Biodistribution of Radiolabeled Lipid–DNA Complexes and DNA in Mice

Abstract

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.

Increased Gene Expression by Cationic Liposomes (TFL-3) in Lung Metastases Following Intravenous Injection

“…It has been reported that incubation in pure serum or serum-containing media could decrease the extent of lipoplex delivery and alter its intracellular fate 19,20) and even cause the lipoplex to disintegrate upon prolonged exposure. 18,21) However, an injected lipoplex is rapidly cleared from the circulation 18,22) and internalized in the lungs, 23) thus ruling out long-term exposure to serum. In addition, Sakurai et al showed the pre-incubation of a DOTAP/Chol lipoplex with serum for as long as 30 min did not significantly impair lipofection in the lungs.…”

“…5). The first pass entrapment (60-80% of the liposomes are entrapped within minutes after intravenous injection) 18,22,28) can be attributed to the highly extended lung capillary bed and was proposed to explain the predominant gene expression in the lungs. 28,29) Ito et al 30) recently reported that vascular endothelial cells, probably under angiogenesis, in the lungs of tumor bearing mice, which demonstrates an increased uptake of cationic liposome-DNA complexes and transgene expression compared to normal mice.…”

“…The morbidity rate of lung cancer has been rising particularly rapidly, and a pressing countermeasure is necessary. 16) Lipoplexes usually accumulate largely in the lung, although the distribution changes with time: lipoplexes are found in the lung shortly after intravenous injection but eventually accumulate in the liver after 24 or 48 h. 17,18) This is because the lipoplex form aggregates in the blood stream, and is captured in the first capillary en-countered. If the pDNA could be quickly transferred from lipoplexes to pulmonary endothelial cells after being captured in a capillary of the lung, an enhanced transgene expression might be obtained.…”

Optimization and Characterization of Dry Powder of Fanhuncaoin for Inhalation Based on Selection of Excipients

“…It was interesting to find that both chitosan and chitosan oligosaccharide can not only influence the particle shape and morphology but enhance the dispersibility of spray-dried powders. Chitosan oligosaccharide, possessing the properties of soluble, biodegradable, biocompatible, bioadhesive and nontoxic, 38) has the ability to enhance mucosal absorption by opening tight junctions, 39) potentially resulting in an increase of drug absorption subsequent to inhalation. In our study, the addition of chitosan oligosaccharide in formulations can achieve higher spray-dried yields (Figs.…”

Synergistic Effect of Cationic Lipids with Different Polarheads, Central Core Structures and Hydrophobic Tails on Gene Transfection Efficiency

“…Since free oligonucleotides and DNA are rapidly degraded by serum nucleases, 4) many efforts are focused on developing the carriers to protect and deliver these materials into targeted cells. Two delivery systems namely viral and non-viral vectors are currently developed to solve the problems.…”