Andrew J. Geall

Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate–DNA interactions for the analysis of lipoplex formation in gene therapy

A rapid and sensitive fluorescent assay method is reported for assessing polyamine conjugate calf thymus DNA binding affinity using cholesterol polyamine carbamates with ethidium bromide as a probe. A reproducible method has been developed with an optimal excitation wavelength. Salt concentration is shown to be a critical parameter for both the observed fluorescence intensity of ethidium intercalated in DNA, and also for the binding of positively charged polyammonium ions to DNA, effecting charge neutralisation. This charge neutralisation precedes DNA condensation, a key first step in gene therapy.

Homologation of polyamines in the rapid synthesis of lipospermine conjugates and related lipoplexes

Lipopolyamine amides are useful cationic lipids, synthetic vectors for non-viral gene delivery. Desymmetrisation of readily available symmetrical polyamines is an important first step in the synthesis of such compounds. The application of trifluoroacetyl as a protecting group allows unsymmetrical polyamine amides to be rapidly prepared. A reductive alkylation homologation strategy allows the sequential, regiocontrolled introduction of additional positive charges. Tetraamine spermine and other polyamine derivatives have been N1-acylated with various single alkyl chains, and their relative binding affinities for DNA determined using an ethidium bromide displacement assay. The important effects on DNA binding affinity of the number of positive charges on the polyamine moiety and also the nature (chain length and degree of unsaturation) of the covalently attached lipid are demonstrated.

Practical synthesis of unsymmetrical polyamine amides

Abstract Desymmetrisation of readily available symmetrical polyamines is an important first step in the synthesis of many polyamine containing natural products. Likewise in the synthesis of polyamine amides which are potentially useful for gene delivery and as neuroprotectants, based upon channel blocking toxins found in certain wasp and spider venoms. The application of trifluoroacetyl as a protecting group allows unsymmetrical polyamine amides to be easily prepared on a gram scale.

The regiochemical distribution of positive charges along cholesterol polyamine carbamates plays significant roles in modulating DNA binding affinity and lipofection

We have quantified the effects of the regiochemical distribution of positive charges along the polyamine moiety in lipopolyamines for DNA molecular recognition. High affinity binding leads to charge neutralisation, DNA condensation and ultimately to lipofection. Binding affinities for calf thymus DNA were determined using an ethidium bromide displacement assay and condensation was detected by changes in turbidity using light scattering. The in vitro transfection competence of cholesterol polyamine carbamates was measured in CHO cells. In the design of DNA condensing and transfecting agents for non‐viral gene therapy, the interrelationship of ammonium ions, not just their number, must be considered.

Effects of Root Extracts of Fagara zanthoxyloides on the In Vitro Growth and Stage Distribution of Plasmodium falciparum

ABSTRACT The development of resistance by Plasmodium falciparum to conventional drugs poses a threat to malaria control. There is therefore a need to find new, effective, and affordable remedies for malaria, including those derived from plants. This study demonstrates that crude, reverse-phase high-pressure liquid chromatography (RP-HPLC)-semipurified, and RP-HPLC-purified root extracts of Fagara zanthoxyloides inhibit the growth of P. falciparum in vitro, with 50% inhibitory concentrations (IC50s) of 4.90, 1.00, and 0.13 μg/ml, respectively. Roots of F. zanthoxyloides, known as chewing sticks, are widely used for tooth cleaning in West Africa. Microscopic examination of Giemsa-stained slides showed a virtual absence of schizonts in ring-stage synchronized cultures treated with crude extracts at concentrations of 30 to 60 μg/ml during 36 to 48 h of incubation. These observations suggest that the active constituent in the extract may be cytotoxic for P. falciparum trophozoites, thereby inhibiting their development to the schizont stage. A pure bioreactive fraction was subsequently obtained from the chromatographic separations. When this fraction was mixed with pure fagaronine, the mixture coeluted as a single peak on the analytical RP-HPLC column, suggesting that fagaronine may be the active antimalarial constituent of Fagara root extracts. Additional experiments showed that fagaronine also inhibited P. falciparum growth, with an IC50 of 0.018 μg/ml. The results of this study suggest that the antimalarial activity of fagaronine deserves further investigation.

Homologation of polyamines in the synthesis of lipo-spermine conjugates and related lipoplexes

Lipopolyamine amides are useful cationic lipids, synthetic vectors for non-viral gene delivery. Desymmetrisation of readily available symmetrical polyamines is an important first step in the synthesis of such compounds. The application of trifluoroacetyl as a protecting group allows unsymmetrical polyamine amides to be rapidly prepared. A reductive alkylation homologation strategy allows the sequential, regiocontrolled introduction of additional positive charges. Tetraamine spermine and other polyamine derivatives have been N1-acylated with various single alkyl chains, and their relative binding affinities for DNA determined using an ethidium bromide displacement assay. The important effects on DNA binding affinity of the number of positive charges on the polyamine moiety and also the nature (chain length and degree of unsaturation) of the covalently attached lipid are demonstrated.

Cheno-, urso- and deoxycholic acid spermine conjugates: Relative binding affinities for calf thymus DNA

Abstract Cationic lipid polyamine amides (cholan-24-amides) have been prepared from chenodeoxycholic (3α,7α-dihydroxy), ursodeoxycholic (3α,7β-dihydroxy), and deoxycholic (3α,12α-dihydroxy) bile acids (5β-cholanes) by acylation of tri-Boc protected spermine. Their relative binding affinities for calf thymus DNA were determined using an ethidium bromide displacement assay. These lipopolyamine amides are synthetic vectors for non-viral gene delivery and models for lipoplex formation with respect to lipofection, a key first step in gene therapy.

Lipopolyamines incorporating the tetraamine spermine, bound to an alkyl chain, sequester bacterial lipopolysaccharide

Lipopolyamines, with high affinity for calf thymus DNA in an ethidium bromide displacement assay, bind with high affinity to bacterial lipopolysaccharide and neutralise in vitro endotoxic activity as determined by Griess nitric oxide and TNF-alpha ELISA assays.

Synthesis of cholesterol-polyamine carbamates: pKa studies and condensation of calf thymus DNA

Novel cholesterol-polyamine carbamates have been prepared and their pKas determined potentiometrically for conjugates substituted with up to five amino functional groups and the binding affinity for calf thymus DNA has also been determined; these polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.

Measurement of polyamine pKa values.

The extent of ionization of the polyamines is an important factor in their interactions with cellular components. The pK(a) is the pH at which a functional group is 50% ionized. For compounds such as polyamines with more than one ionizable center (atom or functional group), there is a pK(a) value for each center of ionization. This chapter describes the pK(a) values for each amine group in many important polyamines, the factors influencing these values and methods for their determination using potentiometric titration and nuclear magnetic resonance spectroscopy.