New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles

In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C_max (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.     

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.     

Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide

The level of adenosine deaminase in various rat tissues has been tested. The enzyme activity of cytosolic fractions decreased in the following order: lung greater than spleen greater than small intestine greater than stomach greater than kidney greater than heart greater than liver greater than skeletal muscle greater than forebrain greater than cerebellum. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and Ki values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.