In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection - PubMed (original) (raw)

In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection

Torben Gjetting et al. Int J Nanomedicine. 2010.

Abstract

Background: DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream.

Method: A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution.

Results: We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing.

Conclusion: The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.

Keywords: DOTAP; biodistribution; gene delivery; lung cancer; polyethylene glycol (PEG); xenograft tumor model.

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Figures

Figure 1

Figure 1

Luciferase reporter gene expression in vivo. After three consecutive daily tail vein injections of DOTAP/chol/DNA lipoplex (A-0*) in nude mice organs were sampled on the fourth day and assayed for luciferase activity, n = 4. Assay background was at 10 pg luc/g protein (indicated with dotted line).

Figure 2

Figure 2

Agarose gel electrophoresis/ethidium bromide staining of DNA/lipoplexes with different content of PEG-lipid compared to the migration of free plasmid DNA (free DNA) and a DNA size marker.

Figure 3

Figure 3

Physical characterization of lipoparticles using a Zetasizer. Each of the four liposome formulations was analyzed before (white columns) and after (grey columns) mixing with plasmid DNA. A) The hydrodynamic size. B) The polydispersity index. C) The zeta potential of the particles. The estimate and the standard error are given for each measurement. Three independent experiments yielded equivalent results.

Figure 4

Figure 4

Reporter gene expression by transfection of NCI-H69 (SCLC) and H1299 (NSCLC) cells in vitro. A) EGFP fluorescence in single cells was measured by FACS flow cytometry and expressed as the percentage of EGFP positive cells. *** indicates a significant difference from A-0 (P < 0.001). B) Luciferase activity was measured in cell lysates and expressed as picogram luciferase per milligram of protein. Data from four independent experiments with two different lipid preparations were collected and the average and standard error of the mean are given. *indicates a significant difference from A-0 (P < 0.05).

Figure 5

Figure 5

Cellular uptake of fluorescently labeled lipoplexes. A) Bright-field and fluorescence microscope pictures taken two days after H1299 cells were transfected with NBD-labeled A-0 (upper panel) and D-10 lipoplexes (lower panel). Scale bar marked in red indicates 15 μm. B) NBD fluorescence in cell lysates was measured at different time points after addition to cell cultures. Non-PEGylated A-0 lipoplex is rapidly taken up by cells to a saturating level within one hour and remain constant hereafter (fitted line [estimate ± standard error]: Fluorescence index = (1001 ± 102) + (2.8 ± 3.6) × time) Slope confidence interval includes 0. In contrast, PEGylated lipoplexes are gradually taken up over two days. Fitted lines: B-2: Fluorescence index = (626 ± 115) + (15 ± 4.3) x time; C-4: Fluorescence index = (487 ± 71) + (15 ± 2.9) × time; D-10: Fluorescence index = (678 ± 92) + (44 ± 3.7) × time. Slope confidence interval exclude 0. Data are normalized and averaged from triplicates of three independent experiments (average ± SD).

Figure 6

Figure 6

Effects of endocytosis inhibitors on luciferase reporter activity measured in (A) NCI-H69 and (B) 1299 cells. Cells were incubated one hour prior to transfection and then exposed to inhibitors for the entire incubation (48 hours). Chloroquine (20 μM (NCI-H69), 30 μM (H1299)), chlorpromazine 10 μM (NCI-H69), 20 μM (H1299), filipin (4 μM), and cytochalasin B (25 μM). Data from 3–5 independent experiments were pooled and log2 transformed to obtain fold change in expression. Error bars indicate standard error of the mean, *means that 0 is not included in confidence interval (P < 0.05), no change: N.S.

Figure 7

Figure 7

A) Blood availability of tritium-labeled lipoplexes with PEGylation. Following tail vein injection of lipoplexes blood samples were drawn by periobital plexus puncture (eye vein blood) after 15 minutes, 2 hours, 5 hours, and 24 hours, and the amount of radioactivity was quantified by scintillation counting. B) Biodistribution of luciferase activity. Mice injected with tritium-labeled DNA/lipoplex with different degrees of PEGylation were sacrificed after 24 hours and samples from organs were analyzed for luciferase activity. Background level in assay is 10 pg/g protein (indicated with dotted line). C) Biodistribution of radioactivity. Tritium counts were expressed as CPM per gram organ sample weight (CPM/g) and the relative distribution in tumor, heart, lung, liver, kidney, and spleen was calculated. Error bars indicate standard deviations. In each group, n = 3–5. A two sided _t_-test of accumulation in heart/lung samples between A-0 and D-10 results in P = 1.9E-05 (***).

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