Dendrisomes: vesicular structures derived from a cationic lipidic dendron.

The behavior of a novel synthetic lipidic cationic lysine-based dendron (partial dendrimer) in aqueous media and its ability, with and without cholesterol, to self-assemble into higher order structures was studied to gain an understanding of these structures as potential drug carriers. The dendron was prepared by solid-phase peptide synthesis. A reverse-phase evaporation (REV) technique was used to prepare cationic vesicular aggregates of the dendron with different molar ratios of cholesterol. The size and zeta potential of these supramolecular aggregates or "dendrisomes" was determined by photon correlation spectroscopy (PCS). Dendrisome morphology and thermotropic properties were studied by transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). Radiolabeled penicillin G was used as a model of a negatively charged water-soluble compound to investigate the encapsulation efficiency of the dendrisomes. In vitro release of the drug was determined using as a comparator a REV liposome formulation. Dendrisomes of all compositions have higher encapsulation efficiencies and slower release rates compared to the comparator. Cholesterol was found both to increase the size of the aggregates from around 310 to 560 nm and to increase shape irregularities, but did not change the positive zeta potential, in the order of +50 mV, of the dendrisomes. Cholesterol decreases penicillin G entrapment efficiency but increases solute leakage at 25 degrees C.

Investigation of the association and flexibility of cationic lipidic peptide dendrons by NMR spectroscopy.

The cationic peptide dendrons synthesized and studied are lower generation polylysine-based partial dendrimers with or without lipid chains in the core. The dendrons with lipidic chains can be utilized as protein and liposomal mimics because of their unique structural properties. The full assignments of three different dendrons (L)7(NH2)8, (C14)1(L)7(NH2)8 and (C14)3(L)7(NH2)8 were obtained in D2O and H2O/D2O using a 500 MHz NMR spectrometer. The hydrophobic lipidic core of branched polylysine dendrons was found to induce aggregation upon increasing concentration. Because non-lipidic dendrons do not self-assemble, the behaviour and internal structural features of two different dendrons with one and three C14 hydrocarbon chains were explored. The critical association concentration clearly depends on the number of core hydrophobic residues and the association starts at 0.025 mM for (C14)1(L)7(NH2)8 and 0.05 mM for (C14)3(L(7(NH2)8. Chemical shift analysis also revealed that the hydrophobic chains of the dendrons associate in the core, whereas the polar head groups (NH2) are mainly located at the surfaces of the aggregates. The T1 relaxation time measurements showed that the mobility of the hydrocarbon chain is greater with the monomeric form of dendron (C14)1(L)7(NH2)8) than that of monomer (C14)3(L)7(NH2)8. The inter-chain hydrophobic interactions restrict the flexibility of the dendron with three hydrocarbon chains. As expected, the flexibility of the monomeric form is higher than that of the aggregated state for both of the dendrons.

Dendriplexes and their characterisation.

The interaction of DNA with partial dendrimers (dendritic polylysine containing seven lysines and eight terminal amino groups with or without a lipidic core) was studied. Compact complexes were formed which we term "dendriplexes". Agarose gel electrophoresis and exclusion of ethidium bromide confirmed the interaction. All the dendrons formed compact complexes above a 2:1 (+/-) charge ratio in water and HBSS. Photon correlation spectroscopy, electron microscopy and zeta potential measurements were used to determine, respectively, the particle size, shape and surface charge of the dendriplexes. The z-average diameter of the dendriplexes were found to be 60-70 nm irrespective of the dendron used and the zeta potential varied from 10 to 35 mV at a 3:1 (+/-) charge ratio depending on the dendron. The protection of the DNA component of these dendriplexes from nuclease degradation was confirmed by DNase protection assays.

Dendrisomes: cationic lipidic dendron vesicular assemblies.

A new lipidic cationic polylysine dendron was prepared by solid-phase peptide synthesis. Its behaviour in aqueous media and its ability, with and without cholesterol, to form higher order structures, "dendrisomes", was studied to further our understanding of how dendrons interact with drug molecules and may be utilised as drug carriers. Dynamics simulations of the dendron show their flexibility. Incorporation of cholesterol increases the hydrodynamic diameter of the aggregates from 311 to 556 nm but does not affect their positive zeta potential (of the order of +50 mV). The dendrisomes encapsulated penicillin G (6.15% w/w) compared to only 1.4% w/w entrapment in REV liposomes of 1:1 distearoyl phosphatidylcholine:cholesterol. Cholesterol, however, decreases the entrapment efficiency. Electrostatic forces and H-bonding between the negatively charged drug and dendron amino groups are likely to be key in determining these interactions.

The interaction of cationic dendrons with albumin and their diffusion through cellulose membranes.

Amphipathic dendrons (partial dendrimers) having three lipidic (C(14)) chains coupled to dendritic lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide synthesis. Their interaction with albumin was studied in the presence of NaCl using dynamic dialysis, the diffusion of the dendrons through regenerated cellulose membranes also being studied. The stoichiometry of dendron: albumin interactions was found to be 1:1.5, 1:4 and 1:5 for the dendrons with 8, 16 and 32 amino groups, respectively. Membrane permeability P, membrane diffusion coefficient D and the membrane partition coefficient K values were calculated for each dendron. P and D values were low but highest for the 8 amino group dendron. The membrane partition coefficient K was greatest for the 8 amino group dendron. This was also the case with octanol/water partition coefficient studies. Considerable adsorption of the dendrons to the cellulose membrane occurred but NaCl decreased adsorption and improved diffusion of the dendrons through the cellulose membrane.

Effect of physiological media on the stability of surface-adsorbed DNA-dendron-gold nanoparticles.

Plasmid DNA was adsorbed onto 87-nm gold nanoparticles to which were adsorbed a layer of novel cationic dendrons. The behaviour of this DNA-dendron-gold system in cell culture media has been described. Adsorption onto the gold nanoparticles of lipophilic cationic dendrons, with either 8 [(C12)(3)Lys7(NH2)(8)] or 16 [(C12)(3)Lys15(NH2)(16)] free amino groups on their outer surfaces and incorporating a nuclear localization signal peptide (NLS), resulted in positively charged nanoparticles with a corresponding small increase in particle size. Evidence suggested that the interaction between the gold nanoparticles and the dendron was mediated by hydrophobic forces. With an increase in ionic strength, the apparent particle size of the dendron-stabilized-gold particles increased, but at higher salt concentrations than plain gold sols. Addition of plasmid DNA did not markedly reduce the surface potential of the dendron-gold complex but resulted in an approximately 10-20% increase in hydro-dynamic diameter. Increasing ionic strength increased the apparent size of the DNA-dendron-gold particles, up to a maximum diameter of approximately 900 nm. Importantly, in cell culture media the size of the DNA-dendron-gold nanoparticles increased markedly, as surface potential was reduced. The presence of serum components partially ameliorated these effects, possibly due to steric stabilization of the particles. Release of the DNA from the complex was compromised in cell culture media (compared with water). This, coupled with the flocculation of the carrier, demonstrated the importance of testing delivery systems in the presence of relevant physiologically based fluids before cell culture or in-vivo studies.