Cell-mediated immune response (cellular immunity) plays a crucially important role in the treatment of chronic infectious disease, elimination of virus-infected cells, prophylactic vaccine, and cancer immunotherapy because cytotoxic T lymphocytes (CTLs) can attack tumor cells and virus-infected cells directly [1
]. For induction of CTL-based cellular immunity, delivery of antigens into the cytosol of antigen-presenting cells, such as macrophages or dendritic cells (DCs), is necessary, which leads to the processing of antigens via proteasome, antigen-loading on major histocompatibility complex (MHC) class I, and antigen presentation to CD8-positive T lymphocytes [5
]. In general, after an exogenous antigen is taken up by antigen-presenting cells via endocytosis it is processed in endo/lysosomes, which leads to MHC class II-mediated antigen presentation to CD4-positive T lymphocytes [5
]. Therefore, promotion of exogenous antigen transfer into cytosol using endosomolytic reagents or intracellular delivery carriers is necessary to achieve antigen-specific cellular immunity. This process is known as “cross-presentation” [7
]. For instance, endosomolytic reagents, such as chloroquine, promote the transfer of antigen into cytosol of dendritic cells in vitro, probably because of its pH-buffering effect in endo/lysosomes, which induce cross-presentation [8
]. However, low-molecular-weight reagents might be diluted to less than active concentration in the body and might have difficulty achieving promotion of cytosolic transfer of antigens under in vivo conditions.
An antigen delivery carrier is another candidate to promote cross-presentation, even under in vivo conditions. Typically, fusogenic protein-incorporated liposomes such as Virosome or Sendai virus fusogenic protein-loaded liposomes are used for the cytoplasmic delivery of antigens [10
]. These antigen-loaded liposomes can deliver antigens into cytosol via fusion with endosomal membranes or plasma membranes. Synthetic endosomolytic materials are also used as cytoplasmic delivery systems. Poly(carboxylic acid)s are well-studied pH-responsive endosomolytic materials because poly(carboxylic acid)s change their structure from a coil to a globule after protonation of carboxyl groups and interact with lipid membranes via hydrogen bond formation with phosphate groups on membranes and hydrophobic interactions [12
]. Poly(acrylic acid) derivatives, such as poly(ethyl acrylic acid) and poly(propylacrylic acid), achieve membrane lysis at acidic pH. Actually, conjugation of poly(propylacrylic acid) to antigenic proteins promotes cross-presentation in vitro and the induction of antigen-specific cellular immune response in vivo [15
]. We also developed pH-responsive polymers using carboxylated polyallylamines, polyglycidols, or polysaccharides [17
]. Particularly, liposomes modified with carboxylated polyglycidols or carboxylated polysaccharides having ether oxygen atoms in their backbone exhibited membrane fusion with the target membrane under weakly-acidic pH. Model antigenic protein, ovalbumin (OVA), was delivered into the cytosol of DCs and OVA-specific cellular immunity was induced, resulting in tumor regression in tumor-bearing mice [20
]. Consequently, pH-responsive polymer-modified liposomes are effective for the induction of cellular immunity by membrane fusion responding to weakly-acidic pH in endosomes of DCs. However, precise quality control of responsiveness and pH where membrane fusion is induced might be difficult because of the polydispersity of synthetic polymers. Therefore, a more versatile cytoplasmic delivery carrier is desired with simple composition from the viewpoint of practical or clinical use.
Recently, we developed a pH-responsive micelle-based intracellular delivery system using dilauroyl phosphatidylcholine (DLPC) and deoxycholic acid (Figure 1
). The molecular assembly behavior of DLPC and deoxycholic acid mixture were evaluated precisely using X-ray scattering analysis. They formed micelles with average size of 12 nm and negative zeta potential [25
]. The DLPC/deoxycholic acid micelles showed membrane disruptive activity under weakly-acidic pH [25
]. At acidic pH, protonation of carboxyl group in deoxycholic acid changed the micelle surface properties from hydrophilic to hydrophobic, which promotes the interaction of micelle with lipid membrane. These micelles also promoted the transfer of co-existing OVA molecules into cytosol of macrophages [25
]. Considering the cytoplasmic delivery performance of DLPC/deoxycholic acid micelles, in this study, the pH-responsive membrane disruptive mechanism of DLPC/deoxycholic acid micelles was further evaluated using a fluorescence resonance energy transfer technique. In addition, the interaction of DLPC/deoxycholic acid micelles with DCs, which are professional antigen-presenting cells and play a crucial role for induction of CTLs, were examined. Furthermore, induction of cellular immunity in vivo by these micelles was investigated.
2. Materials and Methods
Egg yolk phosphatidylcholine (EYPC), l
-dioleoyl phosphatidylethanolamine (DOPE), and 1,2-dilauroyl-sn
-glycero-3-phosphocholine (DLPC) were purchased from NOF Corp. (Tokyo, Japan). N
-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) dioleoyl phosphatidylethanolamine (NBD-PE), lissamine rhodamine B-sulfonyl phosphatidylethanolamine (Rh-PE) and cholesteryl hemisuccinate (CHEMS) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). Deoxycholic acid sodium salt monohydrate, oleic acid, and chlorpromazine hydrochloride were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Pyranine was purchased from Tokyo Chemical Industries Ltd. (Tokyo, Japan). p
-pyridinium bromide (DPX), filipin complex from Streptomyces filipinensis
, OVA, and Triton X-100 were purchased from Sigma (St. Louis, MO, USA). FITC-labeled ovalbumin (FITC-OVA) were prepared as reported previously [24
]. 2-Morpholinoethanesulfomic acid sodium salt (MES) was purchased from Merck (Darmstadt, Germany). Amiloride hydrochloride dihydrate was obtained from LKT Laboratories Inc. (St. Paul, MN, USA). LysoTracker Red was purchased from Lonza (Walkersville, MD, USA). CpG-ODN (ODN-2395) was obtained from Invitrogen Corp. (San Diego, CA, USA). OVA CTL epitope peptide (SIINFEKL) was obtained from PH Japan Co., Ltd. (Hiroshima, Japan).
2.2. Preparation of Micelles or Liposomes
A thin membrane of phospholipid (1 μmol, EYPC or DLPC) and/or deoxycholic acid (1.6 μmol) was dispersed in 1 mL of Dulbecco’s phosphate-buffered saline (dPBS) or MES buffer (25 mM MES and 125 mM NaCl, pH 7.4) using a bath-type sonicator for 2 min (final lipid concentration, 1 μmol/mL). pH-sensitive liposomes composed of DOPE/oleic acid or DOPE/CHEMS were prepared according to earlier reports of the literature [26
]. Briefly, a thin membrane of DOPE (1 μmol) and CHEMS (0.67 μmol) or oleic acid (0.43 μmol) was dispersed in 1 mL of acetate buffer (25 mM acetate and 125 mM NaCl, pH 7.4) using a bath-type sonicator for 2 min.
Pyranine-loaded liposomes were prepared from thin membrane of EYPC dispersed by aqueous 35 mM pyranine, 50 mM DPX, and 25 mM MES solution (pH 7.4). The mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was extruded through a polycarbonate membrane with pore size of 100 nm. The liposome suspension was applied to a G100 column to remove free pyranine from the pyranine-loaded liposomes.
For lipid mixing assay, EYPC liposomes containing NBD-PE and Rh-PE were prepared using the method described above with the membrane composed of EYPC, NBD-PE, and Rh-PE (98.8:0.6:0.6, mol/mol/mol).
For evaluation of cellular association of micelles, DLPC/deoxycholic acid micelles containing Rh-PE were prepared as described above except that mixtures of lipids containing Rh-PE (0.6 mol %) were dispersed in dPBS.
For intracellular distribution of micelles or liposomes, liposomes or micelles containing NBD-PE and Rh-PE were prepared as described above except that mixtures of lipids containing NBD-PE and Rh-PE (each 0.6 mol %) was dispersed in dPBS.
2.3. Atomic Force Microscopy (AFM)
AFM measurements were taken using a probe station and a unit system of the scanning probe microscopy system (SPI3800, SPA400; Seiko Instruments Inc., Chiba, Japan). The silicon cantilever (SI-DF40; Seiko Instruments Inc., Chiba, Japan) had a spring constant of 16 N/m. The micelle suspension was applied to freshly cleaved mica and incubated on the mica for 30 min. Measurements were taken in dynamic force mode (noncontact mode).
2.4. Cell Culture
DC2.4 cells, an immature murine DC line, were provided by Dr. K. L. Rock (Harvard Medical School, Boston, MA, USA). They were grown in RPMI 1640 supplemented with 10% Fetal bovine serum (FBS) (MP Biomedicals Inc., Santa Ana, CA, USA), 2 mM l
-glutamine, 100 μM non-essential amino acids (Gibco, Inc., Billings, MT, USA), 50 μM 2-mercaptoethanol, and antibiotics at 37 °C [28
2.5. Interaction of Micelles with Liposomes
Pyranine-loaded liposomes (final lipid concentration; 6.7 nmol/mL) were mixed with equal amounts of micelles or liposomes in MES buffer of varying pH at 37 °C for 90 min. The fluorescence intensity at 512 nm with excitation at 416 nm was followed using a spectrofluorometer (FP-6500; Jasco Corp., Tokyo, Japan). The percentage of leakage of pyranine from liposomes was defined as:
Leakage (%) = (Lt − Li)/(Lf − Li) × 100
respectively represent the initial and intermediary fluorescence intensities. Lf
is the fluorescence intensity after addition of Triton X-100 (final concentration: 0.1%) to achieve complete membrane disruption.
2.6. Lipid Mixing Assay
Lipid mixing behavior between EYPC liposomes and various lipid suspensions (Deoyxcholic acid suspension, EYPC liposome, EYPC/deoxycholic acid micelle, DLPC liposome, and DLPC/deoxycholic acid micelle) was evaluated using the resonance energy transfer between NBD-PE and Rh-PE on EYPC liposomes. EYPC liposomes containing NBD-PE and Rh-PE (final concentration of lipid 6.6 μM) were mixed with various lipid suspensions (final concentration of lipid 6.6 μM) in 25 mM MES and 125 mM NaCl solution of varying pHs (4.5–7.4) and fluorescence intensities of NBD-PE and Rh-PE followed. Lipid mixing was followed by monitoring the fluorescence intensity ratio of NBD-PE to Rh-PE. The excitation wavelength of NBD-PE was 450 nm. The monitoring wavelengths for NBD-PE and Rh-PE were, respectively, 530 and 580 nm. To achieve complete lipid mixing, samples were dissolved in methanol, dried by evaporation, and resuspended in water.
2.7. Cellular Association of Micelles
The DC2.4 cells (5 × 104
cells) cultured for three days in 24-well plates were washed with Hank’s balanced salt solution (HBSS, Sigma); then they were incubated in serum-free RPMI medium (500 μL). Micelles (25 μL) containing Rh-PE (0.6 mol %) were added gently to the cells and were incubated for 5 h at 4 or 37 °C. After incubation, the cells were washed three times with HBSS. The fluorescence intensity of these cells was found using a flow cytometer (Coulter Epics XL; Coulter Corp., Brea, CA, USA). For inhibition assay, cells were pre-treated with each inhibitor (chlorpromazine hydrochloride: 6.25–25 μg/mL [29
], filipin: 2.5–10 μg/mL [29
], amiloride: 2.5–10 mM [30
]) for 30 min. After washing twice, micelles were added to these cells.
2.8. Detection of Intracellular Lipid Mixing
The DC2.4 cells (1.5 × 105 cells) cultured for three days in 35-mm glass-bottom dishes were washed with HBSS; they were then incubated in serum-free RPMI medium (2 mL). Then, the liposomes or micelles (100 μL) containing NBD-PE and Rh-PE (each 0.6 mol %) were added gently to the medium of the cells and were incubated for 5 h at 37 °C. After incubation, the cells were washed with HBSS three times and were analyzed using confocal laser scanning microscopy (CLSM, LSM 5 EXCITER; Carl Zeiss Co. Inc., Oberkochen, Germany). Fluorescence of NBD-PE and Rh-PE was observed through specific pass filters (λem = 500–530 nm for NBD-PE and λem > 560 nm for Rh-PE) with excitation at 488 nm.
2.9. Cytoplasmic Delivery of Antigenic Proteins
The DC2.4 cells (1.5 × 105 cells) cultured for 3 days in 35-mm glass-bottom dishes were washed with HBSS; then they were incubated in serum-free RPMI medium (2 mL). The FITC-OVA (50 μg) with or without DLPC liposomes or DLPC/deoxycholic acid micelles (100 μL) were added gently to the cells and were incubated for 4 h at 37 °C. After incubation, the cells were washed with HBSS three times. CLSM analysis of these cells was performed. Intracellular acidic compartments were stained with LysoTracker Red according to the manufacturer’s instructions.
Female C57BL/6 mice (H-2b, six weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The experiments were conducted in accordance with the guidelines for animal experimentation of Terumo Corp.
2.11. ELIspot Assay
C57BL/6 mice were immunized intradermally with 16 μg of OVA, a mixture of OVA and CpG-ODN (2 μg), or a mixture of OVA, CpG-ODN and DLPC/deoxycholic acid micelles (1 μmol/mL, 2 μL) (total volume: 20 μL/mouse). After seven days, splenocytes were collected from each group. Splenocytes (2 × 106 cells/well) were stimulated in vitro with 20 μg/mL of OVA peptide (SIINFEKL) or were left unstimulated (negative controls) for 40 h in a 96-well plate coated with anti-murine IFN-γ mAb. The IFN-γ-producing cells in the splenocyte populations were measured using mouse IFN-γ ELISPOT set and AEC substrate set (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. The data were expressed as the mean spot forming units (SFU) per million cells ± standard error (S.E.).
2.12. Statistical Analysis
The Tukey-Kramer method using Microsoft Excel was employed in the statistical evaluation of the results in Figure 3.
Cytoplasmic delivery of antigen is crucial to induce antigen-specific CTL responses, which plays an important role in attacking tumor cells and eliminating virus-infected cells. Promotion of the cytoplasmic delivery of antigen using pH-responsive materials has been studied intensively, such as viral protein-incorporated liposomes, pH-sensitive liposomes, polymeric particles, and nanogels [10
]. However, efficient vaccine delivery carriers with practical levels were only slightly reported. Here, we proposed an antigen-specific CTL induction system using a simple mixture of phospholipid and deoxycholic acid. Reportedly, a mixture of phospholipid with short alkyl chains (C12 or C10) and deoxycholic acid demonstrated pH-sensitive membrane disruptive ability [25
]. Particularly, a combination of DLPC and deoxycholic acid exhibited pH-responsiveness in weakly-acidic pH regions corresponding to endo/lysosomal pH and formed nano-size micelles promoted intracellular delivery of macromolecules into macrophages [25
]. As described in this paper, we specifically examined pH-responsive properties of DLPC/deoxycholic acid micelles and their application to CTL induction system.
Within 20 min, DLPC/deoxycholic acid micelles induced stronger membrane disruption at acidic pH (Figure 2
a) than conventional pH-sensitive liposomes did (Figure 2
b). Such a quick and strong pH-response might be beneficial to achieving membrane disruption of target membranes (endosomes or lysosomes) during the endocytic pathway and endosomal escape. At pH 4.5, almost complete lipid mixing was achieved by DLPC/deoxycholic acid micelles (Figure 2
c), whereas leakage was 65% at the same pH (Figure 2
b). This result suggests that DLPC/deoxycholic acid micelles first induce lipid mixing with the target membrane when micelles approach the target membrane because of high fluidity and flip-flop activity of DLPC [35
]. Mixing of DLPC to the target membrane might decrease packing of the bilayer structure and increase the membrane permeability, causing a leakage of contents.
DLPC/deoxycholic acid micelles were taken up by dendritic cells mainly via macropinocytosis (Figure 3
d) with little absorption in the cells (Figure 3
a). This result might be attributable to the negatively charged surface of DLPC/deoxycholic acid micelles (‒30 mV) and low interaction with the cellular membrane at neutral pH. EYPC/deoxycholic acid micelles, which has almost the same size and surface properties with DLPC/deoxycholic acid micelles, showed relatively low cellular association compared with DLPC/deoxycholic acid micelles (Figure 4
b,c). This might be attributed by less fluidity of EYPC molecules in micelles than DLPC, which further suppresses the interaction with the cellular membrane. After internalization to the cells, micelles induced lipid mixing even inside of the cells (Figure 4
c). However, EYPC/deoxycholic acid micelles exhibited no pH-responsive membrane disruption (Figure 2
c) and intracellular lipid mixing (Figure 4
b), which also supports the importance of DLPC for induction of lipid mixing at acidic pH.
The presence of DLPC/deoxycholic acid micelles completely changed the intracellular fate of antigenic proteins internalized to cells along with micelles (Figure 5
c). FITC-OVA molecules were delivered to cytosol of dendritic cells in the presence of DLPC/deoxycholic acid micelles. Lipid mixing of DLPC/deoxycholic acid micelles with endosomes might promote leakage of the FITC-OVA molecules to cytosol. Furthermore, lipid mixing and cytoplasmic delivery of FITC-OVA were observed even within 1 h (Supplementary Materials Figure S4
), which reflect immediate pH-responsiveness of DLPC/deoxycholic acid micelles (Figure 2
). Considering the cytoplasmic delivery mechanism by DLPC/deoxycholic acid micelles, both micelles and antigenic proteins should exist in the same endo/lysosome when endo/lysosomes are destabilized. Reportedly, endosomes and lysosomes are formed by the fusion of numerous endocytic vesicles during endocytic processes [36
]. For example, gold nanoparticles were first taken up into endosomes as single particles and were then accumulated into the same endocytic vesicles during endocytic transportation [37
]. Additionally, DLPC/deoxycholic acid micelles and FITC-OVA molecules might be taken up by cells independently, but micelles and FITC-OVA molecules might be sorted to the same endo/lysosomes during the endocytosis process, leading to cytoplasmic delivery of FITC-OVA by the pH-response of the micelles.
As expected, cytoplasmic delivery of antigen induced OVA-specific immune response in mice (Figure 6
). By contrast, DLPC/deoxycholic acid micelles did not induce CTL response without CpG-ODN (Figure 6
). Actually, addition of DLPC/deoxycholic acid micelles to dendritic cells hardly induced maturation of the dendritic cells even in high concentration (Supplementary Materials Figure S5
). Such a property might be attributed to the weak interaction of micelles with the cellular membrane because of their negatively-charged surface. In addition, intradermally-injected micelles reached regional lymph node within 30 min and disappeared from injected site for 4 h (Supplementary Materials Figure S6
). Micelles might be taken up by dermal dendritic cells and these dendritic cells might migrate to the lymph nodes. Another possibility is the direct transfer of micelles to lymph nodes because of its quite small particle size. After migration to the lymph nodes, DLPC/deoxycholic acid micelles delivered OVA into cytosol of dendritic cells, leading to induction of OVA-specific CTL responses. These results suggest that DLPC/deoxycholic acid micelles act as an “enhancer” of transfer to lymph node and cytoplasmic antigen delivery. Such characteristics of DLPC/deoxycholic acid micelles might be beneficial as additives of conventional or commercially-available vaccines because the DLPC/deoxycholic acid micelle, itself, does not induce any kind of immune response and does not disturb the intrinsic performance of vaccines. In addition, deoxycholic acid has already been approved by the FDA as an active principal of injectable drugs. DLPC also has a history of clinical use [38
]. Therefore, pH-responsive DLPC/deoxycholic acid micelles are promising as additives of commercially-available vaccines to enhance cross-presentation of antigens for antigen-specific cellular immunity.