Kinetics Of Insulin Release Research Articles

Neuro-fuzzy model for predicting insulin delivery from crosslinked agar-carbomer hydrogels

This study focuses on the innovation of an inhaled sustained release form of insulin and the development of a neuro-fuzzy model specifically tailored to predict insulin release kinetics from polycondensed agar-carbomer hydrogels. These were synthesized by blending agar and carbomer, incorporating propylene glycol and glycerol, and then cross-linking by polycondensation. The structure and morphology of the hydrogel were analyzed via Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy and Proton Nuclear Magnetic Resonance Spectroscopy. The neuro-fuzzy model, a combination of artificial neural networks and fuzzy logic, employs inputs such as concentrations of crosslinking agents, polycondensation time, and release time, with the output being the rate of insulin release. The model demonstrated a strong correlation with experimental data, highlighting its effectiveness and precision in predicting insulin delivery from hydrogel compositions and temporal parameters. This emphasizes the importance of intelligent modelling for forecasting the kinetic release of therapeutic agents from novel drug delivery systems.

The study of galactomannans with different molecular weights and their ability to form microparticles suitable for pulmonary delivery

The influence of locust bean gum (LBG) galactomannans (GMs) molecular weight (Mw) to assemble microparticulate systems was evaluated, and carriers for deep lung delivery were developed. A commercial batch of LBG with a mannose/galactose (M/G) ratio of 2.4 (batch 1) was used to study the influence of different microwave partial acid hydrolysis conditions on carbohydrate composition, glycosidic linkages, and aqueous solutions viscosity. The microwave treatment did not affect the composition, presenting 4-Man (36–42 %), 4,6-Man (27–35 %), and T-Gal (24–25 %) as the main glycosidic linkages. Depolymerization led to a viscosity reduction (≤0.005 Pa·s) with no major impact on polysaccharide debranching. The structural composition of the LBG galactomannans were further elucidated with sequence-specific proteins using carbohydrate microarray technologies. A second batch of LBG (M/G 3.3) was used to study the impact of GMs with different Mw on microparticle assembling, characteristics, and insulin release kinetics. The low-Mw GMs microparticles led to a faster release (20 min) than the higher-Mw (40 min) ones, impacting the release kinetics. All microparticles exhibited a safety profile to cells of the respiratory tract. However, only the higher-Mw GMs allowed the assembly of microparticles with sizes suitable for this type of administration.

Design of molecularly imprinted alginate microgels for topical release of insulin

In this work, pH-sensitive molecularly imprinted (MIP) microgels are developed based on alginate for topical insulin delivery and ocular disease treatment. MIP microgels were synthesized via the emulsification-gelation method with uniform size distribution (9–11 µm) and rough surface structure. Moreover, the impact of insulin concentration, adsorption time, and pH of the drug solution on the adsorption capacity of MIP microgels was studied. Increasing the initial insulin concentration showed enhanced MIP microgel adsorption capacity at pH 5.4, while kinetic analysis revealed insulin binding after 1 h. The kinetics of insulin release at pH 6.4 (normal eye) and 7.4 (dry eye) demonstrated a good fit with the Korsmeyer–Peppas model, indicating controlled drug release. The pH-sensitive MIPs resulted in slower insulin release at pH 6.4 with ∼60% of the drug released within 1 h and reaching 80% after 8 h. In contrast, a higher insulin release rate was observed at pH 7.4 (∼ 80% after 8 h). MIP microgels showed selective adsorption toward the template molecule measured by imprinting factors about 0.60 ± 0.03 and 1.44 ± 0.21 for bovine serum albumin (BSA) and insulin, respectively. The mucoadhesive strength of MIP and NIP microgels, assessed by adsorbing them onto cow mucin, exhibited excellent performance, with values of 82±3% and 91±2%, respectively. In summary, the MIP microgels show sustained, and controlled drug delivery and are good candidates for ocular tissue drug delivery.

Preparation of Chitosan-Tripolyphosphate Formulated Insulin Microparticles, Their Characterization, ANN Prediction, and Release Kinetics

Preparation of Chitosan-Tripolyphosphate Formulated Insulin Microparticles, Their Characterization, ANN Prediction, and Release Kinetics

Hydroxyethylcellulose-Based Hydrogels Containing Liposomes Functionalized with Cell-Penetrating Peptides for Nasal Delivery of Insulin in the Treatment of Diabetes.

Liposomes functionalized with cell-penetrating peptides are a promising strategy to deliver insulin through the nasal route. A hydrogel based on hydroxyethylcellulose (HEC) aqueous solution was prepared, followed by a subsequent addition of liposomes containing insulin solution functionalized with trans-activator of transcription protein of HIV-1 (TAT) or Penetratin (PNT). The formulations were characterized for rheological behavior, mucoadhesion, syringeability, in vitro release and in vivo efficacy. Rheological tests revealed non-Newtonian fluids with pseudoplastic behavior, and the incorporation of liposomes (HLI, HLITAT and HLIPNT) in hydrogels did not alter the behavior original pseudoplastic characteristic of the HEC hydrogel. Pseudoplastic flow behavior is a desirable property for formulations intended for the administration of drugs via the nasal route. The results of syringeability and mucoadhesive strength from HEC hydrogels suggest a viable vehicle for nasal delivery. Comparing the insulin release profile, it is observed that HI was the system that released the greatest amount while the liposomal gel promoted greater drug retention, since the liposomal system provides an extra barrier for the release through the hydrogel. Additionally, it is observed that both peptides tested had an impact on the insulin release profile, promoting a slower release, due to complexation with insulin. The in vitro release kinetics of insulin from all formulations followed Weibull's mathematical model, reaching approximately 90% of release in the formulation prepared with HEC-based hydrogels. Serum insulin levels and the antihyperglycemic effects suggested that formulations HI and HLI have potential as carriers for insulin delivery by the nasal pathway, a profile not observed when insulin was administered by subcutaneous injection or by the nasal route in saline. Furthermore, formulations functionalized with TAT and PNT can be considered promoters of late and early absorption, respectively.

Metal-ion coordinated self-assembly of human insulin directs kinetics of insulin release as determined by preclinical SPECT/CT imaging

Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn2+ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe2+/Zn2+, Eu3+/Zn2+, Fe2+/Co3+) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the PheB1 or LysB29 position to create conjugates that bind either Fe2+ or Eu3+, while its natural binding site (HisB10) preferentially coordinates with either Zn2+ or Co3+. HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at PheB1 or LysB29, respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by 111In-radiolabeling for SPECT/CT imaging. When the 111In-B29Tpy-HI conjugate was coordinated with Fe2+/Zn2+, its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. 111In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded 111In-B1Tpy-HI was observed compared to that of 111In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., PheB1 or LysB29) on HI and its coordination with the added metal ions (i.e., Fe2+/Zn2+ or Fe2+/Co3+) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions.

Repurposing pinacol esters of boronic acids for tuning viscoelastic properties of glucose-responsive polymer hydrogels: effects on insulin release kinetics.

In the era of the diabetes pandemic, injectable hydrogels (HGs) capable of releasing the desired amount of insulin under hyperglycemic conditions will significantly advance smart insulin development. Several smart boronic acid-based polymer HGs release insulin under high-glucose conditions. However, the correlation of insulin release characteristics with rheological properties is not well understood yet. Herein, we report a generalized and facile fabrication strategy of a new class of glucose-responsive hydrogels by crosslinking a biocompatible polymer, poly(vinyl alcohol) with pinacol esters of bisboronic acids via transesterification reactions. We show the versatility of the method by fabricating four hydrogels with diverse rheological properties. The HGs embody more than 70% water amenable for hosting insulin in the matrix. HG with high storage modulus, derived from 1,4-benzenediboronic acid bis(pinacol) ester releases ∼3 fold less insulin compared to softer HGs derived from acetylene-1,2-diyl bis(boronic acid pinacol ester) and bis[(pinacolato)boryl]methane under hyperglycemic conditions. We find that HG derived from the bis[(pinacolato)boryl]methane crosslinker exhibits superior insulin release properties due to the softness of the hydrogel matrix. We further show that the newly formulated gel is injectable without any structural change in the released insulin molecules and does not cause cytotoxicity. We believe that glucose-responsive hydrogels with tunable viscoelastic properties will pave the way for developing a variety of hydrogels with programmable insulin release properties.

Engineered insulin-polycation complexes for glucose-responsive delivery with high insulin loading

Glucose-responsive insulin delivery systems have the potential to improve quality of life for individuals with diabetes by improving blood sugar control and limiting the risk of hypoglycemia. However, systems with desirable insulin release kinetics and high loading capacities have proven difficult to achieve. Here, we report the development of electrostatic complexes (ECs) comprised of insulin, a polycation, and glucose oxidase (GOx). Under normoglycemic physiological conditions, insulin carries a slight negative charge and forms a stable EC with the polycation. In hyperglycemia, the encapsulated glucose-sensing enzyme GOx converts glucose to gluconic acid and lowers the pH of the microenvironment, causing insulin to adopt a positive charge. Thus, the electrostatic interactions are disrupted, and insulin is released. Using a model polycation, we conducted molecular dynamics simulations to model these interactions, synthesized ECs with > 50% insulin loading capacity, and determined in vitro release kinetics. We further showed that a single dose of ECs can provide a glycemic profile in streptozotocin-induced diabetic mice that mimics healthy mice over a 9 h period with 2 glucose tolerance tests.

Pre-Formulation Studies: Physicochemical Characteristics and In Vitro Release Kinetics of Insulin from Selected Hydrogels.

Insulin loaded to the polymer network of hydrogels may affect the speed and the quality of wound healing in diabetic patients. The aim of our research was to develop a formulation of insulin that could be applied to the skin. We chose hydrogels commonly used for pharmaceutical compounding, which can provide a form of therapy available to every patient. We prepared different gel formulations using Carbopol® UltrezTM 10, Carbopol® UltrezTM 30, methyl cellulose, and glycerin ointment. The hormone concentration was 1 mg/g of the hydrogel. We assessed the influence of model hydrogels on the pharmaceutical availability of insulin in vitro, and we examined the rheological and the texture parameters of the prepared formulations. Based on spectroscopic methods, we evaluated the influence of model hydrogels on secondary and tertiary structures of insulin. The analysis of rheograms showed that hydrogels are typical of shear-thinning non-Newtonian thixotropic fluids. Insulin release from the formulations occurs in a prolonged manner, providing a longer duration of action of the hormone. The stability of insulin in hydrogels was confirmed. The presence of model hydrogel carriers affects the secondary and the tertiary structures of insulin. The obtained results indicate that hydrogels are promising carriers in the treatment of diabetic foot ulcers. The most effective treatment can be achieved with a methyl cellulose-based insulin preparation.

Insulin-loaded liposomes functionalized with cell-penetrating peptides: influence on drug release and permeation through porcine nasal mucosa

The nasal route is promising for the delivery of insulin for diabetes treatment. Drug loading into delivery systems such as liposomes can improve the bioavailability of insulin for this route. Herein, we address the development and characterization of insulin-loaded liposomes, functionalized with cell-penetrating peptides (CPPs), such as the TAT and Penetratin (PNT) peptides, which act as promoters of the penetration and absorption of the drug, and could further improve insulin bioavailability. Furthermore, it was evaluated the effect of both encapsulation and functionalization with CPPs on insulin release and permeation through the porcine nasal mucosa. The results obtained revealed that insulin-loaded liposomes functionalized with CPPs presented average values of hydrodynamic diameter in the nanometer scale, with low polydispersion index, and stable zeta potential, during the storage period of 90 days. Liposomes functionalized with CPPs at a concentration of 0.1 mM promoted a decrease in zeta potential values of − 49.6 ± 1.33 mV for insulin-loaded liposomes to − 33.9 ± 1.10 mV when functionalized with TAT and − 20.6 ± 1.30 mV when functionalized with PNT owing to electrostatic interaction between highly positively charged CPPs and negatively charged insulin. Insulin encapsulation with non-functionalized liposome was 72%, but when functionalized with PNT and TAT, it decreased, respectively to 60% and 71%. Photomicrographs obtained by transmission electron microscopy showed a small multilamellar and unilamellar morphology and in nanometric scale (80–150 nm). The spectra of circular dichroism carried out in all formulations demonstrated that the integrity and stability of the insulin were preserved, as observed in the insulin in solution. In vitro release kinetics of insulin from liposomes followed Weibull's mathematical model, which relates the amount of drug accumulated in solution as a function of time. Comparing the systems, it was verified that the addition of CPPs in the formulations decreased permeation through the porcine nasal mucosa, because of electrostatic interaction between insulin and CPPs, which in turn, may be inhibiting the peptide's absorption-promoting activity. Therefore, when associated with liposomes, penetratin and TAT peptides did not show evidence of increasing insulin permeation through the nasal mucosa. However, insulin-loaded liposomes without functionalization with CPPs increased nasal permeability coefficient, suggesting that the developed system can optimize the absorption of insulin through the nasal route, which has not been previously published.

Injectable Biodegradable Polymeric Complex for Glucose-Responsive Insulin Delivery.

Insulin therapy is the central component of treatment for type 1 and advanced type 2 diabetes; however, its narrow therapeutic window is associated with a risk of severe hypoglycemia. A glucose-responsive carrier that demonstrates consistent and slow basal insulin release under a normoglycemic condition and accelerated insulin release in response to hyperglycemia in real-time could offer effective blood glucose regulation with reduced risk of hypoglycemia. Here, we describe a poly(l-lysine)-derived biodegradable glucose-responsive cationic polymer for constructing polymer-insulin complexes for glucose-stimulated insulin delivery. The effects of the modification degree of arylboronic acid in the synthesized cationic polymer and polymer-to-insulin ratio on the glucose-dependent equilibrated free insulin level and the associated insulin release kinetics have been studied. In addition, the blood glucose regulation ability of these complexes and the associated glucose challenge-triggered insulin release are evaluated in type 1 diabetic mice.

Preparation and characterization of insulin-loaded injectable hydrogels as potential adjunctive periodontal treatment

The application of local drugs as adjunctive periodontal treatment is a topic of rapidly increasing interest. Consequently, new discoveries are arising, a noteworthy portion of which employ hydrogels as delivery systems due to their high biocompatibility with and similarity to human tissues. In the search for new therapeutic agents capable of aiding periodontal treatment, authors became interested in a unique concept investigated by very few in vitro or in vivo studies concerning the local application of insulin. These studies concluded that insulin promotes the recovery and regeneration of damaged soft and bone tissues. The aim of the study was an endeavor to design a linear hydrogel that is injectable into periodontal pockets, and is able to carry a small insulin load through physical bonds and provide sustained release. The chitosan hydrogel as well as blends of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) and polyethylene glycol (PEG) were prepared and characterized in terms of the hydrogel texture and injectability. Afterward, to study the insulin release kinetics, a specific amount of each formulation was loaded with insulin, and then incubated in phosphate-buffered saline (PBS). Specimens of the incubated samples were withdrawn daily to measure insulin concentrations by means of the ultraviolet (UV)-absorbance method; ultimately the cumulative release was calculated. Out of the 5 formulations, 4 had homogenous one-phasic texture and their insulin release profiles in vitro ranged from a few hours to about 2 weeks. The blend of 26.5 wt% PVP, 6.6 wt% PVA, 0.03 wt% calcium chloride dihydrate (CaCl2.2H2O), and 66.8 wt% water, loaded with 2 IU/g of insulin, had favorable regular sustained release, approaching 13 days. The composition of the hydrogel, the component ratio and the amount of loaded insulin were found to affect the release profile. A linear hydrogel of copolymerized PVP, PVA and CaCl2.2H2O can serve as a local vehicle for the sustained delivery of insulin inside periodontal pockets.

Pluronic® F127-mediated control of insulin release rates from NPH microcrystals and blood glucose depression in STZ-induced diabetic rats.

Introduction: Neutral protamine Hagedorn (NPH) insulin is an intermediate-acting basal insulin with a long history of clinical use, consisting native human insulin. Its rather undesirable action profile, characterized by a peak release within a few hours, followed by insufficient insulin delivery upon a single subcutaneous (s.c.) dose, is well-documented. This may have been caused by the inherent microcrystal structure involving the basic peptide protamine, as well as the presence of tissue enzyme activities that readily act on protamine at the injection site. This issue may be circumvented by utilizing thermosensitive, erodible Pluronic F127 (PF127) to modulate the kinetics of insulin release from NPH over a period of 24 hours in which the hydrogel is completely eroded. Methods: Previously, we have shown that insulin release rates in vitro from NPH/PF127 formulations (0-25% PF127) markedly reduced the initial insulin release, especially in the presence of enzyme activity that selectively degraded protamine at 1-5 U/mL. Insulin release over the course of 20 hours was better modulated in the presence of increasing PF127 content. In this study, the insulin formulations (0, 20, and 25% PF127) were administered s.c. (4 U/kg) to streptozotocin (STZ)-induced diabetic rats and blood glucose levels were monitored over 24 hours. Results: In vivo blood glucose depression profiles in STZ-induced diabetic rats exhibited a similar pattern of control to in vitro data at the single s.c. dose of 4 U/kg, apparently extending the duration of action of NPH over a 24-hour period in the presence of PF127. Conclusion: Our findings suggest that the undesirable kinetics of insulin release from NPH is significantly influenced by tissue enzyme activity and that the presence of PF127 provided a timely modulation of insulin release from NPH microcrystals in the STZ-induced diabetic rat model.

Preparation and characteristics of W/O microemulsion stabilized with polyglycerylpolyricinoleate as a potential system for oral insulin delivery

Aim. The aim of the present work was to develop the composition and study the characteristics of water-in-oil microemulsion stabilized with polyglycerylpolyricinoleate — Tween 80 — ethanol mixture as a potential system for oral insulin delivery. Materials and methods. To determine the boundaries of the regions of existence of water-in-oil microemulsion in the pseudo-three component systems water — polyglycerylpolyricinoleate (PG-3-PR, Gobiotics BV, Netherlands)/ Tween 80/ethanol — paraffin oil, mixtures of paraffin oil and surfactants with oil — surfactant ratios from 9.5:0.5 to 0.5: 9.5 (wt.) were thoroughly mixed and titrated with an aqueous phase (distilled water).Compositions with the value of hydrophilic-lipophilic balance of the PG-3-PR — Tween 80 mixture equal to 6.15 were studied. Among several types of formed systems, a single-phase region corresponding to a homogeneous, optically transparent, liquid water-in-oil microemulsion was determined. The kinetic and thermodynamic stability of a number of compositions, including those containing insulin (Actrapid HM, Novo Nordisk А/С, Denmark), was studied. The values of the effective viscosity of microemulsions at different ratios of surfactant — oil and surfactant — co-surfactant were determined using a vibration viscometer. Based on the results obtained, a composition was selected to study the kinetics of insulin release into a model environment that simulated the environment of the small intestine. Insulin solution (the control sample) and the insulin-containing microemulsion were placed in the dialysis bags and immersed in 50 mL of PBS (pH 7.4) in a shaking incubator at 180 rpm and 37 ° С. At predetermined intervals, the aliquots of dissolution media were withdrawn, and the concentration of the released peptide was determined by the Bradford assay using a UV spectrophotometer at 595 nm. Results. The composition with 9:1 surfactant — co-surfactant ratio, containing 10 % of the aqueous phase (an insulin solution with a concentration of 100 IU / ml), which remained stable both during three cycles of freezing/thawing and heating/cooling, and after long-term storage at room temperature, was selected to study the kinetics of in vitro release of the peptide into the model medium. The effective viscosity of the sample was 2.4±0.04 Pa.s. The microemulsion sample demonstrated a prolonged release of insulin within 48 hours of the experiment (43 %). Conclusions. As a result, the boundaries of the existence of microemulsion regions in pseudo-three — component systems water — polyglycerylpolyricinoleate / Tween 80 / ethanol — paraffin oil were established, as well as the values of the effective viscosity of a number of compositions were determined. The study of the kinetic and thermodynamic stability of the obtained systems, including those containing insulin, as well as the study of the kinetics of the release of biologically active substance from the microemulsion into the model medium, allowed us to determine the optimal composition for further development of nanoscale dosage forms intended for prolonged delivery of insulin to the gastrointestinal tract.

