Assessing the in vivo biocompatibility of molecularly imprinted polymer nanoparticles

Molecularly imprinted polymer nanoparticles (nanoMIPs) are high affinity synthetic receptors which show promise as imaging and therapeutic agents. Comprehensive analysis of the in vivo behaviour of nanoMIPs must be performed before they can be considered for clinical applications. This work reports the solid-phase synthesis of nanoMIPs and an investigation of their biodistribution, clearance and cytotoxicity in a rat model following both intravenous and oral administration. These nanoMIPs were found in each harvested tissue type, including brain tissue, implying their ability to cross the blood brain barrier. The nanoMIPs were cleared from the body via both faeces and urine. Furthermore, we describe an immunogenicity study in mice, demonstrating that nanoMIPs specific for a cell surface protein showed moderate adjuvant properties, whilst those imprinted for a scrambled peptide showed no such behaviour. Given their ability to access all tissue types and their relatively low cytotoxicity, these results pave the way for in vivo applications of nanoMIPs

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

Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes

The aim of this work is to evaluate whether the size of the analyte used as template for the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) can affect their performance in pseudo-enzyme linked immunosorbent assays (pseudo-ELISAs). Successful demonstration of a nanoMIPs-based pseudo-ELISA for vancomycin (1449.3 g mol) was demonstrated earlier. In the present investigation, the following analytes were selected: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12 kDa) biotin (244.31 g mol) and melamine (126.12 g mol). NanoMIPs with a similar composition for all analytes were synthesised by persulfate-initiated polymerisation in water. In addition, core-shell nanoMIPs coated with polyethylene glycol (PEG) and imprinted for melamine were produced in organics and tested. The polymerisation of the nanoparticles was done using a solid-phase approach with the correspondent template immobilised on glass beads. The performance of the nanoMIPs used as replacement for antibodies in direct pseudo-ELISA (for the enzymes) and competitive pseudo-ELISA for the smaller analytes was investigated. For the competitive mode we rely on competition for the binding to the nanoparticles between free analyte and corresponding analyte-HRP conjugate. The results revealed that the best performances were obtained for nanoMIPs synthesised in aqueous media for the larger analytes. In addition, this approach was successful for biotin but completely failed for the smallest template melamine. This problem was solved using nanoMIP prepared by UV polymerisation in an organic media with a PEG shell. This study demonstrates that the preparation of nanoMIP by solid-phase approach can produce material with high affinity and potential to replace antibodies in ELISA tests for both large and small analytes. This makes this technology versatile and applicable to practically any target analyte and diagnostic field

Сенсорная система для определения сульфаметоксазола на основе молекулярно импринтированных полимерных мембран

Проблематика. Розробка сенсорних систем на основі штучних аналогів біологічних макромолекул є актуальною для сучасної аналітичної біотехнології, оскільки забезпечує нові ефективні експрес-методи детекції малих органічних молекул, в тому числі фармацевтичних препаратів. Мета дослідження. У роботі пропонується аналітична система для високоселективного та чутливого визначення сульфаметоксазолу на основі молекулярно імпринтованих полімерних (МІП) мембран, синтезованих із застосуванням методу полімеризації in situ у комбінації з методом комп’ютерного моделювання. Методика реалізації. Молекули сульфаметоксазолу, селективно адсорбовані штучними рецепторними сайтами у структурі МІП мембран, візуалізували завдяки їх здатності формувати забарвлені у коричневий колір комплекси після реакції з фериціанідом калію та нітропрусидом натрію в лужному середовищі. Результати дослідження. Межа визначення сульфаметоксазолу становила 2 мМ, а лінійний динамічний діапазон роботи сенсорної системи – 2–15 мМ, що дає змогу визначати сульфаметоксазол у фармацевтичних препаратах. Стабільність розроблених сенсорних систем на основі МІП становила принаймні 6 місяців, що значно перевищує стабільність аналогічних пристроїв на основі природних рецепторів. Висновки. Доведено придатність розроблених сенсорних систем для аналізу сульфаметоксазолу як у модельних, так і в реальних зразках (комерційно доступних фармацевтичних препаратах). Розроблені сенсорні системи характеризуються високою селективністю, чутливістю, портативністю та невисокою вартістю.Background. Development of sensor systems based on synthetic mimics of biological molecules will provide new effective express-methods for detection of small organic molecules, including pharmaceuticals, for modern analytical biotechnology. Objective. An analytical system for highly selective and sensitive detection of sulfamethoxazole based on molecularly imprinted polymer (MIP) membranes is proposed, synthesized using the method of in situ polymerization in a combination with the method of computational modeling. Methods. Sulfamethoxazole molecules, that were selectively adsorbed by the synthetic binding sites in MIP membranes structure, were visualized due to their ability to form brown-colored complexes after reaction with potassium ferricyanide and sodium nitroprusside in alkaline media. Results. The limit for sulfamethoxazole detection comprised 2 mM, while the linear dynamic range – 2–15 mM, which allows one to detect sulfamethoxazole in pharmaceutical preparations. Stability of the developed MIP-based sensor systems was estimated as at least 6 months, which significantly increases stability of analogous devices based on natural receptors. Conclusions. Applicability of the developed sensor systems for the analysis of sulfamethoxazole in both model solutions and real samples (commercial pharmaceutical preparations) was proven. The developed systems are characterized with high selectivity, sensitivity, small size and low cost.Проблематика. Разработка сенсорных систем на основе искусственных аналогов биологических макромолекул является актуальной для современной аналитической биотехнологии, поскольку обеспечивает новые эффективные экспресс-методы детекции малых органических молекул, в том числе фармацевтических препаратов. Цель исследования. В работе предлагается аналитическая система для высокоселективного и чувствительного определения сульфаметоксазола на основе молекулярно импринтированных полимерных (МИП) мембран, синтезированных с использованием метода полимеризации in situ в комбинации с методом компьютерного моделирования. Методика реализации. Молекулы сульфаметоксазола, селективно адсорбированные синтетическими рецепторными сайтами в структуре МИП мембран, визуализировали благодаря их способности формировать окрашенные в коричневый цвет комплексы после реакции с феррицианидом калия и нитропруссидом натрия в щелочной среде. Результаты исследования. Предел обнаружения сульфаметоксазола составил 2 мМ, а линейный динамический диапазон сенсорной системы – 2–15 мМ, что дает возможность определять сульфаметоксазол в фармацевтических препаратах. Стабильность разработанных сенсорных систем на основе МИП составила по крайней мере 6 месяцев, что значительно превосходит стабильность аналогичных приборов на основе природных рецепторов. Выводы. Доказана возможность использования разработанных сенсорных систем для анализа сульфаметоксазола как в модельных, так и в реальных образцах (коммерчески доступных фармацевтических препаратах). Разработанные сенсорные системы характеризуются высокой селективностью, чувствительностью, портативностью и невысокой стоимостью

