Article pubs.acs.org/molecularpharmaceutics
New Biotechnological Microencapsulating Methodology Utilizing Individualized Gradient-Screened Jet Laminar Flow Techniques for Pancreatic β‑Cell Delivery: Bile Acids Support Cell Energy-Generating Mechanisms Armin Mooranian,† Rebecca Negrulj,† Ryu Takechi,‡ Emma Jamieson,§ Grant Morahan,§ and Hani Al-Salami*,† †
Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, Perth, Western Australia 6102, Australia ‡ School of Public Health, Curtin Health Innovation Research Institute, Curtin University, Perth, Western Australia 6102, Australia § Centre for Diabetes Research, Harry Perkins Institute of Medical Research, Perth, Western Australia 6009, Australia ABSTRACT: In previous studies, we developed a new technique (ionic gelation vibrational jet flow; IGVJF) in order to encapsulate pancreatic β-cells, for insulin in vivo delivery, and diabetes treatment. The fabricated microcapsules showed good morphology but limited cell functions. Thus, this study aimed to optimize the IGVJF technique, by utilizing integrated electrode tension, coupled with high internal vibration, jet-flow polymer stream rate, ionic bath-gelation concentrations, and gelation time stay. The study also utilized double inner/outer nozzle segmented-ingredient flow of microencapsulating dispersion, in order to form β-cell microcapsules. Furthermore, a microcapsule-stabilizing bile acid was added, and microcapsule’s stability and cell functions measured. Buchi-based built-in system utilizing IGVJF technology was screened to produce best microcapsulecontaining β-cells with or without a stabilizing-enhancing bile acid. Formed microcapsules were examined, for physical characteristics, and encapsulated cells were examined for survival, insulin release, and inflammatory profiles. Optimized microencapsulating parameters, using IGJVF, were: 1000 V voltage, 2500 Hz frequency, 1 mL/min flow rate, 3% w/v ionic-bath gelation concentration, and 20 min gelation time. Microcapsules showed good morphology and stability, and the encapsulated cells showed good survival, and insulin secretion, which was optimized by the bile acid. Deployed IGVJFbased microencapsulating parameters utilizing stability-enhancing bile acid produced best microcapsules with best pancreatic βcells functions and survival rate, which, suggests potential application in cell transplantation. KEYWORDS: biotechnology, cell engineering, microencapsulation, transplantation
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The first process is engineering a delivery device with compatible biomaterials that has appropriate shape and size, where cells can be safely loaded and housed. The delivery device has to possess several important features, which include: (1) excipient-compatibility to ensure that no chemical, thermal, or physical degradation or modulation of excipients is taking place over the transplantation period, (2) shell/outermembrane has to be adequately and uniformly porous to allow diffusion of nutrients and oxygen into inside the device, and for insulin and other cellular proteins and wastes to diffuse outside the device, but at the same time, pores have to prevent β-cells from leaking out or immune and other cells from penetrating into the device, and (3) microarchitecture of the devices has to facilitate long-term survival of cells and ideally
INTRODUCTION Biotechnology is a field of science which includes the technology of delivering living cells and biologically active moieties, by means of encapsulation and specialized targeted delivery methodologies.1 It can be used to encapsulate cells, which are capable of synthesizing and releasing certain enzymes or hormones needed for disease treatment. An example is Type 1 diabetes, which is characterized by loss of pancreatic β-cells and subsequent loss of insulin release and glucose hemostasis. Type 1 diabetes accounts for about 5% of all diabetic cases and its prevalence is sharply on the rise.2 Current treatment relies on insulin delivery by injection or pump, which has many draw backs including route of administration and hypoglycemic episodes. In diabetes and pancreatic β-cell delivery, the design and fabrication of biomaterial devices suitable for cell transplantation rely on two main processes. Both processes are critical for the design and engineering of suitable celldelivery matrices, capable of long-term insulin release and replacement of damaged pancreatic islets. © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
March 22, 2017 June 26, 2017 July 6, 2017 July 6, 2017 DOI: 10.1021/acs.molpharmaceut.7b00220 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
microencapsulator, equipped with air-flow nozzle intermittently with concentric nozzle and both nozzles were tailored to forming best microcapsules using our set techniques.20,21 Optimal microencapsulation parameters were determined as per Table 1.7−18,22−27
