pDNA Complexes

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Bioconjugate Chem. 1999, 10, 406−411

Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes Patrick Midoux* and Michel Monsigny Centre de Biophysique Mole´culaire, Glycobiologie CNRS UPR4301 and University of Orle´ans, rue Charles-Sadron, F-45071 Orle´ans Cedex 02, France. Received September 14, 1998; Revised Manuscript Received December 22, 1998

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ( 5% of the -amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.

INTRODUCTION

Poly-L-lysine, which strongly interacts with DNA, may be used to compact plasmid DNA (pDNA) in order to increase the uptake of foreign genes in mammalian cells with the aim to transfect them. The resulting polyplexess a polyplex is a cationic polymer/DNA complex (1)sare taken up by the cells via either a nonspecific endocytosis or a receptor-mediated endocytosis after polylysine substitution with a recognition signal (for reviews, see refs 2-5). However, one of the main limiting factors for the transfection is the inefficacy of the delivery of plasmids into the cytosol from the endocytotic vesicles. Indeed, upon internalization, most of the plasmid molecules are sequestered for several hours into vesicles from where very few plasmid molecules can escape to reach the nuclear machinery. Polyfectionspolyfection is a polyplexmediated transfectionswas shown to be much more efficient when cells were incubated in the presence of membrane-disrupting agents such as chloroquine (6-8), glycerol (9), or fusogenic peptides which destabilize the membrane of acidic vesicles containing polyplexes (7, 1017). Therefore, the designing of cationic polymers inducing pDNA compaction and membrane disruption in a slightly acidic medium will be good candidates to help the pDNA to enter the cells and to escape the endocytotic pathway leading to the delivery to lysosomes. Knowing that (i) the imidazole group of histidine has a pK around 6.0 and thus becomes cationic in a slightly acidic medium and (ii) poly-L-histidine mediates an acid-dependent fusion and leakage of negative charged liposomes (18, 19), we planned to use a histidylated cationic polymer as a vector for transfecting cells. Because poly-L-histidine is not charged at pH 7.4 and does not form complexes with pDNA at neutral pH, polylysine was partially substituted with histidyl residues. In the present work, we show that (i) histidylated polyL-lysine was suitable to make complexes with pDNA and * To whom correspondence should be addressed. Fax: 33 (0)2 38 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: [email protected].

to efficiently transfect various cell lines in the absence of chloroquine or fusogenic peptides; (ii) the highest gene transfer efficiency was obtained with a polylysine (average degree of polymerization of 190) having 38 ( 5% of the -amino groups substituted with histidyl residues; (iii) the transfection was rather insensitive to the presence of serum in the culture medium; and (iv) the transfection efficiency depended on the protonation of the imidazole groups in the acid lumen of intracellular vesicles. MATERIALS AND METHODS

Preparation of Partially Histidylated Polylysine (His-pLK). Poly-L-lysine and HBr 30000-50000 (pLK,1 DP ) 190) (Bachem Feinchemikalien, Bubendorf, Switzerland) (1 g in 200 mL H2O) were passed through an anion-exchange column (Dowex 2 × 8, -OH form, 20-50 mesh) in order to remove bromide ions (20). The eluate was neutralized with a 10% p-toluenesulfonic acid solution in water and freezed-dried. pLK p-toluenesulfonate salt (50 mg; 0.86 µmol) in 3 mL of dimethyl sulfoxide (Aldrich, Strasbourg, France) in the presence of diisopropylethylamine (42 µL; 288 µmol) (Aldrich) was reacted for 24 h at 20 °C with (Boc)His(Boc)-OH (32 mg; 96 µmol) (Novabiochem, Bad Soden, Germany) in the presence of benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate (Richelieu Biotechnologies, Saint Hyacinthe, Canada) (43 mg; 97 µmol). The N-protecting Boc groups were removed by acidic treatment by adding 20 mL of H2O/trifluoroacetic acid mixture (1:1; v/v) for 24 h at 20 °C. Water and trifluoroacetic acid were removed under reduced pressure. The polymer was precipitated by adding 10 vol of 2-propanol and spun down by centrifugation (1800g for 15 min). The pellet was washed with 2-propanol, collected by centrifugation (1800g for 15 min), solubilized in distilled water, and freeze-dried. The average number of His molecules bound 1 Abbreviations: DP, average degree of polymerization; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; FBS, fetal bovine serum; His, histidyl residue; PBS, phosphate-buffered saline; pLK, poly-L-lysine.

10.1021/bc9801070 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/11/1999

Histidylated Polylysine: A Novel DNA Carrier Polymer

Figure 1. Schematic structure of histidylated polylysine. i ) DP; R ) NH3+ or His.

per pLK molecule was determined by 1H NMR spectroscopy at 300 MHz in D2O according to x ) 6(h8.7/hLys)DP, where h8.7 was the value of the integration of the signal at 8.7 ppm corresponding to the proton (1H C12) of His (Figure 1), hLys was in the range 1.3-1.9 ppm corresponding to the 6 methylene protons (C3, C4 and C5) of lysine residues (Figure 1), and DP was the degree of polymerization of pLK. The number of His residues bound per pLK molecule was 70. Histidylated polylysine was fluoresceinylated by using fluoresceinyl isothiocyanate (FITC isomer I, Molecular Probe, La Jolla, CA) as previously described (7). Cells and Cell Culture. COS-7 (SV40 transformed kidney cells of African green monkey, ATCC CRL 1651, ATCC, Rockville, MA), 293-T7 (human embryonic kidney producing T7 RNA polymerase) (21), and Rb1 (rabbit smooth muscle cells) (22) cells were cultured in DMEM (Gibco, Renfrewshire, U.K.) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco); HeLa cells (ATCC CCL 2.1) in DMEM with 5% FBS; B16 cells (murine melanoma cells, ATCC CRL 6322) in DMEM with 10% heat-inactivated new borne calf serum (Gibco); HepG2 (human hepatoma cells, ATCC HB 8065), MCF-7 cells (human breast adenocarcinoma cells, ATCC HTB22), and HOS cells (human osteosarcoma cells, ATCC CRL 1543) in MEM (Gibco) with 10% heat inactivated FBS; 16HBE cells (human airway epithelial cells) in DMEM with 10% heat-inactivated FBS in fibronectin-coated culture plates; A549 cells (human nonsmall cell lung carcinoma cells, ATCC CCL 185) in RPMI with 10% heatinactivated FBS. All culture media were supplemented with 2 mM L-glutamine (Merck, Darmstadt, Germany) and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin, Eurobio, France). Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Cells were harvested by treatment with PET [PBS with 0.02% (w/v) EDTA and 2.5 µg/mL trypsin] at 37 °C for 5 min. Cells were mycoplasma free as evidenced by bisbenzimidazole (Hoechst 33258) (23). Plasmids. pSV2LUC plasmid (5 kb) was an expression vector encoding the firefly luciferase gene under the control of the SV40 T large antigen promoter (24). pUT650 plasmid (5.15 kb) (CAYLA, Toulouse, France) was an expression vector encoding the firefly luciferase gene under the control of the human cytomegalovirus promoter. Supercoiled DNA plasmids were isolated by a standard alkaline lysis method followed by CsCl gradient centrifugation in the presence of ethidium bromide, extensive extraction with n-butanol, and precipitation with ethanol. Polyfection. Polyplexes (histidylated polylysine or polylysine/pDNA complexes) were prepared by adding, under agitation, polymers in 0.3 mL of serum-free DMEM, pH 7.4 to 5 µg (1.5 pmol) of plasmid in 0.7 mL of serum-free DMEM. The mixed solution was kept for 30 min at 20 °C before use. Adherent cell lines [(2-4) × 105 cells] were plated (day 0) into 4 cm2 culture dishes (12-

Bioconjugate Chem., Vol. 10, No. 3, 1999 407

well culture plates). On day 1, the medium was removed and 1 mL of a solution containing a polyplex supplemented with 1% FBS, unless otherwise specified, was added into each well. After 4 h of incubation at 37 °C in a humidified atmosphere (95% air, 5% CO2), the medium was removed and cells were further incubated at 37 °C in 2 mL of the relevant complete culture medium in a humidified atmosphere (95% air, 5% CO2). When used, chloroquine (100 µM) was added to the transfection solution, which was immediately supplemented with 1% FBS and put into each well. When used, the fusogenic peptide (E5CA, GLFEAIAEFIGGWEGLIEGCA) (7) (10 µM) was added and the transfection solution was immediately supplemented with 1% FBS and put into each well. Polyethylenimine (PEI 25 kDa from Aldrich) (4.5 mg) was dissolved in 10 mL of H2O, neutralized with HCl, and sterilized by filtration. Polyplexes were prepared by mixing 10 µL of PEI in 50 µL of 0.15 M NaCl with 5 µg (1.5 pmol) plasmid in 50 µL 0.15 M NaCl. The mixed solution was kept for 30 min at 20 °C. Then, the solution supplemented with 1 mL of DMEM containing 10% FBS was added into each well. After 4 h of incubation at 37 °C in a humidified atmosphere (95% air, 5% CO2), the medium was removed and cells were further incubated at 37 °C in 2 mL of the relevant complete culture medium in a humidified atmosphere (95% air, 5% CO2). Luciferase Assay. Luciferase gene expression was measured by monitoring its luminescence activity according to De Wet et al. (25). The medium was discarded, and the cells were washed three times with PBS. The homogenization buffer (200 µL of 8 mM MgCl2, 1 mM DTT, 1 mM EDTA, 1% Triton X100, 15% glycerol, 25 mM Tris-phosphate buffer, pH 7.8) was poured into each well, and the tissue culture plates were kept for 15 min at 20 °C. The solution was recovered and further spun down (5 min at 800g). A total of 95 µL of a 2 mM ATP solution in the homogenization buffer without Triton X100 was added to 60 µL of supernatant, and the solution was shaken with a vortex. The luminescence was recorded for 4 s in a Lumat LB 9501 luminometer (Berthold, Wildbach, Germany) upon addition of 150 µL of a 167 mM luciferin solution in water. Measurements were done in duplicate. The data shown correspond to the number of relative light units (RLU) from 106 cells. The number of RLU of 1 pg/mL of luciferase was 2000 under these assay conditions. ζ Potential Measurement. Polyplexes were prepared in 10 mM NaCl, 1 mM MOPS, and 0.1 mM EDTA, at pH 7.2 to a pDNA final concentration of 10 µg/mL. ζ potential was measured by using a ZetaSizer 3000 (Malvern Instruments, Orsay, France) with the following parameters: viscosity, 1.014 cP; dielectric constant, 79; temp, 25 °C; F(Ka), 1.50 (Smoluchowsky); current, 10 mA. RESULTS

Polyfection by Using Histidylated Polylysine. Histidylated polylysine containing 84 histidyl residues (His84-pLK) (Figure 1) was complexed in 1 mL of serumfree DMEM at pH 7.4 with pUT650 plasmid encoding the luciferase gene, and the polyplexes were used to transfect HepG2 cells. The luciferase activity in the cell lysates was compared to that obtained after polyfection with pLK/pUT650 complexes in the absence and in the presence of either chloroquine or E5CA, a fusogenic peptide. Regarding to the luciferase activity, the polyfection was very efficient with His84-pLK/pDNA complexes and very inefficient with pLK/pDNA complexes. The luciferase activity was 4.5 orders of magnitude (107

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Figure 2. Histidylated polylysine mediated polyfection. Polyplexes were prepared by mixing either His84-pLK (40 µg) or pLK (5 µg) in 0.3 mL of serum-free DMEM with 10 µg (3 pmol) of pUT650 plasmid in 0.7 mL of serum-free DMEM. The solution was kept for 30 min at 20 °C. When it is relevant the solution was made 100 µM in chloroquine or 10 µM in E5CA. HepG2 cells (3 × 105 cells plated in a 4 cm2 well) were incubated for 4 h at 37 °C with polyplexes in the presence of 5% FBS. Then cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells. (Inset) Concentration dependence of polyfection with His84-pLK/pDNA complexes.

