Thiagarajan Sakthivel

DNA transfection and transfected cell viability using amphipathic asymmetric dendrimers

Amphipathic asymmetric dendrimers have been investigated for use in delivery of genes into cells, with the objective of optimising transfection efficiency and maintaining cell viability. We have synthesised amphipathic asymmetric dendrimers by solid phase methods. The ability of two of these to transfect BHK cells in culture with beta-galactosidase gene was determined by X-gal staining. Cell viability was measured by the MTT assay for BHK cells, and by spectroscopy for lysis of erythrocytes. Interactions between dendrimer and DNA were investigated by agarose gel electrophoresis. BHK cells were optimally transfected at 5:1 +/- charge ratio yielding 20% cells receiving at least one copy of the plasmid. Cell viability decreased when the dendrimer to DNA ratio exceeded 5:1. Raising the pH significantly affected the electrophoretic mobility of complexes of dendrimer and DNA. We conclude that amphipathic asymmetric dendrimers enable efficient plasmid DNA uptake into BHK cells. Cell viability is maintained at high concentrations of dendrimer when complexed with DNA at a 5:1 +/- charge ratio. Efficiency of transfection and cell viability suggest the system may be suitable for gene delivery in vivo.

Oral uptake and translocation of a polylysine dendrimer with a lipid surface

A series of lipidic peptide dendrimers based on lysine with 16 surface alkyl (C(12)) chains has been synthesised in our laboratories. One of the series, a fourth generation dendrimer with a diameter of 2.5 nm was chosen to study its absorption after oral administration to female Sprague-Dawley rats (180 g, 9 weeks old). It was synthesised as the tritiated derivative (all acetyl portions) and had a molecular weight of 6300 and log P (octanol/water) of 1.24. First a single oral dose 14 mg/kg was administered by gavage. Maximum levels of dendrimer observed were 15% in the small intestine, 5% in the large intestine and 3% in the blood at 6 h after administration, while 1.5% reached the liver, 0.1% the spleen and 0. 5% the kidneys. In a parallel study with a higher dose of 28 mg/kg, approximately 1% was absorbed via Peyers patches of the small intestine at 3 h. The maximum uptake by small intestine enterocytes was 4% of the dose after 3 h. After 12 h, 0.3 and 4% dendrimer was measured respectively in Peyers patches and enterocytes of the large intestine. When calculated on the basis of target tissue weight, the total percentage of the dose absorbed through Peyers patches was greater than through normal enterocytes in the small intestine after 3 and 24 h, but the opposite was true in the large intestine. These levels of uptake and translocation are lower than those exhibited by polystyrene particles in the range from 50 to 3000 nm. This might suggest that there is an optimum size for nanoparticulate uptake by the gut.

Synthesis and Physicochemical Properties of Lipophilic Polyamide Dendrimers

AbstractPurpose. To synthesise symmetrical dendritic macromolecules with external lipid surfaces, to investigate their behaviour at the air-water interface and their ability to form supramolecular aggregates, and to gain an understanding of their potential as drug carriers. Methods. Dendrimeric compounds were synthesised with molecular weights ranging from 737 (1st generation dendrimer) to 25,246 (6th generation dendrimer) with carbon numbers ranging from 40 to 1404. The surface behaviour of these compounds was determined using spread films at the air/water interface on a Langmuir trough, and transmission electron microscopy was used to study the supramolecular aggregates formed by the more hydrophobic members of the series. Results. Dendrimers up to a maximum of 6 generations were synthesised. Surface saturation did not allow the completion of the synthesis of the 7th generation. The limiting surface areas at the air/water interface ranged from 0.4 nm2 to 16.1 nm2 values in good agreement with the areas derived from computer generated molecular models (0.5 nm2 to 14 nm2). Conclusions. The synthesised dendrimers exhibited a linear relationship between area per molecule and the molecular weight of the compounds. A dendrimer with 16 lipoamino acid branches formed tubular supramolecular aggregates with a helical structure and dimensions in the long axis of 140−200 nm.

Interaction of cationic partial dendrimers with charged and neutral liposomes.

Amphipathic partial dendrimers having three lipidic (C(14)) chains coupled to dendritic lysine head groups with eight, 16 or 32 free terminal amino groups have been synthesised by solid-phase peptide synthesis. Liposomes were prepared with positive, negative and neutral charge using the dehydration-rehydration method and their interaction with the partial dendrimers studied. The interaction efficiency of the partial cationic dendrimers studied was greater than 88%. Interaction of the cationic partial dendrimer converted liposomes with very low or negative charge into positively charged species. Apparent vesicle size increased with the head size of the partial dendrimer but, in the case of negatively charged liposomes, large changes in properties were observed after ultracentrifugation due to the formation of myelin figures. To investigate the mode of interaction with the liposomes, adsorption studies were performed by adding the partial dendrimer after the preparation of dehydration-rehydration vesicles. The results indicated that adsorption is inversely proportional to the head size of the partial dendrimer molecules and the extent of adsorption was similar on both positively and negatively charged liposomes. Adsorption produced liposomes with greater or similar zeta potentials to liposomes that incorporated partial dendrimer through the dehydration-rehydration method. Taking account of the different interaction efficiencies, this suggests there is a degree of partial dendrimer entrappment inside the liposomes.

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.

Non-aqueous emulsions: hydrocarbon–formamide systems☆

There are few reports in the literature on formulation of non-aqueous emulsions. This study was designed to evaluate some design criteria for such systems. Formamide is the closest polar solvent that has the ability to replace water in emulsification when employing established non-ionic surfactants as stabilisers. For the majority of studies, linear alkanes (C6-C16) were dispersed in formamide as the continuous phase were stabilised with polysorbate 20. Initial studies involved gentle emulsification and observing mean globule size. The mean globule size varied in a non-linear fashion with alkyl chain length, the minimum being between C10 and C12. Sonication for 30 s led to smaller differences in the mean globule size. The effect of various parameters such as surfactant concentration and solvophilicity of the surfactant was observed. The surface activities of polysorbate 20, 40, 60 and 80 in formamide and critical micellar concentrations were determined. The latter were several orders of magnitude higher in formamide than in water, and the areas per molecule larger. The addition of water to the dodecane formamide systems did not destabilise the emulsion. Release of the model drug dehydroepiandrosterone from dodecane in formamide emulsions was studied in distilled water, the rate of release being dependent on the volume fraction of dodecane.

Adsorption of amphipathic dendrons on polystyrene nanoparticles

Adsorption of dendrons onto nanoparticles may provide new model structures which may be useful in drug and gene delivery. Tritiated amphipathic dendrons having three lipidic (C(14)) chains coupled to branched (dendritic) lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide techniques. The interaction between these tritiated dendrons and 200 nm polystyrene latex nanoparticles was investigated in phosphate buffered saline. The amount of dendron adsorbed increased with increasing concentration of dendrons and then decreased. Maximum adsorption of dendrons per gram of nanoparticles was found to be between 8.2 and 84 x 10(-6)M, the amounts adsorbed being inversely proportional to the number of amino groups present in the molecule. The number of dendron molecules adsorbed per nanoparticle was found to be between 430 and 4421. The degree of adsorption was found to be slightly altered by the temperature.

Distribution of a lipidic 2.5 nm diameter dendrimer carrier after oral administration

The biodistribution of a lipidic peptide dendrimer has been studied after oral administration to female Sprague-Dawley rats (180 g, 9 weeks old). Uptake by gut epithelial tissue of the radiolabelled dendrimer molecule (mol. wt. 6300; diameter 2.5 nm; log P=1.24) was studied in rats after a single oral dose by gavage (14 mg/kg). The maximum levels of dendrimer observed were 3% (blood), 1.5% (liver), 0.1% (spleen), 0.5% (kidneys), 15% (small intestine) and 5% (large intestine). Approximately 6% of a single administered dose (28 mg/kg) was recovered from the entire gastrointestinal tract while 1% was absorbed via the small intestine lymphoid tissue after 3 h; after 12 h, 0.1% was detected. The maximum uptake by the non-lymphoid small intestine was 4% of the dose after 3 h. After 12 h, 0.3 and 4% dendrimer was measured in the lymphoid large intestine and the non-lymphoid large intestine, respectively. The total percentage of the administered dose absorbed through the lymphoid tissue was comparatively greater than through the non-lymphoid tissue of the small intestine with respect to organ weight after 3 and 24 h.

Formulation of oil in oil emulsions: potential drug reservoirs for slow release

Emulsions composed of two immiscible oils are uncommon. In this paper, emulsions of castor oil in silicone oil (dimethylsiloxane) with a range of viscosities from 1–100 cSt were studied using a range of non-ionic surfactants as emulsifiers. Stable emulsions were obtained using an octyl phenyl-poly(10)oxyethylene ether (Triton-X-100) but few of the other non-ionic surfactants studies achieved stable systems. Of the surfactants studied, only polyoxy ethylene aryl-alkyl ethers were useful stabilisers, whereas alkyl ester analogues were not. Optimisation of various process and formulation factors was achieved using a D-optimal response surface experimental design approach yielding a reduced fit quadratic model. The variables selected include phase volume ratio (ϕ), surfactant concentration (C SA ), sonication time (T SO ) and the viscosity (η) of the external phase silicone oil. Formulated emulsions were found to be stable up to around 37°C but the systems are destabilised above 50°C. Dexamethasone and dehydroepiandosterone (DHEA) were solubilized in the internal castor oil phase and their release from the emulsion across a dialysis membrane into an aqueous medium was recorded. Slow release over several hours was observed, suggesting that the oil in oil emulsions may be useful as depot or reservoir systems for drug delivery.

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)3Lys7(NH2)8] or 16 [(C12)3Lys15(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.