Intracellular distribution-based drug selectivity comparisons for geldanamycin and its derivatives with varying pKa.

<p>IDB selectivity is defined as the IC₅₀ in MD-MB-231 cells treated with scrambled shRNA, which have low lysosomal pH, divided by the value obtained from the same cell line treated with shRNA against the V1E1 subunit of the vacuolar proton ATPase, which have elevated lysosomal pH. Bars represent mean±s.d. (n = 3); *, <i>p</i><0.05 compared to GDA.</p

Overall selectivity comparisons for geldanamycin and its derivatives with varying pKa.

<p>Overall selectivity represents the IC₅₀ for a derivative in normal human fibroblasts divided by the value obtained in HL60 human leukemic cells. Bars represent mean±s.d. (n = 3); *, <i>p</i><0.05 compared to GDA.</p

Characterization of MDA-MB-231 cells with reduced V-H⁺-ATPase subunit V1E1 expression and increased lysosomal pH.

<p>A. Western blot analysis of the V1E1 subunit expression is shown along with the actin loading control. Experimentally determined lysosomal pH values in untreated, scrambled shRNA- and V1E1 shRNA-treated cells is shown (values represent the mean±s.d., n = 3) B. V-ATPase subunit knockdown does not alter the growth rate of MDA-MB 231 cells. The filled circles represent cells treated with the scrambled shRNA vector and the open circles represent cells treated with the V-ATPase V1E1 shRNA. Cells were plated at a density of 3×10⁵ cells/well on a 6-well plate and were trypsinized, counted and replated every 24 hours for 3 days (data points represent the mean±s.d, n = 3).</p

Only lysosomotropic anticancer agents possess significant intracellular distribution-based drug selectivity.

<p>Mitoxantrone and daunorubicin are weakly basic with pKa values near 8 and have significant IDB selectivity. Non-lysosomotropic anticancer agents 5-fluorouracil and chlorambucil have IDB selectivity values near 1, which demonstrates that their activity is not influenced by lysosomal pH. Bars represent mean±s.d. (n = 3).</p

Theoretical plot of lysosomal sequestration as a function of weak base pKa and alpha (α).

<p>The equation used for these simulations can be found in the text. The pH of lysosomes and extracellular space were set at 4.4 and 7.4, respectively. The alpha parameter was varied as indicated.</p

Structures and properties of geldanamycin derivatives.

<p>The seven derivatives with different weak base modifications at the 17-position of GDA are shown. The pKa values for derivatives 1, 2, 3, 5 and 6 was measured experimentally using proton NMR (n = 1). The pKa for compound 4 was measured experimentally in an earlier manuscript (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s2" target="_blank">Results</a>). The pKa for compound 7 was estimated using software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s4" target="_blank">Materials and Methods</a>). The binding affinity for each of the inhibitors with rHsp90 is shown (n = 2), and was based on a previously established fluorescence polarization assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s4" target="_blank">Materials and Methods</a>).</p

Phospholipid Composition Modulates Carbon Nanodiamond-Induced Alterations in Phospholipid Domain Formation

The focus of this work is to elucidate how phospholipid composition can modulate lipid nanoparticle interactions in phospholipid monolayer systems. We report on alterations in lipid domain formation induced by anionically engineered carbon nanodiamonds (ECNs) as a function of lipid headgroup charge and alkyl chain saturation. Using surface pressure vs area isotherms, monolayer compressibility, and fluorescence microscopy, we found that anionic ECNs induced domain shape alterations in zwitterionic phosphatidylcholine lipids, irrespective of the lipid alkyl chain saturation, even when the surface pressure vs area isotherms did not show any significant changes. Bean-shaped structures characteristic of dipalmitoylphosphatidylcholine (DPPC) were converted to multilobed, fractal, or spiral domains as a result of exposure to ECNs, indicating that ECNs lower the line tension between domains in the case of zwitterionic lipids. For membrane systems containing anionic phospholipids, ECN-induced changes in domain packing were related to the electrostatic interactions between the anionic ECNs and the anionic lipid headgroups, even when zwitterionic lipids are present in excess. By comparing the measured size distributions with our recently developed theory derived by minimizing the free energy associated with the domain energy and mixing entropy, we found that the change in line tension induced by anionic ECNs is dominated by the charge in the condensed lipid domains. Atomic force microscopy images of the transferred anionic films confirm that the location of the anionic ECNs in the lipid monolayers is also modulated by the charge on the condensed lipid domains. Because biological membranes such as lung surfactants contain both saturated and unsaturated phospholipids with different lipid headgroup charges, our results suggest that when studying potential adverse effects of nanoparticles on biological systems the role of lipid compositions cannot be neglected