A basal-bolus insulin regimen integrated microneedle patch for intraday postprandial glucose control.

Multiple daily insulin injections have been a common regimen worldwide for the management of diabetes mellitus but involved potential safety and compliance problems. In this context, a single integrated microneedle patch (IMP) with multiple release kinetics is demonstrated to provide better physiologic insulin coverage for postprandial glycemic excursion in a convenient and pain-free manner. The combination of rapid separating technique and multiple individual microneedle arrays provides the combined ability to efficiently deliver insulin into the skin within seconds and to independently control insulin release kinetics. In addition, the diabetic rats with a traditional breakfast-lunch-dinner lifestyle exhibit obvious intraday glucose fluctuations, while the hypoglycemic experiments indicate that the IMP is capable of simultaneous bolus and sustained insulin delivery to closely match the glucose rise that occurs in response to meals and efficiently minimize excessive fluctuations, suggesting the potential of this new transdermal insulin delivery system as substitutes for multiple daily injections.

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Introduction Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients [2]. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. For this reason, it is very important to thoroughly understand the kinetics of insulin release in response to various conditions, which can help develop effective therapy. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions. Lab-on-a-chip system In our research we present a Lab-on-a-chip system in which a pancreatic “pseudoislet” model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 6 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader. Methods All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6). Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates (“pseudoislets”). In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37oC, 5% CO2) the viability of the cells was determined using fluorescence microscope. Proliferation of the ells was measured using spectrofluorimetric multi-well plate reader after incubation with AlamarBlue solution. The “pseudoislets” were then stimulated with various glucose concentrations (imitation of health and disease state). After this, the standard ELISA test procedure was adapted to the microfluidic conditions to examine the insulin secretion profile from the obtained “pseudoislets”. Results and Conclusions It was confirmed that 15 spherical α and β-cell aggregates with diameters of 160-180 µm were obtained in one culture chamber using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was confirmed by the use of immunostaining method. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allowed to monitor cell proliferation and viability in situ during the culture. A high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, in the developed model health and disease conditions were simulated and the level of insulin secreted from this model was determined using the ELISA test. At this stage, a research model corresponding to the model in vivo conditions was obtained. The next step will be to develop a method for analyzing insulin secretion in real time (such as Surface Plasmon Resonance). This study presents basic research and in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.

Bottom-Up Fabrication of Multilayer Enteric Devices for the Oral Delivery of Peptides.

To develop a planar, asymmetric, micro-scale oral drug delivery vehicle by i) fabricating microdevice bodies with enteric materials, ii) efficiently and stably loading sensitive drug molecules, and iii) capping microdevices for controlled drug release. Picoliter-volume inkjet printing was used to fabricate microdevices through additive manufacturing via drop-by-drop deposition of enteric polymer materials. Microdevice bodies with reservoirs are fabricated through deposition of an enteric polymer, Eudragit FS 30 D. A model API, insulin, was loaded into each microdevice and retained its stability during printing and release. Eudragit L 100 and/or S 100 were used to cap microdevices and control the kinetics of insulin release in simulated intestinal conditions. Microdevice morphologies and size can be tuned on the fly based on printing parameters to span from the microscale to the mesoscale. Insulin retained its stability throughout device fabrication and during in vitro release in simulated intestinal conditions. Insulin release kinetics, from burst release to no release, can be tailored by controlling the blend of the Eudragit capping material. This approach represents a uniquely scalable and flexible strategy for microdevice fabrication that overcomes limitations in loading sensitive biologics and in the tuneability of device geometries that are inherent to traditional microfabrication strategies.

