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Artifi cial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–8Copyright © 2014 Informa Healthcare USA, Inc.ISSN: 2169-1401 print / 2169-141X onlineDOI: 10.3109/21691401.2014.971806

Advanced bile acid-based multi-compartmental microencapsulated pancreatic β -cells integrating a polyelectrolyte-bile acid formulation, for diabetes treatment

Armin Mooranian 1 , Rebecca Negrulj 1 , Nigel Chen-Tan 2 , Marc Fakhoury 3 , Frank Arfuso 4 , Franca Jones 5 & Hani Al-Salami 1

1 Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Biosciences Research Precinct, Curtin University, Perth, Western Australia, Australia, 2 Faculty of Science and Engineering, Department of Imaging and Applied Physics, Curtin University, Perth, Western Australia, Australia, 3 Faculty of Medicine, Department of Neuroscience, University of Montreal, Montreal, Quebec, Canada, 4 Curtin Health Innovation Research Institute, Biosciences Research Precinct, School of Biomedical Science, Curtin University, Perth, Western Australia, Australia, and 5 Faculty of Science and Engineering, Department of Chemistry, Curtin University, Perth, Western Australia, Australia

Introduction

Insulin injections have been used for decades in diabetes treatment (Rafaelsen 1964). Type 1 diabetic patients rely on insulin injections due to the lack of insulin-producing pancreatic cells, while about 30% of Type 2 diabetic patients resort to injecting insulin due to either the inability of exist-ing β -cells to produce suffi cient insulin and control glycemia, or the absence of functional β -cells as a result of excessive stimulation by anti-diabetic drugs such as sulfonlyureas (Barnett 2010).

Despite strict use of anti-diabetic drugs to control blood glucose levels, hypoglycemic episodes and blood glucose fl uctuations remain common among diabetic patients (Bonadonna et al. 2006, Maji 2004). Recently, there have

been new and robust delivery systems for insulin, such as microchips, that are more sensitive to blood glucose changes and more tailored to patients ’ requirements. How-ever, the long term safety and effi cacy of these new systems remain questionable (Tuch et al. 2011). An ideal treatment for insulin defi ciency would be to rejuvenate the damaged β -cells and permanently control glycemia without any adverse eff ects. Th is can be achieved by either transplanting stem cells, which will diff erentiate into functional β -cells, or by transplanting already diff erentiated β -cells. To date, transplantation of β -cells has not been successful, due to many obstacles (Puri and Hebrok 2012). Th ese obstacles are mainly the immune system of the host, and infl ammation (de Vos et al. 2002, 2006). In order to achieve signifi cant gly-cemic control, the transplanted cells have to be in suffi cient numbers and exhibit adequate biological activity, without triggering any immune response. Th us, there is a need for suitable capsules that can encapsulate and protect the cells permanently, while allowing their biological activity post-transplantation. Th is can be achieved by using the artifi cial cell microencapsulation (ACM) technology, which was pio-neered by Th omas Chang at McGill University in Canada in the 1960s, to deliver drugs and cells (Chang 1971, 1992, Chang et al. 1967).

ACM technology utilizes polymers for cellular microen-capsulation, and forms a range of microcapsules with various measures of thickness and size (Kittel et al. 2013, Whelehan and Marison 2011). In previously published work, we dem-onstrated the potential applications of bile acids, either alone or combined with other agents, in diabetes therapy (Al-Salami et al. 2007, 2008, 2009, 2012, Calasan et al. 2012, Lalic-Popovic et al. 2013a, 2013b, Mikov et al. 2008, 2012). In a recent study, we developed a microcapsule-based platform

Correspondence: Dr Hani Al-Salami, Senior Lecturer of Pharmaceutics, School of Pharmacy, Curtin University, GPO Box U1987 Perth WA, Australia 6845. Tel: � 61 8 9266 9816. Fax: � 61 8 9266 2769. E-mail: hani.al-salami@curtin.edu.au.

(Received 13 September 2014 ; accepted 29 September 2014 )

Abstract This study utilized the Seahorse Analyzer to examine the eff ect of the bile acid ursodeoxycholic acid (UDCA), on the morphology, swelling, stability, and size of novel microencapsulated β -cells, in real-time. UDCA was conjugated with fl uorescent compounds, and its partitioning within the microcapsules was examined using confocal microscopy. UDCA produced microcapsules with good morphology, better mechanical strength ( p � 0.01), and reduced swelling properties ( p � 0.01), but lower cell viability ( p � 0.05) and cell count per microcapsule ( p � 0.01). UDCA reduced the cells ’ biochemical activities, mitochondrial respiration, and energy production, post-microencapsulation. This is the fi rst time biological functions of microencapsulated β -cells have been analyzed in real-time.

Keywords: bile acid , microencapsulation , pancreatic beta cells , Seahorse analyzer

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for β -cells, and we used bile acids as membrane-stabilizing excipients (Mooranian et al. 2014a). However, cell viability remained limited, and thus, in this study, we aimed to incor-porate a polyelectrolyte mixture into our microcapsules. Th is mixture comprises ultrasonic gel, poly-l-ornithine, poly-allylamine, and poly-(4styrene)-sulfonate, and will be added into the polymer, sodium alginate. We anticipate the addition of the polyelectrolyte mixture to improve the physi-cal characteristics of the microcapsules, which may result in better cell survival, post-microencapsulation. We also aim to examine the impact of integrating ursodeoxycholic acid (a bile acid with anti-infl ammatory eff ects) (Martinez-Moya et al. 2013) into the microcapsules, and study the microcap-sules ’ structure, morphology, size, mechanical strength, and stability, as well as cell viability and distribution within the microcapsules, in vitro . We have also examined the parti-tioning of the bile acids within the microcapsules through its conjugation with fl uorescent compounds, followed with studies using spinning disk confocal scanning.

Materials and methods

Cell culture As described previously (Mooranian et al. 2014a), BRIN-BD11 cells were cultured in T-75 cm 2 tissue culture fl asks (Th ermo Fisher Scientifi c ® , Australia) and fed with RPMI 1640 media (Gibco, Life Technologies, USA) supplemented with 5.5 mmol glucose (Sigma Chemical Co, USA), 10% fetal bovine serum (Th ermo Fisher Scientifi c, Australia), and 5% penicillin-streptomycin (Th ermo Fisher Scientifi c, Austra-lia). Th e BRIN-BD11 cells were incubated in an environment of 5% CO 2 in humidifi ed air at 37 ° C, using a NuAire NU-8500 Water Jacket CO 2 Incubator (NuAire, USA). Th e trypsinized cells were added to an equivalent volume of freshly prepared media and centrifuged at 1500 rpm for 5 min at 20 ° C, using a Beckman Coulter Allegra X-12 centrifuge (Beckman Coulter, USA). Th e cells were resuspended in fresh media, ready for microencapsulation under sterile conditions.

Formulation preparation Sodium alginate (SA, � 99%), sterile poly-l-ornithine hydro-chloride (PLO), and ursodeoxycholic acid (UDCA, 99%) were all purchased from Sigma Chemical Co, USA. Calcium chloride dihydrate (CaCl 2 .2H 2 0, 98%) was obtained from Scharlab S.L, Australia. Sterilized poly-(4styrene)-sulfonate (PSS) and poly-allylamine hydrochloride (PAA) were both purchased from Sigma Chemical Co, USA. Ultrasonic gel was purchased from Australian Medical Association (Perth, Australia). Stock solutions were prepared using UltraPure ™ distilled water (Life Technologies, USA), see Table I.

All solutions were mixed well using sterilized laboratory equipment in a Gelaire BH-EN Class II biological safety cabi-net (Gelaire Company, Australia), with independently certi-fi ed sterile working conditions, in a physical containment level 2 laboratory.

Sterilization protocol All reagents were sterile upon purchase, endotoxin-tested, and suitable for cell culture research. All subsequent solutions

and formulations made in our laboratory were sterilized by fi ltration (0.22 μ m) before use.

Microencapsulation by vibrating-jet fl ow method (VJFM) Microencapsulation was carried out using a B ü chi micro-encapsulation system (B Ü CHI Labortechnik, Switzerland) developed in our laboratory (Mooranian et al. 2014b, Negrulj et al. 2013). Th e droplets formed via the B ü chi based vibra-tional-jet fl ow technology were collected in a 2% w/v CaCl 2 hardening bath (Mooranian et al. 2014b, Negrulj et al. 2013). Post-microencapsulation, the microcapsules were stored in T-25 cm 2 tissue culture fl asks (Th ermo Fisher Scientifi c, Australia) containing 5 ml of media, and incubated under sterile conditions (37 ° C with 5% CO 2 in the incubator) for 24 h prior to commencement of experiments.

Characterization of loaded microcapsules Optical microscopy (OM) A fl uorescent microscope (Olympus IX-51 inverted fl uores-cent microscope, Japan) was used to view the samples.

Scanning electron microscopy (SEM) and energy dispersive X-ray (EDXR) spectroscopy MIRA3 FE SEM (Tescan, Czech Republic) with 2.5 nm cali-brated resolution and an accelerating voltage of 3 kV was used to examine the microcapsules ’ surface morphology. EDXR (Oxford Instruments, INCA X-Act, USA) was used to examine the microcapsules ’ surface, as described in our previous studies (Mooranian et al. 2014a).

Spinning disk confocal scanning microscopy of encapsulated islet cells Encapsulated islet cells were stained with the CellTrace ™ carboxyfl uorescein succinimidyl ester (CFSE) Cell Prolif-eration Kit (Life Technologies, USA), post-trypsinization. Confocal scanning was carried out using an UltraVIEW Vox spinning disk confocal microscope (Perkin Elmer, USA) equipped with a Yokogawa CSU-X1 confocal scanning unit (Perkin Elmer, USA) and Hamamatsu ORCA-R2 camera (Hamamatsu Cooperation, Japan) with a 488 nm laser. To ensure optimal viability of the encapsulated islet cells and utmost sterility during imaging, microcapsules were placed

Table I. Th e chemical constituents of the two formulations used to microencapsulate BRIN BD11 pancreatic β - cells.

Formulation type Reagents Proportions (%) Hardening bath

Formula 1 (F1) – Control

SA 1.0 2% w/v Calcium chlorideUSG 4.5

PLO 1PSS 0.7PAA H20

2.3 Up to 100%

Formula 2 (F2) – Test

SA 1.0 2% w/v Calcium chlorideUDCA 1

USG 4.5PLO 1PSS 0.7PAA H20

2.3 Up to 100%

UDCA, ursodeoxycholic acid; SA, sodium alginate; USG, ultrasound gel, PLO, poly-l-ornithine; PSS, polystyrene sulfonate; PAA, polyallylamine.

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Advanced bile acid-based multi-compartmental microencapsulated pancreatic β -cells 3

within a fully incubated environmental control unit (fully enclosed microscope and stage unit) enriched with CO 2 (5%) and temperature controlled (37 ° C). Image analysis was undertaken using the Velocity Multi-Platform 3D Cellular Imaging and Analysis Software (Perkin Elmer, USA) (Moora-nian et al. 2014a).

Spinning disk confocal scanning microscopy of TRITC-UDCA chemical conjugate To determine the partitioning of the bile acid UDCA within all the layers of the microcapsule, a conjugation reaction using a fl uorescent compound (tetramethylrhodamine isothiocyanate; TRITC) was performed, based on the work of Sherman and Fisher (Sherman and Fisher 1986). Th e microcapsules were then analyzed for their morphology and bile acid distribution/partitioning within the microcapsule matrix, using an UltraVIEW Vox spinning disk confocal microscope (Perkin Elmer, USA) equipped with a 532 nm laser. As no islet cells were stained and no other substance displayed any fl uorescent activity, fl uorescent visualization of the microcapsules at 532 nm was determined to be spe-cifi c to the TRITC-UDCA conjugate.

Microencapsulated cell count and the microencapsulation effi ciency To determine the total number of live cells used for micro-encapsulation following re-suspension post-centrifugation, a 40 μ l aliquot was removed and placed in a 65 μ l Eppendorf tube (Eppendorf South Pacifi c, Australia) to which 10 μ l of 0.4% trypan blue solution was added (Sigma Chemical CO, Australia). Th e mixture was mixed well, and two lots of 10 μ l were removed from the mixture and each placed on opposite sides of a Countess cell counter chamber slide (Invitrogen, Korea), and the slide was then placed into the slide port of a Countess Automated Cell Counter (Invitrogen, Korea) for analysis, as described in our previous studies (Mooranian et al. 2014a).

At the end of this protocol, no β -cells remained attached to the surface of the fl ask, or remained within the microcap-sule core, confi rming that all cells were gradually released from the microcapsules. Th e microencapsulation effi ciency was calculated according to the following equation:

Microencapsulation effi ciency �

Live cell count per microcapsule

� 100 (1)Total live cell content microencapsulated

MTT assays (cell mitochondrial activity assessment) A validated method was used, based on the work of Uludag and Sefton, with slight modifi cations (Uludag and Sefton 1990, 1992, Uludag et al. 1994). MTT was prepared as a 5 mg/ml stock solution (Sigma Chemical CO, USA) in phosphate buff er saline of pH 7.4 (Th ermo Fisher Scientifi c, Australia), and the undissolved residues were removed by sterile fi ltra-tion. Th e stock solution was stored in a sterile environment at 4 ° C in the dark, and used within 7 days of preparation. Th e MTT assay was carried out as described previously (Moora-nian et al. 2014a).

Microencapsulated β -cell respiration and metabolic activity (glycolysis) Evaluation of mitochondrial activities of cells within the microcapsules, including oxygen consumption rate (OCR), extracellular acidifi cation rate (ECAR), and proton pro-duction rate (PPR) were undertaken in real-time, using a method developed in-house, using a Seahorse Flux Analyzer XF 96 (Seahorse Bioscience, USA). Various other parameters were also measured: Basal Respiration (BR), ATP production (ATPP), Maximum Respiration (MR), Coupling Effi ciency (CE), Non-Mitochondria-OCR (NM-OCR), and Glycolysis (G).

Th e instrument measures mitochondrial biomarkers by sensing changes in oxygen and proton (pH) content via a fl uorescent biosensor (Nicholls et al. 2010, Gerencser et al. 2009). Measurements of key mitochondrial activities are done in a non-invasive manner, such that the microcap-sule structure is not damaged during the process, and cell viability is measured before and after the run, to ensure accurate results that are based on the biological activi-ties of viable cells (Wu et al. 2007). Th e experiments were designed to determine microencapsulated cellular respira-tion at a glucose concentration of 2.5 mM (Malmgren et al. 2009). For an analysis of mitochondrial respiration and activity to take place, a series of injections (20 min apart) from a multichannel injector port system were required (Gerencser et al. 2009, Nicholls et al. 2010). Th e fi rst injec-tion was of media (no glucose), which served to equilibrate experimental conditions. Th e second injection was of the ATP coupler oligomycin (2 μ M) that inhibits ATP synthesis (Malmgren et al. 2009, Brand and Nicholls 2011, Wikstrom et al. 2012). Th is allows the determination of the percent-age of oxygen consumption devoted to ATP synthesis, and the proportion of oxygen needed to overcome the proton leak across the mitochondrial membrane (Wu et al. 2007, Dranka et al. 2010, Wikstrom et al. 2012). Prior to running the Seahorse XF stress-testing assays, a fi nal cell count was undertaken in order to measure total viable cell count used. Th e microencapsulated β -cells of Formula 1 and Formula 2 were prepared and treated in exactly the same way as per our in-house method (Wu et al. 2007). All microcapsules were examined visually after the end of mitochondrial stress-testing assays, to determine what morphological impact the procedure had on the microcapsules and their encapsulated cellular contents. Data were presented graph-ically over the duration of the assays (Wikstrom et al. 2012, Malmgren et al. 2009).

Zeta-potential and size analysis To determine the electrokinetic stability and uniformity in the size of the microcapsules in the dispersion system, the zeta potential and size distribution for the microen-capsulated formulations were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), and by the Mie and Fraunhofer scattering technique using a Master-sizer 2000 (Malvern Instruments, Malvern, UK), as described in our previous studies (Mooranian et al. 2014a, 2014b). All determinations were performed in triplicate. Results are reported as mean � SD.

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Scanning electron microscopy (SEM) Figure 1 shows SEM surface images of F1 and F2 β -cell microcapsules respectively. F1 and F2 microcapsules were spherically shaped, with similar sizes. F1 microcapsules (Figure 1b) exhibit an uneven surface, which is rough and abundant with ridges, compared with F2 microcapsules (Figure 1f ). Th is is in line with our previous studies that showed membrane-stabilizing eff ects of bile acids on the microcapsules (Takka and Cali 2012).

Energy dispersive X-ray spectroscopy (EDXR) analysis EDXR analysis of β -cells containing F1 and F2 microcap-sule surfaces revealed characteristic elemental composi-tions expected for microcapsules, formed via ionic gelation chemical reactions in the hardening bath (Negrulj et al. 2013, Steele et al. 2013). Specifi cally, prominent elemental atoms Ca, O, and C were abundant on the chemical spectra without interference. Th ese atoms are representative of the calcium alginate membrane (Figure 1c) and the bile acid UDCA (Figure 1g) (Negrulj et al. 2013, Kuhajda et al. 2006). Atoms unique to the polyelectrolytes PAA, PSS, and PLO (such as N and S), were not visible on the membrane surface, suggesting that they are in the inner core. Th is is in line with our previously published work (Mooranian et al. 2014a).

Spinning disk confocal scanning microscopy of encapsulated islet cells Figure 1d shows confocal microscopic images of F1-stained cells, while Figure 1h shows confocal microscopic images of F2-stained cells. Th ere is no obvious diff erence between viable cell distribution within the microcapsules of F1 and F2 formulations, suggesting that UDCA did not aff ect viable cell distribution within the microcapsules.

Spinning disk confocal scanning microscopy of TRITC-UDCA chemical conjugate Figure 1i shows confocal microscopic images of F2 fl uores-cent conjugated-UDCA microcapsules. UDCA is clearly par-titioned throughout all parts of the microcapsules including the surface, and this was supported by the EDXR analysis (Figure 1g).

Microencapsulation effi ciency and cell count Table II shows lower viable cell count per microcapsule in the presence of UDCA ( p � 0.01), while the effi ciency of the microencapsulation process remains similar. Th is suggests that UDCA either reduced cell proliferation or enhanced cell death, post-microencapsulation. A possible explanation is that the presence of UDCA resulted in less porous micro-capsules, as shown in Figure 2a, which brought about less oxygen and nutrient permeation through the membrane, and eventual cell hypoxia and death.

Microcapsule size analysis and zeta potential measurements Table III shows that UDCA did not adversely aff ect the size of β -cell-containing microcapsules. However, UDCA reduced the values of Zeta potential ( p � 0.05) of the dispersion sys-tem, possibly due to its ionic interaction with the alginate

Swelling studies To determine the swelling properties of the microcapsules, 50 mg of dry microcapsules were weighed and placed in 20 ml of phosphate buffer pH 7.4, at a temperature of 37 ° C, for 6 h, and measurements were carried out as per previously published work (Mooranian et al. 2014a, 2014b). All experiments were done in triplicate. The swelling index of the microcapsules was calculated from the following formula (Pal and Nayak 2012, Awasthi and Kulkarni 2013):

Swelling Index (%) � Final weight

(2)Initial weight

Mechanical resistance In order to test the mechanical stability of the microcapsules, mechanical resistance testing was carried out using a Boeco Multishaker PSU 20 (Boeco Company, Germany). Vials containing 20 microcapsules in 20 ml of phosphate buff er (pH 7.4) were placed in a shaker and vibrated/agitated back and forth, at a frequency of 150 rpm for a period of 24 h. At various time intervals, the number of fractured or damaged microcapsules was counted. Th e mechanical strength of the microcapsules was then calculated using the following equation (Jiin 2000):

Mechanical Strength Index (%) �

Number of Intact Microcapsules Remaining in the Vial � 1 (3)

Total Number of Microcapsules Loaded in the Vial

Stability studies Th e 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 stabil-ity chambers) at � 20 ° C, 5 ° C, 25 ° C, and 40 ° C, with relative humidity set at 35%, for 3 days (72 h). Th e experiment was conducted using a stability chamber (Angelantoni Environ-mental and Climatic Test Chamber, Italy) (Mooranian et al. 2014b).

Statistical analysis Th e results were expressed as mean � standard deviation, n � 3. For statistical analysis, two-way ANOVA with Tukey post-hoc analysis was used, setting the level of signifi cance at p � 0.05. All the statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc., USA). Th e results for the p value were only reported where signifi -cance was noted.

Results and discussion

Optical microscopy Figure 1a shows β -cell microcapsules without UDCA (F1, control) while Figure 1e shows β -cell microcapsules with UDCA (F2, test). Th ere is no obvious diff erence in shape or size. Th ere are small agglomerations visible, which we believe to be β -cell populations.

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Advanced bile acid-based multi-compartmental microencapsulated pancreatic β -cells 5

system, which is in line with our previous published work (Mooranian et al. 2014a, 2014b).

MTT and Seahorse mitochondrial respiration and metabolic activity analyses A calorimetric assay of cellular viability for β -cells within intact microcapsules was investigated using an MTT assay (Figure 2a). Th is was complemented by a real-time analy-sis of cellular microenergetics, mitochondrial activity, and cellular respiration (Figure 2b – m) using the Seahorse analyzer. To the best of our knowledge, this is the fi rst time these parameters have been measured, in real-time, for microencapsulated β -cells. Th e results were consistent and

Figure 1. Micrographs of F1 and F2 microcapsules. a: Optical image of F1, b: SEM image of F1, c: Surface sites with corresponding EDXR spectral analysis of F1, d: Confocal images of stained cells of F1 microcapsules, e: Optical image of F2, f: SEM image of F2, g: Surface sites with corresponding EDXR spectral analysis of F2, h: Confocal images of stained cells of F2, and i: Confocal images of fl uorescent UDCA images of F2 microcapsules. a and e: Scale 40 � . d and h: Scale 10 �.

Table II. Microencapsulated live cell count and the microencapsulation effi ciencies for both formulations (F1 and F2).

Formulation Average live cell count

per microcapsule Effi ciency of viable b -cell microencapsulation (%)

F1 185 � 7 23 � 2 F2 105 � 8 * * 20 � 3

Results are mean � SD, n � 3, * * p � 0.01%.

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2009). Th is suggests lower BR and less oxidation of pyruvate to produce NADH, which in turn generates a proton (H � ) gradient. Th e gradient is utilized by ATP synthase to gener-ate ATP (ATPP) from ADP and facilitate less β -cell activity (Wu et al. 2007). For pancreatic β -cells, the ATP:ADP ratio is of high importance as it triggers glucose-stimulated insulin secretion (Malmgren et al. 2009). However, insulin was not measured during this assay, which is a limitation to our fi nd-ings. Glycolysis stress-testing revealed that UDCA resulted in less ECAR and PPR, possibly due to reduced cellular energy production via the glycolysis pathway, and resulting in a subsequent decrease in MR and CE. Th is also resulted in a reduction in the NM-OCR and G activity of the cells, which complements the MTT assay results (Figure 2a).

Previously published data have shown that bile acids enhance β -cell viability via their antioxidant eff ects (Negrulj et al. 2013, Perez and Briz 2009). Th is is contrary to our fi ndings. However, our fi ndings were obtained using micro-encapsulated cells rather than free ones. Th is suggests that diff erent formulations of bile acids will have diff erent eff ects on cell viability and biological functionality, post-microencapsulation.

Swelling and mechanical strength Studies on swelling and mechanical strength were carried out in order to confi rm that UDCA signifi cantly increased the strength of the microcapsules. Figure 3a shows that UDCA

Figure 2 shows that UDCA signifi cantly reduced cell viabil-ity, post-microencapsulation ( p � 0.05). It also shows that UDCA signifi cantly reduced β -cells ’ OCR ( p � 0.05), ECAR ( p � 0.05), PPR ( p � 0.05), NM-OCR ( p � 0.05), BR ( p � 0.05), MR ( p � 0.01), NM-OCR ( p � 0.01), ATPP ( p � 0.01), CE ( p � 0.05), and cellular glycolysis ( p � 0.01). Th is suggests a strong and consistent negative impact of UDCA on the β -cells ’ biological activity and mitochondrial respiration, 48 h post-microencapsulation. Th is may be explained by UDCA causing a signifi cant reduction in the porosity of the micro-capsule membrane, and thereby hindering oxygen/nutrient exchange and resulting in β -cell hypoxia and apoptosis.

Figure 2 shows that UDCA caused a signifi cant decrease in OCR, suggesting reduced mitochondrial respiration (Wik-strom et al. 2012, Dranka et al. 2010). Th is indicates that there are fewer O 2 molecules serving as electron acceptors and facilitating activity within the electron transport chain, with a proportional decrease in oxidative phosphorylation (Brand and Nicholls 2011, Malmgren et al. 2009, Gerencser et al.

Figure 2. Microencapsulated β -cell viability within intact microcapsules without (control) and with (test) UDCA. Pre-MC: pre-microencapsulation β -cells, F1 (without UDCA) and F2 (with UDCA) are empty microcapsules, F1: β -cells microcapsules (without UDCA) and F2: β -cells microcapsules (with UDCA). Values are mean � SD and n � 3.

Table III. Microcapsule size and Zeta potential of F1 and F2 formulations.

Formulation Average microcapsule size ( μ m)

Average zeta potential (mV)

F1 936 � 5 � 31 � 2 F2 940 � 8 � 38 � 3 *

* p � 0.05.

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Advanced bile acid-based multi-compartmental microencapsulated pancreatic β -cells 7

acknowledge the use of equipment, scientifi c and techni-cal assistance of the Curtin University Electron Microscope Facility, which has been partially funded by the University, State, and Commonwealth Governments. Th e authors also acknowledge the Pharmaceutical Technology Laboratory for their valuable assistance (Curtin School of Pharmacy).

Declaration of interest

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

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signifi cantly reduced the swelling properties of the β -cell microcapsules ( p � 0.01), and also signifi cantly enhanced the mechanical strength of these microcapsules when exposed to vibrational stress for 24 h ( p � 0.01).

Th e swelling and mechanical strength data support the SEM data, showing reduced porosity of the microcapsules as a result of UDCA addition (Figure 3). Th e even distribution of UDCA throughout all layers of the microcapsules resulted not only in a stronger microcapsule, but also reduced viable cell count (Table II), while maintaining the same microcap-sule size (Table III). Th us, the combination of UDCA with the polyelectrolytes (USG, PAA, and PSS) resulted in microcap-sules that are too strong to maintain cell viability.

Stability studies At a fi xed humidity of 35%, the change in temperature exerted a signifi cant eff ect on the morphology, appearance and size of the microcapsules. At � 20 ° C and 5 ° C, both types of microcapsules (control and test) maintained their original shape, morphology, color (pale-ivory), and texture (soft and fl exible). At 25 ° C and 40 ° C, both microcapsules experienced a signifi cant reduction in size (30%), and a change in color (yellow-orange) and texture (hard and brittle). Th is sug-gests that UDCA does not have an eff ect on the size, mor-phology, or color of the microcapsules, especially at higher temperature.

Conclusion

Th e addition of UDCA and the polyelectrolyte mixture resulted in stronger β -cell microcapsules, but cell survival and biological functions were compromised. Th us, future studies will explore diff erent polyelectrolyte-bile acid for-mulations in order to obtain microcapsules with good physi-cal properties as well as better cell viability and long-term survival, which may prove to be a viable delivery platform for β -cells for the treatment of diabetes.

Acknowledgments

Th e authors acknowledge the CHIRI at Curtin University, and the Curtin-seeding grant for the support, and also

Figure 3. Swelling characteristics of β -cell-containing microencapsulated formulas F1 and F2 (a) and mechanical strength testing of β -cell-containing microcapsules F1 and F2.

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