min at 3500 rpm). An aliquot of the lighter oil phase was removed with a pipette. The amount of the core material present in the sunflower oil was quantified using a standard curve (460 nm) on a Beckman DU-70 spectrophotometer (Indianapolis, IN, USA); the curve was obtained at 5 concentration levels (0.020 to 0.150 mg/mL), with linearity (R2 = 0.998) was determined using aleast squares regression method in triplicate for each concentration level. The encapsulation efficiency (%) was calculated by comparing the amount of core material in the microparticles relative to the amount of core material used to produce the microparticles.

The wall of the original moist microparticles was strengthened by cross-linking with 3 different concentrations of TG (10, 30 and 50 units (U)/g of protein), using the same pH as that for the complex coacervation of the polymers (pH 4.0). These amounts represent, respectively, 0.1, 0.3 and 0.5 g of transglutaminase/g of protein. Cross- linking with the chemical agent glutaraldehyde (1 mM/g of protein or 0.39 mL of glutaraldehyde solution/g of protein) was performed under the same conditions as a control. The process, which was maintained under constant magnetic stirring, required 15 h at room temperature.The pH, time and temperature conditions were chosen based on previ- ous work conducted by our group (Alvim & Grosso, 2010; Prata, Zanin, Ré, & Grosso, 2008) and other authors (Dong et al., 2008; Zhang, Zhang, Hu, Bao, & Huang, 2012). The cross-linked microparticles were washed with water at the same pH.After evaluating the results obtained with TG, the higher concentra- tion (50 U/g of protein) was used to cross-link the microparticles at pH 6.0. The cross-linking was performed at room temperature, with constant agitation over 15 h, and the same conditions were used to cross-link glutaraldehyde (1 mM/g of protein).
The strength of the cross-linking in the wall was determined by eval- uating the release of the core material in an oily medium. The release of the core material from the freeze-dried microparticles was determined by adding 100 mg of these microparticles to 20 mL of sunflower oil in capped glass tubes covered with an aluminum sheet to avoid the effects of light. The tubes were agitated in a rotary shaker (AP22, Phoenix, SP, Brazil) moving at approximately 30 rpm. After 4, 8, 12 and 24 h, the tube contents were filtered using glass wool and centrifuged for 10 min at 3500 rpm.The amount of core material released into the sunflower oil was quantified using the standard curve. All determinations were performed in triplicate.Thus, the oil release assay was performed with dried particles (i.e., without hydrophilic hindrance) in another oily medium, sunflower oil (i.e., hydrophobic affinity), over a period of 24 h.

To analyze swelling, the morphologies and sizes of all microparticles were determined, with measurements taken both before freeze-drying and after rehydration with distilled water (1 h with constant agitation). The morphologies were observed using an optical microscope (Jenaval, Carl Zeiss, Göttingen, Germany) coupled to a digital camera and record- ed using Image Pro Plus 4.0 software. The mean diameter was deter- mined for at least 500 of the microparticles using the free Scion Image software ( cross-linked microparticles (100 mg of dried particles plus 1 mL of deionized water, 15 min) were submitted to an in vitro gas- trointestinal simulation with pepsin and pancreatin (Bermejo et al., 2002). These samples were incubated in 1 mL of simulated gastric medium (pepsin solution; 2.65 U/mg of protein) in 0.1 M HCl, adjusted to pH 1.2, for 2 h at 37 °C in a water bath with constant agitation. The solution was then neutralized with 1.5 M NaHCO3 at pH 7.0, and 1 mL of intestinal medium (0.15 mg pancreatin/mL of 0.1 M NaHCO3) was added. Incubation for 4 h at 37 °C with agitation in a water bath was followed by immersion in an ice bath to stop the action of the enzyme. The samples were observed using an optical microscope, as described above.To verify the release of the core material under simulated gastric conditions, freeze-dried microparticles were incubated as described above in a solution simulating gastric conditions (pepsin, pH 1.2). After reaching the time of the simulated gastric assay, sunflower oil (20 mL) was added as a solvent to remove the oily core from the aque- ous simulated gastric medium, and the tube was manually stirred for 3 min. The contents of each tube were filtered using glass wool and centrifuged at 3500 rpm for 10 min. The absorbance of the supernatant was measured at 460 nm using a spectrophotometer, and the quantity of core material released was estimated using the standard curve. To measure the amount of core material released under simulated gastric conditions, cross-linking at pH 4.0 and 6.0 was evaluated. Particles with- out cross-linking were not evaluated.Statistical analyses were performed using the SAS program (Version 6.8, SAS Inc.), with differences between means determined by multiple Tukey tests, using a level of confidence of 95% (p b 0.05). All treatments and analyses were performed in triplicate.

