High Density of Octaarginine Stimulates Macropinocytosis Leading to Efficient Intracellular Trafficking for Gene Expression
2005; Elsevier BV; Volume: 281; Issue: 6 Linguagem: Inglês
10.1074/jbc.m503202200
ISSN1083-351X
AutoresIkramy A. Khalil, Kentaro Kogure, Shiroh Futaki, Hideyoshi Harashima,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression. The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression. The human immunodeficiency virus TAT 2The abbreviations used are: TAT, transactivator of transcription; R8, octaarginine peptide; EPC, egg phosphatidylcholine; Chol, cholesterol; Rh-PE, N-(lissaminerhodamine-B-sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; NBD, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); DOPE, dioleoylphosphatidylethanolamine; CHEMS, cholesteryl hemisuccinate; STR-R8, stearylated-octaarginine; R8-Lip, octaarginine-modified liposomes; R8-Lip-HD, R8-Lip modified with a high density of STR-R8 (5.2 mol % of total lipid); R8-Lip-LD, R8-Lip modified with a low peptide density (0.86 mol % of total lipid); R8-Cps, R8-modified DNA-coated particles; HSPG, heparan sulfate proteoglycan; PTD, protein transduction domain; Tf, transferrin, ND, neutral dextran; R8-Lip-LD-RA, R8-Lip-LD containing rhodamine aqueous phase; R8-Lip-HD-E, empty R8-Lip-HD; MF, mean fluorescence; RLU, relative light units; TMR, tetramethylrhodamine; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. -derived peptide is a small basic peptide that has been shown to successfully mediate the efficient cellular uptake of a wide variety of cargos, including proteins, peptides, and nucleic acids (1Schwarze S.R. Hruska K.A. Dowdy S.F. Trends Cell Biol. 2000; 10: 290-295Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 2Lindgren M. Hallbrink M. Prochiantz A. Langel U. Trends Pharmacol Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 3Snyder E.L. Dowdy S.F. Pharm. Res. 2004; 21: 389-393Crossref PubMed Scopus (294) Google Scholar). Torchilin et al. (4Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (707) Google Scholar) recently demonstrated that even certain TAT-linked liposomes with a diameter of 200 nm could be efficiently internalized into a variety of cell lines in intact form. The attachment of TAT directly to the liposome surface without a spacer or the presence of high molecular weight polyethylene glycol spacers abolished liposome internalization, indicating the importance of the direct contact of TAT with the cell surface (4Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (707) Google Scholar). More recently, other groups reported an enhanced cellular uptake of liposomes when modified with TAT peptide (5Fretz M.M. Koning G.A. Mastrobattista E. Jiskoot W. Storm G. Biochim. Biophys. Acta. 2004; 1665: 48-56Crossref PubMed Scopus (103) Google Scholar, 6Marty C. Meylan C. Schott H. Ballmer-Hofer K. Schwendener R.A. Cell Mol. Life Sci. 2004; 61: 1785-1794Crossref PubMed Scopus (15) Google Scholar). Complexes formed between TAT-liposomes and DNA also showed enhanced transfection in vitro and in vivo (7Torchilin V.P. Levchenko T.S. Rammohan R. Volodina N. Papahadjopoulos-Sternberg B. D'Souza G.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1972-1977Crossref PubMed Scopus (427) Google Scholar). Despite the well demonstrated ability of TAT to internalize different cargos, the internalization mechanism of the peptide itself or its cargo remains a controversial issue. According to early studies, none of the classic receptor-, transporter-, or endocytosis-mediated processes seemed to be involved in the uptake of TAT and other similar peptides (8Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2071) Google Scholar, 9Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). Direct penetration- and inverted micelle-driven delivery have been suggested as possible internalization mechanisms (10Trehin R. Merkle H.P. Eur. J. Pharm. Biopharm. 2004; 58: 209-223Crossref PubMed Scopus (189) Google Scholar). However, the mechanism of entry was recently re-evaluated based on possible problems that may occur due to cell fixation prior to microscopy observation, and more evidence appeared to support the involvement of endocytosis (11Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar, 12Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Endocytosis-mediated uptake is also a controversial issue, because various findings showing the involvement of different endocytic mechanisms have been reported (11Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar, 12Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 13Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 14Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1427) Google Scholar, 15Kaplan I.M. Wadia J.S. Dowdy S.F. J. Control. Release. 2005; 102: 247-253Crossref PubMed Scopus (580) Google Scholar, 16Nakase I. Niwa M. Takeuchi T. Sonomura K. Kawabata N. Koike Y. Takehashi M. Tanaka S. Ueda K. Simpson J.C. Jones A.T. Sugiura Y. Futaki S. Mol. Ther. 2004; 10: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, 17Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 18Ferrari A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The TAT sequence, which is critical for translocation, contains several arginine residues (19Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar). Homopolymers of arginine are similar to the TAT peptide in terms of efficiency and uptake mechanism (19Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar, 20Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), making them a possible candidate for mimicking the TAT peptide. The optimum number of arginine residues for efficient internalization was shown to be ∼8 residues (19Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar). The octaarginine (R8) peptide could mediate the efficient intracellular delivery of macromolecules, and similar to the TAT peptide but less studied, the exact uptake mechanism of R8-cargos is still largely unknown. The diversity of results regarding the uptake mechanism of arginine-rich peptides suggests that some factors may affect the entry mechanism. These factors include the type of peptide, its mode of exposure to the cell surface, the nature of the cargo, and the chemical linkage between the peptide and the cargo (21Brooks H. Lebleu B. Vives E. Adv. Drug Deliv. Rev. 2005; 57: 559-577Crossref PubMed Scopus (577) Google Scholar). For example, we have previously shown that the R8 peptide and its complexes with DNA were taken up by different mechanisms, suggesting that the nature of the interaction between the peptide and the cell surface (i.e. the peptide in free or complexed state) affects the uptake mechanism (22Khalil I.A. Futaki S. Niwa M. Baba Y. Kaji N. Kamiya H. Harashima H. Gene Ther. 2004; 11: 636-644Crossref PubMed Scopus (151) Google Scholar). Another possible factor that has been less studied is the effect of peptide density on the internalization mechanism. It was previously shown that a single TAT peptide was sufficient to allow the cellular delivery of an unfolded fusion construct of the same protein (23Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar). Other studies showed that several TAT peptides attached to the surface of the cargo were required to permit efficient cellular delivery (24Eguchi A. Akuta T. Okuyama H. Senda T. Yokoi H. Inokuchi H. Fujita S. Hayakawa T. Takeda K. Hasegawa M. Nakanishi M. J. Biol. Chem. 2001; 276: 26204-26210Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). However, no direct comparison to show the effect of peptide density on the uptake mechanism was conducted. Liposomes are good tools for use in such a comparison, because their surface can be easily modified with different densities of peptide and they can provide localized areas of high peptide density that are available to interact with the cell membrane. Therefore, the main purpose of this study was to investigate the effect of peptide density on the internalization mechanism and intracellular trafficking of cargos modified with arginine-rich peptides. The R8 peptide was chosen as a prototype of arginine-rich peptides and liposomes were chosen as an example of cargos. Here, we present results showing that the mechanism of uptake of liposomes modified with a low R8 density shifted from clathrin-mediated endocytosis to macropinocytosis when the density of R8 was increased. The uptake route influenced intracellular trafficking, resulting in a remarkable difference in gene expression when condensed plasmid DNA was encapsulated into each type of liposome. These results highlight important features concerning the mechanism of entry and intracellular fate of R8-modified nanoparticles and demonstrate the role of the peptide density in determining the cellular uptake pathways of cargos. Furthermore, the data provided here indicate that uptake through macropinocytosis is more efficient in terms of avoiding lysosomal degradation resulting in an enhanced gene expression. Materials—Egg phosphatidylcholine (EPC), cholesterol (Chol), N-(lissaminerhodamine-B-sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Rh-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), and dioleoylphosphatidylethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B, Syto-24, tetramethylrhodamine-labeled neutral dextran (70 kDa), FITC-labeled transferrin, and LysoSensor Green were purchased from Molecular Probes (Eugene, OR). Cholesteryl hemisuccinate (CHEMS), poly-l-lysine, amiloride, and filipin were purchased from Sigma-Aldrich. Stearylated-octaarginine was synthesized as described previously (25Futaki S. Ohashi W. Suzuki T. Niwa M. Tanaka S. Ueda K. Harashima H. Sugiura Y. Bioconjug. Chem. 2001; 12: 1005-1011Crossref PubMed Scopus (408) Google Scholar). Other chemicals were purchased from Wako Chemicals (Osaka, Japan). Plasmid DNA Pcmv-luc-encoding luciferase was prepared by using an EndFree Plasmid Mega Kit (Qiagen, Hilden, Germany). NIH3T3 cells were obtained from the American Type Culture Collection (Manassas, VA). Preparation of the Octaarginine-modified Liposomes—Liposomes were basically composed of EPC and Chol (7:3 molar ratio), and stearylated-R8 (STR-R8) was incorporated at 0-10 mol % of the total lipid. To label the lipid phase, Rh-PE or NBD-phosphoethanolamine was incorporated at 1 mol % of the total lipid. Liposomes were prepared by hydration method followed by extrusion with a Mini-Extruder (Avanti Polar Lipids), through polycarbonate membrane filters (Nucleopore) of 400, 200, and 100 nm. Sulforhodamine B was used as an aqueous phase marker, when required. Liposomes were purified on a Bio-Gel A-1.5m column (100-200 mesh). The particle sizes of the liposomes were measured by a quasi-elastic light scattering method, and the zeta potential was determined by means of an electrophoretic light scattering spectrophotometer (ELS-8000, Photal Otsuka Electronics). Preparation of the Octaarginine-modified DNA-coated Particles—Plasmid DNA was condensed with poly-l-lysine as described previously (26Kogure K. Moriguchi R. Sasaki K. Ueno M. Futaki S. Harashima H. J. Control. Release. 2004; 98: 317-323Crossref PubMed Scopus (235) Google Scholar). A condensed DNA solution was then added to the lipid film, formed by the evaporation of a chloroform solution of the lipids: EPC/Chol/STR-R8 (7:3:0.086 or 0.52 molar ratio), or DOPE/CHEMS (9:2 molar ratio) on the bottom of a glass tube, followed by incubation for 10 min to hydrate the lipid film. The glass tube was then sonicated for ∼1 min in a bath-type sonicator (125 W, Branson Ultrasonics, Danbury, CT). In the case of DOPE/CHEMS, the particles, after sonication, were incubated with an aqueous solution of STR-R8 (0.86 or 5.2 mol % of total lipids) for 30 min at room temperature. The size and zeta potential of the coated particles were measured as described above. Confocal Laser Microscopy—To investigate the cellular uptake of the R8-modified liposomes (R8-Lip), NIH3T3 cells were treated with double-labeled R8-Lip (NBD-labeled lipid and rhodamine-labeled aqueous phase, final concentration of 0.1 mm lipid) in serum-free medium at 37 °C for 1 h. The cells were then washed 3 times with ice-cold PBS and analyzed by confocal laser microscopy (LSM510, Carl Zeiss). To investigate the mechanism of internalization of R8-Lip, cells were incubated in the absence or presence of sucrose (0.4 m) for 30 min or amiloride (5 mm) for 10 min. Different R8-liposomes, containing a rhodamine aqueous phase, were then added, and the incubation was continued for 1 h. The cells were then washed 3 times with ice-cold PBS supplemented with heparin and observed by confocal microscopy. Nuclei were stained with Syto-24 in the last 20 min of incubation. For the colocalization study, in the case of transferrin, cells were first incubated with R8-Lip containing rhodamine aqueous phase for 30 min, the medium was then removed, a new medium containing FITC-labeled transferrin (5 μm) was added, and incubation was continued for 5 min before observation within 5 min. In the case of neutral dextran, NBD-labeled R8-liposomes were added to cells followed by adding tetramethylrhodamine-labeled neutral dextran (5 μm) and incubation continued for 30 min. The medium was then exchanged with liposome-free medium containing neutral dextran followed by incubation for 10 min. Cells were then washed with ice-cold PBS and observed. To investigate the intracellular fate of the liposomes, NIH3T3 cells were treated with different R8-liposomes containing an aqueous rhodamine phase at 37 °C for 30 min. The medium was then removed; the cells were washed with new medium, incubated in fresh liposome-free medium for a total of 1, 3, or 6 h, and then observed with confocal microscopy. Nuclei were stained with Syto-24 in the last 20 min of incubation. The same experiment was repeated using R8-Lip modified with low density of STR-R8 and containing rhodamine aqueous phase in the absence or presence of non-labeled R8-Lip modified with a high density of STR-R8 (a 5-fold increase, calculated as R8 content). To quantify the intracellular fluorescence, the total pixel areas of the fluorescence were measured using AquaCosmos software (Hamamatsu Photonics, Hamamatsu, Japan). To investigate the colocalization of R8-Lip with lysosomes, cells were incubated with different R8-Lip preparations containing rhodamine aqueous phase at 37 °C for 30 min. The medium was then removed; the cells were washed with new medium and incubated in fresh liposome-free medium for a total of 3 h. Thirty minutes before observation by confocal microscopy, the LysoSensor was applied at a final concentration of 1 μm to stain the acidic compartments. Flow Cytometry—To investigate the cellular uptake of R8-Lip, NIH3T3 cells were incubated in serum-free medium containing different R8-Lip (final concentration, 0.1 mm lipids) for 1 h at 37°C. At the end of the incubation, the medium was removed, and the cells were washed once with ice-cold PBS with or without heparin (20 units.ml-1). The cells were then trypsinized and washed twice by centrifugation at 4°C(± heparin), suspended in 1 ml of PBS, and, after passing through a nylon mesh, they were analyzed by flow cytometry (BD Biosciences). To investigate the cellular association in the presence of heparin, NIH3T3 cells were incubated in serum-free medium containing increasing concentrations of heparin for 5 min at 37 °C. Different R8-Lip preparations containing rhodamine aqueous phase were then added, and the incubation was continued for 1 h. At the end of the incubation, the medium was removed, and the cells were washed once with ice-cold PBS, trypsinized, washed twice by centrifugation at 4 °C, suspended in 1 ml of PBS, passed through a nylon mesh, and analyzed by flow cytometry. To examine the mechanism of internalization of R8-Lip, cells were incubated in the absence or presence of a mixture of metabolic inhibitors (sodium azide, 0.1%, sodium fluoride, 10 mm, and antimycin A, 1 μg.ml-1) for 30 min, sucrose (0.4 m) for 30 min, amiloride (5 mm) for 10 min, or filipin (1 μg.ml-1) for 1 h. Different R8-Lip preparations, containing rhodamine aqueous phase, were then added, and the incubation was continued for 1 h. Then cells were analyzed by flow cytometry after washing 3 times with PBS supplemented with heparin, as described above. To investigate the uptake of neutral dextran, cells were incubated with tetramethylrhodamine-labeled neutral dextran (5 μm) in the presence or absence of empty (non-labeled) R8-Lip modified with a high density of STR-R8 for 30 min at 37 °C then analyzed. To investigate the stimulation of macropinocytosis, cells were incubated with R8-Lip modified with low density of STR-R8 and containing a rhodamine aqueous phase mixed with increasing concentrations of empty (non-labeled) R8-Lip modified with a high density of STR-R8 for 1 h at 37 °C. The experiment was performed in the presence or absence of amiloride (5 mm) to inhibit macropinocytic uptake. Transfection Assay—One day before transfection, NIH3T3 cells were seeded into 24-well plates at 4 × 104 cells per well. Cells were incubated for 1 h at 37 °C with 0.25 ml of serum-free medium containing different R8-modified DNA-coated particles containing 0.4 μg of DNA. Next, 1 ml of medium containing 10% fetal calf serum was added, and the incubation continued for an additional 23 h. The cells were then washed and solubilized with reporter lysis buffer (Promega, Madison, WI). Luciferase activity in the cell lysate was then measured by means of a luminometer (Luminescencer-PSN, ATTO, Japan). The amount of protein in the cell lysate was determined using a BCA protein assay kit (Pierce). To investigate the contribution of different uptake pathways in gene expression, cells were pretreated with or without sucrose (0.4 m) or amiloride (2.5 mm) for 10 min and R8-modified DNA-coated particles (lipid composition DOPE/CHEMS) containing 5.2 mol % STR-R8 were then added, and the incubation was continued for 1 h. The medium was then removed, and the cells were washed 3 times with PBS containing 20 units.ml-1 heparin and once with PBS. The cells were then incubated in the presence of serum-free medium for 70 min followed by further incubation in the presence of 1 ml of medium containing 10% FBS for periods of up to 12 h. To control the effects of sucrose or amiloride on intracellular events, cells were first loaded with R8-modified DNA-coated particles for 1 h, then washed and incubated for 70 min in the presence of these reagents, after which, 1 ml of medium containing 10% fetal bovine serum was added, and the cells were further incubated for a total of 12 h. Preparation and Characterization of Octaarginine-modified Liposomes—A series of R8-Lip containing various concentrations of STR-R8 peptide was prepared. In this preparation, the stearyl moiety acts as an anchor to the lipid membrane leaving the R8 peptide freely attached to the surface. The zeta potential of the prepared liposomes was determined as a measure of their net charge (Fig. 1A). Increasing the concentration of STR-R8 peptide caused an initial rapid increase in zeta potential followed by a slower increase for concentrations up to ∼5mol %. No further increase in zeta potential was observed >5 mol % STR-R8, indicating that the liposomal surface was saturated with the cationic peptide. In the next experiments, we chose liposomes modified with a low density (0.86 mol %) of STR-R8 (R8-Lip-LD), which had a low positive charge, and those modified with a high density (5.2 mol %) of STR-R8 (R8-Lip-HD), which had the highest possible positive charge. Data related to the characterization of the different liposomes used in this study are shown in Table 1.TABLE 1Characterization of R8-modified liposomesLipid composition (molar ratio)DiameteraData represent means ± S.D. of at least two different determinations.Zeta potentialaData represent means ± S.D. of at least two different determinations.nmmVR8-Lip-LD-RAbRA, liposomes containing rhodamine-labeled aqueous phase.EPC:Chol:STR-R8 (7:3:0.086)152 ± 1813 ± 11R8-Lip-HD-RAbRA, liposomes containing rhodamine-labeled aqueous phase.EPC:Chol:STR-R8 (7:3:0.52)102 ± 1035 ± 4R8-Lip-LD-FLcFL, liposomes containing NBD-labeled lipid phase.EPC:Chol:NBD-PE:STR-R8 (7:3:0.1:0.085)170 ± 27 ± 1R8-Lip-HD-FLcFL, liposomes containing NBD-labeled lipid phase.EPC:Chol:NBD-PE:STR-R8 (7:3:0.1:0.52)149 ± 340 ± 1a Data represent means ± S.D. of at least two different determinations.b RA, liposomes containing rhodamine-labeled aqueous phase.c FL, liposomes containing NBD-labeled lipid phase. Open table in a new tab Cellular Uptake of R8-liposomes—First, we quantitatively compared the cellular uptake of R8-Lip-LD and R8-Lip-HD containing an aqueous rhodamine phase by flow cytometry. We confirmed that the surface-bound liposomes could be removed by means of heparin wash (data not shown). The measured intracellular fluorescence in the case of R8-Lip-HD was higher than that for R8-Lip-LD by less than one order of magnitude (Fig. 1B). When we measured the total fluorescence (surface-bound plus internalized liposomes) by excluding the heparin washes, we found that the fluorescence in the case of R8-Lip-HD was about one order of magnitude higher than that in the case of R8-Lip-LD (data not shown). Liposomes that were devoid of STR-R8 did not show any intracellular or surface-bound fluorescence (data not shown). The cellular uptake of both liposomes was further confirmed using confocal laser microscopy of living cells (supplemental Fig. S1). Surface-bound fluorescence was higher in the case of R8-Lip-HD. Taken together; these results indicate that both R8-Lip-LD and R8-Lip-HD can bind to the cell surface, especially R8-Lip-HD, and that they are efficiently internalized. Transfection Activities of R8-Lip Containing Plasmid DNA—Because R8-Lip showed a high potential for the intracellular delivery of macromolecules encapsulated in their cores, we investigated the cellular uptake of R8-Lip-containing plasmid DNA, for use in gene delivery. We prepared condensed DNA particles coated with a lipid envelope consisting of EPC and Chol and modified with the R8 peptide (R8-modified coated particles, R8-Cps) as described under "Experimental Procedures." We incubated the cells for 1 h with R8-Cps containing FITC-labeled DNA modified with 0.86 or 5.2 mol % STR-R8 (R8-Cps-LD and R8-Cps-HD) and then observed the cells by confocal microscopy. In both cases, the intracellular fluorescence was similar to the pattern of distribution obtained earlier with R8-Lip (data not shown), indicating the efficient cellular uptake of plasmid DNA encapsulated in the R8-Lip. We next investigated the transfection efficiency of R8-Cps containing plasmid DNA encoding a luciferase reporter gene. R8-Cps-LD did not show a significant gene expression, whereas the R8-Cps-HD produced gene expression levels about 3 orders of magnitude higher (Fig. 1C). This difference in gene expression is not correlated with the difference in cellular uptake as indicated by flow cytometry and confocal microscopy. Therefore, the superiority of the R8-Cps-HD regarding gene expression cannot be explained by differences in the amount of DNA internalized, but that there are intracellular events responsible for this difference. Mechanism of Uptake of Different R8-liposomes—Because the gene expression levels of the R8-Cps were not correlated with the amount of liposomes internalized, we investigated the mechanism of uptake of different R8-Lip as a candidate to explain the difference in the intracellular fate of the particles. It has previously been shown that the cell-surface heparan sulfate proteoglycans (HSPGs) act as nonspecific receptors in the cellular binding of different protein transduction domain (PTD) peptides (27Richard J.P. Melikov K. Brooks H. Prevot P. Lebleu B. Chernomordik L.V. J. Biol. Chem. 2005; 280: 15300-15306Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 28Console S. Marty C. Garcia-Echeverria C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). As mentioned above, several washings with a buffer containing heparin were sufficient to remove the surface-bound liposomes. Furthermore, the addition of heparin to the medium prior to the addition of R8-Lip dramatically inhibited the cellular binding and internalization of R8-Lip especially in the case of R8-Lip-LD (supplemental Fig. S1). In both cases, concentrations as low as 1 unit·ml-1 were sufficient to completely block the interaction of the liposomes with the cell surface. Taken together, these data probably show the involvement of cell-surface HSPGs in the uptake of R8-Lip, similar to other PTD-liposomes (6Marty C. Meylan C. Schott H. Ballmer-Hofer K. Schwendener R.A. Cell Mol. Life Sci. 2004; 61: 1785-1794Crossref PubMed Scopus (15) Google Scholar). To investigate the contribution of the endocytic pathway in the internalization of R8-Lip, we
Referência(s)