Efficient treatment of murine systemic infection with Candida albicans using amphotericin B incorporated in nanosize range particles (emulsomes).

The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg(-1). In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans.

Towards the development of molecularly imprinted artificial receptors for the screening of estrogenic chemicals.

Molecularly imprinted polymers are prepared using various steroid compounds as the templates. The imprinted polymers can selectively re-bind the original print molecules, which leads to versatile potential applications. The feasibility of using these artificial receptors to replace their biological counterparts for preliminary screening of a chemical library is demonstrated. A steroid library composed of 22 closely related compounds is screened with an estrogen specific polymer. The print molecule is identified with accuracy and structural similarities of other members are correlated with normalized retention indices. Molecularly imprinted artificial receptors are envisioned as being useful for screening purposes in drug discovery or for identifying endocrine-disrupting chemicals.

Chemiluminescence imaging ELISA using an imprinted polymer as the recognition element instead of an antibody.

An imaging assay analogous to competitive enzyme immunoassays has been developed using a molecularly imprinted polymer instead of an antibody. The antigen 2,4-dichlorophenoxyacetic acid (2,4-D) was labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. Microtiter plates (96 or 384 wells) were coated with polymer microspheres imprinted with 2,4-D, which were fixed in place by using poly(vinyl alcohol) as glue. In a competitive mode, the analyte-peroxidase conjugate was incubated with the free analyte in the microtiter plate, after which the bound fraction of the conjugate was quantified. After addition of the chemiluminescent substrates, light emission was measured in a high-throughput imaging format with a CCD camera. Calibration curves corresponding to analyte concentrations ranging from 0.01 to 100 microg/mL were obtained.

New configurations and applications of molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are applicable in a variety of different configurations. For example, bulk polymers imprinted with beta-lactam antibiotics are presented to be used as stationary phases for the chromatographic separation of beta-lactam antibiotics with both aqueous and organic mobile phases. However, in some analytical applications, monosized spherical beads are preferred over the currently used ground bulk polymers. A precipitation polymerization technique allows preparation of monosized spherical imprinted beads with diameters down to 200 nm having excellent recognition properties for different target molecules. Nevertheless, with current imprinting protocols a substantial amount of template has to be used to prepare the polymer. This can be problematic if the template is poorly soluble, expensive or difficult to obtain. It is shown that for analytical applications, the functional monomer:template ratio can be drastically increased without jeopardizing the polymer's recognition properties. Furthermore, a substantial reduction of the degree of crosslinking is demonstrated, resulting in much more flexible polymers that are useful for example the preparation of thin imprinted films and membranes for sensors. Apart from analysis, MIPs also are applicable in chemical or enzymatic synthesis. For example, MIPs using the product of an enzyme reaction as template are utilized for assisting the synthetic reaction by continuously removing the product from the bulk solution by complexation. This results in an equilibrium shift towards product formation.

Influence of mobile phase composition and cross-linking density on the enantiomeric recognition properties of molecularly imprinted polymers.

A series of experiments were conducted to investigate elements which affect the enantiomeric recognition properties of molecularly imprinted polymers (MIPs) in the HPLC mode. Our results show that the recognition properties of MIPs are greatly influenced by the mobile phase used. For a polymer prepared in acetonitrile, a good enantiomeric separation was observed when acetonitrile-based mobile phase was used, when the mobile phase was changed to chloroform-based, no enantiomeric recognition was observed although the sample molecule was retarded. This indicates that the specific co-operative binding interactions between the functional groups at the imprinted polymer's recognition sites and the sample molecule were considerably disrupted and only non-specific interactions remained. When the mobile phase was changed back to acetonitrile-based, the recognition was regained. In contrast, for polymers prepared in chloroform, chloroform-based mobile phase gave much better separation than acetonitrile-based mobile phase. When other solvents were tested, significant solvent effects were generally observed. Based on these observations, the recognition properties of the methacrylic acid (MAA)-co-ethylene glycol dimethacrylate (EGDMA) polymers were reinvestigated, and the results show that by simply using an optimised mobile phase system, significantly improved recognition over previously reported results was observed. For a polymer made against Cbz-L-Trp, 100 microg of Cbz-D,L-Trp was separated with a separation factor (alpha) of 4.23 and a resolution (Rs) of 3.87, whereas in the previous report, 10 microg of Cbz-D,L-Trp was only separated with alpha = 1.67 and Rs = 0.1. It is generally realised that the imprinted polymer's recognition property is also very much influenced by the nature of the polymer network. It was shown that the recognition decreased with a decrease in the apparent degree of cross-linking (molar percentage of cross-linker in the polymerisation mixture). Nonetheless, our results show that in our optimised assay system a significant separation could still be obtained on a polymer which was only 22% cross-linked. We consider this to be of importance, since it may suggest a way of imprinting larger molecules because of the possibly improved mass transfer in low cross-linking density polymers. It was reported that when trifunctional cross-linkers [for example: trimethylolpropane trimethacrylate (TRIM)] were used as the cross-linker instead of EGDMA, considerably improved enantiomeric separation and resolving capability were observed. Our results show that the improved performance of the MAA-co-EGDMA MIPs is actually comparable to the performance of the MIPs prepared with those trifunctional cross-linkers. The combination of a hydrogen bonding functional monomer (acrylamide) with TRIM also did not give improved recognition. The results suggest that although the three-dimensional network of these two kinds of polymer may be quite different, the observed recognition improvements were probably largely due to solvent effect.

Synthesis and catalysis by molecularly imprinted materials.

Molecularly imprinted materials have been demonstrated to possess a very high degree of selectivity towards targeted substrates. In addition to such tailor-made molecular recognition, progress has been made in introducing reactive groups into the recognition sites. Putting teeth into imprinted matrices is one method of making true enzyme mimics or plastizymes, which are plastic polymer enzyme mimics.

Imprinted polymer-based sensor system for herbicides using differential-pulse voltammetry on screen-printed electrodes.

A sensor system for the herbicide 2,4-dichlorophenoxy-acetic acid has been developed based on specific recognition of the analyte by a molecularly imprinted polymer and electrochemical detection using disposable screen-printed electrodes. The method involves a competitive binding step with a nonrelated electrochemically active probe. For batch binding assays, imprinted polymer particles are incubated in suspension with the analyte and the probe, followed by centrifugation and quantification of the unbound probe in the supernatant. Two different compounds, namely 2,4-dichlorophenol and homogentisic acid, were tested as potential electroactive probes. Both compounds could be conveniently detected by differential-pulse voltammetry on screen-printed, solvent-resistant three-electrode systems having carbon working electrodes. Whereas 2,4-dichlorophenol showed very high nonspecific binding to the polymer, homogentisic acid bound specifically to the imprinted sites and thus allowed calibration curves for the analyte in the micromolar range to be recorded. An integrated sensor was developed by coating the imprinted polymer particles directly onto the working electrode. Following incubation of the modified electrode in a solution containing the analyte and the probe, the bound fraction of the probe is quantified. This system provides a cheap, disposable sensor for rapid determination of environmentally relevant and other analytes.

Use of molecularly imprinted polymers in a biotransformation process.

Molecularly imprinted polymers are highly stable and can be sterilised, making them ideal for use in biotransformation process. In this communication, we describe a novel application of molecularly imprinted polymers in an enzymatic reaction. The enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to give Z-L-Asp-L-Phe-OMe (Z-aspartame) was chosen as a model system to evaluate the applicability of using molecularly imprinted polymers to facilitate product formation. When the product-imprinted polymer is present, a considerable increase (40%) in product yield is obtained. Besides their use to enhance product yields, as demonstrated here, we suggest that imprinted polymers may also find use in the continuous removal of toxic compounds during biochemical reactions.

Plastic antibodies: developments and applications.

Antibodies are natural receptor molecules produced after contact with an antigen. In an attempt to mimic nature, the technique of molecular imprinting has been developed, which allows specific recognition sites to be formed in synthetic polymers through the use of templates. These recognition sites mimic the binding sites of antibodies and may be substituted for them in applications such as affinity separation, assay systems and biosensors. The stability and low cost of these polymers make them particularly attractive to industry.

Magnetic molecularly imprinted polymer beads for drug radioligand binding assay.

