Synthesis of high functionality and quality mannose-grafted lipids to produce macrophage-targeted liposomes

Article abstract of DOI:10.1016/j.ejps.2018.07.036

The mannose receptor, which is responsible for tumor invasion, proliferation, and metastasis in the tumor microenvironment, is overexpressed in tumor-associated macrophages. Mannose is commonly applied to PEGylated liposomes in macrophage-targeted cancer therapy. To develop a high functionality and quality (HFQ) lipid for macrophage-targeted liposomes, we designed a novel mannosylated lipid with improved mannose receptor binding affinity using serine–glycine repeats (SG)_n. We synthesized Man(S)-(SG)₅-SSK-K(Pal)₂ using only a fluorenylmethyloxycarbonyl (Fmoc) protecting group solid-phase peptide synthesis method, which produced a high-quality lipid at a moderately good yield. We then prepared Man-(SG)₅/PEGylated liposomes using a post-insertion technique to insert Man(S)-(SG)₅-SSK-K(Pal)₂ into the PEGylated liposomes. In vitro cell investigations revealed that the Man-(SG)₅/PEGylated liposomes effectively associated with mouse peritoneal macrophages by interacting with the mannose receptors. The results suggest that we produced a novel high-quality, highly functional mannosylated lipid that is suitable for clinical drug delivery applications.

Full text of DOI:10.1016/j.ejps.2018.07.036

Accepted Manuscript
Synthesis of high functionality and quality mannose-grafted lipids
to produce macrophage-targeted liposomes
Masayori Hagimori, Yorinao Chinda, Tadaharu Suga, Kazuto
Yamanami, Naoya Kato, Tatsuo Inamine, Yuki Fuchigami,
Shigeru Kawakami
PII:
DOI:
Reference:
S0928-0987(18)30334-8
doi:10.1016/j.ejps.2018.07.036
PHASCI 4612
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
2 May 2018
9 July 2018
17 July 2018
Please cite this article as: Masayori Hagimori, Yorinao Chinda, Tadaharu Suga, Kazuto
Yamanami, Naoya Kato, Tatsuo Inamine, Yuki Fuchigami, Shigeru Kawakami , Synthesis
of high functionality and quality mannose-grafted lipids to produce macrophage-targeted
liposomes. Phasci (2018), doi:10.1016/j.ejps.2018.07.036
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ACCEPTED MANUSCRIPT
Synthesis of high functionality and quality mannose-grafted lipids to produce
macrophage-targeted liposomes
Masayori Hagimoria,*, Yorinao Chindaa, Tadaharu Sugaa, Kazuto Yamanamia, Naoya
Katoa, Tatsuo Inamineb, Yuki Fuchigamia, Shigeru Kawakamia,*
aDepartment of Pharmaceutical Informatics, Graduate School of Biomedical Sciences,
Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
bDepartment of Pharmacotherapeutics, Graduate School of Biomedical Sciences,
Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
*Corresponding authors:
Masayori Hagimori (e-mail: hagimori@nagasaki-u.ac.jp)
Shigeru Kawakami (e-mail: skawakam@nagasaki-u.ac.jp)
Keywords: mannose; lipid; serine–glycine repeat spacer; macrophage; liposome
ABSTRACT
The mannose receptor, which is responsible for tumor invasion, proliferation, and
metastasis in the tumor microenvironment, is overexpressed in tumor-associated
macrophages. Mannose is commonly applied to PEGylated liposomes in
macrophage-targeted cancer therapy. To develop a high functionality and quality (HFQ)
lipid for macrophage-targeted liposomes, we designed a novel mannosylated lipid with
improved mannose receptor binding affinity using serine–glycine repeats (SG)n. We
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synthesized Man(S)-(SG)5-SSK-K(Pal)2 using only a fluorenylmethyloxycarbonyl
(Fmoc) protecting group solid-phase peptide synthesis method, which produced a
high-quality lipid at a moderately good yield. We then prepared Man-(SG)5/PEGylated
liposomes using a post-insertion technique to insert Man(S)-(SG)5-SSK-K(Pal)2 into the
PEGylated liposomes. In vitro cell investigations revealed that the
Man-(SG)5/PEGylated liposomes eff ectively associated with mouse peritoneal
macrophages by interacting with the mannose receptors. The results suggest that we
produced a novel high-quality, highly functional mannosylated lipid that is suitable for
clinical drug delivery applications.
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1. Introduction
A number of anticancer drugs have been developed; however, cancer is still a major
threat to human health worldwide. Recent studies have revealed that heterogeneity in
the tumor microenvironment—for example in gene expression, cellular morphology,
metabolic activity, metabolism, motility, or metastatic potential—limits the therapeutic
effects of anticancer drugs, and may cause drug resistance (Marusyk and Polyak, 2010).
To overcome these heterogeneities, the use of nanoparticle-based drug delivery system
(DDS) carriers—such as liposomes, polymeric micelles, and nanogels—has attracted
attention as a rational approach to cancer treatment (Friedman et al., 2013; Srinivasarao
et al., 2015). To date, active tumor-targeting liposomes have been created using
functional lipids grafted with ligands that are directed toward specific cancer cell
receptors, or using polymers that are responsive toward stimuli such as temperature, pH,
and enzymes (Fumoto and Kawakami, 2014; Hagimori et al., 2017). However, the
production of DDS carriers with high functionality for clinical applications presents a
challenge owing to the poor quality of the functional lipids and difficulties arising from
the preparation method with regard to reproducibility (Altınoglu et al., 2015; Gerasimov
et al., 1999). Therefore, there is a great demand for high functionality and quality (HFQ)
lipids for clinical DDS applications that can be prepared using a simple method.
Mannose receptors are C-type lectin receptors that are expressed on the surfaces of
macrophages, and have carbohydrate recognition domains bound by mannose (Allavena
et al., 2010; Irache et al., 2008). They have potential roles in both innate and adaptive
immune responses, and are associated with inflammatory and infectious diseases
(Allavena et al., 2010; Irache et al., 2008). Furthermore, mannose receptors are highly
expressed in tumor-associated macrophages that are related to tumor invasion,
proliferation, and metastasis in tumor microenvironments (Qian and Pollard, 2010).
Several macrophage-targeted liposomes grafted with mannose as a ligand have been
developed for cancer treatment (Kono, et al., 2014; Matsui, et al., 2010; Un et al., 2010a,
b; Vinogradov et al., 2014). Poly(ethylene glycol) (PEG) is generally used as a
polymeric steric stabilizer to reduce uptake by the reticuloendothelial system (RES) and
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prolong circulation in the blood (Allen et al., 1991; Shibata et al., 2013). Using PEG as
a spacer, ligands including mannose have also been conjugated to the ends of
PEG-lipids. However, in some PEGylated nanoparticles, the display of mannose on the
surface of the liposome is hindered because it becomes embedded in the PEG layer; this
inhibits sufficient uptake of the nanoparticles (Hagimori et al., 2017). Moreover, it is
difficult to separate pure ligand-PEG-lipids because PEG has a broad molecular weight
distribution (Shibata et al., 2013). To improve the functionality and quality of
ligand-conjugated lipids, we have recently developed HFQ lipids using a serine–glycine
(SG) repeat peptide instead of the PEG spacer (Suga et al., 2017). We selected a short
peptide (KCCYSL; abbreviated to KCC) for human epidermal growth factor receptor-2
(HER2), and obtained KCC-(SG)5,7-lipids with a single molecular weight. Because the
HER2-targeted peptide ligand was efficiently displayed outside the PEG layer, the
uptake of KCC-(SG)5,7/PEGylated liposomes was dramatically enhanced in
HER2-positive breast cancer cells.
In the present study, we developed a novel mannosylated HFQ lipid with an (SG)5
spacer for clinically available macrophage-targeted liposomes. Microfluidic devices
such as the NanoAssemblr platform can simplify preparation of liposomes for clinical
applications (Dong, et al., 2017). Regarding application for these devices, high
dispersibility of HFQ lipids in water should also be considered. However, there have
been few reports on the dispersibility of HFQ lipids in water. Therefore, we attempted to
design a water-dispersible mannosylated HFQ lipid by simulating the net charge of
lipids,
and
synthesized
Man(S)-(SG)5-SSK-K(Pal)2
from
N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine using only
a
fluorenylmethyloxycarbonyl (Fmoc) protecting group solid-phase peptide synthesis
method. We applied Man(S)-(SG)5-SSK-K(Pal)2 to Man-(SG)5/PEGylated liposomes,
and investigated in vitro its cellular association and intracellular distribution in mouse
peritoneal macrophages.
2. Materials and methods
2.1. Materials
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We purchased D-mannose, CH3ONa, and 1-methyl-2-pyrrolidone (NMP) from Wako
Pure Chemical Industries (Osaka, Japan). We purchased Fmoc-protected amino acids,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),
N,N-diisopropylethylamine (DIPEA), triisopropylsilane (TIS), and Rink Amide AM
resin from Merck (Darmstadt, Germany). We purchased 1-hydroxybenzotriazole
(HOBT) from the Peptide Institute, Inc. (Osaka, Japan). Thioglycolate medium was
purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). We purchased
1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC)
and
rhodamine-
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) from Avanti Polar
Lipids
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000] (NH2-PEG2000-DSPE)
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(Alabaster,
AL,
USA).
We
purchased
and
(polyethylene
glycol)-2000] (mPEG2000-DSPE) from NOF Co. (Tokyo, Japan). Palmitic acid (Pal) and
cholesterol were purchased from Nacalai Tesque Inc. (Kyoto, Japan). All other
chemicals were of the highest purity available. We used a peptide calculator produced
by Bachem (Bubendorf, Switzerland) to determine the theoretical physicochemical
properties of the peptides.
2.2. Synthesis of cyanomethyl-2,3,4,6-tetra-O-acetyl-1-thiomannoside
(CME-thiomannoside)
CME-thiomannoside was prepared as described previously (Lee et al., 1976). Briefly,
70% perchloric acid was added drop-wise to a solution of D-mannose (25 g) in acetic
anhydride (100 mL), and the reaction mixture was stirred overnight. We then added
25% HBr/CH3COOH (50 mL), and stirred the mixture overnight under cool conditions.
We obtained the acetohalo sugar after separation and evaporation. We then added
thiourea to a solution of the acetohalo sugar in dry acetone, and refluxed the mixture for
15 min. Chloroacetonitrile, K2CO3, and NaHSO3 were added to the solution, and the
mixture was stirred for
2
h
over ice. We obtained 8.98
g
of
cyanomethyl-2,3,4,6-tetra-O-acetyl-1-thiomannoside
(CME-thiomannoside)
after
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separation and evaporation. The CME-thiomannoside was confirmed by fast-atom
bombardment mass spectrometry (FAB-MS) analysis. The mass-to-charge ratio (m/z)
according to FAB-MS was 404 [M + H+]. According to high-resolution mass spectrum
(HRMS) FAB, the m/z ratios for C16H21NO9S [M + H+] were 404.1015 (calculated) and
404.1016 (found).
2.3. Synthesis of Man-PEG2000-DSPE
Man-PEG2000-DSPE was prepared according to the method reported previously (Un et
al.,
2010b).
We
obtained
2-imino-2-methoxyethyl-1-thiomannoside
(IME-thiomannoside) by stirring CME-thiomannoside with 0.01 M CH3ONa/MeOH for
24 h at room temperature (RT) (Lee et al., 1976). IME-thiomannoside (10 eq) and
NH2-PEG2000-DSPE (1 eq) were reacted in pyridine containing trimethylamine for 24 h.
After concentrating by evaporation, water was added to the residue. The mixture was
then dialyzed for 24 h (molecular weight (MW) cut-off of 6–8 kDa) and lyophilized.
Man-PEG2000-DSPE was confirmed by matrix-assisted laser desorption/ionization
time-of-flight mass spectroscopy (MALDI-TOF-MS).
2.4. Synthesis of Man-(SG)5-SSR-K(Pal)2
K-(SG)5-SSR-K(Pal)2 was prepared using an Fmoc solid-phase peptide synthesis
method. The Fmoc groups were deprotected with 20% piperidine for 20 min, and
coupling reactions using Fmoc amino acids were performed with HBTU/HOBT/DIPEA
for 30 min. The unreacted amino groups were capped with 25% acetic anhydride.
Following the coupling reaction to form Fmoc-Lys(Fmoc)-OH, the Fmoc groups were
deprotected and palmitic acid (10 eq) was reacted twice with HBTU/HOBT/DIPEA for
4 h. A solution comprising trifluoroacetic acid (TFA)/TIS/H2O (94/2.5/2.5, v/v/v) was
added and reacted for 3 h to cleave from the resin. Dialysis of the crude product for 5
days (MW cut-off of 6–8 kDa) followed by lyophilization yielded (SG)5-SSR-K(Pal)2.
IME-thiomannoside (10 eq) and K-(SG)5-SSR-K(Pal)2 (1 eq) were reacted for 24 h in a
mixture comprising pyridine (2 mL), dimethyl sulfoxide (1 mL), and trimethylamine
(20 µL). The products were concentrated by evaporation and water was added to the
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residue. This was followed by dialysis for 48 h (MW cut-off of 6–8 kDa) and
lyophilization. The reaction mixture was analyzed using a high-performance liquid
chromatography (HPLC) system with a reversed-phase column (COSMOSIL
5C18-AR-II 4.6 × 250 mm) at a flow rate of 0.5 mL/min. The mobile phase comprised a
linear gradient of solvent A (0.1% TFA in acetonitrile) and solvent B (0.1% TFA in
water) that increased from 50% solvent A to 100% solvent A over 40 min. The product
was confirmed by MALDI-TOF-MS.
2.5. Synthesis of N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine
(Kragol & Otvos, 2001)
We synthesized α,β-mannose pentaacetate by reacting α,β-D-mannose (490 mg) with
acetic anhydride in pyridine. We added BF3·Et2O (24.6 mmol) to a solution of
α,β-mannose pentaacetate (2.72 mmol) and Fmoc-serine-OH (3.28 mmol) in CH3CN,
and stirred the mixture for 4 h in the dark. After adding CH2Cl2 (160 mL), the organic
layer was washed with 1 M HCl (20 mL) and H2O (20 mL), dried over MgSO4, and
concentrated by evaporation. The product was then lyophilized. The reaction mixture
was purified using a HPLC system with a reversed-phase column (COSMOSIL
5C18-AR-II 20 × 250 mm) at a flow rate of 5 mL/min. The mobile phase comprised a
linear gradient of solvent A (0.1% TFA in 90% acetonitrile and 10% water) and solvent
B (0.1% TFA in water) that increased from 50% solvent A to 100% solvent A over 30
min. MS (FAB) m/z: 658 [M + H+]; HRMS (FAB) for C32H36NO14 [M + H+] 658.2125
(calculated), 658.2136 (found).
2.6. Synthesis of Man(S)-(SG)5-SSK-K(Pal)2
We prepared Man(S)-(SG)5-SSK-K(Pal)2 using an Fmoc solid-phase peptide synthesis
method.
The
reaction
to
produce
using
N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine
HBTU/HOBT/DIPEA was carried out over 4 h, whereas the reactions involving the
other amino acids were carried out over 30 min. The unreacted amino groups were
capped with 25% acetic anhydride. Following the coupling of Fmoc-Lys(Fmoc)-OH,
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the Fmoc groups were deprotected, palmitic acid (10 eq) was reacted twice with
HBTU/HOBT/DIPEA for 4 h, and deacetylation was preformed four times with 0.01 M
CH3ONa/NMP-MeOH (17:3) for 30 min. Cleavage from the resin and dialysis of the
crude product were carried out according to the methods described above. The reaction
mixture was analyzed using a HPLC system with a reversed-phase column
(COSMOSIL 5C18-AR-II 4.6 × 250 mm) at a flow rate of 0.5 mL/min. The mobile
phase comprised a linear gradient of solvent A (0.1% TFA in acetonitrile) and solvent B
(0.1% TFA in water) that increased from 50% solvent A to 100% solvent A over 60 min.
2.7. Preparation of Man-(SG)5/PEGylated liposomes
We dissolved DSPC, cholesterol, and mPEG2000-DSPE in methanol, and evaporated the
resulting solution to prepare dried lipid films. We then added 0.5 mol%
rhodamine-DOPE to label the liposomes. The dried lipid films were hydrated with 2 mL
of distilled water at 65°C for 10 min. The PEGylated liposomes were sonicated for
3 min in a probe type sonicator, and filtered through a 0.45-µm filter (Nihon-Millipore,
Tokyo, Japan). We added 67.2 µL (6 mol% of the total lipid content) or 100.8 µL (9
mol% of the total lipid content) of 2 mg/mL Man(S)-(SG)5-SSK-K(Pal)2 in H2O to 500
µL of the PEGylated liposomes (2 mg/mL), and incubated the mixture at 60°C for 1 h.
We obtained Man-(SG)5/PEGylated liposomes after diluting with phosphate-buffered
saline (PBS). The particle sizes and ζ-potentials of the Man-(SG)5/PEGylated liposomes
in PBS were measured using a Zetasizer Nano ZS system (Malvern Instruments, Ltd.,
Malvern, United Kingdom).
2.8. Preparation of mannosylated PEGylated liposomes
We dissolved DSPC, cholesterol, mPEG2000-DSPE, and Man-mPEG2000-DSPE in
methanol, and evaporated the resulting solution to prepare dried lipid films. We then
added 0.5 mol% rhodamine-DOPE to label the liposomes. The dried lipid films were
hydrated with 2 mL of PBS at 65°C for 10 min. The Man-PEG2,000/PEGylated
liposomes were sonicated for 3 min in a probe type sonicator, and filtered through a
0.45-µm filter (Nihon-Millipore, Tokyo, Japan). The particle sizes and ζ-potentials of
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the Man-PEG2,000/PEGylated liposomes in PBS were measured using a Zetasizer Nano
ZS system (Malvern Instruments, Ltd., Malvern, United Kingdom).
2.9. Animal experiments
All animal studies were conducted in accordance with the experimental guidelines
provided by Nagasaki University, and the experiments were approved by the
Institutional Animal Care and Use Committee of Nagasaki University.
2.10. Harvest and culture of mouse peritoneal macrophages
Four-week-old male ICR mice were supplied by CLEA Japan Inc. (Tokyo, Japan). We
used the methods reported by Un et al. (2010a) to culture and harvest the mouse
peritoneal macrophages. A solution of 2.9% thioglycolate medium (1 mL) was
intraperitoneally administrated to the mice. Four days later, the collected macrophages
were washed and suspended in Roswell Park Memorial Institute 1640 (RPMI-1640)
medium containing 10% fetal bovine serum, and the mouse peritoneal macrophages (0.5
mL; 1.0 × 106 cells/mL) were applied to a 24-well plate. After incubation for 2 h at 37°C
in a 5% CO2 atmosphere, the floating cells were removed, and the macrophages were
incubated for another 3 days.
2.11. Cellular association study using flow cytometry
Fluorescently labelled liposomes were prepared by adding 0.5 mol% Rhod-DOPE. After
the mouse peritoneal macrophages had been cultured in 24-well plates for 72 h
(2.6 × 105 cells/cm2), they were incubated with serum-free RPMI-1640 containing
liposomes (25 µM) (PEGylated liposomes, Man-PEG2,000/PEGylated liposomes, or
Man-(SG)5/PEGylated liposomes) for 3 h at 37°C in a 5% CO2 atmosphere, and
analyzed by flow cytometry (BD LSRFortessa, BD Biosciences, San Diego, CA, USA).
2.12. Intracellular distribution study using a confocal laser scanning microscope
(CLSM)
The mouse peritoneal macrophages were cultured in 35-mm glass-based dishes (2.5 ×
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105 cells/cm2) for 72 h, and incubated with serum-free RPMI-1640 containing
liposomes (25 µM) (PEGylated liposomes, Man-PEG2,000/PEGylated liposomes, or
Man-(SG)5/PEGylated liposomes) for 3 h at 37°C in a 5% CO2 atmosphere. The nuclei
and lysosomes were stained with 4′,6-diamidino-2-phenylindole (DAPI)
(Sigma-Aldrich Co., Milwaukee, WI, USA) and LysoTracker Green (Invitrogen Co.,
Carlsbad, CA, USA), respectively, in preparation for analysis using a CLSM (LSM 710,
Carl Zeiss, Oberkochen, Germany).
2.13. Investigation of the competitive inhibition of the mannose receptor by mannan
using flow cytometry
After the mouse peritoneal macrophages had been cultured in 24-well plates (2.6 × 105
cells/cm2) for 72 h, the cells were washed twice with PBS, and pre-incubated for 30 min
with serum-free RPMI-1640 containing mannan (1 mg/mL). They were then incubated
with serum-free RPMI-1640 containing liposomes (25 µM) (PEGylated liposomes,
Man-PEG2,000/PEGylated liposomes, or Man-HFQ liposomes) in the presence of
mannan (1 mg/mL) for 3 h at 37°C in a 5% CO2 atmosphere. The cells were analyzed
by flow cytometry (BD LSRFortessa, BD Biosciences, San Diego, CA, USA).
2.14. Investigation of the competitive inhibition of the mannose receptor by mannan
using a CLSM
After the mouse peritoneal macrophages had been cultured in 35-mm glass-based dishes
(2.5 × 105 cells/cm2) for 72 h, the cells were washed twice with PBS, and incubated
with serum-free RPMI-1640 containing mannan (1 mg/mL). They were then incubated
with serum-free RPMI-1640 containing liposomes (25 µM) (PEGylated liposomes,
Man-PEG2,000/PEGylated liposomes, or Man-HFQ liposomes) in the presence of
mannan (1 mg/mL) for 3 h at 37°C in a 5% CO2 atmosphere. The nuclei and lysosomes
were stained with DAPI (Sigma-Aldrich Co., Milwaukee, WI, USA) or LysoTracker
Green (Invitrogen Co., Carlsbad, CA, USA), respectively, in preparation for analysis
using a CLSM (LSM 710, Carl Zeiss, Oberkochen, Germany).
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2.15. Statistical analysis
The data are expressed as the means ±ꢀSD. The statistical significance of differences
between groups was tested by analysis of variance. The Tukey–Kramer test was used to
compare differences between multiple groups.
3. Results
3.1. Synthesis of water-dispersible mannosylated lipids
Peptide sequences including polar amino acids (glutamic acid or arginine) or elongated
SG spacers were used to confer a high degree of water dispersibility on the
mannosylated lipids, and net peptide charges were simulated using a Bachem peptide
calculator (Fig. 1). The net charges of K-(SG)5-SS-E-K(pal)2 and
K-(SG)10-SS-E-K(pal)2 were zero. The introduction of one or two glutamic acid (E)
residues between the C-terminal lysine and (SG)5 conferred a net negative charge. A
positive charge was conferred on K-(SG)5-SS-E-K(pal)2 by replacing the glutamic acid
(E) residue with arginine (R) to form K-(SG)5-SS-R-K(pal)2. These lipids were
produced using an Fmoc solid-phase peptide synthesis method, and their dispersibility
in water was evaluated. Only the positively charged lipid K-(SG)5-SS-R-K(pal)2 had
high dispersibility in water at a concentration of more than 0.2 mM. As shown in Fig. 2,
the liquid-phase conjugation of K-(SG)5-SS-R-K(pal)2 with IME-thiomannoside was
carried out in pyridine containing dimethyl sulfoxide and trimethylamine for 24 h, and a
mannosylated lipid, Man-(SG)5-SSR-K(Pal)2, was obtained at a yield of 8.2%. Analysis
of the product using a HPLC-UV system and mass spectrometry revealed an abundance
of peaks attributable to by-products (Fig. S1, S2).
We included mannose conjugation in the Fmoc solid-phase peptide synthesis method to
improve the yield and quality of the mannosylated lipids. We designed a novel
mannosylated lipid, Man(S)-(SG)5-SSK-K(Pal)2, with a positive net charge (Fig. 3). An
Fmoc-mannose-serine
(Man(S))
derivative,
N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine, was synthesized
via a two-step reaction from mannose, and the yield was 17%. As shown in Fig. 4, we
reacted the carboxyl group of the Fmoc-mannose-serine residue with the amine group of
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the resin using HBTU/HOBT/DIPEA. After removing the Fmoc group using piperidine,
the peptide chain, which included (SG)5-SSK-K, was elongated and ultimately coupled
with palmitic acid. After cleavage from the resin, deprotection of the Fmoc groups of
the amino acids and the acetyl groups of mannose produced the desired
Man(S)-(SG)5-SSK-K(pal)2 at a yield of 40.8%. According to HPLC-UV and mass
spectrometry analysis, the obtained product had high purity (Fig. S3, S4).
3.2. Preparation of Man-(SG)5/PEGylated liposomes
We used sonication and post-insertion methods to prepare mannosylated HFQ
liposomes. We added Man(S)-(SG)5-SSK-K(pal)2 in water to the PEGylated liposomes
and incubated the mixture at 60°C for 1 h to produce Man-(SG)5/PEGylated liposomes
with three different degrees of mannosylation (3, 6, and 9%). We also prepared
mannosylated PEGylated liposomes (Man-PEG2,000/PEGylated liposomes) with three
different degrees of mannosylation (3, 6, and 9%), according to a previously reported
method. The particle sizes and ζ-potentials of the liposomes are summarized in Table 1.
Except for the ungrafted PEGylated liposomes and the 3% Man-(SG)5/PEGylated
liposomes, the size of the liposomes ranged from 93.8 to 123.5 nm. The ζ-potentials of
Man-(SG)5/PEGylated liposomes were maintained at a slightly negative charge,
whereas those of the Man-PEG2,000/PEGylated liposomes had an increasingly positive
charge according to the degree of mannosylation.
3.3. Cellular association study
To compare cellular association in the ungrafted PEGylated liposomes, the
Man-PEG2,000/PEGylated liposomes, and the Man-(SG)5/PEGylated liposomes, the
liposomes were labeled with rhodamine, and the fluorescence of the labeled liposomes
in the cells was investigated by flow cytometry. The flow cytometry profiles and mean
fluorescence intensities are shown in Fig. 5. The Man-(SG)5/PEGylated liposomes
exhibited greater fluorescence intensity than the ungrafted PEGylated and
Man-PEG2,000/PEGylated liposomes in the mouse peritoneal macrophages. In particular,
there was significant enhancement of the cellular association in the 9% grafted
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Man-(SG)5/PEGylated liposomes. In contrast, cellular association in the
Man-PEG2,000/PEGylated liposomes with 6 or 9% mannosylation was reduced
compared with the ungrafted PEGylated liposomes.
3.4. Intracellular distribution study
As shown in Fig. 6, we investigated the intracellular distribution of ungrafted
PEGylated liposomes, Man-PEG2,000/PEGylated liposomes, and Man-(SG)5/PEGylated
liposomes in mouse peritoneal macrophages using a CLSM. Following incubation of
the Man-(SG)5/PEGylated liposomes for 1 h, we observed bright red rhodamine
fluorescence from the labelled Man-(SG)5/PEGylated liposomes, especially in the 9%
grafted Man-(SG)5/PEGylated liposomes. Furthermore, the red fluorescence was
colocalized with green fluorescence from LysoTracker Green (a lysosomal marker). In
contrast, we observed weak rhodamine fluorescence in the ungrafted PEGylated and
Man-PEG2,000/PEGylated liposomes.
3.5. Cellular association and distribution with mannan
We confirmed the target efficacy of the Man-(SG)5/PEGylated liposomes using mannan
as a mannose receptor ligand (Fig. 7). After the mouse peritoneal macrophages had been
incubated with serum-free RPMI-1640 containing 1 mg/mL mannan for 30 min, the
medium was replaced with fresh medium containing Man-(SG)5/PEGylated liposomes
(25 µM) grafted with 6 or 9% mannose and 1 mg/mL mannan. The mixture was then
incubated for 3 h. Flow cytometry quantification studies revealed that cellular
association in the 6 and 9% mannose-grafted Man-(SG)5/PEGylated liposomes was
significantly reduced by the addition of 1 mg/mL mannan (Fig. 7A). We also carried out
CLMS studies with 1 mg/mL mannan using 9% mannose-grafted Man-(SG)5/PEGylated
liposomes in mouse peritoneal macrophages (Fig. 7B), and found that the localization of
the Man-(SG)5/PEGylated liposomes in lysosomes was inhibited by mannan.
4. Discussion
The objective of the present study was to develop a novel and clinically applicable
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liposome that can target the mannose receptor. Therefore, we designed and synthesized
water-dispersible HFQ lipids, prepared Man-(SG)5/PEGylated liposomes as mannose
receptor-targeted HFQ liposomes, and evaluated their in vitro efficacy in mouse
peritoneal macrophages. Several studies have demonstrated that mannosylated
liposomes are promising carriers for anticancer drugs, proteins, NF-κB, and plasmid
DNA (Fukasawa, et al., 1998; Kawakami and Hashida, 2014; Kono, et al., 2014; Matsui
et al., 2010; Un, et al., 2010a, b; Vinogradov et al., 2014). A number of mannosylated
liposomes with PEG surface coatings have been designed because long, flexible PEG
chains stabilize nanoparticles (Allen et al., 1991). It has been reported that the PEG
layer serves as a barrier (Hagimori et al., 2017; Suga et al., 2017), prevents the display
of mannose, and reduces the interaction between the liposome and the target cell. To
overcome these drawbacks, Zhu et al. used an acid-sensitive bond to efficiently target
tumor-associated macrophages (Zhu et al., 2013). In this system, the nanoparticles shed
their PEG coatings in acidic tumor microenvironments; subsequently the mannosylated
nanoparticles associate via mannose–mannose receptor recognition. In the present study,
we used serine–glycine (SG) repeat peptides to efficiently display the mannose ligand
outside the PEG layer. The dispersibility of the lipid in water is a crucial factor in the
post-insertion technique that enables the insertion of lipids into commercially available
liposomes that are suitable for clinical DDSs using microfluidic devices. Therefore, we
attempted to design and synthesize a HFQ lipid with high dispersibility in water.
Previously our group reported mannose receptor-targeted liposomes comprising
Man-PEG2000-DSPE, which were synthesized using the liquid phase synthesis method,
namely the reaction of NH2-PEG2000-DSPE with IME-thiomannoside in pyridine (Un, et
al., 2010b). To apply this liquid phase synthesis method, we designed (SG)n peptide
sequences that included glutamic acid as an acidic amino acid, arginine as a basic amino
acid, and a repeat sequence of SG for hydrophilicity; we simulated net charge using a
Bachem peptide calculator, and synthesized five lipids constructed from (SG)n peptide
sequences and palmitic acid (Fig. 1). Only the positively charged lipid,
K-(SG)5-SSR-K(pal)2, exhibited high dispersibility in water. Because deionized water is
slightly acidic, the γ-carboxyl groups of the glutamic acid residues were protonated,
14

ACCEPTED MANUSCRIPT
resulting in glutamic acid residues that did not enhance the dispersibility of the lipids in
water. Furthermore, the SG repeat sequence did not improve the dispersibility of the
lipids in water. The mannosylated lipid, Man-(SG)5-SSR-K(pal)2, was synthesized
through the liquid phase reaction of K-(SG)5-SSR-K(pal)2 and IME-thiomannoside in
pyridine (Fig. 2). However, there were many byproducts, and the yield of
Man-(SG)5-SSR-K(pal)2 was only 8.2%. Although the imidoester of
IME-thiomannoside is an amine-reactive crosslinking group, side-reactions are likely
below pH 10 (Hand and Jencks, 1962). To suppress side-reactions and improve purity,
we designed a novel lipid, Man(S)-(SG)5-SSK-K(pal)2, which comprised a
mannose–serine residue and (SG)5-SSK-K(pal)2 (Fig. 3). A straightforward Fmoc
solid-phase
N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine as
material produced
peptide
synthesis
method
using
a
starting
a
moderately good yield (40.8%) of high purity
Man(S)-(SG)5-SSK-K(pal)2 (Fig. 4).
Macrophage-targeted Man-(SG)5/PEGylated liposomes with different degrees of
mannosylation (3, 6, and 9%) were prepared by sonication to reduce the size
distribution of liposomes and post-inserting Man(S)-(SG)5-SSK-K(pal)2 into PEGylated
liposomes. Physicochemical investigations of the ungrafted PEGylated liposomes, the
Man-PEG2,000/PEGylated liposomes, and the Man-(SG)5/PEGylated liposomes revealed
that their ultimate diameters were approximately 100 nm (Table 1). With regard to
ζ-potentials, although the 6 and 9% mannosylated Man-PEG2,000/PEGylated liposomes
had positive charges, the Man-(SG)5/PEGylated liposomes had weak negative charges.
It has been reported that a weak negative charge on the nanoparticles can reduce
non-specific binding (Chen, et al., 2010). Therefore, these results suggest that
Man-(SG)5/PEGylated liposomes possess favorable characteristics for in vivo
applications. The flow cytometry studies revealed that cellular association was greater
in the 6 or 9% mannosylated Man-(SG)5/PEGylated liposomes than in the PEGylated
liposomes and 6 or 9% mannosylated Man-PEG2,000/PEGylated liposomes in mouse
peritoneal macrophages (Fig. 5). This suggests that mannose ligands can be displayed
outside the PEG layer of Man-(SG)5/PEGylated liposomes using an (SG)5 spacer. This
15

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finding agrees well with the results of our previous investigations into
KCC-(SG)5/PEGylated
liposomes
(Suga
et
al., 2017).
The
cationic
Man-PEG2,000/PEGylated liposomes exhibited lower cellular association than the
anionic ungrafted PEGylated liposomes (Fig. 5). These quantitative results corroborate
the reports that the uptake of cationic liposomes is lower than that of anionic liposomes
in mouse peritoneal macrophages (Aramaki et al., 1995; Dadashzadeh et al., 2010).
However, because it has been reported that positive charge on the surface of DDS
carriers enhances cellular uptake (Fröhlich, 2012), charge and sequence optimization of
Man-mPEG2,000-DSPE
might
improve
the
cellular
association
of
Man-PEG2,000/PEGylated liposomes. To confirm intracellular distribution, we obtained
CLSM images of ungrafted PEGylated liposomes, Man-PEG2,000/PEGylated liposomes,
and Man-(SG)5/PEGylated liposomes in mouse peritoneal macrophages (Fig. 6). In
accordance with the results shown in Fig. 5, the CLSM images revealed that the
Man-(SG)5/PEGylated liposomes were internalized in macrophages more than the
ungrafted PEGylated liposomes. Furthermore, co-staining with LysoTracker Green
provided evidence that Man-(SG)5/PEGylated liposomes can be localized in lysosomes
via mannose receptor-mediated endocytosis. The lysosomal degradation of liposomes is
one of the problems in drug delivery (Hagimori et al., 2017). Therefore, further studies
will be required to improve the intracellular distribution of Man-(SG)5/PEGylated
liposomes in combination with methods facilitating intracellular lysosomal escape. The
inhibitory study in the presence of excess mannan as a mannose receptor ligand
indicated that the high uptake of Man-(SG)5/PEGylated liposomes by mouse peritoneal
macrophages is mediated by mannose receptors (Fig. 7).
5. Conclusions
We have developed a novel mannosylated HFQ lipid with an (SG)5 spacer,
Man(S)-(SG)5-SSK-K(pal)2, for macrophage-targeted liposomes. The lipid was
synthesized using only
a
Fmoc solid-phase synthesis method with
starting
N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranosyl)-L-serine as
a
material, and the product had high purity and high dispersibility in water.
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ACCEPTED MANUSCRIPT
Man-(SG)5/PEGylated liposomes can be prepared by post-inserting the
Man(S)-(SG)5-SSK-K(pal)2
into
PEGylated
liposomes.
Moreover,
Man-(SG)5/PEGylated liposomes can be taken up by mannose receptor-mediated
endocytosis in mouse peritoneal macrophages. Although further studies are needed to
clarify the anticancer effect of drug loaded Man-(SG)5/PEGylated liposomes, the data
indicate that Man(S)-(SG)5-SSK-K(pal)2 has potential as a HFQ lipid for mannose
receptor–targeted drug delivery systems.
Acknowledgements
This work was partly supported by JSPS KAKENHI Grant Number 18H03535 (S.K)
and Takeda Science Foundation (S.K).
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Fig. 1. The peptide sequence with polar amino acids (glutamic acid and arginine) or
elongated SG spacer, and their net charge
Fig. 2. Liquid-phase synthesis of Man-(SG)5-SSR-K(pal)2
Fig. 3. Chemical structure of Man(S)-(SG)5-SSK-K(pal)2
Fig. 4. Solid-phase synthesis of Man-(SG)5-SSR-K(pal)2
Fig. 5. Cellular association of PEGylated lyposomes, Man-PEG2000/PEGylated
liposomes, and Man-(SG)5/PEGylated-liposome in peritoneal macrophage at a
concentration 25 μM. Cell were incubated with liposomes for 3 h, then analyzed by
flow cytometry. (A) Mean fluorescence intensity, (B) Histogram profiles. Date are
represented as mean value ± SD (n = 5). ANOVA and Tukey’s test. (*P＜0.05,
***P<0.001)
Fig. 6. Intracellular distribution studies of unmodified PEGylated liposomes,
Man-PEG2000/PEGylated liposomes, and Man-(SG)5/PEGylated liposomes in mouse
peritoneal macrophages. Nuclei (DAPI fluorescent signal: blue), Lysosome
(LysoTracker signal: green), liposomes (Rhodamine fluorescent signal: red), and Merge:
yellow signals indicate colocalization of liposomes and LysoTracker.
Fig. 7. Intracellular distribution of Man-(SG)5/PEGylated liposomes with mannan (-)
and mannan (+) in mouse peritoneal macrophages. (A) Quantification studies: Cells
were incubated with PEGylated liposomes, 6% Man-(SG)5/PEGylated liposomes, or 9%
Man-(SG)5/PEGylated liposomes without mannan as control (filled column) or with 1
mg/mL mannan (opened column). Each value represents the mean ± S.D. of three
experiments. Statistically significant differences (**P<0.01, ***P<0.001) from a control
of unmodified-liposomes, (##P<0.01; ###P<0.001) from a control (liposomes alone) for
each group. N.S., not significant. (B) CLMS images: Cells were incubated with 9%
Man-(SG)5/PEGylated liposomes with mannan (-) and mannan (+). Nuclei (DAPI
fluorescent signal: blue), Lysosome (LysoTracker signal: green), liposomes (Rhodamine
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fluorescent signal: red), and Merge: yellow signals indicate colocalization of liposomes
and LysoTracker.
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Table 1.The lipid composition, mean particle size and ζ potential of liposomes
Particle size
(nm)
ζ-potential
(mV)
Lipid composition (molar ratio)
PEGylate -liposomes
157.4 ± 2.3
123.5 ± 1.3
105.3 ± 1.4
93.8 ± 1.4
-4.15 ± 0.32
-0.08 ± 0.97
2.03 ± 0.73
4.62 ± 0.81
-1.82 ± 0.64
-3.29 ± 0.71
-3.57 ± 0.73
(DSPC/Cholesterol/mPEG2000-DSPE = 55/35/10)
3% Man-PEG2,000/PEGylated liposomes
(DSPC/Cholesterol/mPEG2000-DSPE/Man-PEG-DSPE = 55/35/7/3)
6% Man-PEG2,000/PEGylated liposomes
(DSPC/Cholesterol/mPEG2000-DSPE/Man-PEG-DSPE = 55/35/4/6)
9% Man-PEG2,000/PEGylated liposomes
(DSPC/Cholesterol/mPEG2000-DSPE/Man-PEG-DSPE = 55/35/1/9)
3% Man-(SG)5/PEGylated liposomes
213.9 ± 18.5
(DSPC/Cholesterol/mPEG2000-DSPE/Man(S)-(SG)5-SSK-K(Pal)2 = 55/35/7/3)
6% Man-(SG)5/PEGylated liposomes
122.8 ± 3.6
(DSPC/Cholesterol/mPEG2000-DSPE/Man(S)-(SG)5-SSK-K(Pal)2 = 55/35/7/6)
9% Man-(SG)5/PEGylated liposomes
121.7 ± 0.7
(DSPC/Cholesterol/mPEG2000-DSPE/Man(S)-(SG)5-SSK-K(Pal)2 = 55/35/7/9)
Results are expressed as the mean ± S.D. of three experiments.
24

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5