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Dolní Dobrouč, Czech Republic

Hermannova M.,Contipro Pharma | Iordache A.-M.,Palacky University | Slovakova K.,Palacky University | Havlicek V.,Palacky University | And 5 more authors.
Journal of Mass Spectrometry | Year: 2015

Hyaluronic acid is a naturally occurring linear polysaccharide with substantial medical potential. In this work, discrimination of tyramine-based hyaluronan derivatives was accessed by ion mobility-mass spectrometry of deprotonated molecules and nuclear magnetic resonance spectroscopy. As the product ion mass spectra did not allow for direct isomer discrimination in mixture, the reductive labeling of oligosaccharides as well as stable isotope labeling was performed. The ion mobility separation of parent ions together with the characteristic fragmentation for reduced isomers providing unique product ions allowed us to identify isomers present in a mixture and determine their mutual isomeric ratio. The determination used simple recalculation of arrival time distribution areas of unique ions to areas of deprotonated molecules. Mass spectrometry data were confirmed by nuclear magnetic resonance spectroscopy. © 2015 John Wiley & Sons, Ltd. Source

Smejkalova D.,Contipro Pharma | Nesporova K.,Contipro Pharma | Huerta-Angeles G.,Contipro Pharma | Syrovatka J.,Contipro Pharma | And 4 more authors.
Biomacromolecules | Year: 2014

Due to its native origin, excellent biocompatibility and biodegradability, hyaluronan (HA) represents an attractive polymer for superparamagnetic iron oxide nanoparticles (SPION) coating. Herein, we report HA polymeric micelles encapsulating oleic acid coated SPIONs, having a hydrodynamic size of about 100 nm and SPION loading capacity of 1-2 wt %. The HA-SPION polymeric micelles were found to be selectively cytotoxic toward a number of human cancer cell lines, mainly those of colon adenocarcinoma (HT-29). The selective inhibition of cell growth was even observed when the SPION loaded HA polymeric micelles were incubated with a mixture of control and cancer cells. The selective in vitro inhibition could not be connected with an enhanced CD44 uptake or radical oxygen species formation and was rather connected with a different way of SPION intracellular release. While aggregated iron particles were visualized in control cells, nonaggregated solubilized iron oxide particles were detected in cancer cells. In vivo SPION accumulation in intramuscular tumor following an intravenous micelle administration was confirmed by magnetic resonance (MR) imaging and histological analysis. Having a suitable hydrodynamic size, high magnetic relaxivity, and being cancer specific and able to accumulate in vivo in tumors, SPION-loaded HA micelles represent a promising platform for theranostic applications. © 2014 American Chemical Society. Source

Smejkalova D.,Contipro Pharma | Nesporova K.,Contipro Pharma | Hermannova M.,Contipro Pharma | Huerta-Angeles G.,Contipro Pharma | And 6 more authors.
International Journal of Pharmaceutics | Year: 2014

Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (M w = 15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelle's interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX. © 2014 Elsevier B.V. Source

Franke L.,Contipro Pharma | Cozikova D.,Contipro Pharma | Smirnou D.,Contipro Pharma | Hermannova M.,Contipro Pharma | And 3 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2015

Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R2) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. © 2015 Elsevier B.V. Source

Smejkalova D.,Contipro Pharma | Hermannova M.,Contipro Pharma | Buffa R.,Contipro Pharma | Cozikova D.,Contipro Pharma | And 4 more authors.
Carbohydrate Polymers | Year: 2012

The generation of degradation byproducts from hyaluronan during its acid hydrolysis was studied. The degradation byproducts were removed from reaction mixture by ultrafiltration, followed by ionex separation and purification on a reversed-phase HPLC column. Isolated fractions were structurally analyzed by NMR and mass spectrometry. Results indicated that when acid hydrolysis is performed at 100°C for 24 h, about 0.7% of high molar mass hyaluronan (1.2 × 106 g mol-1) was converted into furan-like derivatives. Other major degradation byproducts present in 0.2 wt% amounts were identified as derivatives of cyclopentanone. Cell viability tests indicated that most of the degradation byproducts have negative effect on cell growth. These tests were performed using mouse fibroblast cell line 3T3 and human keranocytes cell line HaCaT. For this reason, it is important to evaluate the amount of degradation byproducts in low molar mass hyaluronan produced by acid hydrolysis, mainly when these products are used in pharmaceutical and cosmetic applications. During this study it was found out that the unwanted degradation byproducts can be completely removed when an ultrafiltration step is introduced after acid hydrolysis prior hyaluronan isolation. © 2012 Elsevier Ltd. All rights reserved. Source

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