Ouzo polymerization: A bottom-up green synthesis of polymer nanoparticles by free-radical polymerization of monomers spontaneously nucleated by the Ouzo effect; Application to molecular imprinting.

HYPOTHESIS: Top-down fabrication of polymer nanoparticles from preformed polymers by spontaneous colloid formation under influence of the Ouzo effect is a widely applied concept whereas bottom-up free-radical polymerization of monomers nucleated under the Ouzo regime have found limited application after a seminal report almost half a century ago. We hypothesized that the approach would be of value today as a versatile method for green synthesis of polymer nanoparticles, including molecularly imprinted ones. EXPERIMENTS: Ternary mixtures of pentaerythritol triacrylate, ethanol, and water were prepared by either a one-pot-one-step batch-wise procedure or a continuous-flow process using a 3D-printed micro-fluidic mixer. The mixtures were subjected to free-radical polymerization. Cortisol was added as a template to generate a molecular memory through molecular imprinting. Characterization of the fabricated nanoparticles was carried out by dynamic light scattering, electron microscopy, and binding studies. FINDINGS: Compositions in the Ouzo region of the ternary phase diagram provided spontaneous and instantaneous formation of nucleated monomer droplets. Free-radical polymerization, promoted by heat or UV light, transformed the droplets into polymer nanoparticles in a green and sustainable route. Addition of cortisol created polymer nanoparticles with high affinity for cortisol in aqueous media. Competition studies showed some cross-reaction with other steroids, comparable to that found with antibodies, and complete discrimination of structurally unrelated drug molecules.

Molecularly imprinted polymer nanocarriers for sustained release of erythromycin.

PURPOSE: To develop and evaluate molecularly imprinted nanocarriers for sustained release of erythromycin in physiological buffer media. METHODS: Erythromycin-imprinted poly(methacrylic acid-co-trimethylolpropane trimethacrylate) nanocarriers and corresponding control nanocarriers were prepared by free-radical precipitation polymerization. The nanocarriers were characterized by transmission electron microscopy, dynamic light scattering, and nitrogen sorption analysis. Binding studies were carried out with erythromycin and five structurally unrelated drugs. Molecular descriptors of the drugs were computed and correlated to measured binding data by multivariate data analysis. Loading with erythromycin and in vitro release studies were carried out in physiological buffer media. Kinetic models were fitted to drug release data. RESULTS: The template affected the size and morphology of the nanocarriers. Binding isotherms showed that erythromycin-imprinted nanocarriers had a higher erythromycin binding capacity than corresponding control nanocarriers. Multivariate data analysis, correlating binding to molecular descriptors of the drugs, indicated a molecular imprinting effect. Erythromycin loading capacity was 76 mg/g with a loading efficiency of 87%. Release studies in physiological buffer showed an initial burst release of a quarter of loaded erythromycin during the first day and an 82% release after a week. The release was best described by the Korsmeyer-Peppas model. CONCLUSIONS: Sustained release of erythromycin in physiological buffer was demonstrated.

QSRR analysis of ß-lactam antibiotics on a penicillin G targeted MIP stationary phase.

The imprinting factors of the ß-lactam antibiotics penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, and piperacillin on a poly(methacrylic acid-co-trimethylolpropane trimethacrylate) molecularly imprinted stationary phase targeted for penicillin G were correlated with molecular descriptors obtained by molecular computation. One-parameter linear regression and multivariate data analysis by principal component analysis and partial least square regression indicated that descriptors associated with molecular topology, shape, size, and volume were highly correlated with the imprinting factor and influential on the derived models.

The use of magnetite nanoparticles for implant-assisted magnetic drug targeting in thrombolytic therapy.

Implant-assisted targeting of magnetic particles under the influence of an external magnetic field has previously been verified through mathematical modeling, in vitro studies, and in vivo studies on rat carotid arteries as a feasible method for localized drug delivery. The present study focuses on the development of nanoparticles for the treatment of in-stent thrombosis. Magnetic nanoparticles in the size-range 10-30 nm were synthesized in a one-pot procedure by precipitation of ferrous hydroxide followed by oxidation to magnetite. The nanoparticles were silanized with tetraethyl orthosilicate in the presence of triethylene glycol and/or polyethylene glycol. The surface coated magnetite nanoparticles were activated with either N-hydroxysulfosuccinimide or tresyl chloride for covalent immobilization of tissue plasminogen activator (tPA). Hysteresis loops showed saturation magnetizations of 55.8, 44.1, and 43.0 emu/g for the naked nanoparticles, the surface coated nanoparticles, and the tPA-nanoparticle conjugates, respectively. The hemolytic activity of the nanoparticles in blood was negligible. An initial in vivo biocompatibility test in pig, carried out by intravascular injection of the nanoparticles in a stented brachial artery, showed no short-term adverse effects. In vitro evaluation in a flow-through model proved that the nanoparticles were captured efficiently to the surface of a ferromagnetic coiled wire at the fluid velocities typical for human arteries. A preliminary test of the tPA-nanoparticle conjugates in a pig model suggested that the conjugates may be used for treatment of in-stent thrombosis in coronary arteries.

