Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles

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Enhanced anti-tumor activity of EGFP-EGF1conjugated nanoparticles by a multi-targeting strategy Bo Zhang, Ting Jiang, Li Ling, Zhonglian Cao, Jingjing Zhao, Yanyan Tuo, Xiaojian She, Shun Shen, Xinguo Jiang, Yu Hu, and Zhiqing Pang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00036 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016

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ACS Applied Materials & Interfaces

Enhanced

Anti-Tumor

Activity

of

EGFP-EGF1-Conjugated

Nanoparticles

by

a

Multi-Targeting Strategy Bo Zhang a,1, Ting Jiang a,1, Li Lingb, Zhonglian Caoc, Jingjing Zhaob, Yanyan Tuob, Xiaojian Sheb, Shun Shenb, Xinguo Jiangb, Yu Hua*, Zhiqing Pangb* a

Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of

Science & Technology, Wuhan, Hubei, 430022, China; b

School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of

Education, 826 Zhangheng Road, Shanghai, 201203, China; c

Instrumental Analysis Center of School of Pharmacy, Fudan University, 826 Zhangheng

Road, Shanghai, 201203, China 1: equal contribution to the work * Corresponding author: Yu Hu, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei, 430022, PR China. Tel.: +86-27-85726335; fax: +86-27-85776343. E-mail address: [email protected] (Y. Hu). Zhiqing Pang, School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of Education, 826 Zhangheng Road, Shanghai, 201203, China Tel.: +86-21-51980069; fax: +86-21-51980069. E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract Tumor stromal cells have been increasingly recognized to interact with tumor parenchyma cells and promote tumor growth. Therefore, we speculated that therapeutics delivery to both parenchyma cells and stromal cells simultaneously might treat tumor more effectively. Tissue factor (TF) was shown to extensively locate in tumor and abundantly site in both tumor parenchyma cells and stromal cells including neo-vascular cells, tumor-associated fibroblasts and tumor-associated macrophages, indicating it might function as a favorable target for drug delivery to multiple cell types simultaneously. EGFP-EGF1 is a fusion protein derived from factor VII, the natural ligand of TF. It retains the specific TF binding capability but does not cause coagulation. In the present study, nanoparticle modified with EGFP-EGF1 (ENP) was constructed as a multi-targeting drug delivery system. The protein binding experiment showed EGFP-EGF1 could bind well to A549 tumor cells and other stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages. Compared with unmodified nanoparticles (NP), ENP uptake by A549 cells and those stromal cells was significantly enhanced but inhibited by excessive free EGFP-EGF1. In addition, ENP induced more A549 tumor cell apoptosis than Taxol and NP when paclitaxel (PTX) was loaded. In vivo, ENP accumulated more specially in TF-overexpressed A549 tumors by in vivo imaging, mainly regions unoccupied by factor VII and targeted tumor parenchyma cells as well as different types of stromal cells by immunofluorescence staining. Treatment with PTX-loaded ENP (ENP-PTX) significantly reduced the A549 tumor growth in nude mice while NP-PTX- and Taxol-treated mice had lower response to the therapy. Furthermore, H&E and TUNEL staining revealed that ENP-PTX induced more severe tumor necrosis and more extensive cell apoptosis. Altogether, the present study


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demonstrated that ENP could target multiple key cell types in tumors through TF, which could be utilized to improve the therapeutic effect of anticancer drugs. Keywords：EGFP-EGF1，TF，tumor，multi-targeting，nanoparticle Introduction Effective tumor targeted drug delivery is of vital importance for the treatment of malignant tumors as conventional chemotherapeutics lack selectivity and are always associated with severe adverse effects.1, 2 The key issue in tumor targeting therapy is to understand the proliferative foundation of tumor and utilize it as the therapeutic target to destroy tumors.3 For a long time, it has been believed that tumor growth mainly depends on tumor parenchyma cells and neo-vascular cells, and accordingly for tumor treatment, most researches are focused on targeting either of these two types of cells or both of them simultaneously via the enhanced permeability and retention (EPR) effect as well as active targeting strategy.2, 4 However, the benefits of these strategies are only modest and clinical success is limited mainly due to the heterogeneous expression of targeted receptors. Therefore, targetable receptors more widely and homogeneously distributed in tumors may enable improved drug efficacy,5, 6 which is still in continued and urgent need. It is now increasingly recognized that tumor stroma contains distinct cell types including tumor-associated fibroblasts, tumor-associated macrophages and neo-vascular cells which interact with each other, exert variable roles in enabling tumor progression and function as the “soil” of tumor growth.3, 7, 8 Furthermore, the tumor parenchymal cell-centric paradigm has been gradually shifted to a stroma-centric paradigm9 and recently some approaches targeting to distinct types of tumor stromal cells have also been designed. For example, fibroblast activation protein (FAP)-responsive pro-drug or docetaxel conjugate nanoparticle has been established to selectively



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kill tumor-associated fibroblasts to produce a therapeutic response.10 Moreover, survival benefits are also obtained by altering macrophages11 or delivering proapoptotic peptides selectively to tumor-associated macrophages.7 However, these approaches targeting therapeutics only to a limited number of cell types in a tumor, which could not eradicate the tumor “soil” totally and there is still large room for improvement. Therefore, we propose that the multi-therapeutic strategy targeting both tumor parenchyma cell and different types of tumor stromal cells and destroying tumor “seeds” and “soil” simultaneously might be a more promising approach for tumor treatment.1 To the best of our knowledge, few studies in this filed has been reported, partly because both targeting moieties capable of targeting multiple cell types in tumors simultaneously and the targetable receptors widely and homogeneously distributed in tumors are scarce. Tissue factor (TF) is a 47 KD trans-membrane protein widely presented in normal tissues12 and its expression gets dramatically up-regulated in pathological conditions, such as thrombosis,13 atherosclerosis,14 and tumor.15 Cancer cell-expressed TF contributes to cancer progression, facilitation of metastasis and induction of neo-vascular formation in a wide spectrum of tumors,16 including lung cancer,17 glioma,18 breast cancer19 and so on. TF not only locates on tumor parenchyma cells but also sites on tumor stromal cells such as tumor-associated fibroblasts, tumor-associated macrophages and neo-vascular cells to varying degrees.20-22 The wide distribution of TF in tumor would enable it function as a favorable target for drug delivery to multiple cell types in tumor simultaneously. EGFP-EGF1 is a fusion protein derived from rat factor VII, the natural ligand of TF. EGFP-EGF1 retains the specific affinity for TF without induction of coagulation.23, 24 It is well established in our previous studies that EFGP-EGF1 could mediate drug delivery system specially to TF highly


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expressed sites, such as rat brain thrombosis25,

26

or C6 glioma,2 showing potential clinical

application in targeted drug delivery. As TF is regarded as an evolutionarily conserved protein,27 EGFP-EGF1 might also efficiently mediate drug delivery system to human tumor where human TF is highly expressed by the multi-targeting strategy as mentioned above, which will open a new avenue for tumor targeting strategy design and according widely extend the disease coverage of EGFP-EGF1. In the present study, EGFP-EGF1-conjugated nanoparticles (ENP) were constructed as the multi-targeting drug delivery system. The multi-targeting ability of ENP in vitro was investigated by protein binding experiment and nanoparticles uptake experiment. In addition, the related endocytosis mechanisms of ENP were investigated in vitro by cellular uptake inhibition and intracellular trafficking experiments. Furthermore, the targeting ability and related mechanisms in vivo were investigated by in vivo imaging and immuno-fluorescence staining experiments. Finally, the pharmacodynamics of paclitaxel（PTX）-loaded ENP （ENP-PTX） was also assessed in vitro and in vivo, compared with that of Taxol and conventional NP-PTX. Methods and materials The EGFP-EGF1 fusion protein was expressed from E. coli BL21 cells as previously reported.2 D, L-lactide (purity: 99.5%) was obtained from PURAC (Arkelsedijk, Holland). Methoxy-poly (ethylene glycol) (MPEG, MW 3000 Da) was from NOF (Tokyo, Japan) and Maleimide-poly (ethylene glycol) (Mal-PEG, MW 3400 Da) was custom-synthesized by Nektar (Huntsville, AL, USA). Methoxy-poly (ethylene glycol)-poly (lactic acid) (MPEG–PLA, Mw 33000 Da) and Maleimide-poly (ethylene glycol)-poly (lactic acid) (Mal–PEG–PLA, Mw 35000 Da) block copolymers were both synthesized by ring-opening polymerization of lactide with MPEG and



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Mal-PEG as the initiator as described elsewhere.28

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Coumarin-6, lipopolysaccharide (LPS),

transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) were from sigma (Saint Louis, MO, USA). PTX was purchased from Xi’ an San jiang Bio-Engineering Co. Ltd. (Xi’an, China). Sodium cholate was purchased from Shanghai Chemical Reagent Company (Shanghai, China). 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindo-tricarbocyanineiodide (DiR), a near-infrared dye, was obtained from Biotium (Invitrogen, CA, USA). The AnnexinV-FITC apoptosis detection kit and Hoechst 33342 were purchased from Beyotime® Biotechnology Co., Ltd. (Nantong, China). TF goat polyclonal primary antibody and F4/80 rabbit polyclonal primary antibodywere from Santa Cruz biotechnology (Santa Cruz, CA, USA), factor VII and fibroblasts activation protein (FAP) rabbit polyclonal primary antibody was bought from BIOSS Biotechnology Co., Ltd. (Beijing, China), CD31 rabbit polyclonal primary antibody was obtained from Abcam (Hong Kong) Ltd. (Hong Kong, China). Cy™ 3-conjugated affinipure donkey anti-goat secondary antibody was from

Jackson

ImmunoResearch

Laboratories,

Inc

(West

Grove,

PA,

USA).

Alexa

fluor®647-conjugated affinipure donkey anti-rabbit was from Invitrogen (Carlsbad, USA). Plastic cell culture dishes were obtained from Corning Incorporation (Corning, NY, USA). Foetal bovine serum (FBS), F-12K cell culture medium, Dulbecco’s Modified Eagle’s Medium (high glucose) (DMEM), RPMI Medium 1640, trypsin-EDTA (0.25%) and penicillin-streptomycin were obtained from Gibco (CA, USA). Puromycin was from Aladdin ® (Shanghai, China). Deionised water from the Millipore Simplicity System (Millipore, Bedford, MA) was used throughout the entire study. All other reagents and chemicals were analytical reagent grade and were obtained from Sinopharm Chemical Reagent (Shanghai, China). Human lung cancer cells A549, primary human umbilical vein endothelial cells (HUVEC), mouse NIH3T3 fibroblasts and THP1 were ordered from the


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Chinese Academy of Sciences Cell Bank (Shanghai, China). A549-mCherry-puro cell lines were bought from the Shanghai SBO Medical biotechnology (Shanghai, China). Cells were cultured at 37℃, 5% CO2 with A549 cultured in F-12K, HUVEC, and NIH3T3 cultured in DMEM, THP1 cultured in RPMI Medium 1640 and A549-mCherry-puro cell lines in F-12K supplemented with 2µg/ml puromycin. Besides, all the cell culture mediums were supplemented with 10% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin. Male Balb/c nude mice (20 ± 2 g) were from the Shanghai Slac Lab Animal Ltd. (Shanghai, China) and housed under specific-pathogen-free (SPF) conditions. All procedures were performed in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. In vitro binding of EGFP-EGF1 A549 cells were seeded at a density of 2×105 cells/well in 12-well plates, 24 h later A549 were incubated in F-12K with 3, 6 and 9 µM of EGFP-EGF1 protein for 6 h. For qualitative analysis, cells were rinsed with PBS (0.01 M, pH=7.4) for three times, fixed with 4% paraformaldehyde and then analyzed using fluorescence microscope (Leica, DMI4000B, Germany). For quantitative analysis, cells were collected by trypsinization and analyzed by flow cytometry (BD, USA). In addition, HUVEC, NIH3T3 and THP1were activated with LPS (1µg/ml for 4 h),23 TGF-β (100 ng/ml for 24 h)29 and IL-4 (20 ng/ml for 24 h),30 respectively for TF induction, and these types without activation served as control to incubate with 9 µM of EGFP-EGF1 protein for 6 h before collected by trypsinization and analyzed by flow cytometry (BD, USA). The characterization of nanoparticles Blank NP and ENP as well as coumarin-6-, DiR- or PTX-loaded NP and ENP were developed as previously described.2 Particle size and zeta potential of both NP and ENP were measured using a



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Malvern Nano ZS (Malvern Instruments, UK). After negative stained with 2% phosphatotungstic acid, the morphology of both nanoparticles was observed under a transmission electron microscope (TEM) (H-600, Hitachi, Japan). The amount of coumarin-6 and PTX in NP or ENP was measured by the high performance liquid chromatography (HPLC) (Agilent, 1200, USA).41 The mobile phase was a mixture of methyl alcohol and water (CH3OH:H2O=96:4) for coumarin-6 and a mixture of acetonitrile and water (CH3CN:H2O =55:45, v/v) for PTX, respectively. The flow rate was 1.0 ml/min and the sample injection volume was 20 µl. The detection wavelength was 465 nm and 227 nm for coumarin-6 and PTX, respectively. The amount of DiR in NP or ENP was determined by a universal fluorescence spectrophotometer (Excitation: 748 nm, Emission: 780 nm) using a Tecan Infinite M200 Pro Multiplate Reader (TECAN Safire2, Switzerland). Cellular uptake experiment A549 cells, HUVEC, NIH3T3 and THP1were seeded into 24-well plates at a density of 1×105 cells /well, 24 h later, coumarin-6-labeled NP and ENP were added into the well and incubated for 1 h. For EGFP-EGF1 inhibition experiment, excessive free EGFP-EGF1 (10 µg/ml) was added 2 h before ENP incubation. For qualitative analysis, cells were rinsed with PBS (0.01 M, pH=7.4) for three times, fixed with 4%paraformaldehyde and then analyzed under fluorescence microscopy (Leica, Germany). For quantitative analysis, cells were harvested by trypsinization and then subject to flow cytometry (BD, USA). Cellular uptake mechanisms A549 cells were seeded into 12-well plates at a density of 2×105 cells/well and incubated for 24 h. After a 30 min pre-incubation in F-12K, the cells were treated with coumarin-6-labeled NP or ENP (coumarin-6 concentration adjusted to 20 ng/ml) and various endocytic inhibitors including:


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F-12K (control), 0.1% w/v sodium azide, 2 µmol/L phenylarsine oxide, 20 µg/ml chlorpromazine, 450 mmol/L sucrose, 10 µg/ml filipin, 50 µmol/L nacodazole, 40 µmol/L cytochalasin B, 20 µg/ml Brefeldin A, 100 nmol/L monensin. After incubation for 1 h, cells were washed with ice-cold PBS (0.01 M, pH=7.4) and subsequently with acid buffer (consisting of 120 mmol/L NaCl, 20 mmol/L sodium barbital, and 20 mmol/L sodium acetate, pH=3) at 4 ℃ for 5 min. Then the cells were collected by trypsinization, suspended in 0.5 ml PBS (0.01 M, pH=7.4) and the mean fluorescence intensity was analyzed by flow cytometry (BD, USA). Subcellular localization of the nanoparticles In order to determine which organelles were involved in the intracellular distribution of the nanoparticles, lysosome and mitochondria staining was performed on A549 cells. A549 cells were seeded at a density of 2×104 cells per dish into a 35-mm glass-bottom culture dish (NEST, China), and cultured at 37 ℃ in the presence of 5% CO2 for 24 h. After incubation with coumarin-6-labeled NP or ENP with coumarin-6 concentration adjusted to 20 ng/ml, cells were further stained with Lyso-tracker Red (50 nmol/L, 30 min) or Mito-tracker Red (200 nmol/L, 30 min) and Hoechst 33342 (1 µg/ml, 10 min) to visualize endosome/lysosome (endolysosomes) or mitochondria and cell nucleus, respectively. After being fixed with 4% paraformaldehyde at room temperature for 10 min, the cells were rinsed with PBS (0.01 M, pH=7.4) and observed under confocal microscopy (ZEISS, 710, LSM, Germany). Cytotoxicity assay A549 cells were seeded into 12-well plates at a density of 1 × 105 cells /well. After 24 h, Taxol, NP-PTX and ENP-PTX were applied with the final PTX concentration in each well 100 ng/ml. Cells without any drug treatment served as control. After incubation for 24 h, cells from each



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group were collected by trypsinization and stained according to procedures of AnnexinV-FITC apoptosis detection kit for quantitative analysis using flow cytometry. (BD, USA). For qualitative analysis, cells were staining with Hoechst 33342 (1 µg/ml) for 10 min at room temperature and the morphology of cell nuclei were observed under fluorescence microscopy (Leica, Germany). In vivo imaging The A549 xenograft-bearing nude mice models were established by subcutaneous injection of 5×106 A549 cells in 100 µl of PBS (0.01 M, pH=7.4). After administration with DiR-labeled NP or ENP with the dose of DiR 0.5 mg/Kg, the mice models were imaged in vivo at various time points (1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h) by the In Vivo IVIS spectrum imaging system (PerkinElmer, USA). 24 h post administration, the mice were sacrificed and subjected to perfusion with 4% paraformaldehyde. Then, tumor tissues and other major organs including liver, spleen, kidney, heart, lung and brain were collected and the corresponding fluorescence signals ex vivo were also analyzed by the in vivo IVIS spectrum imaging system. In vivo distribution The A549 xenograft-bearing nude mice models were injected with coumarin-6-labeled NP and ENP at the dose of coumarin-6 0.05 mg/Kg. 12 h later, the mice were sacrificed, perfused with 4% paraformaldehyde, and prepared for frozen slices

with the

thickness of 20 µm.

Immunofluorescence staining was performed as elsewhere described.31, 32 TF expression in the tumor slices was stained with TF goat polyclone antibody (1:100) at 4 ℃ overnight and then exposed to Cy™3-conjugated donkey anti-goat secondary antibody (1:100) for 1 h at room temperature. A549 tumor parenchymal cells were labeled with red fluorescence protein (RFP) expressed by the A549-mCherry-puro cell lines. Neo-vascular cells, tumor-associated


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macrophages, tumor-associated fibroblasts, and factor VII were stained with primary rabbit polyclone CD 31, F4/80, FAP, or factor VII antibody (1:100) at 4 ℃ overnight, respectively, and then further stained with Alexa fluor®647-conjugated donkey anti-rabbit IgG secondary antibody (1:100) for 1 h at room temperature. Cell nuclei were counterstained by Hoechst 33342 (1 µg/ml) at room temperature for 10 min. Finally, the slices were mounted in Dako fluorescent mounting medium and observed under confocal microscopy (ZEISS, 710, LSM, Germany). In vivo anti-tumor effects 5.0 ×106 A549 cells in 100 µl of PBS (0.01 M, pH=7.4) were subcutaneously injected into nude mice to establish nude mice xenograft tumor models. When the diameters of tumors reached around 5 mm, the mice were randomly divided into four groups (n= 6) to minimize the weight and tumor size differences among the groups: control group treated with saline, Taxol group, NP-PTX group, and ENP-PTX group (PTX dose 5 mg/kg). Therapy was continued every four days through tail vein administration for four times. The tumor size and body weight were measured every other day. The tumor volumes were calculated using the formula: V=1/2×a×b2, where a and b represented the maximum and minimum diameters of tumors, respectively. After four cycles of treatments, the tumor size and body weight were recorded for another three times. When the whole experiment ended, tumors from all mice models were collected and the tumor weight was measured. The tumors were fixed with 4% paraformaldehyde, imbedded in paraffin and sectioned at 5 µm for H&E staining and terminal dUTP-mediated nick-end-labeling (TUNEL) staining as previously described.2 The tumor slices of H&E staining and TUNEL staining were observed under the fluorescence microscope (Leica, Germany) at 200× and 400× magnification, respectively, and the semi-quantitative data of cell apoptosis in vivo were obtained by Image J



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software. Statistical Analysis All data were presented as the mean ± standard deviation. Statistical differences in tumor volumes and tumor weight were determined by one-way analysis of variance (ANOVA), followed by post hoc analysis of Bonferroni for multiple groups comparison. A value of p< 0.05 was considered as significant. Results and discussion In vitro binding of EGFP-EGF1 with A549 cells To assess multi-targeting ability of ENP in vitro, TF-overexpressed human lung cancer cells line A549 was chosen as the parenchyma cell model.33 HUVEC, NIH3T3 and THP1 activated with special stimulating factor were selected as the cell model of tumor-associated neo-vascular cells,2 tumor-associated fibroblasts1 and tumor-associated macrophage,34 respectively. The results in Fig. S 1 showed EGFP-EGF1 protein bound well to TF-overexpressed A549 cells with a concentration-depend manner, which consisted well to previous work.2 In addition, the average fluorescence intensity of activated HUVEC, NIH3T3 and THP1 was 3.34, 2.23 and 2.70 fold higher than that of unstimulated HUVEC, NIH3T3 and THP1, respectively (Fig. S 2). These results indicated that the fusion protein EGFP-EGF1 could also bind with human TF and be applied to human derived tumor cells and other stromal cells. This might be due to the evolutionary conservation of TF.27 The encouraging outcome indicated the protein EGFP-EGF1 could function well as a multi-targeting moiety in the present study. Characterization of nanoparticles As shown in Fig. 1, the diameters for both NP and ENP were around 110 nm with a narrow size


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distribution (Fig. 1 C). Both NP and ENP in the TEM photographs were of regular size and smooth surface (Fig. 1 A & B). The size observed under TEM was slightly smaller than that measured by the DLS method. The targeting moiety EGFP-EGF1 conjugation to nanoparticles slightly increased the diameter and lowered the zeta potential of nanoparticles (Fig. 1 C) as previously described.2 Encapsulation of coumarin-6, DiR or PTX did not obviously influence the size or zeta potential of nanoparticles as previously reported.2, 35 As few changes happened to the physical and chemical parameters of NPs when coumarin-6 or DiR was loaded, furthermore, the amount of coumarin-6 or DiR released from NPs was less than 1% in 24 h,35 which confirmed that the fluorescence tracker including coumarin-6 or DiR could represent NPs. Nanoparticles uptake and related mechanism As shown in Fig. 2, EGFP-EGF1 modification significantly enhanced nanoparticles uptake by A549 cells and stromal cells including activated HUVEC, NHI3T3 and THP-1 as compared with traditional NP, which could be inhibited by free EGFP-EGF1 co-incubation. These results demonstrated the targeting moiety EGFP-EGF1 protein could facilitate nanoparticles uptake by tumor parenchyma cell and different types of stromal cells, indicating EGFP-EGF1-mediated drug delivery system might be used for the treatment of human tumors by multi-targeting mechanism. This was in good agreement with previous study in which EGF1-EGFP conjugation significantly increased the nanoparticles uptake by C6 cells and perturbed HUVEC.2 Results of related uptake mechanism was shown in Fig. 3, sodium azide, an energy depletory, decreased cellular uptake of both NP and ENP to 58.4% and 53.4% of that of control, respectively, indicating both NP and ENP uptake was energy-dependent process. Phenylarsine oxide, the endocytosis inhibitor, also significantly reduced the cellular uptake of NP and ENP to 36.1% and 26.6%, respectively,



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indicating that endocytosis was involved in both NP and ENP uptake. Chlorpromazine and sucrose which prevented clathrin-coated pit formation and functioned as the inhibitors of clathrin-associated endocytosis pathway,36 reduced NP uptake to 64.0% and 69.6% as compared with control group, respectively. Furthermore, chlorpromazine and sucrose also decreased ENP uptake to 78.3% and 66.2%, respectively, indicating clathrin-associated endocytosis pathway was involved in both NP and ENP uptake. Filipin, a special sterol-binding agent that served as an inhibitor of caveolae-dependent endocytosis,37 significantly decreased the ENP uptake to 76.5%, while it had no effect on the NP uptake, suggesting that caveolae-dependent endocytosis was involved in the ENP uptake rather than NP uptake. Cytochalasin B and nocodazole, both as typical inhibitors of the macropinocytosis pathway,38 significantly reduced NP uptake to 89.0% and 75.1% , respectively, but almost had no effect on ENP uptake, suggesting that macropinocytosis was involved in NP uptake but not in ENP uptake. Lysosomes and Golgi apparatus had been shown to play important roles in both intracellular cargo transport and disposition.39, 40 In the present study, the lysosome inhibitor monensin considerably decreased both NP and ENP uptake to 87.0% and 88.4%, respectively. Brefeldin A, which disrupted the Golgi apparatus and intracellular trafficking, significantly decreased the uptake of NP and ENP to 83.8% and 81.2%, respectively, suggesting the intracellular transport of both NP and ENP were related to lysosome and Golgi apparatus. Altogether, the results demonstrated clathrin-and caveolae-associated endocytosis pathway participated in ENP uptake, a different manner as compared with NP uptake, in which clathrin-associated endocytosis pathway and micropinocytosis was involved. The active targeting nanoparticles were always associated with clathrin- and caveolae-mediated endocytosis, such as peptide-22-modified nanoparticles for low-density lipoprotein receptor (LDLR) targeting41


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and angiopep-2-modified nanoparticles for low-density lipoprotein receptor-related protein (LRP) targeting.42 However, the uptake mechanism was also closely associated with the type of targeting moiety, the targeting receptor, the concentration of inhibitors, and the cell line model used.43, 44 To more precisely determine the main uptake pathway of ENP, further studies were still needed by using different concentration of inhibitors, siRNA inhibition of clathrin or caveolae,45 and TEM observation as elsewhere reported.38 Intercellular distribution of nanoparticles Confocal microscopy was utilized to analyze nanoparticles distribution in A549 cells. As shown in Fig. 4, after incubation for 1 h, both NP (B & J) and ENP (F & N) were internalized by A549 cells. Nanoparticles co-localization with endosome/lysosome (endolysosomes) (C & K) or mitochondria (G & O) indicated by yellow color (D, H, L, P) suggested that both NP and ENP uptake by the cells were delivered to lysosome and mitochondria. In addition, the fluorescence intensity of A549 cells incubated with coumarin-6-labeled ENP was stronger than that of coumarin-6-labeled NP, which was consistent with the results of cell uptake experiment analyzed by fluorescence microscopy and flow cytometry (Fig. 2 A & E). Cytotoxicity assay Results from in vitro cytotoxicity assay determined by flow cytometry using Annexin V/propidiumiodide double staining was shown in Table S 1, ENP-PTX induced significantly more cell apoptosis than any other group. The cytotoxicity of NP-PTX was comparable to that of Taxol, presumably because of the added cell-killing effect of the solvent Cremophor EL-ethanol of Taxol. 46

The qualitative analysis displayed similar outcomes (Fig. S 3). The morphology of cell nuclei in

control group was integral with homogenous fluorescence. However, the cell nuclei displayed



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fragmentation and dense staining with varying degrees in drug-treatment groups, among which the most severe impairment was observed in ENP-PTX treatment group. These results indicated ENP-PTX presented the strongest cytotoxicity against A549 cells, which consisted well to the nanoparticles uptake experiment. In vivo imaging In vivo fluorescence imaging was utilized to investigate the targeting potential of ENP in ectopic subcutaneous A549 tumor models, based on the fluorescence signal of DiR encapsulated in nanoparticles. The in vivo three-dimensional reconstituted imaging (Fig. 5 A-C) at 24 h showed the fluorescence signal intensity in the tumor site of ENP-treated mice was much stronger than that of NP. The in vivo imaging at different time points after drug administration also showed consistent results (Fig. S 4). At 24 h, the mice models were sacrificed, tumors and major organs were harvested and the findings were validated ex vivo. In line with the results obtained by in vivo imaging, the semi-quantitative results of ex vivo tumors revealed the tumor fluorescence intensity for ENP-treated mice was 2.3 times stronger than that for NP-treated group (Fig. 5 D). Moreover, the nanoparticles distribution in normal organs of ENP was similar to that of NP (Fig. S 5). These results altogether showed that the targeting ability of EGFP-EGF1 did work in human tumor models after conjugation to nanoparticles surface, leading to more efficient retention of nanoparticles within tumors. The interaction between the targeting moiety EGFP-EGF1 and TF might be the main contributor to the targeting effect. In vivo distribution As the natural ligand for TF, factor VII could reach tumor site by the EPR effect or be produced by tumor cells themselves,19, 21 so how to explain the possible competition between EGFP-EGF1


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protein and factor VII which have already located in tumor tissues prior to EGFP-EGF1 arriving? Immuno-fluorescence staining was conducted to preliminary analyze the issue. As shown in Fig. 6, TF was highly and extensively expressed in A549 tumor tissues (Fig. 6 C & H). As the natural ligand of TF, factor VII also located in tumor tissues in some regions (Fig. 6 D & I).21 For ENP group, nanoparticles mainly distributed in regions unoccupied by factor VII and much less in the factor VII-occupied region (Fig. 6 G). For NP group, there was no significant difference in nanoparticles distribution whether TF was occupied or not (Fig. 6 B). However, NP distribution in a local region occupied by factor VII was relative higher than other regions (Fig. 6 B & D), which might be due to the presence of tumor vascular indicated by the lumen structure nearby (Fig. 6 C). In addition, ENP distribution in the tumor slices was much more prominent than traditional NP. The outcome could be reasonably explained as follows: TF occupied by factor VII in advance would no longer be open to TF-targeting ENP and would accordingly decrease ENP accumulation in these regions. In contrast, the regions unoccupied by factor VII were still open to ENP and thus TF could bind with ENP and augment ENP accumulation in the factor VII-unoccupied regions. As for traditional NP, TF occupied by factor VII or not had no effect on the nanoparticles distribution. Factor VII would certainly exert negative influence on drug delivery system modified by EGFP-EGF1 or those similar targeting moieties including factor VII itself17, 47 or the light chain of factor VII.48 However, even though TF was occupied by factor VII, there were still some other domains of TF open for targeted therapy. To screen targeting moieties with specific affinity for these domains may open another avenue to utilize all forms of TF in tumor and thus might obtain a more satisfactory outcome. Multi-targeting mechanism of ENP in vivo



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It was now believed that there were different types of cells in tumor tissues including tumor parenchymal cells, tumor neo-vascular cells, tumor-associated macrophages and tumor-associated fibroblasts, which were closely associated and compensated for each other to drive tumor progression.3, 6-8 TF was reported to be highly expressed in these types of cells with varying degrees.20-22 And here we tried to observe nanoparticles distribution in these cells to analyze the possible multi-targeting potential of ENP to A549 tumor xenograft in vivo. As shown in Fig. 7, ENP not only resided in neo-endothelial but also reached other types of cells in tumor microenvironment far away from tumor vessels (Fig. 7 H). In contrast, NP mainly sited near tumor vessels and few was observed to reach neo-endothelial (Fig. 7 G). To further reveal the possible multi-targeting mechanism of ENP, other tumor stromal cells were also stained by immune-fluorescence staining and tumor parenchymal cells were tracked by the fluorescence of RFP as A549-mCherry-puro cell lines stably expressed RFP. ENP distribution in these types of cells in tumor xenograft (Fig. 7 B, D & F) was much more prominent than that of NP (Fig. 7 A, C & E), indicating TF sited in all these cells could function as the targeting receptor for ENP to varying degrees. As the Figures showed, ENP distribution in different cell types varied greatly from each other and was not so homogeneously. This might be due to the varying TF levels on different cells and also the possible competition from factor VII distribution (Fig. 6). As tumor parenchymal cells proliferation and neo-vessels formation were the two basic characterizations of many tumors, strategies targeting tumor parenchymal cells35 or destroying tumor-associated vessels to block nutrition supplement for tumor therapy2, 49 have been extensively reported. In contrast, ENP in the present study could target multiple types of cells in tumor tissues. This strategy shifted from the tumor parenchymal cell-centric paradigm9 to a more global paradigm that


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covered almost all important cell types in tumor, which established a new strategy for drug delivery system design and suggested an encouraging anti-tumor outcome when loaded with chemotherapeutics.3 In vivo anti-tumor effect Next, the antitumor effect of ENP-PTX in vivo was evaluated. The final size and weight of tumors from drug-treated mice were significantly reduced, especially for mice treated with ENP-PTX (Fig. 8 A-D), and the tumor growth curve showed that tumor growth was delayed most prominently in the group treated with ENP-PTX (Fig. 8 A). The inhibitive rate calculated based on tumor volume and weight were 60.53% and 55.60% in the ENP-PTX group, respectively, about 2.1 times higher than those in the NP-PTX group and 2.6 times higher than those in the commercial Taxol group. During the whole study, there were no significant differences in the average body weights between the four groups (Fig. 8 B), indicating no obvious side effects arising from the treatments. To more intuitively detect pharmacodynamics of different PTX formulations in tumor tissue after four cycles

