Swelling, mechanical strength, and release properties of probucol microcapsules with and without a bile acid, and their potential oral delivery in diabetes

Abstract

We have demonstrated a permeation-enhancing effect of deoxycholic acid (DCA), the bile acid, in diabetic rats. In this study, we designed DCA-based microcapsules for the oral delivery of the antilipidemic drug probucol (PB), which has potential antidiabetic effects. We aimed to further characterize these microcapsules and examine their pH-dependent release properties, as well as the effects of DCA on their stability and mechanical strength at various pH and temperature values. Using the polymer sodium alginate (SA), we prepared PB-SA (control) and PB-DCA-SA (test) microcapsules. The microcapsules were examined for drug content, size, surface composition, release, Micro-CT cross-sectional imaging, stability, Zeta potential, mechanical strength, and swelling characteristics at different pH and temperature values. The microencapsulation efficiency and production yield were also examined. The addition of DCA resulted in microcapsules with a greater density and with reduced swelling at a pH of 7.8 and at temperatures of 25°C and 37°C (p < 0.01). The size, surface composition, production yield, and microencapsulation efficiency of the microcapsules remained similar after DCA addition. PB-SA microcapsules produced multiphasic PB release, while PB-DCA-SA microcapsules produced monophasic PB release, suggesting more controlled PB release in the presence of DCA. The PB-DCA-SA microcapsules showed good stability and a pH-sensitive uniphasic release pattern, which may suggest potential applications in the oral delivery of PB in diabetes.

Keywords::

• deoxycholic acid
• Micro-CT imaging
• microencapsulation
• production yield
• release characteristics

Introduction

Bile acids are naturally produced in humans and have shown health benefits through their endrocrinological, metabolic, microfloral,and other known and unknown effects (Al-Salami et al. 2007, Calasan et al. 2012, Negrulj et al. 2013). Studies in our laboratory have demonstrated beneficial antidiabetic effects of bile acids, alone or combined with other agents, in vivo (Mikov et al. 2006, 2007, 2008, 2012, Al-Salami et al. 2008a, 2012) and ex vivo (Al-Salami et al. 2008b, 2008c, 2008d, 2008e, 2008f, 2009, Calasan et al. 2012), or as membrane-stabilizing molecules in vitro (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, 2014f). In a recent study, we showed (in vivo) a permeation-enhancing effect of the bile acid deoxycholic acid (DCA), when combined with the antidiabetic drug gliclazide, and administered to diabetic rats (Lalic-Popovic et al. 2013). In a follow-up study, we designed a bile acid-based microcapsule platform for the targeted oral delivery of lipophilic drugs in diabetic animals (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014c, 2014f). One of the applications of this microcapsule platform is in improving the oral delivery of lipophilic drugs with potential antidiabetic effects (Negrulj et al. 2013). A good approach is to optimize the oral delivery of lipophilic drugs that have already shown desirable antidiabetic effects such as lowering of blood cholesterol and reducing the formation of atherosclerotic plaques (Negrulj et al. 2013). A good example is the microencapsulation of the anti-hyperlipidemic drug, probucol (PB) (Mooranian et al. 2014a, 2014d).

PB is a highly lipophilic drug with beneficial antidiabetic effects (Mooranian et al. 2014a, 2014d, Negrulj et al. 2013). These effects include reduction of cholesterol, protection of β-cells of the pancreas, and antiradical and antioxidant effects (Wu et al. 2013, Yamashita and Matsuzawa 2009). Accordingly, this study aimed to study the suitability of newly designed PB-DCA microcapsules for the oral delivery of PB, in vitro. Sodium alginate (SA) was used as a biodegradable, biocompatible polymer (Mooranian et al. 2014a, 2014d, Negrulj et al. 2013). The study examined the morphology, shape, size, drug content, surface composition, Micro-CT cross-sectional imaging, swelling properties, release kinetics, mechanical strength, Zeta potential, and the density of the microcapsules. The production yield and the encapsulation efficiency were also examined. In order to investigate the effect of DCA on the PB microcapsules, the study covered both types of microcapsules, PB-SA (as control) and PB-DCA-SA (as test).

Materials and methods

Materials

Probucol (PB, 99%), sodium alginate (SA, 99%) and deoxycholic acid (DCA, 99%) were purchased from Sigma Chemical Co, USA. Calcium chloride dihydrate (CaCl₂.2H₂O, 98%) was obtained from Scharlab S.L, Australia. All solvents and reagents were supplied by Merck (Australia), and were of HPLC grade and used without further purification or modification.

Drugs preparations

Stock suspensions of PB (20 mg/ml) and DCA (1 mg/ml) were prepared by adding the powder to 10% Ultra water-soluble gel (Mooranian et al. 2014a, 2014d). The CaCl₂ stock solution (2%) was prepared by adding CaCl₂ powder to HPLC water. All preparations were mixed thoroughly at room temperature for 4 h, stored in the refrigerator, and used within 48 h of preparation.

Preparation of microcapsules

Microcapsules of PB-loaded sodium alginate (PB-SA) were prepared using a Büchi-based microencapsulating system (BÜCHI Labortechnik, Switzerland) (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e). Polymer solutions containing SA and PB with or without DCA were made up to a final concentration (of PB-DCA-SA) in a ratio of 1:3:30 respectively, as described in our previous studies (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, Mooranian et al.).

Characterization of loaded microcapsules

Drug content, production yield, microencapsulation efficiency, and stability studies

Drug content, production yield, and microencapsulation efficiency. One gram of microcapsules was carefully weighed, ground, and dissolved in 200 ml of phosphate buffer (pH 7.8), and the suspension was stirred by a magnetic stirrer for 6 h. Aliquots of the dissolution medium (2 ml) were withdrawn at predetermined time points (every 200 s), filtered through a 0.22 μm Millipore filter, and analyzed (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, Mooranian et al.).

PB concentrations were calculated from the calibration curve (Mooranian et al. 2014a). All analyses were carried out in triplicate (n = 3). The drug contents, production yield, and microencapsulation efficiency were calculated as previously described (Mooranian et al. 2014a).

Zeta potential and size analysis. To determine the electrokinetic stability and size uniformity of the microcapsules in the dispersion system, Zeta potential and size distribution for the microencapsulated formulation of PB-SA and PB-DCA-SA were measured by photon correlation spectroscopy using a Nano ZS (Malvern Instruments, Malvern, UK) and by the Mie and Fraunhofer scattering technique using Mastersizer 2000 (Malvern Instruments, Malvern, UK) (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, Mooranian et al.). All determinations were performed in triplicate. Results are reported as mean ± SD.

Optical microscopy (OM). Morphological characteristics and particle size analysis were determined utilizing a Nikon YS2-H mounted with a ToupTek photonics FMA050 fixed calibrated microscope adaptor (Japan) (n = 3) (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, Mooranian et al.).

Energy Dispersive X-Ray (EDXR) spectroscopy studies. The surface composition of the microcapsules was examined using the EDXR (Oxford Instruments, INCA X-Act, USA) (Mooranian et al. 2014c, 2014d, Mooranian et al.).

X-ray microtomography (Micro-CT) scanning studies. The morphology and microstructure of the respective microcapsules were examined using a SkyScan 1172A Micro-CT (Kontich, Belgium) machine running Control software (Version 1.5 Build 8) and assembled to a Hamamatsu 10 Mp camera (Hamamatsu, Japan) with a pixel size of 11.48 μm. The scans were acquired under a current of 100 μA and an accelerating voltage of 100 kV using rotation steps of 0.3 degrees for all scans. Analysis was undertaken using a scan exposure time of 1180 milliseconds, and 16 frames were averaged for each exposure in order to minimize background noise/interference. Freshly made microcapsules from the respective formulations were prepared for analysis, and scanning was conducted in triplicate.

Swelling Studies. To determine the swelling properties of the microcapsules, 50mg of dry microcapsules were weighed and placed in 20 mL of buffer, under conditions of two pH values (3 and 7.8) and two temperatures (25°C and 37°C) for 6 h (Mooranian et al. 2014a, 2014b, 2014e).

Drug release studies (in-vitro dissolution test). A weighed sample (2 g) of PB and DCA-loaded microcapsules were suspended in 200 ml of phosphate buffer solution at pH values of 1.5, 3, 6, and 7.8 for 6 h, as appropriate (Mooranian et al. 2014a, 2014e). The dissolution medium was stirred at 200 rpm. All the experiments were carried out at 25°C. All absorbances of the solution were measured every 30 min using our Hewlett Packard-based Time-Controlled UV-Spec mounted with a close-loop flow system (Mooranian et al. 2014a, 2014e). All analyses were carried out in triplicate (n = 3).

Mechanical resistance testing

In order to test the mechanical stability of the microcapsules, mechanical resistance testing was carried out using a Boeco Multishaker PSU 20 (Boeco Company, Hamburg, Germany) over a 24-h period (Mooranian et al. 2014b).

Physical and chemical stability. The stability test was carried out by placing predetermined amounts of freshly prepared microcapsules onto sterile Petri dishes (30 microcapsules in each) and storing them in thermostatically controlled ovens (environmental stability chambers) at 20°C, 5°C, 25°C, and 40°C, with relative humidity set at 35%, for a period of 72 h. The experiment was conducted using a stability chamber (Angelantoni Environmental and Climatic Test Chamber, Italy) (Mooranian et al. 2014a, 2014b, 2014e).

Statistical analysis

Values are expressed as means ± SD. Drug content, production yield, and microencapsulation efficiency were assessed using the Student's t-test. Statistical analysis was performed using a two-way ANOVA with Tukey HSD post-hoc comparison of means only when the associated main effect or interaction was statistically significant. The best fit model was derived using GraphPad Prism software (V6; GraphPad Software, Inc., USA). Statistical significance was set at p < 0.05.

Results and discussion

Drug content, production yield, microencapsulation efficiency, Zeta potential, and microcapsule size

Table I shows the drug content (%), production yield, encapsulation efficiency, Zeta potential, and size of the PB-SA (control) and PB-DCA-SA (test) microcapsules. Data show that the addition of DCA only affected Zeta potential in reducing the surface charge from − 72.9 ± 2 to − 20.6 ± 1 (p < 0.01). This suggests an ionic interaction between the DCA and the polymer SA. Data also suggest that DCA addition did not have any detrimental effect on the microcapsules’ size or drug content, or the efficiency of the microencapsulation process.

Table I. Drug contents, production yield, encapsulation efficiency, Zeta potential, and mean particle size of PB-SA and PB-DCA-SA microcapsules. n = 3, data are averages ± SD. **p < 0.01.

Formulation code	Formula composition	Drug content (%) ± SD	Production yield (%) ± SD	Encapsulation efficiency (%) ± SD	Zeta potential (mV) ± SD	Mean microcapsule size (μm) ± SD
PB-SA (control)	Probucol-Sodium Alginate microcapsules	2.5 ± 0.3	81 ± 5	72 ± 2	− 72.9 ± 2	870 ± 40 (span 1.42)
PB-DCA- SA (test)	Probucol-Deoxycholic Acid-Sodium Alginate microcapsules	2.2 ± 0.2	75.1 ± 5	70 ± 2	− 20.6 ± 1**	810 ± 70 (span 1.45)

Optical microscopy

The PB-SA (Figure 1a) and PB-DCA-SA microcapsules were analyzed for their particle size and morphology (Figure 1b). Both microcapsules showed sphericity and homogeneity, with similar sizes, and the diameter of both microcapsules averaged around 850 μm. This suggests that the addition of DCA did not adversely change the size or morphology of the microcapsules.

Figure 1. Optical micrograph (a) and SEM images (b) of control (PB-SA) microcapsules, with L1 being the horizontal diameter, L2 the vertical diameter, and L3 the width. Similarly, optical micrograph (c) and SEM images (d) of test (PB-DCA-SA) microcapsules with L1 being the vertical diameter, L2 the horizontal diameter, and L3 the microcapsule width.

EDXR studies

Microcapsule surface analysis was undertaken for both PB-SA and PB-DCA-SA microcapsules using EDXR technology. This analytical technique allows for the determination of the elemental composition of microcapsule surfaces, since each element displays unique energy-dependent X-ray spectroscopic patterns (Tam et al. 2005). Figure 2 shows an example of both PB-SA and PB-DCA-SA microcapsule surfaces with the corresponding surface sites where EDXR analysis was conducted. Surface analysis of both microcapsules showed similar elemental contents. This suggests that the bile acid, DCA, is distributed within the microcapsules and not on the surface, which means the pH-targeted delivery of the PB and DCA will take place only after the microcapsule membrane dissociates and its contents are released.

Figure 2. Energy dispersive X-ray spectra of control (PB-SA) microcapsule surface showing PB deposition and surface composition with corresponding analysis (a), and energy dispersive X-ray spectra of test (PB-DCA-SA) microcapsule surface showing PB deposition and surface composition with corresponding analysis (b).

X-ray microtomography (Micro-CT) scanning studies

Computer-assisted X-ray Micro-CT scanning is often used to determine the microstructure, internal complexity, and morphology of alginate-based microencapsulated systems (Arifin et al. 2011, 2012). Figure 3a–c depicts images of a PB-SA microcapsule, and Figure 3d–f depicts images of a PB-DCA-SA microcapsule at various cross-sectional scan exposure times. Overall, microcapsule shape and morphology were similar across the scanned time points for both microcapsules, but there were notable differences in their internal complexity. Specifically, there was a greater encapsulated density of material in the PB-DCA-SA microcapsules owing to bile acid partitioning within the microcapsule layers. In addition, the PB-DCA-SA microcapsules presented well-defined membrane borders as opposed to the PB-SA microcapsules, and this may be explained by the membrane-stabilizing and structural support properties of the bile acid, which is in line with our previous findings (Mooranian et al. 2014a, 2014b, 2014c, 2014d, 2014e, Mooranian et al., Negrulj et al. 2013).

Figure 3. Micro-CT images of control (PB-SA) microcapsules (a–c) and test (PB-DCA-SA) microcapsules (d–f) depicting the internal complexity and structural morphology of the microcapsules. The visible purple color in PB-DCA-SA (d–f) figures reflects greater density of the microcapsule core caused by incorporation of DCA into the matrix.

Swelling studies

Figures 4a and 4b show that at both 25°C and 37°C, the presence of DCA reduced the swelling properties of the microcapsules only at a high pH (7.8), which suggests that DCA exerts a membrane-stabilizing effect, particularly at higher pH values, thereby exerting a more controlled dissolution pattern. This effect is desirable, as the targeted region of PB release is the lower gastrointestinal tract (Mooranian et al. 2014a, Negrulj et al. 2013).

Figure 4. Microcapsule swelling characteristics at 25°C (a) and 37°C (b) showing the reduced swelling behavior of microcapsules post DCA incorporation. Mechanical strength testing (c) also showed greater membrane stabilization brought about by DCA.

The main mechanisms responsible for water penetration and matrix erosion at higher pH are likely to be from alginate water uptake and subsequent matrix porosity and dissolution (Takka and Cali, 2012, Goh et al. 2012). Clearly, swelling was reduced by the DCA reinforcement. This could be due to the hydrophobic nature of the DCA, repelling water molecules that would otherwise penetrate and rupture the membrane, causing bursts of drug release. On the other hand, at acidic pH 3, alginate does not absorb water, and thus maintains the shape of the microcapsules (Goh et al. 2012, George and Abraham 2006). In order to test the resistance of the alginate matrix to pressure, both with and without DCA, mechanical strength testing was performed.

Mechanical resistance testing

The results of the mechanical strength testing (Figure 4c) were in line with swelling data and showed stronger and more intact microcapsules in the presence of DCA compared with control. By the end of a 24-h period, almost all PB-DCA-SA microcapsules remained intact while a significant percentage of PB-SA microcapsules had lost their contents. This may also suggest that PB-DCA-SA microcapsules will be able to withstand pressure as they move through the gastrointestinal tract and until they reach the lower intestine, where PB is maximally absorbed. The desired outcome of the swelling and mechanical resistance testing is that the PB-DCA-SA microcapsules will withstand gut pressure until reaching the lower intestine, where the pH exceeds 6 and the release of PB and DCA commences (Mooranian et al. 2014a, Negrulj et al. 2013).

Drug release studies

Figures 5a and 5b show PB release from the microcapsules at four different pH values: 1.5, 3, 6, and 7.8. Data show no release at the lower pH values (Figure 5a) while there was little release at pH 6. There is a clear difference in the PB release pattern at pH 6 from both microcapsules, with PB starting to be released at 1.5 h (control) compared with 2.0 h (test). At pH 7.8, PB exhibited a multiphasic release pattern and the addition of DCA caused a more controlled, monophasic release pattern suggesting optimized delivery. This is beneficial as in vivo studies showed that PB has huge inter-individual variation in absorption and this has been associated with its severe side effects and limited clinical applications (Negrulj et al. 2013).

Figure 5. Probucol release over time from PB-SA and PB-DCA-SA microcapsules at pH 3 and 1.5 (a), and at pH 7.8 and 6 (b). n = 3, data are expressed as mean ± SD.

Accelerated stability studies (environmental chamber)

Accelerated stability studies can provide useful information on how the microcapsules react to change in temperature in terms of their physical characteristics (Mooranian et al. 2014a, 2014b, 2014e). The stability testing at various temperatures revealed key differences in the microcapsules’ morphology, size, and texture. While there was no change in morphology, size, or texture of microcapsules at − 20°C and 5°C, there were significant changes at higher temperatures, 25°C and 40°C. At 25^°C, the morphology did not change, but the color changed from creamy white to pale yellow. The texture also became more brittle and stiff. At 40°C, the microcapsules changed color to dark yellow, and became very brittle and stiff while the size became smaller and the microcapsules shrunk significantly. The changes at higher temperature can be explained by the loss of moisture contents resulting in a reduction in size. There was also an increase in surface hardness and color change, suggesting significant dehydration of the alginate matrix, and subsequent smaller, harder, and darker microcapsules. The presence of DCA made no difference to the shape, texture, color, or size of the microcapsules, suggesting that DCA did not change or compromise the reaction of microcapsules to heat, especially at higher temperatures.

Conclusion

The study used a host of methods to examine newly designed bile acid-based microcapsules for the orally targeted delivery of a highly lipophilic drug with potential applications in diabetes. The bile acid used was DCA, which has previously shown permeation-enhancing effects, while the lipophilic drug used was PB, which has previously shown protective effects of pancreatic β-cells. In order to examine the morphological and drug-distribution properties, optical as well as SEM, EDXR, and Micro-CT imaging were used, while, in order to examine the stability and physical properties of the formulations, Zeta potential, swelling, and mechanical strength were tested. In order to examine the orally targeted delivery, release studies were performed at various pH and temperature values. Overall, the data show that DCA incorporation into PB microcapsules did not affect drug contents or the size of the microcapsules, but brought about a more dense core, with DCA accumulating mainly in the inside of the microcapsules. Based upon in vitro examination of the microcapsules’ swelling properties, as well as mechanical strength and PB release, our findings suggest that it would be possible to achieve targeted delivery of PB and DCA into the lower intestine, where both are meant to have best absorption. A future study may examine either the impact of other bile acids on PB release or examine the in vivo effects of the microcapsules, using an animal model of diabetes.

Acknowledgments

The authors acknowledge the CHIRI at Curtin University, and the Curtin-seeding grant for the support provided, and also acknowledge the use of equipment and the scientific and technical assistance of the Curtin University Electron Microscope Facility, which has been partially funded by the University, State, and Commonwealth Governments.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.