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.

An enhanced fluorescent sensor system based on molecularly imprinted polymer chips with silver nanoparticles for highly-sensitive zearalenone analysis.

A novel enhanced fluorescent sensor system for zearalenone (ZON) determination in flour samples is presented. The ZON-selective molecularly imprinted polymer (MIP) films were developed with a computational modelling method and synthesised with cyclododecyl-2,4-dihydroxybenzoate as a "dummy" template and ethylene glycol methacrylate phosphate as a functional monomer acted as the selective recognition elements for ZON fluorescence detection. Spherical silver nanoparticles (AgNPs) were embedded in the MIP films' structure to enhance the sensor sensitivity. The imprinted films showed a high ZON recognition ability compared to non-imprinted films. Various factors that affected the measurement of the analysed sample were investigated and optimised. Embedding the AgNPs in the MIP films' structure led to an enhanced sensitivity (up to a 200-fold decrease of LOD) compared to unmodified MIP films. This fluorescent sensor system provided ZON analysis with high sensitivity, specificity, and a wider linear dynamic range of 5 ng mL-1 to 25 µg mL-1. An enhanced fluorescent sensor system based on MIP chips with embedded AgNPs could detect trace amounts of ZON in foods and feedstuffs with high sensitivity and selectivity.

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation.

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

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.

Molecularly imprinted nanoparticles-based assay (MINA) - detection of leukotrienes and insulin.

A novel molecularly imprinted polymer nanoparticle-based assay (MINA) performed in magnetic microplates was developed as an improved high-quality alternative to existing antibody-based immunoassays. MINA is a generic technology that can be adapted for biomarker detection in biological samples. Herein, we demonstrate the applicability of the MINA assay for the detection of leukotrienes and insulin in biological samples. MINA, used in a competition format, has allowed the detection of LTE4 in urine in a concentration range from 0.45 to 364 pM, with a LOD of 0.73 pM. MINA, used in a competition format, has allowed the detection of insulin in plasma in a concentration range from 25 to 2500 pM, with a LOD of 27 pM. This assay has shown comparable performance for LTE4 and insulin detection to existing chromatographic techniques (LC-MS/MS) and immunoassays in clinically relevant concentrations. The main advantages of this assay are the efficient and low cost fabrication, preparation of synthetic binders without the use of animals, and fewer steps used in the assay protocol as compared to traditional immunoassays.

Application of molecularly imprinted polymers in the anti-doping field: sample purification and compound analysis.

The problem posed by anti-doping requirements is one of the great analytical challenges; multiple compound detection at low ng ml-1 levels from complex samples, with requirements for exceptional confidence in results. This review surveys the design, synthesis and application of molecularly imprinted polymers (MIPs) in this field, focusing on the templating of androgenous anabolic steroids (AASs), as the most commonly abused substances, but also other WADA prohibited substances. Commentary on the application of these materials in detection, clean-up and sensing is offered, alongside views on the future of imprinting in this field.

Sensor Based on Molecularly Imprinted Polymer Membranes and Smartphone for Detection of Fusarium Contamination in Cereals.

The combination of the generic mobile technology and inherent stability, versatility and cost-effectiveness of the synthetic receptors allows producing optical sensors for potentially any analyte of interest, and, therefore, to qualify as a platform technology for a fast routine analysis of a large number of contaminated samples. To support this statement, we present here a novel miniature sensor based on a combination of molecularly imprinted polymer (MIP) membranes and a smartphone, which could be used for the point-of-care detection of an important food contaminant, oestrogen-like toxin zearalenone associated with Fusarium contamination of cereals. The detection is based on registration of natural fluorescence of zearalenone using a digital smartphone camera after it binds to the sensor recognition element. The recorded image is further processed using a mobile application. It shows here a first example of the zearalenone-specific MIP membranes synthesised in situ using "dummy template"-based approach with cyclododecyl 2, 4-dihydroxybenzoate as the template and 1-allylpiperazine as a functional monomer. The novel smartphone sensor system based on optimized MIP membranes provides zearalenone detection in cereal samples within the range of 1-10 µg mL-1 demonstrating a detection limit of 1 µg mL-1 in a direct sensing mode. In order to reach the level of sensitivity required for practical application, a competitive sensing mode is also developed. It is based on application of a highly-fluorescent structural analogue of zearalenone (2-[(pyrene-l-carbonyl) amino]ethyl 2,4-dihydroxybenzoate) which is capable to compete with the target mycotoxin for the binding to zearalenone-selective sites in the membrane's structure. The competitive mode increases 100 times the sensor's sensitivity and allows detecting zearalenone at 10 ng mL-1. The linear dynamic range in this case comprised 10-100 ng mL-1. The sensor system is tested and found effective for zearalenone detection in maize, wheat and rye flour samples both spiked and naturally contaminated. The developed MIP membrane-based smartphone sensor system is an example of a novel, inexpensive tool for food quality analysis, which is portable and can be used for the "field" measurements and easily translated into the practice.

Highly Efficient Abiotic Assay Formats for Methyl Parathion: Molecularly Imprinted Polymer Nanoparticle Assay as an Alternative to Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assay (ELISA) is a widely used standard method for sensitive detection of analytes of environmental, clinical, or biotechnological interest. However, ELISA has clear drawbacks related to the use of relatively unstable antibodies and enzyme conjugates and the need for several steps such as washing of nonbound conjugates and addition of dye reagents. Herein, we introduce a new completely abiotic assay where antibodies and enzymes are replaced with fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) and target-conjugated magnetic nanoparticles, which acted as both reporter probes and binding agents. The components of the molecularly imprinted polymer nanoparticle assay (MINA) are assembled in microtiter plates fitted with magnetic inserts. We have compared the performance of a new magnetic assay with molecularly imprinted polymer (MIP)-based ELISA for the detection of methyl parathion (MP). Both assays have shown high sensitivity toward allowing detection of MP at picomolar concentrations without any cross-reactivity against chlorpyriphos and fenthion. The fully abiotic assays were also proven to detect analyte in real samples such as tap water and milk. Unlike ELISA-based systems, the novel assay required no washing steps or addition of enzyme substrates, making it more user-friendly and suitable for high throughput screening.

NanoMIP-based approach for the suppression of interference signals in electrochemical sensors.

Herein, we describe the use of molecularly imprinted nanoparticles (nanoMIPs) as sequestering (masking) agents, to suppress the signal coming from interfering molecules and facilitate the detection of the target analyte. In this work, ascorbic acid was used as a model interfering molecule in dopamine electrochemical detection. NanoMIPs selective for ascorbic acid demonstrated to be capable of binding and suppressing electrochemical signal from ascorbic acid, enabling the detection of dopamine in the range 100-500 nM, without any need for sample pre-treatment. Tests in real samples (spiked human serum) were also carried out successfully. Due to the generic nature of the imprinting process, the proposed approach can be tailored to suppress potentially any interfering species, by simply varying the type of nanoMIPs used.

Molecularly imprinted polymer nanoparticle-based assay (MINA): application for fumonisin B1 determination.

The enzyme-linked immunosorbent assay (ELISA) has been used as a standard tool for monitoring food and animal feed contamination from the carcinogenic fumonisin B1 (FB1). Unfortunately, ELISA is not always efficient due to the instability of the antibody and enzyme components in the immunoassay, the presence of natural enzyme inhibitors in the samples and the high levels of non-specific protein binding. Additionally, the production of antibodies for ELISA can be time-consuming and costly, due to the involvement of animals in the manufacturing process. To overcome these limiting factors, a molecularly imprinted nanoparticle based assay (MINA) has been developed, where the molecularly imprinted nanoparticles (nanoMIPs) replace the primary antibody used in a competitive ELISA. Herein, computational modelling was used to design the nanoMIPs by selecting monomers that specifically interact with FB1. The affinity of the monomers to FB1 was verified by measuring their binding in affinity chromatography experiments. The nanoMIPs were produced by solid phase synthesis and the results showed that nanoMIPs had a hydrodynamic diameter of around 249 ± 29 nm. The assay tested in model samples is highly selective and does not show cross-reactivity with other mycotoxins such as fumonisin B2 (FB2), aflatoxin B1 (AFB1), citrinin (CTT), zearalenone (ZEA), and deoxynivalenol (DON). The MINA allows the detection of FB1 in the concentration range of 10 pM-10 nM with a detection limit of 1.9 pM and a recovery of 108.13-113.76%.

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.

Modulation of Quorum Sensing in a Gram-Positive Pathogen by Linear Molecularly Imprinted Polymers with Anti-infective Properties.

We describe the development, characterization, and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as an anti-infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae. The LMIP is prepared (polymerized) in presence of a template molecule, but unlike in traditional molecular imprinting approaches, no cross-linker is used. This results in soluble low-molecular-weight oligomers that can act as a therapeutic agent in vitro and in vivo. The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP. These findings provide a proof of principle that LMIPs can be used to block QS, thus setting the stage for the development of LMIPs a novel drug-discovery platform and class of materials to target Gram-positive pathogens.

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.

PEG-stabilized core-shell surface-imprinted nanoparticles.

Here we present a simple technique to produce target-specific molecularly imprinted polymeric nanoparticles (MIP NPs) and their surface modification in order to prevent the aggregation process that is ever-present in most nanomaterial suspensions/dispersions. Specifically, we studied the influence of surface modification of MIP NPs with polymerizable poly(ethylene glycol) on their degree of stability in water, in phosphate buffer, and in the presence of serum proteins. Grafting a polymer shell on the surface of nanoparticles decreases the surface energy, enhances the polarity, and as a result improves the dispersibility, storage, and colloidal stability as compared to those of core (unmodified) particles. Because of the unique solid-phase approach used for synthesis, the binding sites of MIP NPs are protected during grafting, and the recognition properties of nanoparticles are not affected. These results are significant for developing nanomaterials with selective molecular recognition, increased biocompatibility, and stability in solution. Materials synthesized this way have the potential to be used in a variety of technological fields, including in vivo applications such as drug delivery and imaging.

Application of the 'gate effect' of a molecularly imprinted polymer grafted on an electrode for the real-time sensing of heparin in blood.

Heparin is the most important anticoagulant drug used during surgeries and extracorporeal therapies. Although the blood levels of heparin should be monitored continuously during the procedure to ensure the safety of the patient, there is currently no technique for measuring heparin in real time. This study describes the use of a molecularly imprinted polymer (MIP) as a recognition element in the development of a heparin sensor for real-time monitoring. An indium tin oxide (ITO) electrode grafted with a heparin-specific MIP was used as a working electrode to perform cyclic voltammetry of ferrocyanide. The anodic current was found to be dependent on heparin concentration, probably due to the "gate effect", which is a change in the accessibility of the MIP-modified electrode to ferrocyanide, triggered by specific interaction between MIP and heparin. The kinetics of heparin interaction with the MIP-grafted electrode was evaluated using potentiostatic chronoamperometry of ferrocyanide in an electrochemical flow cell. The response time to stepwise changes in heparin concentration between 0 and 0.04 units per mL was estimated at 20 s, which is remarkably shorter than that achieved using conventional methods for monitoring heparin. The MIP-grafted electrode demonstrated exceptional sensitivity and could detect heparin in whole blood samples (0-6 units per mL) diluted 100-fold with physiological saline containing ferrocyanide. Therefore, the MIP-grafted electrode is suitable for real-time monitoring of heparin in blood. Another advantage is that a very small volume of blood is needed, which is very important, especially when regular measurements are required.

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.

Evading and overcoming AAV neutralization in gene therapy.

Adeno-associated virus (AAV)-derived viral vectors are a promising platform for the delivery of curative, life-changing therapies to a huge number of patients with monogenic disorders. There are currently over 250 clinical trials ongoing worldwide. However, for these therapies to benefit as many patients as possible, techniques must be developed to treat those with pre-existing immunity and to potentially allow re-administration of a dose in the future, should efficacy wane over time. This review discusses the current state and prospects of technologies to evade and overcome these immune responses and allow successful treatment of the greatest number of patients possible.