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


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.


CONCLUSIONS
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.
de Wolf
Holger K
HK
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 80082, 3508 TB, Utrecht, The Netherlands.
de Raad
Markus
M
Snel
Cor
C
van Steenbergen
Mies J
MJ
Fens
Marcel H A M
MH
Storm
Gert
G
Hennink
Wim E
WE
D000818:Animals; D002449:Cell Aggregation; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000628:therapy*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D009994:Osmolar Concentration; D010316:Particle Size; D011108:Polymers / Q000737:chemistry*; D055672:Static Electricity; D014162:Transfection / Q000379:methods
D046911:Macromolecular Substances; D009943:Organophosphorus Compounds; D011108:Polymers; C508109:poly(2-dimethylamino ethylamino)phosphazene; D004247:DNA; D008156:Luciferases