Dorota Kowalczuk

Characterization of the developed antimicrobial urological catheters

Antimicrobial urological catheters were developed by the mixed, covalent and non-covalent binding of sparfloxacin (SPA) to heparin (HP) film which was first deposited on the latex surface of biomaterial. The SPA-HP modified surface was characterized by SEM analysis and ATR-Fourier transform infrared spectroscopy. For the antimicrobial prevention, SPA as an antibiotic with a broad antimicrobial spectrum was chosen. Antimicrobial activity of antibiotic-modified catheter against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli strains was assessed using various procedures. On the basis of the inhibition zone and diffusion assays the efficacy around the modified catheters was demonstrated. The test samples clearly showed an antibacterial activity against all tested bacterial stains for a least one month. Inhibition of the bacterial colonization on the modified catheter surface was proved by the biofilm test. Antimicrobial activity of SPA-treated catheter surface was also quantitatively evaluated according to standard method of ISO based on JIS. The R-values were found to be higher than 3.8. The performed research indicated that the immobilization of SPA on the catheter surface by means of the mixed-type bonds resulted in stable antibacterial protection of the urological catheters for a long time.

Covalent coating of hydroxyapatite by keratin stabilizes gentamicin release

A novel hybrid hydroxyapatite (HAP) matrix, covalently coated with rarely applied, hardly degradable keratin and effectively modified by gentamicin immobilized in mixed-type mode (via interactions of diverse strength), was created. This hybrid showed a remarkably high drug immobilization yield and the most sustainable antibiotic release among all tested composites. It was also able to inhibit bacterial growth, both in surrounding liquid and on matrix surface, much longer (for at least 121 days of experiment) than analogous gelatin-modified and nonmodified matrices. Gentamicin-keratin-coated-HAP granules were nontoxic to human osteoblasts and enabled their proliferation with a rate similar as noncoated HAP. Presence of keratin on HAP granules seemed to slightly enhance the osteoblast proliferation. The results indicate that newly created HAP hybrid with covalently immobilized keratin and gentamicin--nontoxic and osteoblast-friendly--is a promising biomaterial of significantly prolonged antibacterial activity.

Normal- and Reversed-Phase Thin-Layer Chromatography of Seven Oral Antidiabetic Agents

The chromatographic behavior of seven oral antidiabetic drugs -chlorpropamide, tolbutamide, glibenclamide, metformin, pioglitazone, rosiglitazone, and repaglinide - has been investigated. Normal-phase chromatography was performed on silica gel and alumina layers with mixtures of chloroform, diethyl ether, and ethyl acetate as mobile phases. For more effective resolution aqueous ammonia or acetic acid was added to the mobile phases. Silica gel enabled better separation than alumina. Reversed-phase chromatography was performed on octadecyl-bonded silica gel (RP-18) with mixtures of acetonitrile or 2-propanol with phosphate buffer as mobile phases. The effect of pH on the separation of the drugs was also examined. For separation of these drugs reversed-phase chromatography was more effective than use of normal-phase mode.

Prevention of biofilm formation on urinary catheters: Comparison of the sparfloxacin-treated long-term antimicrobial catheters with silver-coated ones†

Urinary catheters are widely used for hospitalized patients and are often associated with high risk of urinary tract infection. The agar and broth diffusion tests, visual TTC (triphenyltetrazolium chloride) method, and confocal scanning laser microscopic (CSLM) observations have shown highly satisfactory antimicrobial and antibiofilm activity of the novel sparfoxacin (SPA)-treated urinary catheters compared with the controversial effectiveness of silver(Ag)-coated catheters against a background of untreated catheters used as controls. SPA-treated catheters were significantly less likely to become colonized (less than 0.01%; inner and outer surfaces against Escherichia coli and Staphylococcus aureus) than both silver-coated (from 0.01% to 39.3 %; outer surface against E. coli and inner surface against S. aureus, resp.) and untreated catheters (from 88.43% to 99.72%; outer and inner surfaces, resp., against S. aureus), and maintained their broad spectrum of antimicrobial and antibiofilm activity during storage for at least 6 months.

The cytotoxicity assessment of the novel latex urinary catheter with prolonged antimicrobial activity

The purpose of this study was to evaluate the in vitro biocompatibility of the novel Sparfoxacin (SPA)-treated latex catheter with previously performed prolonged antimicrobial activity. Rectangular-shaped test samples of silicone latex catheter were fabricated according to patented procedure permitting the immobilization of SPA on heparin (HP)-coated catheter by means of mixed, covalent and non-covalent bonds. Samples subjected to cytotoxicity assay were divided into four groups: (1) the untreated catheter, (2) HP-coated catheter, (3) HP-coated catheter with SPA immobilized in low SPA concentration solution (SPA-L treated sample), and (4) high SPA concentration solution (SPA-H treated sample). Then the samples were placed directly into green monkey kidney (GMK) cell monolayer for 24 h. After the incubation period, cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The degree of cytotoxicity of each sample was evaluated according to the reference value represented by the control cells cultured without catheter sample. Statistical significance was determined by repeated t-test (P < 0.05). The cytotoxic effect of treated and untreated catheters was also estimated by microscopic observations of GMK cells morphological changes. SPA-treated catheters demonstrated high survival rates in MTT assay (>93%) on the contrary to the untreated catheters (6.13%) and HP-coated catheters (80.90%). Moreover, microscopic observation of GMK cells exposed to SPA-treated samples revealed no morphological changes and no cell growth reduction. We suggest that SPA-treated latex catheters are biofilm formation resistant (as we revealed in our previous work), considerably less toxic than untreated ones, and can be undoubtedly used in urological practice.

