Suppression of Phagocytic Activity Leads to the Efficient Surface Modification of Macrophages with Liposomes for Developing a Biomimetic Drug Delivery System

Abstract

Macrophages selectively infiltrate the lesion sites of several diseases, including cancers, and, thus, have attracted attention as a biomimetic drug delivery carrier. To achieve the efficient drug loading of macrophages with minimal cytotoxicity, drugs are preferably encapsulated into nanoparticles, such as liposomes, and modified on the surface of macrophages rather than being incorporated into cells. However, liposomes are rapidly taken up by macrophages after binding to the cell surface because of their strong phagocytic activity. To overcome this, we herein attempted to modify the surface of macrophages with liposomes by suppressing their phagocytic activity using a pretreatment with anionic liposomes. We confirmed that 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG)- and cholesterol-rich anionic liposomes were efficiently taken up by RAW264.7 murine macrophage-like cells. Furthermore, the cellular uptake of anionic liposomes by RAW264.7 cells was higher in the absence of fetal bovine serum (FBS) than in its presence. Moreover, the viability of RAW264.7 cells was maintained above 90% when cells were incubated with anionic liposomes for 3 h, whereas viability was markedly decreased after a 24-h incubation. Based on these results, we pretreated RAW264.7 cells by an incubation with DSPG- and cholesterol-rich liposomes for 3 h in the absence of FBS. This pretreatment significantly inhibited the internalization of other liposomes, which subsequently bound to the cell surface. Therefore, we succeeded in modifying the surface of macrophages with liposomes, and liposome-modified macrophages have potential as a biomimetic active drug delivery carrier.

INTRODUCTION

Immune cells, such as macrophages, neutrophils, and T cells, have an intrinsic ability to migrate to specific disease sites across physical barriers.^1,2) Therefore, they have attracted attention as biomimetic drug delivery carriers. Macrophages have the potential to selectively accumulate in the lesion sites of several diseases, such as cancer,^3,4) Parkinson’s disease,⁵⁾ and human immunodeficiency virus (HIV) encephalitis.⁶⁾ Moreover, they possess a high phagocytic capacity, and, thus, the efficient drug loading of macrophages is feasible. Therefore, macrophages are increasingly being used as a cellular carrier for targeted drug delivery.⁷⁾

However, since the incorporation of a large amount of drugs into macrophages may reduce cell viability and activity, difficulties are still associated with loading macrophages with a sufficient amount of drugs with minimal cytotoxicity. One of the approaches used to achieve an abundant, but harmless, drug payload is to load drugs onto the cell surface. Previous studies successfully modified cell surfaces with drug-incorporated nanoparticles without any significant cytotoxicity.^8,9) We also developed surface-modified mesenchymal stem cells with liposomes, a widely used drug carrier, using magnetic anionic liposome (Mag-AL)/atelocollagen (ATCOL) complexes.¹⁰⁾ We recently attempted to apply this method to macrophages by modifying their surface with liposomes. However, contrary to expectations, approximately 80% of Mag-AL/ATCOL complexes were internalized by macrophages within 3 h of their binding to the cell surface. This finding was attributed to the strong phagocytic ability of macrophages. Therefore, other approaches need to be developed for the efficient surface modification of phagocytic cells with liposomes.

The hepatic clearance of liposomes in rats was previously shown to be markedly lower in a repeated dose study than in a single dose study.^11,12) This finding was attributed to the saturation of the phagocytic hepatic uptake of liposomes by Kupffer cells. Based on these findings, we postulated that the phagocytic ability of macrophages may be suppressed by a pretreatment with empty liposomes, leading to the efficient surface modification of macrophages with Mag-AL/ATCOL complexes.

In the present study, we attempted to optimize the conditions of the pretreatment of macrophages with empty liposomes for the efficient suppression of their phagocytic activity. Moreover, we investigated whether the surface modification of macrophages with liposomes may be achieved using the combined application of an empty liposome pretreatment and Mag-AL/ATCOL complexes.

MATERIALS AND METHODS

Cell Culture

RAW264.7 murine macrophage-like cells were supplied by the European Collection of Authenticated Cell Cultures (Salisbury, U.K.). RAW264.7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 °C under 5% CO₂/95% air.

Preparation of Liposomes

Anionic liposomes composed of 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG) (NOF Inc., Tokyo, Japan), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), cholesterol (Nacalai Tesque, Kyoto, Japan), and DiOC18(3) (Biotium Inc., Fremont, CA, U.S.A.) were prepared using the previously reported hydration method.¹³⁾ The lipid composition of each anionic liposome was listed in Table 1. Large or small anionic liposomes were obtained by the sonication for 30 s or 3 min using a probe-type sonicator, respectively.

Table 1. Physicochemical Properties of Anionic Liposomes Used in This Study

	Lipid composition DSPG : DSPC : cholesterol	Size	Particle size (nm)	Zeta potential (mV)	Polydispersity index (PDI)
Liposome A	5 : 1 : 4	Small	118.9 ± 8.3	−34.7 ± 2.6	0.18 ± 0.03
		Large	854.4 ± 44.8	−34.2 ± 1.4	0.33 ± 0.04
Liposome B	5 : 3 : 2	Small	107.9 ± 4.1	−28.6 ± 1.5	0.19 ± 0.01
		Large	905.5 ± 19.6	−28.1 ± 1.5	0.34 ± 0.06
Liposome C	1 : 5 : 4	Small	101.0 ± 6.4	−21.4 ± 1.6	0.24 ± 0.02
		Large	926.3 ± 26.6	−22.4 ± 2.8	0.47 ± 0.07
Liposome D	1 : 7 : 2	Small	110.9 ± 11.6	−12.2 ± 1.8	0.28 ± 0.02
		Large	898.0 ± 12.7	−12.7 ± 1.1	0.49 ± 0.05

Each value represents the mean ± S.D. (n = 3).

Mag-AL composed of DSPG, cholesterol, and DiIC18(3) (Wako Pure Chemical Corporation, Osaka, Japan) was also prepared by the hydration method described in our previous study.¹³⁾ Mag-AL contained 0.1 mg/mL of iron oxide (II, III) magnetic nanoparticles (Sigma-Aldrich, St. Louis, MO, U.S.A.) in its internal aqueous phase. Mag-AL/ATCOL complexes were formed by gently mixing 200 µg lipid/mL of Mag-AL with 40 µg/mL of ATCOL (Koken Co., Ltd. Tokyo, Japan). The particle sizes and ζ-potentials of the prepared liposomes were measured using Zetasizer Pro (Malvern Instrument, Worcestershire, U.K.).

Cellular Uptake of Anionic Liposomes in RAW264.7 Cells

RAW264.7 cells were seeded on 24-well culture plates at a density of 1 × 10⁵ cells/cm² and cultured for 48 h. The culture medium was removed and 500 µL of DMEM containing 5–200 µg lipids of each anionic liposome with or without 10% FBS was added to the well. After an incubation at 37 °C for 3 or 24 h, cells were washed twice with PBS and cell viability was measured using Cell Counting Reagent SF (Nacalai Tesque) and the Synergy HTX multimode microplate reader (Agilent Technologies Japan, Ltd., Tokyo, Japan). Cells were then lysed using lysis buffer (0.5% Triton X-100, 2 mM ethylenediaminetetraacetic acid, 0.1 M Tris, pH 7.8), and the resultant lysates were centrifuged at 10000 × g at 4 °C for 10 min. The amount of anionic liposomes in the supernatant was quantified by measuring fluorescence intensity with a Synergy HTX multimode microplate reader. The amount of anionic liposomes was normalized to the protein content of the cells measured using a protein assay BCA kit (Nacalai Tesque).

Cellular Association and Internalization of Mag-AL in RAW264.7 Cells

RAW264.7 cells were seeded on 35-mm culture dishes at a density of 1 × 10⁵ cells/cm² and cultured for 48 h. The culture medium was removed and 2 mL of DMEM containing 200 µg lipid of small liposome A or 400 µg lipid of large liposome A was added, followed by an incubation at 37 °C for 3 h. Liposomes were then removed, and 2 mL of Hank’s balanced salt solution (HBSS) containing 100 µg lipid of Mag-AL/ATCOL complexes was added to the wells. Following a 30-min incubation at 37 °C on a magnetic plate (OZ Biosciences, San Diego, CA, U.S.A.), cells were washed twice with ice-cold HBSS. To measure the total associated amount of Mag-AL, cells were lysed using lysis buffer. To measure internalized anionic liposomes, cells were treated with 0.25% trypsin for 5 min, followed by an incubation in 0.1% collagenase at 37 °C for 30 min. Cells were then collected by centrifugation at 250 × g at 4 °C for 5 min and lysed using lysis buffer. The resultant lysates were centrifuged at 10000 × g at 4 °C for 10 min and the amount of Mag-AL in the supernatant was quantified by measuring fluorescence intensity with the Synergy HTX multimode microplate reader. In the fluorescence microscopy study, cells were fixed with 4% paraformaldehyde and observed using the fluorescence BZ-X810 microscope (KEYENCE Corporation, Tokyo, Japan).

Statistical Analysis

Results are presented as the mean ± standard deviation (S.D.) of three or four experiments. Two-group comparisons were performed using the Student’s t-test.

RESULTS AND DISCUSSION

Optimization of Pretreatment Conditions of RAW264.7 Cells with Anionic Liposomes for the Efficient Suppression of Their Phagocytic Activity

Effects of Particle Sizes and the Lipid Composition of Anionic Liposomes on Their Cellular Uptake by RAW264.7 Cells

Negatively charged nanoparticles are efficiently internalized into macrophages via scavenger receptor-mediated uptake.¹⁴⁾ Therefore, we used anionic liposomes in the pretreatment of macrophages to suppress their phagocytic activity. On the other hand, the in vitro uptake of anionic liposomes by macrophages is markedly affected by the physicochemical properties of liposomes.^15–17) Therefore, we initially investigated the effects of the lipid composition and particle size of anionic liposomes on their uptake by RAW264.7 cells. The particle size, zeta potential, and polydispersity index of each liposome type are listed in Table 1. As shown in Fig. 1, the efficiency by which DSPG-rich liposomes (liposomes A and B) were taken up by RAW264.7 cells was slightly higher than that of DSPG-poor liposomes (liposomes C and D). This result is in accordance with previous findings showing that the amount of anionic liposomes composed of 50% DSPG taken up by J774 murine macrophage-like cells was markedly larger than that of liposomes composed of a lower DSPG content (9 and 30%).¹⁸⁾ Moreover, among DSPG-rich liposomes, the cellular uptake of liposome A was higher than that of liposome B. This may be attributed to differences in the content of cholesterol. Huong et al. reported that the binding and subsequent uptake of liposomes by rat peritoneal macrophages increased with a change in the content of cholesterol in liposomes from 22 to 44%.¹⁶⁾ It currently remains unclear why cholesterol-rich liposomes are more efficiently taken up by macrophages than cholesterol-poor liposomes. However, cholesterol-rich (50%) liposomes were previously shown to be more stable than cholesterol-poor (22%) liposomes both in vitro and in vivo (Kirby et al.).¹⁹⁾ Therefore, we assumed that liposome A remained more stable during the incubation with RAW264.7 cells than liposome B, resulting in greater cellular internalization. Regarding particle sizes, we found that the amount of lipids taken up by RAW264.7 cells was higher for small liposomes than for large liposomes.

Fig. 1. Cellular Uptake of Anionic Liposomes with Different Lipid Compositions and Particle Sizes by RAW264.7 Cells

Small or large anionic liposomes with different lipid compositions (50 µg/mL) were added to each well and incubated at 37 °C for 3 h in the presence or absence of FBS. The amount of anionic liposomes in the supernatant was quantified by measuring fluorescence intensity, and normalized to the protein content of the cells. Each value represents the mean + S.D. (n = 4). * p < 0.05; ** p < 0.01, significantly different from the absence of FBS.

In addition, since the components of the incubation medium have been shown to affect the in vitro uptake of anionic liposomes by macrophages,^16,17) we also investigated whether the presence of FBS affected the uptake of anionic liposomes by RAW264.7 cells. The results obtained showed that the cellular uptake of anionic liposomes was slightly lower in the presence of FBS (Fig. 1). Johnstone et al. also reported that the cellular uptake of anionic liposomes by mouse bone marrow macrophages was decreased in the presence of serum proteins.¹⁷⁾ Regarding this phenomenon, the adsorption of serum proteins on the surface of liposomes has been suggested to increase the hydrophilicity of liposomes, resulting in limited interactions between liposomes and cell membranes or receptors. Therefore, among the various liposomes tested, the uptake of liposome A by RAW264.7 cells in the absence of FBS was the most efficient.

Effects of the Concentration and Incubation Time of Anionic Liposomes on the Viability of RAW264.7 Cells

We examined the effects of the lipid concentrations and incubation times of liposome A on the viability of RAW264.7 cells. The viability of RAW264.7 cells was maintained above 90% when cells were incubated with 200 µg/mL of small liposome A or 400 µg/mL of large liposome A for 3 h (Fig. 2A). On the other hand, lipid concentration-dependent cytotoxicity was observed in RAW264.7 cells after a 24-h incubation with liposome A, regardless of its particle size (Fig. 2B). Therefore, we decided that the incubation of RAW264.7 cells with 200 µg/mL of small liposome A or 400 µg/mL of large liposome A for 3 h was an adequate pretreatment condition to suppress the phagocytic activity of RAW264.7 cells.

Fig. 2. Viability of RAW264.7 Cells Incubated with Anionic Liposomes

Small or large anionic liposome A (5–400 µg/mL) were added to each well and incubated at 37 °C for 3 h (A) or 24 h (B) in the absence of FBS. Cell viability was measured by WST-8 assay. Each value represents the mean ± S.D. (n = 4).

Effects of the Pretreatment of RAW264.7 Cells with Anionic Liposomes on the Internalization of Mag-AL/ATCOL Complexes after Binding on the Cell Surface

We confirmed that small liposome A was more efficiently taken up by RAW264.7 cells than large liposome A based on the lipid amount (Fig. 1). However, it is impossible to decide which particle size is more suitable for suppressing the phagocytic activity of RAW264.7 cells based on this result because the volume and particle number per unit weight of lipid markedly differs between small and large liposomes.²⁰⁾ Therefore, we comparatively assessed the effects of the pretreatment of RAW264.7 cells with small or large liposome A on the internalization of the subsequently added Mag-AL/ATCOL complexes. The particle size and ζ-potential of Mag-AL was 118.2 ± 9.2 nm and −42.4 ± 5.5 mV, respectively. When Mag-AL was complexes with ATCOL, both particle size and ζ-potential were increased to 223.6 ± 10.1 nm and 3.8 mV, respectively. As shown in Fig. 3A, approximately 50% of surface-bound Mag-AL/ATCOL complexes were immediately internalized by RAW264.7 cells not pretreated with liposome A. Moreover, the majority of complexes were internalized into cells 24 h after binding. Similar results were obtained from fluorescence microscopic images (Fig. 3B). On the other hand, the initial intensive internalization of complexes was strongly suppressed to approximately 15% by the pretreatment with small liposome A (Fig. 3C). However, the internalization of the complexes eventually reached approximately 50% 24 h after binding. When cells were pretreated with large liposome A, the cellular associated amount of Mag-AL/ATCOL complexes (284.2 ± 7.6 µg lipid/mg protein) were not significantly different from when cells were pretreated with small liposome A (280.2 ± 14.3 µg lipid/mg protein), whereas only 6% of complexes were initially internalized into cells, and only 15% were internalized 24 h after binding (Fig. 3D). Fluorescence microscopic images demonstrated that a large percentage of Mag-AL/ATCOL complexes were located on the surface of cells (Fig. 3E). It has been reported that macrophages significantly migrate into tumor tissue within 24 h, and therefore the prepared surface-modified macrophages with liposomes could serve as a tumor-targeted drug delivery carrier.

Fig. 3. Cellular Association and Internalization of Mag-AL/ATCOL Complexes in RAW264.7 Cells Pretreated with Anionic Liposomes

Mag-AL/ATCOL complexes were added to RAW264.7 cells without a pretreatment (A) or pretreated with small (C) or large (D) liposome A, and then incubated at 37 °C for 30 min in the presence of a magnetic field. To measure the total associated amount of Mag-AL, cells were lysed at predetermined time points using lysis buffer. To measure internalized anionic liposomes, cells were treated with 0.25% trypsin for 5 min, followed by an incubation in 0.1% collagenase. The amount of Mag-AL in each sample was quantified by measuring fluorescence intensity, and normalized to the protein content of the cells. Each value represents the mean ± S.D. (n = 4). Fluorescence microscopic images of Mag-AL/ATCOL complex-modified RAW264.7 cells without a pretreatment (B) or pretreated with large liposome A (E). Cells were fixed with 4% paraformaldehyde and observed using a fluorescence microscope. Scale bar: 10 µm.

A previous study demonstrated that the lipid amount of large liposomes (160 nm) taken up by macrophages was approximately 3-fold lower than that of small liposomes (25 nm), whereas the aqueous volume introduced into macrophages was approximately 10-fold larger in large liposomes than in small liposomes.²⁰⁾ Since the estimated volume of large liposome A is more than 300-fold larger than that of small liposome A, we assumed that the aqueous volume taken up by RAW264.7 cells was larger in large liposome A than in small liposome A, resulting in the efficient suppression of the phagocytic activity of cells. Moreover, we confirmed that the release rate of large liposome A from RAW264.7 cells was slower than that of small liposome A (Supplementary Fig. S1). This longer retention property of large liposome A in RAW264.7 cells may contribute to longer suppression of cellular phagocytic activity.

In conclusion, we herein demonstrated that the pretreatment of macrophages with DSPG- and cholesterol-rich anionic liposomes enabled the efficient surface modification of macrophages with Mag-AL/ATCOL complexes. Macrophages with surfaces modified with liposomes have potential as a biomimetic active drug targeting carrier. We are now preparing surface-modified macrophages with anti-cancer drugs-encapsulated Mag-AL/ATCOL complexes, and evaluating their tumor-targeting and anti-tumor efficiency in tumor-bearing mice.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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