Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

Combinatorial screening of polymer nanoparticles for their ability to recognize epitopes of AAV-neutralizing antibodies.

A library of 17 nanoparticles made of acrylate and methacrylate copolymers is prepared, characterized, and screened against six epitopes of adeno-associated viruses (AAV)-neutralizing antibodies to assess their affinity and specificity. Peptide epitopes are immobilized onto the surface of glass beads, packed in filtration microplates, and incubated with fluorescein-labelled nanoparticles. Following intense washing, the affinity of nanoparticles to immobilized epitopes is assessed by measuring the fluorescence of captured nanoparticles. The results show that polar monomers, acrylic acid in particular, have a positive impact on polymer affinity towards all peptides used in this study. The presence of hydrophobic monomers, on other hand, has a negative impact on polymer binding. The composition of peptides used in this study has no noticeable impact on the affinity of synthesized nanoparticles. The affinity of nanoparticles with the highest affinity to peptide targets does not exceed millimolar level. Overall, it is found that the synthesized library showed modest affinity but lacked specificity, which should be further "tuned," for example, by using molecular imprinting to achieve an acceptable level of affinity and specificity for practical application.

Probing Peptide Sequences on Their Ability to Generate Affinity Sites in Molecularly Imprinted Polymers.

An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

Development of a computationally-designed polymeric adsorbent specific for mycotoxin patulin.

Patulin is a toxic compound which is found predominantly in apples affected by mould rot. Since apples and apple-containing products are a popular food for the elderly, children and babies, the monitoring of the toxin is crucial. This paper describes a development of a computationally-designed polymeric adsorbent for the solid-phase extraction of patulin, which provides an effective clean-up of the food samples and allows the detection and accurate quantification of patulin levels present in apple juice using conventional chromatography methods. The developed bespoke polymer demonstrates a quantitative binding towards the patulin present in undiluted apple juice. The polymer is inexpensive and easy to mass-produce. The contributing factors to the function of the adsorbent is a combination of acidic and basic functional monomers producing a zwitterionic complex in the solution that formed stronger binding complexes with the patulin molecule. The protocols described in this paper provide a blueprint for the development of polymeric adsorbents for other toxins or different food matrices.

Magnetic high throughput screening system for the development of nano-sized molecularly imprinted polymers for controlled delivery of curcumin.

Curcumin is a versatile anti-inflammatory and anti-cancer agent known for its low bioavailability, which could be improved by developing materials capable of binding and releasing drug in a controlled fashion. The present study describes the preparation of magnetic nano-sized Molecularly Imprinted Polymers (nanoMIPs) for the controlled delivery of curcumin and their high throughput characterisation using microtitre plates modified with magnetic inserts. NanoMIPs were synthesised using functional monomers chosen with the aid of molecular modelling. The rate of release of curcumin from five polymers was studied under aqueous conditions and was found to correlate well with the binding energies obtained computationally. The presence of specific monomers was shown to be significant in ensuring effective binding of curcumin and to the rate of release obtained. Characterisation of the polymer particles was carried out using dynamic light scattering (DLS) technique and scanning electron microscopy (SEM) in order to establish the relationship between irradiation time and particle size. The protocols optimised during this study could be used as a blueprint for the development of nanoMIPs capable of the controlled release of potentially any compound of interest.

Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin.

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

Solid-Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable Template - "Plastic Antibodies".

Molecularly Imprinted Polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics and separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and affinity, localized binding domain) whilst maintaining the advantages of MIPs (low-cost, short development time and high stability) hence the interest in MIP nanoparticles. Herein we report a reusable solid-phase template approach (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30-400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, Kd = 6.3 × 10-8 m), vancomycin (d = 250 nm, Kd = 3.4 × 10-9 m), a peptide (d = 350 nm, Kd = 4.8 × 10-8 m) and proteins have been produced. Our instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time we demonstrate the reliable re-use of molecular templates in the synthesis of MIPs (≥ 30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template-free and the solid-phase acts both as template and affinity separation medium.

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents.

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g(-1) ), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC-MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 µg mL(-1) . Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step-purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high-purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process.

Development of a new microtiter plate format for clinically relevant assays.

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

Rational design of molecularly imprinted polymer: the choice of cross-linker.

The paper describes a rational approach for the selection of cross-linkers during the development of molecularly imprinted polymers (MIPs). As a model system for this research MIPs specific for the drug zidovudine (AZT) were designed and tested. Three cross-linkers trimethylolpropane trimethacrylate (TRIM), ethylene glycol dimethacrylate (EGDMA) and divinylbenzene (DVB) were studied. The analogue of zidovudine (AZT) ester (AZT-ES) was used as a dummy template. The imprinting factors for all of the polymers in the static adsorption experiments were calculated. The data on the AZT adsorption by control polymers (CP), which were prepared with different cross-linkers without a functional monomer, was also analyzed. DVB was found to be more inert towards zidovudine than EGDMA and TRIM, which was confirmed by both molecular modelling and adsorption experiments. It was demonstrated that DVB-based polymers had a higher imprinting factor (I = 1.85) compared with other tested cross-linked polymers. It was suggested that the selection of the cross-linker should be based on the strength of the interaction with the template: the cross-linker which displays lower binding of the template should be preferential because it generates MIPs with lower non-specific binding and a higher imprinting factor, and therefore specificity. Which cross-linker to use for the preparation of any particular MIP can be determined by analysis of the interactions between the cross-linker and template. This could be done either virtually using computational modelling or by template adsorption using a small library of polymers prepared using different cross-linkers.

Nanoparticle-induced enhancement of cholinesterase activity in the presence of malathion: A potential nerve agent therapeutic.

Organophosphate nerve agents are associated with assassination, terrorism and chemical warfare, but there has been slow progress in developing a broad-spectrum response to poisoning. For some nerve agents the oxime component of the therapy may not be effective, limiting the effectiveness of emergency treatment that is desperately needed. An alternative therapy may be possible based on accelerating enzyme (acetylcholinesterase) catalysis in unaffected adjacent enzymes. Herein we demonstrate a restoration of acetylcholinesterase activity in malathion-inhibited cell membrane preparations by the administration of functional nanoparticles. The molecularly imprinted polymer nanoparticles were designed to bind selectively to designated enzyme epitopes. Enzyme activity of membrane-bound acetylcholinesterase was measured in the presence of the organophosphate malathion and the selected nanoparticles. Enzymatic acceleration of the cholinesterase was observed at 162 ± 17 % the rate of erythrocyte ghosts without bound nanoparticles. This may restore sufficient acetylcholine hydrolysis to mitigate the effects of poisoning, offsetting the acetylcholine accumulation resulting from enzyme inhibition.

Modulation of acetylcholinesterase activity using molecularly imprinted polymer nanoparticles.

Modulation of enzyme activity allows for control over many biological pathways and while strategies for the pharmaceutical design of inhibitors are well established; methods for promoting activation, that is an increase in enzymatic activity, are not. Here we demonstrate an innovative epitope mapping technique using molecular imprinting to identify four surface epitopes of acetylcholinesterase (AChE). These identified epitopes were then used as targets for the synthesis of molecularly imprinted nanoparticles (nanoMIPs). The enzymatic activity of AChE was increased upon exposure to these nanoMIPs, with one particular identified epitope nanoMIP leading to an increase in activity of 47× compared to enzyme only. The impact of nanoMIPs on the inhibited enzyme is also explored, with AChE activity recovering from 11% (following exposure to an organophosphate) to 73% (following the addition of nanoMIPs). By stabilizing the conformation of the protein rather than targeting the active site, the allosteric nature of MIP-induced reactivation suggests a new way to promote enzyme activity, even under the presence of an inhibitor. This method of enzyme activation shows promise to treat enzyme deficiency diseases or in medical emergencies where an external agent affects protein function.

Passive control of quorum sensing: prevention of Pseudomonas aeruginosa biofilm formation by imprinted polymers.

Here we present the first molecular imprinted polymer (MIP) that is able to attenuate the biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa through specific sequestration of its signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-AHL). The MIP was rationally designed using computational modeling, and its capacity and specificity and that of a corresponding blank polymer toward signal molecule of P. aeruginosa (3-oxo-C(12)-AHL) and its analogue were tested. The biofilm formation in the presence of polymers and without polymers was studied using scanning confocal laser microscopy. Staining with crystal violet dye was used for the quantification of the biofilm formation. A significant reduction of the biofilm growth was observed in the presence of MIP (>80%), which was superior to that of the resin prepared without template, which showed a reduction of 40% in comparison with biofilm, which was grown without polymer addition. It was shown that 3-oxo-C(12)-AHL-specific MIP prevented the development of quorum-sensing-controlled phenotypes (in this case, biofilm formation) from being up-regulated. The developed MIP could be considered as a new tool for the elimination of life-threatening infections in a multitude of practical applications; it could, for example, be grafted on the surface of medical devices such as catheters and lenses, be a component of paints, or be used as a wound adsorbent.

Size matters: influence of the size of nanoparticles on their interactions with ligands immobilized on the solid surface.

The correlation between the size of biotinylated nanoparticles and their affinity in relation to interactions with the solid surface was investigated. The silica particles with a diameter of 50-200 nm containing amino groups on the surface were labeled with different quantities of biotin. The affinity properties of biotinylated nanoparticles were studied using a Biacore 3000 instrument equipped with a streptavidin-coated sensor chip (SA chip). It was shown that the increase in the particle size from 50 to 200 nm reduced the affinity (K(D)) of biotin-streptavidin interactions from 1.2 x 10(-12) to 1.2 x 10(-10) M. It was found that the particles with higher concentrations of immobilized biotin on particle surfaces demonstrated stronger binding with streptavidin.

Attenuation of Vibrio fischeri quorum sensing using rationally designed polymers.

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri , using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri , N-(beta-ketocaproyl)-l-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters.

Electrochemical sensor for catechol and dopamine based on a catalytic molecularly imprinted polymer-conducting polymer hybrid recognition element.

One of the difficulties with using molecularly imprinted polymers (MIPs) and other electrically insulating materials as the recognition element in electrochemical sensors is the lack of a direct path for the conduction of electrons from the active sites to the electrode. We have sought to address this problem through the preparation and characterization of novel hybrid materials combining a catalytic MIP, capable of oxidizing the template, catechol, with an electrically conducting polymer. In this way a network of "molecular wires" assists in the conduction of electrons from the active sites within the MIP to the electrode surface. This was made possible by the design of a new monomer that combines orthogonal polymerizable functionality; comprising an aniline group and a methacrylamide. Conducting films were prepared on the surface of electrodes (Au on glass) by electropolymerization of the aniline moiety. A layer of MIP was photochemically grafted over the polyaniline, via N,N'-diethyldithiocarbamic acid benzyl ester (iniferter) activation of the methacrylamide groups. Detection of catechol by the hybrid-MIP sensor was found to be specific, and catechol oxidation was detected by cyclic voltammetry at the optimized operating conditions: potential range -0.6 V to +0.8 V (vs Ag/AgCl), scan rate 50 mV/s, PBS pH 7.4. The calibration curve for catechol was found to be linear to 144 microM, with a limit of detection of 228 nM. Catechol and dopamine were detected by the sensor, whereas analogues and potentially interfering compounds, including phenol, resorcinol, hydroquinone, serotonin, and ascorbic acid, had minimal effect (< or = 3%) on the detection of either analyte. Non-imprinted hybrid electrodes and bare gold electrodes failed to give any response to catechol at concentrations below 0.5 mM. Finally, the catalytic properties of the sensor were characterized by chronoamperometry and were found to be consistent with Michaelis-Menten kinetics.

Chimeric polymers formed from a monomer capable of free radical, oxidative and electrochemical polymerisation.

A new monomer, which incorporates both aniline and methacrylamide functional groups, was shown to possess orthogonal polymerisation behaviour to produce conjugated polyaniline suitable for a wide range of applications.

Development of a piezoelectric sensor for the detection of methamphetamine.

A computationally designed molecularly imprinted polymer (MIP) specific for methamphetamine was used as a synthetic receptor for the development of a piezoelectric sensor. Several different protocols were tested for the immobilisation of the MIP onto the gold sensor surface. The developed MIP sensor had a detection limit for methamphetamine as low as 1 microg mL(-1). The effect of the addition of poly(vinyl acetate) (PVA) on the pre-polymerisation mixtures, which increases the porosity of the polymer layer, was also studied using an Atomic Force Microscope (AFM). PVA seemed to affect both the porosity and the binding kinetics of the polymers prepared in dimethylformamide (DMF). However, no clear effect on porosity and binding kinetics was observed when polymers were prepared in diglyme. Moreover, PVA did not appear to improve the amplitude of the sensor response. In conclusion, because of its excellent recognition ability in aqueous solutions, the sensor described in this work could be an ideal starting point for the development of a commercial device for fast, on-site or road-side testing of drugs of abuse in body fluids such as saliva.

Optimisation of the preservation conditions for molecularly imprinted polymer nanoparticles specific for trypsin.

The influence of lyophilisation, autoclaving and sonication on the stability and performance of trypsin-specific molecularly imprinted polymer nanoparticles (MIP NPs) has been studied in order to improve their long-term physical stability. Glucose, glycine, sorbitol and trehalose were tested as cryoprotectant agents during the lyophilisation treatment. The effect of lyophilisation and sterilisation on affinity of trypsin-specific NPs was assessed using Biacore 3000 instrument. The results have demonstrated that MIP NPs successfully withstood the lyophilisation and autoclaving conditions without a reduction of their recognition properties and affinity. It is possible to conclude that both tested lyophilisation and sterilisation treatments were suitable for a long-term storage of the prepared MIP NPs and could be used to store MIP NPs in dry state and hence reduce the chance of the bacterial contamination. An effective preservation of the MIP NPs is a crucial requirement for their future applications in the clinical diagnostics and bioimaging.

Influence of continuous magnetic field on the separation of ephedrine enantiomers by molecularly imprinted polymers.

A set of polymers was imprinted with (-)-ephedrine using UV initiation, under the influence of a constant external magnetic field with intensities ranging from 0 to 1.55 T. Synthesised materials were characterised by X-ray crystallography, infrared spectroscopy, swelling and surface area. Recognition properties were assessed by the ability to discriminate between (+) and (-)-ephedrine and by Scatchard analyses on chromatographic mode. It was shown that polymer morphology and recognition properties are affected by the magnetic field. This resulted in considerable improvements in the chromatographic resolution of ephedrine enantiomers by materials synthesised under the influence of magnetic field. Apparently the magnetic field improved the ordering of the polymer structure and facilitated the formation of more uniform imprinting sites.