Biodistribution of radiolabeled lipid-DNA complexes and DNA in mice.

The tissue biodistribution and expression of [33P]DNA-1-[2-[9-(Z)-octadecenoyloxy]ethyl]]-2-[8](Z)-heptadece nyl]-3 -[hydroxyethyl]imidazolinium chloride (DOTIM):cholesterol complexes and 33P-radiolabeled DNA expressing chloramphenicol acetyl transferase (CAT; 4.7 kB) were studied after intravenous (iv) injection in ICR mice. Mice were injected with 200 microL of complex containing DNA at 3 mg/kg or DNA alone. One group received 8 microCi of radioactivity and were sacrificed at 5 and 20 min, and 1, 2, 4 and 24 h post-dose (n = 4/time point). A second group received the equivalent of 3.9 microCi of radioactivity and were sacrificed at 20 min, and 2 and 24 h for subsequent whole body autoradiographic analysis (WBA; n = 2/time point). The tissue distribution of intact DNA was assessed by Southern blot at 24 h post-dose, whereas the integrity of complexes and DNA incubated in heparinized whole blood was studied separately. In further studies, the time course of expression in lung tissue over a 48-h period was examined, and the relative lung-expression of purified open circular (OC) versus supercoiled (SC) DNA at 24 h was evaluated. Approximately 42% of the radioactivity was found in the lungs 5 min after injection and about half this percentage was found in the liver. By 2 h, only 5% remained in the lungs, but 48% was present in the liver. No other tissue accumulated >5% of the dose throughout the duration of the study. WBA radiograms confirmed the tissue distribution results and highlighted significant accumulation of radioactivity in bone over time. Southern Blot analysis demonstrated intact DNA in many tissues 24 h after dosing. In contrast, the majority of DNA incubated in blood was degraded within 2 h, although the complexes afforded some protection relative to DNA alone. The OC DNA expressed equivalently to SC DNA in lung tissue (OC = 1035 +/- 183 pg; SC = 856 +/- 257 pg/mg soluble protein, n = 6, mean +/- SEM) at 24 h, and detectable levels of CAT were present within 2 h of dosing (21.3 +/- 7.2 pg, n >/= 8, mean +/- SD). The results confirm that DNA-DOTIM:cholesterol complexes are initially deposited in the lungs after iv administration.

Characterization and in vivo testing of a heterogeneous cationic lipid-DNA formulation.

PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..