Presence Of Cell Culture Media Research Articles

Formulation and stability of surface-tethered DNA–gold–dendron nanoparticles

The formulation of plasmid DNA on 100 nm gold nanoparticles surface-tethered via cationic dendrons, and the behaviour of the complex in cell culture media, is described in this communication. Adsorption of dendrons onto gold nanoparticles in water resulted in the generation of positively charged nanoparticles with a corresponding small increase in particle size. Addition of plasmid DNA did not markedly reduce the surface potential but resulted in a ∼10–20% increase in hydrodynamic diameter. More dramatic effects were seen in the presence of cell culture media that, overall, drastically increased the apparent size of the gold–dendron–DNA nanoparticles and reduced the surface potential of the colloids, the presence of serum components partially ameliorating these effects possibly due to steric stabilisation. Release of the surface-tethered DNA was reduced in cell culture media compared to water. This reduced detachment of DNA coupled with the flocculation of the carrier which would likely inhibit endocytosis, demonstrates the importance of testing drug delivery systems with relevant physiologically based fluids prior to their use in vivo studies.

Influence of cell culture medium on the photosensitizing effectiveness of bacteriochlorin a

To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich environment, the photosensitizing efficacy was tested by clonogenic survival of Chinese hamster ovary and T24 (human bladder carcinoma) cells. Confluent cell layers were incubated with 2.5 μg ml −1 BCA in cell culture medium for 1, 4, 6, 18 and 24 h. Upon illumination with red light it was found that BCA was not effective as a photosensitizer in this medium. Extraction methods showed that this lack of photosensitization could not be explained by the inability of the dye to enter the cells in the presence of cell culture medium. The presence of cell culture medium did not change the spectral properties of BCA to an appreciable extent. Standard KBr density gradient ultracentrifugation showed that in the presence of cell culture medium approximately 20% of the BCA was sedimented with low density lipoprotein (LDL) and 60% with high density lipoprotein (HDL). Incubating T24 cells 18 h before the clonogenic cell survival assay in serum-deficient medium restored the photosensitizing properties of BCA. It is proposed that in a protein-rich (in vivo) environment BCA associates with lipoproteins and can be taken up by malignant neoplasms via the LDL pathway.

Reactive oxygen species produced from chromate pigments and ascorbate.

The reactions of various chromate pigments and ascorbate were investigated by an ESR spin trapping technique. Production of Cr(V) was detected directly and productions of very electrophilic reactive oxygen species (ROS) was detected via the oxidation of formate. We demonstrated previously that both dissolved oxygen and Cr (V) were essential in the production of ROS in this system, and that ROS production was inhibited by catalase. We studied here the effect of solubility of different chromate pigments: sodium, calcium, strontium, basic zinc, basic lead supported on silica, and lead and barium chromates on the production of ROS in buffered medium and cell culture medium (Dublecco's Modified Eagle medium + fetal calf serum). Sodium, calcium, basic zinc, and basic lead chromates were active in the production of ROS in presence of cell culture medium, whereas lead and barium chromates were inactive.

Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase

Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared. The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative. The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively. This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis. The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant. The proteinase activity is assessed by measuring the fluorescence of the supernatant. This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range. The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors.