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Liposomes Coated with Isolated Macrophage Membrane Can Target Lung Metastasis of Breast Cancer Haiqiang Cao, Zhaoling Dan, Xinyu He, Zhiwen Zhang, Haijun Yu, Qi Yin, and Yaping Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b03148 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016
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Liposomes Coated with Isolated Macrophage Membrane Can Target Lung Metastasis of Breast Cancer Haiqiang Cao†,‡, Zhaoling Dan†, Xinyu He†,‡, Zhiwen Zhang†, *, Haijun Yu†, Qi Yin†, Yaping Li†,*
†State
Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ‡ University
*
of Chinese Academy of Sciences, Beijing 100049, China
Corresponding author: Prof. Zhiwen Zhang (
[email protected]) and Prof. Yaping
Li (
[email protected]) Tel/Fax: +86-21-2023-1979
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KEYWORDS: Macrophage; Liposome; Drug delivery; Metastasis; Breast cancer.
ABSTRACT: Cancer metastasis leads to high mortality of breast cancer and is difficult to treat because of poor delivery efficiency of drugs. Herein, we report wrapping of drug carrying liposome with an isolated macrophage membrane to improve delivery to metastatic sites. The macrophage membrane decoration increased cellular uptake of emtansine liposome in metastatic 4T1 breast cancer cells and had inhibitory effects on cell viability. In vivo, the macrophage membrane enabled the liposome to target metastatic cells, and produced a notable inhibitory effect on lung metastasis of breast cancer. Our results provide a biomimetic strategy via the biological properties of macrophages to enhance the medical performance of a nanoparticle in vivo for treating cancer metastasis.
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Cancer metastasis refers to the spread of cancer cells from a primary tumor to seed secondary tumors in distant sites and causes high mortality of breast cancer.
1-5
In clinic, the
metastatic breast cancer is largely incurable and the five-year survival rate is only about 20%.6, 7
Despite recent advances in current clinical modalities, the therapeutic efficacy is still limited
and the overall survival is improved only a few months at most.
5-8
The lack of efficacy of
current therapy on cancer metastasis is the result of poor drug delivery efficiency where many current therapeutic agents can’t reach the metastatic sites. 1, 5 Nanotherapeutics can effectively target tumor by the enhanced permeability and retention (EPR) effects, which suggests potential for metastasis targeting.
9-16
However, the
metastases are often small clusters of cancer cells with high multiplicity and dispersion in the invaded organs.
1, 4
Moreover, the metastases foci has poor vasculature and angiogenic
dormancy when the diameter of metastatic lesions is less than 1-2 mm.
1, 3
Unfortunately, the
EPR effect is limited to vascularized tumors with the diameter larger than ~4.6 mm, which prevents the use of nanotherapeutics for targeting small, unvascularized metastases. 1 Macrophages, one of the most abundant cells in tumor microenvironments, are directly associated with tumor progression and metastasis.
2-4, 17-19
During lung metastasis of breast
cancer, macrophage can actively bind to metastatic cancer cells via interactions between the α4 integrins of macrophage and the vascular cell adhesion molecule-1 (VCAM-1) of cancer cells, thereby priming the metastatic cells for survival and promoting their outgrowth to form metastatic lesions.
2-5, 20, 21
Recently, bioinspired strategies are gaining attention for drug
delivery application because the nanoparticles are engineered to mimic the cellular functions. Nanoparticle carriers have biomimicked erythrocyte, leukocyte, tumor cells, macrophage and stem cells, etc. 22-34 Here we hypothesized that liposomes coated with macrophage membranes could enhance delivery to metastatic sites via the α4 integrin-VCAM-1 interactions between macrophages and metastatic cancer cells.
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Herein, we explore the use of macrophage membrane decorated liposome to improve specific metastasis targeting capability and suppress lung metastasis of breast cancer (Scheme 1). Macrophage membrane was isolated from a murine monocyte/macrophage cell line of RAW 264.7 cells with high expression of α4 and β1 integrins. We first encapsulated the cytotoxic anti-cancer drug of emtansine into pH-sensitive liposome, and then coat emtansine liposome with isolated macrophage membrane to generate macrophage membrane coated emtansine liposome (MEL) for targeting metastases foci in lung (Scheme 1). The decoration of emtansine liposome with macrophage membrane was performed by extrusion method and characterized. In particular, the specific metastasis targeting ability and anti-metastatic activity of MEL were evaluated in lung metastatic breast cancer model.
RESULTS AND DISCUSSION Characterization of MEL. MEL was prepared with the following three steps: (i) preparation of emtansine liposome, (ii) isolation of macrophage membrane, and (iii) camouflaging emtansine liposome with macrophage membrane (Scheme 1). Emtansine liposome was prepared with distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG), 1,2-dioleoyl-sn-glycero-3-phoshoethanolamine (DOPE) and emtansine. The high performance liquid chromatography (HPLC) analysis showed that the encapsulation efficiency of emtansine in liposome was 96.7%, which indicated that the lipophilic emtansine was almost entirely entrapped into liposome. The field emission transmission electronic microscope (FE-TEM) measurements showed that emtansine liposome was nanometer-sized spherical particles (Figure 1A). The dynamic light scattering (DLS) analysis indicated that the hydrodynamic diameter of emtansine liposome was 64.5 nm with a polydispersity index (PDI) of 0.281 (Figure S1). The zeta potential was -28.0±1.8 mV. The mean diameter of emtansine liposome was hardly changed within 24 h in phosphate buffered solution (PBS) at pH 7.4, suggesting good stability of emtansine liposome over time (Figure S2). In the 4 ACS Paragon Plus Environment
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intracellular acidic environments, the DOPE in emtansine liposome could undergo a phase transition from a lamellar phase to a fusogenic hexagonal phase, thereby leading to the destabilization of lipid membrane and rapid release of encapsulated cargos.
11, 35
The mild
acidic environments in endosomes (pH 5 ∼ 6) and lysosomes (pH 4 ∼5) could trigger the ondemand drug release.
9, 11, 36
To determine the pH-responsive properties, the drug release
profile of emtansine liposome was monitored in PBS at different pH values of 7.4, 5.5 and 4.7 (Figure 1B). At predetermined time intervals, the drug amount of emtansine in the release media was measured by HPLC analysis. With the extending of time, the accumulative drug release was rapidly raised at pH 5.5 and pH 4.7, but slowly increased at pH 7.4. At 8.0 h of incubation, the cumulative drug release percentage of emtansine liposome reached 51% at pH 5.5 and 58% at pH 4.7, which was much higher than that at pH 7.4 (Figure 1B). As a result, emtansine liposome presented pH-sensitive drug release profiles in the intracellular acidic environments. Then, RAW 264.7 macrophage cells were disrupted by direct extrusion, and centrifuged to isolate the macrophage membrane. The protein content in the purified membrane was determined by bicinchonininc acid (BCA) protein assay. The purified macrophage membrane was mixed with emtansine liposome and serially extruded through a series of polycarbonate membrane with pore sizes of 400 nm and 200 nm to produce MEL. The decoration of macrophage membrane onto emtansine liposome was determined by various in vitro physicochemical characterizations. Following the membrane decoration, the hydrodynamic diameter of MEL was increased to 115.4 nm with a PDI of 0.154 (Figure S3-4), which was bigger than that of emtansine liposome. The particle size of MEL was unchanged within 24 h in PBS at pH 7.4, indicating good stability of MEL over time (Figure S5). The zeta potential of MEL was 26.2±2.9 mV, which was comparable with emtansine liposome. The FE-TEM observations showed that MEL was nanometer-sized and had a core-shell yolk-like structure (Figure 1A), which was smaller than the hydrodynamic diameter. Then, the protein profiles 5 ACS Paragon Plus Environment
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in macrophage membrane and MEL were determined by SDS-PAGE electrophoresis assay. The protein composition in macrophage membrane was mostly retained in MEL, but no protein signal was detected in emtansine liposome (Figure 1C). It was reported that macrophage could bind to metastatic cancer cells via the α4β1 integrins-VCAM-1 interactions. 20, 37
In order to clarify the possible mechanism of MEL mediated specific metastasis targeting,
the expression of α4 and β1 integrins on RAW 264.7 cells was measured by immunofluorescence detection and fluorescence-activated cell sorting (FACS) analysis (Figure 1D-E). The captured images under laser confocal scanning microscope (LCSM) showed strong fluorescence on the cell membrane (Figure 1D), which effectively confirmed the expression of α4 and β1 integrins on cell membrane of RAW 264.7 cells. The expressions of α4 and β1 integrins on RAW 264.7 cells were also verified by FACS analysis (Figure 1E). Moreover, the α4 and β1 integrins markers on RAW 264.7 cells, purified macrophage membrane and MEL were further detected by western-blotting measurements to determine the quality of purified macrophage membrane and the effective decoration of macrophage membrane in MEL (Figure 1F). By contrast, the β-actin signals in these samples were measured as control. The specific protein signals of α4 and β1 integrins were obviously observed in RAW 264.7 cells, macrophage membrane and MEL, which validated the presence of these integrin markers. In addition, the β-actin signals were readily detected in the samples of RAW 264.7 cells, but barely observed in purified macrophage membrane and MEL, which denoted the high purity of isolated macrophage membrane and no interference of cell actins on the decoration in MEL (Figure 1F). Collectively, these evidences suggested the successful decoration of emtansine liposome with macrophage membrane in MEL. In Vitro Therapeutic Efficacy. The effect of macrophage membrane decoration on cellular uptake was evaluated in metastatic 4T1 breast cancer cells (Figure 2). The high expression of VCAM-1 on 4T1 cell membrane was verified (Figure S6). To ensure the specificity of the biomimetic interactions, the α4β1 integrins markers in MEL were blocked 6 ACS Paragon Plus Environment
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by their specific anti-integrin α4β1 monoclonal antibody (ab80944, abcam) for subsequent measurements. The blocked MEL was prepared according to the manufacturer’s protocol and performed as control. The cellular uptake of emtansine liposome, MEL and blocked MEL was visualized by LCSM and further quantified by FACS analysis. Emtansine liposome was labeled with hydrophobic fluorescence dye DiI by physical encapsulation. Then, fluorescent MEL and blocked MEL were prepared from the fluorescent emtansine liposome for further measurements. The LCSM images showed that MEL displayed a higher internalization into metastatic 4T1 cells than emtansine liposome or blocked MEL group, which was denoted by the stronger red fluorescence signals (Figure 2A and Figure S7). Moreover, the FACS analysis showed the cellular uptake of MEL in 4T1 cells had a 2.0-fold higher signal than that of emtansine liposome, which effectively verified the enhanced effect of macrophage membrane decoration in MEL on cellular uptake (Figure 2C and Figure S8). However, when the α4β1 integrins markers were blocked, the cellular uptake was reduced by 25% in comparison to MEL group (p
