Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose‐responsive polymer‐based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15–31 mM (K_d), which match physiological glucose fluctuations. The dose‐responsive glucose‐mediated release of the novel insulins was demonstrated using glucamine‐derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%–122%) which is unusual for insulin LysB29 derivatives. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of stimulus responsive multiple emulsions by membrane emulsification using con a as biochemical sensor

In this work, a novel strategy for the controlled fabrication of biomolecular stimulus responsive water-in-oil-in-water (W/O/W) multiple emulsion using the membrane emulsification process was investigated. The emulsions interface was functionalized with a biomolecule able to function as a receptor for a target compound. The interaction between the biomolecular receptor and target stimulus activated the release of bioactive molecules contained within the structured emulsion. A glucose sensitive emulsion was investigated as a model study case. Concanavalin A (Con A) was used as the biomolecular glucose sensor. Various physicochemical strategies for stimulus responsive materials formulation are available in literature, but the preparation of biomolecule-responsive emulsions has been explored for the first time in this paper. The development of novel drug delivery systems requires advanced and highly precise techniques to obtain their particular properties and targeting requirements. The present study has proven the flexibility and suitability of membrane emulsification for the preparation of stable and functional multiple emulsions containing Con A as interfacial biomolecular receptor able to activate the release of a bioactive molecule as a consequence of interaction with the glucose target molecule. The influence of emulsion interfacial composition and membrane emulsification operating conditions on droplets stability and functional properties have been investigated. The release of the bioactive molecule as a function of glucose stimulus and its concentration has been demonstrated.     

Synthesis and characterization of alginate coated zinc calcium phosphate nanoparticles for intestinal delivery of insulin

Nanosized calcium phosphates studied as drug delivery systems are highly compatible with the various drugs like insulin, antibiotics etc. Zinc is an essential trace element that plays a crucial role in the synthesis, storage and release of insulin in a human body. Therefore, an attempt has been made to develop zinc modified calcium phosphate nanoparticles (less than 100 nm) as carriers for intestinal delivery of insulin. The insulin loaded nanoparticles were coated with pH sensitive alginate. These pH sensitive nanoparticles released insulin in the intestinal medium, and the conformation of released insulin was stable. The blood glucose level of diabetic rats came to normal on administration of the formulation. With the beneficial effect of zinc reported on diabetic patients, the present system seems to be an excellent carrier for intestinal delivery of insulin.     

A reversible hydrogel membrane for controlling the delivery of macromolecules

Glucose-sensitive hydrogel membranes have been synthesized and characterized for their rate-of-delivery of macromolecules. The mechanism for changing this rate is based on variable displacement of the affinity interaction between dextran and concanavalin A (con A). Our main objective was to characterize the diffusion of model proteins (insulin, lysozyme, and BSA) through the membrane, in response to changes in environmental glucose concentrations. Membranes were constructed from crosslinked dextrans to which con A was coupled via a spacer arm. Changes in the porosity of the resulting hydrogel in the presence of glucose led to changes in the diffusion rate observed for a range of proteins. Gels of specified thickness were cast around to nylon gauze support (pore size, 0.1 mm) to improve mechanical strength. Diffusion of proteins through the gel membrane was determined using a twin-chamber diffusion cell with the concentrations being continuously monitored using a UV-spectrophotometer. Changes in the transport properties of the membranes in response to glucose were explored and it was found that, while 0.1M D-glucose caused a substantial, but saturateable, increase in the rates of diffusion of both insulin and lysozyme, controls using glycerol or L-glucose (0.1M) had no significant effect. Sequential addition and removal of external glucose in a stepwise manner showed that permeability changes were reversible. As expected, diffusion rates were inversely proportional to membrane thickness. A maximum increase in permeability was observed at pH 7.4 and at 37 degrees C. The results demonstrate that this hydrogel membrane functions as a smart material allowing control of solute delivery in response to specific changes in its external environment.     

A Review of Multi-Responsive Membranous Systems for Rate-Modulated Drug Delivery

Membrane technology is broadly applied in the medical field. The ability of membranous systems to effectively control the movement of chemical entities is pivotal to their significant potential for use in both drug delivery and surgical/medical applications. An alteration in the physical properties of a polymer in response to a change in environmental conditions is a behavior that can be utilized to prepare ‘smart’ drug delivery systems. Stimuli-responsive or ‘smart’ polymers are polymers that upon exposure to small changes in the environment undergo rapid changes in their microstructure. A stimulus, such as a change in pH or temperature, thus serves as a trigger for the release of drug from membranous drug delivery systems that are formulated from stimuli-responsive polymers. This article has sought to review the use of stimuli-responsive polymers that have found application in membranous drug delivery systems. Polymers responsive to pH and temperature have been extensively addressed in this review since they are considered the most important stimuli that may be exploited for use in drug delivery, and biomedical applications such as in tissue engineering. In addition, dual-responsive and glucose-responsive membranes have been also addressed as membranes responsive to diverse stimuli.     

Leptin administration improves skeletal muscle insulin responsiveness in diet-induced insulin-resistant rats

In addition to suppressing appetite, leptin may also modulate insulin secretion and action. Leptin was administered here to insulin-resistant rats to determine its effects on secretagogue-stimulated insulin release, whole body glucose disposal, and insulin-stimulated skeletal muscle glucose uptake and transport. Male Wistar rats were fed either a normal (Con) or a high-fat (HF) diet for 3 or 6 mo. HF rats were then treated with either vehicle (HF), leptin (HF-Lep, 10 mg. kg(-1). day(-1) sc), or food restriction (HF-FR) for 12-15 days. Glucose tolerance and skeletal muscle glucose uptake and transport were significantly impaired in HF compared with Con. Whole body glucose tolerance and rates of insulin-stimulated skeletal muscle glucose uptake and transport in HF-Lep were similar to those of Con and greater than those of HF and HF-FR. The insulin secretory response to either glucose or tolbutamide (a pancreatic beta-cell secretagogue) was not significantly diminished in HF-Lep. Total and plasma membrane skeletal muscle GLUT-4 protein concentrations were similar in Con and HF-Lep and greater than those in HF and HF-FR. The findings suggest that chronic leptin administration reversed a high-fat diet-induced insulin-resistant state, without compromising insulin secretion.     

New route for synthesizing poly(ethylene glycol)-acrylic acid hydrogels using γ-irradiation for drug delivery carriers

Biocompatible and pH-responsive poly(ethylene glycol) (PEG)-acrylic acid (AAc) hydrogels were prepared by new technique using γ-irradiation for controlled oral drug delivery. The gel fraction was over 80% and the equal amounts of PEG and AAc blended hydrogel had efficient insulin loading using equilibrium swelling. These hydrogels exhibited unique pH-responsive characteristics in which interpolymer complexes were formed in acidic media and dissociated in neutral or basic environments. The insulin release from the gel was significantly retarded in acidic media while rapid release occurred under neutral/basic conditions. At the high pH solution, the gels swelled rapidly and over 70% of the insulin loaded was released over a period of 10 h. Within 2 h of administration of the insulin-containing gels, significant blood glucose reduction effects were observed in diabetic rats. The blood glucose reduction lasted for up to 10 h following administration.     

Numerical simulation of a glucose sensitive composite membrane closed-loop insulin delivery system

Closed-loop insulin delivery system works on pH modulation by gluconic acid production from glucose, which in turn allows regulation of insulin release across membrane. Typically, the concentration variation of gluconic acid can be numerically modeled by a set of non-linear, non-steady state reaction diffusion equations. Here, we report a simpler numerical approach to time and position dependent diffusivity of species using finite difference and differential quadrature (DQ) method. The results are comparable to that obtained by analytical method. The membrane thickness directly determines the concentrations of the glucose and oxygen in the system, and inversely to the gluconic acid. The advantage with the DQ method is that its parameter values need not be altered throughout the analysis to obtain the concentration profiles of the glucose, oxygen and gluconic acid. Our work would be useful for modeling diabetes and other systems governed by such non-linear and non-steady state reaction diffusion equations.     

Investigation of insulin loaded self-assembled microtubules for drug release

Self-assembled microtubules were used to entrap insulin for the preparation of new drug delivery devices. The interactions of insulin with the microtubules were probed by circular dichroism, zeta potential analysis, as well as FTIR spectroscopy. The morphologies of the insulin-loaded tubules were examined by AFM and TEM. We found that insulin loading was both pH- as well as concentration-dependent. The circular dichroism analysis indicated that, at pH range 6-7, the conformation change in the presence of the microtubules was minimal and hence would be the most appropriate conditions for insulin loading. The entrapment efficiency and release of insulin was found to be pH-dependent. Further, the controlled drug release studies indicated that, under acidic conditions, insulin release was extremely slow, and it is likely that the insulin is protected inside the microtubules. Thus, the microtubules may potentially protect the insulin from aggregation and release at lower pH (gastric pH) in ViVo. However, at pH 6.5 (closer to intestinal pH) a sustained release was observed. Such new materials may inhibit the aggregation of peptides under suitable conditions and potentially be used for drug delivery, in particular, for other peptide-based drugs.     