Nutritional recovery from a low-protein diet during pregnancy does not restore the kinetics of insulin secretion and Ca2+ or alterations in the cAMP/PKA and PLC/PKC pathways in islets from adult rats.

We investigated the insulin release induced by glucose, the Ca2+ oscillatory pattern, and the cyclic AMP (cAMP)/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC) pathways in islets from adult rats that were reared under diets with 17% protein (C) or 6% protein (LP) during gestation, suckling, and after weaning and in rats receiving diets with 6% protein during gestation and 17% protein after birth (R). First-phase glucose-induced insulin secretion was reduced in LP and R islets, and the second phase was partially restored in the R group. Glucose stimulation did not modify intracellular Ca2+ concentration, but it reduced the Ca2+ oscillatory frequency in the R group compared with the C group. Intracellular cAMP concentration was higher and PKA-Cα expression was lower in the R and LP groups compared with the C group. The PKCα content in islets from R rats was lower than that in C and LP rats. Thus, nutritional recovery from a low-protein diet during fetal life did not repair the kinetics of insulin release, impaired Ca2+ handling, and altered the cAMP/PKA and PLC/PKC pathways.

Towards the Development of a Real‐time Insulin Biosensor

Disease management for Type 1 Diabetes (T1D) patients can be challenging, and more than two-thirds of the diabetic population in the United States fails to maintain optimal blood glucose levels. The artificial pancreas (AP), automates disease management by automatically delivering insulin with dosage adjusted in real-time as a response to blood glucose levels. However, despite automation using advanced control algorithms, patients reliant on the AP risk hyper and hypoglycemia, which can result in fatality or other less serious complications. Such mis-dosing can result from discrepancies between the administered and the circulating insulin levels. As such, a direct continuous monitoring of circulating insulin could tighten glucose regulation while improving safety and patient response to treatment. Thus, we aim to develop a real-time insulin biosensor for APs using nature's own insulin sensor, the insulin receptor (InR). We plan to engineer our biosensor modeled upon the InR ectodomain using phage-displayed libraries with 109 different InR variants and iterative rounds of selections. FRET and polarization assays will be used to assess InR conformational change, binding kinetics of insulin to InR variants, and device sensitivity. Support or Funding Information Juvenile Diabetes Research Foundation, The Helmsley Charitable Trust Insulin binding (kon) and release (koff) kinetics determine sensitivity of insulin monitoring over a dynamic range of concentrations. Conformational changes of the native InR are used to develop a FRET-based optical detection system for insulin binding using fusion proteins covalently attached to fluorophores. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGA composite scaffold.

Insulin is widely considered as a classical hormone and drug in maintaining energy and glucose homeostasis. Recently, insulin has been increasingly recognized as an indispensable factor for osteogenesis and bone turnover, but its applications in bone regeneration have been restricted because of the short periods of activity and uncontrolled release. In this study, we incorporated insulin-loaded poly lactic-co-glycolic-acid (PLGA) nanospheres into nano-hydroxyapatite/collagen (nHAC) scaffolds and investigated the bioactivity of the composite scaffolds in vitro and in vivo. Bioactive insulin was successfully released from the nanospheres within the scaffold, and the release kinetics of insulin could be efficiently controlled by uniform-sized nanospheres. The physical characterizations of the composite scaffolds demonstrated that incorporation of nanospheres in nHAC scaffolds using this method did not significantly change the porosity, pore diameters, and compressive strengths of nHAC. In vitro, the insulin-loaded nHAC/PLGA composite scaffolds possessed favorable biological function for bone marrow mesenchymal stem cells adhesion and proliferation, as well as the differentiation into osteoblasts. In vivo, the optimized bone regenerative capability of this composite scaffold was confirmed in rabbit mandible critical size defects. These results demonstrated successful development of a functional insulin–PLGA–nHAC composite scaffold that enhances the bone regeneration capability of nHAC.