Supplementary figure 1. Characterization of the ability of pegylated peptides to form complexes with pDNA at CR1. (A) Ability of pegylated peptides to form complexes at increasing range of pegylation rate was assessed with gel shift assay. (B) The ability of pegylated peptides to condense pDNA at different pegylation rates was evaluated using EtBr exclusion assay. Pegylation rate 0% represents PF14/pDNA complexes formed at CR1. Results are represented as relative fluorescence, where 100% is naked pDNA fluorescence
Supplementary figure 2. Characterization of the ability of pegylated peptides to form complexes with pDNA at CR3. (A) Ability of pegylated peptides to form complexes at increasing range of pegylation rate was assessed with gel shift assay. (B) The ability of pegylated peptides to condense pDNA at different pegylation rates was evaluated using EtBr exclusion assay. Pegylation rate 0% represents PF14/pDNA complexes formed at CR3. Results are represented as relative fluorescence, where 100% is naked pDNA fluorescence
Supplementary figure 3. Characterization of the ability of pegylated peptides to form complexes with pDNA at CR4. (A) Ability of pegylated peptides to form complexes at increasing range of pegylation rate was assessed with gel shift assay. (B) The ability of pegylated peptides to condense pDNA at different pegylation rates was evaluated using EtBr exclusion assay. Pegylation rate 0% represents PF14/pDNA complexes formed at CR4. Results are represented as relative fluorescence, where 100% is naked pDNA fluorescence. Heparin displacement assay was used to analyze the stability and dissociation profile of peptide/pDNA complexes made with (C) PF14 and pegylated peptides at pegylation rate 40% or (D) PF14, PF141, PF142, PF143 and pDNA complexes. For that a different concentrations of heparin was added to preformed complexes and incubated 30 min at 37°C. Thereafter heparin-treated complexes were analyzed by EtBr exclusion assay. Results are represented as relative fluorescence, where 100% is naked pDNA fluorescence.
Supplementary figure 4. The stability of peptide/pDNA complexes in the presence of serum components. Preformed complexes at CR2 with PF14 or pegylated peptides (PF141, PF142 and PF143) were incubated with 10% serum (lanes with +) for 2 hours (A) or 4 hours (B) at 37°C. Lanes with (-) represent complexes not treated with serum. After incubation with serum, all complexes and naked pDNA were treated with proteinase K (1mg/ml) for 30 min at 37°C and thereafter analyzed by gel shift assay. Lanes named by – or + correspond to untreated or serum treated complexes. The pegylation rate of complexes were 10% and 20% . To evaluate the effect of serum treatment to transfection efficiency, preformed complexes (CR2, pegylation rate 10% and 20%) were incubated with serum for 2 hours (C) and 4 hours (D). After incubation with serum peptide/pLuc2 complexes were used to transfect CHO cells and after 24 hours luciferase activity was measured and normalized against protein content.
Supplementary figure 5. Characterization of the ability of PF14 and pegylated peptides to form complexes with Cy5 labeled pDNA. Heparin displacement assay was used to evaluate if complexes made with fluorescently labeled pDNA had similar stability as complexes made with pDNA without label. For that preformed complexes (CR2, pegylation rate 40%) with pDNA (A) and fluorescently labeled pDNA (B) was mixed with different concentrations of heparin and incubated 30 min at 37°C. Thereafter heparin-treated complexes were analyzed by EtBr exclusion assay. Results are represented as relative fluorescence, where 100% is naked pDNA fluorescence. (C) The ability of peptides to condense Cy5-labeled pDNA as efficiently as pDNA without label was analyzed with EtBr exclusion assay. For that pDNA or Cy5-pDNA was mixed with peptides at CR2 and pegylation rate 40% was used for pegylated complexes. Fluorescence of complexes was measured and results are represented as relative fluorescence, where 100% corresponds to naked pDNA fluorescence.
Supplementary figure 6. Gene induction by pegylated complexes in time. To assess gene expression profile induced by pegylated peptide/pDNA complexes in time, 20 µg of pLuc2 was mixed with PF142 at pegylation rate 10%, at CR4 and administered i.v. via tail vein. Tissues were harvested after 24 hours (A) and 48 hours (B), thereafter luciferase activity was measured and normalized against protein content. Data is represented at fold increase over pDNA treated group.
Supplementary figure 7. Cleavage assay of the MMP2-cleavable and non-cleavable peptides. The peptides were treated with active human MMP2 (30 ng/μL), at 37°C for 40 minutes. The reaction was followed using RP-UPLC (C18 reverse-phase column, eluted with 20–100% acetonitrile in water with 0.1% TFA). Figure shows the RP-UPLC chromatograms of the MMP2cleavable and non-cleavable peptides with and without MMP2 incubation. The cleavage products were confirmed by MALDI-TOF mass spectroscopy. The mass spectrum indicated that the peptide was cut between glycine and leucine residues of the MMP2 cleavage site.
Supplementary figure 8. Toxicity profile and biological activity of pegylated peptide/pDNA complexes. (A) Toxicity profile of PF141, PF142 and PF143 complexes were evaluated in CHO cells using MTS assay. Briefly, 24 hours before experiment 1×104 CHO cell were seeded in 96-well plates. Cells were treated with complexes formed at CR2 and at different pegylation rate, ranging from 10-100%. 24 hours later MTS reagent was added and after 1 hour incubation absorbance was measured. Viability of treated cells is represented as percentage of untreated cells. (B) Toxicity profile of PF144 and PF147 complexes were evaluated in U87 cells using MTS assay. Cells were treated with complexes which were formed at CR2, at 100% pegylation rate and preincubated with MMP2 enzyme. 24 hours later MTS was added and absorbance was measured. Viability of treated cells is represented as percentage of pDNA treated cells. In order to evaluate transfection ability of pegylated MMP2 sensitive peptide/pDNA complexes, 3×104 Neuro2a cells or 3×104 or 5×104 U87 cells were seeded 24 hours before experiment into 24 well plates. Cells were treated with PF144 or PF147 and pLuc2 complexes (C) or different percentages of PF145 (D) and PF146 (E) and pLuc2 complexes at CR2 for 4 h in serum containing media followed by addition of 1 ml 10% serum containing medium and incubated for another 20 hours. To study the effect of MMP2 cleavage upon transfection efficiency, peptide/pDNA complexes were pre-incubated with active recombinant MMP2 (0.1 mg/ml) for 30 min at 37 °C. Thereafter, cells were lysed and luciferase activity was measured and analyzed as described earlier.
Supplementary table 1. Average size of pegylated peptide/pDNA complexes at CR4.
Supplementary table 2. Average surface charge of pegylated peptide/pDNA complexes at CR4.