promote their mass and functions, through allowing efficient glucose diffusion and insulin flow within the device, into systematic circulation, and mimicking physiological functions of host own pancreas.3,4 Accordingly, the first process aims to design appropriate cell delivery system that forms a suitable house for the cells to function, long-term. The second process is developing robust, efficient, and reproducible biotechnological methodologies, using advanced techniques, which can load pancreatic β-cells into a device (e.g., a capsule), uniformly, and with optimum cell survival, and high loading dose.3,5,6 Accordingly, the second process aims to design appropriate biotechnological methodology for best cell encapsulation and optimum cell load, within the delivery device. Over the last several years, our laboratory has focused on engineering delivery microcapsules with optimum biomaterialcompatibility and best size and shape for supporting β-cell functions.7−16 Our studies examined a range of excipients and microcapsule-stabilizing agents as well as biological compounds, such as bile acids, in order to optimize the morphology and physical features of the microcapsules for best cell functions. In one recent study, we developed a new microencapsulating biotechnological method for cell encapsulation, which produced good (consistent in size distribution and surface features) microcapsules that have high cell-loading capacity.17 The new method (ionic gelation vibrational jet flow; IGVJF) has produced microcapsules with good size uniformity but the encapsulated cells exhibited limited biological functions. Thus, this study aimed to incorporate and screen biophysical microencapsulating parameters integrated into the IGVJF methodology, and examine microcapsules’ morphology, stability, and uniformity with and without a microcapsulestabilizing bile acid. The study also aimed to examine effects of the microencapsulating method on survival and functions of the microencapsulated cells. The biophysical parameters include electrode voltage tension, internal frequency and vibration, polymer stream rate, ionic bath concentration, and gelationtime stay.
Table 1. Biophysical Microencapsulation Parameters and Corresponding Value Range Screened for Optimum Microencapsulating Parameters Based on the Ionic Gelation Vibrational Jet Flow Methodology17 parameter
screening range
integrated electrode voltage tension internal frequency/vibration polymer stream rate with microencapsulating dispersion ionic bath concentration gelation-time stay
250−1000 V 1000−2500 Hz 0.5−1.2 mL/min 1−10% w/v 5−60 min
Confocal Microscopy Analyses and Stability Measurements. Confocal analysis was done after staining NIT-1 cells with CellTrace carboxyfluorescein succinimidyl ester using Cell Proliferation Kit (Life Technologies, Australia).28−30 Microcapsules’ images were taken using an UltraVIEW Vox spinning disk confocal microscopy (PerkinElmer, USA) equipped with Yokogawa CSU-X1 confocal scanning unit (PerkinElmer, USA), as per our established-methods.20,21,31 Ursodeoxycholic acid distribution within the layers of the microcapsule was analyzed by conjugating it with fluorescent tetramethylrhodamine isothiocyanate (TRITC) before forming the microcapsules then analyzing them using UltraVIEW Vox spinning disk confocal microscopy. Thermal and chemical stability measurements were done using differential scanning calorimetric analysis (PerkinElmer DSC 8000, USA) and Fourier transform infrared spectroscopy (PerkinElmer Spectrum 2, USA), using our established methods.28−30 Mechanical stability was measured in order to assess if bile acid incorporation changes the ability of microcapsules to withstand stress by testing the ability of the microcapsules to remain intact, over 2.5 days, in a shaker (150 rpm), using a Boeco Multishaker PSU 20 (Boeco Company, Germany). The data was presented as the percentage of intact microcapsules remaining at the end of the 2.5 days, as per our established methods.32 Osmotic stability was assessed by measuring the microcapsules’ ability (under static incubation) to maintain their weight and resist loss of moisture, over 2.5 days. Dry microcapsules were weighed and placed in 35 mL of phosphate buffer pH 7.35 at a temperature of 37.5 °C for 2.5 days and the swollen microcapsules were removed and the % swelling of microcapsules was calculated as per established methods.32−34 Electrokinetic stability was measured via measuring Zeta-potential followed by size analysis, in order to determine consistency of the microcapsules’ formation and size distribution. Zetasizer 3000HSa (Malvern Instruments, Malvern, UK) and Mastersizer 2000 (Malvern Instruments, Malvern, UK) were used for Zeta-potential and size distribution measurements, as per our well-established methods.33,34 Sample volume and number of microcapsules per ml of vehicle were maintained constant and analyses was carried out on freshly prepared microencapsulating formulation (1 mL/gram each aliquot). Viability, Insulin Secretion, and Energy Hemostasis of Microencapsulated β-Cells. MTT calorimetric assay was
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MATERIALS AND METHODS Excipients and Microencapsulation Procedures. Sodium alginate ≥99% (stock used, 20 mg/mL), poly L-ornithine hydrochloride (stock used, 20 mg/mL), ursodeoxycholic acid ≥98% (stock used, 4 mg/mL), poly-4styrene-sulfonate (stock used, 30 mg/mL), and poly allylamine hydrochloride (stock used, 10 mg/mL) were purchased from Sigma-Aldrich (New South Wales, Australia). Cell preparation and microencapsulation were prepared using our established methods.17,18 NIT-1 cells were maintained and grown using sterile tissue culture flasks (Thermo Fisher Scientific, Australia) and fed with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies, USA) supplemented with D-glucose (Sigma Chemical Co, USA), fetal bovine serum (Thermo Fisher Scientific, Australia), and penicillin-streptomycin (Thermo Fisher Scientific, Australia).19 Both microencapsulating formulations were prepared as 2% sodium alginate (25−30 ca% mannuronic acid content), 2% polylornithine, 3% poly styrene sulfate, and 3% polyallylamine with test formulation containing an additional 4% ursodeoxycholic acid. The alginate (Mwt 216), polylornithine (Mt 30k), poly styrene (Mwt 70k), and polyallylamine (Mwt 60k) were excipients while the bile acid was a stabilizing agent. Cells were placed in a syringe set up with specialized pump-flow controlled system, and cells were microencapsulated as a scattered suspension of cells. BüchiB
DOI: 10.1021/acs.molpharmaceut.7b00220 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics used to assess viability of microencapsulated cells using our published methods.13,15 Briefly 5 mg/mL stock solution of MTT (Sigma Chemical CO, USA) was prepared in buffer and pH adjusted to 7.4, before the undissolved residues were removed by sterile filtration and used within 48 h. Microencapsulated β-cells were first incubated in Krebs-Ringer buffered HEPES (pH 7) with 0.5% w/v bovine serum albumin and the microcapsules were then incubated in either a lowglucose (2.5 mM) or high-glucose (17.5 mM) solution at 37.5 °C for 90 min. The amount of insulin secreted into low- and high-glucose solutions was measured using an Ultrasensitive Mouse Insulin ELISA Kit (Mercodia Cooperation, Uppsala, Sweden). Evaluation of metabolic and mitochondrial activities of microencapsulated β-cells, included assessing bioenergetic biomarkers basal respiration, aerobic metabolism and ATP production, maximum respiration, respiratory capacity, and glycolysis. This was carried out in Real-Time, using an in-house developed method via the Seahorse Flux Analyzer XF 96 (Seahorse Bioscience, USA).9 Seahorse instrumentation measures the biomarkers by measuring changes in oxygen and proton content via a fluorescent biosensor.35,36 A series of injections from a multichannel port were done in order to analyze mitochondrial respiration and activities.35,36 The injections started with only media (without glucose), and this was used to equilibrate the experimental conditions. This was followed by another injection for ATP-coupler oligomycin in order to inhibit ATP-synthesis.37−39 A final cell count was carried out before running the instrument and all microcapsules were examined visually after the end of the mitochondrial stress testing to determine if they are intact and ready for analysis. All data was normalized for the viable microencapsulated cell count within each microcapsules. Cytokine Secretion Analysis. Microcapsules were incubated for 2.5 days and media was measured for Tumor Necrosis Factor alpha, Interferon gamma, Interleukin 1β, Interleukin 6, Interleukin 12, and Interleukin 17 using Cytokine Bead Array technology (BD Biosciences, USA) using our established methods.31 Briefly, freshly prepared cell-containing microcapsules (control and test) were cultured in media for 2.5 days, and 100 μL (n = 3) aliquots removed and stored at −4 °C overnight prior to analysis. Aliquots were prepared for Cytokine Bead Array analysis using appropriate BD Flex Sets according to the manufacturer’s protocols and in-house developed methods. Aliquots were analyzed using Attune Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, USA) and data were processed using software FlowJo (FlowJo LLC, Ashland, Oregon, USA). Statistical Analysis. Parametertic/nonparametric student t test or ONE-WAY ANOVA followed by Tuckey analysis were performed, as appropriate, setting the level of significance at p < 0.05. All the statistical analysis was conducted using the GraphPad Prism software, version 4.03. The p value was only reported where significance was noted (p < 0.05 significant and p < 0.01 highly significant).
Table 2. Optimum Biophysical Microencapsulation Parameters, which Produced Best Most Stable Alginate and Alginate-Bile Acid Microcapsules Containing Pancreatic βCells parameter
optimal value
integrated electrode voltage tension internal frequency/vibration polymer stream rate with microencapsulating dispersion ionic bath concentration gelation-time stay
1000 V 2500 Hz 1.0 mL/min 3% w/v 20 min
membrane was maintained
