Hyaluronan/Diethylaminoethyl Chitosan Polyelectrolyte Complexes as Carriers for Improved Colistin Delivery.

Improving the therapeutic characteristics of antibiotics is an effective strategy for controlling the growth of multidrug-resistant Gram-negative microorganisms. The purpose of this study was to develop a colistin (CT) delivery system based on hyaluronic acid (HA) and the water-soluble cationic chitosan derivative, diethylaminoethyl chitosan (DEAECS). The CT delivery system was a polyelectrolyte complex (PEC) obtained by interpolymeric interactions between the HA polyanion and the DEAECS polycation, with simultaneous inclusion of positively charged CT molecules into the resulting complex. The developed PEC had a hydrodynamic diameter of 210-250 nm and a negative surface charge (ζ-potential = -19 mV); the encapsulation and loading efficiencies were 100 and 16.7%, respectively. The developed CT delivery systems were characterized by modified release (30-40% and 85-90% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro experiments showed that the encapsulation of CT in polysaccharide carriers did not reduce its antimicrobial activity, as the minimum inhibitory concentrations against Pseudomonas aeruginosa of both encapsulated CT and pure CT were 1 µg/mL.

Supramolecular Sensors for Opiates and Their Metabolites.

The present study highlights a sensing approach for opiates using acyclic cucurbituril (aCBs) sensors comprising four glycouril units terminated on both ends with naphthalene fluorophore walls. The connectivity between the glycourils and naphthalene rings largely defines the opening size of the cucurbituril cavity and its diameter. The large hydrophobic binding cavity is flexible and is able to adapt to guests of various size and topology. The recognition event between the aCBs and guests results in modification of the fluorescence of the terminal walls, a fluorescence response that can be used to sense the drugs of abuse morphine, heroin, and oxycodone as well as their metabolites. Molecular dynamics is employed to understand the nature of the binding interactions. A simple three sensor cross-reactive array enables the determination of drugs and their metabolites in water with high fidelity and low error. Quantitative experiments performed in urine using a new three-way calibration model allows for determination of drugs and their metabolites using one sensor from a single fluorescence reading.

High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening.

A simple and efficient method for determination of the yield, enantiomeric/diasteriomeric excess (ee/de), and absolute configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach utilizes a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan methyl ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophysical properties and allow for fluorescence-based ee determination of molecules containing a 1,2- or 1,3-diol moiety. This method has been utilized for the screening of ee in a number of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asymmetric hydrogenation of benzil was performed in a highly parallel fashion with errors <1 % ee confirming the feasibility of the systematic examination of crude products from the parallel asymmetric synthesis in real time and in a high-throughput screening (HTS) fashion.

Toward Fluorescence-Based High-Throughput Screening for Enantiomeric Excess in Amines and Amino Acid Derivatives.

A highly accurate and reliable screening method for enantiomeric excess of amine derivatives in the presence of water is reported. The fluorescence-based screening system has been realized by self-assembly of chiral diol-type dyes (BINOL, VANOL and VAPOL), 2-formylphenylboronic acid, and chiral amines forming iminoboronate esters. The structure and chirality of the amine analytes determine the stability of the diastereomeric iminoboronate esters, which in turn display differential fluorescence. The fluorescence signal reflects the enantiomeric purity of the chiral amines and was utilized in high-throughput arrays. The arrays were able to recognize enantiomeric excess of amines, amino esters, and amino alcohols. In addition to qualitative analysis, quantitative experiments were successfully performed. Studies of the role of additives such as water or citrate were carried out to gain insight into the stability of the iminoboronate esters. It is shown that the above additives destabilize less stable esters while the stable esters remain unchanged. Thus, the presence of water and citrate leads to increased difference between the diastereomeric iminoboronates and contributes to the enantiodiscrimination of the chiral amines.

Determination of enantiomeric excess in amine derivatives with molecular self-assemblies.

We report the first fluorescence-based assay for the rapid determination of the ee value of amines, amino alcohols, and amino acid esters. The method uses the self-assembly of 2-formylphenylboronic acid with a chiral diol and a chiral amine or derivatives (of unknown chirality) to produce two diastereomeric iminoboronates that differ in their fluorescence intensity and polarization. The approach allows for the accurate determination of the ee value of chiral amines with errors of just 1-2%. We believe that this application of orthogonal dynamic covalent self-assembly in the determination of the enantioselectivity will lead to the development of high-throughput procedures for the determination of chirality.

Hyaluronan/colistin polyelectrolyte complexes: Promising antiinfective drug delivery systems.

Nanotechnology-based modification of known antimicrobial agents is a rational and straightforward way to improve their safety and effectiveness. The aim of this study was to develop colistin (CT)-loaded polymeric carriers based on hyaluronic acid (HA) for potential application as antimicrobial agents against multi-resistant gram-negative microorganisms (including ESKAPE pathogens). CT-containing particles were obtained via a polyelectrolyte interaction between protonated CT amino groups and HA carboxyl groups (the CT-HA complex formation constant [logKCT-HA] was about 5.0). The resulting polyelectrolyte complexes had a size of 210-250 nm and a negative charge (ζ-potential -19 mV), with encapsulation and loading efficiencies of 100% and 20%, respectively. The developed CT delivery systems were characterized by modified release (45% and 85% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro tests showed that the encapsulation of CT in polymer particles did not reduce its pharmacological activity; the minimum inhibitory concentrations of both encapsulated CT and pure CT were 1 µg/mL (against Pseudomonas aeruginosa).

High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening.

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.

Polypeptide Self-Assembled Nanoparticles as Delivery Systems for Polymyxins B and E.

Polymyxins are peptide antibiotics that are highly efficient against many multidrug resistant pathogens. However, the poor stability of polymyxins in the bloodstream requires the administration of high drug doses that, in turn, can lead to polymyxin toxicity. Consequently, different delivery systems have been considered for polymyxins to overcome these obstacles. In this work, we report the development of polymyxin delivery systems based on nanoparticles obtained from the self-assembly of amphiphilic random poly(l-glutamic acid-co-d-phenylalanine). These P(Glu-co-dPhe) nanoparticles were characterized in terms of their size, surface charge, stability, cytotoxicity, and uptake by macrophages. The encapsulation efficiency and drug loading into P(Glu-co-dPhe) nanoparticles were determined for both polymyxin B and E. The release kinetics of polymyxins B and E from nanoformulations was studied and compared in buffer solution and human blood plasma. The release mechanisms were analyzed using a number of mathematical models. The minimal inhibitory concentrations of the nanoformulations were established and compared with those determined for the free antibiotics.

A dual chromophore sensor for the detection of amines, diols, hydroxy acids, and amino alcohols.

The determination of enantiomeric excess (ee) in various groups of chiral compounds, namely amines, amino alcohols, diols, and hydroxy acids is performed using a dual chromophore FRET/PET based sensor ensemble. The sensing ensemble utilizes fluorescence changes from two chromophores (indicators) to classify 13 pairs of enantiomers as well as allows for the qualitative and quantitative determination of ee of various classes of chiral compounds with high accuracy (<2% error).