Chloride Accumulation and Swelling in Endosomes Enhances DNA Transfer by Polyamine-DNA Polyplexes

Abstract

The “proton sponge hypothesis” postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mm over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mm at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mm versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery. The “proton sponge hypothesis” postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mm over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mm at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mm versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery. Although gene delivery using non-viral vectors offers potential advantages over virus-based delivery systems, the relatively low transfection efficiency of non-viral vectors has been their major limitation for in vivo applications (1Midoux P. Monsigny M. Bioconjugate Chem. 1999; 10: 406-411Crossref PubMed Scopus (483) Google Scholar, 2Pack D.W. Putnam D. Langer R. Biotechnol. Bioeng. Symp. 2000; 67: 217-223Crossref Scopus (259) Google Scholar, 3Putnam D. Gentry C.A. Pack D.W. Langer R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1200-1205Crossref PubMed Scopus (466) Google Scholar, 4Abdallah B. Sachs L. Demeneix B.A. Biol. Cell. 1995; 85: 1-7Crossref PubMed Google Scholar). The archetypal non-viral gene delivery system is the cationic polymer-DNA complex (polyplex), 1The abbreviations used are: polyplex, cationic polymer-DNA complex; POL, polylysine; PAM, polyamidoamine; PEI, polyethylenimine; PBS, phosphate-buffered saline; TMR, tetramethylrhodamine; TMR-POL, TMR-labeled polylysine; TMR-PAM, TMR-labeled PAM; TMR-PEI, TMR-labeled PEI; BAC, 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate; BAC-dextran-S-S-2Py, BAC-dextran carrying dithian-2-pyridyl linker; FITC, fluorescein isothiocyanate; FITC-PAM-TMR, PAM labeled with FITC and TMR; FITC-PEI-TMR, PEI labeled with FITC and TMR; FITC-POL-TMR, POL labeled with FITC and TMR; N/P, amine-to-phosphate; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BAC-dextran-PAM-TMR, tetramethylrhodamine-labeled polyamidoamine conjugated to BAC-labeled dextran; BAC-dextran-PEI-TMR, tetramethylrhodamine-labeled polyethylenimine conjugated with BAC-labeled dextran; BAC-dextran-POL-TMR, tetramethylrhodamine-labeled polylysine conjugated with BAC-labeled dextran.1The abbreviations used are: polyplex, cationic polymer-DNA complex; POL, polylysine; PAM, polyamidoamine; PEI, polyethylenimine; PBS, phosphate-buffered saline; TMR, tetramethylrhodamine; TMR-POL, TMR-labeled polylysine; TMR-PAM, TMR-labeled PAM; TMR-PEI, TMR-labeled PEI; BAC, 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate; BAC-dextran-S-S-2Py, BAC-dextran carrying dithian-2-pyridyl linker; FITC, fluorescein isothiocyanate; FITC-PAM-TMR, PAM labeled with FITC and TMR; FITC-PEI-TMR, PEI labeled with FITC and TMR; FITC-POL-TMR, POL labeled with FITC and TMR; N/P, amine-to-phosphate; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BAC-dextran-PAM-TMR, tetramethylrhodamine-labeled polyamidoamine conjugated to BAC-labeled dextran; BAC-dextran-PEI-TMR, tetramethylrhodamine-labeled polyethylenimine conjugated with BAC-labeled dextran; BAC-dextran-POL-TMR, tetramethylrhodamine-labeled polylysine conjugated with BAC-labeled dextran. in which plasmid DNA and a cationic carrier are condensed into a tight complex suitable for cellular internalization by endocytosis (5Ogris M. Wagner E. Drug Discov. Today. 2002; 7: 479-485Crossref PubMed Scopus (153) Google Scholar, 6Thomas M. Klibanov A.M. Appl. Microbiol. Biotechnol. 2003; 62: 27-34Crossref PubMed Scopus (439) Google Scholar). Transgene delivery to the nucleus is thought to require escape of the polyplex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (5Ogris M. Wagner E. Drug Discov. Today. 2002; 7: 479-485Crossref PubMed Scopus (153) Google Scholar, 7Xu Y. Szoka F.C. Biochemistry. 1996; 35: 5616-5623Crossref PubMed Scopus (1068) Google Scholar, 8Zuber G. Dauty E. Nothisen M. Belguise P. Behr J.P. Adv. Drug Deliv. Rev. 2001; 52: 245-253Crossref PubMed Scopus (201) Google Scholar). The low efficiency of polyplex escape from endosomes is thought to be an important determinant of the overall efficiency of non-viral gene transfer. Polyamines are useful polycationic macromolecules for non-viral gene transfer because of their high density of positive charges, ease of synthesis, and efficient polyplex formation (8Zuber G. Dauty E. Nothisen M. Belguise P. Behr J.P. Adv. Drug Deliv. Rev. 2001; 52: 245-253Crossref PubMed Scopus (201) Google Scholar, 9Rolland A. Felgner P.L. Adv. Drug Deliv. Rev. 1998; 30: 1-3Crossref PubMed Scopus (24) Google Scholar, 10Luo D. Saltzman W.M. Nat. Biotechnol. 2000; 18: 33-37Crossref PubMed Scopus (370) Google Scholar). Cationic polyamines with fixed, non-titratable charges such as polylysine (POL) are substantially less efficient at gene transfer than polyamines with titratable amines such as polyamidoamine (PAM) (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar) and polyethylenimine (PEI) (12Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar, 13Kichler A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar). The lower efficiency is not caused by differences in morphology of the complexes or cell association (14Tang M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). To explain this observation it has been postulated without direct evidence that the high H+ buffer capacity of polyamines containing titratable amines results in endosomal Cl- accumulation during acidification with presumed osmotic endosome swelling and enhanced polyplex escape (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar, 15Behr J.P. Bioconjugate Chem. 1994; 5: 382-389Crossref PubMed Scopus (395) Google Scholar). This “proton sponge hypothesis” if correct, would have important consequences in defining the barriers to non-viral gene delivery and in the design of improved non-viral vectors. Here, we test the proton sponge hypothesis by comparing the kinetics of acidification, Cl- accumulation, and swelling in endosomes containing polyplexes composed of DNA and fluorescent probe-labeled polyamines (POL, PEI, or PAM). We previously developed long-wavelength, Cl--sensing fluorescent probes for the measurement of [Cl-] in cellular endosomal and Golgi compartments and showed that inward H+ pumping by the bafilomycin-sensitive vacuolar ATPase was coupled quantitatively to Cl- entry in endosomes produced by fluid-phase (16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) or receptor-mediated (17Sonawane N.D. Verkman A.S. J. Cell Biol. 2003; 160: 1129-1138Crossref PubMed Scopus (63) Google Scholar) endocytosis. We find here that “macropinocytic” endosomes containing DNA polyplexes with titratable amines have remarkably increased buffer capacity, reduced acidification, increased Cl- accumulation, and increased swelling/lysis compared with endosomes containing polyplexes with fixed charges, providing direct evidence in support of the proton sponge hypothesis. Cell Culture—Chinese hamster ovary (CHO)-K1 cells (American Type Culture Collection, Manassas, VA) were grown in Ham's F12K medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured on 18-mm diameter round coverglasses at 37 °C in 95% air/5% CO2 and used just prior to confluence. Fluorescently Labeled Polyamine Conjugates for [Cl-] Measurements—The polymers POL (Aldrich, 25 kDa), PAM (43 kDa, 6th generation fractured dendrimer) (18Tang M. Redemann C.T. Szoka Jr., F.C. Bioconjugate Chem. 1996; 7: 703-714Crossref PubMed Scopus (855) Google Scholar), and PEI (Aldrich, 25 kDa) were purified and characterized as described previously (14Tang M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). An aqueous solution of polyamine (POL, PAM, or PEI) (5 μmol in 5 ml of PBS) was stirred slowly with tetramethylrhodamine succinimidyl ester (15 μmol, from Me2SO stock) at room temperature for 1 h (Fig. 1A). Unreacted dye was removed by gel filtration (Sephadex G25, PBS). Using molar absorbance data, TMR/polyamine mol ratios were 3.3:1 (TMR-POL), TMR/polyamidoamine mol ratios were 2.8:1 (TMR-PAM), and TMR/polyethylenimine mol ratios were 4.1:1 (TMR-PEI). Equal volumes of each TMR-polyamine (5 μm in degassed PBS) and iminothiolane (13 μm in PBS containing 5 mm EDTA, pH 8) were incubated for 1 h at room temperature in the dark under N2. Unreacted iminothiolane was removed by gel filtration. Sulfhydryl (SH) group/ligand mol labeling ratios were 0.96:1 (for TMR-POL), 0.93:1 (for TMR-PAM), and 0.95:1 (for TMR-PEI). TMR-polyamine-SH (5 μm) was then incubated with BAC-dextran-S-S-2Py (2 μm in 4 ml of PBS containing 5 mm EDTA, pH 8, synthesized as described in Ref. 16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) for 18 h at room temperature under N2. Unreacted TMR-polyamine-SH and BAC-dextran-S-S-2Py were removed by gel filtration, and the product was lyophilized and stored at -20 °C. Molecular masses of the conjugates (before and after cell internalization) were determined by column chromatography (Sephacryl 300HR). Eluted fractions were assayed for BAC and TMR fluorescence. In fluorescence-quenching studies, microliter aliquots of NaCl (1 m stock) were added to 3 ml of fluorescent ligand (10 μM, pH 7.4). Stern-Volmer constants (K sv) were calculated from the slope of F0/F - 1 versus [Cl-] plots (F0/F - 1 = K sv [Cl-]), where F0 is BAC fluorescence in absence and F is the presence of Cl-. Fluorescently Labeled Polyamines for pH Measurements—TMR-POL, TMR-PAM, and TMR-PEI (5 μmol) prepared as above were gently stirred for 1 h with fluorescein isothiocyanate (35 μmol from Me2SO stock) in 10 ml of aqueous NaHCO3 (pH 8). Reaction products (FITC-POL-TMR, FITC-PAM-TMR, and FITC-PEI-TMR) were purified by gel filtration chromatography. Thin layer chromatography showed no free dye contamination. FITC/polymer molar labeling ratios were 5.5, 6.0, and 6.8 for POL, PAM, and PEI, respectively. Polyamine-DNA Polyplexes—DNA complexes containing Cl- or pH-sensing fluorescent polyamines (or non-fluorescent polyamines) were prepared by mixing plasmid DNA (pCDNA3 encoding GFP, 3.8 kb; 80 μg/ml in 10 mm HEPES, 10 mm NaCl, 10% glucose) and polyamine (in distilled water) at specified primary amine/phosphate (N/P) ratios (2.2, 5.5, and 6.0 for PEI, PAM, and POL, respectively). Polyplexes were dispersed in PBS for endosomal uptake. For measurements of transfection efficiency, CHO cells were incubated with polyplexes at different N/P in 96-well plates for 30 min in serum-free medium and then for 36 h in serum-containing medium. GFP fluorescence was quantified by a fluorescence plate reader. Endosome Labeling, [Cl-], and pH Measurements—Cells were incubated in serum-free medium for 15 min at 37 °C and then pulse-labeled by incubation with DNA polyplex (50 μg/ml) in PBS for 5 min at 4 °C. Coverslips were washed twice at 4 °C with PBS containing dextran sulfate (10 kDa, 1 mg/ml) and washed once in PBS, then transferred to a pre-cooled perfusion chamber. Sets of BAC and TMR images (for Cl-) or FITC and TMR images (for pH) were acquired at specified times after perfusion with PBS at 37 °C. In some experiments, the perfusate contained NH4Cl (5-40 mm) or chloroquine (100 μm). For in vivo calibration of BAC/TMR fluorescence ratio versus [Cl-], perfusate and endosomal [Cl-] were equalized by the incubation of cells for 15-20 min at 37 °Cin 120 mm KCl/KNO3 (0-120 mm Cl-), 20 mm NaCl/NaNO3, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 7.4) containing the ionophores nigericin, valinomycin, carbonyl cyanide p-chlorophenylhydrazone, monensin (all 10 μm), and bafilomycin (200 nm). For calibrations of TMR/FITC fluorescence ratio versus pH, cells were incubated with high K+ solutions containing nigericin, valinomycin, and bafilomycin (pH adjusted to 4-8). Endosomal Buffer Capacity—Buffer capacity (β) was determined from the rapid increase in endosomal pH in response to the addition of 5-40 mm NH4Cl to the perfusate: β = ([NH4Cl]/ΔpH)·10(pHout-pHfinal), where ΔpH is the pH increase just after NH4Cl addition, pHout is perfusate pH, and pHfinal is endosomal pH after the addition of NH4Cl. Perfusate [NH4Cl] was chosen to increase endosome pH from ∼6.1 to 6.7. Fluorescence Microscopy—Cells were imaged using a Leitz upright epifluorescence microscope equipped with Nipkow-wheel confocal attachment and a 14-bit cooled (-30 °C) charge-coupled device camera (19Zen K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; PubMed Scopus Google Scholar). Fluorescence was using a = a BAC = 5 = = 20 and TMR and FITC was using to (16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Endosome volumes were determined from of endosomes In Endosome 30 or min after internalization of polyplex and 37 °C cells were in mm K 10 mm NaCl, 1 mm and MgCl2, 15 mm HEPES pH low for 10 the containing endosomes was added to of to an osmotic The of polyplex was determined by from the versus increase in FITC fluorescence after the addition of 1 m stock) to increase pH to of polyamines PEI and PAM titratable and amines in the pH whereas POL not (Fig. 1A). PEI, POL, and PAM were labeled with TMR and conjugated with BAC-dextran using a (Fig. BAC fluorescence is green and whereas TMR fluorescence is and the absence of DNA gel in at the ratios used to the was an of fluorescent polymers the (Fig. fluorescent as is in transfection filtration chromatography of polyamines and after cellular internalization of Cl- showed at masses of and for PAM, PEI, and POL, The BAC and TMR fluorescence and no fluorescence was seen at lower of the fluorescent polyamine conjugates in Fluorescence of polyplexes showed at and and at for for TMR, was at and was at BAC fluorescence in the polyplexes was by Cl- by a with a Stern-Volmer of 36 whereas TMR fluorescence was not BAC fluorescence was not by polyamine and DNA of the polyplexes were measured to N/P for Cells were incubated for 30 min at 37 °C with polyplexes containing or fluorescently labeled efficiency, measured as cellular GFP fluorescence at 36 was times for PAM versus POL polyplexes (Fig. transfection and N/P were for versus fluorescent polyamines and the transfection are with results (12Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar, M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). Cl- and pH in fluorescence of cells labeled m with polyplexes containing and The cell was after incubation for 5 min at 4 °C labeled at which the green BAC fluorescence was relatively because of the high Polyplexes were to 37 °C. labeled with polyplexes containing PAM and PEI, but not POL, substantially over of fluorescence versus [Cl-] were by cells with endosomes with a high K+ buffer containing ionophores and versus [Cl-] for in aqueous solution and in cells with a Stern-Volmer of 37 constants of were determined for polyplexes containing and The versus [Cl-] calibration of [Cl-] in endosomes by ratio as in the in which was for images at min after calibration of fluorescence versus pH for the pH-sensing polyamine was in cells using high K+ buffer containing ionophores and bafilomycin (Fig. versus pH was in cells and solution a results were for and pH in endosomes at min after at 4 °C and rapid to 37 [Cl-] in endosomes containing PAM polyplexes increased from to mm during the and then decreased to mm at 75 min (Fig. the in endosomal [Cl-] results from of a of [Cl-] kinetics were measured in endosomes containing PEI containing POL polyplexes less Cl- mm at Endosomal acidification was measured in (Fig. pH in endosomes containing PAM and PEI polyplexes decreased slowly from to over 60 to at 75 of endosomes containing POL polyplexes was relatively with pH to 5.9 in 15 Cl- accumulation and acidification in endosomes containing each of the polyplexes We postulated that the increased Cl- accumulation and slowed acidification in endosomes containing PAM or PEI versus POL polyplexes was caused by their greater H+ buffer capacity Using the β (in the pH was and for endosomes containing PAM and PEI which was greater than that of 46 for endosomes containing POL polyplexes (Fig. The increased buffer capacity in endosomes containing PAM or PEI versus POL polyplexes for the increased Cl- accumulation 80 versus mm over 60 min) and slowed acidification versus 1 over 15 and of greater volume of endosomes compared with endosomes by fluid-phase or receptor-mediated of their volume by that the volume of fluorescent endosomes containing PAM polyplexes increased with after with a increase for POL The chloroquine increased volume and Cl- accumulation in endosomes