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Back to Journal »Drug Design, Development and Treatment» Volume 15

The effect of primary unconjugated bile acids on hydrophilic drug nanocapsule drug formulations: pharmacological significance

Authors: Mooranian A, Foster T, Ionescu CM, Carey L, Walker D, Jones M, Wagle SR, Kovacevic B, Chester J, Johnstone E, Kuthubutheen J, Brown D, Atlas MD, Mikov M, Al-Salami H 

Published on October 23, 2021, the 2021 volume: 15 pages 4423-4434

DOI https://doi.org/10.2147/DDDT.S328526

Single anonymous peer review

Editor approved for publication: Dr. Anastasios Lymperopoulos

Armin Mooranian,1,2 Thomas Foster,1,2 Corina M Ionescu,1,2 Louise Carey,1,2 Daniel Walker,1,2 Melissa Jones,1,2 Susbin Raj Wagle,1,2 Bozica Kovacevic,1,2 Jacqueline Chester,1,2 Edan Johnstone,1,2 Jafri Kuthubutheen,3 Daniel Brown,4 Marcus D Atlas,2 Momir Mikov,5 Hani Al-Salami1,2 1Biotechnology and Drug Development Research Laboratory, Curtin School of Medicine and Curtin Institute of Health Innovation Curtin University, Bentley, Perth, 6102, Washington State, Australia; 2Hearing Therapeutics, Ear Science Institute Australia, Queen Elizabeth II Medical Centre, Nedlands, Perth, 6009, WA, Australia; 3Australia Fiona Stanley Hospital, Perth, Western Australia; 4 Curtin School of Medicine and Curtin Health Innovation Institute, Curtin University, Perth, Australia, Australia; 5 Department of Pharmacology, Toxicology and Clinical Pharmacology, Novi Sad University of Medicine Hospital, Novi Sad, 21101, Serbia Mailing address: Hani Al-Salami Hearing Therapy, Biotechnology and Pharmaceutical Sciences, Curtin University, Bentley, Perth, 6102, Australia, Western Australia Tel 61 8 9266 9816 Fax 61 8 9266 2769 Email [Email protection] Introduction: In a recent study in our laboratory, the common primary unconjugated bile acid chenodeoxycholic acid (CDCA) in humans has been shown to improve the stability of nano-encapsulated lipophilic drugs , And it is possible to improve the release profile after oral administration through electrokinetic stability. Therefore, this study aims to examine the effect of CDCA on similar effects of hydrophilic drugs. Methods: Various CDCA-based formulations were produced for the orally administered hydrophilic drug metformin. The analysis of these preparations includes electromotive force, morphology, drug and CDCA preparation content, nano-size distribution, thermally induced deformation and outer core expansion index, release curve, shell resistance ratio, and thermal and chemical index. Since the main target of the drug is pancreatic β cells, the effect of the preparation on cell viability, function and inflammatory characteristics was also studied. Results and conclusions: CDCA-based metformin formulations exhibit improved stability and release profiles through thermal, chemical and electrokinetic effects, depending on the formulation, indicating the potential application of CDCA in the oral targeted delivery of hydrophilic drugs. Keywords: microencapsulation, diabetes, glycerol monooleate, Eudragit, chenodeoxycholic acid

Bile acids are naturally produced in the human liver and play a key role in cholesterol metabolism and maintaining bile acid homeostasis. 1 In addition, bile acids are increasingly recognized as a complex integrator that plays an important role in immune function and signaling pathways, and has been proven to have anti-diabetic effects through signaling pathways. 2 In addition to these direct physiological effects, lincholic acid incorporated into microcapsules and nanocapsules has been shown to improve the stability and release characteristics of lipophilic drugs, and may improve the stability of targeted oral administration due to electrokinetics. 3–5

CDCA is a major bile acid that reduces cholesterol levels by increasing intestinal excretion and interfering with bile acid synthesis, thereby reducing cholesterol secretion and cholesterol saturation. 6,7 It is commonly used to treat constipation and gallstones and lipid storage disorders. 8 However, liver toxicity and gastrointestinal diseases have been shown to have adverse effects. 6 External pharmacological applications Recent studies have found that CDCA interacts with vitamin D receptors, which play a role in the excretion of bile acids. 9

Lipophilic drugs have been encapsulated with CDCA, which improves stability. Mathavan et al. added CDCA to microcapsules containing the lipophilic drug gliclazide, which improved the physical properties, stability and release profile. The change in zeta potential is hypothesized to be due to the surfactant properties of CDCA reducing the charge on the outer surface of the molecule10.

Metformin is known as a biguanide drug and has been widely used to treat type 2 diabetes. Although metformin has a high efficacy, long-term intake of metformin is associated with a variety of adverse events and exhibits a long list of side effects, including weight loss, diarrhea, and vomiting, which can be serious.

The addition of bile acid has been shown to change the surface charge, which is measured in the same way as the zeta potential, and subsequently has benefits in terms of microcapsule properties and drug release. Mooranian et al. incorporated ursodeoxycholic acid (UDCA) into nano- and microcapsules to encapsulate the lipophilic drug probucol. The bile acid preparation showed enhanced electrokinetic stability and enhanced drug absorption. 11 In another study, the inclusion of UDCA for β cell encapsulation improves the physical properties of the capsule and protects the encapsulated cells by enhancing static electricity and free charge. 12

Changes in electromotive force have been shown to improve drug delivery systems and the release of a range of drugs. The binding of bile acids to antibiotics shows changes in free charge and subsequent interactions between functional groups and micelles, allowing characterization of the distribution of solutes in micelles, and methods for optimizing drug delivery systems, thereby increasing drug solubility. 13 Gallardo et al. proved that the stability is mainly controlled by the zeta potential in the polymer drug delivery system, modified to a more favorable zeta potential, which produces a more uniform suspension and is not easy to flocculate. 14 The ophthalmic application of aspirin has shown that the protein that is improved and stabilized by the encapsulation of the drug leads to the sustained release of the drug and significantly reduces hemolysis. 15

Eudragit (Eud) is a polymer based on polymethacrylate. Like BA, it can be used as a useful excipient in the delivery of various pharmaceutical products. There are several different types of Eud polymers, each with different pH dependence and independent solubility. Generally, Euds is classified according to charge (anionic, cationic, or neutral). Cationic Eud can be used to change taste, while non-ionic Eud can be used for GIT targeting. 16 Neutral Eud has been previously explored in ketoprofen capsule formulations, resulting in delayed and pH-independent drug release. 17

Electrokinetic stabilization improves the drug delivery system, especially the physical properties, stability and release profile of encapsulated drugs. In the encapsulation process, the physical and chemical stability of bile acids can be used to increase the resistance to mechanical stress, as well as improved surface properties and resistance to water penetration. The resulting change in the incorporation of bile acid into electromotive force and subsequent improvement in drug delivery are desirable. In addition, the use of EU provides a new method to further improve the release characteristics of drugs in microcapsules. This study aims to investigate the effect of incorporating CDCA and Eud types (NM, RL, RS) into microcapsules to deliver the hydrophilic drug metformin (M) to determine whether the same as seen in lipophilic drug delivery can be established Improve.

Metformin (M, 98%), sodium alginate (SA, 99%) and chenodeoxycholic acid (CDCA, 99%) were purchased from Sigma Chemical Company. Calcium chloride dihydrate (CaCl2.2H2O 98%) was purchased from Scharlab SL in Australia. All reagents and solvents are of HPLC grade and are used without further purification or modification, provided by Merck (Australia).

Prepare a suspension or pretreatment by adding 1.6 g SA, 0.6 g bile acid, 0.1 g glyceryl monooleate, 3 g poloxamer, 0.2 g M powder, and 1 g Eud to 100 mL ultrapure water and mixing manually. Encapsulation material. The suspension was then placed on a magnetic stirrer to thoroughly mix at room temperature for 24 hours, and then stored in a refrigerator at 4°C. The stock solution is within 48 hours after preparation. Prepare a CaCl2 (2%) stock solution by adding 2 g of CaCl2 powder to 100 mL of HPLC water and mixing thoroughly until completely dissolved.

The capsules are prepared using a well-defined and rigorously improved method established in our laboratory. 18-25 According to Table 1, add different types of Eud to prepare pre-encapsulated materials. Each formula also contains 1.6% SA, 0.1% glycerol monooleate, 0.2% M and 3% poloxamer. Formulations F2, F4, and F6 contained 0.6% of CDCA, and each solution contained 1% of the appropriate Eud, as shown in Table 1. Table 1 List of formulations used to prepare the pre-encapsulated mixture

Table 1 List of recipes for preparing pre-packaged mixtures

The Büchi encapsulator system is used to perform ion gel vibrating jet (IGVJF) encapsulation. 26-30 Adjust settings including vibration frequency, voltage, and nozzle diameter to produce uniform droplet sizes of pre-encapsulated material. Then the microcapsules were cross-linked into spheres of uniform size in a 2% CaCl2 bath. 31-34 Then rinse the capsules in deionized water for at least one minute before drying or performing any testing.

In order to evaluate the preliminary size and quantity, the capsules were imaged through an optical microscope. A glass slide with a calibrated scale is loaded with freshly encapsulated material. Then use the 4X objective lens with the Olympus IX-51 inverted microscope to evaluate the capsule. 19,35–37

A scanning electron microscope (SEM) was also performed to check the morphology of the capsule. The freshly formed capsules were dried in a stabilization chamber at 40°C and 40% humidity for three days. The capsule is coated with 5nm platinum under vacuum for imaging with MIRA3 FibSEM SEM.

In addition to microscopic examination, the capsule size is also determined by two other methods. Mie scattering and Fraunhofer diffraction are fast techniques that use laser light to determine the size of particles by using laser light. 38 Malvern Instruments' Mastersizer 2000 is used to record the laser light scattering from prepared particles to determine their size diffraction using Mie scattering and Fraunhofer. The size was also evaluated by spectral evaluation performed by Zetasizer 3000HSa, also from Malvern Instruments. Both instruments operate using standardized procedures that the laboratory has established. 21 Electromotive force, also called zeta potential, is also evaluated using Zetasizer 3000HSa. The measurement was performed at 25°C, and each sample was repeated three times. 18,21

A stability study was conducted to evaluate the temperature resistance of the capsule. Prepare a predetermined amount of capsules and place them in a sterile petri dish for analysis. Then store the capsules at -20°C, 5°C, 25°C, and 40°C. The humidity is set to 35%, and the capsule is stored for 3 days. Adjust humidity and temperature to ensure consistent conditions. After 3 days of incubation, the capsules were evaluated for signs of decomposition.

The drug loading and efficacy were determined by high performance liquid chromatography (HPLC) using established methods for determining M content. 39–41 In short, break up the 100 mg capsule and dilute to 200 mL with phosphate buffer (pH 7.4). The samples were centrifuged and filtered for HPLC evaluation. Then the M concentration was quantified by a UV detector set at a wavelength of 227 nm in accordance with the laboratory method. The M load is determined as a percentage of the weight of M in the sample. The microencapsulation efficiency is determined as the percentage of the actual weight of M relative to the theoretical weight. The sample was repeated three times.

Evaluate capsule expansion as a means of determining mechanical strength. The capsules are stored at 25°C and 37°C with pH values ​​of 3 and 7.4 to simulate the conditions inside the GIT. Determine the capsule size before and after storage to determine the% expansion.

The buoyancy of the capsule was also evaluated. Buoyancy is calculated as the percentage of microcapsules that can float in 100 mL of phosphate buffer. 42

In order to determine the physical and chemical compatibility, Fourier Transform Infrared (FTIR) and Differential Scanning Calorimetry (DSC) analyses were performed according to our previous methods. 19,37 FTIR evaluated the absorption spectra of individual ingredients, combination mixtures, and medicated capsules. Infrared evaluation was performed on the PerkinElmer FTIR spectrometer TWO in the scanning range of 650–4000 cm-1, and the results were recorded at 25°C. DSC analysis of individual capsule components, mixtures and crushed capsules was also performed on a DSC instrument (DSC 8000; PerkinElmer Inc., Waltham, MA, USA). Prepare a 5 mg sample in an aluminum container and heat it at a rate of 20°C/min in a nitrogen atmosphere. The determination was repeated in triplicate.

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay is a technique used to observe the effects of capsules on mitochondrial activity and cell viability. 43,44 Stock MTT solution should be prepared at 5mg/mL within 24 hours after use. All groups were treated in exactly the same way, including the control group. The cell concentration per volume was set to 106/mL, and the cells were randomly assigned to different groups.

The prepared MIN6 cells were added to each well of a 96-well plate and incubated for 24 hours. The dried microcapsules were also stored in pH 7.4 medium at 37°C for 24 hours, and then added to each well. 45 Capsules without cells were used as a negative control, and the plates were incubated for another 24 hours. After incubation, add 20 µL of the prepared MTT to each well. MTT is converted to MTT-formazan by mitochondrial reductase. Then remove MTT-formazan and dissolve it in 100 µL of Sigma Chemicals' dimethyl sulfoxide (DMSO). The resulting purple solution was then analyzed by 550nm photometry. The results are reported in triplicate.

MIN6 cells are also used to show the inflammation markers produced after exposure to microcapsules. As described above, the capsules were incubated with the cells for 48 hours, then the medium was removed and analyzed using the BD Cell Counting Bead Array (CBA). BD Mouse Flex Sets are used to detect tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 6 (IL-6), interleukin 10 (IL-10) and interleukin 1 (IL-1). Flow Cytometry. The results are reported in triplicate.

Although as shown in Figure 1, the shape, size and morphology of the microcapsules seem to be consistent. As shown in Figure 1A, all capsules have the same size, oval and 3D shape. Formulas F1, F2, and F5 seem to have smooth surface textures, while F4 and F6 have obvious crystal structures on their surfaces, while F3 may have a less obvious crystal-like structure. Although no further analysis was performed on the surface of the microcapsules, these may be M on the surface of the capsule. 46 This is consistent with other studies in our laboratory, that is, anti-diabetic drug crystals are found on the surface of the microcapsules. In these studies, these crystals were identified by X-ray spectroscopy and confirmed as drugs. 20,21,35 Other atoms were also found on the surface of the capsule, including Ca and Cl, which may cause crystals to form here. These crystals are commonly found in ionic gel-treated microcapsules and can only be seen by drying the capsules before imaging with SEM. 35 Figure 1 Microcapsule microscope (A), drug content before and after encapsulation (B), and acid content before and after bile encapsulation (C). Data are mean ± SEM.

Figure 1 Microcapsule microscope (A), drug content before and after packaging (B), and bile acid content before and after packaging (C). Data are mean ± SEM.

When comparing the samples before and after the stability test, the drug content of all formulations (F1-F6) remained the same, as shown in Figure 1B. Figure 1C shows that the bile acid content is also consistent in the capsules containing CDCA. This indicates that neither the drug nor the bile acid has been decomposed or significantly chemically modified by the encapsulation process. This is important, as if they have been broken down, and the microcapsules may not have the effect they should have. Other studies have shown that CDCA does not interact strongly with the drugs in the microcapsules, and therefore does not damage the drugs in the microcapsules. 10

Figures 1A and 2A show all capsules (F1-F6) although their composition maintains a consistent size and a mass of about 800 μg. This indicates that the capsules are of similar size and quality and therefore have similar densities despite the different formulations. Figure 2 Microcapsule size distribution (A), electrokinetic (B) and stability (C). The stability is determined as the thermal deformation index at temperatures A.-20°C B. 5°C, 25°C and D. 40°C, with a range of 0-2. Data are mean ± SEM, n=3. *P<0.05, **P<0.01.

Figure 2 Microcapsule size distribution (A), electrokinetic (B) and stability (C). The stability is determined as the thermal deformation index at temperatures A.-20°C B. 5°C, 25°C and D. 40°C, with a range of 0-2. Data are mean ± SEM, n=3. *P<0.05, **P<0.01.

Figure 2 also shows the electromotive force of the formulation. Figure 2B shows that F1 and F2 containing EudNM30D have negative electromotive force ~ -20mv. F3-F6 all have a positive electromotive force between 22-33mv. Except for F3 and F4 (preparations containing EudRL30D), CDCA did not seem to significantly change the potential in the capsule, where the potential changed significantly from 22 to 30 mv (P<0.05) after adding CDCA. In previous studies, CDCA has also been shown to increase the magnitude of electromotive force. 18

The increase of the electromotive force (positive or negative) will cause the repulsive force between the molecules in the microcapsule to increase. The increased repulsive force is related to the increased stability and strength. 47 The increased electromotive force can also be used to reduce particle aggregation, which helps improve drug delivery. A charge of about 30 mv is considered the best choice for drug delivery. 48

Thermal deformation is shown in Figure 2C. The capsule formulations F1 and F2 decomposed at the lowest temperature, starting at 5°C. Compared with F1, as shown by F2, CDCA slightly contributes to stability by preventing premature decomposition at 25°C. Formula F3-F4 has consistent structural integrity, will not decompose before 25°C, and will not be further damaged at 40°C. Ideally, the capsule will not decompose until it reaches body temperature.

Given that the only difference between F1/3 and F3-4 is the use of different Eud, EudNM30D is likely to contribute to the early destruction of these capsules and their net negative electromotive force. EudNM30D is considered a swellable polymer, which may be the reason for its early decomposition. 16

The expansion of the outer core of the capsule and the release of the drug are shown in Figure 3. Outer core expansion is an indicator of microcapsule penetration. The expansion of the outer core indicates that more water enters the capsule and causes it to expand. Generally, increased swelling indicates an increased risk of capsule rupture. Figures 3A-D show that the swelling of the capsules remains consistent between the capsules at different pHs. In the past, CDCA has been shown to improve capsule stability and increase capsule strength. Mainly, CDCA can achieve this by reducing water penetration to reduce swelling, increasing non-covalent bonds to increase strength, and deprotonating carboxylic acids to increase electronegativity. 18 Figure 3 Microcapsule expansion (AD) and drug release curve (E) at pH 1.5 [E-1], pH 3.5 [E-2], pH 6 [E-3] and pH 7.4 [E-4]. Data are mean ± SEM, n=3. *P<0.05, **P<0.01.

Figure 3 Microcapsule expansion (AD) and drug release curve (E) at pH 1.5 [E-1], pH 3.5 [E-2], pH 6 [E-3] and pH 7.4 [E-4]. Data are mean ± SEM, n=3. *P<0.05, **P<0.01.

As shown in Figure 3E, the drug release profile varies from formulation to formulation. At pH 1.5, all formulations except F1 had consistent drug release. At pH 3.5, formulation F4 released significantly more drug than F3 (P<0.05) (CDCA negative), indicating that the presence of CDCA affects drug release. At pH 6, F4, F5, and F6 released statistically significantly more drugs than F1, F2, and F3, as well as at pH 7.4 (P<0.01). The difference between F3 and F4 indicates that in formulations containing EudRL30D, CDCA is affecting drug release. However, in other formulations, CDCA does not seem to significantly affect the drug release profile. The pH-dependent release profile seen in formulations F4-F6, where the drug load reaches >90%, can be used to deliver drugs via GIT.

Previous work has shown that the introduction of bile acids does improve the drug release profile. 4,5,49–53 Bile acids have been shown to be effective in controlling the release profile of drugs. The bile acid deoxycholic acid has been shown to increase the drug release of the microcapsules at low concentrations, and decrease the drug release at higher concentrations. 54 Eud formulations have been found to effectively tolerate changes in the pH of the simulated colon and improve drug delivery in vivo. 55 The introduction of Eud into bile acid-containing capsules has also been shown to improve the drug release profile. 56

As shown in Figure 4B, regardless of the excipient content, the buoyancy (intestinal sinkability) between the microcapsules is consistent. This indicates that the density between the capsules is similar. It is important to keep the buoyancy low so that the capsule will be able to maintain significant contact with the surface of the intestine instead of floating on top. This gives the capsule the highest chance of interacting with epithelial cells to release its contents. Figure 4 Shell resistance (%) (A), intestinal submergence (%) (B, the main thermal indicators of microcapsules (C and D) and the main chemical formation indicators of microcapsules (E and F).

Figure 4 Shell resistance (%) (A), intestinal submergence (%) (B, the main thermal indicators of microcapsules (C and D) and the main chemical formation indicators of microcapsules (E and F).

Regardless of the formulation, the shell resistance in Figure 4A remains consistent between capsules. It shows that Eud type and CDCA do not affect shell resistance.

The DSC and FTIR results in Figure 3C-F show that metformin has not been chemically modified. This shows that the encapsulation process did not significantly change the chemical structure of metformin, which means that it will retain its biological activity. This is consistent with our previous work. There is almost no compromise in the chemical structure of the drug or excipient during the packaging process (Figure 4). 19,37,57.

The cell survival rate is shown in Figure 5A. In general, F1-F6 cell survival research is poor. Compared with untreated cells, F1-F6 all showed a reduced survival rate (P<0.01), but F4 showed no significant change in cell survival rate. CDCA did not significantly change the cell survival rate in the F1/F2 or F5/F6 groups. However, compared with F3, CDCA did statistically significantly increase the survival rate of CDCA-EudRL30D containing F4 capsules. Consistent with the findings in F4, previous studies have shown that CDCA improves the survival rate of CDCA-encapsulated cells. 18 In previous studies, CDCA has been found to be toxic in the liver of rhesus monkeys, however, this only shows that it is significantly higher 58 without reducing the survival rate and is still positive because it shows no adverse effects on the cells. Figure 5 Cell survival rate (A), insulin production (B), inflammation markers (CG). Data are mean ± SEM, n=3. *P<0.05, **P<0.01. Abbreviation: UT, untreated.

Figure 5 Cell survival (A), insulin production (B), inflammation markers (CG). Data are mean ± SEM, n=3. *P<0.05, **P<0.01.

Figure 5B shows data on the production of insulin by capsule-treated cells. All encapsulated cells produce insulin, while untreated cells do not. Although the preparation F4 containing CDCA did cause the highest insulin production, it did not statistically increase significantly, indicating that CDCA does not affect the insulin production of cells. This is related to the discovery that stimulation of the bile acid farnesol X receptor (FXR) has been shown to increase the production and secretion of cellular insulin. 59 Diabetes animal models treated with Eud-Bile acid capsules also previously showed no change in insulin concentration. Nevertheless, they did show improved blood sugar control. 60 It is not clear why CDCA alone can alter insulin release, but with Eud, it cannot significantly affect its release.

Figure 5C-G shows the inflammation markers produced by the cells. Compared with untreated cells, all treated cells showed no statistically significant changes in TNF-α, IFN-γ, or IL-1 (Figure 5C, D, and F). Although not statistically significant, F4 does have the lowest levels of TNF-α and IFN-γ, and the second lowest level of IL-1. IL-6 increased in all treated cells (Figure 5E); however, it had the least significant increase in formulas F1, F2, and F4 (P<0.05). As shown in Figure 5G, IL-10 in Formulation F4 was only statistically significantly reduced (P<0.05).

In the body, CDCA is thought to stimulate the secretion of inflammatory cytokines including TNF-α, IL-1 and IL-6 through NLRP3 activation, thereby triggering liver fibrosis and inflammation. 61 However, previous studies have shown that CDCA treatment of cells leads to a reduction in liver fibrosis inflammation. The production of TNF-α. 18 High levels of TNF-α have been shown to significantly impair cell viability. 62 Other Eud-based formulations have previously shown reduced TNF-α production. 63

IFN-γ alone cannot induce pancreatic cell apoptosis. This combined with TNF-α can induce apoptosis through STAT1 activation. 64 Previous studies repeated the finding that Eud-Bile acid-based formulations did not significantly change IFN-γ levels. 42 Interestingly, other bile has been shown to increase IFN-γ levels by acid. 65

As seen in this study, activation of FXR by bile acids (including CDCA) has been shown to stimulate IL-6 levels. 66 However, previous work in our laboratory has shown that CDCA reduces the expression of IL-6 and IL-1. Treated cells. 18, 67–70

CDCA-Eud-based metformin formulations exhibit improved stability and release profiles through thermal, chemical, and electrokinetic effects, which depend on the formulation, indicating the potential application of CDCA in the oral targeted delivery of hydrophilic drugs.

Formula F4 containing EudRL30D CDCA shows the greatest potential. F4 produces a smooth surface morphology with the same size as other capsules. There were no statistically significant changes in the drug and bile acid content before and after the test. F4 also shows a larger electromotive force, with good shell resistance and intestinal sinking. F4 also showed a cell survival rate comparable to that of untreated cells, but increased insulin production and decreased inflammatory cytokine production. Compared with F3 (EudRL30D, no CDCA), F4 also resulted in a statistically significant improvement, indicating that the bile acid content contributes to its improvement parameters. Therefore, the research results prove the advantages of CDCA-based metformin preparations in terms of better stability and release profile, which may improve safety and potential metformin efficacy. Further research on Eud-CDCA-based microcapsules is needed to gain a deeper understanding of their effectiveness as pharmaceutical excipients. F4 in particular will benefit from further investigation.

The authors thank the Australian Postgraduate Award and Curtin Research Scholarship for their support. The authors thank Curtin University for the use of the electron microscope facility and scientific and technical assistance, which is partially funded by the university, state, and federal governments. MIN-6 cells were provided by the University of Western Australia, and their acquisition and use were approved by Curtin University Institutional Guidelines.

H Al-Salami has been and is currently receiving funding from Beijing Nat-Med Biotechnology Co., Ltd. This work was partially supported by the EU Horizon 2020 Research Project and Innovation Program, which was carried out in accordance with Marie Skłodowska-Curie Grant Agreement No. 872370. Curtin College ORS-WAHAI Alliance and Australian National Health and Medical Research (APP9000597).

Hani Al-Salami reported on funding from the Beijing Nat-Med Biotechnology Co., Ltd., the European Union Horizon 2020 research project, the Curtin College ORS-WAHAI Alliance, and the Australian National Health and Medical Research Center during the research period. Jafri Kuthubutheen, Hani Al-Salami, Armin Mooranian and Daniel Brown reported on the Australian Provisional Patent Application No. 2020901933 "Therapeutic Methods and Formulations" patent. Hani Al-Salami and Armin Mooranian reported on patented encapsulation technology for oral administration of cannabinoids. The authors report that there are no other potential conflicts of interest for this work.

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