Cyclodextrin Nanosponges Research Articles

Ester-Based Hydrophilic Cyclodextrin Nanosponges for Topical Ocular Drug Delivery.

Cyclodextrin-based nanosponges are hyper-cross-linked supramolecular architectures. Similar to the parent compound they are derived from, they have been shown to possess the ability to form inclusion and non-inclusion complexes with drugs of different polarities, enzymes, proteins, peptides and macromolecules. Cyclodextrins only form inclusion complexes with hydrophobic guest molecules, while nanosponges extend this function by forming inclusion and non-inclusion complexes with both hydrophobic and hydrophilic compounds. This host-guest interaction provides enhancement in stability, solubility and sometimes in membrane permeability. The potential of ester-based hydrophilic nanosponges as drug carriers in topical ocular drug delivery was assessed by undertaking drug loading, drug release, phase solubility and ex vivo pig corneal drug permeation studies on the nanosponges. The ester-based cyclodextrin-based nanosponges were synthesized through cross-linking β-cyclodextrin by pyromellitic dianhydride. Various concentrations of pyromellitic dianhydride, the cross-linker, were initially tested for their effects on the drug loading capacity of the nanosponges. Thereafter, varying ratios of drug to nanosponge complexes were studied and analyzed for their drug release kinetics, and solubility and permeation effects. A cyclodextrin-based nanosponge drug delivery system with potential to improve corneal permeation and drug solubility was formulated. Their ability to improve pig corneal permeation was shown; where up to 75% permeation of the model drug was achieved in the presence of the nanosponges as compared to the 28% permeation of the pure drug. The nanosponges provided drug release for approximately an hour. This is a common behavior for hydrophilic complexes due to their high solubility in aqueous media. The ester-based hydrophilic cyclodextrin-based nanosponge derivative can be used as a drug delivery system in topical ocular drug delivery, to improve stability, solubility and corneal permeation.

A novel hybrid catalytic system based on immobilization of phosphomolybdic acid on ionic liquid decorated cyclodextrin-nanosponges: Efficient catalyst for the green synthesis of benzochromeno-pyrazole through cascade reaction: Triply green

Abstract An efficient hybrid catalyst was developed by immobilization of Keggin type heteropolyacid, phosphomolybdic acid, in the nanocavitis of ionic liquid-modified cyclodextrin nanosponges. This immobilized species was characterized by SEM/EDX, FTIR, TGA, ICP-AES and XRD. The catalytic activity of the hybrid catalyst was confirmed in the synthesis of benzochromeno-pyrazole derivatives via cascade reaction of hydrazine hydrate, ethyl acetoacetate, α or β-naphthol and benzaldehyde. To establish the synergetic effects of conjugation of heteropolyacid and ionic liquid, the catalytic activity of the hybrid catalyst was compared with that of bare ionic liquid-modified cyclodextrin nanosponges. The obtained results indicated the superior activity of the former, from different points of view such as product yield and reaction time. Noteworthy, the catalyst was reusable and could be recovered and reused for several reaction runs. The comparison of obtained results with previous reports was performed. Moreover, the potential utility of the catalyst under ultrasonic and microwave irradiation has been proved.

SANS investigation of water adsorption in tunable cyclodextrin-based polymeric hydrogels.

The focus of this work is on addressing how the adsorption properties of cyclodextrin (CD) based polymeric hydrogels (cyclodextrin nanosponges, CDNS) can be regulated by a precise control of a crucial parameter such as the characteristic pore size of the polymer network. With this aim, Small Angle Neutron Scattering (SANS) experiments are performed on different CDNS polymer formulations, differing by (i) the chemical structure of the cross-linking agent used for the polymerization of CD, which affects the flexibility of the strands between two crosslinking junctions, (ii) the relative molar ratio of cross-linker to monomer, affecting the cross-linking density, and (iii) the dimension of the CD macrocycle, regulating the steric hindrance and number of available reactive sites on the monomer. The analysis of the experimental data in terms of a two-correlation-length model allows one to extract a correlation length ζ that is interpreted as an experimental assessment of the average mesh-size of the cross-linked polymer, confirming the effective nano-size of the cavities formed in the network of CDNS. The hydration-dependence of ζ is modelled by an empirical functional form, that provides as key parameters the swelling rate of the polymeric network and the upper limit for the mesh size of the material. These parameters are useful for the characterization of the dynamic response of the hydrogel to swelling and the maximum mesh size compatible with the chemical structure of the polymer.

Ordered and disordered cyclodextrin nanosponges with diverse physicochemical properties

Herein, in addition to conventional β-CD-NSPs, the NSPs of α, γ, HP-β, methyl-β, and SBE-β cyclodextrins were synthesized by a simple modified approach and thoroughly characterized.

Molecularly imprinted cyclodextrin nanosponges for the controlled delivery of L-DOPA: perspectives for the treatment of Parkinson’s disease

ABSTRACTBackground: L-DOPA is an amino acid precursor to the neurotransmitter dopamine that is extensively used as a prodrug for the treatment of Parkinson’s disease. However, L-DOPA is an unstable compound: when exposed to light or added to aqueous solutions, it may degrade, compromising its therapeutic properties.Methods: In this work, a new type of drug-loaded cyclodextrin-based nanosponge, obtained using molecular imprinting, is described for the prolonged and controlled release of L-DOPA. The molecularly imprinted nanosponges (MIP-NSs) were synthesized by cross-linking β-cyclodextrin with 1,1ʹ-carbonyldiimidazole in DMF in the presence of L-DOPA as a template molecule. TGA, DSC and FTIR analyses were performed to characterize the interactions between L-DOPA and the two nanosponge structures. Quantitative NMR spectroscopy was used to determine the amount and the affinity of L-DOPA entrapped in the nanosponges. The in vitro L-DOPA release kinetics from the NSs were quantitatively determined by HPLC analysis.Results: The MIP-NSs show a slower and more prolonged release profile than the non-imprinted nanosponges. No degradation of the L-DOPA hosted in the MIP-NSs was observed after long-term storage at room temperature.Conclusions: The MIP-NSs are a promising alternative for the storage and controlled delivery of L-DOPA.

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study.

The chemical cross-linking of β-cyclodextrin (β-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels. The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of β-cyclodextrin (β-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels. The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

Cyclodextrin nanosponges: a potential catalyst and catalyst support for synthesis of xanthenes

The nanoporous framework of a cyclodextrin nanosponge was used as catalyst for accelerating the one-pot, three-component reaction of dimedone, aldehyde, and phenols for synthesis of xanthene derivatives. Moreover, the nanocavities of cyclodextrin nanosponges were exploited for immobilization of heteropolyacids through the wet impregnation method. This catalyst exhibited superior catalytic performance compared to the bare cyclodextrin nanosponge. Despite the good catalytic activity, the leaching of the catalytic species did not allow efficient recovery and reusability. To circumvent this problem, the cyclodextrin nanosponge was amine-functionalized prior to heteropolyacid immobilization. The results proved that the amine functionalities had an effective role in preserving the catalytic species and improving the reusability through decreasing the leaching time. This catalyst was used for synthesis of a variety of xanthenes in aqueous media. The catalytic amount of catalyst afforded the desired product in excellent yields and with a relatively short reaction time. The results suggested cyclodextrin nanosponge-based catalysts as potential candidates for promoting chemical reactions.

GSH-targeted nanosponges increase doxorubicin-induced toxicity “in vitro” and “in vivo” in cancer cells with high antioxidant defenses

Several reports indicate that chemo-resistant cancer cells become highly adapted to intrinsic oxidative stress by up-regulating their antioxidant systems, which causes an increase of intracellular GSH content. Doxorubicin is one of the most widely used drugs for tumor treatment, able to kill cancer cells through several mechanisms. However, doxorubicin use is limited by its toxicity and cancer resistance. Therefore, new therapeutic strategies able to reduce doses and to overcome chemo-resistance are needed. A new class of glutathione-responsive cyclodextrin nanosponges (GSH-NS), is able to release anticancer drugs preferentially in cells having high GSH content. Doxorubicin-loaded GSH-NS, in the cancer cells with high GSH content, inhibited clonogenic growth, cell viability, topoisomerase II activity and induced DNA damage with higher effectiveness than free drug. Moreover, GSH-NS reduced the development of human tumor in xenograft models more than free drug. These characteristics indicate that GSH-NS can be a suitable drug delivery carrier for future applications in cancer therapy.

Guest-matrix interactions affect the solvation of cyclodextrin-based polymeric hydrogels: a UV Raman scattering study.