containing POL polyplexes (Fig. and providing evidence Cl- accumulation and To endosome swelling is with the kinetics of pH in endosomes was that endosome or lysis would in endosome or pH in of endosomes containing PAM polyplexes at min after some PAM endosomes as as 30 min after as a of lysis and polyamine (Fig. In under the no POL endosomes or strongly (Fig. that endosomes containing PAM or PEI polyplexes be to osmotic lysis than endosomes containing POL Cell containing labeled endosomes, prepared at min after cellular internalization of fluorescent were to osmotic and the of polyamine was from the versus fluorescence increase after addition (Fig. remarkably greater in endosomes containing PAM versus POL polyplexes after osmotic as The here were to test the proton sponge hypothesis, which postulates that enhanced transgene delivery by polyplexes containing titratable amines is caused by increased endosomal Cl- accumulation and swelling/lysis. Cl- accumulation was to be remarkably greater in endosomes containing PEI and PAM versus POL was swelling was and buffer capacity was and lysis were seen at min in PAM and PEI but not POL endosomes, and osmotic fragility was POL endosomes be to PAM endosomes by in the solution of a that in We that increased polyplex buffer capacity results in increased endosomal Cl- entry because of osmotic swelling and endosome results provide direct evidence in support of the proton sponge hypothesis. Our fluorescence labeling of Cl- and to polyamines for with plasmid of of the BAC direct to the Cl- of BAC-labeled dextran with TMR-labeled of fluorescent polyplexes in in and in vivo Cl- and pH absence of and transfection of fluorescent and containing DNA polyplexes are remarkably greater in than endosomes relatively fluid-phase such as fluorescent or high such as and M. Klibanov A.M. Appl. Microbiol. Biotechnol. 2003; 62: 27-34Crossref PubMed Scopus (439) Google Scholar, Rev. Cell Biol. 1996; PubMed Scopus Google Scholar). of DNA polyplexes into endosomes Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and thus to be a different than fluid-phase or receptor-mediated endocytosis. gene transfer in cell a of the cationic such that with We as for free was and that endosomes containing free acidification kinetics and accumulation as endosomes containing DNA polyplexes is not the and of endosomes are different from of endosomes by fluid-phase or receptor-mediated results that their endosomes a H+ ATPase and Cl- Our of PAM and PEI polyplexes gene transfer in cultured cells (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar, O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar). A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar) showed that of cells to PEI polyplexes with bafilomycin in a in gene that the transfection efficiency of PEI is because of proton during endosomal acidification and that gene transfer 4 h of polyplex the of endosomal acidification and the proton sponge hypothesis have been M.A. P. J. 2000; PubMed Scopus Google Scholar) that gene delivery not by of the endosomal and S. A. J. 2002; PubMed Scopus Google Scholar) that gene transfer from the and not the The in the measurements here to in a that 75 min of to the are with the of gene because gene is 4 h after cell to DNA polyplexes and at h after A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar). Cl- and acidification in the 60 min of uptake and are with endosomal The of fluorescence from but not POL-containing endosomes that the endosome has in polyplex into the because DNA into the not from the of D. L. M. D. R. J. C. N. J. Cell Biol. 2000; PubMed Scopus Google Scholar, D. Gene Ther. 2002; PubMed Scopus Google Scholar), the of fluorescence that the fluorescent has from the DNA and the it that a polymer-DNA complex is from the of into the nucleus J. D. J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100 PubMed Scopus Google Scholar). Our results provide the direct that endosomal pH, and endosomes that are to osmotic The that the fluorescence has an for the design of non-viral vectors for efficient transgene the to into the polyplex to the transfer of the DNA into the