Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging.

Materials DOTAP, DOPC and DOPE-PEG2000 (referred to here as PEG2K-lipid) were purchased as solutions in chloroform from Avanti Polar Lipids (Alabaster, AL). The RGD-PEG2K-lipid contains a GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro-OH) covalently attached to the distal end of the PEG-chain of a custom PEG2000-lipid. It was synthesized via Fmoc solid phase synthesis, employing a lipid-PEG-acid building block in the final coupling step. The chemical structures of the lipids are shown in the Supplementary Material (Fig. S2) . TRITC-DHPE ( N -(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphatidylethanolamine ) was purchased from Invitrogen and has an excitation and emission maximum of 555 nm and 580 nm, respectively. The luciferase plasmid (pGL3) used in transfection experiments was purchased from Promega. The GFP-tubulin (Clontech) and pGL3 plasmids were propagated in E. coli and purified using a Qiagen Plasmid Mega Prep Kit. For live-cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol.

Liposome preparation

Lipid solutions in chloroform/methanol (3:1, v:v; for the RGD-PEG2K-lipid) or chloroform were combined at the desired molar ratio of lipids in glass vials. Lipid molar ratios of DOTAP/DOPC/PEG2K-lipid were 80/20–x/x for high-σ M complexes and 30/70–x/x for low-σ M complexes. To liposomes for live-cell imaging, 0.5 wt% (of total lipid) TRITC-DHPE lipid label were added. After mixing, the lipid solutions in organic solvent were dried, first by a stream of nitrogen and then in a vacuum for 12 h. The appropriate amount of sterile, high resistivity (18.2 MΩ cm) water to achieve a final concentration of 1 mM lipid was then added to the dried lipid films, and the resulting mixtures were incubated at 37°C for 16 h to form liposomes. Following this incubation, the liposome solutions were sonicated using a tip sonicator to form small unilamellar vesicles.

Cell culture and transfection Mouse L-cells

(ATCC number: CCL-1) were cultured in DMEM (Invitrogen) supplemented with 5% fetal bovine serum (HyClone) and 1% Penicillin/Streptomyocin (Invitrogen). Cells were kept at 37 °C in a humidified atmosphere containing 5% CO 2 and were reseeded every 72 h to maintain subconfluency. For transfection studies, cells were seeded in 24 well-plates such that confluency at transfection was 60–80%. CL–DNA complexes were formed by diluting 1 μg of DNA and the appropriate amount of liposome solution to 250 μL each with Optimem (Invitrogen) and mixing. Complexes were incubated for 20 minutes at room temperature before addition to cells. Cells were washed once with PBS and then incubated with 200 μL of complex suspension (0.4 μg of DNA per well) for 6 h. After 6 h, the transfection medium was removed, and cells were rinsed once with PBS and then incubated in supplemented DMEM for 18 h. Cells were harvested in 150 μL of Passive Lysis Buffer (Promega) and subjected to one freeze-thaw cycle. Luciferase expression was measured using a Perkin-Elmer 1420 Victor3 V multilabel counter following the assay manufacturer’s (Promega) instructions. TE results are normalized to total cellular protein as measured by a Bradford Assay (BioRad). Data points represent an average of two measurements with error bars showing the standard deviation. All experiments were performed at least two times to ensure reproducibility. Dynamic light scattering and zeta potential The size and effective charge measurement of CL–DNA complexes and nanoparticles (NPs) was measured using a Malvern Nanosizer ZS ( Fig. 1D, E ) or Brookhaven Goniometer Light Scattering system ( Fig. 2A ). CL-DNA particles were prepared in light-scattering vials at the same concentration used in the transfection assay. A total of 2 μg of DNA and the appropriate amount of liposome (to achieve the desired lipid/DNA charge ratio) were mixed in 1 mL of the appropriate buffer and incubated at room temperature for 20 minutes. Dynamic light scattering was performed in both DMEM and high resistivity water as indicated below. Plots show the z-average diameter. All zeta potential measurements were performed in high resistivity water. All data points for dynamic light scattering and zeta potential are the average of two measurements performed on the same sample. Error bars show the standard deviation.

Cyro-electron microscopy

CL–DNA complexes (lipid molar ratios of DOTAP//DOPC=80//20 and DOTAP//DOPC//PEG2K-lipid=80//15//5) were formed in 50 mM NaCl at a lipid/DNA charge ratio of ρ=10 and a final DNA concentration of 3 mg/mL from a 30 mM liposome stock solution. The sample containing 5 mol% PEG2K-lipid was centrifuged at 5000 rpm for 15 minutes after mixing. Samples were preserved in vitrified ice supported by holey carbon films on 400-mesh copper grids. Each sample was prepared by applying a 3 μL drop of sample suspension to a freshly plasma cleaned grid, blotting with filter paper, and immediately proceeding with vitrification in liquid ethane. Grids were stored under liquid nitrogen until transferred to the electron microscope for imaging. Electron microscopy was performed using an FEI Tecnai F20 electron microscope, operating at 120 keV equipped with a Gatan 4kχ4k CCD camera. Vitreous ice grids were transferred into the electron microscope using a cryo-stage that maintains the grids at a temperature below –170 °C. Images were acquired using the automated acquisition software Leginon [ 39 ] using settings of ~2 μm defocus, electron dose of ~20 e - /Å 2 , and a pixel size at the image of 0.22 nm.

Live cell imaging

Live-cell imaging was performed using dually labeled (see Materials) complexes at ρ=10. The concentration of CL–DNA NPs was the same as that used in the transfection assay. Cells were grown to 60% confluency on poly(L-lysine)-coated coverslips (22 mm) and maintained at 37 °C using a Harvard Warner flow chamber (Harvard Apparatus Model #P2 and RC21-B). Images were taken on a Nikon Diaphot 300 using a Nikon 1.4 NA 60χ Plan Apo DIC Objective and Sensicam QE CCD. Brightfield images were captured at a magnification of 60χ in differential interference contract (DIC) mode. Fluorescent images are composed of two merged channels where one channel shows lipid (TRITC-DHPE) and the other DNA (Cy5). Images were analyzed using a Matlab routine that measured the intracellular spatial distribution of fluorescently labeled DNA. Data points and error bars represent the average and standard deviation of 10 to 20 representative cells. The Matlab routine first locates the cell boundary and nuclear membrane using the DIC image. Next, all intracellular fluorescent particles are located by fitting a 2D Gaussian to all fluorescent spots contained within the cell boundary. Finally, the closest distance to the nuclear membrane is measured and recorded for each intracellular particle.

Materials DOTAP, DOPC and DOPE-PEG2000 (referred to here as PEG2K-lipid) were purchased as solutions in chloroform from Avanti Polar Lipids (Alabaster, AL). The RGD-PEG2K-lipid contains a GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro-OH) covalently attached to the distal end of the PEG-chain of a custom PEG2000-lipid. It was synthesized via Fmoc solid phase synthesis, employing a lipid-PEG-acid building block in the final coupling step. The chemical structures of the lipids are shown in the Supplementary Material (Fig. S2) . TRITC-DHPE ( N -(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphatidylethanolamine ) was purchased from Invitrogen and has an excitation and emission maximum of 555 nm and 580 nm, respectively. The luciferase plasmid (pGL3) used in transfection experiments was purchased from Promega. The GFP-tubulin (Clontech) and pGL3 plasmids were propagated in E. coli and purified using a Qiagen Plasmid Mega Prep Kit. For live-cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol.

Supplementary Material 01