RLU/106 cells) higher than with pLK/pDNA complexes (200 RLU/106 cells); it was 3.5 (2 × 107 RLU/106 cells) and 3 (107 RLU/106 cells) orders of magnitude higher than with pLK/pDNA complexes in the presence of chloroquine and the fusogenic peptide, respectively (Figure 2). The luciferase activity was maximal when the cells were incubated for about 3-4 h in the presence of His84-pLK/ pDNA complexes (data not shown); increasing the incubation period (up to 24 h) did not increase the luciferase activity. It is noteworthy to emphasize that no cytoxicity was observed whatever the incubation duration (from 4 to 24 h) in the presence of the polyplexes. The amount of plasmid was varied between 0.05 and 10 µg, keeping constant the histidylated polylysine/pDNA ratio equal to 4:1 (weight by weight), the number of cells per well [(23) × 105 cells in a 4 cm2 well] and the volume (1 mL) of the solution containing the polyplexes. The polyfection was maximal in the presence of 5 µg of plasmid. Increasing the amount of plasmid did not increase the luciferase activity (Figure 2 inset). Increasing the number of cells per well did not increase the luciferase activity. Optimal Number of Histidyl Residues on One Polylysine Molecule. HepG2 cells were transfected by using polyplexes made with pUT650 and polylysine substituted with different number of histidyl residues. The luciferase activity in cell lysates was maximal with polylysine substituted with 72 ( 9 histidyl residues/pLK molecule corresponding to 38 ( 5% substitution of the polylysine -amino groups (Figure 3). When polyfection was performed with pLK containing 20 histidyl residues, the luciferase activity was 10-fold lower than with pLK substituted with 70 histidyl residues.

Midoux and Monsigny

Figure 3. Histidylated polylysine mediated polyfection:influence of the His/pLK ratio. Polyplexes were prepared by mixing pLK substituted with histidyl residues in 0.3 mL of serum-free DMEM with 10 µg (3 pmol) of pUT650 plasmid in 0.7 mL of serum-free DMEM. The solution was kept for 30 min at 20 °C. HepG2 cells (3 × 105 cells plated in a 4 cm2 well) were incubated for 4 h at 37 °C with polyplexes in the presence of 1% FBS. Then, cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells.

Influence of the Histidylated Polylysine/pDNA Molar Ratio. The luciferase activity was maximal with polyplexes made by complexing 10 µg of plasmid with 30 (polymer/pDNA molar ratio of 183) to 40 µg (polymer/ pDNA molar ratio of 244) of His84-pLK (Figure 4). Increasing the molar ratio did not increase the luciferase activity. The efficiency decreased 10-fold when the molar ratio was down to 122 (20 µg of His84-pLK) and was completely inefficient when the molar ratio was lower than 60 (10 µg of His84-pLK). Under conditions optimal for transfection, polyplexes were positively charged with a ζ potential of +24 mV (Figure 4). Influence of the Serum Concentration. Polyfection with His70-pLK was not impaired in the presence of serum. The luciferase activity was 2-fold greater in the presence of 5 and 10% FBS than in the presence of 1% FBS, and it was as large in the presence of 20% FBS as in the presence of 1% FBS (Figure 5). Gene Transfer into Various Cell Lines. The transfection of different mammalian cell lines including human hepatoma cells (HepG2), human breast adenocarcinoma cells (MCF-7), human epidermoid carcinoma cells (HeLa), human osteosarcoma cells (HOS), human airway epithelial cells (16HBE), murine melanoma cells (B16), and simian kidney cells (COS) was assessed by using His70-pLK/pUT650 complexes and rabbit smooth muscle cells (Rb-1) by using His70-pLK/pSV2LUC complexes. All the cell lines (except COS cells) were efficiently transfected with luciferase activity ranging from 2 × 106 to 107 RLU/106 cells (Figure 6). The transfection was compared with that obtained with PEI/pDNA complexes (Table 1). Depending on the cell line, the transfection by His-pLK/pDNA complexes was as effective or less effective (up to 20-fold) than with PEI/pDNA complexes.

Histidylated Polylysine: A Novel DNA Carrier Polymer

Figure 4. Histidylated polylysine mediated polyfection:influence of the polymer/pDNA ratio and ζ potentials of polyplexes. Polyplexes were prepared by mixing 10 µg (3 pmol) of pUT650 plasmid in 0.7 mL of serum-free DMEM with different amounts of histidylated polylysine (His84-pLK) in 0.3 mL of serum-free DMEM. The solution was kept for 30 min at 20 °C. HOS cells (2 × 105 cells/4 cm2) were incubated for 4 h at 37 °C with polyplexes in the presence of 5% FBS. Then cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells.

Bioconjugate Chem., Vol. 10, No. 3, 1999 409

Figure 6. Histidylated polylysine mediated polyfection of various cell lines. Polyplexes were prepared by mixing His70pLK (30 µg) in 0.3 mL serum-free DMEM with 10 µg (3 pmol) of plasmid (pUT650 or pSV2LUC for Rb-1 cells) in 0.7 mL of serum-free DMEM. The mixed solution was kept for 30 min at 20 °C. Cell lines [(2-3) × 105 cells/4 cm2 well] were incubated for 4 h at 37 °C with polyplexes in the presence of 10% FBS. Then, cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells. Table 1. Comparison of the Transfection Efficiency by Using Either Histidylated Polylysine or Polyethylenimine as DNA Carriersa cells

PEI/His-pLK

cells

PEI/His-pLK

B16 293

5.8 0.6

A549 Rb-1

21 17

a Cells (2 × 105 cells/4 cm2 well) were transfected for 4 h at 37 °C with 5 µg/mL (pUT650 or pSV2LUC for Rb-1 cells) complexed with either His-pLK or PEI in the presence of 10% FBS. Then, cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. PEI/HispLK represents the ratio of the luciferase activity into cells transfected by PEI polyplexes to the luciferase activity into cells transfected by His-pLK polyplexes.

Figure 5. Histidylated polylysine mediated polyfection:influence of the serum. HepG2 cells (3 × 105 cells/4 cm2 well) were incubated for 4 h at 37 °C with 5 µg/mL of the polyplexes (His70pLK/pUT650) in the presence of various amount of FBS. Then, cells were washed and incubated in complete culture medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells.

Influence of the Endosomal pH. Histidylated polylysine is supposed to increase the delivery of pDNA into the cytosol via membrane destabilization of acidic endocytotic vesicles containing His70-pLK/pDNA complexes following the protonation of the imidazole groups. This was assessed by transfecting cells in the absence or in the presence of bafilomycin A1, an inhibitor of vacuolar ATPase endosomal proton pump (26). When the polyfection of Rb-1 cells by His70-pLK/pSV2LUC complexes was

performed in the presence of 200 nM bafilomycin A1, the luciferase activity into cells was about 3 order of magnitude lower than in the absence of bafilomycin A1 (Figure 7). A flow cytometry analysis showed that, in the absence of bafilomycin A1 , the polyplexes were in acidic compartment as evidenced by a 1.8-fold increase of the cell fluorescence intensity after a postincubation in the presence of monensin (27, 28); conversely, the fluorescence intensity of cells incubated in the presence of bafilomycin A1 did not increase upon a postincubation in the presence of monensin (data not shown). Therefore, the decrease of the transfection efficiency of histidylated polylysine/pDNA complexes in the presence of bafilomycin A1 , indicated that the protonation of the imidazole groups in acidic medium was involved in the polyfection process. DISCUSSION

The introduction in mammalian cells of a foreign gene as a plasmid DNA by nonviral vectors requires the entry of the plasmid into the cell and then into the nucleus.