Separation of fluoroquinolone antibiotics by TLC on silica gel, cellulose and silanized layers

Separation of the fluoroquinolone antibiotics has been examined using numerous mobile phases and commercially available TLC plates precoated with silica gel, cellulose, and chemically bonded silica gel (RP-C18). The best separation of the antibiotic standards was achieved on silica gel with methanol–acetone–1 mol L–1 citric acid–triethylamine, 2.8 + 2 + 0.2 + 0.5 (v/v) as mobile phase, on cellulose with dichloromethane–isopropanol–THF–25% ammonia, 4 + 6 + 3 + 3 (v/v), as mobile phase, and on silanized silica gel RP-C18 with methanol–0.07 mol L–1 phosphate buffer, pH 6–10 mmol L–1 benzyldimethyltetradecylammonium chloride, 6 + 3 + 1 (v/v), as mobile phase. The separated compounds were detected under UV irradiation at λ = 254 nm or by treatment of the plate surface with different dyeing agents.

Application of solid-phase extraction of fluoroquinolone derivatives from a biological matrix to their biodetermination by liquid chromatography

Abstract A solid‐phase extraction (SPE) procedure was developed for extraction of relatively hydrophilic amphoteric drugs from plasma coupled with their determination by high‐performance liquid chromatography (HPLC). The extraction columns filled with 500 mg of reversed phase octadecyl‐bonded silica phase were conditioned with methanol, water, and ammonium acetate solution. After sample application, the sorbent was washed with the same solution. The analytes were then desorbed with the methanol–acetate buffer (pH 2.8) mixture. A composition, volume, and flow‐rate of the eluting solvent as SPE parameters were optimised. A method for the determination of fleroxacin and sparfloxacin in plasma using the optimal SPE conditions was validated. The absolute recovery of fluoroquinolones mentioned above was ca. 96% and 92%, respectively.

Normal-phase TLC separation of some antiarrhythmics. Densitometric determination of mexiletine hydrochloride in capsules

A normal-phase (NP) TLC method has been established for separation of the five antiarrhythmics - disopyramide, flecainide, mexile-tine, tocainide, and verapamil. The analysis was performed in horizontal chambers on aluminum oxide 60 F254 and silica gel 60 F254 TLC plates. The best mobile phases for separation of the compounds were tetrahydrofuran-hexane-25% ammonia, 5 + 4.8 + 0.2 (v/v), on the alumina plates and chloroform-tetrahydrofuranethanol-25% ammonia, 8.1 + 1.9 + 2 + 0.1 (v/v), on the silica plates. The substances were identified by use of different reagents and under UV irradiation at λ= 254 nm. Quantification of mexiletine hydrochloride in Mexicord capsules was performed densitometrically at λ= 210 nm. A good correlation coefficient (r = 0.9974) was obtained for the calibration plot constructed in the concentration range 20–45 μg per band. The active substance was extracted from the formulation with methanol (recovery 97.01 ± 2.39%, mean ± SD). The RSD expressing the precision of the proposed method was 5.23%. The procedure was simple and rapid and the results were reliable.

Novel thiosemicarbazide derivatives with 4-nitrophenyl group as multi-target drugs: α-glucosidase inhibitors with antibacterial and antiproliferative activity

A series of thiosemicarbazides with 4-nitrophenyl group was obtained in the reaction of carboxylic acid hydrazides with isothiocyanates. All compounds were checked for their antibacterial and antiproliferative activity. Our results have shown that derivatives 6-8 possessed antibacterial activity against S. aureus, S. epidermidis, S. mutans and S. sanguinis, moderate cytotoxicity and good therapeutic safety in vitro. Additionally, compounds 1 and 4 significantly inhibited A549, HepG2 and MCF-7 cell division. Moreover, PASS software indicated that newly obtained compounds are potential α-glucosidase inhibitors. This was confirmed by in vitro studies. To investigate the mode of interaction with the molecular target compounds were docked to glucose binding site of the enzyme and exhibited a similar binding mode as glucose.

Biological safety evaluation of the modified urinary catheter.

The purpose of this study was to evaluate in vitro safety of the novel tosufloxacin (TOS)-treated catheters with the prolonged antimicrobial activity. The test samples of silicone latex catheter were prepared by the immobilization of TOS on chitosan (CHIT)-coated catheter by means of covalent bonds and non-covalent interactions. Each step of the modification process of catheter surface was observed using ATR-Fourier transform infrared spectroscopy. In vitro cytotoxicity of the modified and unmodified catheters was assessed by direct and indirect tests in accordance with ISO standards using green monkey kidney (GMK) cell line. The MTT, lactate dehydrogenase activity (LDH), WST-8, Sulforhodamine B (SRB) test results and microscopic observation clearly indicated that unmodified silicone latex catheters decrease cell metabolic activity, act as a cytotoxic agent causing cell lysis and induce cell death through necrotic or apoptotic process. We suggest that chitosan coat with TOS immobilized limits leaching of harmful agents from silicone latex material, which significantly enhances survivability of GMK cells and therefore is quite a good protection against the cytotoxic effect of this material.