PLL/calf thymus DNA complexes

Fig. 4.9 shows the variation in the mean particle size of PLL/calf thymus DNA complexes with solution salt concentration (ionic strength) and pH. The polyplexes were prepared using the two-syringe mixing method with a fixed mixing rate of 6 ml/min. The data in Fig. 4.9 show that ionic strength and pH of the suspension critically determine the physical stability of the PLL/DNA complexes. PLL of molecular weight 25,250 was used. With the exception of the suspension at a concentration of 10 mM NaCl at the lowest pH, all systems examined showed an increase in the z-average hydrodynamic diameter as a function of time. The rate of aggregation increased with increasing ionic strength and increasing pH. At a pH of 8.0, even the suspension containing 10 mM NaCl exhibited a degree of aggregation as shown in Fig. 4.9C.

A

I

<u 13 Ah B Ph

c

Ah □ 10 mM N aCl 1500 4 O 50 mM N aC l ■ 100 mM N aC l ^ □ 150 mM N aC l 1000 - 1500 - y 1000 - 500 - 1500 ^ 1000 - 500 - 10 10 15 15 20 20 20 25 25 25 5 10 15 Time (mm)

Figure 4.9 Particle size o f poly-L-lysine/calf thymus DNA complexes as a function of time for different salt concentrations and pH. The polyplexes were prepared at a mixing rate o f 6 ml/min and a charge ratio o f 2.0 in 20 mM HEPES: (A) pH 7.2,

(B) pH 7.7 and (C) pH 8.0. The data are expressed as the mean hydrodynamic diameter and standard deviation obtained from two independent experiments.

As shown in Fig. 4.9, the larger sizes (greater than 500 nm) of polyplexes formed immediately after mixing (t = 0) at higher ionic strengths (100 and 150 mM NaCl) confirmed that rapid aggregation occurred in the initial five minutes following preparation of the polyplexes, i.e. during the data acquisition time. Although DNA delivery systems of such sizes have been used in the transfection of cultured cells with satisfactory gene expression levels, in vivo gene delivery would impose limitations on the size of the polyplexes so that they may pass through biological barriers such as the vascular endothelium and blood-brain barrier, diffuse through tissues, and be efficiently internalised by the cells (Pouton, 1999). Furthermore, higher DNA concentrations (> 330 p,g/ml) are essential for clinical applications (Zelphati et al., 1998a). It will be shown later (Chapter 5) that the aggregation of DNA complexes increases dramatically as the DNA concentration is increased.

Fig. 4.10 summarises the comparison of particle size distributions for two different samples of PLL/DNA complexes as a function of time after preparation. The polyplexes were prepared by the two-syringe mixing method at a 2.0 charge ratio in 20 mM HEPES: (A) pH 7.2, 10 mM NaCl and (B) pH 8.0, 150 mM NaCl. The data refer to the systems shown in Fig. 4.9: (A) 10 mM NaCl and (C) 150 mM NaCl, respectively. Comparison of Figs. 4.10A and B demonstrates that the physical stability of the polyplexes is acutely sensitive to the salt concentration. The extreme instability of the system at physiological ionic strength, as shown by samples at 100 and 150 mM NaCl in Fig. 4.9, is well supported by the distinct shift and broadening in the size distributions shown in Fig. 4.10B.

A <?3 *0 .3 ::S 5 mm 10 min 15 mm 20 mm 25 min 10 5 0 10000 100 1000 10 B 10 - 10000 10 100 1000

Log mean diameter (nm)

Figure 4.10 Particle size distributions o f poly-L-lysine/calf thymus DNA complexes at a charge ratio o f 2.0 in 20 mM HEPES: (a) pH 7.2, 10 mM NaCl [corresponds to Eig. 4.9(A) 10 mM NaCl] and (B) pH 8.0, 150 mM NaCl [corresponds to Fig. 4.9(C) 150 mM NaCl], as a function o f time after preparation. Size distributions were determined as mean o f diameter on the basis o f intensity o f scattered light at 90°. The data shown were obtained from a single representative sample.

As previously show n in Fig. 4.2, the results from the M alvern Z etasizer gives intensity, m ass and num ber w eighted size distributions (Fig. 4.2A ), as w ell as z-average diam eter and polydispersity index (Fig. 4.2B ). T he broadening o f the size distribution as a function of tim e corresponds to increasing polydispersity o f the sam ple, w hich is associated with

the variance o f the population (R ustem eier and K illm ann, 1997). T he polydispersity index, estim ated by cum ulants analysis o f the DLS data, has a m axim um value o f 1.0, with a nom inal value o f 0.7 representing a highly polydisperse distribution. T he polydispersity indices o f about 0.2-0.3, for all size distributions o f the system at a low salt concentration (Fig. 4.10A ) and for the initial distribution (“5 m in”) o f the system at the high salt concentration (Fig. 4.10B ), indicate fairly narrow size distributions o f the polyplexes. In contrast, the polydispersity values for subsequent m easured distributions in Fig 4.10B increased from 0.4 at 10 m in to beyond 0.7 for a sam ple analysed nearly 30 m inutes after polyplex form ation. It will be show n later (in S ection 4.2.2.3) that aggregation in polydisperse system s is particularly m arked.

lO m M N a C l 50 m M N aC l 100 m M N aC l 150 m M N aC l P 20 - a.

I

OJ N 7.2 7.7 8.0 pH

Figure 4.11 Effect o f buffer pH and sodium chloride concentration on the measured zeta potential of complexes formed between polylysine and calf thymus DNA. Polyplexes were prepared using the two-syringe mixing method (6 ml/min mixing rate) in 20 mM HEPES buffer at a charge ratio o f 2.0. Bars indicate the value of standard deviation o f two separate experiments. For the sake o f clarity only deviations for polyplex samples at 10 mM NaCl are shown.

The surface charge of the polyplexes can be quantitatively inferred from mobility measurements and is given by the measured zeta potential (see Sections 3.8 and 4.2.1.1). The zeta potential of the PLL/calf thymus DNA complexes whose particle size distributions are shown in Figs. 4.9 and 4,10 were tested. The zeta potentials of the polyplexes were observed to decrease with increasing solution pH, as shown in Fig. 4.11. The results indicate that at higher pH values, the polyplexes are less positively charged, and consequently, more likely to interact with each other. These observations (Fig. 4.11) are in good agreement with the physical instability of polyplexes in buffers of higher pH as shown previously (Fig. 4.9). Polyplexes prepared at higher pH values had zeta potentials close to zero, indicating the tendency of the system to aggregate in a basic environment (pH > 8.0 in this case), and the presence of more titratable groups close to the isoelectric point (lEP, the pH at which the zeta potential becomes zero). The zeta potentials remained fairly constant over a range of ionic strengths for a given pH, which is in disagreement with findings by other groups, where an increase in the salt concentration resulted in a decrease in the zeta potential (Son et al., 2000; Xu et al., 1998). The zeta potential measurements in Fig. 4.11 were performed after the size measurements, i.e. when most of the polyplexes were observed to have aggregated (Fig. 4.9), hence possibly contributing to this dissimilarity.

In document Biophysical studies of synthetic gene delivery systems (Page 85-90)