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Figure 7. Influence of bafilomycin A1 on the polyfection efficiency. Rb-1 cells (2 × 105 cells/4 cm2 well) were incubated at 37 °C in the presence of 1 mL His70-pLK/pSV2LUC complexes (5 µg of pDNA) and in the absence or in the presence of 200 nM bafilomycin A1. After 4 h, the cells were washed and incubated in complete culture medium containing 10%. The gene expression was determined 48 h later by assaying the luciferase activity in cell lysates. RLU, the number of relative light units, represents the luciferase activity in 106 cells.

The compaction of a pDNA in the presence of polylysine increases its uptake by the cells via making the endocytotic process more easy. Once complexed and taken up by cells, plasmids are sequestered for several hours in vesicular compartments from where they cannot escape to reach the nucleus. The delivery of the pDNA into the cytosol should be obtained from early acidic vesicles formed during polyplex uptake under the condition that the endosomal membrane is disrupted by devices bound to the plasmid-associated carrier working at a weakly acidic medium. In this study, we show that partial substitution of poly-L-lysine with histidyl residues results in a cationic polymer that forms complexes with a plasmid DNA at pH 7.4. These complexes actively transfect cells in culture in the absence of any helper compounds such as chloroquine or a fusogenic peptide. Optimal conditions for transfection were found when polylysine was substituted with 72 ( 9 histidyl residues, corresponding to a polylysine substitution level of 38 ( 5%; the efficiency was 10-fold lower when polylysine substitution was down to 10%. The polyfection was efficient when the global charge of the polyplexes was slightly positive (ζ potential ) +24 mV), which likely increased their binding on the cell surface and facilitated their uptake by the cells. The polyplexes are internalized by the cells in acidic compartments. The mechanism of polyfection mediated by histidylated polyplexes is not yet known, but it is supposed that a subsequent delivery of pDNA into the cytosol occurs by the membrane destabilization of acidic vesicles containing polyplexes, upon the protonation of imidazole groups. Although, there is no evidence that the DNA leaves endocytotic vesicles, this statement is supported by (i) the fact that poly-L-histidine induces fusion of lipid bilayers (18, 19) and (ii) the fact that bafilomycin A1 which is known to impair the acidification of the lumen of the endosomes, made polyfection inefficient. Poly-L-histidine destabilizes lipid bilayers in a slightly acidic medium and induces fusion upon protonation of the imidazole groups by increasing interactions between

Midoux and Monsigny

this polymeric polycation and the membrane phospholipids (18). In acidic medium, poly-L-histidine is more fusogenic than polylysine: efficient fusion of negatively charged liposomes (phosphatidylserine liposomes) occurs at a ratio of the positive charge of poly-L-histidine to the negative charge of the liposomes equal to 0.2 while it occurs at a ratio equal to 1 in the case polylysine. Recently, we have reported that the amphipatic peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) containing five histidyl residues, which does not adopt a helical structure in acidic medium, permeabilizes cell membranes in such an acidic medium (pH 6.4) (17). In addition, this peptide increased the expression efficiency when a gene is transferred into cells as glycosylated polylysine/pDNA complexes; the transfection was also inhibited in the presence of bafilomycin A1 (17). These results suggest that the generation of cationic charges via the protonation of several imidazole groups bound to pDNA inside the endosomes consecutively to the acidification of their lumen is a suitable approach to modify the intracellular routing of pDNA, especially its delivery into cytosol. Therefore, the mechanism involved for polyfection with histidylated polylysine could be different from that of polyethylenimine which is supposed to act as a buffer, preventing acidification of the endosomal lumen and inducing the swelling of these vesicles, leading to a membrane destabilization (29). The transfection efficiency by using histidylated polylysine/pDNA complexes was greater than by using polylysine/pDNA complexes in the presence of either chloroquine or the fusogenic peptide E5CA. Chloroquine concentrates in the acidic compartments and prevents the delivery of the endosomal content to lysosomes; it induces the formation of large endosome-derived vacuoles. In addition, it allows the dissociation of pDNA/ polylysine conjugate complexes (8). Anionic permeabilizing peptides, adopting an R-helical structure at pH