Cancer Cell MembraneâBiomimetic Nanoparticles for Homologous

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Cancer Cell Membrane-Biomimetic Nanoparticles for HomologousTargeting Dual-Modal Imaging and Photothermal Therapy Ze Chen, Pengfei Zhao, Zhenyu Luo, Mingbin Zheng, Hao Tian, Ping Gong, Guanhui Gao, Hong Pan, Lanlan Liu, Aiqing Ma, Haodong Cui, Yifan Ma, and Lintao Cai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b04695 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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Cancer Cell Membrane-Biomimetic Nanoparticles for Homologous-Targeting Dual-Modal Imaging and Photothermal Therapy Ze Chen1†, Pengfei Zhao1†, Zhenyu Luo1,3†, Mingbin Zheng1,2*, Hao Tian2, Ping Gong1, Guanhui Gao1, Hong Pan1,3, Lanlan Liu1, Aiqing Ma2, Haodong Cui1, Yifan Ma1, and Lintao Cai1*

1

Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics,

Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P.R. China 2

Guangdong Key Laboratory for Research and Development of Natural Drugs,

Guangdong Medical University, Dongguan 523808, P.R. China 3

University of Chinese Academy of Sciences, Beijing 100049, P.R. China

†

These authors contributed equally to this work.

* Corresponding Authors: L. Cai ([email protected]), M. Zheng ([email protected])

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ABSTRACT Active cell membrane-camouflaged nanoparticle, owning to membrane antigens and membrane structure, can achieve special properties such as specific recognition, long blood circulation and immune escaping. Herein, we reported a cancer cell membrane-cloaked nanoparticles system as a theranostic nanoplatform. The biomimetic nanoparticles (indocyanine green (ICG)-loaded and cancer cell membrane-coated nanoparticles, ICNPs) exhibit a core-shell nanostructure consisting of ICG-polymeric core and cancer cell membrane shell. ICNPs demonstrated specific homologous targeting to cancer cells with good monodispersity, preferable photothermal response, and excellent fluorescence/photoacoustic (FL/PA) imaging properties. Benefited from the functionalization of the homologous binding adhesion molecules from cancer cell membrane, ICNPs significantly promoted the cell endocytosis and homologous-targeting tumor accumulation in vivo. Moreover, ICNPs were also good at disguising as cells to decrease the interception by liver and kidney. Through near infrared (NIR)-FL/PA dual-modal imaging, ICNPs could realize real-time monitor in vivo dynamic distribution with high spatial resolution and deep penetration. Under NIR Laser irradiation, ICNPs exhibited the highly efficient photothermal therapy to eradicate the xenografted tumor. The robust ICNPs with homologous properties of cancer cell membrane can be served as a bionic nanoplatform for cancer targeted imaging and phototherapy. KEYWORDS: Cancer cell membrane, homologous-targeting, dual-modal imaging, biomimetic nanoparticle, photothermal therapy.


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Conventional

nanoparticles

(NPs)-mediated

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drug

delivery

has

dramatical

contribution to the development of clinical cancer therapy.1-3 However, NPs are exogenous materials, early recognition by immune system and clearance by the liver and kidney severely restricted the clinical applications of NPs.4, 5 To overcome the predicament, design and preparation of the nanosystems are becoming biocompatible and complicated. Meanwhile, functional groups must be modified on NPs, which is laborious and may increase potential uncontrollable factors.6 Biomimetic NPs coated with active cell membrane are getting more and more attention.7,

8

As a top-down approach, the biomimetic NPs bypass the laborious

groups-modified engineering.6, 9 Owing to the reserved antigens and cell-membrane structure, biomimetic NPs can acquire special functions, such as ligand recognition and targeting, long blood circulation and immune escaping, offering a promising drug nanoplatform for drug delivery, detoxification, and vaccination.10-12 By incorporating different cell types (such as leukocytes, platelets, and red blood cells) onto synthetic NPs (such as polymeric NPs, Au NPs and silica NPs), a variety of cell membrane-cloaked nanosystems with excellent features and functions have been developed.13-19 Examples include leukocyte membrane-cloaked silica microparticles with endothelium traversing properties, platelet membrane-coated nanovehicles with cancer targeting capabilities, and red blood cell membrane-cloaked NPs capable of long-circulating.14, 15, 19 Currently, tumor targeting agents (e.g., aptamers, peptides and antibodies) primarily recognized the overexpressed surface antigens of cancer cells and based on


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ligand-receptor interactions.20-22 However, we rarely investigated and exploited the inherent homologous adhesion property of cancer cells for tumor targeting. In tumors, the cancer cells expressing surface adhesion molecules (e.g., N-cadherin, galectin-3, epithelial cell adhesion molecule (EpCAM)) with homologous adhesion domains had been demonstrated to be responsible for multicellular aggregation formation.23-25 Cancer cells possessed intercellular homologous binding capability with membrane proteins, which could be utilized for NPs surface functionalization to offer the advantage of complete replication of surface antigenic diversity. Cancer cell membrane-coated NPs, therefore, are expected to obtain homologous-targeting, which are especially appropriate for targeting drug delivery and effective cancer therapy. Based on mature technology of indocyanine green (ICG) loaded lipid-polymer NPs,22 we reported cancer cell membrane-coated NPs with indocyanine green (ICG) / poly (lactic-co-glycolic acid) (PLGA) core and cancer cell membrane shell (ICNPs) to synchronously recognize and eradicate tumor. The morphology, photothermal response and

dual-modal

imaging

capability

of

ICNPs

were

characterized.

The

homologous-targeting, biodistribution, fluorescence (FL)/photoacoustic (PA) imaging and photothermal efficiency of ICNPs were systematically evaluated in vitro/vivo. Our results indicated that ICNPs not only had the homologous targeting effect at the cellular level, but also demonstrated the specific-targeting ability at the animal level with high spatial resolution and deep penetration. The ICNPs can be served as an excellent

nanoplatform

for

homologous-targeting

dual-modal

imaging-guided photothermal therapy.


imaging

and

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RESULTS AND DISCUSSION Preparation and Characterization of the ICNPs The fabrication of ICNPs consists of the following steps: extracting cancer MCF-7 cell membranes, forming PEGylated cell membrane vesicles, assembling ICG loaded PLGA-polymeric cores, and fusing membrane-PEG vesicles on the surface of ICG loaded polymeric cores by extrusion (Figure 1).26 Cell membrane can endow with targeting ability, and PEG functionalized on the cell membrane is able to diminish the nonspecific bindings between the NPs and serum proteins, and shield the surface of NPs from aggregation, opsonization, and phagocytosis in vivo.

27, 28

Transmission

electron microscopy (TEM) images demonstrated that ICNPs exhibited a typical core-shell structure and were spherical in shape with good monodispersity (Figure 2A). The hydrodynamic sizes of the ICNPs and INPs were respectively 200.4 and 197.3 nm with similar size distribution (Figure S1). The INPs were prepared by the same procedures with ICG-PLGA core, PEGylated phospholipid and soybean lecithin shell. The only difference between the ICNPs and the INPs was the cancer cell membrane of ICNPs instead of the soybean lecithin of INPs. The Ultraviolet-visible absorption or fluorescence spectra of INPs and ICNPs were showed that the absorption/emission peak of INPs and ICNPs were both located at 780 or 815 nm that was basically consistent with absorption/emission peak of ICG (Figure S2, Figure 2B). The encapsulation efficiency (EE) of ICG in cancer cell membrane vesicle was 19.33 ± 0.33 %, while the EE of ICG in ICNPs rose to 36.65 ± 0.02%. Drug release test showed that, 95.17 ± 0.42% of ICG was released from cancer cell membrane vesicle


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after 24 h, while the ICG release percentage of ICNPs was only 72.52 ± 0.10%. These results demonstrated that the polymer core had advantages in improving the drug EE and controlling the drug release. As shown in Figure 2C, the PA signal of ICNPs showed a concentration-dependent increase of ICG. The results illustrated that ICNPs demonstrated a great potential for FL/PA dual-modal imaging. The photothermal response of ICNPs was measured by infrared thermal imaging (Figure 2D). Under continuous laser irradiation (1 W/cm2, 8 min), the maximum temperature (Tmax) of free ICG, INPs and ICNPs reached to 68.4°C, 74.1 °C and 74.2°C, respectively. Whereas, the Tmax of phosphate buffered saline (PBS) only increased to 27.6 °C with the same laser irradiation (Figure 2D, S3). The results indicated that ICNPs could achieve enough temperature increase for photothermal therapy. It was worth pointing out that the temperature increase of free ICG was slightly lower than that of INPs and ICNPs. The temperature increase was attributed to the ICG encapsulation in the INPs or ICNPs possessing highly condense concentration than that of free ICG, and the enclosure of NPs entrapped the photothermal radiation, resulting in the lower heat dissipation and higher energy efficiency in the INPs or ICNPs after laser irradiation.29, 30 Validating Cell Adhesion Molecules and Homologous Targeting of ICNPs To confirm the successful ICNPs functionalization with adhesion molecules of cancer cell, the content of various proteins on the surface of ICNPs was investigated systematically. Protein gel electrophoresis exhibited the modulation of protein profile, indicating that ICNPs and cancer cell membrane vesicles both possessed the similar


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protein profile compared with MCF-7 cell lysate (Figure 3A). Cellular adhesion molecules (e.g., EpCAM, N-cadherin, and galectin-3) on cancer cell membrane played important roles in cell adhesion. Through specific recognition of adhesion molecules, they formed adhesion junctions to bind cells together within tumor tissues.23, 24, 31 Western blotting analysis was further conducted to confirm the presence of specific homologous binding adhesion molecules on ICNPs. The results demonstrated that ICNPs also possessed these cellular adhesion molecules (EpCAM, N-cadherin, and galectin-3) for source cell-specific targeting via the homologous binding mechanism, which could realize specific recognition and binding between ICNPs and cancer cells (Figure 3B).32, 33 To investigate the specificity of ICNPs to target to homologous MCF-7 human breast cells, the targeting ability of ICNPs to MCF-7 cells, non-tumor cells (e.g., 293T human embryonic kidney cells and MCF-10A human breast cells) and other tumor cells (e.g., HepG2 hepatocellular carcinoma cells, A549 human lung cancer cells and MDA-MB-231 human breast cancer cells) has also been evaluated (Figure 3C-E). The results demonstrated that the MCF-7 group exhibited the highest uptake efficiency and fluorescence intensity than other cell groups, which further confirmed the specific binding ability of ICNPs to homologous MCF-7 cells. In Vitro FL Imaging and Photothermal Cytotoxicity of ICNPs To investigate the FL imaging ability of ICNPs, the confocal microscopy was used to observe the ICNPs cellular uptake of MCF-7 cells. After 2 h incubation, cells treated with ICNPs presented much higher ICG fluorescence intensity in cellular


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cytoplasm compared with that of free ICG and INPs (Figure 4A). The results were also evaluated by ICG fluorescence imaging in 8-well chamber slide through the ex/in vivo imaging system (Figure 4B). Furthermore, the different cellular uptake induced by homologous-targeting was quantitatively investigated by the flow cytometry analysis. The ICG uptake by ICNPs encapsulation achieved 1.8-fold and 3.2-fold increase compared with that of INPs and free ICG (Figure S4A, S4B). The in vitro photothermal toxicity of homologous-targeting ICNPs to MCF-7 cells was evaluated in subsequence. The cells treated with free ICG + laser (containing 40 µg/mL ICG) caused about 12% of cell death, and the viability of cells treated with

INPs + laser (containing 40 µg/mL ICG) decreased to 55% (Figure 4C). While ICNPs + laser (containing 40 µg/mL ICG) induced up to 93% of cell death (Figure 4C). It suggested that the photothermal efficacy was greatly enhanced with the increased intracellular ICG concentration, due to the homologous-targeting of ICNPs. Fluorescence staining of living/dead cells simultaneously further proved the excellent photothermal efficacy of ICNPs + laser (Figure 4D). In Vivo Biodistribution of Homologous Targeting ICNPs Therefore, we investigated the biodistribution of ICNPs in tumor-carried nude mice by ICG fluorescence imaging. As shown in Figure 5A, free ICG was quickly cleared out from the body and no obvious signals were detected after 12 h. Compared with that of free ICG, INPs enhanced retention in vivo by enhanced penetration and retention (EPR) effect of NPs, and a small amount tumor accumulation was observed at 24 h.34 Furthermore, relying on cancer cell membrane coating, ICNPs possessed


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both passive targeting of EPR effect and homologous active targeting, which acquired a significantly enhanced tumor accumulation after 24 h injection (Figure 5A). The PA signals visually revealed the accordant distribution of ICG within and outside the tumor microvessels.35 The ICNPs-based PA imaging provided a high spatial resolution, which was prone to understand the superb accumulation of ICG in tumor tissue and get the information on tumor microstructure (Figure 5A). After 24 h injection, the tumors and major organs of mice were collected to further investigate the distribution of free ICG, INPs and ICNPs, respectively. As shown in Figure 5B and 5C, INPs achieved small amount of tumor accumulation. The amount of ICNPs accumulated in liver and kidneys was much lower than that of INPs, reducing by 51% and 34%, respectively (Figure 5C). Because of multiple cell membrane components, ICNPs can disguise as cells to decrease the interception of liver and kidney.36-38 Meanwhile, ICG in major organs and tumor of mice was separately extracted and quantified, and the total amount of ICG was consistent with the biodistribution result of FL imaging (Figure S5). Relying on cancer cell membrane coating, ICNPs possessed homologous tumor targeting and reduced interception of the liver and kidney, and finally obtained a 3.1-fold and 4.75-fold increased tumor accumulation compared with that of INPs and free ICG (Figure5B, 5C). It proved that ICNPs not only has the homologous targeting effect at the cellular level, but also demonstrate the homologous targeting ability at the animal level which can be further applied in the real-time dual-modal imaging with high spatial resolution and deep penetration.


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The blood circulation time curve revealed that the ICG blood concentration when loaded in ICNPs was 7-14 times higher than when given as free ICG at the first 60 min after injection (Figure S6). The AUC24h (the area under the plasma drug concentration-time curve over the period of 24 h) of ICNPs was 411 µg·min·mL-1, which was significantly higher (11.7 times) than that of free ICG (35 µg·min·mL-1) (Figure S6). These data indicated that ICNPs could significantly improve the bioavailability of free ICG. To investigate the excretion pathway of ICNPs from the body, we measured ICG content in urine and feces of BALB/c mice at 24 h after injection according to method of reference.39 The excretion of urine ICG was 0.26 ± 0.04 µg, and fecal ICG excretion was 20.50 ± 1.10 µg in 24 h, which suggested that ICNPs had the excretion pathways mainly from the liver into the small intestine and subsequently into fecal excretion. In Vivo Photothermal Therapy of ICNPs Enhanced by Homologous Targeting After intravenous injection of PBS, ICG, INPs or ICNPs (both containing 350 µg/mL ICG) with 150 µL for 24 h, the temperature increase in tumor region during

laser irradiation (1W/cm2, 5 min) was investigated. Treated with free ICG + laser or PBS + laser for 5 min, tumors only increased to 37 °C or 35 °C, which were not high enough to destroy tumors (43°C caused irreversible tumor damage) (Figure 6A, S7). While INPs achieved a small amount of tumor accumulation via the EPR effect, and tumors treated with INPs + laser exhibited a temperature rise to 48.2 °C. Combining with active homologous targeting and passive targeting of EPR effect, ICNPs achieved


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high accumulation in tumor and induced a maximum temperature up to 55.3 °C, which was much higher than 43°C (Figure 6A, S7). We evaluated photothermal efficacy of ICNPs to MCF-7 tumors. There was no obvious variation on mice weight in all treated groups, suggesting that the experimental treatments were well tolerated (Figure S8). As shown in Figure 6B, MCF-7 tumors treated with PBS plus laser grew rapidly, illustrating that laser irradiation had no effect on tumor growth. There was also no apparent efficacy to restrict the growth of tumors on the Free ICG + laser, which was due to the rapid clearance of free ICG and few tumor accumulation in vivo (Figure 6B, 6C). INPs + laser could only inhibit tumor growth within 6 d, while the tumors were eventually recurred (Figure 6B, 6C). Consequently, the survival rate of this group was 40.0% on 18 d post-treatment (Figure S8). It is notable that, because high amount of ICNPs accumulated in tumors, ICNPs + laser induced hyperthermia in tumor to obtain complete remission, and no tumor relapse was observed within 18 d (Figure 6B, 6C). The survival rate (ICNPs + laser) was 100% on day 18 (Figure S8), Hematoxylin and eosin (H&E) was used to stain MCF-7 tumor tissues at 24 h post treatments. Tumors treated with ICNPs + laser, INPs + laser and ICG + laser showed common features of thermal damage (Figure 6D). The ICNPs + Laser treatment led to most significant tumor tissue damage (Figure 6D). Benefited from specific homologous targeting and preferable photothermal response, a single dose of ICNPs + laser treatment resulted in a complete tumors remission without tumor relapse (Figure 6D). The ICNPs toxicity to major organs was investigated by H&E staining, and


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liver/kidney function index (liver functions index: alanine aminotransferase (ALT), aspartate aminotransferase (AST); kidney function index: creatinine (CRE), blood urea nitrogen (BUN)) by blood biochemistry test. Concentration of ALT/AST/CRE/BUN showed no obvious change compared with PBS control group (Figure S9). H&E staining indicated that treatment with ICNPs was biocompatible and safe to nude mice (Figure S9). The results of both blood biochemistry test and H&E staining suggested that ICNPs had good biocompatibility in vivo. CONCLUSION In conclusion, we developed cancer cell membrane-biomimetic ICNPs for homologous-targeting polymer-membrane

dual-modal core-shell

imaging ICNPs

and

exhibited

photothermal excellent

therapy.

The

monodispersity,

photothermal response, and FL/PA dual-modal imaging properties. The cloak of cancer cell membrane on ICNPs successfully camouflaged to reduce the interception of liver and kidney, and the cell adhesion molecules on the surface of disguised NPs possessed homologous-targeted binding to achieve highly tumor accumulation. The longstanding ICNPs in tumor realize real-time dual-modal imaging with high spatial resolution and deep penetration, and significantly enhanced photothermal therapy. Finally, the tumors were completely ablated with one single dose of ICNPs + laser treatment. By mimicking homologous cancer cells, ICNPs could open up a versatile strategy for safe and effective cancer therapy.

MATERIALS AND METHODS


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Materials ICG,

PLGA

(lactide,

glycoli-de

(50:50);

MW,

5000–15000),

methylthiazoletetrazolium (MTT), hematoxylin and eosin (H&E) were purchased from Sigma–Aldrich

(USA).

Soybean

lecithin

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-folate glycol) ) (PEG) 2000

(DSPE)

and (poly(ethylene

(DSPE-PEG2000) were obtained from Avanti (USA).

Calcein-AM/ propidium iodide (PI) and hoechst 33258 were purchased from Invitrogen (USA). Penicillin–streptomycin, fetal bovine serum, DMEM/F12 medium, trypsin EDTA were acquired from Gibco Life Technologies (USA). RIPA lysis buffer was obtained from Beyotime biotechnology (CHN). The monoclonal antibody against EpCAM was purchased from Thermo Fisher Scientific (USA). Antibody against N-cadherin was provided by BD Biosciences (USA). Galectin-3 Antibody (H-160) was

obtained

from

Santa

Cruz

Biotechnology

(USA). Antibody

against

Na+/K+-ATPase was purchased from GenScript (USA). Horseradish peroxidase -conjugated anti-rabbit IgG and anti-mouse IgG were acquired from Biolegend (USA). Amicon ultra-4 centrifugal filter device (molecular weight cut off 10 kDa) and 220 nm polycarbonate membrane were provided by Merck Millipore (Germany). Cancer Cell Membrane Extraction To obtain membrane material, a previously reported extrusion approach was used.14 By hypotonic lysis, mechanical membrane disruption (VCX130 ultrasonics processor, USA) and followed differential centrifugation (Beckman OptimaTM MAX-XP


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Ultracentrifuge, USA), intracellular contents of cancer cells (107) were emptied and then cancer membrane was achieved. Formulation of ICNPs Briefly, Then 750 µg ICG was dissolved into 1 mL of ethanol solution (4%), and 1 mL PLGA solution (dissolved in acetonitrile, 2 mg/mL) was added dropwise under 3 min sonication at a power of 39 W and frequency of 20 kHz to form ICG-PLGA core. To prepare cancer cell membrane vesicles, membrane material and 180 µg DSPE-PEG2000 were physically extruded through a 220 nm polycarbonate membrane for 7 passes. The membrane vesicles were then coated onto PLGA cores by co-extruding vesicles and cores through a 220 nm polycarbonate membrane to form ICNPs. The INPs were prepared by the same procedures with ICG-PLGA core, and PEGylated phospholipid but using soybean lecithin instead of cancer cell membrane to make sure the only difference between the ICNPs and the INPs was the cancer cell membrane. Characterization of the ICNPs The size distribution of the NPs was detected by a Zetasizer Nano ZS (Malvern, U.K.). The TEM (FEI Tecnai G2 F20 S-Twin, USA) was used to observe the particle size and morphologic of NPs. The absorption spectra was tested by UV/vis spectrometry (Lambda25, Perkin–Elmer, USA), and the fluorescence spectra were performed using fluorescence spectroscopy at 740 nm excitation (F900, Edinburgh Instruments, Ltd., U.K.). ICG concentration of ICNPs, INPs and free ICG was quantified by testing 808 nm fluorescent intensity. In vitro PA imaging and


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photoacoustic signal intensity of ICNPs with different concentrations was obtained with commercialized the photoacoustic imaging system (Endra Nexus 128, Ann Arbor, MI). Quartz cuvettes with the ICNPs, INPs, free ICG and PBS were treated with 1 W/cm2 808 nm laser irradiation (Leimai, China) for 8 min. The infrared thermographic maps were obtained by the infrared thermal imaging camera (Fluke Ti27, USA). The ICG EE in NPs was measured by isolating fresh NPs via ultracentrifuge (25000 r/min, 30 min) (MAX-XP, Beckman, USA). FL spectrometry (F900, Edinburgh Industries, UK) was used to determine the non-entrapped ICG in the supernatant. To investigate drug release profile, 1 mL of NPs were taken into dialysis bag (MW: 3500), which was placed in 2 L of PBS at 37°C. The dialysate was collected to determine ICG content by FL spectrometry. Cancer Cell Membrane Protein Characterization Cancer cell membrane protein of ICNPs was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. The membrane protein was extracted from the MCF-7 cells, MCF-7 cells membrane or ICNPs with RIPA lysis buffer and further determined by the BCA assay kit (Beyotime, CHN). All samples with SDS-PAGE sample loading buffer were heated to 100°C for 10 min. Samples (100 µg/well) with equal protein amount were added into the wells of 10% SDS-PAGE gel, using SDS-PAGE electrophoresis buffer as running buffer in Mini-PROTEAN Tetra System (BIO-RAD, CA) based on the instructions of manufacturer. Protein was stained by coomassie blue, and imaged after water destaining for 12 h. Protein was transferred to polyvinylidene fluoride (PVDF)


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membranes for western blot analysis, using Mini-PROTEAN Tetra System (BIO-RAD, CA) at 300 mA for 2h. Membranes were treated with anti-EpCAM, anti-N-cadherin, anti-galectin-3

and

anti-Na+/K+-ATPase,

followed

with

either

horseradish

peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein signals were measured by the enhanced chemiluminescence method using the ChemiDoc MP gel imaging system (Bio-Rad, USA). Tumor Cells Culture MCF-7 cells were used for the cell research. DMEM medium was supplemented with 10% heat-inactivated fetal bovine serum, 1% streptomycin and 1% penicillin. Cells were cultivated at 37 °C in humidified atmosphere of 5% CO2. In Vitro Cellular Uptake Near-infrared (NIR) imaging and flow cytometer were using for further quantitatively analysis of cell uptake. MCF-7 cells, non-tumor cells (293T, MCF-10A) and other types of tumor cells (HepG2, A549 and MB231) were seeded in Eight-well chambered coverglasses (4 × 105 cells/well) and 24-well plate, and cultured for 12h. Cells were then changed by the medium containing ICNPs (10 µg/mL of ICG), INPs (10 µg/mL of ICG) or free ICG (10 µg/mL of ICG). Cells were washed thrice with PBS after 2 h incubation, and the fluorescence signals of ICG were collected by the CRI maestro ex/in vivo imaging system (USA) (704 nm excitation and 735 nm filter). Then the cells were harvested and the fluorescence histograms of ICG were recorded by flow cytometer (BD Accuri C6, USA).


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MCF-7 cells (200 µL of medium, 2 × 104 cells/well) were seeded in eight-well chambered coverglasses (Lab-Tek, Nunc, USA). The old medium was replaced with the medium containing ICNPs (10 µg/mL of ICG), INPs (10 µg/mL of ICG) or free ICG (10 µg/mL of ICG) at 24 h. After 2 h incubation, cells were washed thrice with PBS, fixed with 4% paraformaldehyde solution for 20 min, then stained with Hoechst 33258 for 10 min and rinsed thrice with PBS. Cellular uptake was finally observed by TCS SP5 confocal laser scanning microscope (Leica, GER). In Vitro Photothermal Toxicity of ICNPs MCF-7 cells (1 × 104 cells/well) were seeded in a 96-well plate (100 µL of medium with ICNPs, INPs, or free ICG). After 2 h, cells were washed with PBS and replaced with fresh medium. Then, the digital dry bath incubator (Labnet Accublock, USA) was used to keep the plate in the environment of 37 °C. The cells were irradiated for 5 min with a 1 W/cm2 808 nm laser, and the cell viability was quantified with MTT assay. The cells were irradiated with 808 nm laser, and then fixed with 4% paraformaldehyde solution. After staining with calcein-AM and PI, the Olympus IX71 biological inverted microscope (JPN) was used to observe the viable and dead cells. Animals and Tumor Model Animals received care in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals. The procedures were approved by Animal Care and Use Committee (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences). Four-to-six weeks old female BALB/c mice or nude mice (Vital River


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Laboratory Animal Technology Co. Ltd, CHN) were subcutaneously injected with MCF-7 cells (1 × 106) at the flank region. Tumor volume = length × (width) 2/2. In Vivo Imaging and Biodistribution Analysis When the volumes of MCF-7 tumors reached to 100-200 mm3, nude mice were divided into three groups randomly. Mice were injected with ICNPs, INPs or free ICG (200 µL, 200 µg/mL ICG) via tail vein. The fluorescence signals of ICG were obtained by the ex/in vivo imaging system (CRI maestro, USA) (ex: 704 nm and filter: 735 nm). The preclinical photoacoustic computerized tomography scanner (Endra Nexus 128, Ann Arbor, MI) was used to obtain the in vivo PA imaging. The mice were executed at 24 h after injection. The major organs (heart, liver, spleen, lung, kidneys) and tumor were collected for semiquantitative biodistribution analysis and imaging through the ex/in vivo imaging system (CRi maestro, USA). The FL signal of plasma or tissue samples that were treated with ICG were > 20 times higher than that of the non-treated samples (i.e. background), FL spectrometry was successfully used for in vivo quantification of ICG.39, 40 To extract ICG, the major organs and urine/feces in 5 mL dimethyl sulfoxide (DMSO) were homogenized, and then centrifuged for 15 min at 9000 rpm. The ICG content of each sample was determined by FL spectrometry. Blood samples were centrifuged for 5 min at 16000 g, and then the plasma was utilized for evaluation of blood circulation time curve. Temperature Measurement during Laser Irradiation


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The nude mice bearing MCF-7 tumor were injected intravenously with free ICG, INPs or ICNPs (350 µg/mL ICG, 150 µL), and control mice were treated with 150 µL PBS. At 24 h, the laser (808 nm, 1 W/cm2) irradiated the tumors for 5 min. The region maximum temperatures and infrared thermographic maps were obtained through the Ti27 infrared thermal imaging camera (Fluke, USA). To detect the effect of photothermal therapy in vivo, hematoxylin and eosin stained tumors at 24 h after treatment. Anti-tumor Effect and Biosafety of ICNPs in Vivo The nude mice were injected with ICNPs (150µL, 350 µg/mL ICG), INPs (150µL, 350 µg/mL ICG), ICG (150µL, 350 µg/mL ICG) and PBS (150µL) via tail vein. At 24 h post-injection, laser (808 nm, 1 W/cm2) was used to irradiate the tumors for 5 min. The body weight of mice and tumor volumes were recorded. According to the animal protocol, mice with tumor sizes exceeding 2000 m3 were euthanized. Nude mice (5 per group) were injected with ICNPs (150µL, containing 350 µg/mL ICG) and PBS (150µL) via tail vein. At 24 h post-injection, the ALT/AST or BUN/CRE was evaluated by liver or renal function activity Assay Kit (JianCheng Biotech, CHN).

Major organs were excised and cut into 6 µm sections for H&E

staining, which was observed using Olympus microscope (Olympus BX53, JPN). Statistical Analysis Results were expressed as mean ± standard error of the mean. The differences among groups were analyzed using one-way ANOVA analysis followed by Tukey' s


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post-test. P < 0.05 was indicated by the single asterisk (*), and P < 0.01 was indicated by the double asterisk (**). Conflict of Interest: The authors declare no competing financial interest.

Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website.

Corresponding Author *Correspondence and requests for materials should be addressed to L.C. ([email protected]), M.Z. ([email protected]). Acknowledgment. The work was supported by the National Natural Science Foundation of China (No. 31571013, 81371679, 81501580, 81401509，21375141, and 81401520), Instrument Developing Project of CAS (YZ201439), Key International S&T Cooperation Science Foundation Project (2015DFH50230), Shenzhen Science and Technology Program (GJHS20140610152828690, JSGG20160331185422390, JCYJ20160429191503002, KQCX20140521115045447, JCYJ20130402092657771), China Postdoctoral Science Foundation Project (2015M572386) and Dongguan Project on Social Science and Technology Development (2015108101019).

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Figure captions Figure 1. Illustration of the cancer cell membrane-biomimetic nanoparticles for targeting recognition of source cancer cell, dual-modal imaging and photothermal therapy. (A) Preparation procedure of ICNPs. Extracting cancer MCF-7 cell membrane hybridized with PEGylated phospholipids (DSPE-PEG), and then coated onto ICG loaded polymeric cores by extrusion. (B) Schematic of homologous targeting ICNPs for dual-modal imaging guided photothermal therapy. Through specific homologous targeting and EPR effect (passive targeting), ICNPs realized perfect tumor accumulation, dual-modal FL/PA imaging and effective photothermal therapy after intravenous injection. Figure 2. Characterization of biomimetic ICNPs. (A) TEM images of ICNPs. (B) The fluorescence spectra of the ICNPs, INPs and free ICG. (C) The PA imaging and the quantitative curve of the PA intensity of ICNPs with different concentrations. (D) The infrared thermographic images of ICNPs, INPs, free ICG and PBS after continuous laser irradiation (1 W/cm2, 8 min). Figure 3. Validating cell adhesion molecules and homologous targeting of ICNPs. (A) SDS-PAGE protein analysis. Samples were stained with coomassie blue. I: Cancer cell lysate, II: Cancer cell membrane vesicles, III: ICNPs. (B) Western blotting analysis of membrane-specific protein markers. Samples were run at equal protein concentrations and were immune-stained against membrane markers including EpCAM, N-cadherin, galectin-3 and Na+/K+-ATPase. I: Cancer cell lysate, II: Cancer cell membrane vesicles, III: ICNPs. (C) Flow cytometric profiles of six cell lines


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including 293T, MCF-10A, HepG2, A549, MB231 and MCF-7 cells upon 2 h incubation with ICNPs. (D) The cellular uptake of different cells. (E) Mean fluorescence intensity of different cells. Figure 4. In vitro FL imaging and photothermal cytotoxicity of ICNPs. (A) Confocal microscopy images of MCF-7 cells treated with free ICG, INPs, or ICNPs. (B) NIR images of MCF-7 cells treated with free ICG, INPs, or ICNPs. (C) Survival of MCF-7 cells treated with free ICG + laser, INPs + laser or ICNPs + laser under different ICG concentrations. (D) Fluorescence images of MCF-7 cells treated with laser, ICG + laser, INPs + laser, or ICNPs + laser. Viable cells were stained green with calcein-AM, and dead cells were stained red with PI. (Scale bar = 100 µm) Figure 5. In vivo biodistribution of homologous-targeting ICNPs after intravenous injection. (A) Time-lapse NIR FL images and PA images of nude mice in vivo. ICNPs exhibited obvious homologous-targeting effect. (B) Ex vivo NIR FL images of major organs and tumors after injection of free ICG, INPs and ICNPs at 24 h, ICNPs realized perfect tumor accumulation meanwhile reduced interception by liver and kidney. (C) Semiquantitative biodistribution of free ICG, INPs and ICNPs in nude mice determined by the averaged fluorescence intensity of each organ. The data are shown as mean ± SD (n = 3); a single asterisk (*) indicates P < 0.05, and a double asterisk (**) indicates P < 0.01. Figure 6. In vivo photothermal therapy efficacy of homologous-targeting ICNPs to nude mice bearing MCF-7 tumors. (A) IR thermal images of MCF-7 tumor-bearing mice exposed to 808 nm laser for 5 min (1 W/cm2); (B) MCF-7 tumor


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growth curves of different groups after treatments (n = 5). ICNPs obtain higher photothermal therapy effect and no tumor recurrence was noted over a course of 18 days. (**)P < 0.01; (C) Representative photos of mice bearing MCF-7 tumors and excised tumors on 18 d after treatments. (D) Histological staining of the excised tumors at 24 h after injection of PBS, free ICG, INPs, and ICNPs under laser irradiation. Thermal damage was identified in tumors treated with free ICG, INPs, and ICNPs. (*) P < 0.05, (**) P < 0.01.


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Figure 1 209x151mm (300 x 300 DPI)


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Figure 3 208x125mm (300 x 300 DPI)


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Figure 5 159x99mm (300 x 300 DPI)


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