Interfacial Molecular Imprinting in Nanoparticle-Stabilized Emulsions

A new interfacial nano and molecular imprinting approach is developed to prepare spherical molecularly imprinted polymers with well-controlled hierarchical structures. This method is based on Pickering emulsion polymerization using template-modified colloidal particles. The interfacial imprinting is carried out in particle-stabilized oil-in-water emulsions, where the molecular template is presented on the surface of silica nanoparticles during the polymerization of the monomer phase. After polymerization, the template-modified silica nanoparticles are removed from the new spherical particles to leave tiny indentations decorated with molecularly imprinted sites. The imprinted microspheres prepared using the new interfacial nano and molecular imprinting have very interesting features: a well-controlled hierarchical structure composed of large pores decorated with easily accessible molecular binding sites, group selectivity toward a series of chemicals having a common structural moiety (epitopes), and a hydrophilic surface that enables the MIPs to be used under aqueous conditions

Bacteria clustering by polymers induces the expression of quorum sense controlled phenotypes

Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one mean by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are thus a potential means to control bacterial population responses. Here we report how polymeric "bacteria sequestrants", designed to bind to bacteria through electrostatic interactions and thus inhibit bacterial adhesion to surfaces, induce the expression of quorum sensing controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterise the feedback between bacteria clustering and quorum sensing signaling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population leve

Iodo silanes as superior substrates for the solid phase synthesis of molecularly imprinted polymer nanoparticles

Current state-of-the-art techniques for the solid phase synthesis of molecularly imprinted polymer (MIP) nanoparticles typically rely on amino silanes for the immobilisation of template molecules prior to polymerisation. An investigation into commonly used amino silanes identified a number of problematic side reactions which negatively affect the purity and affinity of these polymers. Iodo silanes are presented as a superior alternative in a case study describing the synthesis of MIPs against epitopes of a common cancer biomarker, epidermal growth factor receptor (EGFR). The proposed iodo silane outperformed the amino silane by all metrics tested, showing high purity and specificity, and nanomolar affinity for the target peptide

Biotin–specific synthetic receptors prepared using molecular imprinti

The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido- 2-methyl-propanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic "receptor" sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. A good correlation was found between the modelling results and the performance of the materials in the template rebinding study. The dissociation constants for all MIPs were 1.4-16.8 nM, which is sufficient for most analytical applications where biotin is used as a label

Development of a sensor prepared by entrapment of MIP particles in electrosynthesised polymer films for electrochemical detection of ephedrine

A voltammetric sensor for (−)-ephedrine has been prepared by a novel approach based on immobilisation of an imprinted polymer for ephedrine (MIPE) in an electrosynthesised polypyrrole (PPY) film. Composite films were grown potentiostatically at 1.0V vs. Pt (QRE) on a glassy carbon electrode using an unconventional “upside-down” (UD) geometry for the three-electrode cell. As a consequence, a high MIP loading was obtained, as revealed by SEM. The sensor response was evaluated, after overoxidation of PPY matrix, by cyclic voltammetry after pre-concentration in a buffered solution of analyte in 0.5–3mM concentration range. An ephedrine peak at ≈0.9V increasing with concentration and saturating at high concentrations was evident. PPY-modified electrode showed a response, which was distinctly lower than the MIP response for the same concentration of the template. The effect of potential interferences including compounds usually found in human fluids (ascorbic acid, uric acid, urea, glucose, sorbitol, glycine, dopamine) was examined