Effects of Complement Depletion on the Pharmacokinetics and Gene Delivery Mediated by Cationic Lipid–DNA Complexes

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Abstract
We show that lipoplexes activate complement in human serum in vitro and deplete complement when administered intravenously (i.v.) to mice. This raised the possibility that complement proteins might alter gene expression mediated by lipoplex in animals. To investigate this phenomenon, complement levels were depleted to less than 5% in ICR mice by intraperitoneal (i.p.) injection of cobra venom factor and anti-C3 antibodies. The pharmacokinetics and distribution of radio labeled DOTAP–cholesterol (1.0:0.9 molar ratio)–DNA (5:1 positive charge ratio) complexes containing 131I-labeled p-hydroxybenzamidine phosphatidylethanolamine and 125I-labeled DNA were measured in mice after i.v. administration. Greater than 75% of the injected lipoplex appeared in the lungs 5 min following injection. The lipid and DNA were eliminated from the lungs at a constant ratio. Distribution in various organs was not affected by complement depletion. Expression of luciferase was highest in the lungs and showed a dose-dependent increase as the amount of DNA injected increased from 3 to 60 μg. Reporter gene expression was not affected by complement depletion. In addition, complement depletion had no effect on either the distribution or gene expression in the heart, spleen, or liver. We conclude that cationic lipid–DNA complexes interact with serum complement proteins upon i.v. injection in mice, but this interaction does not influence the lipofection efficiency or systemic distribution of the lipoplex. The mechanism of lipoplex-mediated gene delivery in vivo is not fully understood. Serum complement proteins provide a multifaceted defense system which, upon activation, may have important consequences for the use of lipoplexes as gene delivery vehicles. The decoration of lipoplex with complement proteins may decrease the stability and/or plasma half-life of intravenously (i.v.) injected lipoplex. To investigate this hypothesis, we injected positively charged lipoplexes into mice and found that serum complement levels were decreased. Complement depletion of mice using cobra venom factor and anti-C3 antibodies prior to injection of complex did not affect transgene delivery or expression. These results suggest that other blood components may be responsible for the characteristic high lung and low liver expression profiles we observe upon i.v. administration of lipoplexes possessing a net positive charge.