ABSTRACT
The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.
Subject(s)
Biopolymers/chemistry , Cyclodextrins/chemistry , DNA/chemistry , Polymers/chemistry , Spectrum Analysis, Raman/methods , Animals , CHO Cells , Cations , Cell Line , Cell Line, Tumor , Colloids/chemistry , Cricetinae , Electrophoresis, Agar Gel , Genetic Vectors , Humans , Luciferases/metabolism , Magnetic Resonance Spectroscopy , Models, Biological , Models, Chemical , Models, Molecular , Nucleic Acid Conformation , Plasmids/metabolism , Transfection , Ultraviolet RaysABSTRACT
Surface plasmon resonance (SPR) technique is used to follow, both in real time and in situ, the association between a physically adsorbed polymer of beta-cyclodextrin (pbetaCD) and different hydrophobically modified poly(N-isopropylacrylamide) (PNIPAM) copolymers containing either adamantyl or dodecyl groups. This association is due to the complex formation between the hydrophobic groups and the betaCD cavities. Therefore, the adsorbed amount of PNIPAM onto the pbetaCD layer depends on the substituent and on its substitution level. The association and dissociation rate constants are evaluated from the kinetics of PNIPAM adsorption. An estimation of the association constants leads to values higher than 10(4) M(-1), reflecting the strong interaction between these polymers.
Subject(s)
Acrylic Resins/chemistry , Cellulose/chemistry , Cyclodextrins/chemistry , Surface Plasmon Resonance , Adsorption , Binding Sites , Hydrophobic and Hydrophilic Interactions , KineticsABSTRACT
The photochemistry of 2-methylbenzil (1) was investigated by steady state and laser flash photolysis techniques. Laser excitation of 1 in benzene leads to a transient, which shows absorption at 370 and 470 nm and is quenched by known triplet quenchers, whereas in methanol or acetonitrile a different transient absorption spectrum was observed. For the latter solvent the absorption bands are located at 350 and 410 nm when the spectrum is recorded 30 micros after the laser pulse. The kinetics associated with these two bands shows biexponential decay, from which lifetimes of 200 and >200 micros were determined. These transients were attributed to a mixture of two possible photoenols. Similar behavior was observed for 2,2'-dimethylbenzil (3), whereas 2,4,6-trimethylbenzil (4) did not show any detectable enol. Lamp irradiation of 1 in benzene leads to 2-hydroxy-2-phenylindan-1-one (2) as the major product, with a quantum yield of 0.09. Compound 3 also gives the corresponding hydroxyindanone as the main product (phi = 0.25), whereas for 4 the cyclization product is formed in a very low quantum yield, i.e., <0.004. From these data two possible mechanisms were suggested to explain the formation of hydroxyindanones from methylbenzils, depending on the nature of the solvent.
ABSTRACT
A new photochromic family of tetrahydroazepinoisoquinolines (THAI) 4a-i has been prepared. It undergoes light-induced ring opening from spiro compounds 4 to betaines 3 that decolorize in a very fast cyclizing reaction. Depending on substitution of the precursors 1 and 6, the photochromic styryltetrahydroindolizines (THI) 5k-q are formed in a periselective way. The conformation and configuration of the new photochromic THAI 4 and THI 5 were investigated by NMR and the structure of both compounds was proven by X-ray analysis. The photochromic properties were studied by laser flash photolysis, which afforded the lifetime of the colored form 3 and 3' in the micro- or nanosecond range.