The focus of the present work is to shed light on possible modifications of the molecular properties of polysaccharide hydrogels induced by the establishment of specific non-covalent interactions during the loading of a guest compound inside the gel phase. With this aim, a case study of the encapsulation of caffeine (Caf) inside cyclodextrin-based hydrogels, namely, cyclodextrin nanosponges (NS), is systematically investigated here by using UV Raman scattering experiments. The UV Raman spectra of the hydrogels, analysed as a function of temperature, concentration of the guest molecule loaded in the gel phase and pH, prove particularly informative both on the structural rearrangements of the hydrophobic/hydrophilic groups of the polymeric network and on the breaking/formation of specific guest-matrix interactions. Analysis of the temperature dependence of dynamical parameters, i.e., the dephasing time associated with specific vibrational modes of the polymer backbone, enables the proposal of a molecular picture in which the loading of Caf in NS hydrogels tends to favour access of the water solvent to the more hydrophobic portions of the polymer matrix, which is in turn reflected in a marked increase in the solvation of the whole system. The achievements of this work appear of interest with respect to the design of new possible strategies for controlling the diffusion/release of bioactive molecules inside hydrogel networks, besides corroborating the potential of UV Raman scattering experiments to give new molecular insights into complex phenomena affecting hydrogel phases.

Beneficial effects of microwave assisted heating versus conventional heating in synthesis of cyclodextrin based nanosponges

The effects of microwave heating versus conventional heating on particle-size distribution, morphology and crystallinity of cyclodextrin nanosponges have been investigated. FTIR-ATR, XRPD, DSC and DTA studies outlined the structural differences between the nanosponges prepared by different methods. The microwave-synthesized nanosponges exhibited higher degree of crystallinity and narrow size distribution. HR-TEM indicates that nanosponges obtained from microwave method are highly crystalline and faceted versus their conventionally-prepared counterparts. Microwave-synthesized nanosponges were measured with approximately twice the loading capacity towards a model drug than similar-sized conventionally prepared particles. Extensive structural characterisation studies revealed the distinct and preferential effects of the microwave heating method.

Cyclodextrin nanosponges to enhance the dissolution profile of quercetin by inclusion complex formation

ABSTRACTThe therapeutic potential of quercetin is mostly hampered by its low water solubility and poor absorption. The aim of this study was to enhance the dissolution rate of quercetin by molecular encapsulation with cyclodextrin nanosponges. The inclusion complexes were obtained by freeze-drying method. Inclusion complexes were characterized by Fourier Transformed Infrared Spectroscopy (FTIR), x-ray diffraction (XRD), Differential Scanning Calorimetry (DSC), transmission electron microscopy (TEM), and dissolution testing. FTIR, XRD, and DSC studies confirmed the interactions of quercetin with nanosponges. TEM images revealed the spherical morphology of complexes. The dissolution of the drug nanosponge complex was significantly higher compared with the pure drug in simulated intestinal fluid (SIF; pH 6.8). The results indicated that the degree of cross-linking has the significant influence on dissolution. Nanosponges with lower degree of cross-linking released 91% of the drug within 45 min, whereas nanosponges with higher degree of cross-linking resulted in sustained release up to 24 h.

Water and polymer dynamics in a model polysaccharide hydrogel: the role of hydrophobic/hydrophilic balance.

The molecular dynamics of water and a polymer matrix is here explored in a paradigmatic model of a polysaccharide hydrogel, by the combined use of UV Raman scattering and infrared measurements. The case example of cyclodextrin nanosponges (CDNS)/hydrogel is chosen since the simultaneous presence in the structure of the polymer matrix of both hydrophilic and hydrophobic sites mimics the complexity of polysaccharide hydrogels. In this way, the contributions provided by the balance between the hydrophilicity/hydrophobicity and the grade of entanglement of the polymer hydrogel to lead to the formation of the gel phase are separately accounted and evaluated. As main results, we found that the hydrophobic CH groups inserted on the aromatic ring of CDNS experience a more pronounced dynamic perturbation with respect to the carbonyl groups due to the collision between the solvent and vibrating atoms of the polymer. The overall results provide a detailed molecular picture of the swelling phenomena occurring when a chemically cross-linked polymer contacts with water or biological fluids and exploits the potentiality of UV Raman spectroscopy to retrieve dynamic information besides their structural counterpart obtained by the classical analysis of the basic features of vibrational spectra.

Anomalous diffusion of Ibuprofen in cyclodextrin nanosponge hydrogels: an HRMAS NMR study.

Ibuprofen sodium salt (IP) was encapsulated in cyclodextrin nanosponges (CDNS) obtained by cross-linking of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn) in two different preparations: CDNSEDTA 1:4 and 1:8, where the 1:n notation indicates the CD to EDTAn molar ratio. The entrapment of IP was achieved by swelling the two polymers with a 0.27 M solution of IP in D2O, leading to colourless, homogeneous hydrogels loaded with IP. The molecular environment and the transport properties of IP in the hydrogels were studied by high resolution magic angle spinning (HRMAS) NMR spectroscopy. The mean square displacement (MSD) of IP in the gels was obtained by a pulsed field gradient spin echo (PGSE) NMR pulse sequence at different observation times td. The MSD is proportional to the observation time elevated to a scaling factor α. The α values define the normal Gaussian random motion (α = 1), or the anomalous diffusion (α < 1, subdiffusion, α > 1 superdiffusion). The experimental data here reported point out that IP undergoes subdiffusive regime in CDNSEDTA 1:4, while a slightly superdiffusive behaviour is observed in CDNSEDTA 1:8. The transition between the two dynamic regimes is triggered by the polymer structure. CDNSEDTA 1:4 is characterized by a nanoporous structure able to induce confinement effects on IP, thus causing subdiffusive random motion. CDNSEDTA 1:8 is characterized not only by nanopores, but also by dangling EDTA groups ending with ionized COO− groups. The negative potential provided by such groups to the polymer backbone is responsible for the acceleration effects on the IP anion thus leading to the superdiffusive behaviour observed. These results point out that HRMAS NMR spectroscopy is a powerful direct method for the assessment of the transport properties of a drug encapsulated in polymeric scaffolds. The diffusion properties of IP in CDNS can be modulated by suitable polymer synthesis; this finding opens the possibility to design suitable systems for drug delivery with predictable and desired drug release properties.

Optimization of microwave-assisted synthesis of cyclodextrin nanosponges using response surface methodology

The reaction of polymerization of cyclodextrins with suitable polyfunctional agents leads to highly cross linked porous structures, referred to as cyclodextrin nanosponges. The conventional heating approach for the synthesis of nanosponges can lead to nonuniform reaction conditions caused by sharp thermal gradients in the bulk solution. In this work, we present a facile method for the synthesis of cyclodextrin nanosponges by microwave irradiation with significant reduction in reaction time. Response surface methodology and Box–Behnken design were used for the optimization of the process parameters including microwave power level (A), reaction time (B) and stirring speed (C). Two dependent variables practical yield and particle size were measured as responses. Mathematical equations and response surface plots were used to relate the dependent variables with independent variables. The optimization model predicted a yield of 96.5287 % and particle size of about 152.355 nm with A, B and C levels of 508.22, 83.55 and 1,471.55 respectively. The observed responses of the optimized process were in close agreement with the predicted values. Three confirmation batches of cyclodextrin nanosponges were synthesized under optimized conditions. The Fourier transformed infrared spectra of nanosponges showed a characteristic peak of the carbonate group at around 1,750 cm−1 which confirms the formation of nanosponges. TGA revealed the stability of obtained nanosponges up to 325 °C. The nanosponges obtained by microwave irradiation exhibited a higher degree of crystallinity. Dimension of the crystal lattice was found to be equal to 0.70 nm. TEM image confirmed the spherical shape and the particle size range of about 153 nm. Compared to conventional heating method, microwave method resulted in four folds reduction in reaction time and provided with particles of homogeneous size distribution and uniform crystallinity.

Fabrication of cyclodextrin nanosponges for quercetin delivery: physicochemical characterization, photostability, and antioxidant effects

Quercetin is a flavonoid widely distributed in vegetables and fruits and exhibits strong antioxidant activity, but the poor solubility and stability of quercetin limit its function and application. The purpose of this study was to enhance the dissolution rate and stability of a poorly water-soluble drug quercetin by complexation with cyclodextrin-based nanosponges. Nanosponges are recently developed sponge-like structures and have the capacity to interact with small molecules in its matrix. In this study, five types of nanosponges were purposely designed by varying the molar ratio of β-cyclodextrin and diphenyl carbonate. Quercetin was loaded into nanosponges by freeze-drying method. The particle sizes of plain and quercetin-loaded nanosponges are in between 40 and 100 nm with low polydispersity indices. Zeta potential is sufficiently high to obtain a stable colloidal nanosuspension. Fourier transformed infrared, Raman spectroscopy, differential scanning calorimetry, and X-ray powder diffraction studies confirmed the interaction of quercetin with nanosponges. Particle sizes measured from TEM images were in agreement with DLS results. The dissolution of the quercetin nanosponges was significantly higher compared with the pure drug. The stability of encapsulated quercetin nanosponge was tracked in a simulated intestinal fluid. A marked improvement in the photostability was also observed. In addition, the antioxidant activity of the quercetin nanosponges was more effective than pure quercetin on DPPH scavenging, anti-superoxide formation, and superoxide anion scavenging. These results signify that nanosponge formulations can be used as effective nanocarriers for the delivery of quercetin.

Hydrogen-bond dynamics of water confined in cyclodextrin nanosponges hydrogel

Cyclodextrin nanosponges (CDNS) are a very promising class of cross-linked polymers, made up of cyclodextrins. CDNS swollen in aqueous solution give rise to cyclodextrin-based hydrogel in different states—gel or liquid suspension—depending on the hydration level of the system. Here we present a thorough inspection of the vibrational dynamics of these hydrogel by Raman scattering experiments, with the aim of clarifying the role played by the hydrogen-bond dynamics of water molecules confined into the nano-sized pores of nanosponges in determining the rigidity of the hydrogel network and their maximum water-holding capacity. Changes occurring in the spectral shape of the OH stretching band of water were interpreted by accounting the connectivity pattern of water molecules concurring to the gelation process. Spectral deconvolution analysis gives evidence of the existence of a characteristic cross-over hydration level associated to the rearrangement of water molecules in more cooperative, bulk-like networks as a consequence of saturation sites of water confinement of nanosponges. This interpretation is further confirmed by the inspection of the estimated collective intensities. These findings also support the existence of a specific phase diagram of the cyclodextrin nanosponges hydrogel, where the molecular structure of the cross-linking agent used during the synthesis of nanosponge plays a fundamental role in defining the nano- and microscopic properties of the system.

Glass-like dynamics of new cross-linked polymeric systems: Behavior of the Boson peak

Cyclodextrin nanosponges (CDNS) provide a very promising class of cross-linked polymers consisting of cyclodextrins as building blocks and showing a characteristic nanoporous structure capable of effectively encapsulating and carrying a variety of both lipophilic and hydrophilic compounds. Hereafter, we investigate the vibrational dynamics of CDNS in the low-wave number region, with the aim to provide physical descriptors correlated to the elastic properties of this innovative glass-forming material. By means of Raman scattering measurements, we explore the modifications occurring to the boson peak (BP) as a function of the cavity size of the cyclodextrin and the relative amount of the cross-linking agent with respect to the monomer cyclodextrin, which can be varied during the synthesis of the polymer. The shift and intensity variations of the BP are discussed in terms of the modification of the elastic properties of CDNS, and a master curve for the boson peak can therefore be obtained. The experimental approach adopted here is a useful tool for investigating the structural and physicochemical properties of novel nanoporous soft materials of interest for biolife applications.

Direct evidence of gel–sol transition in cyclodextrin-based hydrogels as revealed by FTIR-ATR spectroscopy

The phase transition from gel to liquid suspension in cyclodextrin (CD)-based hydrogels is in depth monitored by using Fourier transform infrared spectroscopy in attenuated total reflectance geometry. Cyclodextrin nanosponges (CDNS) synthesized by polymerization of CD with the cross-linking agent ethylenediaminetetraacetic dianhydride at different cross-linking agent/CD molar ratios have been left to evolve from gel phase into liquid suspension upon gradual increase of the hydration level. Measurements of the changes occurring in the vibrational dynamics of the system during this transition provide direct evidence of the gel-sol progress of the CNDS hydrogel, by accounting for the connectivity pattern of water molecules concurring to the gelation process. The experimental results clearly indicate that the increase of the hydration level is accompanied by the corresponding increase of the population of H2O molecules engaged in high-connectivity hydrogen-bond networks. The water tetrahedral arrangement is thus dominant above a characteristic cross-over hydration level, experimentally determined for all the investigated samples. The observation of this characteristic cross-over point for the CDNS hydrogel and its correlation with other parameters of the system (e.g. the absorption ability of CDNS and elasticity of the polymer matrix) is, once again, modulated by the cross-linking agent/CD molar ratio. The latter seems indeed to play a key role in defining the nano- and microscopic properties of nanosponge hydrogels.

Peroxidase-encapsulated cyclodextrin nanosponge immunoconjugates as a signal enhancement tool in optical and electrochemical assays

Cyclodextrin nanosponges bearing carboxylate groups have been prepared by crosslinking β-cyclodextrin with pyromellitic dianhydride to form a carboxylic acid terminated nanoporous material. The surface of the particles was covalently modified with an anti-IgG antibody and then loaded with horseradish peroxidase. The structures of unmodified and protein modified nanosponge particles were investigated by Raman spectroscopy and imaging methods. Confocal microscopy indicates that the antibody is located in the outside of the particle while HRP is encapsulated in the inner part. The possibility to use these modified nanosponges as a signal enhancement tool in enzyme-linked colorimetric and electrochemical assays was evaluated using a sandwich format comprising immobilised gliadin as an antigen, a target anti-gliadin antibody and an anti-IgG antibody conjugated to the enzyme-loaded nanosponge immunoconjugates.