Alginate microparticles as novel carrier for oral insulin delivery

Alginate microparticles produced by emulsification/internal gelation were investigated as a promising carrier for insulin delivery. The procedure involves the dispersion of alginate solution containing insulin protein, into a water immiscible phase. Gelation is triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size is controlled through the emulsification parameters, yielding insulin-loaded microparticles. Particle recovery was compared using several washing protocols. Recovery strategies are proposed and the influence on particle mean size, morphology, recovery yield (RY), encapsulation efficiency, insulin release profile, and structural integrity of released insulin were evaluated. Spherical micron-sized particles loaded with insulin were produced. The recovery process was optimized, improving yield, and ensuring removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone/hexane/isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a RY around 70%. In vitro release studies showed that alginate itself was not able to suppress insulin release in acidic media; however, this strategy preserves the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery.     

Radiation crosslinked psyllium and polyacrylic acid based hydrogels for use in colon specific drug delivery

In order to utilize the psyllium husk, a medicinally important natural polysaccharide, to develop the hydrogels meant for the drug delivery, we have prepared psyllium and polyacrylic acid based polymeric networks by radiation-induced crosslinked copolymerization. Polymeric networks (hydrogels) thus formed were characterized with SEMs, FTIR and swelling studies. Swelling behavior of the hydrogels was studied as a function of monomer concentration in the hydrogels and temperature, pH and [NaCl] of the swelling medium. This paper discusses the swelling kinetics of the hydrogels and release dynamics of anticancer model drug 5-fluorouracil from the hydrogels for the evaluation of swelling and drug release mechanisms. It has been observed from the release dynamics of drug that diffusion exponent ‘n’ have 0.7, 0.8 and 0.7 values and gel characteristics constant ‘k’ have 9.13 × 10⁻³, 6.22 × 10⁻³ and 9.01 × 10⁻³ values for the release of 5-fluorouracil, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer. The values of the diffusion exponent show that the release of drug from drug-loaded hydrogels has occurred through Non-Fickian diffusion mechanism. It has also been observed from the swelling and release of drug in the different pH buffer that the polymer matrix is pH responsive and can be exploited for the delivery of anticancer drug to the colon.     

Oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-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.     

Human Insulin Microcrystals with Lactose Carriers for Pulmonary Delivery

Dry powder formulations for pulmonary delivery are attractive because many issues of solubility and stability can be minimized. Human insulin microcrystals with lactose carriers were produced for pulmonary delivery. The average particle diameter was 2.3 μm, with a narrow, monodispersed size distribution. The percentages of high molecular weight proteins (%HMWPs), other insulin-related compounds (%OIRCs), and A-21 desamido insulin (%D_es) were very low throughout the microcrystal preparation process. Administration of the microcrystal powder by intratracheal insufflation significantly reduced the blood glucose levels of Sprague-Dawley rats. The percent minimum reductions of the blood glucose concentration (%MRBG) produced by the insulin microcrystal powder and by an insulin solution reached 40.4% and 33.4% of the initial glucose levels respectively, and their bioavailability relative to subcutaneous injection (F) was 15% and 10% respectively. These results confirm that the insulin microcrystal powder prepared is suitable for pulmonary delivery in an effective dosage form.     

Eudragit S100 entrapped insulin microspheres for oral delivery

The purpose of this research was to investigate whether Eudragit S100 microspheres have the potential to serve as an oral carrier for peptide drugs like insulin. Microspheres were prepared using water-in oil-in water emulsion solvent evaporation technique with polysorbate 20 as a dispersing agent in the internal aqueous phase and polyvinyl alcohol (PVA)/polyvinyl pyrrolidone as a stabilizer in the external aqueous phase. The use of smaller internal aqueous-phase volume (50 μL) and external aqueous-phase volume (25 mL) containing PVA in the manufacturing process resulted in maximum encapsulation efficiency (81.8%±0.9%). PVA-stabilized microspheres having maximum drug encapsulation released 2.5% insulin at pH 1.0 in 2 hours. In phosphate buffer (pH 7.4), microspheres showed an initial burst release of 22% in 1 hour with an additional 28% release in the next 5 hours. The smaller the volumes of internal and external aqueous phase, the lower the initial burst release. The release of drug from microspheres followed Higuchi kinetics. Scanning electron microscopy of PVA-stabilized microspheres demonstrated spherical particles with smooth surface, and laser diffractometry revealed a mena particle size of 32.51±20 μm. Oral administration of PVA stabilized microspheres in normal albino rabbits (equivalent to 6.6 IU insulin/kg of animal weight) demonstrated a 24% reduction in blood glucose level, with maximum plasma glucose reduction of 76±3.0% in 2 hours and effect continuing up to 6 hours. The area under the percentage glucose reduction-time curve was 93.75%. Thus, our results indicate that Eudragit S100 microspheres on oral administration can protect insulin from proteolytic degradation in the gastrointestinal tract and produce hypoglycemic effect.     

The influence of the copolymer composition on the diltiazem hydrochloride release from a series of pH-sensitive poly[(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide)-<Emphasis Type="Italic">co</Emphasis>-(methacrylic acid)] hydrogels

A series of poly[(N-isopropylacrylamide)-co-(methacrylic acid)] (P[(N-iPAAm)-co-(MAA)]) hydrogels was investigated to determine the composition that exhibits a better pH-modulated release of diltiazem hydrochloride (DIL.HCl). For this purpose hydrogel slabs were loaded with DIL.HCl by the immersion method, and its release under acidic medium (0.1N HCl, pH 1.2) and in phosphate buffer pH 7.2, using United States Pharmacopeia (USP) 24 Apparatus 1, was investigated. According to the results from the slabs, copolymers with 85% mol N-iPAAm content were selected to prepare tablets with different particle size. The effect of pH and particle size changes on DIL.HCl release from these last hydrogel tablets was investigated by a stepwise pH variation of the dissolution medium. The amount of DIL.HCl released from high N-iPAAm content copolymer slabs under acidic pH medium was not only very low but it was also released at a slow rate. In the 85% N-iPAAm tablets, significant differences between and within release profiles were found as a function of particle size and pH, respectively. A relationship between particle size and release rate has been found. The lower DIL.HCl release at acidic pH from enriched N-iPAAm copolymers is interpreted by a cooperative thermal- and pH-collapse. Although for the whole range of copolymer composition a dependence of the equilibrium of swelling on the pH was found, DIL.HCl release experiments indicated that hydrogels with 85% mol N-iPAAm are the more adequate to be used for modulated drug delivery systems. Additionally, the particle size of the tablet can be used to tailor the release rate.     

Carbohydrate inhibitors of concanavalin A that inhibit binding of insulin-sepharose to fat cells and antagonize and mimic insulin's bioactivity. A possible role for membrane carbohydrate in insulin's action.

A consistent pattern of insulin-like properties is expressed by a variety of glycoside inhibitors of concanavalin A (Con A), and is suggestive of a common mechanism of action to explain these effects. Various exogenously added glycoside derivatives inhibit the binding of insulin-Sepharose beads to insulin receptors on isolated intact rat fat cells with a specificity resembling that for Con A-Sepharose binding to these cells. A more limited number of glycosides tested were also found to inhibit the binding of 125I-insulin, although some enhancement of binding that preceded the inhibition was observed for some of these saccharides. The glycosides also antagonize insulin-stimulated glucose utilization by the cells, but in some cases also mimic the hormone by stimulating glucose utilization. A few glycosides mimic insulin without appearing to antagonize its bioactivity. Radiolabeled glycoside inhibitors fail to bind to insulin in equilibrium dialysis experiments although they readily bind to Con A, indicating that the glycosides act directly on the cell rather than on the insulin molecule. The latter observation is consistent with the ability of those glycosides that act like insulin to do so independent of the hormone. In view of the known insulin-like properties of Con A, the effects of the glycosides seen in the present study suggest roles for a membrane carbohydrate and a carbohydrate binding site in the mechanisms of action of both insulin and Con A. In addition to various alternative explanations, a working hypothesis is presented to rationalize the present observations. It proposes that the effects of the exogenously added glycosides (and Con A) may reflect the presence on the membrane of a native carbohydrate moiety by either mimicking or competitively inhibiting its ability to interact reversibly with a lectin-like carbohydrate binding site associated with the function of the insulin receptor.20     

Substitutes for the endocrine pancreas

Since it is generally accepted that a tight metabolic control might prevent the chronic complications of diabetes mellitus, several systems have been developed in which insulin is administered in a manner that mimicks the characteristics of insulin secretion. 1) In the so-called closed-loop systems, insulin delivery is regulated by the concomitant plasma glucose level. These systems, which involve continuous measurement of plasma glucose concentration are not to date implantable and have been used so far only for short-term studies. 2) By contrast, in the "open-loop systems", no glucose sensor is needed and insulin delivery is pre-programmed to achieve a constant basal infusion with peaks of insulin delivery during the meal periods. These systems have been used in man for several months. The present achievement and limitations of both kinds of systems are discussed in this review.     

Glucose-responsive vehicles containing phenylborate ester for controlled insulin release at neutral pH

This study is devoted to developing amphiphilic block polymers based on phenylborate ester, which can self-assemble to form nanoparticles, as a glucose-sensitive drug carrier. Poly(ethylene glycol)-block-poly[(2-phenylboronic esters-1,3-dioxane-5-ethyl) methylacrylate] (MPEG5000-block-PBDEMA) was fabricated with MPEG5000-Br as a macroinitiator via atom transfer radical polymerization (ATRP). Using the solvent evaporation method, these block polymers can disperse in aqueous milieu to self-assemble into micellar aggregates with a spherical core-shell structure. Zeta potential and fluorescence techniques analysis showed a good purification effect, high encapsulation efficiency, and loading capacity of fluorescein isothiocyanate (FITC)-insulin-loaded polymeric micelles under optimal conditions. The in vitro insulin release profiles revealed definite glucose-responsive behavior of the polymeric micelles at pH 7.4 and 37 °C, depending on the environmental glucose concentration and the chemical composition of the block polymers. Further, circular dichroism spectroscopy demonstrated that the overall tertiary structure of the released insulin was in great agreement with standard insulin. (1)H NMR results of the polymeric micelles during glucose-responsive process supposed one possible insulin release mechanism via the polymer polarity transition from amphiphilic to double hydrophilic. The analysis of L929 mouse fibroblast cells viability suggested that the polymeric micelles from MPEG5000-block-PPBDEMA had low cell toxicity. The block polymers containing phenylborate ester that responded to changes in the glucose concentration at neutral pH are being aimed for use in self-regulated insulin delivery.     

pH and reduction dual-sensitive copolymeric micelles for intracellular doxorubicin delivery

This study develops novel pH and reduction dual-sensitive micelles for the anticancer drug doxorubicin (DOX) delivery owing to the fact that the tumor tissues show low pH and high reduction environment. These sub-100 nm micelles present a core-shell structure under physiological conditions, but quickly release the loaded drugs responding to acidic and reductive stimuli. With disulfide bonds in each repeat unit of poly(β-amino ester)s, the novel copolymer was synthesized via Michael addition polymerization from 2,2'-dithiodiethanol diacrylate, 4,4'-trimethylene dipiperidine, and methoxy-PEG-NH(2). DOX released faster from micelles in a weakly acidic environment (pH 6.5) than at pH 7.4 or in the presence of a higher concentration (5 mM) of reducing agent (DTT). The release is even more effective in a scenario of both stimuli (pH 6.5 and 5 mM DTT). MTT assay showed that the DOX-loaded micelles had a higher cytotoxicity for HepG2 tumor cells than DOX at higher concentrations, and that blank micelles had a very low cytotoxicity to the tumor cells. Confocal microscopy observation showed that the micelles can be quickly internalized, effectively deliver the drugs into nuclei, and inhibit cell growth. These results present the copolymer as a novel and effective pH and reduction dual-responsive nanocarrier to enhance drug efficacy for cancer cells.     

Coupling of concanavalin A to cellulose hollow fibers for use in glucose affinity sensor

This article describes a method for the chemical immobilization of concanavalin A (Con A) on the inside wall of a single hollow cellulose fiber for use in glucose affinity sensor. Periodate oxidation of cellulose fiber followed by a spacer for Con A attachment was deemed to be the most optimal procedure for achieving the highest sensitivity of the sensor without compromising its physical integrity. The effects of variables like the duration of periodate oxidation and its concentration and pH of the spacer coupling step and its duration have been examined. The mechanical strength of the hollow fiber as well as its permeability to the analyte (glucose) have been evaluated prior to and after Con A coupling process.It has been demonstrated that Con A bound hollow fiber prepared according to the procedure outlined here can be successfully used to construct glucose affinity sensor for operation in the physiological range of glucose concentrations.