Influence of salt ions on binding to molecularly imprinted polymers.

Salt ions were found to have an influence on template binding to two model molecularly imprinted polymers (MIPs), targeted to penicillin G and propranolol, respectively, in water-acetonitrile mixtures. Water was detrimental to rebinding of penicillin G whereas propranolol bound in the entire water-acetonitrile range tested. In 100% aqueous solution, 3-M salt solutions augmented the binding of both templates. The effects followed the Hofmeister series with kosmotropic ions promoting the largest increase. Binding was mainly of a non-specific nature under these conditions. In acetonitrile containing low amounts of water, the specific binding to the MIPs increased with the addition of salts. Binding of penicillin G followed the Hofmeister series while an ion-exchange mechanism was observed for propranolol. The results suggest that hydration of kosmotropic ions reduces the water activity in water-poor media providing a stabilizing effect on water-sensitive MIP-template interactions. The effects were utilized to develop a procedure for molecularly imprinted solid-phase extraction (MISPE) of penicillin G from milk with a recovery of 87%.

Development and evaluation of spherical molecularly imprinted polymer beads.

The majority of studies on molecularly imprinted polymers has until now been carried out on irregularly shaped particles prepared by grinding of polymer monoliths. The preparation procedures are time- and labor-consuming and produce particles of wide size distributions. To answer the need for fast and straightforward routes to spherical molecularly imprinted polymer beads, we have developed a method comprising the formation of droplets of pre-polymerization solution directly in mineral oil by vigorous mixing followed by transformation of the droplets into solid spherical beads by photoinduced free-radical polymerization. No detergents or stabilizers were required for the droplet formation. Factors influencing the bead synthesis have been investigated and are detailed here. The beads were evaluated in parallel with corresponding irregularly shaped particles prepared from polymer monoliths. Conditions for the synthesis of propranolol-imprinted poly(methacrylic acid-co-trimethylolpropane trimethacrylate) beads in the size range of 1-100 microm in almost quantitative yield are described. The beads were applied as the recognition element in a 96-well plate format radioligand assay of propranolol in human serum.

Molecular engineering of fluorescent penicillins for molecularly imprinted polymer assays.

The interaction of seven novel fluorescent labeled beta-lactams with a library of six polymer materials molecularly imprinted (MI) with penicillin G (PenG) has been evaluated using both radioactive and fluorescence competitive assays. The highly fluorescent competitors (emission quantum yields of 0.4-0.95) have been molecularly engineered to contain pyrene or dansyl labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers. Pyrenemethylacetamidopenicillanic acid (PAAP) is the tagged antibiotic that provides the highest selectivity when competing with PenG for the specific binding sites in a MI polymer prepared with methacrylic acid and trimethylolpropane trimethacrylate (10:15 molar ratio) in acetonitrile in the presence of PenG. Molecular modeling shows that recognition of the fluorescent analogues of PenG by the MI material is due to a combination of size and shape selectivity and demonstrates how critical the choice of label and tether chain is. PAAP has been applied to the development of a fluorescence competitive assay for PenG analysis with a dynamic range of 3-890 muM in 99:1 acetonitrile-water solution. Competitive binding studies demonstrate various degrees of cross-reactivity for some antibiotics derived from 6-aminopenicillanic acid, particularly amoxicillin, ampicillin, and penicillin V (but not oxacillin, cloxacillin, dicloxacillin, or nafcillin). Other antibiotics, such as chloramphenicol, tetracycline, or cephapirin, do not compete with PAAP for binding to the imprinted polymer. The MI assay has successfully been tested for PenG analysis in a pharmaceutical formulation.

Synthesis and screening of a molecularly imprinted polymer library targeted for penicillin G.

A library of molecularly imprinted polymers (MIPs) was synthesized by radical bulk polymerization using the beta-lactam antibiotic penicillin G as the template. Diversity of the library was obtained by combining various functionalized monomers and cross-linkers and by varying the stoichiometry and the concentration of the components in the prepolymerization mixtures. The library was screened for selectivity to penicillin G by a radioligand binding assay and was compared to a corresponding control library. The best MIP candidate, showing the highest selectivity for penicillin G, was prepared from methacrylic acid and trimethylolpropane trimethacrylate as the functionalized monomer and cross-linker, respectively. Cross-reactivity studies with other beta-lactam antibiotics showed a low cross-reactivity of penicillin V (15%), ampicillin (16%), and amoxicillin (19%). Nafcillin and oxacillin showed less cross-reactivity (<1%). Cross-reaction with a cephalosporin antibiotic (cephapirin) and structurally nonrelated antibiotics (chloramphenicol, tetracycline, dapsone, and erythromycin) was less than 0.01%.