Efficient MHC Class I Presentation by Controlled Intracellular Trafficking of Antigens in Octaarginine-modified Liposomes
Authors: Takashi Nakamura, Rumiko Moriguchi, Kentaro Kogure, Nilabh Shastri, Hideyoshi Harashima
Journal: Molecular Therapy (2008)

Recently, much attention has been paid to cell-penetrating peptides (CPPs) as an antigen-delivery tool for presentation through the major histocompatibility complex class I (MHC-I) pathway. However, escape of CPPs from the endosome is inefficient and therefore a bottleneck for antigen delivery. Previously, we showed the importance of topological control of octaarginine (R8) peptides on the liposome surface for regulating cellular uptake as well as intracellular trafficking, especially endosomal escape. In this study, we hypothesized that efficient MHC-I presentation could be achieved by controlled intracellular trafficking of antigen encapsulated in R8-modified liposomes (R8-Lip). The mechanism of uptake of both R8-Lip and cationic liposomes was shown to be by macropinocytosis in dendritic cells. However, confocal laser scanning microscopy (CLSM) revealed that R8-Lip are able to release significantly more antigen to the cytosol than are cationic liposomes. Processing of the antigens delivered by R8-Lip was shown to be proteasome-dependent, which is consistent with selective antigen presentation by R8-Lip via MHC-I. According to antigen-presentation analysis, R8-Lip can induce significantly higher MHC-I presentation at lower doses than either soluble ovalbumin (OVA) or OVA in pH-sensitive or cationic liposomes. Moreover, R8-Lip showed an efficient antitumor effect in vivo. Therefore, R8-Lip is a promising new carrier for MHC-I-specific antigen presentation. Recently, much attention has been paid to cell-penetrating peptides (CPPs) as an antigen-delivery tool for presentation through the major histocompatibility complex class I (MHC-I) pathway. However, escape of CPPs from the endosome is inefficient and therefore a bottleneck for antigen delivery. Previously, we showed the importance of topological control of octaarginine (R8) peptides on the liposome surface for regulating cellular uptake as well as intracellular trafficking, especially endosomal escape. In this study, we hypothesized that efficient MHC-I presentation could be achieved by controlled intracellular trafficking of antigen encapsulated in R8-modified liposomes (R8-Lip). The mechanism of uptake of both R8-Lip and cationic liposomes was shown to be by macropinocytosis in dendritic cells. However, confocal laser scanning microscopy (CLSM) revealed that R8-Lip are able to release significantly more antigen to the cytosol than are cationic liposomes. Processing of the antigens delivered by R8-Lip was shown to be proteasome-dependent, which is consistent with selective antigen presentation by R8-Lip via MHC-I. According to antigen-presentation analysis, R8-Lip can induce significantly higher MHC-I presentation at lower doses than either soluble ovalbumin (OVA) or OVA in pH-sensitive or cationic liposomes. Moreover, R8-Lip showed an efficient antitumor effect in vivo. Therefore, R8-Lip is a promising new carrier for MHC-I-specific antigen presentation. IntroductionCell-penetrating peptides (CPPs) are useful for cytosolic delivery of various molecules, including peptides, proteins, and nucleic acids.1Gupta B Levchenko TS Torchilin VP Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides.Adv Drug Deliv Rev. 2005; 57: 637-651Crossref PubMed Scopus (563) Google Scholar In vaccine studies, CPPs have been applied to major histocompatibility complex class I (MHC-I) presentation for delivery of exogenous antigens to the cytosol of antigen-presenting cells.2Wang RF Wang HY Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells.Nat Biotechnol. 2002; 20: 149-154Crossref PubMed Scopus (104) Google Scholar,3Tanaka Y Dowdy SF Linehan DC Eberlein TJ Goedegebuure PS Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells.J Immunol. 2003; 170: 1291-1298Crossref PubMed Scopus (36) Google Scholar,4Shibagaki N Udey MC Dendritic cells transduced with TAT protein transduction domain-containing tyrosinase-related protein 2 vaccinate against murine melanoma.Eur J Immunol. 2003; 33: 850-860Crossref PubMed Scopus (59) Google Scholar,5Shibagaki N Udey MC Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity.J Immunol. 2002; 168: 2393-2401Crossref PubMed Scopus (124) Google Scholar,6Schutze-Redelmeier MP Gournier H Garcia-Pons F Moussa M Joliot AH Volovitch M et al.Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo.J Immunol. 1996; 157: 650-655PubMed Google Scholar,7Mitsui H Inozume T Kitamura R Shibagaki N Shimada S Polyarginine-mediated protein delivery to dendritic cells presents antigen more efficiently onto MHC class I and class II and elicits superior antitumor immunity.J Invest Dermatol. 2006; 126: 1804-1812Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,8Kim DT Mitchell DJ Brockstedt DG Fong L Nolan GP Fathman CG et al.Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.J Immunol. 1997; 159: 1666-1668PubMed Google Scholar The TAT peptide derived from human immunodeficiency virus is the most widely used CPP for antigen delivery.3Tanaka Y Dowdy SF Linehan DC Eberlein TJ Goedegebuure PS Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells.J Immunol. 2003; 170: 1291-1298Crossref PubMed Scopus (36) Google Scholar,4Shibagaki N Udey MC Dendritic cells transduced with TAT protein transduction domain-containing tyrosinase-related protein 2 vaccinate against murine melanoma.Eur J Immunol. 2003; 33: 850-860Crossref PubMed Scopus (59) Google Scholar,5Shibagaki N Udey MC Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity.J Immunol. 2002; 168: 2393-2401Crossref PubMed Scopus (124) Google Scholar,8Kim DT Mitchell DJ Brockstedt DG Fong L Nolan GP Fathman CG et al.Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.J Immunol. 1997; 159: 1666-1668PubMed Google Scholar Kim et al.8Kim DT Mitchell DJ Brockstedt DG Fong L Nolan GP Fathman CG et al.Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.J Immunol. 1997; 159: 1666-1668PubMed Google Scholar reported that TAT-conjugated ovalbumin (OVA) significantly enhanced MHC-I presentation in vitro, and induced cytotoxic T-lymphocyte (CTL) activity in mice after immunization with TAT-OVA-treated dendritic cells (DCs). However, although TAT peptides are efficiently taken up by antigen-presenting cells via macropinocytosis or endocytosis,9Wadia JS Stan RV Dowdy SF Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.Nat Med. 2004; 10: 310-315Crossref PubMed Scopus (1395) Google Scholar,10Kaplan IM Wadia JS Dowdy SF Cationic TAT peptide transduction domain enters cells by macropinocytosis.J Control Release. 2005; 102: 247-253Crossref PubMed Scopus (568) Google Scholar TAT peptide requires a functional device for endosomal escape, such as a fusogenic peptide derived from influenza virus.9Wadia JS Stan RV Dowdy SF Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.Nat Med. 2004; 10: 310-315Crossref PubMed Scopus (1395) Google Scholar Therefore, cytosolic release is a "bottleneck" in TAT-mediated antigen delivery.We previously reported that the mechanism of uptake of plasmid DNA complexed with octaarginine (R8), a kind of CPP, is clathrin-mediated endocytosis, whereas uptake of the free form of R8 peptide is endocytosis independent.11Khalil IA Futaki S Niwa M Baba Y Kaji N Kamiya H et al.Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation.Gene Ther. 2004; 11: 636-644Crossref PubMed Scopus (149) Google Scholar,12Akita H Ito R Khalil IA Futaki S Harashima H Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy.Mol Ther. 2004; 9: 443-451Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar On the other hand, we found that liposomes modified with a high density of R8 had significantly higher transfection activities through efficient internalization via macropinocytosis, which avoids lysosomal degradation,13Khalil IA Kogure K Futaki S Harashima H High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.J Biol Chem. 2006; 281: 3544-3551Crossref PubMed Scopus (341) Google Scholar i.e., the rate-limiting step (cytosol release) was improved by presentation of R8 peptide on the surface of liposomes. From these results, it was shown that topological control of CPPs, such as optimization of the presentation state and density, is required for maximizing their function, while simple conjugation or complexation is not sufficient for efficient delivery of the cargo.In this study, we propose and test the hypothesis that MHC-I presentation can be induced by efficient delivery of antigen to the cytosol by R8-modified liposomes (R8-Lip). We show that antigens encapsulated into R8-Lip were taken up by bone marrow–derived DCs (BMDCs) via macropinocytosis, escaped from the macropinosomes efficiently and were degraded by proteasomes. Antigen presentation by R8-Lip was MHC-I selective and much more efficient than pH-sensitive liposomes or cationic liposomes. Furthermore, R8-Lip induced a strong antitumor immune response in vivo.ResultsCellular entrance mechanism of R8-LipTo clarify the cellular entrance mechanism of R8-Lip in BMDCs, we prepared R8-Lip encapsulating sulforhodamine B as an aqueous-phase marker. As a comparison, conventional cationic liposomes (Cat-Lip) were also prepared. We measured the cellular uptake by fluorescence-activated cell sorter analysis. The amount of R8-Lip taken up by the cells was 1.2-times higher than that of Cat-Lip, although this difference was not significant (Figure 1a).We then examined the effects of two inhibitors of cellular entrance pathways, namely the macropinocytosis inhibitor amiloride13Khalil IA Kogure K Futaki S Harashima H High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.J Biol Chem. 2006; 281: 3544-3551Crossref PubMed Scopus (341) Google Scholar,14Moron VG Rueda P Sedlik C Leclerc C In vivo, dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.J Immunol. 2003; 171: 2242-2250Crossref PubMed Scopus (98) Google Scholar,15Khalil IA Kogure K Akita H Harashima H Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery.Pharmacol Rev. 2006; 58: 32-45Crossref PubMed Scopus (1047) Google Scholar and the clathrin-mediated endocytosis inhibitor chlorpromazine,14Moron VG Rueda P Sedlik C Leclerc C In vivo, dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.J Immunol. 2003; 171: 2242-2250Crossref PubMed Scopus (98) Google Scholar,15Khalil IA Kogure K Akita H Harashima H Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery.Pharmacol Rev. 2006; 58: 32-45Crossref PubMed Scopus (1047) Google Scholar on cellular uptake of R8-Lip. First, we confirmed the absence of cytotoxicity of the inhibitors on BMDCs. Under the present conditions, no cytotoxicity was observed with these inhibitors (data not shown). In the presence of 500 μmol/l amiloride, the uptake of R8-Lip and Cat-Lip was inhibited 60 and 80%, respectively (Figure 1b). On the other hand, 5 μg/ml chlorpromazine reduced the cellular uptake of R8-Lip and Cat-Lip by only 30 and 20%, respectively (Figure 1c). These results confirmed that both liposomes were taken up mainly via macropinocytosis. Therefore, it is concluded that the entrance route of R8-Lip into BMDCs is the same as that of conventional Cat-Lip. In addition, no nonspecific toxicity was observed after simultaneous treatment with the inhibitors and liposomes (Supplementary Figure S1).Analysis of intracellular trafficking of R8-LipTo confirm whether R8-Lip can deliver encapsulated substances to the cytosol of BMDCs, we analyzed intracellular trafficking of R8-Lip using confocal laser scanning microscopy (CLSM). We prepared R8-Lip and Cat-Lip, which were double-labeled with the lipid marker N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dioleoyl phosphatidylethanolamine (N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-DOPE) and the aqueous marker tetramethylrhodamine-labeled neutral dextran (70 kd), and observed intracellular trafficking of the labeled liposomes in BMDCs. Both R8-Lip and Cat-Lip were internalized into BMDCs 30 minutes after exposure of the cells to the liposomes, since green signals (lipid) and red signals (dextran) colocalized as yellow signals (Figure 2a and b). As shown in Figure 2c, however, at 3 hours after treatment with R8-Lip, dextran red signals were widely distributed in the cytosolic space, and not in the nucleus (blue). In addition, only dextran (red) was seen; no signal from the lipid membrane (green) was detected in the CLSM images of BMDCs 3 hours after treatment with R8-Lip. On the other hand, in the case of Cat-Lip, lipid and dextran were still colocalized at 3 hours (Figure 2d). In addition, we estimated the ratio of diffusion into the cytosol by counting cell numbers. Although the diffusion ratio of Cat-Lip was 10%, R8-Lip showed a diffusion ratio of >50% (Figure 2e, P < 0.001). These results confirm that R8-Lip can efficiently release encapsulated substances to the cytosol. Therefore, it was expected that R8-Lip could deliver antigen to the cytosol and induce MHC-I antigen presentation efficiently.Figure 2Intracellular trafficking of R8-modified liposomes (R8-Lip) and cationic-Lip (Cat-Lip) in bone marrow–-derived dendritic cell. (a–d) Confocal laser scanning microscopy (CLSM) analysis of R8-Lip and Cat-Lip. The cells were observed by CLSM at (a, b) 30 minutes and (c, d) 3 hours after incubation with (a, c) R8-Lip and (b, d) Cat-Lip. The liposomes encapsulated tetramethylrhodamine-labeled 70 kd neutral dextran (red signal), and were labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dioleoyl phosphatidylethanolamine (green signal) as a lipid marker. Nuclei of cells were stained with Hoechst 33342 (blue) for 10 minutes before observation. Bars = 10 μm. (e) Diffusion and trap ratio of encapsulated dextran. After incubation with R8-Lip and Cat-Lip for 30 minutes and 3 hours, the number of cells in which dextran had diffused or became trapped was counted. The vertical axis is expressed as the percentage of the cell number: n = 218 (R8-Lip 30 minutes); n = 228 (R8-Lip 3 hours); n = 187 (Cat-Lip 30 minutes); and n = 145 (Cat-Lip 3 hours). The P value in the χ2-square test was <0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MHC-I and MHC-II antigen presentation by R8-LipWe prepared R8-Lip and Cat-Lip encapsulating OVA as a model antigen. Then, BMDCs were exposed to R8-Lip, Cat-Lip, or soluble OVA. Antigen-presentation efficiencies via MHC-I and MHC-II were analyzed after coculture with B3Z T-cell hybridoma cells and OTIIZ T-cell hybridoma cells (Figure 3). Encapsulation of antigen into either type of liposome increased the antigen-presentation efficiency compared to soluble OVA. As expected, R8-Lip showed a significantly higher presentation efficiency for MHC-I antigen presentation compared to Cat-Lip (Figure 3a). In addition, the MHC-II presentation efficiency of R8-Lip was significantly lower than that of Cat-Lip (Figure 3b). Thus, R8-Lip have high specificity for MHC-I presentation. This result shows that R8-Lip can significantly augment the low efficiency of MHC-I presentation achieved by conventional liposomes.Figure 3Antigen-presentation activities via major histocompatibility complex class I (MHC-I) and MHC-II by soluble ovalbumin (OVA), cationic liposomes (Cat-Lip), and R8-modified liposomes (R8-Lip). Bone marrow–-derived dendritic cells were incubated with antigens for 5 hours at 37 °C, and were cocultured with (a) B3Z T-cell hybridoma for the MHC-I presentation assay or with (b) OTIIZ T-cell hybridoma for the MHC-II presentation assay overnight at 37 °C. Then, the cocultured cells were incubated with chlorophenol red β-D-galactopyranoside buffer for 4 hours at 37 °C, and the absorbance at 595 nm was measured as the antigen-presentation activity. The absorbance of soluble OVA was set to 1. The vertical axis shows relative value per OVA 1 μg. Values are the mean ± SD of at least three different experiments (*P < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of inhibitors on antigen presentation by R8-LipAs shown in Figure 3, R8-Lip and Cat-Lip mainly induce MHC-I presentation and MHC-II presentation, respectively. To confirm that the main antigen presentation was due to major uptake of the antigen by the cell, we examined the effect of inhibition of macropinocytosis by amiloride on antigen presentation. We report the results for the major antigen-presentation pathway, because the presentation activities of the minor pathway were very low and hence difficult to evaluate. As shown in Figure 4a and b, antigen presentations by R8-Lip and Cat-Lip were inhibited dose-dependently by amiloride. These results confirm that R8-Lip taken up via macropinocytosis was mainly responsible for the efficient MHC-I presentation. Furthermore, amiloride itself had no effect on presentation ability or MHC molecule expression of BMDCs, because presentation of soluble OVA257–264 peptide or OVA323–339 peptide was not affected by the drug (data not shown).Figure 4Antigen-presentation analysis of the cellular uptake mechanism of R8-modified liposomes (R8-Lip) and cationic liposomes (Cat-Lip) into bone marrow–-derived dendritic cells (BMDCs). Effect of amiloride on (a) major histocompatibility complex class I (MHC-I) antigen presentation by R8-Lip and on (b) MHC-II antigen presentation by Cat-Lip. BMDCs were treated with amiloride for 30 minutes, and then the cells were incubated with R8-Lip and Cat-Lip for 5 hours. The % presentation is expressed as the percentage of the antigen presentation relative to that in the absence of inhibitors. Values are the mean ± SD of at least three different experiments (*P < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We examined the effect of nocodazole, which is an inhibitor of microtubule polymerization,16Lutz MB Rovere P Kleijmeer MJ Rescigno M Assmann CU Oorschot VM et al.Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells.J Immunol. 1997; 159: 3707-3716PubMed Google Scholar,17Wang X Uto T Sato K Ide K Akagi T Okamoto M et al.Potent activation of antigen-specific T cells by antigen-loaded nanospheres.Immunol Lett. 2005; 98: 123-130Crossref PubMed Scopus (35) Google Scholar on antigen presentation by Cat-Lip and R8-Lip to investigate involvement of a microtubule-dependent MHC-I and MHC-II presentation processes. As shown in Figure 5a, MHC-II presentation by Cat-Lip was inhibited dose-dependently. On the other hand, no significant inhibition was observed in the case of MHC-I presentation by R8-Lip even at a high dose of nocodazole (Figure 5b). In addition, we examined the effect of the proteasome inhibitor lactacystin18Craiu A Gaczynska M Akopian T Gramm CF Fenteany G Goldberg AL et al.Lactacystin and clasto-lactacystin beta-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation.J Biol Chem. 1997; 272: 13437-13445Crossref PubMed Scopus (349) Google Scholar,19Okada N Saito T Mori K Masunaga Y Fujii Y Fujita J et al.Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen-presentation pathway in murine dendritic cells and on dendritic cell maturation.Biochim Biophys Acta. 2001; 1527: 97-101Crossref PubMed Scopus (23) Google Scholar,20Potter NS Harding CV Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes.J Immunol. 2001; 167: 2538-2546Crossref PubMed Scopus (109) Google Scholar on MHC-I presentation by R8-Lip to confirm a contribution of proteasome-mediated antigen processing. In general, antigens in the cytosol are degraded by proteasomes and presented on MHC-I molecules. As expected, lactacystin inhibited R8-Lip-mediated MHC-I presentation dose-dependently (Figure 5c), indicating that MHC-I presentation by R8-Lip depends on proteasomal degradation. Neither lactacystin nor nocodazole affected the presentation ability of MHC molecules of BMDCs, because presentation of soluble OVA257–264 peptides or OVA323–339 peptides was not altered by these inhibitors (data not shown).Figure 5Effects of the microtubule polymerization inhibitor nocodazole and the proteasome inhibitor lactacystin on major histocompatibility complex class I (MHC-I) presentation by R8-modified liposomes (R8-Lip) and cationic liposomes (Cat-Lip). (a, b) Bone marrow–-derived dendritic cells (BMDCs) were treated with nocodazole for 30 minutes, and then the cells were incubated with (a) Cat-Lip or (b) R8-Lip for 5 hours. (c) BMDCs were treated with lactacystin for 1 hour and then incubated with R8-Lip for 5 hours. After incubation, the cells were fixed with 0.5% paraformaldehyde for 15 minutes. Then, the MHC-I and MHC-II antigen-presentation activities were measured as described in the legend to Figure 3. The % presentation is expressed as the percentage of the antigen presentation relative to that in the absence of inhibitors. Values are the mean ± SD of at least three different experiments (*P < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of MHC-I presentation between R8-Lip, Cat-Lip, pH-Lip, and soluble OVATo investigate the relationship between MHC-I presentation and OVA dose, BMDCs were exposed to R8-Lip at various doses of OVA (Figure 6). Also, we compared R8-Lip with Cat-Lip, soluble OVA, and pH-sensitive liposomes (pH-Lip). pH-Lip is a liposome without R8, consisting of DOPE/cholesteryl hemisuccinate/egg phosphatidylcholine (EPC) (7.5:1:1.5). Cat-Lip induced MHC-I presentation at lower doses compared to soluble OVA. This result was inferred to be due to increased cellular uptake mediated by the positive surface charge of Cat-Lip.21Foged C Arigita C Sundblad A Jiskoot W Storm G Frokjaer S Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition.Vaccine. 2004; 22: 1903-1913Crossref PubMed Scopus (159) Google Scholar On the other hand, the antigen presentation efficiency of pH-Lip was lower than that of the soluble antigen. Thus, it seems that mere encapsulation of antigen into liposomes is not enough to improve MHC-I presentation. However, R8-Lip showed significantly higher MHC-I antigen-presentation activity than Cat-Lip. We conclude that R8-Lip have remarkably higher MHC-I presentation activity compared with conventional Cat-Lip and pH-Lip.Figure 6Major histocompatibility complex class I (MHC-I) antigen-presentation activities of soluble ovalbumin (OVA), pH-sensitive liposomes (pH-Lip), cationic liposomes (Cat-Lip), and R8-modified liposomes (R8-Lip). Bone marrow–-derived dendritic cells (BMDCs) were incubated with antigens for 5 hours at 37 °C, and were cocultured with B3Z T-cell hybridoma overnight at 37 °C. Then, the cocultured cells were incubated with chlorophenol red β-D-galactopyranoside buffer for 4 hours at 37 °C, and the absorbance at 595 nm was measured as the antigen-presentation activity. The absorbance of the no-treatment BMDCs used as control. The vertical axis represents MHC-I antigen-presentation activities which are normalized to the control value.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Antitumor effect of R8-LipFinally, to evaluate the effectiveness of R8-Lip in vivo, we examined an antitumor effect in mice bearing E.G7-OVA tumor. C57BL/6 mice were immunized twice with antigens. Then, 1 week after the second immunization, the mice were inoculated with E.G7-OVA cells and the tumor growth was monitored. For this in vivo experiment, we chose stearylated-R8 (STR-R8)/OVA complex as a reference. The R8 peptide is a synthetic CPP based on the amino acid sequence of TAT peptide,22Futaki S Suzuki T Ohashi W Yagami T Tanaka S Ueda K et al.Arginine-rich peptides: an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.J Biol Chem. 2001; 276: 5836-5840Crossref PubMed Scopus (1418) Google Scholar and the stearylated moiety of STR-R8 should form a tight complex with OVA due to its hydrophobicity. As shown in Figure 7, E.G7-OVA tumor inoculated into the left flank of the mice grew day by day. The empty R8-Lip and OVA solution did not have any significant effect. On the other hand, both R8-Lip and STR-R8/OVA had a significant inhibitory effect on tumor growth compared to no treatment, OVA solution, or empty R8-Lip. The difference in antitumor effect between R8-Lip and STR-R8/OVA was not statistically significant, because the effect of STR-R8/OVA was much more variable compared to R8-Lip (Figure 7b). However, the results suggested that R8-Lip tended to inhibit tumor growth efficiently because the mean tumor volumes of R8-Lip were smaller than those of STR-R8/OVA.Figure 7Antitumor effect of R8-modified liposomes (R8-Lip). (a) C57BL/6 mice were subcutaneously immunized twice with R8-Lip, stearylated-R8 (STR-R8)/ovalbumin (OVA), soluble OVA, or empty R8-Lip. One week after the second immunization, mice were inoculated in the left flank with 8 × 105 E.G7-OVA cells. The tumor volume was measured and analyzed statistically. Values are the mean ± SD (n = 4–5). (b) The tumor volume of individual mice on day 20.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionCPPs are found in various viral protein sequences, such as TAT of human immunodeficiency virus, and can effectively penetrate cell membranes.1Gupta B Levchenko TS Torchilin VP Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides.Adv Drug Deliv Rev. 2005; 57: 637-651Crossref PubMed Scopus (563) Google Scholar Recently, we found that the cellular entrance pathway and intracellular trafficking of liposomes can be controlled by changing the topological properties of the CPP R8 peptide, such as its density and flexibility, on the liposomal surface.13Khalil IA Kogure K Futaki S Harashima H High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.J Biol Chem. 2006; 281: 3544-3551Crossref PubMed Scopus (341) Google Scholar By controlling the topology of R8 in this way, high-density R8-Lip can avoid lysosomal degradation and instead be transferred to the cytosol. Thus, in this study, we hypothesized that efficient MHC-I presentation should be achievable by governing the intracellular trafficking of antigen-encapsulating liposomes through topological control of CPP presentation on the liposomal surface. We tested this hypothesis by evaluating the antigen-presentation ability, intracellular trafficking, and in vivo antitumor effect of R8-Lip.Previously, we showed that high-density R8 modification induces macropinocytosis in NIH3T3 cells.13Khalil IA Kogure K Futaki S Harashima H High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.J Biol Chem. 2006; 281: 3544-3551Crossref PubMed Scopus (341) Google Scholar In addition, Nakase et al.23Nakase I Niwa M Takeuchi T Sonomura K Kawabata N Koike Y et al.Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.Mol Ther. 2004; 10: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar reported that free R8 peptide is taken up through macropinocytosis. Thus, we expected that R8-Lip would be internalized into BMDCs via macropinocytosis, whereas other liposomes should be taken up by classical endocytosis. Contrary to this expectation, cellular uptake of both R8-Lip and Cat-Lip was by the same mechanism, macropinocytosis (Figure 1b). Macropinocytosis is one of the constitutive uptake pathways in DCs, and it can internalize large substances (up to 1 μm).24Norbury CC Drinking a lot is good for dendritic cel