Dope peg2000

DOTAP, DOPC and DOPE-PEG2000 were purchased from Avanti Polar lipids as chloroform solutions. MVL5 was synthesized as described previously [39 (link)]. The fluorescent lipids TRITC-DHPE and Texas Red-DHPE (Invitrogen, Carlsbad, California) have excitation and emission maxima of 555/589 nm and 580/615 nm, respectively. The RGD-PEG2K-lipid contained a GRGDSP sequence covalently bound to the distal end of PEG2K. It was custom synthesized on solid phase using Fmoc-amino acids and a lipid-PEG2K acid. The pGL3-control vector coding the Luciferase gene (Promega, Fitchburg, Wisconsin) was propagated in E. coli, and purified using a Qiagen Plasmid Mega Prep Kit. The GFP-Rab5-Q79L plasmid was a gift from the Weimbs lab (UCSB) and propagated and purified as decribed above for pGL3. For 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. For labeling of early endosomes, the CellLights Early Endosome-GFP BacMam 2.0 (Life Technologies, Carlsbad, California) reagent was used according to manufacturer’s protocol.

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.

The lipids used to prepare the investigated NPs are DOTAP, DOPC and DOPE-PEG2000 (referred to here as PEG2K-lipid), which were purchased as chloroform solutions from Avanti Polar Lipids (Alabaster, AL). For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). Four distinct types of DNA were used to form nanoparticles; UltraPure Salmon Sperm DNA Solution (S-DNA) (Invitrogen (Carlsbad, CA)), Lambda Phage DNA (λ-DNA) (Thermo Scientific (Waltham, MA)), pGL3 Luciferase Reporter plasmid DNA (pGL3) (Promega (Fitchburg, Wisconsin)), which was propagated via Qiagen Plasmid Plus Mega Kit (Venlo, Limburg) and 11 bp DNA (purchased as single strands from Sigma-Genosys (Sigma-Aldrich (St. Louis, MI) and delivered as a lyophilized film). Complementary single strands were mixed at an equimolar ratio, diluted to a final concentration of 10 mg/mL, heated in a water bath and held at 90 °C for 15 min and slowly cooled to room temperature to allow complete hybridization. For fluorescence studies, DNA was labeled using YOYO-1 (Invitrogen (Carlsbad, CA)) according to the manufacturer’s protocol.

DOPC, DOPE-PEG₂₀₀₀, and NBD-PE lipids were purchased from Avanti Polar Lipids (AL, USA). All lipids were used without further purification. A stock solution of lipids—either DOPC or 92 mol% DOPC + 8 mol% DOPE-PEG₂₀₀₀—dissolved in chloroform was dried under a stream of nitrogen in a glass vial and then vacuum-dried overnight. Milli-Q water was added to the glass vial to achieve the required lipid concentration (62.5 mM) and the vial was vortexed. After at least 30 min, the solution was extruded 11 times with a mini-extruder using a polycarbonate membrane of 200-nm pore size. For experiments with confocal microscopy, 0.2 mol% of NBD-PE was also mixed in before drying the chloroform. The concentration of lipids in the final liposome solutions used was 12.5 mM.