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The particle growth data of LID complexes formed at various charge ratios in the transfection buffer Opti-MEM are plotted in Fig. 5.4. Extensive aggregation of the lipopolyplexes at all charge ratios was observed. Particle size measurements were also carried out for lipopolyplexes (LKD) in which the integrin-targeting peptide (peptide 6) was substituted for a similar peptide incorporating a longer oligolysine domain (K]^). The supplementary LKD data showed that a longer DNA-binding lysine tail neither conferred stability nor increased aggregation. The aggregation profiles of lipopolyplexes at charge ratios 0.5 when the lipopolyplex was not yet fully formed, and 1.0 when the lipopolyplex was theoretically neutral, appeared no different than those at net positive charge ratios. This suggests that at these charge ratios free plasmid DNA was present in the formulation. On the other hand, since the aggregation profiles at charge ratios higher than

1.0 were not significantly different, excess Lipofectin and integrin-targeting peptide may have been present in the solution.

200 0

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(U ■73 Ph 1500 - 1000 - 500 - A 50 X — ♦ 0.5 ■ 1.0 ♦ 2.0 X 3.0 X 4.0 • 5.0 + 6.7 0 7.0 - 8.0 0 10.0 A LK D (3.0) 100 150 Time (mm) 200 250

Figure 5.4 Mean particle size o f LID and LKD complexes as a function o f the charge ratio. LKD complexes were prepared at a charge ratio o f 3. All lipopolyplexes were prepared in Opti-MEM by the two-syringe mixing method (2 ml/min mixing rate) and at a final DNA concentration o f 5 pg/ml. This is a representative data plot from three experiments.

The P icoG reen assay (Section 3.12) was used as a highly sensitive and accurate m ethod to quantify free D N A , i.e. D N A not com plexed by L ipofectin or integrin-targeting peptide. Fig. 5.5 show s the effect o f the com plex charge ratio on the particle surface charge, given by the zeta potential (open sym bols), and the accessibility o f plasm id D N A to P icoG reen intercalation (closed sym bols). T he data refer to experim ents carried out with pC I-luc, the 5.7 kb plasm id D N A. T he lipopolyplexes w ere prepared at a D N A concentration o f 5 pg/m l using the tw o-syringe m ixing m ethod as described in Section 3.7. In contrast to the constant aggregation profiles for all charge ratios in Fig. 5.4, the zeta potentials at charge ratios less than 1.0 w ere strongly negative, show ing incom plete

charge neutralisation o f the D N A (Fig. 5.5). T he zeta potentials reached a value of approxim ately 25 m V at a charge ratio o f 3-4 and rem ained relatively unchanged even with the addition o f m ore peptide. A ccordingly, the fluorescence progressively decreased through the range o ver w hich the zeta potentials w ere negative, and reached a plateau value also at the charge ratio 3-4, at w hich point the D N A becam e inaccessible to the dye.

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1000 -O .o-’ - 100 -10 -20 10 2 4 6 8 0 o Charge ratio

Figure 5.5 Zeta potentials and DNA accessibility o f the LID complexes as a function o f the charge ratio (varying amounts o f integrin-targeting peptide). The complexes were prepared in PBS at a final DNA concentration o f 5 pg/ml by the two-syringe mixing method (2 ml/min mixing rate), and their zeta potentials were immediately measured. For the fluorescence assay, the complexes were prepared in Opti-MEM at 5 pg/ml DNA concentration and diluted in TE buffer, as described in Section 3.12. Representative data plots from two experiments are shown.

Note that the maximum zeta potential was reached at a charge ratio greater than that of charge neutrality (Fig. 5.5). This may be due to the masking of charges on the oligolysine domain of the peptide by the cyclic integrin-targeting ligand (Kwoh et aL,

1999). In addition, it has been shown in other fluorescence dye exclusion assays that the ionic strength of the solution may increase the accessibility of DNA in the complex to the dye (Eastman et aL, 1997; Ferrari et aL, 2001). Eastman et aL suggested that the salt concentration influenced both lipoplex formation and pre-existing lipoplexes by enhancing extension of the DNA from the complex structure into the solution environment (Eastman et aL, 1997). This may explain the results shown in Fig. 5.5, where the lipopolyplexes were formed in PBS. The presence of charge shielding effects brought about by polyanions in the buffer may also account for the weaker electrostatic interaction between the DNA and the cationic components (LI), leading to an increased amount of peptide required to reach the maximum zeta potential.

Since in vitro transfection experiments, subsequently shown in Section 5.3, were performed in Opti-MEM, it would have been relevant to measure the zeta potentials and fluorescence signal with LED complexes in Opti-MEM. Nevertheless, this was not possible because zeta potentials of complexes in Opti-MEM could not be measured accurately; the quality of data obtained from the zeta potential measurements was very poor and hence unreliable (data not shown). This has been observed elsewhere for poly- L-lysine/DNA complexes in Opti-MEM and is thought to be due to an artefact of the method rather than a property of the complexes (Pouton et aL, 1998). For the PicoGreen assay, the LED complexes were diluted into a simple buffer, i.e. TE buffer (10 mM TrisCl pH 8.0, 1 mM EDTA), rather than a transfection buffer such as Opti-MEM, because this was recommended in the Product Information Sheet supplied with PicoGreen reagent. Furthermore, it was important to minimise contamination by any salts, enzymes and other compounds found in Opti-MEM that may influence the PicoGreen signal (Singer et aL, 1997).

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