Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells

We investigated the ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) in Ewing sarcoma cells, in the perspective of in vivo anti-cancer nucleic acid drug delivery. siRNA was adsorbed onto NDs previously coated with cationic polymer. Cell uptake of NDs has been demonstrated by taking advantage of NDs intrinsic fluorescence coming from embedded color center defects. Cell toxicity of these coated NDs was shown to be low. Consistent with the internalization efficacy, we have shown a specific inhibition of EWS/Fli-1 gene expression at the mRNA and protein level by the ND vectorized siRNA in a serum containing medium

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations

Self-curing super-stretchable polymer/microgel complex coacervate gels without covalent bond formation

Elastic physical gels are highly desirable because they can be conveniently prepared and readily shaped. Unfortunately, many elastic physical gels prepared in water require in situ free-radical polymerization during the gel formation stage. In contrast, complex coacervate gels are physical gels that can be prepared by simply mixing two pre-formed oppositely-charged polyelectrolytes. However, as far as we are aware, highly elastic complex coacervate gels have not yet been reported. Herein, we combine polyanionic microgel particles with a well-known commercially-available cationic polyelectrolyte to prepare polymer/microgel complex coacervate (PMCC) physical gels. This new family of gels requires annealing at only 37 °C and behaves like a covalent gel but does not form covalent bonds. Thermal reconfiguration of the dynamic ionic bonds transforms the shapeable pre-gel into a highly elastic gel that is super-stretchable, adhesive, self-healing, highly swellable and can be further toughened using Ca2+ as an ionic crosslinker. Our PMCC gels have excellent potential for applications as engineering gels and structural biomaterials, as well as for wound healing and water purification

Structural dynamics and physicochemical properties of pDNA/DODAB:MO lipoplexes : effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases

Dioctadecyldimethylammoniumbromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In thiswork,we intend to study the effect of highMOcontent on the structure and physicochemical properties of pDNA/DODAB:MOlipoplexes to achieve some correlationwith their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/ morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/−). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the β-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/−) = 6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.FEDER (037291) through POFC — COMPETEFundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/46968/2009 (PhD grant), PEst-C/BIA/UI4050/2011(CBMA), PEst-C/FIS/UI0607/2011 (CFUM)

Voltammetric behaviour of oligonucleotide lipoplexes adsorbed onto glassy carbon electrodes

The voltammetric behaviour of oligonucleotide lipoplexes (ODN-lipoplexes) prepared from short oligodeoxynucleotides (ODN), with different base compositions, and liposomes of the cationic lipid DOTAP, was studied by differential pulse voltammetry with a glassy carbon mini-electrode. It was found that the ODN base composition influences the ODN-lipoplex voltammetric response. Differential pulse voltammograms for ODN-lipoplexes of the ODN adenosine nucleotides present two different features when compared with the differential pulse voltammograms obtained for free ODN: a new peak appeared and the peak attributed to oxidation of adenosine diminished or was absent, depending on whether the ODN sequence had guanosine nucleotides or not. The presence of guanosine nucleotides in the ODN-lipoplex led to a peak due to guanosine oxidation with similar potential and current to the peak obtained for guanosine oxidation in free ODN. No detectable peaks were recorded in the voltammograms obtained with lipoplexes composed of ODN containing only pyrimidine bases. It was possible to show by voltammetry the occurrence of partial denaturation of short double helices of ODN when mixed with DOTAP liposomes to generate lipoplexes. The extent of denaturation was observed to increase with lipoplex (+/-) charge ratio as shown by the increase in the differential pulse voltammetry peak currents. The electrochemical characterisation of lipoplex properties at a charged interface can be important for understanding and development of these gene therapy vectors.http://www.sciencedirect.com/science/article/B6TGB-4B4RVT3-2/1/80478920c615683b2439ef8a2980898

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed