The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA.

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.

Synthesis of multifunctional PAMAM-aminoglycoside conjugates with enhanced transfection efficiency.

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.

Separation of enantiomers of chiral sulfoxides in high-performance liquid chromatography with cellulose-based chiral selectors using acetonitrile and acetonitrile-water mixtures as mobile phases.

The separation of 14 chiral sulfoxides was systematically studied on 12 cellulose-based chiral columns in acetonitrile and acetonitrile-water mobile phases. Out of all monosubstituted methylphenylcarbamates of cellulose the one having a methyl moiety in position 3 showed more universal chiral resolving ability compared to 2- and 4-substituted derivatives. Out of disubstituted phenylcarbamates of cellulose the ones with methyl substituents showed higher enantiomer resolving ability compared to chloro-substituted ones and substitution in positions 3 of the phenyl moiety was clearly advantageous. From disubstituted derivatives those possessing a combination of methyl- and chloro-substituents were advantageous compared to the ones having dimethyl- or dichloro-substituents. Chiral recognition ability of most chiral selectors towards studied sulfoxides was higher in pure acetonitrile compared to previously studied methanol. The effect of water addition to the mobile phase on analyte retention and enantioseparation was also quite different from that observed with methanol. In particular, with aqueous methanol by increasing the water content in the mobile phase retention increased in most cases and the separation factor improved. In contrast, with aqueous acetonitrile retention and separation factors decreased up to a certain water content in the mobile phase and then started to recover again for most of the studied analytes.

Separation of enantiomers of chiral sulfoxides in high-performance liquid chromatography with cellulose-based chiral selectors using methanol and methanol-water mixtures as mobile phases.

The interplay between structural details of chiral analytes and selectors in the separation of 14 chiral sulfoxides was systematically studied on 18 different polysaccharide-based chiral columns. Retention and enantioselectivity of a set of chiral sulfoxides were of primary interest. Several of chiral columns studied exhibited quite powerful chiral recognition ability in pure methanol. With addition of water to the mobile phase retention increased in the most cases and the separation factor improved. However, several exceptions were also noted. Of monosubstituted phenylcarbamates of cellulose as chiral selectors, chlorosubstituted ones did not show better enantiomer resolving ability compared to unsubstituted cellulose tris(phenylcarbamate). Out of disubstituted phenylcarbamates of cellulose the ones with methylsubstituents showed higher enantiomer resolving ability compared to chloro-substituted ones and substitution in positions 3 and 5 of the phenyl moiety was clearly advantageous. From disubstituted derivatives those possessing a combination of methyl- and chloro-substituents were advantageous compared to the ones having dimethyl- or dichloro-substituents. Interesting examples of reversal in enantiomer elution order (EEO) were observed on cellulose tris(4-chloro-3-methylphenylcarbamate)- and cellulose tris(3-chloro-4-methylphenylcarbamate)-based chiral stationary phases (CSPs) function of the water content in the mobile phase.

Separation of enantiomers of selected chiral sulfoxides with cellulose tris(4-chloro-3-methylphenylcarbamate)-based chiral columns in high-performance liquid chromatography with very high separation factor.

The present study reports successful separations of enantiomers of selected chiral sulfoxides with very high separation factor in high-performance liquid chromatography by using chiral columns prepared with the chiral selector cellulose tris(4-chloro-3-methylphenylcarbamate). High separation factors were observed in polar organic, as well as in hydrocarbon-alcohol-type mobile phases. The key structural components of the solute for obtaining high chiral recognition are discussed as well as thermodynamic quantities of analyte adsorption on the chiral stationary phase were determined. Experiment aimed at the enantioselective extraction of racemates from solution are also described.

Application of cellulose 3,5-dichlorophenylcarbamate covalently immobilized on superficially porous silica for the separation of enantiomers in high-performance liquid chromatography.

Our earlier studies have demonstrated the applicability of polysaccharide-based chiral selectors in combination with superficially porous (or core-shell) silica (SPS) particles for the preparation of highly efficient chiral stationary phases (CSP). In earlier studies, CSPs were prepared by coating (adsorption) of the chiral selector onto the surface of silica. In this study we report for the first time the CSP obtained by covalent immobilization of a chiral selector onto the surface of SPS particles. The applicability of this CSP for the separation of enantiomers in pure methanol and acetonitrile, as well as in n-hexane/2-propanol mobile phases is shown. The effect of the injected sample amount, mobile phase flow rate and detection frequency on separation performance were studied, as well as high efficiency separation of enantiomers with the analysis time less than 30 s was attempted.

Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior.

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.