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Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid exerted no hypoglycaemic effects
Accepted Manuscript Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid exerted no hypoglycaemic effects
Sangeetha Mathavan, Momir Mikov, Svetlana Golocorbin-Kon, Hani Al-Salami PII: DOI: Reference:
S0928-0987(17)30269-5 doi: 10.1016/j.ejps.2017.05.041 PHASCI 4055
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
3 March 2017 11 May 2017 17 May 2017
Please cite this article as: Sangeetha Mathavan, Momir Mikov, Svetlana Golocorbin-Kon, Hani Al-Salami , Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid exerted no hypoglycaemic effects, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.05.041
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Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid
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exerted no hypoglycaemic effects
Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin
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a
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Sangeetha Mathavana, Momir Mikovb, Svetlana Golocorbin-Konc, Hani Al-Salamia *
Health Innovation Research Institute, Curtin University, Perth WA, Australia Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of
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Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
Dr Hani Al-Salami
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*Corresponding author
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c
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Medicine, University of Novi Sad, Novi Sad, Serbia
Senior Lecturer of Pharmaceutics, School of Pharmacy
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Curtin University, GPO Box U1987 Perth, Western Australia 6845, Australia. |
+ 61 8 9266 9816
Fax
|
+ 61 8 9266 2769
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Tel
Email | [email protected] Profile | http://healthsciences.curtin.edu.au/teaching/pharmacy_people.cfm/Hani.Al-Salami
Short Title: Microencapsulated taurocholic acid in Type 1 diabetes
ACCEPTED MANUSCRIPT 2 Abstract Context: The bile acid taurocholic acid (TCA) is endogenously produced, and has shown formulationstabilising effects when incorporated into microcapsules containing potential antidiabetic drugs.
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This study aimed to develop and characterise TCA-microcapsules, and test their antidiabetic effects, in an animal model of Type 1 diabetes (T1D).
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Methods:
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Using the polymer sodium alginate (SA), SA-microcapsules (control) and TCA-microcapsules
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(test) were prepared, and assessed for morphology, surface composition, chemical and thermal stability, swelling, buoyancy, mechanical, release and rheological properties. TCA-microcapsules
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were gavaged as a single dose (1.2mg/300g) to alloxan-induced diabetic rats, and blood glucose and TCA concentrations in serum, tissues (ileum, liver and pancreas) and faeces, were measured.
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One healthy and one diabetic group were used as control and gavaged SA-microcapsules.
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Results:
TCA-microcapsules showed consistent size, TCA presence on surface and all layers of
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microcapsules, chemical and thermal stability, enhanced swelling, buoyancy and targeted-release properties and rheological analysis showed Non-Newtonian flow properties. TCA serum
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concentrations were lower in the healthy group, compared with the diabetic and diabetic-treated groups, but there was no significant difference between diabetic control and diabetic treated groups, in terms of TCA levels, and blood glucose concentrations. Conclusions: The developed TCA-microcapsules showed good stability and release properties, but did not lower blood glucose levels in T1D, which suggests absence of insulin-mimetic effects, when using a single 1.2 mg/rat oral dose.
ACCEPTED MANUSCRIPT 3 Keywords: Taurocholic acid, bile acids, microencapsulation, diabetes mellitus, inflammation, Type 1 diabetes Abbreviations: Taurocholic acid
T1D
Type 1 diabetes
SA
Sodium alginate
TCA-SA
Taurocholic acid-sodium alginate
CaCl2
Calcium chloride
OM
Optical microscopy
SEM
Scanning electron microscopy
EDXR
Energy dispersive X-ray spectroscopy
DSC
Differential scanning calorimetry
FTIR
Fourier transform infrared spectroscopy
USP
United States Pharmacopoeia
UV-Vis
Ultraviolet-Visible spectroscopy
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LC-MS
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TCA
Liquid chromatography-mass spectroscopy
C18 column
Octadecyl carbon chain (C18) bonded silica
ABC-protein
ATP-binding cassette transporter protein
ANOVA
Analysis of variance
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Introduction
Taurocholic acid (TCA) is a dominant conjugate form of bile acids in humans. It is chemically
known
as
2-(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholn-24-yl,amino,
ethanesulfonic acid, and has a molecular formula of C26H45NO7S and molecular weight of 515.70g/mol. TCA is a tri-hydroxy bile acid that has an OH group at the C-3, C-7 and C-12
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positions. TCA is also known as cholaic acid, and it is a taurine conjugate of cholic acid,
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and constitutes more than 30% of bile acid pool and extensively metabolised in the small
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intestine by gut microflora. It is hydrophilic in nature and has a melting point of 125°C (1). Being an acid, TCA has a pKa value of 1.4 and is partially soluble in water but is soluble
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in alcohol and ether (2).
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Although TCA is not a globally registered drug with clinical indications to treat a disease, it has shown potential applications in inflammatory disease models. It has shown
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substantial antiinflammatory and immunoregulatory effects in healthy mice treated with
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proinflammatory endotoxins (3). It has also shown permeation-enhancing capabilities, when combined with other drugs. In recent studies, TCA enhanced the permeation of
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heparin-docetaxel conjugates, in the lower intestine via transcellular and/or paracellular pathways (4, 5). Moreover, in one study, TCA exerted protective and desirable effects on
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muscle and pancreatic β-cells, which suggests potential antidiabetic effects (6). Since TCA metabolism starts in the duodenum, it needs to be encapsulated in targeted-delivery matrices, which target the lower part of the intestine, and maximise its absorption (7).
Accordingly, this study aimed to develop, characterise and test antidiabetic effects of new microcapsules with oral targeted-delivery properties, which can deliver TCA to the lower part of the ileum for maximum absorption. The study also aimed at investigating changes
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of TCA concentrations in serum, tissues and faeces, associated with diabetes development and TCA-microcapsules treatment. Thus, in this study, sodium alginate (SA) was used as a polymer, to form microcapsules without (control) and with TCA (test). The microcapsules were characterised in vitro, then gavaged as a single dose to one diabetic group. Two other groups (one healthy and one diabetic) were gavaged SA-microcapsules and used as
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control. Blood, tissues and faeces were collected 10 hours postdose for blood glucose and
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TCA measurements. The in-vitro studies include imaging and morphological studies, surface elemental-analysis, stability, rheological properties, swelling, mechanical strength, and buoyancy
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analysis and release profiles at various temperature and pH values. The in-vivo studies include
2.1
Materials
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Materials and methods
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blood and tissues concentrations measurements of glucose and the bile acid, TCA.
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Taurocholic acid (96.0%) and alloxan (>98%) were purchased from Sigma Chemical Co, USA. Sodium alginate was obtained as low viscosity, from Acros Organics, USA.
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Anhydrous calcium chloride (CaCl2) was purchased from Scharlab S.L, Australia. Ultrasonic gel was obtained from Australian Medical Association, Australia. All other
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solvents and reagents used in this study were supplied by Merck, NSW, Australia and were of analytical grade. 2.2
Drug stock preparation
The stock suspension of TCA (4mg/ml) was prepared by solubilising the crystalline powder of TCA with the ultra water-soluble gel (10%). The calcium chloride stock solution (10% w/v) was prepared by dissolving the salt to deionized water. Alloxan was
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mixed with saline prior to injection. All preparations were mixed for 3 hours at room temperature to ensure uniform mixing and were used within 24 hours of preparation. 2.3
Formation of microcapsules
The microencapsulation of TCA was carried out by combining a mixture of SA and TCA
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1.8% and 4 mg/ml and dispersing in deionized water (prior to use by microencapsulation
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process) using our well-established microencapsulating methodologies (6, 8, 9).
Microencapsulation efficiency, analyte content, production yield and
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microcapsules characterisations
0.1 g of microcapsules were weighed, and dissolved in 200 ml of phosphate buffer (pH 7.8) and the suspension was stirred and aliquots of the dissolution medium (2 ml) were
Spectroscopic
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withdrawn and TCA measured using established Liquid Chromatography Mass methods
(10).
The
analyte
contents,
production
yield
and
microencapsulation efficiency were calculated using our established methods, as described
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elsewhere (11-14) .
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The characterisation studies were done using Optical microscopy (OM)- Nikon YS2-H, Japan; Scanning electron microscopy (SEM)- Zeiss Neon 40EsB FIBSEM (Cambridge, MA) and Energy dispersive X-ray spectra (EDXR)- Oxford Instruments, INCA X-Act, Concord, MA), as per our established methods. Briefly, OM was used to evaluate the shape of the formulated microcapsules, while SEM and EDXR were used to investigate the surface morphological features of the microcapsules and elemental distribution of the atoms distributed across the surface of the microcapsules. The analysed microcapsule
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samples were coated with 5nm platinum under vacuum. The mean diameter of the particle size was calculated using the software associated with the instrument (15-17).
2.5
Thermal, chemical and accelerated stability analyses and mechanical strength
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and swelling properties measurements
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Thermal analytical studies were carried out using the Differential Scanning Calorimeter
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(DSC-8000 Perkin Elmer Inc., Waltham, MA, USA). A dried sample weight equivalent to 5mg of the microcapsules was utilized for examining the melting behaviour using the
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sealed aluminium pans, while, Fourier transform infrared spectroscopic analysis (FTIR spectrometer TWO, Perkin Elmer Inc., Waltham, MA) was used in the analysis of the
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functional groups present on the microcapsules using a frequency range of 450 to 4000cm-1 (18).
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The microencapsulating formulation and the microcapsules’ physical-stability, and membrane integrity were assessed at different pH and temperature values. Microcapsules’ physical stability was assessed by placing 1 gram of microcapsules onto sterile petri dishes
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and storing that in thermostatically controlled ovens (accelerated-stability chambers,
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Angelantoni Environmental and Climatic Test Chamber, Italy) at different temperature ranges of -15°C, 5°C, 25°C and 40°C with relative humidity set at 35% for three days. The colour, texture and size of microcapsules were visually assessed for changes in appearance. In order to assess mechanical resistance and strength, microcapsules were placed in a Boeco Multishaker (Boeco Company, Germany) in 20 ml of phosphate buffer and agitated for 2 days, and the number of damaged microcapsules was recorded and mathematically assessed, using our well-established methods (12, 13).
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In order to assess microcapsules’ membrane strength and integrity, their swelling properties were assessed, at four pH values (1.5, 3.0, 6.0 and 7.8) and at two temperatures (25°C and 37°C) for 4 hours, as per our established methods (19).
Rheological properties, buoyancy and dissolution release studies
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Basic rheological measurements were analysed using Visco-88 viscometry (Bohlin- Visco
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88, ATA Scientific Pty. Ltd., NSW, Australia), where 20 ml of each sample were placed in the instrument-cap and various measurements taken by altering rotation-speed and
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recording parameters, as appropriate. Buoyancy testing was carried out by placing 200 microcapsules in 100 ml solution of phosphate buffer. The buffer was stirred by rotating
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paddles at 50 rpm for 4 hours (USP dissolution apparatus 24, type II) at a temperature of 37°C ± 0.2, which was regulated by a thermostat. Every hour, the number of floating
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microcapsules was counted and buoyancy index was calculated using the ratio of the
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number of floating microcapsules to that of the total number of microcapsules.
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Analyte dissolution release testing was carried out using 0.3g of freshly prepared microcapsules, placed in 500 ml of phosphate buffer at pH values of 1.5, 3.0, 6.0 and 7.8
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over 6 hrs period at two different temperatures of 25°C and 37°C. The dissolution medium buffer was stirred at 200 rpm at a flow rate of 30ml/min. The test was performed using a closed loop flow system developed for microcapsule drug release testing via UV-Visible spectrophotometric
analysis
using
a
Perkin
Elmer
LAMBDA
spectrophotometer (Perkin Elmer, Waltham, Massachusetts, USA) (16, 19).
25
UV-Vis
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2.7
Diabetic induction and bile acid analysis
Male Wistar rats (250 ± 45g) were maintained in an animal facility and given free access to water and food, and temperature and light were controlled to mimic their natural habitat. The rats were randomly divided into different groups and one of the group (TCA-SA) was
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made diabetic by intravenously administering the drug alloxan at a dose of 25mg/kg/body weight (20-25). Three days following the alloxan administration, rats that showed blood
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glucose levels higher than 18 mmol/L along with signs and symptoms of diabetes were
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considered diabetic (23, 26-29). For the study design, see Figure 1. The experiments were approved by the Animal Ethics Committee at Curtin University and all experiments were
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performed according to the Australian Code of Practice for the care and use of animals for
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scientific purposes. This particular group (TCA-SA) was part of a larger study where blood glucose levels and gliclazide levels from the same control groups have been published (8).
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The tertiary bile acid, TCA was analysed by LC-MS. The method involved the use of 40µl
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BA sample acidified with ammonium acetate buffer (100µl) at pH 4.0. It was extracted twice with 800µl of diethyl ether and the final aliquot (500µl) was evaporated to dryness.
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The residue was reconstituted in the isocratic mixture of methanol: water (65:35) and 10µl was injected into the LC-MS system.
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The LC-MS system consisted of a Shimadzu LC-20AD pump attached to a Shimadzu LCMS-2020 mass detector. The analytical column was a C18 Symmetry (3.9 x 150mm, 5µm particle size) with a C18 (3.9 x 2mm) guard column. Analysis was performed using an isocratic flow with a flow rate 0.2 ml/min and under these conditions; the retention time for TCA was observed at 8.7 min, while the run time was kept to 15 min. 2.8
Statistical analysis
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All the results are expressed as mean ± standard error of mean (SEM) using triplicate samples of the same batch unless otherwise stated. All the animal experimental analysis was carried out using n=7 or 8 rats per group ± SEM. Statistical comparisons data were assessed using one way ANOVA or parametric/non-parametric t-test, as appropriate, using V6.01, GraphPad Prism Software; Inc., San Diego, CA. Values falling below the
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detectable limit of assay were recorded as zero. A probability of p<0.05 was regarded as
3.1
Results: Microencapsulation
efficiency,
analyte
content
and
production
yield
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measurements
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significant and p<0.01 as highly significant.
Drug content and total production yield of TCA-SA microcapsules was measured, and
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showed consistent TCA content per microcapsules and good production yield with high
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microencapsulation efficiency suggesting method adequacy and robustness (Figure 2 a).
Size analysis, morphological and surface composition studies
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Both microcapsules had similar and consistent size, averaging around 930 µm, in diameter. The mean particle size was not significantly affected by presence of TCA, which suggests that TCA incorporation did not compromise microcapsules size or shape. Microscopic and SEM imaging revealed opaque and uniform shape with homogenous particle size distribution and similar surface characteristic features in both microcapsules. The surface of the microcapsules appeared rough, in particular TCA-SA microcapsules, but consistent from one microcapsule to another for all analysed batches (Figures 2 b to k).
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This suggests that the addition of TCA into SA formulation did not compromise microcapsules’ formation or significantly alter the morphology of the microcapsules. The EDXR analysis of TCA-SA microcapsules showed the presence of atoms characteristics of TCA such as ‘Sulphur (S)’ atoms on the surface of the microcapsules,
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which suggests that TCA did not confine into the core of the microcapsules but is likely to be distributed throughout the different layers of the microcapsules (Figure 3 (a) and (b)).
Thermal and chemical analysis
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This may affect the release profile of TCA from the microcapsules.
Analysis of TCA powder, and SA and TCA-SA excipients pre and post
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microencapsulation revealed that the melting point of each excipient remained consistent and when excipients were physically mixed, they maintained their main peaks and thermal
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features suggesting thermal compatibility. Unlike G-SA microcapsules, which showed
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slightly higher melting points post-microencapsulation, TCA and SA melting points postmicroencapsulation were slightly lower, which may suggest ionic interaction or possible
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change in the lattice phase (Table I). This may be due to a shift in their thermal capacity within the 30-245°C range or due to interactions between all ingredients and potential mild
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loss of crystallinity (30).
The FTIR spectrum of SA (Table I) showed the characteristic peaks at 3265cm-1 corresponding to the OH stretching and two medium intensity peaks at 1592cm-1 and 1028cm-1 relating to the COOH stretching and C-O-C stretching. Similarly, TCA-SA microcapsules exhibited three major peaks, from which two strong intense peaks were observed at wavelengths of 2890cm-1 and 1661cm-1 corresponding to C-H stretching and
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conjugated aliphatic C=C and C=N respectively. In addition, another medium or low intensity peak at 1025cm-1 was observed corresponding to C=O stretching. Accordingly, FTIR analysis of SA and TCA microcapsules showed FTIR spectra similar to that of the physical mixture and suggests the absence of new chemical identity molecules formed and thus, suggests chemical compatibility and also the absence of chemical alterations due to
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Accelerated stability testing, swelling, buoyancy, dissolution-release and
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the microencapsulation process.
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mechanical strength analysis
Results from the accelerated stability studies showed that analyte contents per
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microcapsules were not significantly altered at the end of the experiment, and also no specific changes in texture, colour and shape associated with different temperatures were
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noticed. TCA maintaining its content per microcapsule suggests stability within the
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microcapsules’ matrix, while the consistent effects of different temperatures on microcapsules’ appearance and texture suggests robustness of the microencapsulating
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method and that TCA addition did not alter stability or appearance of the SAmicrocapsules. This is not in line with previous studies in our laboratory that showed
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changes in microcapsules shape and texture associated with high temperature (9, 31) and suggests variation in microcapsules’ response to high temperature, especially when microencapsulating different drugs. TCA increased the swelling index of SA-microcapsules at temperatures, 25°C (p< 0.05) and 37°C (p< 0.05) (Figure 4-a&b), enhanced the buoyancy (p< 0.05 and p< 0.01) (Figure 4-c), showed good and targeted dissolution release profile (p< 0.01) (Figure 4-d), and
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better mechanical strength (p< 0.01) (Figures 4-e). This suggests that TCA incorporation into SA-microcapsules improved physical properties of microcapsules with stable and more solid matrix and stronger membrane exhibiting improved ability to interact with gutcontents and possess pH-targeted delivery at pH>7, where gut-maximum absorption for
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bile acids is (7). This is consistent with previous studies showing bile acid membrane-stabilising effects,
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when microencapsulated in SA-based microcapsules (19). In addition, the release profile
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shows immediate TCA release at higher pH values and thus, is consistent with the observed distribution of TCA within the microcapsules’ outer membrane, as shown by the
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EDXR analysis (Figure 3).
Rheological studies
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TCA incorporation into SA-microcapsules did not significantly affect the behaviour of
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rheological parameters (viscosity, shear rate, Torque and shear stress; Table II). As shear stress increased, viscosity decreased and share rate increased while Torque increased,
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which suggests Non-Newtonian Thixotropic behaviour, regardless of addition of the bile acid, TCA. This rheological behaviour is common and desirable in pharmaceutical
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formulation and suggests that the microcapsules exhibit thermodynamic, mechanical and deformational flexibility, and are appropriate for drug delivery and TCA targeted and controlled release (32-34).
3.6
Blood glucose and bile acid studies
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Blood glucose levels were significantly elevated by diabetes induction (p< 0.01) and TCAmicrocapsules did not significantly change the elevated blood glucose levels (Figure 5-a). In addition, TCA serum concentrations were increased due to diabetes induction (p< 0.01), and TCA-microcapsules did not significantly alter TCA concentrations in either serum (Figure 5-b), ileum (Figure 5-c), pancreas (Figure 5-e) or faeces (Figure 5-f), which
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suggests significant modulation of the bile acid profile in diabetic animals, that was not Interestingly, TCA
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normalised by an acute treatment with TCA-microcapsules.
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concentrations were lower (p< 0.01) in diabetic rats treated with TCA-SA, compared with
Discussion
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healthy (Figure 5-d).
This study encompasses 1) in vitro methods designed to characterise the bile acid TCA pre
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and post microencapsulation, the shape, size, surface-elemental compositions, excipient
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thermo-chemical properties, physical and mechanical stability under various temperatures, rheological profiles under various stress conditions, buoyancy and release properties of
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TCA-microcapsules, as well as 2) in vivo methods designed to examine if oral administration of a single dose of TCA-microcapsules to T1D rats will change their blood
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glucose levels or bile acid profile.
Recent studies in other labs have demonstrated unique formulation properties and biological activities of TCA. Khatun Z. et al., showed efficacy of TCA in the oral delivery of nanoparticles as targeted-delivery systems (5), while Mooranian A. et al., showed that TCA optimised the release profile of lipophilic drugs, when incorporated into alginatebased microcapsules (35). In addition, Wang C. et al., demonstrated beneficial immune-
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regulatory effects of TCA in mice (36), while Wu T. et al., demonstrated significant haemostatic effects of TCA on glycaemic control and insulin release (37). Accordingly, in this study, TCA microcapsules were examined in-vitro and in- vivo, for anti-diabetic activities.
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Microscopic imaging and size distribution analysis showed consistent size and shape with
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small variations between different batches, regardless of TCA presence. This illustrates
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that the incorporation of the bile acid, TCA into SA-based formulations did not affect the formation of microcapsules or their shape and size. Elemental and atomic surface analysis
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of microcapsules’ outer membrane revealed that when TCA was added to the SAformulation it did not only distribute into the core and internal layers but it also distributed
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throughout all layers of the microcapsules including the surface. TCA distribution on the surface of the TCA-SA microcapsules was evident in its immediate release from the
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microcapsules at pH 7.8 (p<0.01), with significant and visible release profile. The in-vivo
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implications of TCA distribution within the microcapsules, in terms of their absorption
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profile and tissue accumulation and potential anti-diabetic effects were also studied.
Physicochemical and stability measurements of the microencapsulating formulation and
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the microcapsules revealed that analytes and excipients (SA and TCA) are thermally and chemically compatible and are stable pre and post microencapsulation. The addition of TCA into SA-based formulation did not compromise stability or alter rheological and flow properties of formulation, but rather enhanced physical strength of the microcapsules. The importance of enhanced stability of microcapsules has been examined in the literature. Neubauer MP. et al., have demonstrated applications of self-assembly methodology and rheological features and deformation, on drug contents, release patterns and morphological
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features and have shown direct relationship with shear stress and hydrogel architecture (38). In addition, Yan J. et al., (39) and Zheng G. et al., (40) have shown that covalent linking and stabilisation of microcapsules matrix can be utilised to optimise stability and thermal and chemical compatibilities and enhance morphological features of the microcapsules. This is in line with the results and suggest good potential for the in-vivo
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delivery of microencapsulated TCA-SA. This was illustrated by visible TCA absorption
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and systemic concentrations in blood, tissues and faeces. However, TCA-SA
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microcapsules did not result in direct insulin-mimetic effects and significant reduction of the elevated blood glucose of diabetic animals, independent of insulin. Nevertheless,
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diabetes induction showed significant increase in TCA levels in serum, and liver of diabetic treated animals which suggests significant alterations in bile acid haemostasis and
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feedback mechanisms and gut re-absorption (enterohepatic recirculation), due to potential alterations of changes of expression and functionality of ABC-protein transporters
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responsible for bile acid gut-uptake. This is in line with previous studies in our laboratory
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that have shown significant alterations of protein-expressions in the ileal mucosa and subsequent alterations in bile acids metabolism and gut-absorption as well as systemic
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concentrations and overall bile acid profile (25, 28).
Conclusion
The current study aimed to develop and test microcapsules with targeted-delivery properties that can deliver the bile acid, TCA, orally and test its hypoglycaemic effects in a rat model of T1D. The study examined physicochemical properties of microcapsules containing TCA and tested their morphology, stability, physical properties and release profiles in vitro. The study also examined TCA-microcapsule’s hypoglycaemic effects and
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the microcapsule’s effects on bile acid profile. Results showed good physicochemical properties and targeted-delivery characteristics of TCA-microcapsules, but showed no hypoglycaemic effects in T1D rats. Results also showed changes in TCA concentrations as a result of diabetes development. Thus, diabetes has direct effects on bile acid profile, but TCA-microcapsules failed to normalise the change in bile acid profile caused by diabetes
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development, which suggests lack of effectiveness in diabetes treatment.
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Disclosures
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The authors acknowledge that they have no declaration of interest to declare.
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Acknowledgements
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The authors acknowledge the Curtin CUPS Scholarship for the support and also would like
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to acknowledge the use of equipment, scientific and technical assistance of the Curtin University Electron Microscope Facility, which has been partially funded by the
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12. Mooranian A, Negrulj R, Chen-Tan N, Al-Sallami HS, Fang Z, Mukkur T, et al. Novel artificial cell microencapsulation of a complex gliclazide-deoxycholic bile acid formulation: a characterization study. Drug Des Devel Ther. 2014;8:1003-12.
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13. Mooranian A, Negrulj R, Chen-Tan N, Al-Sallami HS, Fang Z, Mukkur TK, et al. Microencapsulation as a novel delivery method for the potential antidiabetic drug, Probucol. Drug Des Devel Ther. 2014;8:1221-30.
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14. Mooranian A, Negrulj R, Chen-Tan N, Fakhoury M, Arfuso F, Jones F, et al. Advanced bile acid-based multi-compartmental microencapsulated pancreatic beta-cells integrating a polyelectrolyte-bile acid formulation, for diabetes treatment. Artif Cells Nanomed Biotechnol. 2014:1-8. 15. Mooranian A, Negrulj R, Chen-Tan N, Watts GF, Arfuso F, Al-Salami H. An optimized probucol microencapsulated formulation integrating a secondary bile acid (deoxycholic acid) as a permeation enhancer. Drug Des Devel Ther. 2014;8:1673-83. 16. Mooranian A, Negrulj R, Mathavan S, Martinez J, Sciarretta J, Chen-Tan N, et al. Stability and Release Kinetics of an Advanced Gliclazide-Cholic Acid Formulation: The Use of ArtificialCell Microencapsulation in Slow Release Targeted Oral Delivery of Antidiabetics. J Pharm Innov. 2014;9:150-7. 17. Mooranian A, Negrulj R, Al-Sallami HS, Fang Z, Mikov M, Golocorbin-Kon S, et al. Release and swelling studies of an innovative antidiabetic-bile acid microencapsulated formulation, as a novel targeted therapy for diabetes treatment. J Microencapsul. 2015;32(2):151-6.
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18. Mooranian A, Negrulj R, Mathavan S, Martinez J, Sciarretta J, Chen-Tan N, et al. An advanced microencapsulated system: a platform for optimized oral delivery of antidiabetic drugbile acid formulations. Pharm Dev Technol. 2015;20(6):702-9. 19. Mathavan S, Chen-Tan N, Arfuso F, Al-Salami H. The role of the bile acid chenodeoxycholic acid in the targeted oral delivery of the anti-diabetic drug gliclazide, and its applications in type 1 diabetes. Artificial cells, nanomedicine, and biotechnology. 2016;44(6):150819.
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20. Carvalho ENd, Carvalho NA, Ferreira LM. Experimental model of induction of diabetes mellitus in rats. Acta Cirurgica Brasileira. 2003;18(SPE):60-4.
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21. Korec R. Treatment of alloxan and streptozotocin diabetes in rats by intrafamiliar homo (allo) transplantation of neonatal pancreases. Endocrinol Exp. 1980;14(3):191-8.
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22. Štětinová V, Květina J, Pastera J, Polášková A, Pražáková M. Gliclazide: pharmacokinetic–pharmacodynamic relationships in rats. Biopharmaceutics & drug disposition. 2007;28(5):241-8.
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23. Al-Salami H, Butt G, Tucker I, Golocorbin-Kon S, Mikov M. Probiotics decreased the bioavailability of the bile acid analog, monoketocholic acid, when coadministered with gliclazide, in healthy but not diabetic rats. Eur J Drug Metab Pharmacokinet. 2012;37(2):99-108.
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24. Lalić-Popović M, Vasović V, Milijašević B, Goločorbin-Kon S, Al-Salami H, Mikov M. Deoxycholic acid as a modifier of the permeation of gliclazide through the blood brain barrier of a rat. Journal of Diabetes Research. 2013;2013.
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25. Al‐Salami H, Butt G, Tucker I, Skrbic R, Golocorbin‐Kon S, Mikov M. Probiotic Pre‐treatment Reduces Gliclazide Permeation (ex vivo) in Healthy Rats but Increases It in Diabetic Rats to the Level Seen in Untreated Healthy Rats. Archives of drug information. 2008;1(1):35-41. 26. Al-Salami H, Butt G, Tucker I, Fawcett PJ, Golo-Corbin-Kon S, Mikov I, et al. Gliclazide reduces MKC intestinal transport in healthy but not diabetic rats. Eur J Drug Metab Pharmacokinet. 2009;34(1):43-50.
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27. Alam MJ, Rahman MA. Changes in the saccharoid fraction in rats with alloxan-induced diabetes or injected with epinephrine. Clin Chem. 1971;17(9):915-20.
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28. Al-Salami H, Butt G, Tucker I, Mikov M. Influence of the semisynthetic bile acid (MKC) on the ileal permeation of gliclazide in healthy and diabetic rats. Pharmacological reports. 2008;60(4):532. 29. Mikov M, Al-Salami H, Golocorbin-Kon S, Skrbic R, Raskovic A, Fawcett JP. The influence of 3α, 7α-dihydroxy-12-keto-5β-cholanate on gliclazide pharmacokinetics and glucose levels in a rat model of diabetes. Eur J Drug Metab Pharmacokinet. 2008;33(3):137-42. 30. Takka S, Çali AG. Bile salt-reinforced alginate-chitosan beads. Pharm Dev Technol. 2012;17(1):23-9. 31. Mooranian A, Negrulj R, Arfuso F, Al-Salami H. Multicompartmental, multilayered probucol microcapsules for diabetes mellitus: Formulation characterization and effects on production of insulin and inflammation in a pancreatic beta-cell line. Artif Cells Nanomed Biotechnol. 2016;44(7):1642-53.
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32. Tengamnuay P, Mitra AK. Bile salt-fatty acid mixed micelles as nasal absorption promoters of peptides. II. In vivo nasal absorption of insulin in rats and effects of mixed micelles on the morphological integrity of the nasal mucosa. Pharm Res. 1990;7(4):370-5. 33. de Celis Alonso B, Rayment P, Ciampi E, Ablett S, Marciani L, Spiller RC, et al. NMR relaxometry and rheology of ionic and acid alginate gels. Carbohydrate Polymers. 2010;82(3):6639.
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34. Bahramian B, Motlagh GH, Majidi SS, Kaffashi B, Nojoumi SA, Haririan I. Evaluation of melt rheology of lactose-filled polyethylene glycol composites by means of capillary rheometery. Pharm Dev Technol. 2013;18(1):98-105.
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35. Mooranian A, Negrulj R, Arfuso F, Al-Salami H. The effect of a tertiary bile acid, taurocholic acid, on the morphology and physical characteristics of microencapsulated probucol: potential applications in diabetes: a characterization study. Drug Deliv Transl Res. 2015;5(5):51122. 36. Wang C, Li L, Guan H, Tong S, Liu M, Liu C, et al. Effects of taurocholic acid on immunoregulation in mice. Int Immunopharmacol. 2013;15(2):217-22.
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37. Wu T, Bound MJ, Standfield SD, Gedulin B, Jones KL, Horowitz M, et al. Effects of rectal administration of taurocholic acid on glucagon-like peptide-1 and peptide YY secretion in healthy humans. Diabetes Obes Metab. 2013;15(5):474-7. 38. Neubauer MP, Poehlmann M, Fery A. Microcapsule mechanics: from stability to function. Adv Colloid Interface Sci. 2014;207:65-80.
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39. Yang J, Gao C. Fabrication of diverse microcapsule arrays of high density and good stability. Macromol Rapid Commun. 2010;31(12):1065-70.
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40. Zheng G, Liu X, Wang X, Chen L, Xie H, Wang F, et al. Improving stability and biocompatibility of alginate/chitosan microcapsule by fabricating bi-functional membrane. Macromol Biosci. 2014;14(5):655-66.
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Figure captions Figure 1: Study design detailing animal work Figure 2: a) Microencapsulation efficiency, analyte content and production yield (n=3, data expressed as average ± SEM); light microscopic images of b) SA microcapsules and
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scanning electron micrographic images of SA microcapsules at c) 100µm, d) 20µm, e) 10µm and f) 1µm; and light microscopic images of g) TCA-SA microcapsules and
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scanning electron micrographic images of TCA-SA microcapsules at h) 100µm, i) 20µm, j)
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10µm and k) 1µm
Figure 3: a) Energy dispersive X-ray spectra of SA microcapsule
and b) TCA-SA
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microcapsule at four locations with its corresponding analysis (1, 2, 3 and 4) respectively Figure 4: a) Swelling index (at 25°C), b) Swelling index (at 37°C), c) Buoyancy index, d)
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Dissolution release and e) Mechanical strength index of TCA-SA from microcapsules. Data are expressed as mean± SEM, n=3
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Figure 5: a) Blood glucose of TCA; Concentration of TCA (Healthy, Diabetic-control and
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Table legends
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TCA-SA) in b) serum, c) ileum, d) liver, e) pancreas and f) faeces. Data are mean ± SEM
Table I: DSC and FTIR spectral analysis of polymer and bile acid microcapsules Table II: Viscosities and related rheological parameter of both microencapsulated formulations: SA and TCA-SA. Values expressed as mean ± SEM, n=3. UD: undetected (below the instrument limit of detection)
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Differential scanning calorimetric analysis Dominant Peak(s)
SA powder
227 ± 0.5°C
TCA powder
220°C ± 3.7°C
TCA-SA powder
219°C ± 1.2°C and 225°C ± 2.2°C
SA microcapsules
230°C ± 0.1°C
TCA-SA microcapsules
216°C ± 0.9°C and 226°C ± 1.2°C
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Chemical excipients
Main λ (cm-1)
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Chemical excipients
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Fourier transform infrared analysis
Functional groups
3265, 1592, 1409, 1028
TCA powder
3368, 2932, 1635, 1543, 1165
O-H/ N-H, C-H, C=C, C=C, C-O
TCA-SA powder
3372, 1628, 1486, 1100
O-H/ N-H, C=C, C=C, C-O
SA microcapsules
3374, 1597, 1029
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SA powder
2890, 1661, 1025
O-H/N-H, C=C, C=O C-H, C=C, C=O
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TCA-SA microcapsules
O-H, C=C, C-H, C=O
Table I: DSC and FTIR spectral analysis of polymer and bile acid microcapsules
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VISCOSITY MEASUREMENTS BEFORE MICROENCAPSULATION
20 35 61 107 187 327 572 1000 20 35 61 107 187 327 572 1000
UD UD 30.4±3.0 27±1.0 25±0.4 17±0.3 8±0.2 7±0.1 UD UD 18±3 15±2 14±3 11±3 8±0.2 5±0.1
Torque (mNm)
Shear stress (mPa)
UD UD UD 0.01±0.0 0.02±0.01 0.05±0.02 0.06±0.02 0.08±0.02 UD UD UD UD 0.03±0.01 0.06±0.01 0.08±0.02 0.16±0.04
UD UD UD UD 1300±100 2000±100 3600±200 5000±100 UD UD UD UD 2200±20 4000±40 5000±30 7000±70
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1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Shear rate (s-1) 22±1 44±2 74±4 120±6 225±15 400±16 600±22 1000±38 UD UD UD 52±8 100±10 125±12 275±14 563±20
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Viscosity (mPas)
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TCA-SA
RPM
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SA
Set speed
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Formula Code
Table II: Viscosities and related rheological parameter of both microencapsulated
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Graphical abstract
Sangeetha Mathavan, Momir Mikov, Svetlana Golocorbin-Kon, Hani Al-Salami PII: DOI: Reference:
S0928-0987(17)30269-5 doi: 10.1016/j.ejps.2017.05.041 PHASCI 4055
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
3 March 2017 11 May 2017 17 May 2017
Please cite this article as: Sangeetha Mathavan, Momir Mikov, Svetlana Golocorbin-Kon, Hani Al-Salami , Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid exerted no hypoglycaemic effects, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.05.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid
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exerted no hypoglycaemic effects
Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin
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a
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Sangeetha Mathavana, Momir Mikovb, Svetlana Golocorbin-Konc, Hani Al-Salamia *
Health Innovation Research Institute, Curtin University, Perth WA, Australia Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of
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b
Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
Dr Hani Al-Salami
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*Corresponding author
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c
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Medicine, University of Novi Sad, Novi Sad, Serbia
Senior Lecturer of Pharmaceutics, School of Pharmacy
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Curtin University, GPO Box U1987 Perth, Western Australia 6845, Australia. |
+ 61 8 9266 9816
Fax
|
+ 61 8 9266 2769
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Tel
Email | [email protected] Profile | http://healthsciences.curtin.edu.au/teaching/pharmacy_people.cfm/Hani.Al-Salami
Short Title: Microencapsulated taurocholic acid in Type 1 diabetes
ACCEPTED MANUSCRIPT 2 Abstract Context: The bile acid taurocholic acid (TCA) is endogenously produced, and has shown formulationstabilising effects when incorporated into microcapsules containing potential antidiabetic drugs.
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This study aimed to develop and characterise TCA-microcapsules, and test their antidiabetic effects, in an animal model of Type 1 diabetes (T1D).
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Methods:
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Using the polymer sodium alginate (SA), SA-microcapsules (control) and TCA-microcapsules
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(test) were prepared, and assessed for morphology, surface composition, chemical and thermal stability, swelling, buoyancy, mechanical, release and rheological properties. TCA-microcapsules
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were gavaged as a single dose (1.2mg/300g) to alloxan-induced diabetic rats, and blood glucose and TCA concentrations in serum, tissues (ileum, liver and pancreas) and faeces, were measured.
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One healthy and one diabetic group were used as control and gavaged SA-microcapsules.
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Results:
TCA-microcapsules showed consistent size, TCA presence on surface and all layers of
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microcapsules, chemical and thermal stability, enhanced swelling, buoyancy and targeted-release properties and rheological analysis showed Non-Newtonian flow properties. TCA serum
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concentrations were lower in the healthy group, compared with the diabetic and diabetic-treated groups, but there was no significant difference between diabetic control and diabetic treated groups, in terms of TCA levels, and blood glucose concentrations. Conclusions: The developed TCA-microcapsules showed good stability and release properties, but did not lower blood glucose levels in T1D, which suggests absence of insulin-mimetic effects, when using a single 1.2 mg/rat oral dose.
ACCEPTED MANUSCRIPT 3 Keywords: Taurocholic acid, bile acids, microencapsulation, diabetes mellitus, inflammation, Type 1 diabetes Abbreviations: Taurocholic acid
T1D
Type 1 diabetes
SA
Sodium alginate
TCA-SA
Taurocholic acid-sodium alginate
CaCl2
Calcium chloride
OM
Optical microscopy
SEM
Scanning electron microscopy
EDXR
Energy dispersive X-ray spectroscopy
DSC
Differential scanning calorimetry
FTIR
Fourier transform infrared spectroscopy
USP
United States Pharmacopoeia
UV-Vis
Ultraviolet-Visible spectroscopy
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LC-MS
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TCA
Liquid chromatography-mass spectroscopy
C18 column
Octadecyl carbon chain (C18) bonded silica
ABC-protein
ATP-binding cassette transporter protein
ANOVA
Analysis of variance
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Introduction
Taurocholic acid (TCA) is a dominant conjugate form of bile acids in humans. It is chemically
known
as
2-(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholn-24-yl,amino,
ethanesulfonic acid, and has a molecular formula of C26H45NO7S and molecular weight of 515.70g/mol. TCA is a tri-hydroxy bile acid that has an OH group at the C-3, C-7 and C-12
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positions. TCA is also known as cholaic acid, and it is a taurine conjugate of cholic acid,
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and constitutes more than 30% of bile acid pool and extensively metabolised in the small
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intestine by gut microflora. It is hydrophilic in nature and has a melting point of 125°C (1). Being an acid, TCA has a pKa value of 1.4 and is partially soluble in water but is soluble
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in alcohol and ether (2).
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Although TCA is not a globally registered drug with clinical indications to treat a disease, it has shown potential applications in inflammatory disease models. It has shown
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substantial antiinflammatory and immunoregulatory effects in healthy mice treated with
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proinflammatory endotoxins (3). It has also shown permeation-enhancing capabilities, when combined with other drugs. In recent studies, TCA enhanced the permeation of
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heparin-docetaxel conjugates, in the lower intestine via transcellular and/or paracellular pathways (4, 5). Moreover, in one study, TCA exerted protective and desirable effects on
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muscle and pancreatic β-cells, which suggests potential antidiabetic effects (6). Since TCA metabolism starts in the duodenum, it needs to be encapsulated in targeted-delivery matrices, which target the lower part of the intestine, and maximise its absorption (7).
Accordingly, this study aimed to develop, characterise and test antidiabetic effects of new microcapsules with oral targeted-delivery properties, which can deliver TCA to the lower part of the ileum for maximum absorption. The study also aimed at investigating changes
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of TCA concentrations in serum, tissues and faeces, associated with diabetes development and TCA-microcapsules treatment. Thus, in this study, sodium alginate (SA) was used as a polymer, to form microcapsules without (control) and with TCA (test). The microcapsules were characterised in vitro, then gavaged as a single dose to one diabetic group. Two other groups (one healthy and one diabetic) were gavaged SA-microcapsules and used as
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control. Blood, tissues and faeces were collected 10 hours postdose for blood glucose and
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TCA measurements. The in-vitro studies include imaging and morphological studies, surface elemental-analysis, stability, rheological properties, swelling, mechanical strength, and buoyancy
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analysis and release profiles at various temperature and pH values. The in-vivo studies include
2.1
Materials
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Materials and methods
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blood and tissues concentrations measurements of glucose and the bile acid, TCA.
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Taurocholic acid (96.0%) and alloxan (>98%) were purchased from Sigma Chemical Co, USA. Sodium alginate was obtained as low viscosity, from Acros Organics, USA.
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Anhydrous calcium chloride (CaCl2) was purchased from Scharlab S.L, Australia. Ultrasonic gel was obtained from Australian Medical Association, Australia. All other
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solvents and reagents used in this study were supplied by Merck, NSW, Australia and were of analytical grade. 2.2
Drug stock preparation
The stock suspension of TCA (4mg/ml) was prepared by solubilising the crystalline powder of TCA with the ultra water-soluble gel (10%). The calcium chloride stock solution (10% w/v) was prepared by dissolving the salt to deionized water. Alloxan was
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mixed with saline prior to injection. All preparations were mixed for 3 hours at room temperature to ensure uniform mixing and were used within 24 hours of preparation. 2.3
Formation of microcapsules
The microencapsulation of TCA was carried out by combining a mixture of SA and TCA
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1.8% and 4 mg/ml and dispersing in deionized water (prior to use by microencapsulation
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process) using our well-established microencapsulating methodologies (6, 8, 9).
Microencapsulation efficiency, analyte content, production yield and
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microcapsules characterisations
0.1 g of microcapsules were weighed, and dissolved in 200 ml of phosphate buffer (pH 7.8) and the suspension was stirred and aliquots of the dissolution medium (2 ml) were
Spectroscopic
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withdrawn and TCA measured using established Liquid Chromatography Mass methods
(10).
The
analyte
contents,
production
yield
and
microencapsulation efficiency were calculated using our established methods, as described
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elsewhere (11-14) .
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The characterisation studies were done using Optical microscopy (OM)- Nikon YS2-H, Japan; Scanning electron microscopy (SEM)- Zeiss Neon 40EsB FIBSEM (Cambridge, MA) and Energy dispersive X-ray spectra (EDXR)- Oxford Instruments, INCA X-Act, Concord, MA), as per our established methods. Briefly, OM was used to evaluate the shape of the formulated microcapsules, while SEM and EDXR were used to investigate the surface morphological features of the microcapsules and elemental distribution of the atoms distributed across the surface of the microcapsules. The analysed microcapsule
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samples were coated with 5nm platinum under vacuum. The mean diameter of the particle size was calculated using the software associated with the instrument (15-17).
2.5
Thermal, chemical and accelerated stability analyses and mechanical strength
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and swelling properties measurements
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Thermal analytical studies were carried out using the Differential Scanning Calorimeter
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(DSC-8000 Perkin Elmer Inc., Waltham, MA, USA). A dried sample weight equivalent to 5mg of the microcapsules was utilized for examining the melting behaviour using the
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sealed aluminium pans, while, Fourier transform infrared spectroscopic analysis (FTIR spectrometer TWO, Perkin Elmer Inc., Waltham, MA) was used in the analysis of the
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functional groups present on the microcapsules using a frequency range of 450 to 4000cm-1 (18).
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The microencapsulating formulation and the microcapsules’ physical-stability, and membrane integrity were assessed at different pH and temperature values. Microcapsules’ physical stability was assessed by placing 1 gram of microcapsules onto sterile petri dishes
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and storing that in thermostatically controlled ovens (accelerated-stability chambers,
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Angelantoni Environmental and Climatic Test Chamber, Italy) at different temperature ranges of -15°C, 5°C, 25°C and 40°C with relative humidity set at 35% for three days. The colour, texture and size of microcapsules were visually assessed for changes in appearance. In order to assess mechanical resistance and strength, microcapsules were placed in a Boeco Multishaker (Boeco Company, Germany) in 20 ml of phosphate buffer and agitated for 2 days, and the number of damaged microcapsules was recorded and mathematically assessed, using our well-established methods (12, 13).
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In order to assess microcapsules’ membrane strength and integrity, their swelling properties were assessed, at four pH values (1.5, 3.0, 6.0 and 7.8) and at two temperatures (25°C and 37°C) for 4 hours, as per our established methods (19).
Rheological properties, buoyancy and dissolution release studies
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Basic rheological measurements were analysed using Visco-88 viscometry (Bohlin- Visco
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88, ATA Scientific Pty. Ltd., NSW, Australia), where 20 ml of each sample were placed in the instrument-cap and various measurements taken by altering rotation-speed and
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recording parameters, as appropriate. Buoyancy testing was carried out by placing 200 microcapsules in 100 ml solution of phosphate buffer. The buffer was stirred by rotating
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paddles at 50 rpm for 4 hours (USP dissolution apparatus 24, type II) at a temperature of 37°C ± 0.2, which was regulated by a thermostat. Every hour, the number of floating
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microcapsules was counted and buoyancy index was calculated using the ratio of the
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number of floating microcapsules to that of the total number of microcapsules.
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Analyte dissolution release testing was carried out using 0.3g of freshly prepared microcapsules, placed in 500 ml of phosphate buffer at pH values of 1.5, 3.0, 6.0 and 7.8
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over 6 hrs period at two different temperatures of 25°C and 37°C. The dissolution medium buffer was stirred at 200 rpm at a flow rate of 30ml/min. The test was performed using a closed loop flow system developed for microcapsule drug release testing via UV-Visible spectrophotometric
analysis
using
a
Perkin
Elmer
LAMBDA
spectrophotometer (Perkin Elmer, Waltham, Massachusetts, USA) (16, 19).
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UV-Vis
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2.7
Diabetic induction and bile acid analysis
Male Wistar rats (250 ± 45g) were maintained in an animal facility and given free access to water and food, and temperature and light were controlled to mimic their natural habitat. The rats were randomly divided into different groups and one of the group (TCA-SA) was
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made diabetic by intravenously administering the drug alloxan at a dose of 25mg/kg/body weight (20-25). Three days following the alloxan administration, rats that showed blood
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glucose levels higher than 18 mmol/L along with signs and symptoms of diabetes were
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considered diabetic (23, 26-29). For the study design, see Figure 1. The experiments were approved by the Animal Ethics Committee at Curtin University and all experiments were
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performed according to the Australian Code of Practice for the care and use of animals for
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scientific purposes. This particular group (TCA-SA) was part of a larger study where blood glucose levels and gliclazide levels from the same control groups have been published (8).
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The tertiary bile acid, TCA was analysed by LC-MS. The method involved the use of 40µl
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BA sample acidified with ammonium acetate buffer (100µl) at pH 4.0. It was extracted twice with 800µl of diethyl ether and the final aliquot (500µl) was evaporated to dryness.
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The residue was reconstituted in the isocratic mixture of methanol: water (65:35) and 10µl was injected into the LC-MS system.
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The LC-MS system consisted of a Shimadzu LC-20AD pump attached to a Shimadzu LCMS-2020 mass detector. The analytical column was a C18 Symmetry (3.9 x 150mm, 5µm particle size) with a C18 (3.9 x 2mm) guard column. Analysis was performed using an isocratic flow with a flow rate 0.2 ml/min and under these conditions; the retention time for TCA was observed at 8.7 min, while the run time was kept to 15 min. 2.8
Statistical analysis
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All the results are expressed as mean ± standard error of mean (SEM) using triplicate samples of the same batch unless otherwise stated. All the animal experimental analysis was carried out using n=7 or 8 rats per group ± SEM. Statistical comparisons data were assessed using one way ANOVA or parametric/non-parametric t-test, as appropriate, using V6.01, GraphPad Prism Software; Inc., San Diego, CA. Values falling below the
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detectable limit of assay were recorded as zero. A probability of p<0.05 was regarded as
3.1
Results: Microencapsulation
efficiency,
analyte
content
and
production
yield
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measurements
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significant and p<0.01 as highly significant.
Drug content and total production yield of TCA-SA microcapsules was measured, and
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microencapsulation efficiency suggesting method adequacy and robustness (Figure 2 a).
Size analysis, morphological and surface composition studies
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Both microcapsules had similar and consistent size, averaging around 930 µm, in diameter. The mean particle size was not significantly affected by presence of TCA, which suggests that TCA incorporation did not compromise microcapsules size or shape. Microscopic and SEM imaging revealed opaque and uniform shape with homogenous particle size distribution and similar surface characteristic features in both microcapsules. The surface of the microcapsules appeared rough, in particular TCA-SA microcapsules, but consistent from one microcapsule to another for all analysed batches (Figures 2 b to k).
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This suggests that the addition of TCA into SA formulation did not compromise microcapsules’ formation or significantly alter the morphology of the microcapsules. The EDXR analysis of TCA-SA microcapsules showed the presence of atoms characteristics of TCA such as ‘Sulphur (S)’ atoms on the surface of the microcapsules,
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which suggests that TCA did not confine into the core of the microcapsules but is likely to be distributed throughout the different layers of the microcapsules (Figure 3 (a) and (b)).
Thermal and chemical analysis
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This may affect the release profile of TCA from the microcapsules.
Analysis of TCA powder, and SA and TCA-SA excipients pre and post
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microencapsulation revealed that the melting point of each excipient remained consistent and when excipients were physically mixed, they maintained their main peaks and thermal
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features suggesting thermal compatibility. Unlike G-SA microcapsules, which showed
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slightly higher melting points post-microencapsulation, TCA and SA melting points postmicroencapsulation were slightly lower, which may suggest ionic interaction or possible
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change in the lattice phase (Table I). This may be due to a shift in their thermal capacity within the 30-245°C range or due to interactions between all ingredients and potential mild
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loss of crystallinity (30).
The FTIR spectrum of SA (Table I) showed the characteristic peaks at 3265cm-1 corresponding to the OH stretching and two medium intensity peaks at 1592cm-1 and 1028cm-1 relating to the COOH stretching and C-O-C stretching. Similarly, TCA-SA microcapsules exhibited three major peaks, from which two strong intense peaks were observed at wavelengths of 2890cm-1 and 1661cm-1 corresponding to C-H stretching and
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conjugated aliphatic C=C and C=N respectively. In addition, another medium or low intensity peak at 1025cm-1 was observed corresponding to C=O stretching. Accordingly, FTIR analysis of SA and TCA microcapsules showed FTIR spectra similar to that of the physical mixture and suggests the absence of new chemical identity molecules formed and thus, suggests chemical compatibility and also the absence of chemical alterations due to
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Accelerated stability testing, swelling, buoyancy, dissolution-release and
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the microencapsulation process.
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mechanical strength analysis
Results from the accelerated stability studies showed that analyte contents per
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microcapsules were not significantly altered at the end of the experiment, and also no specific changes in texture, colour and shape associated with different temperatures were
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noticed. TCA maintaining its content per microcapsule suggests stability within the
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microcapsules’ matrix, while the consistent effects of different temperatures on microcapsules’ appearance and texture suggests robustness of the microencapsulating
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method and that TCA addition did not alter stability or appearance of the SAmicrocapsules. This is not in line with previous studies in our laboratory that showed
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changes in microcapsules shape and texture associated with high temperature (9, 31) and suggests variation in microcapsules’ response to high temperature, especially when microencapsulating different drugs. TCA increased the swelling index of SA-microcapsules at temperatures, 25°C (p< 0.05) and 37°C (p< 0.05) (Figure 4-a&b), enhanced the buoyancy (p< 0.05 and p< 0.01) (Figure 4-c), showed good and targeted dissolution release profile (p< 0.01) (Figure 4-d), and
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better mechanical strength (p< 0.01) (Figures 4-e). This suggests that TCA incorporation into SA-microcapsules improved physical properties of microcapsules with stable and more solid matrix and stronger membrane exhibiting improved ability to interact with gutcontents and possess pH-targeted delivery at pH>7, where gut-maximum absorption for
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bile acids is (7). This is consistent with previous studies showing bile acid membrane-stabilising effects,
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when microencapsulated in SA-based microcapsules (19). In addition, the release profile
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shows immediate TCA release at higher pH values and thus, is consistent with the observed distribution of TCA within the microcapsules’ outer membrane, as shown by the
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EDXR analysis (Figure 3).
Rheological studies
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TCA incorporation into SA-microcapsules did not significantly affect the behaviour of
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rheological parameters (viscosity, shear rate, Torque and shear stress; Table II). As shear stress increased, viscosity decreased and share rate increased while Torque increased,
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which suggests Non-Newtonian Thixotropic behaviour, regardless of addition of the bile acid, TCA. This rheological behaviour is common and desirable in pharmaceutical
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formulation and suggests that the microcapsules exhibit thermodynamic, mechanical and deformational flexibility, and are appropriate for drug delivery and TCA targeted and controlled release (32-34).
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Blood glucose and bile acid studies
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Blood glucose levels were significantly elevated by diabetes induction (p< 0.01) and TCAmicrocapsules did not significantly change the elevated blood glucose levels (Figure 5-a). In addition, TCA serum concentrations were increased due to diabetes induction (p< 0.01), and TCA-microcapsules did not significantly alter TCA concentrations in either serum (Figure 5-b), ileum (Figure 5-c), pancreas (Figure 5-e) or faeces (Figure 5-f), which
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suggests significant modulation of the bile acid profile in diabetic animals, that was not Interestingly, TCA
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normalised by an acute treatment with TCA-microcapsules.
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concentrations were lower (p< 0.01) in diabetic rats treated with TCA-SA, compared with
Discussion
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healthy (Figure 5-d).
This study encompasses 1) in vitro methods designed to characterise the bile acid TCA pre
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and post microencapsulation, the shape, size, surface-elemental compositions, excipient
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thermo-chemical properties, physical and mechanical stability under various temperatures, rheological profiles under various stress conditions, buoyancy and release properties of
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TCA-microcapsules, as well as 2) in vivo methods designed to examine if oral administration of a single dose of TCA-microcapsules to T1D rats will change their blood
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glucose levels or bile acid profile.
Recent studies in other labs have demonstrated unique formulation properties and biological activities of TCA. Khatun Z. et al., showed efficacy of TCA in the oral delivery of nanoparticles as targeted-delivery systems (5), while Mooranian A. et al., showed that TCA optimised the release profile of lipophilic drugs, when incorporated into alginatebased microcapsules (35). In addition, Wang C. et al., demonstrated beneficial immune-
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regulatory effects of TCA in mice (36), while Wu T. et al., demonstrated significant haemostatic effects of TCA on glycaemic control and insulin release (37). Accordingly, in this study, TCA microcapsules were examined in-vitro and in- vivo, for anti-diabetic activities.
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Microscopic imaging and size distribution analysis showed consistent size and shape with
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small variations between different batches, regardless of TCA presence. This illustrates
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that the incorporation of the bile acid, TCA into SA-based formulations did not affect the formation of microcapsules or their shape and size. Elemental and atomic surface analysis
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of microcapsules’ outer membrane revealed that when TCA was added to the SAformulation it did not only distribute into the core and internal layers but it also distributed
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throughout all layers of the microcapsules including the surface. TCA distribution on the surface of the TCA-SA microcapsules was evident in its immediate release from the
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microcapsules at pH 7.8 (p<0.01), with significant and visible release profile. The in-vivo
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implications of TCA distribution within the microcapsules, in terms of their absorption
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profile and tissue accumulation and potential anti-diabetic effects were also studied.
Physicochemical and stability measurements of the microencapsulating formulation and
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the microcapsules revealed that analytes and excipients (SA and TCA) are thermally and chemically compatible and are stable pre and post microencapsulation. The addition of TCA into SA-based formulation did not compromise stability or alter rheological and flow properties of formulation, but rather enhanced physical strength of the microcapsules. The importance of enhanced stability of microcapsules has been examined in the literature. Neubauer MP. et al., have demonstrated applications of self-assembly methodology and rheological features and deformation, on drug contents, release patterns and morphological
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features and have shown direct relationship with shear stress and hydrogel architecture (38). In addition, Yan J. et al., (39) and Zheng G. et al., (40) have shown that covalent linking and stabilisation of microcapsules matrix can be utilised to optimise stability and thermal and chemical compatibilities and enhance morphological features of the microcapsules. This is in line with the results and suggest good potential for the in-vivo
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delivery of microencapsulated TCA-SA. This was illustrated by visible TCA absorption
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and systemic concentrations in blood, tissues and faeces. However, TCA-SA
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microcapsules did not result in direct insulin-mimetic effects and significant reduction of the elevated blood glucose of diabetic animals, independent of insulin. Nevertheless,
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diabetes induction showed significant increase in TCA levels in serum, and liver of diabetic treated animals which suggests significant alterations in bile acid haemostasis and
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feedback mechanisms and gut re-absorption (enterohepatic recirculation), due to potential alterations of changes of expression and functionality of ABC-protein transporters
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responsible for bile acid gut-uptake. This is in line with previous studies in our laboratory
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that have shown significant alterations of protein-expressions in the ileal mucosa and subsequent alterations in bile acids metabolism and gut-absorption as well as systemic
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concentrations and overall bile acid profile (25, 28).
Conclusion
The current study aimed to develop and test microcapsules with targeted-delivery properties that can deliver the bile acid, TCA, orally and test its hypoglycaemic effects in a rat model of T1D. The study examined physicochemical properties of microcapsules containing TCA and tested their morphology, stability, physical properties and release profiles in vitro. The study also examined TCA-microcapsule’s hypoglycaemic effects and
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the microcapsule’s effects on bile acid profile. Results showed good physicochemical properties and targeted-delivery characteristics of TCA-microcapsules, but showed no hypoglycaemic effects in T1D rats. Results also showed changes in TCA concentrations as a result of diabetes development. Thus, diabetes has direct effects on bile acid profile, but TCA-microcapsules failed to normalise the change in bile acid profile caused by diabetes
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development, which suggests lack of effectiveness in diabetes treatment.
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Disclosures
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The authors acknowledge that they have no declaration of interest to declare.
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Acknowledgements
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The authors acknowledge the Curtin CUPS Scholarship for the support and also would like
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to acknowledge the use of equipment, scientific and technical assistance of the Curtin University Electron Microscope Facility, which has been partially funded by the
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References
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University, State and Commonwealth Governments.
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18. Mooranian A, Negrulj R, Mathavan S, Martinez J, Sciarretta J, Chen-Tan N, et al. An advanced microencapsulated system: a platform for optimized oral delivery of antidiabetic drugbile acid formulations. Pharm Dev Technol. 2015;20(6):702-9. 19. Mathavan S, Chen-Tan N, Arfuso F, Al-Salami H. The role of the bile acid chenodeoxycholic acid in the targeted oral delivery of the anti-diabetic drug gliclazide, and its applications in type 1 diabetes. Artificial cells, nanomedicine, and biotechnology. 2016;44(6):150819.
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37. Wu T, Bound MJ, Standfield SD, Gedulin B, Jones KL, Horowitz M, et al. Effects of rectal administration of taurocholic acid on glucagon-like peptide-1 and peptide YY secretion in healthy humans. Diabetes Obes Metab. 2013;15(5):474-7. 38. Neubauer MP, Poehlmann M, Fery A. Microcapsule mechanics: from stability to function. Adv Colloid Interface Sci. 2014;207:65-80.
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40. Zheng G, Liu X, Wang X, Chen L, Xie H, Wang F, et al. Improving stability and biocompatibility of alginate/chitosan microcapsule by fabricating bi-functional membrane. Macromol Biosci. 2014;14(5):655-66.
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Figure captions Figure 1: Study design detailing animal work Figure 2: a) Microencapsulation efficiency, analyte content and production yield (n=3, data expressed as average ± SEM); light microscopic images of b) SA microcapsules and
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scanning electron micrographic images of SA microcapsules at c) 100µm, d) 20µm, e) 10µm and f) 1µm; and light microscopic images of g) TCA-SA microcapsules and
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scanning electron micrographic images of TCA-SA microcapsules at h) 100µm, i) 20µm, j)
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10µm and k) 1µm
Figure 3: a) Energy dispersive X-ray spectra of SA microcapsule
and b) TCA-SA
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microcapsule at four locations with its corresponding analysis (1, 2, 3 and 4) respectively Figure 4: a) Swelling index (at 25°C), b) Swelling index (at 37°C), c) Buoyancy index, d)
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Dissolution release and e) Mechanical strength index of TCA-SA from microcapsules. Data are expressed as mean± SEM, n=3
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Figure 5: a) Blood glucose of TCA; Concentration of TCA (Healthy, Diabetic-control and
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Table legends
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TCA-SA) in b) serum, c) ileum, d) liver, e) pancreas and f) faeces. Data are mean ± SEM
Table I: DSC and FTIR spectral analysis of polymer and bile acid microcapsules Table II: Viscosities and related rheological parameter of both microencapsulated formulations: SA and TCA-SA. Values expressed as mean ± SEM, n=3. UD: undetected (below the instrument limit of detection)
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Differential scanning calorimetric analysis Dominant Peak(s)
SA powder
227 ± 0.5°C
TCA powder
220°C ± 3.7°C
TCA-SA powder
219°C ± 1.2°C and 225°C ± 2.2°C
SA microcapsules
230°C ± 0.1°C
TCA-SA microcapsules
216°C ± 0.9°C and 226°C ± 1.2°C
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Chemical excipients
Main λ (cm-1)
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Chemical excipients
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Fourier transform infrared analysis
Functional groups
3265, 1592, 1409, 1028
TCA powder
3368, 2932, 1635, 1543, 1165
O-H/ N-H, C-H, C=C, C=C, C-O
TCA-SA powder
3372, 1628, 1486, 1100
O-H/ N-H, C=C, C=C, C-O
SA microcapsules
3374, 1597, 1029
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SA powder
2890, 1661, 1025
O-H/N-H, C=C, C=O C-H, C=C, C=O
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O-H, C=C, C-H, C=O
Table I: DSC and FTIR spectral analysis of polymer and bile acid microcapsules
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VISCOSITY MEASUREMENTS BEFORE MICROENCAPSULATION
20 35 61 107 187 327 572 1000 20 35 61 107 187 327 572 1000
UD UD 30.4±3.0 27±1.0 25±0.4 17±0.3 8±0.2 7±0.1 UD UD 18±3 15±2 14±3 11±3 8±0.2 5±0.1
Torque (mNm)
Shear stress (mPa)
UD UD UD 0.01±0.0 0.02±0.01 0.05±0.02 0.06±0.02 0.08±0.02 UD UD UD UD 0.03±0.01 0.06±0.01 0.08±0.02 0.16±0.04
UD UD UD UD 1300±100 2000±100 3600±200 5000±100 UD UD UD UD 2200±20 4000±40 5000±30 7000±70
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Shear rate (s-1) 22±1 44±2 74±4 120±6 225±15 400±16 600±22 1000±38 UD UD UD 52±8 100±10 125±12 275±14 563±20
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RPM
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Set speed
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Table II: Viscosities and related rheological parameter of both microencapsulated
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Graphical abstract