Polyamines and novel polyamine conjugates interact with DNA in ways that can be exploited in non-viral gene therapy.

As a part of our continuing studies on 'Polyamines and their role in human disease' we are investigating how polyamines, and especially how novel polyamine conjugates, interact with DNA. We are studying how these conjugates interact with circular plasmids in order to produce nanometre-sized particles suitable for transfecting cells. Our considerations of structure--activity relationships (SAR) within naturally occurring and synthetic polyamines have shown the significance of the inter-atomic distances between the basic nitrogen atoms. As these atoms are typically fully protonated under physiological conditions, they exist in equilibrium as polyammonium ions. The covalent addition of a lipid moiety, typically one or two alkyl or alkenyl chains, or a steroid, allows much greater efficiency in DNA condensation and in the cellular transfection achieved. Thus efficient DNA condensation and subsequently drug delivery (i.e. with DNA as the drug) can be brought about using novel polyamine conjugates. Taking further advantage of the functionalization of specific steroids (e.g. cholesterol and certain bile acids), we have designed and prepared novel fluorescent molecular probes as tools to throw light on the problematic steps in non-viral gene delivery which still impede efficient gene therapy. Thus, the current aims of our research are to understand, design and prepare small-molecule lipopolyamines for non-viral gene therapy (NVGT). The rational design and practical preparation of non-symmetrical polyamine carbamates and amides, based on steroid templates of cholesterol and the bile acid lithocholic acid as the lipid moiety, provides fluorescent molecular probes that condense DNA. These novel lipopolyamine conjugates mimic the positive charge distribution found in the triamine spermidine and the tetra-amine spermine alkaloids. After optimizing their SAR, these fluorescent probes will be useful in monitoring gene delivery in NVGT.

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.

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.

Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA.

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.

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.