In vitro and in vivo testing of glucose-responsive insulin-delivery microdevices in diabetic rats

We have developed glucose-responsive implantable microdevices for closed-loop delivery of insulin and conducted in vivo testing of these devices in diabetic rats. The microdevices consist of an albumin-based bioinorganic membrane that utilizes glucose oxidase (GOx), catalase (CAT) and manganese dioxide (MnO(2)) nanoparticles to convert a change in the environmental glucose level to a pH stimulus, which regulates the volume of pH-sensitive hydrogel nanoparticles and thereby the permeability of the membrane. The membrane is integrated with microfabricated PDMS (polydimethylsiloxane) structures to form compact, stand-alone microdevices, which do not require tethering wires or tubes. During in vitro testing, the microdevices showed glucose-responsive insulin release over multiple cycles at clinically relevant glucose concentrations. In vivo, the microdevices were able to counter hyperglycemia in diabetic rats over a one-week period. The in vitro and in vivo testing results demonstrated the efficacy of closed-loop biosensing and rapid response of the 'smart' insulin delivery devices.     

Injectable dual glucose-responsive hydrogel-micelle composite for mimicking physiological basal and prandial insulin delivery

For type 1 and advanced type 2 diabetic patients, insulin replacement therapy with simulating on-demand prandial and basal insulin secretion is the best option for optimal glycemic control. However, there is no insulin delivery system yet could mimic both controlled basal insulin release and rapid prandial insulin release in response to real-time blood glucose changes. Here we reported an artificial insulin delivery system, mimicking physiological basal and prandial insulin secretion, to achieve real-time glycemic control and reduce risk of hypoglycemia. A phenylboronic acid(PBA)/galactosyl-based glucose-responsive insulin delivery system was prepared with insulin-loaded micelles embedded in hydrogel matrix. At the hyperglycemic state, both the hydrogel and micelles could swell and achieve rapid glucose-responsive release of insulin, mimicking prandial insulin secretion.When the glucose level returned to the normal state, only the micelles partially responded to glucose and still released insulin gradually. The hydrogel with increased crosslinking density could slow down the diffusion speed of insulin inside, resulting in controlled release of insulin and simulating physiological basal insulin secretion. This hydrogel-micelle composite insulin delivery system could quickly reduce the blood glucose level in a mouse model of type 1 diabetes, and maintain normal blood glucose level without hypoglycemia for about 24 h. This kind of glucose-responsive hydrogel-micelle composite may be a promising candidate for delivery of insulin in the treatment of diabetes.     

A gold@silica core–shell nanoparticle-based surface-enhanced Raman scattering biosensor for label-free glucose detection

The gold nanostar@silica core–shell nanoparticles conjugated with glucose oxidase (GOx) enzyme molecules have been developed as the surface-enhanced Raman scattering (SERS) biosensor for label-free detection of glucose. The surface-immobilized GOx enzyme catalyzes the oxidation of glucose, producing hydrogen peroxide. Under laser excitation, the produced H₂O₂ molecules near the Au nanostar@silica nanoparticles generate a strong SERS signal, which is used to measure the glucose concentration. The SERS signal of nanostar@silica∼GOx nanoparticle-based sensing assay shows the dynamic response to the glucose concentration range from 25 μM to 25 mM in the aqueous solution with the limit of detection of 16 μM. The sensing assay does not show any interference when glucose co-exists with both ascorbic acid and uric acid. The sensor can be applied to a saliva sample.     

Gold nanoparticles-based nanoconjugates for enhanced enzyme cascade and glucose sensing

We have coupled gold nanoparticles with horseradish peroxidase (HRP) to assemble catalytic nanoconjugates (HRP-AuNPs) for glucose detection. We found that a proper mixing ratio of HRP/AuNPs can significantly improve catalytic activity for the cascade reaction, an effect arising from increased spatial coupling between enzymes. Such gold nanoparticle-based nanoconjugates are shown to be promising nanosensors for glucose.     

Insulin Delivery from Glucose-Responsive,Self-Assembled,Polyamine Nanoparticles: Smart “Sense-and-Treat” Nanocarriers Made Easy

Polyamine–salt aggregates (PSA) are biomimetic soft materials that have attracted great attention due to their straightforward fabrication methods, high drug-loading efficiencies, and attractive properties for pH-triggered release. Herein, a simple and fast multicomponent self-assembly process was used to construct cross-linked poly(allylamine hydrochloride)/phosphate PSAs (hydrodynamic diameter of 360 nm) containing glucose oxidase enzyme, as a glucose-responsive element, and human recombinant insulin, as a therapeutic agent for the treatment of diabetes mellitus (GI-PSA). The addition of increasing glucose concentrations promotes the release of insulin due to the disassembly of the GI-PSAs triggered by the catalytic in situ formation of gluconic acid. Under normoglycemia, the GI-PSA integrity remained intact for at least 24 h, whereas hyperglycemic conditions resulted in 100 % cargo release after 4 h of glucose addition. This entirely supramolecular strategy presents great potential for the construction of smart glucose-responsive delivery nanocarriers.     

Analysis of peptides and proteins affinity-bound to iron oxide nanoparticles by MALDI MS

Iron oxide nanoparticles modified with oleate have been employed for the extraction of peptides and proteins from aqueous solution before matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Adsorption of peptides and proteins onto the nanoparticles were mainly through electrostatic attraction and hydrophobic interaction. The analyte-adsorbed iron oxide nanoparticles could be efficiently collected from solution using a magnet. No elution step was needed. With this preconcentration strategy, the lowest detectable concentration of angiotensin I, insulin, and myoglobin in 500 microL of aqueous solution were 0.1 nM, 0.1 nM, and 10.0 nM, respectively. In addition, the nanoparticles could extract the analytes from solution with a high content of salt and surfactant, thus eliminating suppression effect during MALDI MS analysis. This method was successfully applied to concentrate the tryptic digest products of cytochrome c. In addition, the tryptic digestion of cytochrome c can be directly conducted on the iron oxide nanoparticles.     

A glucose-responsive controlled release of insulin system based on enzyme multilayers-coated mesoporous silica particles

A novel glucose-responsive controlled release of insulin system is constructed through coating enzyme multilayers on mesoporous silica particles (MSPs). The MSPs serve as the drug reservoir, and the enzyme multilayers cross-linked with glutaraldehyde act as a valve to control the release of insulin in response to the external glucose level.     

Designed glucose-responsive microgels with selective shrinking behavior

We report on the synthesis of various glucose-responsive microgels based on N-alkylacrylamide derivatives and phenylboronic acid (PBA) as a glucose sensing moiety. Depending on their chemical composition, the microgels exhibit opposite behaviors in response to glucose concentration increase: they can either swell or shrink, using two different mechanisms for glucose recognition. Both behaviors may be suitable for glucose sensing and insulin delivery. When glucose binds a single boronate receptor, the microgel swells as glucose concentration increases. This mechanism can be used to deliver a drug by diffusion through the network. In other cases, glucose binds specifically to two boronates, which creates additional cross-links within the network and provokes shrinkage. Such systems are promising for the development of sensors with improved selectivity and also as potential "intelligent" valves in microfabricated delivery systems. By a rational choice of the constituting units of the network structure, we show how to favor one or the other type of response to glucose variation. Therefore, glucose-swelling microgels operating under physiological conditions have been obtained by copolymerization with an appropriate choice of alkylacrylamide monomer and boronate derivative. At a pH above the pK(a) of the boronic acid derivative, the same structures shrink in response to glucose concentration. The nature of the cross-linker is a key parameter to enable this dual behavior. In other microgels, an amine group is introduced in the vicinity of the boronic acid, which lowers its pK(a) and favors microgel contraction at physiological pH. This work has allowed us to give some general rules to control the swelling/shrinking behavior of glucose-responsive microgels.     

基于纳米粒子的比色检测及其在化学传感及生物检测的应用

基于纳米粒子的比色检测由于其简单性、多样性而备受关注。在该体系中,目标分析物直接或间接诱发纳米粒子的聚集和分散,从而可通过溶胶的颜色改变实现分析检测。该综述介绍了纳米粒子分散及聚集过程的物理现象,简单讨论了纳米粒子间的作用力,对两种作用机制(粒子间交联和非粒子间交联来调节纳米粒子的分散和聚集)进行了介绍。并对纳米粒子在重金属离子检测、生物检测等方面的应用及方法的分析特性进行了概述,对其未来发展趋势进行了展望。     

Insulin-Loaded SLN Prepared with the Emulsion Dilution Technique: In Vivo Tracking of Nanoparticles after Oral Administration to Rats

Insulin-loaded solid lipid nanoparticles (SLN) were prepared according to a solvent dilution method from O/W emulsions using isovaleric acid as organic phase. Insulin was derivatized with fluorescein isothyocianate (FITC) obtaining a fluorescent marker to be used in in vivo experiments. FITC-insulin and native insulin–loaded SLN were quite similar with regard to their mean sizes and encapsulation efficiency. SLN intestinal uptake was then investigated administering FITC-insulin loaded SLN on healthy male Wistar rats. Significant drug accumulation within intestinal lymphatic system was recovered, but the immune system seems to play an important role in SLN degradation: further studies are necessary to improve the results on blood glucose level.     

Magnetoswitchable electrochemistry gated by alkyl-chain-functionalized magnetic nanoparticles: control of diffusional and surface-confined electrochemical processes

Magnetic nanoparticles consisting of undecanoate-capped magnetite (average diameter ca. 5 nm) are used to selectively gate diffusional and surface-confined electrochemical reactions. A two-phase system consisting of an aqueous buffer solution and a toluene phase that includes the suspended undecanoate-capped magnetic nanoparticles is used to control the interfacial properties of the electrode surface. Two different phenomena are controlled by attraction of the magnetic nanoparticles to the electrode by means of an external magnet: (i) The attracted magnetic nanoparticles form a hydrophobic layer on the electrode surface resulting in the blocking of diffusional electrochemical processes, while retaining the redox functions of surface-confined electrochemical units. (ii) For certain surface-immobilized redox species (e.g., quinones), the attraction of the magnetic nanoparticles to the electrode surface alters the mechanism of the process from an aqueous-type electrochemistry to a dry organic-phase-type electrochemistry. Also, bioelectrocatalytic and electrocatalytic transformations at the electrode are controlled by means of attraction of the magnetic nanoparticles to the electrode surface. Controlling the catalytic functions of the modified electrode by means of the magnetic nanoparticles attracted to the electrode is exemplified in two different directions: (i) Blocking of the bioelectrocatalyzed oxidation of glucose by glucose oxidase (GOx) using a surface-confined ferrocene monolayer as electron-transfer mediator. (ii) Activation of the microperoxidase-11 electrocatalyzed reduction of cumene hydroperoxide. In the latter system, the hydrophobic magnetic nanoparticles adsorb toluene, and the hydrophobic matrix acts as a carrier for cumene hydroperoxide to the electrode surface modified with the microperoxidase-11 catalyst.     

Wired-enzyme core-shell Au nanoparticle biosensor

We report a fully integrated core-shell nanoparticle system responsive to glucose. The system is comprised of self-assembled glucose oxidase and an osmium molecular wire on core-shell Au nanoparticles. Characterization of the functional nanoparticles by spectroscopy, quartz crystal microbalance and electrochemical techniques has shown that the catalytically active shell has a structure as designed and all components are active in the self-assembled multilayer shell. Furthermore, amperometric reagentless detection of glucose and contactless photonic biosensing by the Os(II) resonant Raman signal have been demonstrated. The enzymatic reduction of FAD by glucose and further reduction of the Raman silent Os(III) by FADH 2 yields a characteristic enzyme-substrate calibration curve in the millimolar range. Furthermore, coupling of electronic resonant Raman of the osmium complex with the SERS amplification by Au NPs plasmon resonance has been demonstrated which leads to an extra enhancement of the biosensor signal. We present a proof of concept extending the work done with planar surfaces to core-shell NPs as an advance in the design of glucose-responsive chemistry detected by SERS-like methods.     

Regular and irregular deswelling of polyacrylate and hyaluronate gels induced by oppositely charged surfactants

New multiresponsive core-shell microgels have been synthesized, with a thermoresponsive core and a glucose-responsive shell, made respectively of poly(N-isopropylacrylamide) (pNIPAM) and pNIPAM-co-acrylamidophenylboronic acid (pNIPAM-co-APBA). The structure of the particles was elucidated by means of dynamic light scattering. Their thermal properties were investigated and compared to those of the core alone. Without glucose, the hydrophobic shell prevented the core from swelling in a certain temperature range where the shell was shown to be collapsed. This core compression vanished upon glucose addition, when the shell became hydrophilic and swelled. Therefore, the extent of core swelling was regulated by two processes: its own internal stimulus, i.e. temperature, and shell compression, which is proportional to glucose concentration, even at physiological salinity. The concept was applied to a selected chemical composition. Core-shell microgels with a response to glucose at physiological pH were obtained and used to encapsulate insulin. Insulin release was shown to be regulated by the presence of glucose.     

Gold nanoparticles as carriers for efficient transmucosal insulin delivery

Nanomaterials have gained tremendous importance in biology and medicine because they can be used as carriers for delivering small molecules such as drugs, proteins, and genes. We report herein the binding of the hormone insulin to gold nanoparticles and its application in transmucosal delivery for the therapeutic treatment of diabetes mellitus. Insulin was loaded onto bare gold nanoparticles and aspartic acid-capped gold nanoparticles and delivered in diabetic Wistar rats by both oral and intranasal (transmucosal) routes. Our principle observations are that there is a significant reduction of blood glucose levels (postprandial hyperglycemia) when insulin is delivered using gold nanoparticles as carriers by the transmucosal route in diabetic rats. Furthermore, control of postprandial hyperglycemia by the intranasal delivery protocol is comparable to that achieved using the standard subcutaneous administration used for type I diabetes mellitus, thus showing considerable promise for further development.     

Nanoparticle-based pseudostationary phases in CEC: a breakthrough in protein analysis?

This review focuses on major trends in nanoparticle-based pseudostationary phase (PSP) CEC since the publication of our previous reviews within nanoparticle-based CEC [Nilsson, C., et al., Electrophoresis 2006, 27, 76-83; Nilsson, C., et al., J. Chromatogr. A 2007, 1168, 212-224.]. Special attention is given to the development toward protein analysis, which is driven by the strong emergence of protein drug development in the pharmaceutical industry. Furthermore, we discuss the development in coupling different detection techniques with nanoparticle-based PSP CEC, which were originally predicted to be particularly cumbersome. However, at present, direct UV, LIF and ESI-MS have been used without any severe complications. Different types of nanoparticles used as PSP during the period include gold nanoparticles, carbon nanostructures and lipid-based nanoparticles. New materials (for example, different types of carbon nanostructures and self assembled lipid-based nanostructures) are a strong driving force for development in separation science. Finally, future trends in nanoparticles-based CEC are envisioned.     

Nanostructured platinum-lipid bilayer composite as biosensor

The present work describes the preparation of supported bilayer lipid membrane (s-BLM) doped with metal nanoparticles for the design of biosensors. Platinum (Pt) nanoparticles were deposited through s-BLM to build a hybrid device of nanoscale electrode array by potential cycling in 1 mM K(2)PtCl(6) solution containing 0.1 M KCl. The properties of Pt nanoparticle-doped s-BLM composite were then characterized by cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). Our results showed that Pt nanoparticles grew in voids of the s-BLMs, through which the underlying glassy carbon (GC) electrode was connected, with maximum length extended out of the lipid membrane around 40 nm. Doping of Pt nanoparticles through s-BLM increased the membrane capacitance and decreased the membrane resistance of s-BLM. Pt nanoparticles array in s-BLM electrocatalyzed the reduction of oxygen (O(2)) in phosphate buffer solution (PBS). Practical application of Pt nanoparticle-doped s-BLM for the construction of glucose biosensor was also demonstrated in terms of its dose-response curve, stability and reproducibility. Thus, lipid membrane doped with Pt nanoparticles is a novel electrode system at nanoscale that can penetrate through the insulating membrane to probe molecular recognition and catalytic events at the lipid membrane-solution interface.     

Rapid and highly sensitive protein extraction via cobalt oxide nanoparticle-based liquid-liquid microextraction coupled with MALDI mass spectrometry

A new approach for rapid and highly sensitive protein extraction using cobalt oxide nanoparticles modified with cetyltrimethylammonium (Co(3)O(4)/CTA(+) NP) using nanoparticle-based liquid-liquid microextraction (NP-LLME) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully demonstrated. For the first time, the metal oxide NPs (Co(3)O(4)/CTA(+) NP) prepared in the organic phase (toluene) have been successfully applied for the extraction and preconcentration of proteins from sample solutions and complex samples via electrostatic forces involved between the metal oxide NP and proteins. Lysozyme was used as the model protein to investigate the optimal extraction parameters of the current approach. The optimal conditions were obtained at pH > pI for 10 min of incubation time (extraction time) with 3% salt (NaCl) addition. The Co(3)O(4)/CTA(+) NP was successfully applied for the highly sensitive analysis of an array proteins such as insulin, chymotrypsinogen and lysozyme from aqueous solution, protein mixture and milk samples in nanoparticle-based liquid-phase microextraction coupled with MALDI-MS. The potentiality of the NP-LLME using Co(3)O(4)/CTA(+) NP for the extraction of proteins was also compared with other types of NP-liquid phase microextraction (LPME) methods. The current approach offers distinct advantages including rapidity, straightforwardness, high sensitivity for washing- and separation-free MALDI-MS analysis of proteins.     

Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles

Sensitive DNA detection is extremely important in clinical diagnostics, gene therapy, and a variety of biomedical studies. We have developed a novel DNA bioanalysis method with a 0.8 fM (0.8 x 10-15 M) detection limit using a bioconjugated fluorescent nanoparticle-based sandwich assay. An organic dye silica nanoparticle is synthesized using a modified reverse microemulsion. The nanoparticles are highly fluorescent, extremely photostable, and easy for bioconjugation for bioanalysis. They exhibit an excellent signaling ability in the presence of trace amounts of DNA targets. With an effective surface modification, nonspecific binding and nanoparticle aggregation are minimized. In addition, the nanoparticle-based DNA bioanalysis assay can effectively discriminate one-base mismatched DNA sequences. We expect this nanoparticle-based assay to be widely useful in a number of biomedical applications where reproducible, selective, and ultrasensitive gene analysis is critical.     

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.     

Rapid room temperature synthesis of electrocatalytically active Au nanostructures

We describe a facile route for the one-pot room temperature synthesis of anisotropic Au nanostructures in aqueous solution in the absence of seeds or surfactants and their electrocatalytic activity. The Au nanostructures were synthesized using piperazine derivatives 1-(2-hydroxyethyl)piperazine and 1,4-Bis(2-hydroxyethyl)piperazine as reducing agents. The Au nanostructures were characterized by spectral, transmission electron microscopic (TEM), X-ray diffraction and electrochemical measurements. The absorption spectrum of colloidal nanoparticles displays two bands ~580 and ~930 nm, corresponding to the dipole and quadrupole plasmon resonance, respectively. TEM measurements show that the Au nanostructures have penta-twined polyhedral shape with an average size of 52 nm. X-ray and selected area electron diffraction patterns reveal the existence of face centered cubic nanocrystalline Au. The concentration of Au(III) controls the stability of the nanoparticles. The nanoparticles were immobilized on 3-D silicate network pre-assembled on a conducting support to examine their electrocatalytic activity. The nanoparticle-based electrochemical interface was characterized by spectral, voltammetric and impedance measurements. The nanoparticle shows high catalytic activity in the oxidation of NADH and reduction of oxygen. Unique inverted 'V' shape voltammogram was obtained for the oxidation of NADH at less positive potential. The nanoparticle-based interface favors two-step four-electron reduction of oxygen to water in neutral pH. Significant decrease in the overpotential for the oxidation of NADH and reduction of oxygen with respect to the polycrystalline Au electrode was observed. The electrocatalytic performance of the polyhedral nanoparticle is compared with the conventional citrate stabilized spherical nanoparticles.