Insulin Release Profile Research Articles

Biodegradable oligo(amidoamine/β-amino ester) hydrogels for controlled insulin delivery

An injectable biodegradable hydrogel system based on oligo(amidoamine/β-amino ester) (OAAAE) was designed and synthesized for controlled release of insulin under the physiological conditions. OAAAE was prepared in one step via the Michael-addition oligomerization of the secondary amine groups of 4,4-trimethylene dipiperidine (TMDP) with the vinyl groups of 1,8-octylene diacrylamide (ODA) and 1,6-hexane diol diacrylate (HDA). The formed oligomer was characterized by 1H NMR and gel permeation chromatography (GPC). OAAAE in aqueous solution (20 wt%) underwent a gel–sol transition in the pH range of 6.8–7.4. A complex hydrogel was formed when mixing insulin with the oligomer solution followed by increasing pH and temperature. Degradation of the hydrogel, influence of insulin on gel phase and gel strength, in vitro cytotoxicity of OAAAE, in vitro and in vivo release of insulin from the complex hydrogel were investigated. Furthermore, the in vivo release profile of insulin from the complex hydrogel was compared with that from the neutral hydrogel.

Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

BackgroundA variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.Principal FindingsTotal internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K+-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca2+ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K+ (KATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.Conclusions/SignificanceWe provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, KATP channel responsiveness and the subsequent activity of Ca2+ channels through pathways other than CDK5-mediated regulation.

Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds

Human pancreatic islet in vitro culture is very challenging and requires the presence of various extra cellular matrix (ECM) components in a three-dimensional environment, which provides mechanical and biological support. The development of such an environment is vital in providing favourable conditions to preserve human islets in long-term culture. In this study, we investigated the effects of human islet culture within various three-dimensional environments; collagen I gel, collagen I gel supplemented with ECM components fibronectin and collagen IV, and microfabricated scaffold with ECM-supplemented gel. The cultured human islets were analyzed for functionality, gene expression and hormone content following long-term in vitro culture. It was clear the incorporation of ECM components within the three-dimensional support improved prolonged culture. However, long-term and highly uniform human islet culture within a microfabricated scaffold, with controlled pore structures, coupled with the presence of ECM components, displayed an insulin release profile similar to freshly isolated islets, yielding a stimulation index of ∼1.8. Moreover, gene expression was markedly increased for all pancreatic genes, giving a ∼50-fold elevation of insulin gene expression with respect to suspension culture. The distribution and presence of pancreatic hormones was also highly elevated. These findings provide a platform for the long-term maintenance and preservation of human pancreatic islets in vitro.

In vitro evaluation of N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan for oral insulin delivery

The present investigation explores an oral insulin delivery system based on the modification of chitosan, N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan (HTCC). Synthesis of HTCC was carried out by coupling glycidyl trimethylammonium chloride (GTAC) to chitosan in aqueous medium. Quaternization was confirmed by TNBS assay, FTIR, NMR, SEM studies and zeta potential analysis. Cytotoxicity studies of the derivative were carried out by MTT assay and release profile of insulin from HTCC matrix was monitored under in vitro experimental conditions. Further biological activity and conformational stability of released insulin were confirmed using ELISA and circular dichroism studies. Adhesion studies on mucin and freshly excised rat intestinal sections were carried out to evaluate the mucoadhesive nature of the matrix. Confocal microscopy observations showed that these microparticles were capable of opening tight junctions. By exploiting the mucoadhesive and controlled drug releasing capabilities, HTCC particles seems to be a promising candidate for oral insulin delivery.

D‐Psicose, a Sweet Monosaccharide, Ameliorate Hyperglycemia, and Dyslipidemia in C57BL/6J db/db Mice

D-psicose has been implicated in glycemic control in recent animal and human studies. In this study, the effects of D-psicose on glycemic responses, insulin release, and lipid profiles were compared with those of D-glucose and D-fructose in a genetic diabetes model. C57BL/6J db/db mice were orally supplemented with 200 mg/kg BW of D-psicose, D-glucose, or D-fructose, respectively, while diabetes control or wild type mice were supplemented with water instead. D-psicose sustained weight gain by about 10% compared to other groups. The initial blood glucose level maintained from 276 to 305 mg/dL during 28 d in the D-psicose group, whereas a 2-fold increase was found in other groups (P < 0.05) among diabetic mice. D-psicose significantly improved glucose tolerance and the areas under the curve (AUC) for glucose among diabetes (P < 0.05), but had no effect on serum insulin concentration. The plasma lipid profile was not changed by supplemental monosacchrides, although the ratio of LDL-cholesterol/HDL-cholesterol was ameliorated by D-psicose. The administration of D-psicose reversed hepatic concentrations of triglyceride (TG) and total cholesterol (TC) by 37.88% and 62.89%, respectively, compared to the diabetes control (P < 0.05). The current findings suggest that D-psicose shows promise as an antidiabetic and may have antidyslipidemic effects in type 2 diabetes.

Synthesis and characterization of novel pH-, ionic strength and temperature- sensitive hydrogel for insulin delivery

A series of novel silane crosslinked hydrogel was prepared from kappa carrageenan (KC), acrylic acid (AA) using vinyltriethoxysilane (VTESi). Potassium persulphate initiated the grafting and copolymerization reactions between reactants. In addition, the condensation of the hydroxyl groups of KC and VTESi resulted into crosslinking. Novelty of this work is the use of VTESi as crosslinker for such a composition of hydrogel. The structure of prepared hydrogels was characterized by Fourier transform infrared spectroscopy. The analysis of spectra confirmed the presence of feed components in the prepared hydrogels. Thermogravimetric analysis showed an increase in the stability of the hydrogels either having high AA contents or crosslinker amount. The effect of feed components, pH (buffer, non-buffer), electrolytic media and temperature on the swelling behaviour of the hydrogels is reported here. Most promising results with high swelling ratio were observed in hydrogel having low monomeric ratio (KC:AA = 1:7). pH response of this hydrogel in acidic and neutral pH makes it suitable for drug delivery application. Insulin, a protein based drug was selected as a model drug. It requires its delivery in small intestine for proper action; therefore its release behaviour was studied in-vitro in simulated stomach and intestinal fluids. The release profile of insulin showed negligible release in simulated gastric fluid (SGF) and sustained release in simulated intestinal fluid (SIF). The obtained results are in good agreement with the swelling response of this hydrogel. The weak structure of this hydrogel makes it preferable for drug delivery, as it is able to get crumbled after releasing the drug for 6 h at neutral pH.

Controlling the degradation of pH/temperature-sensitive injectable hydrogels based on poly(β-amino ester)

Poly (ethylene glycol) (PEG) — poly(e-caprolactone(CL)-co-D,L-lactide (LA)) (PCLA-PEG-PCLA) was synthesized by ring-opening polymerization to form a temperature sensitive hydrogel triblock copolymer. Poly(β-amino ester) (PAE) obtained from 1,4-butanediol diacrylate (BDA), and 4,4′-trimethylene dipiperidine (TMDP) was used as a pH sensitive moiety to conjugate to the triblock copolymer. The physicochemical properties of the temperature-sensitive triblock and pH/temperature-sensitive pentablock copolymers (PAE-PCLA-PEG-PCLA-PAE) were characterized by 1H NMR spectroscopy and gel permeation spectroscopy. The sol-gel phase transition behavior of the PAE-PCLA-PEG-PCLA-PAE block copolymers was investigated. An aqueous solution of the copolymers (20 wt%) changed from a sol phase at pH 6.4 and 10°C to a gel phase at pH 7.4 and 37°C. In addition, the degradation of PAE-PCLA-PEG-PCLA-PAE was compared with that of poly(ethylene glycol) — poly(e-caprolactone) — poly (β-amino ester) (PAE-PCL-PEG-PCL-PAE) using both in vitro and in vivo experiments. The relationship between the insulin release profile from the matrix and the degradation of these materials also investigated.

Glucose-responsive composite microparticles based on chitosan, concanavalin A and dextran for insulin delivery

Glucose-responsive systems are very useful for self-regulated insulin delivery. In this study, glucose-responsive composite microparticles based on chitosan, concanavalin A and dextran were prepared by first fabricating chitosan microparticles subsequently coupling concanavalin A via Schiff-base reaction and dextran layer via specific affinity. SEM, fluorescence microscope, XPS and TGA were used to characterize the morphology and composition of the microparticles. SEM results indicated that the microparticles could exist individually and in a spherelike shape. The fluorescence image showed that the microparticles were well dispersed with an average diameter of 2.5 μm. The XPS and TGA data confirmed the composite layers of the microparticles. Insulin could be loaded into the microparticles due to electrostatic and intermolecular interaction with high entrapment efficiency of 92.2% and loading capacity of 9.1%. The release profiles of insulin revealed that the insulin release was in response to the glucose concentration in the medium and the glucose sensitivity was reversible. The released insulin was proved to remain active through the whole process. The results suggest that this composite microparticle may be a promising system for self-regulated insulin delivery.

Release profile of insulin entrapped on mesoporous materials by freeze–thaw method

Adsorption profiles of insulin on porous materials and release profiles of insulin entrapped on folded-sheet mesoporous silica (FSM) were studied. Three types of FSM with different pore sizes (3.0, 6.1, and 9.2 nm) were used as candidates. A simple technique of repeated freezing and thawing resulted in effective adsorption of insulin on mesoporous structures. The amount of adsorbed insulin, estimated by protein assay, increased with an increase in the pore sizes of FSM used. Nitrogen sorption analysis showed that the specific surface area and pore volume decreased according to the insulin adsorption. On the release profile of insulin, the smallest pore size of FSM (3.0) was found to be a suitable material for a fast release of insulin, whereas the medium-pore FSM (6.1) held the insulin inside the pores for a longer time. Consequently, the desired release of insulin could be achieved by selecting the appropriate pore size of FSM.

Preparation, characterization, and in vivo evaluation of insulin-loaded PLA–PEG microspheres for controlled parenteral drug delivery

Aim: The aim of this study was to prepare insulin-loaded poly(lactic acid)–polyethylene glycol microspheres that could control insulin release at least for 1 week and evaluate their in vivo performance in a streptozotocin-induced diabetic rat model. Methods: The microspheres were prepared using a water-in-oil-in-water double emulsion solvent evaporation technique. Different formulation variables influencing the yield, particle size, entrapment efficiency, and in vitro release profiles were investigated. The pharmacokinetic study of optimized formulation was performed with single dose in comparison with multiple dose of Humulin® 30/70 as a reference product in streptozotocin-induced diabetic rats. Results: The optimized formulation of insulin microspheres was nonporous, smooth-surfaced, and spherical in structure under scanning electron microscope with a mean particle size of 3.07 ×μm and entrapment efficiency of 42.74% of the theoretical amount incorporated. The in vitro insulin release profiles was characterized by a bimodal behavior with an initial burst release because of the insulin adsorbed on the microsphere surface, followed by slower and continuous release corresponding to the insulin entrapped in polymer matrix. Conclusions: The optimized formulation and reference were comparable in the extent of absorption. Consequently, these microspheres can be proposed as new controlled parenteral delivery system.

Preparation and characterization of insulin nanoparticles using chitosan and Arabic gum with ionic gelation method

In the past decade, many strategies have been developed to enhance oral protein delivery. The aim of the current work was to develop a nanoparticulate system based on ionic gelation between chitosan and Arabic gum for loading of insulin. Various formulations were prepared using 2 3 factorial designs. The optimum association efficiency was obtained for formulations F2, F5, and F8. The release profile of insulin in phosphate buffer solutions (pH 6.5 and pH 7.2) is completely different than that in acidic medium (pH 1.2). Increased solubility of chitosan in acidic medium and better swelling of Arabic gum chains at pH >6.5 resulted in lower insulin release of nanoparticles at pH 6.5 in comparison with that of the other pH mediums. The values of the exponent n were 0.49 and 0.82 for formulations F8 and F5, respectively, indicating a non-Fickian transport. This suggests that release is possibly controlled by diffusion or relaxation of the polymer chains. From the Clinical Editor This paper summarizes the development of a nanoparticulate system based on ionic gelation between chitosan and gum Arabic for oral delivery of insulin. If preclinical studies in animal models will indicate reliable and quantifiable delivery of insulin, this method may pave the way to a novel and less invasive way of administering insulin to diabetes patients.

Amphiphilic Random Glycopolymer Based on Phenylboronic Acid: Synthesis, Characterization, and Potential as Glucose-Sensitive Matrix

This study is devoted to developing amphiphilic, random glycopolymers based on phenylboronic acid, which self-assemble to form nanoparticles (NPs), as a glucose-sensitive agent. Maleimide-glucosamine was copolymerized with 3-acryl aminophenylboronic acid in methanol at 70 degrees C. Using the nanoprecipitation method, NPs with a narrow size distribution were successfully generated. Transmission electron microscopic analysis showed that the NPs were well dispersed as individual, spherically shaped particles. The swelling behavior of the NPs and the in vitro release profiles of insulin at different glucose concentrations revealed definite glucose sensitivity of the glycopolymers. Further, circular dichroism spectroscopy demonstrated that the overall tertiary structure of the released insulin was not altered compared with standard insulin. The analysis of relative cell proliferation suggested that the glycopolymer NPs had good biocompatibility. The glycopolymers that responded to changes in the glucose concentration of the surrounding environment are being aimed for use in self-regulated insulin delivery.

Quantitative Monitoring of Insulin Secretion from Single Islets of Langerhans in Parallel on a Microfluidic Chip

Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.

Controlled release of insulin from pH/temperature-sensitive injectable pentablock copolymer hydrogel

A pH- and temperature-sensitive hydrogel of poly(β-amino ester)-poly(ε-caprolactone)-poly (ethylene glycol)-poly(ε-caprolactone)-poly(β-amino ester) (PAE-PCL-PEG-PCL-PAE) pentablock copolymer was evaluated as a sustained injectable insulin delivery system. Insulin was readily loaded into the matrix, forming an ionically linked insulin–PAE complex. Complex mixtures containing various concentrations of insulin and copolymer were subcutaneously injected into male Sprague–Dawley rats to study the profile of insulin release in vivo. The insulin-release profile showed that insulin was maintained at a constant steady-state level for 15 days, and further demonstrated that insulin levels were controlled by the amount of insulin loaded into the copolymer and the copolymer concentration in the hydrogel. The effect of the insulin–gel complex was further investigated in the streptozotocin (STZ) diabetic rat model. After subcutaneously injecting complex mixtures into STZ-induced diabetic rats, blood glucose and plasma insulin levels were measured. The results showed that the diabetic rats could be treated for more than 1 week with a single injection of the complex mixture containing 10 mg/mL insulin in a 30 wt.% copolymer solution, suggesting that this pH/temperature-sensitive insulin–hydrogel complex system may have therapeutic potential.

Oral insulin delivery using nanoparticles based on microemulsions with different structure-types: Optimisation and in vivo evaluation

The purpose of this study was to optimise entrapment of insulin in poly(alkylcyanoacrylate) nanoparticles prepared from microemulsions with different microstructure containing isopropyl myristate, caprylocaproyl macrogolglycerides, polyglyceryl oleate and insulin solution and to investigate the in vitro release and bioactivity of insulin in nanoparticles dispersed in the microemulsion templates. Entrapment efficiency and release of insulin were studied using a reverse-phase HPLC assay. Morphology of the nanoparticles was examined with scanning electron microscopy. Bioactivity of insulin was studied using a streptozotocin-diabetic rat model. Nanoparticles were spherical with 200–400 nm in size without significant difference between different microemulsion templates, types and amounts of monomer. Entrapment efficiency increased significantly with increasing monomer concentration but decreased with increasing aqueous fraction in the microemulsion template. Insulin loading however, showed an opposite trend. In vitro release profiles of insulin from the nanoparticles dispersed in the microemulsion templates were controlled by the monomer concentration only. In vivo, a consistent and significant hypoglycemic effect over controls was found for up to 36 h depending on the type of monomer. No significant serum insulin levels were detectable. This study showed that the strategy of delivering insulin orally, entrapped in nanoparticles and dispersed in a biocompatible microemulsion is promising and highlights the importance of optimisation studies in combination with in vivo experiments.

Oral Delivery of Peptide Drugs Using Nanoparticles Self-Assembled by Poly(γ-glutamic acid) and a Chitosan Derivative Functionalized by Trimethylation

In the study, chitosan (CS) was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) polymers with different degrees of quaternization. Nanoparticles (NPs) self-assembled by the synthesized TMC and poly(gamma-glutamic acid) (gamma-PGA, TMC/gamma-PGA NPs) were prepared for oral delivery of insulin. The loading efficiency and loading content of insulin in TMC/gamma-PGA NPs were 73.8 +/- 2.9% and 23.5 +/- 2.1%, respectively. TMC/gamma-PGA NPs had superior stability in a broader pH range to CS/gamma-PGA NPs; the in vitro release profiles of insulin from both test NPs were significantly affected by their stability at distinct pH environments. At pH 7.0, CS/gamma-PGA NPs became disintegrated, resulting in a rapid release of insulin, which failed to provide an adequate retention of loaded insulin, while the cumulative amount of insulin released from TMC/gamma-PGA NPs was significantly reduced. At pH 7.4, TMC/gamma-PGA NPs were significantly swelled and a sustained release profile of insulin was observed. Confocal microscopy confirmed that TMC40/gamma-PGA NPs opened the tight junctions of Caco-2 cells to allow the transport of insulin along the paracellular pathway. Transepithelial-electrical-resistance measurements and transport studies implied that CS/gamma-PGA NPs can be effective as an insulin carrier only in a limited area of the intestinal lumen where the pH values are close to the p K a of CS. In contrast, TMC40/gamma-PGA NPs may be a suitable carrier for transmucosal delivery of insulin within the entire intestinal tract.

Polyelectrolyte Biomaterial Interactions Provide Nanoparticulate Carrier for Oral Insulin Delivery

Nanospheres are being developed for the oral delivery of peptide-based drugs such as insulin. Mucoadhesive, biodegradable, biocompatible, and acid-protective biomaterials are described using a combination of natural polyelectrolytes, with particles formulated through nanoemulsion dispersion followed by triggered in situ gel complexation. Biomaterials meeting these criteria include alginate, dextran, chitosan, and albumin in which alginate/dextran forms the core matrix complexed with chitosan and albumin coat. Smaller size and higher albumin-based acid-protective formulation was orally administered to diabetic rats and glucose reduction and physiological response analyzed. Insulin encapsulation efficiency was 90, 82, and 66% for uncoated, chitosan-coated, and albumin-chitosan-coated alginate nanospheres, respectively. The choice of coating polymer seems to influence insulin release profile and to be crucial to prevent peptic digestion. Physiological response following oral delivery showed that insulin albumin-chitosan-coated alginate nanospheres reduced glycemia ∼ 72% of basal values. Albumin serves as an important enteric coating providing acid- and protease protection enabling uptake of active drug following oral dosage.

Insulin-loaded alginate microspheres for oral delivery – Effect of polysaccharide reinforcement on physicochemical properties and release profile

Oral administration of insulin requires protein protection from degradation in the gastric environment and its absorption improvement in the intestinal tract. To achieve this objective several types of microspheres composed of alginate, chitosan and dextran sulphate have been prepared by ionotropic gelation. Parameters such as the mean particle size, swelling behaviour, insulin encapsulation efficiency, loading capacity and release profiles in simulated gastric and intestinal fluids have been compared for the systems developed. In this study, attempts have been made to increase insulin protection and to improve its release from microspheres by reinforcing the alginate matrix with chitosan and/or dextran sulphate. Dextran sulphate was able to avoid insulin release at pH 1.2, protecting the protein from the acidic environment and reducing the total insulin released at pH 6.8. This effect was explained by an interaction between the permanent negatively charged groups of dextran sulphate and insulin molecules.

Preparation and Characterization of Nanoparticles Shelled with Chitosan for Oral Insulin Delivery

Nanoparticles (NPs) composed of chitosan (CS) and poly(gamma-glutamic acid) (gamma-PGA) were prepared by a simple ionic-gelation method for oral insulin delivery. Fourier transform infrared (FT-IR) spectra indicated that CS and gamma-PGA were ionized at pH 2.5-6.6, while X-ray diffractograms demonstrated that the crystal structure of CS was disrupted after it was combined with gamma-PGA. The diameters of the prepared NPs were in the range of 110-150 nm with a negative or positive surface charge, depending on the relative concentrations of CS to gamma-PGA used. The NPs with a positive surface charge (or shelled with CS) could transiently open the tight junctions between Caco-2 cells and thus increased the paracellular permeability. After loading of insulin, the NPs remained spherical and the insulin release profiles were significantly affected by their stability in distinct pH environments. The in vivo results clearly indicated that the insulin-loaded NPs could effectively reduce the blood glucose level in a diabetic rat model.

Modelling the effect of CSII on the control of glucose concentration in type 1 diabetes

The profile of insulin release in continuous subcutaneous insulin infusion (CSII) plays a major role in regulating blood glucose in type 1 diabetes. We develop a mathematical model to incorporate the effect of an intermittent insulin supply on glucose concentration, as a function of time for the dual wave bolus technique. The rate of exogenous insulin release is controlled by a threshold of glucose concentration, determined by the characteristics of the patient’s body. The results of model simulations provide the optimal profile of insulin release over a specific period of time. Only marginal decrease in the time required for lowering blood glucose level is observed when the total daily dosage (TDD) is greatly increased. This may also be of less benefit in terms of the amount of insulin secreted in the body and will in turn lead to a glucose level outside the physiological range, due to a delay in the glucose metabolism. We also analyze the model to determine the conditions under which oscillatory dynamics of glucose concentration can be observed. Consistent with other modelling studies, the results suggest that an oscillatory insulin supply will be more efficient in producing a sustained physiological response than a constant supply.