Biodegradable poly(2-dimethylamino ethylamino)phosphazene for in vivo gene delivery to tumor cells. Effect of polymer molecular weight

Abstract

Purpose: Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity.

Materials and methods: p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity.

Results: Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25-29 mV), and had a size of 80-90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz's (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression.

Conclusion: Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection.

Fig. 1

Structure of poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz).

Fig. 2

SEC chromatogram of the starting polymer (a) and polymer fraction ppz290 (b). Left axis: low angle light scattering (bright grey), viscosity (medium grey), refractive index (dark grey). Right axis: calculated molar mass dependency (black).

Fig. 3

Aggregation of DNA/p(DMAEA)-ppz complexes at high ionic strength. Particle size of complexes based on the starting polymer (circles) and ppz130 (diamonds), after 1 h of incubation in HBS (20 mM Hepes, 150 mM NaCl, pH 7.4) (mean, n = 3).

Fig. 4

DNA binding of p(DMAEA)-ppz polymers. DNA binding was measured in Hepes buffer and quantified as percentage residual fluorescence, 1 h after addition of polymer to EtBr labeled DNA (mean ± SD, n = 3).

Fig. 5

Cytotoxicity of DNA/p(DMAEA)-ppz complexes towards Neuro 2 A cells, 24 h after incubation (mean ± SD, n = 3).

Fig. 6

In vitro gene expression of Neuro 2 A cells after transfection with DNA/p(DMAEA)-ppz complexes. Relative light units per well, 24 h after transfection (mean ± SD, n = 3).

Fig. 7

Effect of ionic strength on the in vitro transfection activity of DNA/p(DMAEA)-ppz complexes. Transfection values of polyplexes based on the starting polymer, prepared at high ionic strength (HBS) relative to values determined for polyplexes prepared at low ionic strength (HBG), 24 h after transfection of Neuro 2 A cells (mean, n = 3).

Fig. 8

DNA/p(DMAEA)-ppz complex-induced erythrocyte aggregation (representative example at an N/P ratio of 12). Light microscopic image of erythrocytes, after 1 h of incubation with polyplexes based on the starting polymer (a), ppz950 (b), ppz570 (c), ppz290 (d), ppz130 (e).

Fig. 9

Luciferase expression, 24 h after i.v. administration of pDNA/ppz290 complexes (a) and pDNA/ppz130 complexes (b) into mice bearing a Neuro 2 A s.c. tumor (30 μg DNA/mouse). White bars: N/P ratio 12, hatched bars: N/P ratio 20. Luciferase expression is plotted as relative light units per organ (RLU/organ) (mean + SD, n = 4). Average tumor weight was 600 mg.