Some Pluronics, particularly F127, are known to stabilize nanospheres and prolong their circulation time in vivo. However, these copolymers of poly(ethylene glycol) (PEG) and poly(propylene glycol) are not biodegradable, and despite the long history, there is no approved commercial product using F127 for parenteral administration until now. Meanwhile, hydroxyethyl starch (HES) is a biodegradable polymer that is currently investigated as a substitute for PEG. In order to produce a fully biodegradable amphiphilic polymer, we esterified different molar masses of HES with lauric acid to get different molar substitutions. These polymers, as well as Pluronic F68 and F127, were used to stabilize poly(lactic-co-glycolic acid) (PLGA) nanospheres prepared by nanoprecipitation. For physicochemical characterization, the particle size, zeta potential, and the thickness of the adsorbed polymer layer were measured. The ability of the polymer coating to prevent the adsorption of human serum albumin (HSA) and fibrinogen (FBG) was evaluated. Finally, the phagocytosis of the stabilized nanospheres by a monocyte macrophage cell line (J774.2) was assessed. Results show that the PLGA nanospheres had an average particle size of 110-140 nm. The thickness of the adsorbed polymer layer increases with the increase in molar mass, and is generally higher for HES laurates than the studied Pluronics. Pluronic F68, F127 as well as the HES laurates with low molar substitution prevented the adsorption of HSA. HES laurates with low molar substitution and F127, but not F68, prevented the adsorption of FBG. The phagocytosis experiments showed that the HES laurates, particularly the one with the highest molar mass, could reduce the uptake of the nanospheres better than F68 and comparable to F127. Finally, these results warrant in vivo experiments to evaluate how the HES laurates can affect the pharmacokinetics and fate of the nanospheres.
The bis-barbituric acid oxonol, DiBAC 4 (3) is used as a standard potentiometric probe in human cells. However, its fluorescence depends not only on membrane potential but also varies with nonpotential related changes in the amount of intracellular free and bound dye. This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC 4 (3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC 4 (3), the logarithmic relation between cell fluorescence and dye concentration turned into a virtually linear function intersecting with zero. Consequently, calibration can then be confined to determination of the fluorescence of depolarized cells stained with the same concentration as used for the actual measurement of membrane potential. Unexpectedly, quenching of fluorescence occurred in totally depolarized cells at concentrations higher than 6,250 nM. Linearity and quenching could be confirmed by additional experiments on Chinese hamster ovary CHO-K1 and B lymphoblastoid LCL-HO cells. ' International Society for Advancement of CytometryKey terms bis-barbituric acid oxonol; flow cytometry; membrane potential; mV-scale calibration PATCH-clamp recording is considered to be the reference method to quantitatively measure the plasma membrane potential V p in human cells. A disadvantage in respect of routine use is that this method is time-consuming and laborious thus usually resulting in measuring values for only a small number of cells (1,2). Therefore, voltage-sensitive anionic and cationic dyes, which partition between the extracellular staining buffer and the cytoplasm under the influence of V p according to a Nernstian distribution, have been applied as an alternative (3-12).Due to their strong accumulation in mitochondria (13,14), cationic dyes report V p alone only when the mitochondrial membrane potential V m remains constant during the measurement. To ensure this condition, V m was specifically abolished by ionophores in some studies (15)(16)(17). With increasing concentration, the monomer form of cationic dyes, among them carbocyanines as the most popular dye class, is usually more and more displaced by dimers or higher aggregates. This process takes place both with dye dissolved in aqueous solution or bound to cell compartments, and is accompanied by distinct spectral shifts in absorbance and fluorescence maxima leading to quenching of the fluorescence of the monomers (18-21). For example, DiSC 3 (5) monomers show red flu...
In the present study, the distribution of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA)-receptor type was immunohistochemically demonstrated in healthy human skin (n = 22) and healthy buccal mucosa (n = 20). Moreover, the intracellular calcium concentration of HaCaT-cells and native human keratinocytes were studied under the influence of the selective agonist NMDA and the selective NMDA-antagonist MK-801. Immunohistochemical imaging of NMDA receptors in healthy epidermis showed a positive reaction in the stratum basale, spinosum and granulosum, whereby the greatest expression was observed in the granular layer. In the mucosal preparations, the distribution of NMDA receptors was observed to be equal in all cell layers. In the cell culture (HaCaT-cells), NMDA concentrations between 25 microM and 1 mM resulted in a significant increase in the number of cells showing elevated intracellular calcium concentration. This effect could be significantly reduced by prior application of MK-801 (100 micro M). In supplementary tests on HaCaT-keratinocytes, blockade of the keratinocytic NMDA receptors with MK-801 suppressed the differentiation of the cells (expression of cytokeratin 10). The proliferation of cells was not influenced by NMDA. The investigations showed that glutamate receptors of the NMDA type have an influence on keratinocytic calcium concentration. This appears especially important for the differentiation of keratinocytes.
Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed a-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.
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