3.Results and discussion
Fig. 1 presents the morphologies of the coacervates produced and of those subsequently subjected to cross-linking. Both types of microparti- cles, namely, those cross-linked at pH 4.0 and uncross-linked, were spherical with a well-defined wall. The core material was dispersed in a multinuclear formation and homogeneously distributed throughout the matrix. No morphological differences between the cross-linked and uncross-linked particles were observed. Similarly, the encapsula- tion efficiency was high for both uncross-linked microparticles (79.0%) and those cross-linked with transglutaminase (85.0%) and glutaralde- hyde (89.9%).The encapsulation efficiency reported for coacervates produced using gum Arabic and gelatin as the emulsifier is generally high (Liu, Low, & Nickerson, 2010; Qv, Zeng, & Jiang, 2011; Zhang et al., 2011). In the present study, gum Arabic was used as the emulsifier, and the homogeneity of the distribution and the presence of a well-defined wall suggest that the use of gum Arabic as an emulsifier does not affect the cross-linking process, producing microparticles similar to those produced when gelatin is used for this purpose (Zhang et al., 2011).The high water content (greater than 80%) and the presence of protein (~33% in dry basis) in these coacervate microparticles led to a limited shelf life, which can be increased by drying the microparticles. Freeze-dried microparticles were thus investigated for their ability to rehydrate and release the core in an oily medium. The ability of the freeze-dried microparticles cross-linked with different concentrations of transglutaminase (0, 10, 30, and 50 U/g of protein) to regain their original form after rehydration was compared to those cross-linked with glutaraldehyde, and the results with 0 and 50 U/g are shown in Fig. 1. Size variations were determined by optical microscopy (Table 1), including average size determinations in the initial moist particles and in the rehydrated particles after drying.Initially, the uncross-linked microparticles were significantly smaller than those subjected to cross-linking. However, after drying and rehy- dration (1 h soaking in water), these particles swelled to a diameter that was 21.5% greater than their initial size. The cross-linked micropar- ticles resisted swelling after drying and rehydration, resulting in micro- particles that were 11.4% and 12.6% smaller than their initial size for those cross-linked with transglutaminase and glutaraldehyde, respec- tively. These results suggest that both cross-linking agents were efficient in hardening the wall.Additionally, the denser the bridges formed between the hydrocol- loid chains, the more packed the structure and the more difficult it is for water to enter. The cross-linked microparticles did not regain their original size after drying and rehydration. However, the rehydration that was observed occurred almost immediately (b 10 s), and the micro- particles regained their original spherical shape, independent of the type or amount of cross-linking agent.

One of the measures for determining the efficiency of cross-linking is the rate of release of the core material. The release of the core material from microparticles produced without cross-linking or with different quantities of transglutaminase and glutaraldehyde (pH 4.0) is shown in Fig. 2. The release of the hydrophobic core occurred only when freeze-dried microparticles were dispersed in a hydrophobic medium (sunflower oil) because non-dried micropar- ticles contain large amounts of water, which represents a barrier to the diffusion of the hydrophobic compound into the hydrophobic release medium (Prata, 2006).Irrespective of the type or amount of cross-linking agent, similar release patterns were observed for the multinuclear microparticles pro- duced in this work (Fig. 2), with most of the active compound released during the first 4 h, followed by a period of slow but sustained release that was maintained until the end of the 24 h test. This pattern of release is suitable for various target applications, particularly for the sustained release of drugs.The amount of core material released varied as a function of the type of cross-linking agent and the amount of enzyme used. Approximately 46% of the core material was released by uncross-linked microparticles after 24 h. Cross-linking with transglutaminase decreased the amount released proportionately to the amount of the enzyme used, i.e., the cross-linked system with the highest concentration of transglutaminase (50 U/g of protein) was more protective although less effective than glutaraldehyde (1 mM/g of protein). After this time, only 21.7% and 16.3% of the core material had been released for cross-linked micropar- ticles prepared with these concentrations of transglutaminase and glutaraldehyde, respectively.

The greater efficiency of glutaraldehyde as a cross-linking agent may be due to its polymeric nature, which allows access to reactive groups of various lengths, leading to a larger number of cross-links (Charulatha & Rajaram, 2003). This efficiency as a cross-linking agent results in reduced mobility of macromolecular chains and the consequent forma- tion of a more rigid structure (Dinarvand, Mahmoodi, Farboud, Salehi, & Atyabi, 2005). Thus, less diffusion of the active encapsulated compound through the polymer wall would be expected. Given the restrictions regarding the use of chemical cross-linking agents for food and pharma- ceutical purposes, however, the results obtained in this study suggest that transglutaminase can be used as an alternative for cross-linking gelatin structures.Although only 21.7% of the core material was released after 24 h when transglutaminase was used at the highest concentration (50 U/g of protein), these results can be improved by modifying the cross- linking conditions because the activity of enzymes is highly sensitive to the pH, hardening time and temperature of the reaction. In this study, the hardening time and temperature of the reaction were fixed based on the findings of our group. In the literature, the effects of vari- ous cross-linking parameters on the rate of release of microalgal oil from gelatin-gum Arabic coacervates, including the hardening time, temperature, concentration of cross-linking agent and pH, have been in- vestigated (Zhang et al., 2012). The authors found that a cross-linking temperature above 15 °C and a longer hardening time did not affect the action of the enzyme, thus confirming the experimental conditions that our group has utilized (Alvim & Grosso, 2010; Prata et al., 2008) and defining the temperature and time of cross-linking used in this work. However, those authors found that transglutaminase concentra- tions above 15 U/g did not improve the efficiency of cross-linking, in contrast to the results obtained in the present study. The pH can also be modified to improve the transglutaminase activity, and modification of pH will be discussed in the following.

For microparticles to deliver drugs to the intestinal environment, the microparticles must first resist the acidic conditions of the stomach and the enzymatic action of pepsin. The microparticles must then release the active compound under intestinal conditions. Minimizing the re- lease of the active ingredient under acidic conditions is thus necessary; this core retention can be enhanced by controlling both the pH of the cross-linking and the concentration of the (enzymatic) cross-linking agent.The pH used to produce the microparticles in this study (pH 4.0) is far from optimal for enzymatic action (pH 5–8) (Motoki & Seguro, 1998) or chemical cross-linking (pH 9) (Thies, 1995). The efficiency of cross-linking should therefore improve if the reactions are conducted at a higher pH. A pH of 6.0 was thus attempted for the cross-linking of the wall, while maintaining the other parameters of temperature, time and amount of chemical or enzyme used.The effectiveness of microparticles cross-linked at pH 4.0 and 6.0 for enteric use was evaluated by assessing their morphologies after incubation under simulated gastrointestinal conditions (Fig. 3). The uncross-linked microparticles and those cross-linked with the lowest transglutaminase concentration (10 U/g of protein) were severely dam- aged by the acidic medium used to simulate gastric conditions. Such damage, however, was not observed for those cross-linked with glutar- aldehyde or for either of the higher transglutaminase concentrations (30 and 50 U/g of protein), irrespective the pH of cross-linking involved. Under simulated intestinal passage, all microparticles, irrespective of the amount of enzyme or type of cross-linking agent, dissolved, resulting in the complete release of the core material.The effects of the pH of cross-linking with glutaraldehyde and the highest concentration of transglutaminase on the release of the core material from microparticles submitted to a gastric in vitro assay are shown in Fig. 4. Even at pH 4.0, which is considered not ideal for maxi- mum cross-linking activity of GLU or TG, both cross-linking agents were relatively effective in the retention of the core material. Approximately 54.2% of the core material was released from uncross-linked micropar- ticles after 2 h of suspension in simulated gastric conditions, whereas in those cross-linked with TG, only 32.3% was released.

Cross-linking with GLU led to the release of only one third of this value (10.3%).Increasing the cross-linking pH to 6.0 increased the efficiency of oleoresin retention (Fig. 4). The performance of microparticles cross-transglutaminase; Colloids Naturels for gum Arabic and Gelita South America for gelatin. linked with GLU increased only marginally, with the release decreasing from 10.3% to 9.1%, whereas for TG, the performance improved more, with retention of the core material increasing by 47% (a decrease from 32.3% to 17.0%). This improvement may be due to the more expanded conformation of the gelatin molecules at a pH above the isoelectric point (Kawanishi, Christenson, & Ninham, 1990), under which more amino groups accessible for enzymatic cross-linking.The effect of the TG concentration on the release of the core material under simulated gastric conditions after cross-linking at pH 6.0 was also evaluated. An increase in the enzyme concentration from 10 U/g of protein to 50 U/g of protein led to a substantial delay in the release of the encapsulated core material (Fig. 5). After 2 h under simulated gastric conditions, approximately 25% of the core material was released when microparticles had been cross-linked with lower concentrations of TG, and this release was reduced to approximately 17% for the higher con- centration. This behavior was similar to the behavior observed for the release from freeze-dried microparticles cross-linked at pH 4.0 after the same length of time in an oily medium (Fig. 2), with approximately 22% of the core material being released for the lower concentration and 15% for the higher. These results demonstrate that release of the core material from TG-cross-linked microparticles can be modulated by the control of its concentration, thus providing opportunities for fine- tuning for specific uses.

The release of core material from dried microparticles cross-linked with different concentrations of TG at pH 4.0 was inversely to the amount of TG used. Cross-linking with TG (N 10 U/g of protein) proved to be effective in maintaining the integrity of the wall under simulated gastric conditions. All microparticles, regardless of the degree or agent of cross-linking, dissolved under simulated intestinal conditions. In- creasing the pH to 6.0 improved the enzymatic action, and the micro- particles were more resistant to the simulated gastric conditions. Such TG-cross-linked microparticles should be suitable for use in food or pharmaceutical applications in which the core material is to be released in the intestines, even though the enzyme may be somewhat less efficient than glutaraldehyde as a cross-linking agent.