In vitro antitumor activity of free and nano-encapsulated Na5[PMo10V2O40]·nH2O and its binding properties with ctDNA by using combined spectroscopic methods.

Free and nanosized starch and lipid encapsulated Na5[PMo10V2O40]·nH2O complexes (abbreviated as PMoV, SEP and LEP, respectively) have been prepared and structurally characterized by Fourier transform infrared (FT-IR) spectroscopy, inductively coupled plasma (ICP) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. The results show that the PMoV retains its parent structure after encapsulation by starch and lipid nanoparticles. The in vitro antitumor activity of PMoV in its free and nano-encapsulated forms was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay that was carried out on two types of human cancer cells, MCF-7 (breast cancer cells) and HEK-293 (Human Embryonic Kidney). The results represent the enhancement of cell penetration and antitumor activity of PMoV due to its encapsulation in starch or lipid nanoparticles. However, this observed enhancement for the lipid relative to the starch nanocapsule can be attributed to its smaller size. In order to investigate the molecular nature of antitumor activity, the binding properties of PMoV with calf thymus DNA (ctDNA) were also comprehensively evaluated using UV-vis absorption spectroscopy, fluorescence quenching and fluorescence Scatchard plots. The results rule out the intercalating binding mode and propose the groove or outside stacking binding for PMoV. However, a biphasic binding behavior that is due to the change in the binding mode was observed by varying of [PMoV]/[ctDNA] mole ratio. The results of cell culture assay and DNA binding experiments represent that the rate of cell penetration is more important than DNA binding affinity in the antitumor activity for POM.

Gemini surfactants affect the structure, stability, and activity of ribonuclease Sa.

Gemini surfactants have important advantages, e.g., low micromolar CMCs and slow millisecond monomer ↔ micelle kinetics, for membrane mimetics and for delivering nucleic acids for gene therapy or RNA silencing. However, as a prerequisite, it is important to characterize interactions occurring between Gemini surfactants and proteins. Here NMR and CD spectroscopies are employed to investigate the interactions of cationic Gemini surfactants with RNase Sa, a negatively charged ribonuclease. We find that RNase Sa binds Gemini surfactant monomers and micelles at pH values above 4 to form aggregates. Below pH 4, where the protein is positively charged, these aggregates dissolve and interactions are undetectable. Thermal denaturation experiments show that surfactant lowers RNase Sa's conformational stability, suggesting that surfactant binds the protein's denatured state preferentially. Finally, Gemini surfactants were found to bind RNA, leading to the formation of large complexes. Interestingly, Gemini surfactant binding did not prevent RNase Sa from cleaving RNA.

Thermal stability and enzymatic activity of RNase A in the presence of cationic gemini surfactants.

The thermal stability and enzymatic activity of bovine pancreatic ribonuclease A (RNase A) have been investigated in the presence of a homologous series of cationic gemini surfactants (alkanediyl-α,ω-bis(hydroxyethyl methyl hexadecyl ammonium bromide)). UV, circular dichorism and fluorescence spectroscopies have been used for this study. The denaturation curves at various surfactant concentrations were analyzed on basis of a two-transition model to obtain values of T(m) (temperature at the midpoint of denaturation) and ΔH(m) (enthalpy change at T(m)) of each transition. The main conclusion of this study is that these cationic gemini surfactants slightly activate and stabilize RNase A below their critical micelle concentrations at pH 5.0. The cationic gemini surfactant with the shorter spacer interacts more efficiently with RNase A than those with longer spacers.

Interactions of gemini surfactants with two model proteins: NMR, CD, and fluorescence spectroscopies.

Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (µM vs. mM) critical micelle concentrations and possess slower (ms vs. µs) monomer <-- / --> micelle kinetics. The structure of the gemini surfactants studied is [HOCH(2)CH(2)-, CH(3)-, CH(3)(CH(2))(15)-N(+)-(CH(2))(s)-N(+)-(CH(2))(15)CH(3),-CH(3),-CH(2)CH(2)OH]·2Br(-) where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D (1)H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins.