Effect of Lipid Composition on the Interaction of Liposomes with THP-1-Derived Macrophages

Abstract

Recently, liposomal formulations that target macrophages have been used to treat lung diseases. However, the detailed mechanism of the cellular uptake must be elucidated to identify a formulation with excellent cellular uptake efficiency to treat non-tuberculous mycobacterial lung disease. We studied the effect of lipid composition on the cellular uptake of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Chol) liposomes with a size of approximately 200 nm into THP-1-derived macrophages. The amount of DPPC/Chol liposomes (80/20 mol%) was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes. The anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane fluidity of the DPPC/Chol (80/20 mol%) liposomes was higher than that of the other two liposomes. DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes were taken up via clathrin- and caveolae-mediated endocytosis and phagocytosis. However, proteins involved in cellular uptake through ligand–receptor interactions were adsorbed to a greater extent on DPPC/Chol (80/20 mol%) liposomes than on DPPC/Chol (67/33 mol%) liposomes. Pretreatment of cells with antibodies against the low-density lipoprotein receptor and scavenger receptor type B1 largely inhibited the uptake efficiency of DPPC/Chol (80/20 mol%) liposomes. Our results indicate that the membrane fluidity of DPPC/Chol liposomes, which is controlled by the Chol ratio, is an important factor in controlling protein adsorption and the subsequent uptake efficiency of liposomes.

INTRODUCTION

Nontuberculous mycobacteria (NTM) are found throughout the environment and can cause pulmonary infections in patients with structural lung damage, immune deficiency, or other risk factors. These infections cause lung disease with symptoms such as nodular bronchiectasis and fibrocavitary disease.^1,2) Mycobacterium avium complex (MAC) is the most common cause of NTM lung disease.¹⁾

NTM can persist intracellularly within macrophages, and studies have indicated that MAC species can live and replicate within macrophages.^3,4) Therefore, an effective treatment strategy for MAC lung disease is to deliver antibiotics to macrophages. Recently, amikacin, an aminoglycoside antibiotic, was approved worldwide as an amikacin liposome inhalation suspension (ALIS) for patients with MAC lung disease.⁵⁾ ALIS is designed to deliver large amounts of amikacin to the lungs while maintaining low systemic concentrations to avoid systemic adverse events. However, resistance to amikacin remains a problem,⁶⁾ and developing new antibiotics with various efficient drug delivery routes is imperative.⁷⁾

Liposomes are often used to model the plasma membrane.⁸⁾ Phosphatidylcholine is the main phospholipid of the outer cell membrane leaflet⁹⁾ and the major lung surfactant component in the human body.¹⁰⁾ 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is a saturated phospholipid that comprises approximately 30–60% of the phosphocholine contained in lung surfactants.¹¹⁾ Cholesterol (Chol) is the main sterol found in mammalian plasma membranes and regulates membrane fluidity.¹²⁾ The liposomes used in the ALIS are composed of DPPC and Chol.¹³⁾

THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for human macrophages¹⁴⁾ and are used not only as macrophage models in the study of lung infections¹⁵⁾ but also in the study of inflammatory arthritis.¹⁶⁾ Knowledge of the cellular uptake mechanism is important in the design of nanoparticles, such as liposomes, for effective cellular uptake into lung macrophages. The intracellular uptake mechanism differs among different cell types.¹⁶⁾ Although the mechanism of the cellular uptake of polyethylene glycol (PEG)-modified liposomes has been reported¹⁶⁾ in various cell types, including THP-1 cells, the detailed factors involved in regulating the cellular uptake of liposomes composed of DPPC and Chol in THP-1 cells have not been studied.

We thus studied the effect of the lipid composition of DPPC/Chol liposomes on the cellular uptake efficiency and investigated the major mechanism of liposome uptake into THP-1-derived macrophages, to achieve the efficient cellular uptake of DPPC/Chol liposomes. The particle size of liposomes used in this study was approximately 200 nm, based on the reports for inhalation of liposomal amikacin to achieve efficient delivery of liposomes to the lungs, distribution of liposomes throughout the lungs, and penetration into biofilms and macrophages to reach the sites of infection.^14,17)

MATERIALS AND METHODS

Materials

DPPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DPPE) were obtained from Avanti Polar Lipids (Birmingham, AL, U.S.A.). Chol was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). PMA was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Recombinant human apolipoproteins A-1 and E3 were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Human plasma apolipoprotein B and human complement C3b were obtained from Sigma-Aldrich.

Liposome Preparation

The lipid compositions of the liposomes used in this study were as follows: DPPC/Chol (60/40 mol%), DPPC/Chol (67/33 mol%), and DPPC/Chol (80/20 mol%). To prepare fluorescently labeled liposomes, NBD-DPPE was incorporated into 1 mol% of the total lipid. Liposomes were fabricated using a modified version of the Bangham method.¹⁸⁾ Table 1 shows the lipid composition and physicochemical characteristics. Briefly, the desired amounts of lipids were mixed in chloroform and dried by rotatory evaporation in a 70 °C water bath to create a thin homogeneous lipid film. The dried film was hydrated with 0.5 mL of 250 mM ammonium acetate at 70 °C for 5–10 min. The solution was then passed 21 times through a mini-extruder (Avanti Polar Lipids) equipped with a 200-nm polycarbonate filter.

Table 1. Hydrodynamic Diameters and Zeta Potentials of the Liposomes

Lipid composition	Diameter (nm)	Polydispersity index	Zeta potential (mV)
DPPC/Chol (60/40)	184.9 ± 1.57	0.085 ± 0.008	−23.9 ± 0.737
DPPC/Chol (67/33)	189.0 ± 0.31	0.11 ± 0.015	−20.6 ± 1.249
DPPC/Chol (80/20)	179.6 ± 0.87	0.12 ± 0.044	−17.2 ± 6.202

Values are the mean ± standard deviation (n = 3). Values in parentheses are in mol%. Chol, cholesterol; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.

Dynamic Light Scattering Measurements

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the liposomes were measured at 25 °C by dynamic light scattering (DLS) using a Horiba nanoPartica SZ-100V2 (Horiba, Kyoto, Japan). Samples (0.5 mM) were diluted in 10 mM phosphate buffer (pH 7.0). Data were analyzed using the software package SZ-100 for Windows, version 2.4. The means and standard deviations were obtained from three measurements. These data are presented in Table 1.

Internalization of Liposomes into Cells

The internalization of liposomes into THP-1 cells (RIKEN Bioresource Research Center, Tsukuba, Ibaraki, Japan) was measured using a protocol according to our previous report.¹⁹⁾ Initially, THP-1 cells (0.5 × 10⁶ per well) were seeded in 12-well plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.5 µg of PMA for the differentiation of THP-1 monocytes into macrophages.²⁰⁾ The medium was replaced with a medium without PMA the next day, followed by further incubation for 48 h.

After incubation for 48 h (37 °C, 5% CO₂), fluorescently labeled liposomes suspended in 500 µL of RPMI-1640 supplemented with 10% FBS were added to the cells (final lipid concentration was approximately 50 µg/mL, adjusted to ensure that all liposome solutions had equal fluorescence intensity before addition to cells). The cells were then incubated for an additional 24 h (37 °C, 5% CO₂). After incubation, the medium was replaced with fresh phosphate-buffered saline (PBS). Cells were collected, washed with Hank’s balanced salt solution (HBSS; Thermo Fisher Scientific, Waltham, MA, U.S.A.) thrice, and lysed with 0.1% (v/v) Triton X-100 in PBS. The resulting cell suspension was sonicated for 10 min in a bath sonicator, mixed thoroughly for 5 min, and centrifuged (15000 × g, 4 °C, 5 min).

The fluorescence intensity of the supernatant was measured using a fluorescence spectrophotometer (F-2500, Hitachi High-Technologies, Tokyo, Japan) at excitation and emission wavelengths of 490 and 540 nm, respectively. The fluorescence values were normalized to the protein content of the cell lysate (Protein Assay Dye, Bio-Rad Laboratories, Hercules, CA, U.S.A.).

For the inhibition study, endocytosis was inhibited using 30 µg/mL chlorpromazine (a clathrin-mediated endocytosis inhibitor),¹⁹⁾ 10 µM cytochalasin D (a phagocytosis inhibitor),²¹⁾ and 150 µM genistein (a caveolae-mediated endocytosis inhibitor).¹⁹⁾ We confirmed that these inhibitors did not cause appreciable toxicity (Supplementary Fig. 1a) and that each inhibitor inhibited the appropriate endocytosis pathway using markers^19,22) (Supplementary Figs. 1b–d). THP-1 cells (0.5 × 10⁶ per well) were seeded in 12-well plates in RPMI-1640 and differentiated into macrophages as described above. After incubation for 48 h (37 °C, 5% CO₂), each endocytosis inhibitor in 500 µL of RPMI-1640 supplemented with 10% FBS was added to the culture medium; 30 min later, DPPC/Chol (80/20 or 67/33 mol%) liposomes were added to the treated cells (final concentration, 50 µg/mL) and incubated for 24 h (37 °C, 5% CO₂) with inhibitors. The cytotoxicity of the inhibitors was evaluated using the cytotoxicity assay kit, CKK-8 (DOJIN, Kumamoto, Japan), per the manufacturer’s instructions.

Fluorescence Anisotropy Measurement

For the anisotropy measurement,^23,24) 1.4 µM 1,6-diphenyl-1,3,5-hexatriene (DPH, Sigma-Aldrich) was mixed with a 350 µM liposome solution. Following the sample incubation in the dark for 1 h, the fluorescence anisotropies of the samples were determined at 37 °C using a Spectra Max M5 microplate reader (Molecular Devices Japan, Tokyo, Japan). The samples were excited at 355 nm and the fluorescence intensity was monitored at 460 nm. Anisotropy I was calculated as follows:

  

where I_V and I_H are the fluorescence intensities parallel and perpendicular to the excitation plane, respectively. The G value was 1.000 per the manufacturer’s recommendation.

Confocal Imaging

The localization of the NBD-labeled liposomes was determined using a confocal microscope (LCM710; Zeiss, Oberkochen, Germany). Data were collected using the Zen software (2012). THP-1 cells (2 × 10⁵ cells per well) were plated in 35-mm glass-bottom dishes coated with poly-L-lysine (Matsunami, Osaka, Japan), each containing 1.5 mL of RPMI-1640 supplemented with 10% FBS, and were differentiated with PMA into macrophages, as described above. After incubation for 48 h (37 °C, 5% CO₂), cells were exposed to the fluorescent-labeled liposomes, DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) (final lipid concentration was approximately 50 µg/mL, adjusted to ensure that the two liposome solutions had equal fluorescence intensity before addition to cells). After overnight incubation, the cells were washed twice with HBSS, late endosomes/lysosomes were labeled with LysoTracker RED DND-99 (Thermo Fisher Scientific), and nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific).

Interaction of Liposomes with Apolipoproteins and C3b

The mixture of apolipoproteins A, B, and E, or C3b (each 0.94 µg) was incubated with 0.2 mM fluorescent-labeled DPPC/Chol liposome in RPMI-1640 (total 500 µL) for 1 h at 37 °C.¹⁹⁾ Thereafter, the samples were loaded onto Sepharose CL-4B columns (1.0 × 15 cm) and eluted with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (10 mM HEPES, 150 mM NaCl, pH 7.4).²⁵⁾ The effluent was collected in batches of 1 mL per tube. The fluorescence intensity of each fraction was measured using a SpectraMax M5 with excitation at 485 nm and emission at 538 nm.²⁵⁾ The isolated liposomal fractions were concentrated using a centrifugal concentrator (VC-96N; TAITEC, Saitama, Japan). The apolipoproteins adsorbed onto the liposomes were detected by Western blotting using antibodies against apolipoproteins A–I (FL-267; Santa Cruz Biotechnology, Dallas, TX, U.S.A.), anti-apolipoprotein B (ab7616; Abcam, Cambridge, U.K.), anti-apolipoprotein E (Abcam, D6E10), and anti-C3/C3b (Abcam, 755).

Low-Density Lipoprotein Receptor (LDLR) and Scavenger Receptor Type B1 (SR-B1) Blocking Studies

Monoclonal antibodies (mAbs) were used to block the ligand binding domains of the LDLR and SR-B1 receptors.¹⁹⁾ The mAbs against the LDLR and SR-B1 were obtained from Calbiochem (15C8, San Diego, CA, U.S.A.) and Abcam (EP1556Y), respectively. THP-1 cells (0.5 × 10⁶ per well) were seeded and differentiated as described above. Cells were incubated with or without 5 µg/mL anti-LDLR or anti-SR-B1 mAb for 1 h at 4 °C.^19,26–28) After washing with PBS, NBD-labeled liposomes (final lipid concentration, 50 µg/mL) in 500 µL of culture medium were added to the cells, and the cells were incubated for 1 h (37 °C, 5% CO₂). Because LDLR recycling occurs within 2 h,²⁸⁾ we used a shorter incubation time (i.e., 1 h) to obtain the data before the anti-LDLRs bound to LDLRs were replaced by liposomes.¹⁹⁾ Isotype controls [anti-LDLR, purified mouse immunoglobulin G (IgG)2a (BioLegend, San Diego, CA, U.S.A.); anti-SR-B1, normal rabbit IgG (Jackson Immuno Research, West Grove, PA, U.S.A.)] were used as reference controls. The fluorescence intensity of the cells was measured as described in Internalization of Liposomes into Cells.

RESULTS AND DISCUSSION

Effect of the Lipid Composition on Cellular Internalization

The cellular internalization of liposomes containing different ratios of DPPC and Chol was studied. Chol molar ratios of 40%, which are similar to that of ALIS,¹³⁾ 33, and 20% were employed. As shown in Fig. 1, the amount of cellular internalization varied among the liposomes. Although the internalization of the DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes was not significantly different, the amount of DPPC/Chol (80/20 mol%) liposomes was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes.

Fig. 1. Effect of the Lipid Composition of DPPC/Chol Liposomes on the Intracellular Fluorescence Intensity

Values are expressed as mean ± standard deviation (n = 3). Statistical analyses were performed using a one-way ANOVA with Tukey’s post-hoc test. N.S., not significant. ****, p < 0.0001.

Investigation of Fluorescence Anisotropy

We investigated the mechanisms underlying the different cellular uptake efficiencies of the liposomes. The phase state of the membrane of DPPC/Chol liposomes has been well studied.^29,30) The DPPC/Chol binary liposome system, at 37 °C, which is lower than DPPC chain melting temperature, has been shown to have only one liquid order phase, but below 20% Chol, coexistence of solid (gel) and liquid phases has been reported.^29,30)

DPH is a widely used rotational probe for estimating the structural order of membrane lipids.^23,24,31) In the ordered phase state, the rotation of DPH is limited and the anisotropy (r) is larger than that in the less ordered phase. As shown in Fig. 2, the anisotropy (r) of DPPC/Chol (80/20 mol%) liposomes was significantly higher than those of DPPC/Chol (67/33 mol%) and DPPC/Chol (60/40 mol%) liposomes. The higher r values of the DPPC/Chol (80/20 mol%) liposomes indicated that the lipid phase was more ordered. This result coincided with those of previous report.^29,30)

Fig. 2. Anisotropy (r) of DPPC/Chol Liposomes by DPH

Values are expressed as mean ± standard deviation (n = 3). Statistical analyses were performed using a one-way ANOVA with Tukey’s post-hoc test. N.S., not significant. **, p < 0.01.

Spectrin protein molecules have been reported to penetrate the phospholipid monolayer in the presence of up to 20% cholesterol, in a manner different from the changes resulting from alterations in membrane fluidity because of the fatty acid chain composition.³²⁾ One study has shown that erythroid and brain spectrin bound to dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylethanolamine (DMPE) membranes 10- and 40-fold stronger, respectively, in the presence of 20% cholesterol, where both the gel (Lβ) and liquid crystalline (Lα) phases coexisted, at 15 °C, compared with DMPC/DMPE liposomes without Chol.³³⁾ The authors presumed that this strong binding of proteins was because of the coexistence of gel and liquid order phases of the membrane, which created a defect on the boundary of the domain through which proteins could penetrate.³³⁾ As the size and zeta potential, which may affect the cellular internalization of nanoparticles,³⁴⁾ were similar between our DPPC/Chol liposomes (Table 1), the difference in membrane fluidity of the liposomes and the possible protein adsorption on the liposomes can affect the efficiency of internalization.

Endocytosis Mechanism

Some endocytosis mechanisms involve protein ligands and specific receptor interactions.^35,36) Therefore, we studied the involvement of clathrin- and caveolae-mediated endocytosis and phagocytosis, where ligand-receptor interactions are involved,^36–38) to investigate the uptake mechanism in THP-1-derived macrophages. We used DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes because between Chol ratios 20 and 33%, the tendency of cellular internalization and membrane fluidity drastically changed.

First, we used confocal microscopy to investigate the intracellular trafficking of liposomes after exposure to THP-1-derived macrophages. The particle size in culture medium was measured because the size reflects the stability of the liposomes and affects the cellular uptake efficiency.³⁴⁾ The particle sizes were similar before and after incubation for 1 h at 37 °C and the particles sizes of both liposomes were similar (183.5 and 178.1 nm) (Fig. 3a). Therefore, these liposomes showed a tendency to be stable in the culture medium. Although the final lipid concentrations were adjusted to ensure that both liposome solutions had equal fluorescence intensities before addition to the cells, the intracellular fluorescence intensity of DPPC/Chol (80/20 mol%) was markedly higher than that of DPPC/Chol (67/33 mol%) based on confocal images (Fig. 3b). As a result, the possibility of transferring only labeled lipids to the cells was excluded. Notably, these imaging results are consistent with the quantitative results shown in Fig. 1. Overall, fluorescence intensity reflects the intracellular uptake of liposomes. Moreover, both fluorescently labeled liposomes were co-stained with dotted fluorescence derived from late endosome/lysosome-specific staining (Fig. 3b). These results suggest that both liposomes were internalized by endocytosis.

Fig. 3.  (a) Changes in the Particle Sizes of DPPC/Chol (67/33 mol%) and DPPC/Chol (80/20 mol%) Liposomes during Incubation at 37 °C in Culture Medium Containing FBS and (b) Confocal Images of DPPC/Chol Liposomes

(a) Percentages are the ratios of the particle size before and after incubation. Values are expressed as mean ± standard deviation (n = 3). Statistical analyses were performed using the unpaired t-test. N.S., not significant. (b) Confocal images showing the intracellular distribution of fluorescently labeled liposomes (final lipid concentration, approximately 50 μg/mL) in THP-1-derived macrophages. Liposomes were labeled with NBD-Chol (green), late endosomes were labeled with LysoTracker (red), and nuclei were labeled with Hoechst dye (blue). Bar, 10 μm.

We further investigated the mechanism of endocytosis using endocytosis inhibitors (Fig. 4). As shown in Figs. 4a and b, clathrin- and caveolae-mediated endocytosis and phagocytosis were involved in the endocytosis of DPPC/Chol liposomes. The involvement of multiple endocytosis mechanisms has also been reported for Chol/L-α-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphorylethanolamine-PEG-maleimide liposomes.¹⁶⁾ In DPPC/Chol (67/33 mol%) liposomes, phagocytosis was relatively major.

Fig. 4. Effect of Endocytosis Inhibitors on the Intracellular Fluorescence Intensity of (a) DPPC/Chol (67/33 mol%) Liposomes and (b) DPPC/Chol (80/20 mol%) Liposomes

Values are expressed as the mean ± standard deviation (n = 3). Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test. ***, p < 0.001 ****, and p < 0.0001 compared with the corresponding untreated control. CytD, cytochalasinD; Chlor, chlorpromazine.

Effect of Protein Adsorption onto the Liposome Surface

Plasma proteins adsorbed onto liposomes affect the internalization of liposomes.^19,35) Apolipoprotein A-1, apolipoprotein B, and apolipoprotein E have been shown to affect the uptake of liposomes by hepatocytes.^22,39,40) Multiple receptors have been shown to be associated with apolipoprotein-mediated uptake, including LDLR^35,40) and SR-B1.^19,35) Macrophages have various receptors that determine the control of recognition, endocytosis, and secretion of internalized objects.⁴¹⁾ We confirmed that THP-1 cells express LDLR⁴²⁾ and scavenger receptors⁴³⁾ (Fig. 5a). Therefore, we studied the adsorption of apolipoproteins A, B, and E on liposomes. After incubation with a mixture of apolipoproteins A, B, and E, the liposomes were isolated via size-exclusion chromatography, and the adsorbed proteins were analyzed via Western blotting. As shown in Fig. 5b, all apolipoproteins were largely adsorbed onto the DPPC/Chol (80/20 mol%) liposomes compared to the DPPC/Chol (67/33 mol%) liposomes. The other representative ligand proteins, which are ligands of the phagocytic cell receptor of THP-1 cells, are complement proteins such as C3b.⁴⁴⁾ We also obtained a similar difference in the amount of adsorbed protein by incubating C3b with liposomes (Fig. 5b).

Fig. 5. (a) Expression of LDLR and SR-B1 in the THP-1 Cells, (b) Adsorption of the Apolipoproteins onto the DPPC/Chol Liposomes and (c) the Effect of Pretreatment with Anti-LDLR and Anti-SR-B1 Monoclonal Antibodies (mAbs) on Intracellular Fluorescence Intensity of DPPC/Chol (80/20 mol%) Liposomes

(b) The liposomes were incubated with apolipoproteins A, B, and E and isolated from the unbound apolipoproteins using size exclusion chromatography. Liposome fractions were collected and the adsorbed apolipoproteins were analyzed by Western blotting. (c) Data are presented as mean ± standard deviation (n = 3). Statistical analyses were performed using the unpaired t-test. **, p < 0.01.

We investigated the effects of LDLR and scavenger receptor inhibition on the internalization of DPPC/Chol (80/20 mol%) liposomes by apolipoproteins. Figure 5c shows that internalization was largely inhibited by the pre-incubation with LDLR and SR-B1 antibodies. We used a mixture of LDL-R and SR-B1 antibodies because some apolipoproteins are recognized by both LDL-R and SR-B1³⁵⁾ and these two receptors act cooperatively.^19,45) These results indicated that the apolipoproteins contained in FBS were involved in the internalization of the DPPC/Chol liposomes through recognition by LDL-R and SR-B1, and differences in the amounts of adsorbed proteins may have been the main reason for the differences in the uptake efficiency between DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes, rather than a direct effect of liposome fluidity on internalization. Further research is needed to clarify the profiles of FBS proteins adsorbed onto the liposomes using quantitative and comprehensive measurement techniques, such as nano LC-MS/MS, and to study the involvement of complement receptors in the DPPC/Chol liposome internalization.

Controlling the amount of protein adsorbed onto the liposomes may be important for efficient cargo delivery using lipid-based particles. For example, besides endocytosis through receptors,^22,39,40) apolipoprotein E adsorbed onto lipid nanoparticles can rearrange lipids and contribute to endosomal escape.⁴⁶⁾ It has been shown that the protein corona is important for regulating cellular interactions and has been investigated to facilitate efficient nanoparticle uptake using lung surfactant or bronchoalveolar lavage fluid for its pulmonary applications.^47,48) Apolipoprotein E is synthesized by alveolar epithelial cell type I in the lungs.⁴⁹⁾ Therefore, the attachment of apolipoprotein E onto liposomes may be an attractive approach for the efficient cargo delivery to the lungs. The lipid composition affects protein adsorption onto liposomes.^50,51) Our results show that the contribution of the internalization mechanism is different depending on the Chol ratio (Fig. 4), which might be caused by the different profiles of adsorbed proteins. Similar results have been reported that complement dependence on liposome internalization is different depending on the Chol content of liposomes, although the lipid composition and sizes of the liposomes are different from those of our studies.⁵²⁾ The resultant different profiles might change the binding to the specific receptor, resulting in different contributions of the endocytosis modes.

In the case of clathrin, caveolae endocytosis or phagocytosis, liposome internalization, fusion of the liposome with the lysosome through endosomes or phagosomes, liposome degradation, and active substance release were involved. Moreover, in the case of clathrin- or caveolae-mediated endocytosis, some of the internalized liposomes are fused with the endosomal membrane, and the encapsulated active substances are released into the cytosol. Therefore, in the case of clathrin, caveolae endocytosis, or phagocytosis, the active substances can play roles to combat pathogens residing in the lysosome or cytosol,⁵³⁾ and either mechanism could be a strategy for treating NTM lung disease. It is presumed that the adsorption of liposomes onto the cell surface is the rate-limiting step for internalization into macrophage.⁴¹⁾ In this study, we found that some proteins that are the ligands of representative receptors of macrophages⁴¹⁾ adsorbed onto the DPPC/Chol (80/20) liposomes much more than onto the DPPC/Chol (67/33) liposomes. In fact, DPPC/Chol (80/20) liposome internalization was much greater than that of the other liposomes. A possible advantage of achieving high concentrations of active substances at the infection site is the decreased chance of developing antibiotic resistance.¹⁵⁾ Therefore, our results indicated that the DPPC/Chol (80/20 mol%) lipid composition could serve as a suitable treatment for NTM lung disease.

To date, the physicochemical properties of liposomes have been investigated as factors affecting cellular uptake efficiency using various lipid compositions. These physicochemical properties include particle size, shape, and charge.³⁴⁾ To the best of our knowledge, the present study is the first to report that the fluidity of DPPC/Chol liposomal membranes, which is controlled by different Chol ratios, can be an important factor in controlling the adsorption of proteins on liposomes and the subsequent cellular uptake efficiency. Further research is required to elucidate the effects of the membrane state of liposomes on protein adsorption as well as the relationship between adsorbed proteins and related receptors in cellular uptake.

CONCLUSION

We demonstrated that the cholesterol ratio of DPPC/Chol liposomes affected the cellular uptake efficiency of THP-1-derived macrophages and proposed that liposomal fluidity is important for controlling the adsorbed proteins on liposomes and cellular uptake efficiency. This knowledge will contribute to the efficient design of liposomal formulations, particularly for treating NMT lung diseases.

Acknowledgments

This work was partially supported by JSPS Kakenhi (Grant No. 22H02754 [K.S.-K]) and Japan Agency for Medical Research and Development (AMED, Grant No. 21fk0108429h0001 [K.S.-K. H.T. and K.H.]).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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