Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells

Accepted Manuscript Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells

Kumiko Sakai-Kato, Mari Sakurai, Yuki Takechi-Haraya, Kunie Nanjo, Yukihiro Goda PII: DOI: Reference:

S0005-2736(17)30279-1 doi: 10.1016/j.bbamem.2017.09.005 BBAMEM 82578

To appear in: Received date: Revised date: Accepted date:

16 January 2017 6 August 2017 5 September 2017

Please cite this article as: Kumiko Sakai-Kato, Mari Sakurai, Yuki Takechi-Haraya, Kunie Nanjo, Yukihiro Goda , Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells, (2017), doi: 10.1016/j.bbamem.2017.09.005

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ACCEPTED MANUSCRIPT

Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells

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Kumiko Sakai-Kato*, Mari Sakurai, Yuki Takechi-Haraya, Kunie Nanjo, Yukihiro Goda

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Division of Drugs, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo

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158-8501, Japan

*Corresponding author

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Kumiko Sakai-Kato, PhD

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Tokyo 158-8501, Japan

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Division of Drugs, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku,

Phone: +81-3-3700-9662

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Fax: +81-3-3700-9662

E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract In this study, HepG2 cells, an in vitro model system for human hepatocytes, were used to evaluate the interaction of lipoprotein receptors with liposomes carrying fluorescently labeled cholesterol and their subsequent intracellular uptake. In these experiments, two

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lipoprotein receptors, scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein

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receptor (LDLR), accounted for approximately 20% and 10%, respectively, of the

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intracellular uptake of the labeled liposomes. These findings indicate that additional

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mechanisms contributed to liposomal internalization. Liposomes modified with both

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apolipoproteins A-I and E were internalized in HepG2 cells in FBS-depleted culture medium at the same levels as unmodified liposomes in FBS-containing culture medium, which

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indicates that apolipoproteins A-I and E were the major serum components involved in

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liposomal binding to SR-B1 or LDLR (or both). These results increase our understanding of

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the disposition of liposomes, processes that can directly affect the efficacy and safety of drug

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products.

Keywords: liposome; lipoprotein receptor; apolipoprotein; internalization; HepG2 cell

Abbreviations:

DMEM,

Dulbecco's

Modified

Eagle's

medium;

EDTA,

ethylenediaminetetraacetic acid; FBS, fetal bovine serum; HBSS, Hanks' Balanced Salt Solution; HDL, high-density lipoprotein; HSPC, hydrogenated soy phosphatidylcholine; LDL, 2

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low-density lipoprotein receptor; LDLR, low-density lipoprotein receptor; mAbs, monoclonal antibodies;

NBD-Chol,

25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol; PEG2000-DSPE, 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine;

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N-(carbonyl-methoxyPEG

PBS,

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phosphate buffered saline; siRNA, small inhibitory RNA; SR-B1, scavenger receptor class B

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type 1

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1. Introduction Nanotechnology-based drug formulations have been developed to improve the in vivo stability and pharmacokinetics of the encapsulated active substances or to modulate

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their release rate or active-site targeting [1,2]. Lipid-based formulations, such as

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liposomes, are being developed worldwide, and some are in clinical use [3–5]. Various

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types of drugs, including anticancer drugs, can be encapsulated within liposomes or in

of

liposomes

is

very

important

because

it

determines

their

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disposition

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their intramembrane space and then delivered to the target location in the body. The

blood-concentration profile, which in turn influences the safety and efficacy of

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encapsulated drug products. Moreover, for drugs with repeated dosing, the accumulation

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of lipid carriers needs to be considered because it can affect drug safety. Although the

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disposition of encapsulated drugs and liposomal uptake by macrophages have been elucidated in detail [6–9], little is known about the disposition of liposomes or

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liposomal constituents, such as cholesterol, by hepatocytes. According to one of the few reports available, a commercially available cholesterol-containing liposomal product is eliminated primarily through the feces (33%, compared with <2% in urine), presumably as a result of biliary excretion of intact liposomes or cholesterol (or both) [10]. Cholesterol appears to be especially important for stabilizing liposomes against the effects of various plasma components [11]. 4

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Low-density lipoproteins (LDLs), which are endogenous lipid-based particles, are incorporated into cells by binding to the low-density lipoprotein receptor (LDLR), a cell-surface glycoprotein that removes LDL from plasma by receptor-mediated endocytosis

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[12–15]. In addition to expressing LDLR, hepatocytes also express scavenger receptor class

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B type 1 (SR-B1), a multiligand receptor that binds a variety of lipoproteins, including

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high-density lipoprotein (HDL) and LDL [16,17]. Furthermore, SR-B1 is presumed to transfer additional cellular cholesterol to growing lipoprotein particles [18]. Apolipoproteins,

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which are ligands for several lipoprotein receptors, are thought to be involved in liposomal

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internalization, but the mechanism underlying the involvement of LDLR, SR-B1, and

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apolipoproteins in this process has not yet been clarified [18–22]. Therefore, in this study, we through which lipoprotein receptors

and

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investigated the molecular mechanism

apolipoproteins are involved in the internalization of PEGylated liposomes. The PEGylated

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liposomes that we used were approximately 100 nm in diameter and were composed of lipids,

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PEGylated lipids, cholesterol, and fluorescently labeled cholesterol (to enable the liposomes to be traced). The lipid composition and particle size, which were approximately the same as those of liposomal products in clinical use [3], minimized recognition of the liposomes by macrophages in the liver or spleen, thus prolonging their circulation [3,8]. Using HepG2 cells as an in vitro model system for human hepatocytes, we studied the interaction of our PEGylated liposomes with LDLR and SR-B1. 5

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2. Materials and Methods 2.1. Materials and cells Hydrogenated soy phosphatidylcholine (HSPC) and N-(carbonyl-methoxyPEG

NOF

Corporation

(Tokyo,

Japan);

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from

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2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG2000-DSPE) were purchased

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25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (NBD-Chol) was purchased from Avanti Polar Lipids (Alabaster, AL, USA); Dulbecco's Modified Eagle's

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medium (DMEM), RPMI-1640, penicillin–streptomycin, and Opti-MEM I were purchased

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from Life Technologies (Brooklyn, NY, USA); and fetal bovine serum (FBS) was obtained

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from Nichirei Biosciences (Tokyo, Japan). Recombinant human apolipoproteins A-I and E

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were purchased from Wako Pure Chemical Industries (Osaka, Japan). Apolipoprotein B from human plasma was purchased from Sigma-Aldrich (Tokyo, Japan). All other chemicals used

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in this study were of the highest purity available. HepG2 cells (American Type Culture

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Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin–streptomycin. Cells were grown in a humidified incubator at 37 °C and 5% CO2.

2.2. Preparation of liposomes The

lipid

composition

of

the

liposomes 6

used

in

our

experiments

was

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HSPC/PEG2000-DSPE/Chol/NBD-Chol

(56.3/5.3/37.4/1

mol

%).

NBD-Chol–labeled

liposomes were prepared by using a modified extrusion method [23]. Briefly, the desired amounts of lipids (60 µmol total lipids) was mixed in chloroform, and the mixture was dried

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by evaporation at 70 °C to create a thin homogeneous lipid film. The film was further dried

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overnight under vacuum desiccation to remove residual solvent. The dried film was hydrated

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with 2 mL of 5% (w/w) glucose aqueous solution under mechanical agitation at 70 °C for 5–10 min, and the hydrated film was dispersed by sonication for 5–10 min in a bath type

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sonicator (Sharp, Tokyo, Japan). The resulting dispersion was sonicated for an additional 1

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min with a tip-type sonicator (Sonics, Newtown, CT, USA). After sonication, the dispersion

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was passed 21 times through a mini-extruder (Avanti Polar Lipids) equipped with

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polycarbonate filters with 100-nm pores. The particle size, polydispersity index, and ζ-potential of the resulting liposomes were determined (Zetasizer Nano ZS, Malvern

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Instruments, Malvern, Worcestershire, United Kingdom). Hereafter, we refer to these

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NBD-Chol–labeled, PEGylated liposomes simply as “liposomes.”

2.3 Confocal microscopy The intracellular trafficking of the liposomes in HepG2 cells was followed by using confocal microscopy (A1, Nikon, Tokyo, Japan). Data were collected by using the manufacturer-supplied software and were exported as tagged image files. HepG2 cells (5 × 7

ACCEPTED MANUSCRIPT 105 cells per well) were plated in 35-mm glass-bottom dishes coated with poly-L-lysine (Matsunami, Osaka, Japan), each containing 1 mL of DMEM supplemented with 10% FBS (2 mL each well). After incubation for 72 h (37 °C, 5% CO2), the cells were exposed to

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liposomes (final lipid concentration, 50 μg/mL) in culture medium. After 4 h, the cells were

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washed with and kept in Hank’s balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA,

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USA) for confocal imaging. The nuclei were labeled with Hoechst 33342 (Life Technologies)

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according to the manufacturer’s instructions.

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2.4. Inhibition of endocytosis

Endocytosis was inhibited with 10 μg/mL chlorpromazine (an inhibitor of

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clathrin-mediated endocytosis), 150 μM genistein (which inhibits caveolae-mediated endocytosis), and 50 μM 5-(N-ethyl-N-isopropyl)amiloride (an inhibitor of macropinocytosis)

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[24,25]. We confirmed that these inhibitors did not cause significant toxicity (Supplementary

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Figure 1a) and that each effectively inhibited the appropriate endocytosis pathway (Supplementary Figure 1b–d). HepG2 cells (5 × 105 per well) were seeded in 12-well plates in DMEM containing 10% FBS (1 mL per well). After incubation for 48 h (37 °C, 5% CO2), one of the endocytosis inhibitors was added to the culture medium; 30 min later, liposomes in 500 μL of DMEM containing 10% FBS were added to the treated cells (final lipid concentration, 50 μg/mL), and the cells were incubated for 2 h (37 °C, 5% CO2). After 8

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incubation, the medium was replaced with PBS. Cells were trypsinized with 0.25% (v/v) trypsin–EDTA (Life Technologies), washed with HBSS three times, and suspended in lysis buffer (1.0% [v/v] Triton X-100 in HBSS). The resulting cell suspension was sonicated for 10

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min in a bath-type sonicator (Sharp), vortexed for 5 min, and centrifuged (15,000 × g, 4 °C,

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10 min).

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The fluorescence intensity of the supernatant was measured with a fluorescence spectrophotometer (F-7000, Hitachi High-Technologies, Tokyo, Japan). The excitation and

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emission wavelengths were 490 and 540 nm, respectively. The fluorescence intensity was

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normalized to the protein content of the cell lysate (Protein Assay Kit, Bio-Rad Laboratories,

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Hercules, CA, USA).

2.5 LDLR and SR-B1 blocking studies

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Monoclonal antibodies (mAbs) were used to block the ligand-binding domains of

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LDLR (located in the extracellular region of the N terminus [26]) and SR-B1. The mAbs to the two receptors were obtained from Calbiochem (San Diego, CA, USA) and Abcam (Cambridge, UK), respectively. HepG2 cells (5 × 105 per well) were seeded on 12-well plates in DMEM containing 10% FBS (1 mL per well) and incubated for 48 h (37 °C, 5% CO2). Cells were incubated with or without 5 μg/mL anti-LDLR or anti-SR-B1 mAb for 1 h at 4 °C [20,27,28], and then NBD-Chol–labeled liposomes (final lipid concentration, 50 μg/mL) in 9

ACCEPTED MANUSCRIPT 500 μL of culture medium were added to the treated cells, which were incubated for 1 h (37 °C, 5% CO2). In preliminary experiments, we evaluated mAb doses from 2.5 to 10.0 μg/mL (LDLR) and from 2.5 to 5.0 μg/mL (SR-B1). Anti-LDLR mAb doses from 5.0 to 10.0

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μg/mL yielded similar inhibitory efficiencies, as did anti-SRB1 mAb doses from 2.5 to 5.0

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μg/mL. We therefore decided to use a dose of 5 μg/mL for both mAbs in the experiments. We

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chose the 1-h incubation time because LDLR recycling occurs—reportedly reaching a steady state, during which the amounts of receptor-bound and intracellular LDL remain

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constant—within 2 h [28]. Therefore, we used a shorter incubation time (i.e., 1 h) to capture

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the data before anti-LDLRs that were bound to LDLRs were replaced by the liposomes. Cells

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incubated with an isotype control (anti-LDLR, purified mouse IgG2a [BioLegend, San Diego,

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CA, USA]; anti-SR-B1, normal rabbit IgG [Santa Cruz Biotechnology, Dallas, TX, USA]) were used as reference controls. After incubation, the medium was replaced with PBS, and

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the fluorescence intensity of the cells was measured as described in section 2.4.

2.6 Treatment with small inhibitory RNAs Small inhibitory RNA (siRNA) 25mer oligonucleotides for SR-B1 and LDLR and their negative controls were obtained from Life Technologies (Stealth RNAi and Stealth RNAi Negative Control Medium GC Duplex, respectively). The siRNAs were transfected into cells by using Lipofectamine RNAiMAX (Life Technologies) according to the 10

ACCEPTED MANUSCRIPT manufacturer’s protocols; 48 h after transfection, the culture medium was replaced with fresh medium containing liposomes, and incubation was continued for a further 2 h. After incubation, the medium was replaced with PBS, and the fluorescence intensity of the cells

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was measured as described in section 2.4.

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2.7 Effect of FBS on internalization of liposomes

HepG2 cells (5 × 105 per well) were seeded in 12-well plates in DMEM containing

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10% FBS (1 mL per well). After incubation for 48 h (37 °C, 5% CO2), the cells were washed

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with PBS; liposomes (final lipid concentration, 50 μg/mL) either in 500 μL of culture

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medium supplemented with 10% FBS or in 500 μL of FBS-depleted culture medium were

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added; and the cultures were incubated for an additional 2 h at 37 °C. Similarly, to clarify the role of apolipoproteins in the internalization of liposome into the cells, the liposomes (final

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lipid concentration, 50 μg/mL) were preincubated with apolipoprotein A-I, E, or B (final

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concentration of each apolipoprotein, 33 μg/mL) at 4 °C overnight [29]. Then the apolipoprotein-modified liposomes were added to the HepG2 cells in 500 μL of FBS-depleted culture medium, and the cells were incubated for an additional 2 h at 37 °C. After incubation, the intracellular fluorescence intensity (derived from NBD-Chol) was quantified as described in section 2.4.

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2.8 Western blotting The expression of human LDLR and SR-B1 was detected by western blotting, as

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described previously [30], by using mAbs to LDLR and SR-B1 (both obtained from Abcam).

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2.9 Statistical analyses

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Results are presented as means ± SDs of at least three experiments. Two-group comparisons were performed by using Student’s t-test. A P value less than 0.05 was

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considered significant.

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3. Results and Discussion

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3.1 Physicochemical properties of liposomes The mean particle size of the liposomes was 115 nm; the polydispersity index value

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was 0.12, indicating a homogenous size distribution; and the ζ-potential was –2.3 mV. We

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confirmed that the mean particle size and ζ-potential of the liposomes in culture medium containing 10% FBS were 117 nm and –2.6 mV, which are equivalent to the values measured in PBS without FBS. Furthermore, the liposome particles were stable even in FBS-containing culture medium at 37 °C for 3 h (Figure 1a).

3.2 Intracellular distribution of liposomes 12

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We used confocal microscopy to study the intracellular distribution of the inherently fluorescent liposomes in HepG2 cells. The expected dot-like staining pattern was localized to the perinuclear regions but not to the nucleus (Figure 1b,c). In addition, the plasma

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membrane remained unstained, thus demonstrating that nonspecific binding of the liposomes

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to the plasma membrane was negligible.

3.3 Mechanism of liposome internalization into HepG2 cells

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Treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis,

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significantly suppressed the fluorescence intensity of the liposomes in HepG2 cells (P < 0.01;

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Figure 2). In contrast, treatment with either 5-(N-ethyl-N-isopropyl)amiloride or genistein did

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not alter the amount of intracellular liposomes. Given that the contributions of caveolae-mediated endocytosis and macropinocytosis to liposomal internalization were

transport

into

the

hepatocytes

was

clathrin-mediated

endocytosis.

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liposomes

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negligible in our system (Figure 2), these observations suggest that a key mechanism of

Clathrin-mediated endocytosis reportedly is the primary mechanism by which nanoparticles are internalized into cells [31]. In particular, clathrin-mediated endocytosis is reported to be involved in the uptake of nanometer-sized liposomes into A549 and HeLa cells [32]. In contrast, cationic liposomes have been reported to be internalized in HepG2 cells by lipid-raft-mediated endocytosis [33]. Because the endocytotic pathway can vary depending 13

ACCEPTED MANUSCRIPT on the nanoparticle’s physicochemical properties (e.g., size, charge) [34], we cannot compare our current findings with the previous studies in detail, but our results are consistent with

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those supporting clathrin-mediated endocytosis of liposomes into hepatocytes.

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3.4 Binding of liposomes to lipoprotein-related receptors on HepG2 cells

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Clathrin-mediated endocytosis is mediated by cell-surface receptors [35]. To examine the involvement of lipoprotein-related receptors in the endocytosis of liposomes, we

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first confirmed the expression of LDLR and SR-B1 in the HepG2 cells used in our study. As

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shown in Figure 3a, western blotting confirmed that both proteins were expressed in HepG2

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cells, as indicated by an LDLR-specific band at approximately 120 kDa and an

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SR-B1-specific band at approximately 76 kDa; these values are close to the reported values [20,36].

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We then examined the involvement of LDLR and SR-B1 in the intracellular transfer

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of the liposomes. Both receptors are known to be involved in the internalization of cholesterol to maintain intrinsic cholesterol homeostasis [12–15,36]. The intracellular fluorescence intensity derived from NBD-Chol decreased to 92% of that of the control when anti-LDLR mAb was used and to 84% when anti-SR-B1 mAb was used; the reduction due to the anti-SR-B1 mAb was significant (P < 0.01) (Figure 3b). These results show that LDLR and SR-B1 were involved in the intracellular transfer of the liposomes. When both receptors 14

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were inhibited simultaneously, the inhibition efficiency was 63% (P < 0.05, Figure 3c). These data show that LDLR and SR-B1 acted cooperatively; as reported previously, SR-B1 mediates LDL association with and uptake by cells that do not express any LDLR [37].

interactions

[32,34].

Our

findings

imply

that

in

addition

to

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nonspecific

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Nanoparticles reportedly are internalized through multiple mechanisms, including

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lipoprotein-receptor–mediated endocytosis, other processes (such as clathrin- and caveolae-independent endocytosis through receptor–ligand binding, and endocytosis through

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nonspecific interactions [34]) were involved in the internalization of the liposomes by

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hepatocytes.

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We used receptor-specific siRNAs to confirm the significant inhibitory effect of

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knockdown of lipoprotein receptor production on intracellular fluorescence due to NBD-Chol. The efficiency of siRNA knockdown was confirmed through ImageJ analysis of the

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individual bands in the western blots [38] (as described in section 2.8), which indicated that

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LDLR and SR-B1 expression levels were decreased to 28% and 23%, respectively (Figure 4a). Treatment with the LDLR-specific siRNA decreased the fluorescence inside HepG2 cells to 91% of that when an siRNA negative control was used (P < 0.01, Figure 4b). When HepG2 cells were exposed to the SR-B1-specific siRNA, NBD-Chol–derived intracellular fluorescence decreased to 66% of the control value (P < 0.01, Figure 4b). These results confirm those of the receptor-blocking experiment (Figure 3) and indicate that both LDLR 15

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and SR-B1 were specifically involved in the internalization of the liposomes into hepatocytes. The difference between the inhibition levels observed in the antibody blocking and siRNA knockdown experiments (Figures 3 and 4, respectively) likely reflects the differing

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mechanisms of antibody inhibition and siRNA knockdown. Regardless, both methods of

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receptor inhibition show that the internalization of the liposomes by HepG2 cells was

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mediated specifically by LDLR and SR-B1.

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3.5 Involvement of apolipoproteins in liposomal binding to receptors

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The binding of lipoproteins such as LDL and HDL to LDLR and SR-B1 reportedly

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is mediated by apolipoproteins, including apolipoproteins A-I, B, and E in serum [39,40]. In

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addition, PEGylated liposomes administered to mice were shown to bind to serum apolipoproteins, but whether these proteins are involved in the clearance of PEGylated

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liposomes from the bloodstream has not yet been clarified [41].

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As shown in Figure 5, when liposomes in FBS-depleted culture medium were added to HepG2 cells, the observed intracellular NBD-Chol fluorescence intensity was about 40% less than that observed when the liposomes were added in culture medium containing 10% FBS (control). However, the decrease in intensity was significantly attenuated when liposomes modified with apolipoprotein A-I (P < 0.05, Figure 5a) or apolipoprotein E (P < 0.01, Figure 5c) were added. In contrast, apolipoprotein B did not attenuate the decrease in 16

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intensity (Figure 5b). This suggests some interesting size-dependent characteristics of apolipoprotein adsorption or opsonization of liposomes and their subsequent cellular uptake. Interestingly, when liposomes modified with both apolipoproteins A-I and E were added in

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FBS-depleted medium, there was no decrease in fluorescence intensity relative to that in the

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control experiment (Figure 5d). These results show that apolipoproteins A-I and E played a

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key role in the internalization of the liposomes into HepG2 cells. Apolipoproteins A-I and E have high affinity for SR-B1 and LDLR, respectively [39, 42], and our results indicate that

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liposome-adsorbed apolipoproteins A-I and E were recognized by SR-B1 and LDLR. These

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results are consistent with our results indicating that cooperation between LDLR and SR-B1

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was involved in the internalization of the liposomes (Figure 3c). Therefore, we suggest that

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the liposomes were taken up by endocytic process involving LDLR and SR-B1. SR-B1 is known to promote selective uptake of HDL–cholesteryl ester by cells without particle uptake.

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In addition, SR-BI reportedly binds HDL with high affinity and mediates the selective uptake

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of HDL–cholesteryl ester into the liver [43,44]. SR-BI also mediates the bidirectional flux of free cholesterol between cells and HDL [45–47]. Therefore, some portion of the cholesterol contained in the liposomes may have entered HepG2 cells via SR-B1 in the same manner as cholesterol or cholesteryl ester in HDL.

4. Conclusions 17

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We have clarified the contributions of LDLR and SR-B1 to the internalization of liposomes into HepG2 cells. Our results suggest that apolipoproteins A-I and E are bound to the surface of liposomes and are involved in their recognition by the lipoprotein receptors on

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hepatocytes. Our findings imply that in addition to lipoprotein-receptor–mediated endocytosis,

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other processes (such as clathrin- and caveolae-independent endocytosis through

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receptor–ligand binding, and endocytosis through nonspecific interactions [34]) are involved in the internalization of liposomes by hepatocytes. Further research is necessary to clarify the

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correlation between the physicochemical properties of liposomes (including lipid

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composition) and receptor specificity, with the goals of controlling liposome disposition and

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targeting liposomes to specific tissues. We surmise that the knowledge obtained from our

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current study with HepG2 cells will facilitate the development of hepatocyte-targeting liposomes and will provide insights into the accumulation and metabolism of liposomes,

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given that drug products are predominantly metabolized and excreted through the liver.

Acknowledgement

This work was supported in part by a JSPS KAKENHI (grant no. 15K07915 [K.S.-K.]) and by funding for Research on Regulatory Harmonization and Evaluation of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics, and Research on Development of New Drugs from the 18

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Japan Agency for Medical Research and Development, AMED. The authors would like to

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thank R. Takizawa and T. Mano for their technical assistance with the experiments.

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Figure Legends

Figure 1. (a) Temporal dependence of liposomal particle size during incubation of liposomes

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in culture medium containing 10% FBS.

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(b and c) Confocal images showing the intracellular distribution of fluorescently labeled

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liposomes (final lipid concentration, 50 μg/mL) in HepG2 cells: (b) negative control (no

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nuclei were labeled with Hoechst dye (blue).

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liposomes) and (c) liposomes added. Liposomes were labeled with NBD-Chol (green), and

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Figure 2. Effect of endocytosis inhibitors on the intensity of intracellular NBD-Chol

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fluorescence. HepG2 cells were treated with each inhibitor for 30 min before the labeled liposomes were added; intracellular uptake of the labeled liposomes was evaluated 2 h after

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they were added to the cells. **, P < 0.01 compared with the corresponding untreated control.

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Data are presented as means  SDs (n = 4).

Figure 3. Effect of pretreatment with anti-LDLR and anti-SR-B1 monoclonal antibodies (mAbs) on intracellular NBD-Chol fluorescence. (a) Detection of LDLR and SR-B1 in HepG2 cells by western blotting.

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the respective isotype control. Data are presented as means  SDs (n = 4).

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(c) HepG2 cells were pretreated for 1 h at 4 °C with 5 μg/mL anti-LDLR and 5 μg/mL

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anti-SR-B1 mAb before labeled liposomes were added. Intracellular NBD-Chol fluorescence was measured 1 h after the addition of the liposomes to the cells. *, P < 0.05 compared with

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the mixture containing both isotype controls. Data are presented as means  SDs (n = 4).

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Figure 4. Effect of LDLR and SR-B1 knockdown on the intensity of intracellular NBD-Chol

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fluorescence.

Knockdown of LDLR and SR-B1 expression by using specific siRNAs. HepG2 cells

Effect of LDLR and SR-B1 knockdown on the intensity of intracellular NBD-Chol

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were also treated with siRNA negative controls. The arrows indicate the target bands.

fluorescence. Fluorescence was evaluated 2 h after labeled liposomes were added to HepG2 cells. **, P < 0.01 compared with the control group (that is, cells treated with negative-control siRNAs). Data are presented as means  SDs (n = 4).

Figure 5. Effect of apolipoproteins on the intensity of intracellular NBD-Chol fluorescence. 29

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Intracellular NBD-Chol fluorescence was evaluated 2 h after the addition of labeled liposomes to HepG2 cells in culture medium containing 10% FBS (control group) and in culture medium without FBS (FBS(-)). Intracellular NBD-Chol fluorescence was also

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evaluated 2 h after the addition of apolipoprotein-modified labeled liposomes to HepG2 cells

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in culture medium without FBS (FBS(-) + Apo).

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(a) Apolipoprotein A-I, (b) apolipoprotein B, (c) apolipoprotein E, and (d) apolipoproteins A-I and E.

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**, P < 0.01 compared with the corresponding groups. *, P < 0.05 compared with the

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corresponding groups. Data are presented as means  SDs (n = 3).

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights We used fluorescent NBD-Chol-liposomes to study liposomal transit into hepatocytes.

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SR-B1 and LDLR participated in liposome uptake by HepG2 cells.

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Apolipoprotein A-I and E mediated the interactions among SR-B1, LDLR, and liposomes.

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