of

drug

administration,

excised

tumors

were

subsequently

assessed

by

immunohistochemistry (IHC). The H&E staining and TUNEL staining of the tumor slices were displayed in Fig. 9 & S6. There were negligible necrosis and sporadic cell apoptosis in saline treated group. Tumor tissue cell necrosis including pyknosis, karyorrhexisor and karyolysis and apoptosis in the three PTX treated groups were much more obvious than control group, among which ENP-PTX group showed the most extensive necrosis and most significant cell apoptosis. The hemi-quantitative data of cell apoptosis (Fig. 9 I) consisted well to the qualitative representative image (Fig. 9 E-H). In addition, the necrosis scope consisted well with the cell apoptosis region in all groups (Fig. 9 & S6). These data altogether indicated that multi-targeting



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parenchymal cells and stromal cells simultaneously could contribute to the significant therapeutic benefit. Conclusion The present study successfully established ENP as a multi-targeting drug delivery system to human tumor. In vitro experiments demonstrated that EGFP-EGF1 could bind well to A549 tumor parenchyma cells and other different types of stromal cells including tumor-associated neo-vascular cells, tumor-associated fibroblasts and tumor-associated macrophages and enhanced nanoparticles uptake by those types of cells. In vivo experiments displayed ENP targeted multiple types of key cells in tumor tissues, accumulated more specially in TF-expressing region unoccupied by factor VII and thus achieved the most promising therapeutic benefits. Acknowledgement The work was supported by the National Natural Science Foundation of China (81472757, 81301974, 81302043, 81302714), the State Scholarship Fund, the Doctoral fund of Ministry of Education of China (20100071120050), and “Zhuoxue” program of Fudan University. Supporting Information Available: EGFP-EGF1 protein binding experiment (Fig. S1-2), the pharmacodynamics experiment in vitro (Table S 1 and Fig. S 3), in vivo imaging of A549 tumor xenograft treated with DIR-labeled NP or ENP and ex vivo imaging of major normal organs (Fig. S 4-5). HE staining and TUNEL staining of tumor slice in different treatment group at 200× (Fig. S 6). This information is available free of charge via the Internet at http://pubs.acs.org/. Reference (1) Prakash, J.; de Jong, E.; Post, E.; Gouw, A. S.; Beljaars, L.; Poelstra, K., A Novel Approach to Deliver Anticancer Drugs to Key Cell Types in Tumors Using a PDGF Receptor-Binding Cyclic


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Fig. 1 The characterizations of NP and ENP. TEM images of NP (A) and ENP (B) negatively stained by 2% phosphotungstic acid. Particle size distribution and zeta potential of NP and ENP (C). The bar represented 200 nm.



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Fig. 2 Cellular uptake of coumarin-6-labeled NP (the first row), ENP (the middle row) and ENP with EGFP-EGF1 protein incubation in advance (the third row) by A549 cells (A & E), activated HUVEC (B & F), activated NIH3T3 (C & G) and activated THP-1 (D & H) analyzed by fluorescence microscopy (A, B, C, D) and flow cytometry (E, F, G, H). Blue: Hoechst 33342 counterstained nucleus. Green: coumarin-6-labeled nanoparticles. The bar was 200 µm. *p< 0.01 vs ENP.


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Fig. 3 Cellular uptake of coumarin-6-labeled NP and ENP by A549 cells in the presence of different endocytic inhibitors including NaN3 (0.1%), phenylarsine oxide (2 µmol/L), chlorpromazine (20 µg/ml), sucrose (450 mmol/L), filipin (10 µg/ml), nacodazole (50 µmol/L), cytochalasinB (40 µmol/L),brefeldin A (20 µg/ml), monensin (100 nmol/L), respectively (n = 3). The concentration of coumarin-6 were 20 ng/ml in each well. *p< 0.05 vs control.



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Fig. 4 Intracellular distribution of coumarin-6-labeled NP (A, B, C, D, I, J, K, L) and ENP (E, F, G, H, M, N, O, P) in A549 cells observed by confocal microscope after incubation for 1 h. Image D was merged by image A, B and C. Image H was merged by image E, F and G. Image L was merged by image I, J and K. Image P was merged by image M, N and O. Blue: cell nuclei. Green: nanoparticles. Red: Lysotracker Red (C & K) or Mitotracker Red (G & O). Yellow: Red co-localized with green. The bar was 200 µm.

Fig. 5 In vivo three-dimensional reconstruction reconstructed imaging (with individual bars) of A549 xenograft-bearing nude mice 24 h after injection with DiR-labeled NP (A) or ENP (B).


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Whole body fluorescence imaging (C), ex vivo fluorescence imaging of tumors at 24 h and the corresponding semi-quantitative results of tumors (D) **p

Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles

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