1. Volume 11, Issue 6, Pages 990-995 (June 2005)
A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy 
S. Moein Moghimi, Peter Symonds, J. Clifford Murray, A. Christy Hunter, Grazyna Debska, Adam Szewczyk 
Molecular Therapy 
Volume 11, Issue 6, Pages (June 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Time-dependent lactate dehydrogenase (LDH) release from (A) Jurkat T cells, (B) THLE-3 hepatocytes, and (C) human umbilical vein endothelial cells (HUVECs) following treatment with different concentrations of branched-PEI (25 kDa) in free form and bound to DNA. DNA–PEI complexes were formed in 1.5-ml Eppendorf tubes by adding calf thymus DNA (Sigma, UK), in 10 mM Hepes buffer (pH 7.2), to vortexing PEI (Aldrich, UK) solutions at different weight ratios (1:1 and 1:3) and were left for 15 min at room temperature. Complexes were diluted with filtered Hepes buffer for size and electrophoretic mobility measurements at 25°C by using a Zetasizer 3000 system (Malvern Instruments, UK). The size of all complexes was in the range of 60–90 nm in diameter. Free PEI and PEI–DNA complexes were diluted with serum-free medium and added to 1 × 106 Jurkat T cells (Clone E6-1; American Type Culture Collection, Manassas, VA, USA), THLE-3 hepatocyte cells (American Type Culture Collection), or HUVECs in 1 ml of serum-free RPMI 1640 (Invitrogen, UK). Final concentration of DNA was 10 μg per sample. As a negative control, untreated cells were diluted with the same volume of medium used to prepare the polycations. Cells were also challenged with 10 μg calf thymus DNA in the absence of PEI. Cells were then incubated at 37°C and 5% CO2. Samples were taken at appropriate times and centrifuged at 2000g for 5 min to pellet. The released LDH was measured using Promega's (Southampton, UK) CytoTox 96 Non-Radioactive Cytotoxicity Assay. Samples were read using a plate reader set to 492 nm. Total cytotoxicity was calculated by comparing the levels of released LDH in the experimental samples to total levels of cellular LDH obtained by lysing 1 × 106 corresponding cells with Promega cell lysis buffer. Similar results were also obtained with a linear form of PEI (750 kDa), data not shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
PEI is an apoptotic agent. (A) The effect of branched PEI (25 kDa) on time-dependent PS externalization in Jurkat T cells is shown. Similar results were obtained with linear PEI (750 kDa) and PEI–DNA complexes (supplementary data). Following incubation with PEI and PEI–DNA complexes cells were pelleted and then resuspended in 250 μl of binding buffer (DAKO, Glostrup, Denmark) for double staining with annexin V–FITC and propidium iodide according to the manufacturer's protocol. For a positive control, doxorubicin hydrochloride (50 μg/ml final concentration), a potent proapoptotic agent, was used. Cell-associated fluorescence distributions were obtained from 20,000 events per cell sample through a FL-1 band-pass filter (annexin V–FITC binding) and FL-3 band-pass filter (propidium iodide) using a Becton–Dickinson FACScan. Data analysis was performed with Becton–Dickinson's CellQuest software. (B) The effect of free PEI concentration on the activity of caspase-3 in Jurkat T cells is shown. The Caspase-3 Colorimetric Assay Kit (Sigma, Poole, UK) was used. A specific caspase-3 inhibitor, Ac-DEVD-Chinese hamster ovary (Ac-DEVD-CHO), was included in matched assay samples. Caspase-3 activity was detectable 24 h post-PEI treatment but not in the presence of the inhibitor Ac-DEVD-CHO (+I); doxorubicin (Dox) is the positive control. Similar results were obtained with linear PEI and PEI–DNA complexes and in different primary human cell lines (see supplementary data). (C and D) The effect of PEI on cytochrome c release from isolated mitochondria is shown. Mitochondria were obtained by cell disruption and differential centrifugation in a medium containing 225 mM mannitol, 75 mM sucrose, 3 mM Tris–HCl (pH 7.4), and 0.1 mM EGTA. The final wash was in the same medium but without EGTA. Each measurement was performed at a mitochondrial protein concentration of 1.5 mg/ml. Mitochondrial swelling was monitored continuously as changes in A540 at 25°C; 250 μM Ca2+ was used as the positive control. Oxygen consumption by isolated mitochondria was measured using a Clark-type oxygen electrode (Yellow Spring Instrument Co., Yellow Spring, OH, USA) at 25°C in treatment buffer (125 mM KCl, 25 mM Hepes (pH 7.4), 5 mM NaCl, 1 mM KH2PO4, 1 mM MgCl2, 5 mM malate, 10 mM glutamate, and 0.5 mM Na2EDTA). For uncoupling of oxidative phosphorylation, dinitrophenol was used. The mitochondrial membrane potential was estimated using 8 μM safranine at 25°C in a 3-ml cuvette of a Shimadzu RF 5000 spectrofluorimeter (Tokyo, Japan) in the treatment buffer. (C) Western blot analysis was used to determine cytochrome c release from isolated mitochondria following treatment (10 min) with different quantities of polycations, using established procedures [23]. For positive control, 0.5 mM Ca2+ was used. In (D) the extent of cytochrome c release is compared to the positive control (calcium) and all values are subtracted from the background (Con incubation).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Time-dependent changes in MMP in Jurkat T cells by free and DNA-complexed (at different weight ratios) branched PEI, as determined by the specific MMP fluorescent probe JC-1. Following treatment with PEI and DNA–PEI complexes cells were centrifuged and resuspended in 1 ml of Mitosensor buffer containing 1 μl Mitosensor reagent (BD Biosciences, Oxford, UK). The Mitosensor reagent is a cationic dye (JC-1), which fluoresces differently in apoptotic and nonapoptotic cells. JC-1 forms aggregates in cells with a high FL-2 (585 nm) fluorescence, indicating a normal MMP. Loss of the MMP results in a reduction in FL-2 fluorescence with a concurrent gain in FL-1 (530 nm) fluorescence as the dye shifts from an aggregate to monomeric state. Retention of the dye in cells can be monitored through increase in FL-1 fluorescence. Following incubation at 37°C and 5% CO2 for 20 min cells were then analyzed using Becton–Dickinson FACScan and CellQuest software. Apoptotic change was calculated by comparing changes between control and treated cells in the green FL1 (530 nm) and red/orange FL2 (560–585 nm) fluorescent channels. (A, B, C, D) There is no change at MMP at 1 h, but (E, F, G, H) MMP loss is dramatic at 24 h posttreatment with both free and DNA-complexed PEI (regardless of PEI:DNA ratio). DNA in free form has no effect on MMP.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions