Cancer Cell Membrane–Biomimetic Nanoparticles for Homologous

Nov 7, 2016 - Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Dongguan 523808, People's Republic...
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Cancer Cell Membrane−Biomimetic Nanoparticles for Homologous-Targeting 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*,† †

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, People’s Republic of China ‡ Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Dongguan 523808, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: An 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 nanoparticle 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 an ICGpolymeric 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 membranes, ICNPs significantly promoted cell endocytosis and homologous-targeting tumor accumulation in vivo. Moreover, ICNPs were also good at disguising as cells to decrease interception by the liver and kidney. Through nearinfrared (NIR)-FL/PA dual-modal imaging, ICNPs could realize real-time monitored in vivo dynamic distribution with high spatial resolution and deep penetration. Under NIR laser irradiation, ICNPs exhibited highly efficient photothermal therapy to eradicate xenografted tumor. The robust ICNPs with homologous properties of cancer cell membranes can serve as a bionic nanoplatform for cancer-targeted imaging and phototherapy. KEYWORDS: cancer cell membrane, homologous targeting, dual-modal imaging, biomimetic nanoparticle, photothermal therapy 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

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onventional nanoparticle (NP)-mediated drug delivery has made a dramatic contribution to the development of clinical cancer therapy.1−3 However, NPs are exogenous materials, and early recognition by the immune system and clearance by the liver and kidney severely restrict the clinical applications of NPs. 4,5 To overcome this predicament, the design and preparation of 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 an active cell membrane are getting more and more attention.7,8 As a top-down approach, the biomimetic NPs bypass the laborious group-modified © 2016 American Chemical Society

Received: July 14, 2016 Accepted: November 7, 2016 Published: November 7, 2016 10049

DOI: 10.1021/acsnano.6b04695 ACS Nano 2016, 10, 10049−10057

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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 homologoustargeting ICNPs for dual-modal imaging guided photothermal therapy. Through specific homologous targeting and the EPR effect (passive targeting), ICNPs realized perfect tumor accumulation, dual-modal FL/PA imaging, and effective photothermal therapy after intravenous injection.

On the basis of the mature technology of indocyanine green (ICG)-loaded lipid-polymer NPs,22 we report cancer cell membrane-coated NPs with an ICG/poly(lactic-co-glycolic acid) (PLGA) core and cancer cell membrane shell (ICNPs) to synchronously recognize and eradicate tumors. 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 a homologous targeting effect at the cellular level but also demonstrated specific targeting ability at the animal level with high spatial resolution and deep penetration. The ICNPs can serve as an excellent nanoplatform for homologous-targeting dual-modal imaging and imagingguided photothermal therapy.

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 circulation times.14,15,19 Currently, tumor-targeting agents (e.g., aptamers, peptides, and antibodies) primarily recognize the overexpressed surface antigens of cancer cells and are based on ligand−receptor interactions.20−22 However, the inherent homologous adhesion property of cancer cells for tumor targeting is rarely investigated and exploited. 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 possess intercellular homologous binding capability with membrane proteins, which could be utilized for NP 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 is especially appropriate for targeting drug delivery and effective cancer therapy.

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 10050

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Figure 2. Characterization of biomimetic ICNPs. (A) TEM images of ICNPs. (B) Fluorescence spectra of the ICNPs, INPs, and free ICG. (C) PA imaging and quantitative curve of the PA intensity of ICNPs with different concentrations. (D) Infrared thermographic images of ICNPs, INPs, free ICG, and PBS after continuous laser irradiation (1 W/cm2, 8 min).

temperature (Tmax) of free ICG, INPs, and ICNPs reached 68.4, 74.1, and 74.2 °C, respectively, whereas the Tmax of phosphatebuffered saline (PBS) only increased to 27.6 °C with the same laser irradiation (Figures 2D and S3). The results indicated that ICNPs could achieve enough of a temperature increase for photothermal therapy. It is 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 a highly condensed concentration over 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 cells, the content of various proteins on the surface of ICNPs was investigated systematically. Protein gel electrophoresis exhibited the modulation of the protein profile, indicating that ICNPs and cancer cell membrane vesicles both possessed a similar protein profile compared with MCF-7 cell lysate (Figure 3A). Cellular adhesion molecules (e.g., EpCAM, N-cadherin, and galectin-3) on cancer cell membranes 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, Ncadherin, and galectin-3) for source-cell-specific targeting via the homologous binding mechanism, which could realize

membranes can provide 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 exhibit 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 a similar size distribution (Figure S1). The INPs were prepared by the same procedures with an ICG-PLGA core and 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 showed that the absorption/emission peak of INPs and ICNPs were both located at 780 or 815 nm, which was basically consistent with absorption/emission peak of ICG (Figure S2, Figure 2B). The encapsulation efficiency (EE) of ICG in cancer cell membrane vesicles was 19.33 ± 0.33%, while the EE of ICG in ICNPs rose to 36.65 ± 0.02%. A drug release test showed that 95.17 ± 0.42% of ICG was released from the cancer cell membrane vesicle 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 10051

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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 the six cell lines 293T, MCF-10A, HepG2, A549, MB231, and MCF-7 upon 2 h incubation with ICNPs. (D) Cellular uptake of different cells. (E) Mean fluorescence intensity of different cells.

The in vitro photothermal toxicity of homologous-targeting ICNPs to MCF-7 cells was next evaluated. The cells treated with free ICG + laser (containing 40 μg/mL ICG) caused about 12% 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% cell death (Figure 4C). This 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-carrying 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 the enhanced penetration and retention (EPR) effect of NPs, and a small amount of tumor accumulation was observed at 24 h.34 Furthermore, relying on the cancer cell membrane coating, ICNPs possessed both passive targeting of the 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 ICNPsbased PA imaging provided a high spatial resolution, which

specific recognition and binding between ICNPs and cancer cells (Figure 3B).32,33 To investigate the specificity of ICNPs to target homologous MCF-7 human breast cells, the targeting ability of ICNPs to MCF-7 cells, nontumor 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, confocal microscopy was used to observe the ICNPs’ cellular uptake of MCF-7 cells. After 2 h of incubation, cells treated with ICNPs presented much higher ICG fluorescence intensity in the cellular cytoplasm compared with that of free ICG and INPs (Figure 4A). The results were also evaluated by ICG fluorescence imaging in eight-well chamber slides through the ex/in vivo imaging system (Figure 4B). Furthermore, the different cellular uptake induced by homologous targeting was quantitatively investigated by flow cytometry analysis. The ICG uptake by ICNP encapsulation was increased by 1.8-fold and 3.2-fold compared with that of INPs and free ICG (Figure S4A,B). 10052

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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 an 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 with reduced interception by the 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.

showed the superb accumulation of ICG in tumor tissue and provided information on tumor microstructure (Figure 5A). After 24 h of 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 C, INPs achieved a small amount of tumor accumulation. The amount of ICNPs accumulated in the liver and kidneys was much lower than that of INPs, reduced by 51% and 34%, respectively (Figure 5C). Because of multiple cell membrane components, ICNPs can be disguised as cells to decrease 10053

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Figure 6. In vivo photothermal therapy efficacy of homologously targeting ICNPs to nude mice bearing MCF-7 tumors. (A) IR thermal images of MCF-7 tumor-bearing mice exposed to a 808 nm laser for 5 min (1 W/cm2). (B) MCF-7 tumor growth curves of different groups after treatments (n = 5). ICNPs obtain a higher photothermal therapy effect, and no tumor recurrence was noted over the course of 18 days. (**) P < 0.01. (C) Representative photos of mice bearing MCF-7 tumors and excised tumors 18 d after treatments. (D) Histological staining of the excised tumors 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.

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) at 150 μL for 24 h, the temperature increase in the tumor region during laser irradiation (1 W/cm2, 5 min) was investigated. Treated with free ICG + laser or PBS + laser for 5 min, tumors only increased to 37 or 35 °C, which was not high enough to destroy the tumors (43 °C caused irreversible tumor damage) (Figures 6A and 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. Combined with active homologous targeting and passive targeting of the EPR effect, ICNPs achieved high accumulation in tumor and induced a maximum temperature up to 55.3 °C, which was much higher than 43 °C (Figures 6A and S7). We evaluated the photothermal efficacy of ICNPs on MCF-7 tumors. There was no obvious variation in 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 in restricting the growth of tumors on the free ICG + laser, which was due to the rapid clearance of free ICG and small tumor accumulation in vivo (Figure 6B,C). INPs + laser could inhibit tumor growth only within 6 d, and the tumors eventually recurred (Figure 6B,C). Consequently, the survival rate of this group was 40.0% on 18 d post-treatment (Figure S8). It is notable that, because a high amount of ICNPs accumulated in tumors, ICNPs + laser-induced hyperthermia in

interception by the liver and kidney.36−38 Meanwhile, ICG in major organs and tumors of mice was separately extracted and quantified, and the total amount of ICG was consistent with the biodistribution result of FL imaging (Figure S5). Due to the 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 (Figure 5B,C). This proved that ICNPs not only have a homologous targeting effect at the cellular level but also demonstrate a homologous targeting ability at the animal level, which can be further applied in real-time dual-modal imaging with high spatial resolution and deep penetration. 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 a 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 the method in ref 39. The ICG excretion in urine was 0.26 ± 0.04 μg, and fecal ICG excretion was 20.50 ± 1.10 μg in 24 h, which suggested that ICNPs had an excretion pathway mainly from the liver into the small intestine and subsequently into fecal excretion. 10054

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sonics processor, USA) and then differential centrifugation (Beckman OptimaTM MAX-XP ultracentrifuge, USA), intracellular contents of cancer cells (107) were emptied and then the cancer membrane was achieved. Formulation of ICNPs. Briefly, 750 μg of ICG was dissolved in 1 mL of ethanol solution (4%), and 1 mL of 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 an ICG-PLGA core. To prepare cancer cell membrane vesicles, membrane material and 180 μg of DSPE-PEG2000 were physically extruded through a 220 nm polycarbonate membrane for seven passes. The membrane vesicles were then coated onto PLGA cores by coextruding vesicles and cores through a 220 nm polycarbonate membrane to form ICNPs. The INPs were prepared by the same procedures with an 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 morphology of the NPs. The absorption spectra were obtained 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.). The ICG concentration of ICNPs, INPs, and free ICG was quantified by testing with 808 nm fluorescent intensity. In vitro PA imaging and photoacoustic signal intensity of ICNPs with different concentrations were obtained with a commercial photoacoustic imaging system (Endra Nexus 128, Ann Arbor, MI, USA). 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 an infrared thermal imaging camera (Fluke Ti27, USA). The ICG EE in NPs was measured by isolating fresh NPs via ultracentrifuge (25 000 r/min, 30 min) (MAX-XP, Beckman, USA). FL spectrometry (F900, Edinburgh Industries, UK) was used to determine the nonentrapped ICG in the supernatant. To investigate the drug release profile, 1 mL of NPs was taken into a dialysis bag (Mw 3500), which was placed in 2 L of PBS at 37 °C. The dialysate was collected to determine the ICG content by FL spectrometry. Cancer Cell Membrane Protein Characterization. Cancer cell membrane protein of ICNPs was characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. The membrane protein was extracted from the MCF-7 cells, MCF-7 cell membrane, or ICNPs with RIPA lysis buffer and further determined by the BCA assay kit (Beyotime, China). All samples with the SDS-PAGE sample loading buffer were heated to 100 °C for 10 min. Samples (100 μg/well) with equal protein amounts were added into the wells with 10% SDS-PAGE gel, using SDS-PAGE electrophoresis buffer as running buffer in the Mini-PROTEAN Tetra System (BIO-RAD, CA, USA) based on the manufacturer’s instructions. Protein was stained by Coomassie Blue and imaged after water destaining for 12 h. Protein was transferred to polyvinylidene fluoride (PVDF) membranes for Western blot analysis, using the Mini-PROTEAN Tetra System (BIORAD) at 300 mA for 2 h. 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 antimouse IgG. The protein signals were measured by the enhanced chemiluminescence method using the ChemiDoc MP gel imaging system (Bio-Rad). Tumor Cell 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 a humidified atmosphere of 5% CO2. In Vitro Cellular Uptake. Near-infrared (NIR) imaging and flow cytometry were used for further quantitative analysis of cell uptake. MCF-7 cells, nontumor 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 a 24-well plate and cultured for 12 h. Cells were then changed with a medium containing

tumor resulted in complete remission, and no tumor relapse was observed within 18 d (Figure 6B,C). 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 the most significant tumor tissue damage (Figure 6D). Benefiting from specific homologous targeting and preferable photothermal response, a single dose of ICNPs + laser treatment resulted in complete tumor remission without tumor relapse (Figure 6D). The ICNPs’ toxicity to major organs was investigated by H&E staining, and 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 in nude mice (Figure S9). The results of both blood biochemistry tests 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 dual-modal imaging and photothermal therapy. The polymer-membrane core−shell ICNPs exhibited excellent monodispersity, photothermal response, and FL/PA dual-modal imaging properties. The cloak of a cancer cell membrane on ICNPs successfully camouflaged them and reduced interception of the liver and kidney, and the cell adhesion molecules on the surface of disguised NPs possessed homologous targeted binding to achieve high tumor accumulation. The longstanding ICNPs in tumors 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 Materials. ICG, PLGA (lactide, glycolide (50:50); MW, 5000− 15 000), methylthiazoletetrazolium (MTT), and hematoxylin and eosin (H&E) were purchased from Sigma−Aldrich (USA). Soybean lecithin and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-folate (DSPE) poly(ethylene glycol) (PEG) 2000 (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, and trypsin EDTA were acquired from Gibco Life Technologies (USA). RIPA lysis buffer was obtained from Beyotime Biotechnology (China). 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 ultra10055

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ACS Nano ICNPs (10 μg/mL of ICG), INPs (10 μg/mL of ICG), or free ICG (10 μg/mL of ICG). Cells were washed three times with PBS after 2 h of 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). 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 of incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde solution for 20 min, and then stained with Hoechst 33258 for 10 min and rinsed three times with PBS. Cellular uptake was finally observed by a 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 at 37 °C. The cells were irradiated for 5 min with a 1 W/cm2 808 nm laser, and the cell viability was quantified with an MTT assay. The cells were irradiated with an 808 nm laser and then fixed with 4% paraformaldehyde solution. After staining with calcein-AM and PI, an Olympus IX71 biological inverted microscope (Japan) 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 the Animal Care and Use Committee (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences). Four- to 6-week-old female BALB/c mice or nude mice (Vital River Laboratory Animal Technology Co. Ltd., China) were subcutaneously injected with MCF-7 cells (1 × 106) in the flank region.

μL, 350 μg/mL ICG), ICG (150 μL, 350 μg/mL ICG), and PBS (150 μL) via the tail vein. At 24 h postinjection, the 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 (150 μL, containing 350 μg/mL ICG) and PBS (150 μL) via the tail vein. At 24 h postinjection, the ALT/AST or BUN/CRE was evaluated by a liver or renal function activity assay kit (JianCheng Biotech, China). Major organs were excised and cut into 6 μm sections for H&E staining, which was observed using an Olympus microscope (Olympus BX53, Japan). Statistical Analysis. Results are expressed as mean ± standard error of the mean. The differences among groups were analyzed using one-way ANOVA analysis followed by Tukey’s post-test. P < 0.05 is indicated by a single asterisk (*), and P < 0.01 is indicated by a double asterisk (**).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04695. Additional figures (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Lintao Cai: 0000-0002-2461-6390 Author Contributions ⊥

2

tumor volume = length × (width) /2

Z. Chen, P. Zhao, and Z. Luo contributed equally to this work.

Notes

The authors declare no competing financial interest.

In Vivo Imaging and Biodistribution Analysis. When the volumes of MCF-7 tumors reached 100−200 mm3, the nude mice were divided into three groups randomly. Mice were injected with ICNPs, INPs, or free ICG (200 μL, 200 μg/mL ICG) via the tail vein. The fluorescence signals of ICG were obtained by an ex/in vivo imaging system (CRI Maestro, USA) (ex: 704 nm; filter: 735 nm). A preclinical photoacoustic computerized tomography scanner (Endra Nexus 128, Ann Arbor, MI, USA) was used to obtain the in vivo PA imaging. The mice were euthanized 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 signals of plasma or tissue samples that were treated with ICG were >20 times higher than those of the nontreated 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 of 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 16000g, and then the plasma was utilized for evaluation of the blood circulation time curve. Temperature Measurement during Laser Irradiation. The nude mice bearing MCF-7 tumors were injected intravenously with free ICG, INPs, or ICNPs (350 μg/mL ICG, 150 μL), and control mice were treated with 150 μL of PBS. At 24 h, the laser (808 nm, 1 W/cm2) irradiated the tumors for 5 min. The regions of maximum temperatures and infrared thermographic maps were obtained using a Ti27 infrared thermal imaging camera (Fluke, USA). To detect the effect of photothermal therapy in vivo, H&E was used to stain tumors 24 h after treatment. Antitumor Effect and Biosafety of ICNPs in Vivo. The nude mice were injected with ICNPs (150 μL, 350 μg/mL ICG), INPs (150

ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (Nos. 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, and JCYJ20130402092657771), China Postdoctoral Science Foundation Project (2015M572386), and Dongguan Project on Social Science and Technology Development (2015108101019). REFERENCES (1) Bardhan, R.; Lal, S.; Joshi, A.; Halas, N. J. Theranostic Nanoshells: From Probe Design to Imaging and Treatment of Cancer. Acc. Chem. Res. 2011, 44, 936−946. (2) Schutz, C. A.; Juillerat-Jeanneret, L.; Mueller, H.; Lynch, I.; Riediker, M.; Consortium, N. Therapeutic Nanoparticles in Clinics and Under Clinical Evaluation. Nanomedicine 2013, 8, 449−467. (3) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F. Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 1029−1038. (4) Dobrovolskaia, M. A.; Neun, B. W.; Man, S.; Ye, X.; Hansen, M.; Patri, A. K.; Crist, R. M.; McNeil, S. E. Protein Corona Composition does not Accurately Predict Hematocompatibility of Colloidal Gold Nanoparticles. Nanomedicine 2014, 10, 1453−1463. 10056

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DOI: 10.1021/acsnano.6b04695 ACS Nano 2016, 10, 10049−10057