Influence of Endosomal Escape and Degradation of α

Article pubs.acs.org/molecularpharmaceutics

Influence of Endosomal Escape and Degradation of α‑Galactosylceramide Loaded Liposomes on CD1d Antigen Presentation Takashi Nakamura, Moeka Kuroi, and Hideyoshi Harashima* Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan

ABSTRACT: Alpha-galactosylceramide (GC), a lipid antigen present on CD1d molecules, is a unique adjuvant that enables a strong antitumor effect to be induced via activation of natural killer T cells. We previously reported that a liposomal formulation of GC significantly enhanced GC presentation via CD1d and antitumor immunity. However, the influence of the intracellular fate of liposomes controlled by the lipid composition on GC presentation using GC-loaded liposomes (GC-Lip) remains unclear. In this study, we prepared a GC-Lip formulation by incorporating dioleoyl-phosphatidylethanolamine (DOPE)/cholesterol, egg phosphatidylcholine (EPC)/cholesterol, and distearoyl phosphocholine (DSPC)/cholesterol, and investigated the relationship between the intracellular trafficking of GC-Lip and GC presentation in antigen-presenting cells. When GC-Lip was prepared using DOPE, a fusogenic lipid, the endosomal escape of liposomes was enhanced, resulting in a decrease in GC presentation of CD1d, compared to the EPC based GC-Lip (EPC/GC-Lip). The stability of liposomes in endosomes/lysosomes had no influence on GC presentation. The DSPC based GC-Lip (DSPC/GC-Lip) induced GC presentation without any detectable degradation in liposomal structure, although the EPC/GC-Lip induced GC presentation with degradation of liposomal structure. The efficiency of GC presentation between EPC/GC-Lip and DSPC/GC-Lip was comparable. These GC presentations that were independent of the degradation of liposomes were dominated by saposins, sphingolipid activator proteins. Our findings reveal that GC presentation on CD1d from the fluid liposomes involves the action of saposins, regardless of whether liposome degradation occurs. This insight can be of use in terms of developing GC-Lip formulation for efficient GC presentation. KEYWORDS: liposomes, lipid antigen presentation, CD1d, intracellular fate of liposomes

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INTRODUCTION Alpha-galactosylceramide (GC), a synthetic glycolipid, is a unique adjuvant that enables the activation of both specific and nonspecific immunity. GC is presented by CD1d molecules, lipid antigen-presenting molecules, in antigen-presenting cells (APCs) to invariant natural killer T (NKT) cells.1−3 The activated NKT cells produce large amounts of interferon (IFN) γ, resulting in the activation of killer T cells and natural killer (NK) cells. The activation of NKT cells by GC results in the development of antimetastatic tumor activity.1,4 This protective activity is dependent on the production of IFN-γ by NKT cells and on the activation of killer T cells and NK cells.5 The unique and strong antitumor effects of GC make it promising for use in clinical applications. GC-loaded dendritic cells are often used in clinical trials, because the intravenous injection of a GC does not result in measurable clinical benefits.6−10 These results © XXXX American Chemical Society

suggest that GC is not taken up efficiently by APCs when intravenously administered. Hence, it becomes necessary to control the disposition and cellular uptake of intravenously administered GC via appropriate delivery systems. In a previous study, we investigated the use of stearylated octaarginine (STR-R8) modified liposomes (R8-Lip) as vaccine delivery systems.11−16 Octaarginine (R8) is a cell-penetrating peptide and can be useful for delivering various molecules to cells.17 To apply GC to direct intravenous administration, we incorporated GC into R8-Lip and investigated the GCmediated antitumor immunity of the preparation.15 The Received: October 18, 2014 Revised: May 25, 2015 Accepted: June 24, 2015

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DOI: 10.1021/mp500704e Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

was purchased from R&D Systems, Inc. (Minneapolis, MN). The anti-PSAP siRNAs (siRNA-1:5′-TAGATCTATTTGACTTAATAA-3′, 5′-TTATTAATCAAATAGATCTA-3′; siRNA-2:5′-CACTGAGGAGCTCCTAGTTAA-3′, 5′-TTAACTAGGAGCTCCTCAGTG-3′; siRNA-3:5′-CAAGAACTACGTGGACCAGTA-3′, 5′-TACTGGTCCACGTAGTTCTTG-3′; siRNA-4:5′-CAGAATGATCAGGCCTTAGA-3′, 5′TCTAAGGCCTGATCATTCTG-3′) were purchased from QIAGEN (Hilden, Germany). Control siRNA (5′-AGAuCACCCuCCUUAAAuAUU-3′, 5′-UAUUUAAGGAGGGUGAuCUUU-3′, 2′-OMe-modified nucleotides are shown in lowercase letters) was purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan). Cell. JAWSII cells (the murine dendritic cell line) were purchased from the American Type Culture Collection (Manassas, VA) and cultured in α-MEM containing 20% fetal bovine serum (FBS), 100 U/mL penicillin−streptomycin, 4 mM L-glutamine, 1 mM sodium pyruvate, and 5 ng/mL GMCSF. Preparation of Liposomes. The GC-Lip was prepared as reported previously.15 The EPC/GC-Lip was composed of EPC, Chol, and STR-R8 (molar ratio: 70:30:5). DOPE/GCLip was composed of DOPE, Chol, EPC, and STR-R8 (molar ratio: 70:20:10:5). DSPC/GC-Lip was composed of DSPC, Chol, and STR-R8 (molar ratio: 70:30:5). These lipids (total 400 nmol) and STR-R8 (20 nmol) in chloroform solutions were initially mixed in a test tube, and 50 μg of GC was then added. For the fluorescent labeling of the GC-Lip, 4 nmol of NBD-DOPE was added to the chloroform solutions. The solvent was removed with a stream of nitrogen to produce a lipid film, after which 200 μL of 10 mM HEPES (pH 7.4) was added to the lipid film. The resulting mixture was then incubated for 30 min at room temperature or 60 °C in the case of EPC/GC-Lip and DOPE/GC-Lip or DSPC/GC-Lip, respectively, to hydrate the film. The hydrated lipid film was then sonicated in a bath type sonicator to produce the GC-Lip. EPC based liposomes encapsulating Alexa Fluor 488 labeled 10,000 kDa dextran (Dx) (Life Technologies, Carlsbad, CA) (EPC-Lip/Dx) were composed of EPC, Chol, and STR-R8 (70:30:5 molar ratio). DOPE based liposomes encapsulating Dx (DOPE-Lip/Dx) were composed of DOPE, Chol, EPC, and STR-R8 (70:20:10:5 molar ratio). These lipids (total 5000 nmol) and STR-R8 (250 nmol) in chloroform solutions were initially mixed in a test tube. The solvent was removed with a stream of nitrogen to produce a lipid film, after which 500 μL of 10 mM HEPES (pH 7.4) containing 0.25 mg/mL Dx was added to the lipid film. The resulting mixture was then incubated for 15 min at 60 °C to hydrate the film. The hydrated lipid film was then mixed to produce liposomes. The liposomal solution was subjected to five freeze−thaw cycles followed by extrusion through a 400 nm filter (Nucleopore, AVANTI Polar Lipids, Inc., Alabaster, AL). Unencapsulated Dx was removed by ultracentrifugation at 80000g for 30 min at 4 °C. NBD-labeled EPC-Lip encapsulating sulforhodamine B (RhoB) (Life Technologies, Carlsbad, CA) (EPC/NBD-Lip/ RhoB) was composed of EPC, Chol, and STR-R8 (70:30:5 molar ratio). NBD-labeled DSPC-Lip encapsulating RhoB (DSPC/NBD-Lip/RhoB) was composed of DSPC, Chol and STR-R8 (70:30:5 molar ratio). These lipids (total 5000 nmol) and STR-R8 (250 nmol) in chloroform solutions were initially mixed in a test tube, and 50 nmol of NBD-DOPE was then added. The solvent was removed with a stream of nitrogen to produce a lipid film, after which 500 μL of 10 mM HEPES (pH

liposomal formulation of GC (GC-Lip) resulted in a dramatic enhancement in NKT cell expansion, IFN-γ production, and antitumor effects against the metastasis of B16-F10 melanoma in lungs, compared to a treatment of soluble GC. In addition, the enhancement in GC-mediated antitumor immunity can be attributed to the control of biodistribution of GC by liposomes, i.e., GC was efficiently delivered to APCs. On the other hand, GC must be present on CD1d molecules after being taken up by APCs. GC is present on CD1d molecules in endosomes and lysosomes.18 However, little is known concerning the influence of the intracellular fate of liposomes on GC presentation in APCs. It is generally thought that the intracellular trafficking of cargo in liposomes has an influence on the function of cargo.19,20 In addition, more detailed information regarding the influence of the intracellular fate of liposomes on GC presentation would be of help in the development of liposomal formulations of GC in the future. To address this issue, we investigated the effect of the intracellular fate of liposomes mediated by lipid composition on GC presentation in APCs. We focused on the influence of endosomal escape and endosomal degradation of GC-Lip on GC presentation, since GC is present on CD1d molecules in endosomes and lysosomes. In this study, three types of fluid liposomes in which GC was incorporated were prepared as follows: liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol (Chol) (DOPE-Lip), liposomes composed of egg phosphatidylcholine (EPC) and Chol (EPC-Lip), and liposomes composed of 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) and Chol (DSPC-Lip). All of the liposomes used in this study contained STR-R8. DOPE is generally used as a fusogenic lipid, which adopts an inverted hexagonal phase and promotes the fusion of lipid bilayers. Thus, DOPE-Lip appears to induce fusion with endosomal/ lysosomal membranes and enhances endosomal escape, resulting in a decrease in GC presentation. The EPC-Lip was used in a previous study.15 EPC and DSPC are nonfusogenic, although the phase transition temperature of each is different. The phase transition temperature of DSPC is higher than that of EPC. Therefore, the DSPC-Lip would be expected to be more stable than EPC-Lip in endosomes/lysosomes, resulting in a decrease in GC presentation. We examined the effect of liposomal composition on GC presentation by comparing the degree of intracellular fate of each type of liposome. Recent studies have shown that saposins, a family of small, nonenzymatic lipid-binding proteins, facilitate CD1 lipid loading in the endosomal compartment.21−24 We also examined the effect of saposin by silencing the prosaposin (PSAP) gene with small interfering RNA (siRNA).

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MATERIALS AND METHODS Materials. EPC and DSPC was purchased from the NOF Corporation (Tokyo, Japan). DOPE, N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-DOPE (NBD-DOPE), and Chol were obtained from AVANTI Polar Lipids, Inc. (Alabaster, AL). STR-R8 was synthesized by KURABO industries (Osaka, Japan). GC (KRN7000) was obtained from Funakoshi Co. Ltd. (Tokyo, Japan). Anti-mouse GC:CD1d complex and anti-mouse alpha GC:CD1d complex PE were purchased from eBioscience (San Diego, CA). Anti-mouse CD107a (LAMP-1) was obtained from AbD Serotec (Oxford, U.K.). Brilliant Violet421 goat antimouse IgG and DyLight594 goat anti-rat IgG were purchased from Biolegend (San Diego, CA). Mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) B

DOI: 10.1021/mp500704e Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics 7.4) containing 0.1 mM RhoB was added to the lipid film. The resulting mixture was then incubated for 15 min at 60 °C to hydrate the film. The hydrated lipid film was then mixed to produce liposomes. The liposomal solution was subjected to five freeze−thaw cycles followed by extrusion through a 400 nm filter. Unencapsulated RhoB was removed by ultracentrifugation at 80000g for 30 min at 4 °C. The diameter of the liposomes was determined by dynamic light scattering, and zeta potentials were determined by laserDoppler velocimetry with a ZETASIZER Nano (ZEN3600, Malvern Instruments Ltd., Malvern, WR, U.K.). The characteristics of the liposome formulations are shown in Table 1.

Quantification of GC Presentation on CD1d in JAWSII Cells. 2 × 105 JAWSII cells were cultured in a 6-well plate for 48 h. The cells were washed with PBS, and the medium was then replaced with 1 mL of FBS-free medium containing each GC-Lip. The cells were then incubated for 1 h. After the incubation, the cells were washed twice with PBS containing 20 U/mL heparin. Thereafter, the medium was replaced with fresh medium containing 20% FBS, and the cells were incubated for an additional 23 h. After the incubation, the cells were washed with PBS and collected into tubes. The cells were stained with an anti-mouse alpha GC:CD1d complex PE. The fluorescence of the stained cells was measured by FACSCalibur and then analyzed using the Cell Quest software. Analysis of Intracellular Trafficking of Liposomes and GC Presentation on CD1d in JAWSII Cells by CLSM. 2 × 105 JAWSII cells were cultured in a 35 mm glass base dish for 48 h. The cells were washed with PBS, and the medium was then replaced with 1 mL of FBS-free medium containing NBDlabeled GC-Lip (GC concentration was 3 μg/mL). The cells were then incubated for 1 h. After the incubation, the cells were washed twice with PBS containing 20 U/mL heparin and collected into tubes. For the 1 h observation, the fixation and permeabilization of the cells was then performed with Cytofix/ Cytoperm Buffer (BD Biosciences, Mountain View, CA) at room temperature for 20 min. The cells were incubated in PBS containing 0.1% bovine serum albumin, after the cells were washed with PBS. For the 5 or 24 h observations, the cells were incubated for an additional 4 or 23 h. The fixation and permeabilization of the cells was then performed with Cytofix/ Cytoperm Buffer in a similar manner. The fixed and permeabilized cells were stained by an anti-mouse CD107a (LAMP-1) and an anti-mouse GC:CD1d complex at 37 °C for 1 h. After staining with the primary antibodies, the cells were stained with DyLight594 goat anti-rat IgG and Brilliant Violet421 goat anti-mouse IgG at 37 °C for 1 h. The resulting stained cells were observed by Fluoview FV10i. Quantification of Residual Amount of Liposomes in JAWSII Cells. 2 × 105 JAWSII cells were cultured in a 6-well plate for 48 h. The cells were washed with PBS, and the medium was then replaced with 1 mL of FBS-free medium containing each of the NBD-labeled GC-Lip formulations. The cells were then incubated for 1 h. After the incubation, the cells were washed twice with PBS containing 20 U/mL heparin. The cells were cultured for an additional 23 h and were collected into tubes. The fluorescence was measured by FACSCalibur and was then analyzed using the Cell Quest software. Integrity of Liposomes in Endosomes/Lysosomes of JAWSII Cells. 2 × 105 JAWSII cells were cultured in a 35 mm glass base dish for 48 h. The cells were washed with PBS, and the medium was then replaced with 1 mL of FBS-free medium containing EPC/NBD-Lip/RhoB or DSPC/NBD-Lip/RhoB. The cells were then incubated for 1 h. After the incubation, the cells were washed twice with PBS containing 20 U/mL heparin and were observed. For the 24 h observation, the cells were incubated for an additional 23 h. The cells were observed by Fluoview FV10i after staining with Hoechst33342. Knockdown of PSAP Gene by siRNA. 2 × 105 JAWSII cells were cultured in a 6-well plate for 48 h. At day 0, after the cells were washed with PBS, the cells were transfected with anti-PSAP siRNA or control siRNA at 80 nM with Lipofectamine RNAi MAX (Life Technologies, Carlsbad, CA) in OPTIMEM for 2 h. The anti-PSAP siRNA was a mixture of four different siRNA sequences (siRNA 1−4). Culture medium was

Table 1. Characteristics of Liposomesa liposomes EPC/GC-Lip EPC-Lip/Dx EPC/NBD/GC-Lip DOPE/GC-Lip DOPE-Lip/Dx DOPE/NBD/GCLip DSPC/GC-Lip DSPC/NBD/GC-Lip a

diameter (nm) 107.9 166.1 109.4 128.0 177.9 137.3

± ± ± ± ± ±

2.6 1.0 2.7 5.0 2.6 2.4

112.3 ± 3.2 113.2 ± 2.5

PDI 0.219 0.132 0.226 0.224 0.137 0.231

± ± ± ± ± ±

0.006 0.016 0.009 0.009 0.017 0.009

0.236 ± 0.011 0.234 ± 0.004

zeta-potential (mV) 37.6 41.7 37.6 42.4 47.1 47.0

± ± ± ± ± ±

0.8 0.5 0.7 1.4 1.3 1.0

47.8 ± 1.5 49.6 ± 0.5

Data are the mean ± SEM of at least three independent experiments.

Quantification of the Residual Amount of Liposomes in Endosomes/Lysosomes. 1 × 105 JAWSII cells were cultured in a 35 mm glass base dish for 48 h. The cells were washed with phosphate buffer saline (PBS), and the medium was then replaced with 1 mL of FBS-free medium containing EPC-Lip/Dx or DOPE-Lip/Dx (lipid concentration was 24 μM). In the case of the 30 min observation, the cells were washed twice with PBS containing 20 U/mL heparin after 30 min after adding liposomes and were observed by confocal laser scanning microscopy (CLSM) (Fluoview FV10i, Tokyo, Japan) after staining with LysoTracker Red. In the case of the 5 h observation, the cells were washed twice with PBS containing 20 U/mL heparin after 1 h after adding liposomes and were then incubated for an additional 4 h. The cells were then observed by CLSM after staining with LysoTracker Red. For quantification of the residual amount of liposomes in endosomes/lysosomes, each 8-bit TIFF image was transferred to an Image-Pro Plus version 4.0 (Media Cybernetics Inc., Silver Spring, MD) to quantify the total brightness and pixel area of each region of interest. The pixel areas of clusters in endosomes/lysosomes, colored yellow, were summed for each XY-plane. The value represents the total amount of dextran in the endosomes/lysosomes in an individual cell. Quantification of Cellular Uptake of GC-Lip in JAWSII Cells. 2 × 105 JAWSII cells were cultured in a 6-well plate for 48 h. The cells were washed with PBS, and the medium was then replaced with 1 mL of FBS-free medium containing each of the NBD-labeled GC-Lip formulations. The cells were then incubated for 1 h. After the incubation, the cells were washed twice with PBS containing 20 U/mL heparin and collected into tubes. The fluorescence in the cells was measured by FACSCalibur (BD Biosciences, Mountain View, CA) and was then analyzed using the Cell Quest software (BD Biosciences, Mountain View, CA). C

DOI: 10.1021/mp500704e Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics added to the cells, followed by a further incubation for 22 h, after which 2 × 105 cells were seeded in a 6-well plate and the resulting preparation was then cultured for 24 h (day 1). At day 2 and day 4, siRNA transfection was performed, similar to that for day 0. At day 3, cell passage was performed, similar to that for day 1. At day 5, the cells were collected and isolated using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instruction. To eliminate DNA contamination, the total RNA was treated with DNase I and 500 ng of total RNA for each sample was then reverse transcribed using a PrimeScript reverse transcription (RT) reagent kit (Takara Bio Inc., Shiga, Japan) with an oligo-dT primer. Quantitative polymerase chain reactions (PCR) were carried out on a 7500 RealTime PCR System (Life Technologies, Carlsbad, CA) in 25 μL aliquots of reaction mixture containing cDNA, with appropriate pairs of primers and SYBER Green Realtime PCR Master Mix (TOYOBO Co., Osaka, Japan). PSPA levels were calculated by the comparative Ct method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous housekeeping gene. The following primer pairs were used. GAPDH: 5′-AACTTTGGCATTGTGGAAGG-3′, 5′-GTCTTCTGGGTGGCAGTGAT3′, synthesized by Life Technologies; PSAP primer pairs were purchased from QIAGEN. The sequences were unreleased. GC Presentation in PSAP-Silenced JAWSII Cells. At day 5 (24 h after the final siRNA transfection), the cells were treated with each of the GC-Lip formulations (GC concentration was 5 μg/mL) and GC presentation was measured by flow cytometry, as described above. Statistical Analysis. Comparisons between two treatments were performed by an unpaired t test. Comparisons between multiple treatments were made using one-way or two-way ANOVA, followed by Tukey−Kramer test. A P-value of