Molecularly imprinted polymer-magnetic iron oxide composite materials which exhibit recognition properties and can be withdrawn from solution by application of a magnetic field were prepared for the first time. Magnetic iron oxide was incorporated using a suspension polymerisation methodology with a perfluorocarbon liquid as the dispersing phase for the preparation of methacrylic acid-1,1,1-trimethylolpropane trimethacrylate copolymer beads molecularly imprinted with the beta-blocker (S)-propranolol. The resulting superparamagnetic imprinted polymer beads were capable of binding [3H]-(S)-propranolol more strongly than a non-imprinted, otherwise identical, polymer. In a competitive radioligand binding assay using a magnet to separate polymer from solution, (R)-propranolol and (R,S)-metoprolol exhibited cross-reactivities of 19 and 0.7%, respectively, compared with (S)-propranolol.

Novel peptides binding to the Fc-portion of immunoglobulins obtained from a combinatorial phage display peptide library.

Peptides interacting with the Fc portion of human IgG (IgG Fc) were selected from a phage display decapeptide library. The library was selected five times and interacting phage peptides were eluted either with Staphylococcal protein A or at low pH. Individual peptide phage clones were found to interact more strongly with IgG Fc than did either the original library or the wild-type phage. Increasing concentrations of protein A could competitively reduce the interaction of a peptide phage clone (FARLVSSIRY) eluted with protein A to the same level as the original library. Furthermore, when immunoglobulins from chicken, donkey, human, mouse, swine, rabbit, and sheep were included, peptide phage clones FGRLVSSIRY and TWKTSRISIF interacted strongly with human IgG Fc and porcine IgG and weakly with the immunoglobulins obtained from the other species.

Some new developments and challenges in non-covalent molecular imprinting technology.

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules via the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include the use of molecularly imprinted polymers as tailor-made separation materials, antibody and receptor binding site mimics in recognition and assay systems, enzyme mimics for catalytic applications and as recognition elements in biosensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial-scale production and application.

Insights into the origins of binding and the recognition properties of molecularly imprinted polymers prepared using an amide as the hydrogen-bonding functional group.

Molecularly imprinted polymers (MIPs) prepared using an amide hydrogen-bonding functional monomer (acrylamide) exhibited efficient enantiomeric recognition properties in both organic and aqueous media in the HPLC mode. The results indicate that the amide functional groups formed strong hydrogen-bonding interactions with the template molecule, and specific recognition sites were created within the polymer matrix during the imprinting process. When Boc-L-Trp was used as the template, an MIP prepared in a polar organic solvent (acetonitrile) using acrylamide as the functional monomer showed better enantiomeric recognition of Boc-Trp than the MIPs prepared in the same solvent using an acidic (methacrylic acid) or a basic (2-vinylpyridine) functional monomer or a combination of an acidic and a basic functional monomer (methacrylic acid + 2-vinylpyridine). Our results indicate that in organic media the degree of retention of the sample molecule on the imprinted polymer was controlled by hydrogen-bonding interactions between the sample molecule and the polymer, while in aqueous media it was determined to a considerable extent by hydrophobic interactions. In both media the shape, size and the nature of the hydrogen-bonding groups of the sample molecules were all important factors in determining the enantiomeric and substrate selectivity. In the aqueous media, however, the hydrophobicity of the sample molecules was also found to play an important role.

A new application of molecularly imprinted materials.

We have studied the possibility of shifting a thermodynamically unfavourable enzymatic equilibrium towards product formation via the addition of a highly specific adsorbent. The commercially interesting enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to the sweetener aspartame was chosen as the model system. Extremely stable and specific adsorbents for the product Z-L-Asp-L-Phe-OMe (Z-aspartame) were prepared using the emerging technique of molecular imprinting. A considerable increase (40%) in the yield of product was obtained when such adsorbents were present during the enzymatic reaction. The message of this investigation is that the use of such specific, sterilizable adsorbents should be considered for enzymatic processes to increase the yield. Finally, the direct isolation of a product formed by the retrieval of the adsorbents carrying the product can be envisaged, especially if the adsorbents are magnetic.

Assay system for the herbicide 2,4-dichlorophenoxyacetic Acid using a molecularly imprinted polymer as an artificial recognition element.

Noncovalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex relied on hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometer-sized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated.