Abstract: The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.
Abstract: A Raman spectroscopy method for determining the drug content of street samples of amphetamine was developed by dissolving samples in an acidic solution containing an internal standard (sodium dihydrogen phosphate). The Raman spectra of the samples were measured with a CDD-Raman spectrometer. Two Raman quantification methods were used: (1) relative peak heights of characteristic signals of the amphetamine and the internal standard; and (2) multivariate calibration by partial least squares (PLS) based on second derivative of the spectra. For the determination of the peak height ratio, the spectra were baseline corrected and the peak height ratio (h(amphetamine at 994 cm(-1) )/h(internal standard at 880 cm(-1) )) was calculated. For the PLS analysis, the wave number interval of 1300-630 cm(-1) (348 data points) was chosen. No manual baseline correction was performed, but the spectra were differentiated twice to obtain their second derivatives, which were further analyzed. The Raman results were well in line with validated reference LC results when the Raman samples were analyzed within 2 h after dissolution. The present results clearly show that Raman spectroscopy is a good tool for rapid (acquisition time 1 min) and accurate quantitative analysis of street samples that contain illicit drugs and unknown adulterants and impurities.
Abstract: An efficient synthetic route was developed for the mild chloroacylation of chitosan with different chloroacyl chlorides. Full N-chloroacylation was obtained with this procedure without any O-acylation, and products having lower degrees of substitution can also be produced. Organo-soluble 6-O-triphenylmethylchitosan was used as a starting material for the acylation reactions. The resulting N-chloroacyl-6-O-triphenylmethylchitosan intermediates were also organo-soluble and characterized by FT-IR. N-Methylpiperazine moieties were attached to make end products that were sufficiently soluble for characterization by NMR and also to prove that the present intermediates could be used for further modifications. The end products were fully characterized by 1H NMR, 13C NMR, and 2D 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. The degrees of substitution were determined by 1H NMR. Molecular weight determination by GPC-LS displayed a significant degradation of the polymer. The weight-average molar masses (M(w)) of the end products ranged from 29.6 to 49.4 kDa, when the M(w) of the starting material was 144.2 kDa.
Abstract: Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.
Abstract: The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.
Abstract: The influence of shape, molecular weight and pegylation of linear, grafted, dendritic and branched poly-L-lysines on their DNA delivery properties were investigated. DNA binding, condensation, complex size and morphology, cell uptake and transfection efficiency were determined. Most polylysines condense DNA, linear polymers being more efficient than most dendritic ones. At low molecular weights of PLL DNA binding and condensation were less efficient, particularly with dendrimers. Pegylation did not decrease DNA condensation of PLLs at less than 60% (fraction of M(w)) of PEG. Pegylation stabilized the complexes sterically, but did not protect them from interaction with polyanionic chondroitin sulfate. Cell uptake of polylysine/DNA complexes was high and pegylation increased the transfection efficacy. However, overall transfection level of polylysines is low possibly due to inadequate escape of the complexes from endosomes or poor release of DNA from the complexes. Physicochemical and biological structure-property relationships of poly-L-lysines were demonstrated, but no clear correlations between the tested physicochemical determinants (size of complexes, zeta-potentials, condensation of DNA and the shape of complexes) and biological activities were seen. Transfection activity may be ultimately determined by intracellular factors and/or still unknown features of DNA complexation with the carriers.
