Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal

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Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active Targeting Efficiency of Tumor Hong Yeol Yoon, Min Lee Shin, Man Kyu Shim, Sangmin Lee, Jin Hee Na, Heebeom Koo, Hyukjin Lee, Jong-Ho Kim, Kuen Yong Lee, Kwangmeyung Kim, and Ick Chan Kwon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01083 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Molecular Pharmaceutics

Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active Targeting Efficiency of Tumor †

Hong Yeol Yoon , Min Lee Shin

†, ‡

, Man Kyu Shim

†, §

†

, Sangmin Lee , Jin Hee Na#,

§

‡

†

Heebeom Koo⊥, Hyukjin Lee#, Jong-Ho Kim , Kuen Yong Lee , Kwangmeyung Kim and Ick Chan Kwon

†, ǁ,*

†

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea ‡

Department of Bioengineering, Hanyang University, 222, Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea

§

Department of Pharmacy, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea

ǁ

KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea #

College of Pharmacy, Graduate School of Pharmaceutical Science, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Republic of Korea

⊥Department

of Medical Lifescience, College of Medicine, The Catholic University of Korea

222, Banpo-daero, Seocho-gu, Seoul 06591, Republic of Korea

*Corresponding author Ick Chan Kwon, Ph.D. Tel: +82-2-958-5912; fax: +82-2-958-5909; e-mail: [email protected]

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Abstract Biological ligands such as aptamer, antibody, glucose and peptide have been widely used to bind specific surface molecules or receptors in tumor cells or subcellular structures to improve tumor-targeting efficiency of nanoparticles. However, this active targeting strategy has limitations for tumor targeting due to inter- and intra-heterogeneity of tumors. In this study, we demonstrated an alternative active targeting strategy using metabolic engineering and bioorthogonal click reaction to improve tumor-targeting efficiency of nanoparticles. We observed that azide-containing chemical reporters were successfully generated onto surface glycans of various tumor cells such as lung cancer (A549), brain cancer (U87), and breast cancer (BT-474, MDA-MB231, MCF-7) via metabolic engineering in vitro. In addition, we compared tumor targeting of artificial azide reporter with bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-CNPs) and integrin αvβ3 with cyclic RGD-conjugated CNPs (cRGD-CNPs) in vitro and in vivo. Fluorescence intensity of azide reporter-targeted BCN-CNPs in tumor tissues was 1.6 fold higher and with a more uniform distribution compared to that of cRGD-CNPs. Moreover, even in the isolated heterogeneous U87 cells, BCN-CNPs could bind artificial azide reporters on tumor cells more uniformly (~92.9 %) compared to cRGD-CNPs. Therefore, the artificial azide reporter-targeting strategy can be utilized for targeting heterogeneous tumor cells via bioorthogonal click reaction and may provide an alternative method of tumor targeting for further investigation in cancer therapy.

Keywords: Metabolic glycoengineering, Bioorthogonal click reaction, Active tumor targeting, Heterogeneity

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1. Introduction For the last 20 years, nanoparticles have been widely studied for diagnosis as well as drug delivery due to their unique properties, such as high surface-to-volume ratio, improved pharmacokinetics of drugs, and multi-functionality, compared to drugs or imaging agents with small molecular weight.1-3 In addition, nanoparticles have high accumulation property in angiogenesis-related disease lesions such as arthritis and tumors via enhanced permeation and retention (EPR) effect.4-5 The ligand-mediated targeting (known as active targeting) strategy of drugs or nanomaterials has played an important role in improving their tumor-targeting efficiency.6 For this purpose, various biological ligands such as aptamers,7-9 antibodies,10 glucoses,11-12 and peptides,13 have been utilized as pilots to bind specific surface molecules or receptors in tumor cells or subcellular structures. This approach has provided increasing interactions between the tumor cells and nanomaterials, resulting in improved internalization of nanomaterials in in vitro and in vivo conditions. However, tumor tissues and tumor cells have different properties in morphology, phenotypic expression, drug resistance, and potential for generation of new tumor, which is well known as “tumor heterogeneity.”14-15 Tumor heterogeneity, based on genetic information of tumor cells, can be observed even within the same tissue as well as the same host. Furthermore, cancer cells develop various subpopulations expressing different types and levels of receptors resulting in formation of tumor tissue (Scheme 1a).16 In addition, the rapid and uncoordinated proliferation property of tumors can induce genetic diversity17. Therefore, conventional single-molecule-targeting, such as aptamer-, antibody-, glucose-, and peptide-targeting strategies might be confronted with limitations for tumor-targeting. In this respect, we designed an artificial azide-reportertargeting strategy using metabolic engineering and bioorthogonal click chemistry to improve 3



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tumor-targeting efficiency by making tumor cells more uniform (Scheme 1b). Metabolic engineering is a biological technique that takes the glycan biosynthetic pathways to generate unnatural glycans containing various chemical groups, such as azide, alkyne, thiol, and ketone, onto the cell surface.18 These unnatural glycans can be specifically targeted using bioorthogonal molecules without any side reactions via bioorthogonal click reaction in vitro and in vivo. A number of studies have utilized metabolic engineering and bioorthogonal click reaction in biological or biomedical research fields for specific labeling of proteins, DNA, monitoring of zebrafish growth, and delivery of nanoparticles.19-22 Recently, we have demonstrated that the biodistribution and tumor-targeting efficacy of nanoparticles after administration can be modulated via metabolic engineering and bioorthogonal click chemistry in vitro and in vivo.23-24 Through our research, we have established an artificial tumor-targeting strategy in which azide-containing artificial chemical reporters can be generated and targeted by metabolic engineering and bioorthogonal click reaction. Importantly, the generation of artificial azide reporters could be controlled by delivery of azide-containing metabolite to the tumor cells. Herein, we propose an artificial azide-reporter-targeting strategy using metabolic engineering and bioorthogonal click chemistry to improve tumor-targeting ability of nanoparticles to overcome tumor heterogeneity. This technique can make tumor cells more uniform, which can increase the tumor-targeting efficiency of nanoparticles using artificial azide reporters. These artificial azide reporters can be used as a target of tumor cells vial bioorthogonal click reaction (Scheme 1c). To compare tumor-targeting efficiency of artificial azide reporter with the conventional active targeting strategy, we prepared bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-CNPs) as artificial azide-reporter-targeting nanoparticles, 4


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and cyclic RGD-conjugated glycol chitosan nanoparticles (cRGD-CNPs) were prepared as conventional active targeting nanoparticles. In vitro artificial azide reporter generation was observed using various tumor cells, such as lung cancer (A549), brain cancer (U87) and breast cancer (BT-474, MDA-MB231, MCF-7) cells, after treatment with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz). In addition, we compared targeting efficiency of BCN-CNPs with cRGD-CNPs in U87 (integrin αvβ3 high) and MCF7 (integrin αvβ3 low) cells and analyzed distribution of BCN-CNPs and cRGD-CNPs in vitro. For in vivo study, azide reporters were generated in U87 tumor tissues via intratumoral injection of Ac4ManNAz. Then, BCN-CNPs and cRGD-CNPs were injected intravenously and their in vivo distribution was measured using near infrared fluorescence (NIRF) imaging. Tumor heterogeneity and targeting efficiency of BCN-CNPs and cRGD-CNPs were precisely analyzed by isolating the U87 tumor cells from the U87 tumor-bearing mice. Our approach makes tumor cells “more uniform” using azide reporters on the surface of target tumor cells, which improves the targeting efficiency of nanoparticles via bioorthogonal click reaction. In addition, our approach is advantageous because azide reporters can be generated on the cell surface in large amounts, regardless of the types or subpopulations of tumor cells.

2. Materials and methods 2.1. Materials. Glycol chitosan (average molecular weight 2.5 x 105 Da, degree of deacetylation = 82.7%), 5β-cholanic acid, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC), fluorescein isothiocyanate (FITC), anhydrous dimethyl sulfoxide (DMSO) and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicyclononyne (BCN) NHS ester II was purchased 5



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from Berry &Associates, Inc. (Dexter, MI, USA). The NIRF dye Cy5.5-NHS and IR800CWNHS were purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA) and LI-COR Biotechnology (Lincoln, NE, USA), respectively. Dimethyl sulfoxide-d6 (DMSO-d6, 99% D) was purchased from Cambridge Isotope Laboratories. Inc. (Andover, MA, USA). Azide functionalized cyclic RGDyK (cRGD-N3) was purchased from FutureChem CO., Ltd. (Seoul, Korea). The cancer cell isolation kit was purchased from Affymetrix, Inc. (Santa Clara, CA, USA). Fluorescein (FITC)-conjugated mouse monoclonal anti-integrin αvβ3 antibody (23C6) was purchased from Santa Cruz Biotechnology, Inc. (Texas, USA). Human glioblastoma cell line (U87), human lung adenocarcinoma epithelial cell line (A549), and human breast adenocarcinoma epithelial cell line (MCF7, MDA-MB-231, BT-474) were purchased from the American Type Culture Collection (ATCC, Rochkville, MD, USA). Murine bone marrow stromal cell (M2-10B4) was obtained from the Korean Cell Line Bank (Seoul, Korea). For cell culture, Roswell Park Memorial Institute (RPMI-1640) medium, antibiotics (penicillin with streptomycin), trypsin-EDTA, fatal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Welgene. Inc. (Daegu, Korea). All other chemicals were purchased as reagent grade and used without further purification.

2.2. Preparation of glycol chitosan nanoparticles (CNPs, BCN-CNPs, cRGD-CNPs). To prepare BCN-CNPs and cRGD-CNPs, glycol chitosan was chemically modified with 5βcholanic acid via amide formation as described in a previous report.25. In brief, 75 mg of 5βcholanic acid (208 µmol) in 62.5 ml of methanol was mixed with 60 mg of EDC and 36 mg of NHS for 30 min to activate carboxylic acid in 5β-cholanic acid. The activated 5β-cholanic acid solution was slowly dropwised into glycol chitosan solution (250 mg of glycol chitosan 6


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(1 µmol) in 62.5 ml of distilled water/methanol mixture) to form amide linkages. The solution was vigorously stirred for 24 h at room temperature, and then purified by dialysis against methanol/distilled water mixture (1:0, 1:1, 0:1 v/v) for 3 days using a cellulose membrane (MWCO = 12 – 14,000 Da, Spectrum®, Rancho Dominquez, CA, USA). The resulting solution was lyophilized to obtain white powder, CNPs. To obtain BCN conjugated CNPs, CNPs were chemically modified with BCN-NHS ester II. In brief, 50 mg of CNPs (195.5 µmole of NH2) were dissolved in 10 ml of distilled water/dimethylsulfoxide (DMSO). Then, 5.22 mg of BCN-NHS ester II (9.75 µmole of NH2) was dissolved in 2.5 ml of DMSO and then slowly dropwised into the CNPs solution. The solution was vigorously stirred for 12 h at room temperature and purified by dialysis against DMSO/distilled water mixture (1:0, 1:1, 0:1 v/v) for 3 days using a cellulose membrane (MWCO = 12 – 14,000 Da, Spectrum®, Rancho Dominquez, CA, USA). The resulting solution was lyophilized to obtain BCN-CNPs. To obtain cRGD-conjugated CNPs, BCN-CNPs were chemically modified with cRGD-N3 via copper free click chemistry. In brief, 100 mg of BCN-CNPs (4.8 µmole of BCN) was dissolved in 20 ml of DMSO. Then, 8.6 mg of azide-cRGD (9.6 µmole of N3, 2 equiv.) was dissolved in 5 ml of DMSO and then dropwised into the BCN-CNPs solution. The solution was vigorously stirred for 12 h at 37 oC and purified by dialysis against DMSO/distilled water mixture (1:0, 1:1, 0:1 v/v) for 3 days using a cellulose membrane (MWCO = 12 – 14,000 Da, Spectrum®, Rancho Dominquez, CA, USA). The resulting solution was lyophilized to obtain cRGD-CNPs.

2.3. In-vitro characterization of CNPs. Conjugation ratio of BCN molecule was determined using azide-quenched fluorescence molecule, 3-azido-7-hydroxycoumarin (Baseclick GmbH, 7



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Germany). In brief, 1 mg of BCN-CNPs was dissolved in 1 ml of DMSO (1:1 v/v) co-solvent. Then, 0.2 µmol of 3-azido-7-hydroxycoumarin solution was added into the BCN-CNPs solution and the mixed solution was incubated for 3 h at 37 oC. The fluorescence intensity from 3-azido-7-hydroxycoumarin (λex = 404 nm and λem = 480 nm) was measured using fluorescence spectrophotometer (F-7000, Hitachi, Japan), and conjugation ratio of BCN molecule was analyzed based on a standard curve of BCN and 3-azido-7-hydroxycoumarin. Conjugation ratio of cRGD molecule was determined using UV-Vis absorption at 280 nm. In brief, 1 mg of cRGD-CNPs was dissolved in 1 ml distilled water/DMSO (1:1 v/v) co-solvent. Conjugation ratio of cRGD molecule was analyzed based on UV-Vis absorption at 280 nm with standard curve of cRGD-N3. To analyze the size distribution and zeta-potential (ξ) of CNPs, BCN-CNPs, and cRGD-CNPs, the all samples were dispersed in phosphate buffered saline (PBS, pH 7.4) and were measured size with zeta-potential using Zeta-sizer (Nano ZS, Malvern, UK). The morphologies of CNPs, BCN-CNPs, and cRGD-CNPs were observed by transmission electron microscope (TEM, Tecnai F20, FEI, Netherlands) at an accelerating voltage of 200 ekV. For TEM images, all samples were dispersed in distilled water and were negative stained by 2 % uranyl acetate.

2.4. In vitro azide generation analysis. For the analysis of azide generation in various tumor cells, MCF7, MDA-MB-231, BT-474, U87, A549 and M2-10B4 cells were cultured in 10 % fatal bovine serum and 1% penicillin-streptomycin containing RPMI 1640 medium at 37 oC in a 5 % CO2 incubator. To determine azide generation in various cancer cells, 1 x 105 each of MCF7, MDA-MB-231, BT-474, U87, A549 and M2-10B4 cells were seeded into a 35-mm cover glass-bottom dish. For the azide generation, the cells were incubated with 20 µM of 8


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tetraacetyl azidomannosamine (Ac4ManNAz)-, tetraacetyl azidogalactosamine (Ac4GalNAz)-, and tetraacetyl azidoglucosamine (Ac4GlcNAz)-containing medium for 48 h at 37 oC CO2 incubator. For visualization of azide in the cells, the cells were washed twice with DPBS and further incubated with 20 nM of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5)containing fresh medium for 1 h at 37 oC. Then, the cells were washed twice with DPBS and fixed with formaldehyde-glutaraldehyde combined fixative for 10 min in dark condition. After fixation, the cells were washed twice with DPBS and mounted with 4,6-diamidino-2phenylindole (DAPI). The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Germany) with 405 diode (405 nm), Ar (458, 488, 514 nm) and He-Ne (633nm) lasers. To quantification of fluorescence on tumor cells, they were analyzed by flow cytometer (Guava easy CyteTM, Merck Millipore, Germany). To quantify generated azide groups in glycoproteins in vitro, 2 x 104 each of MCF7, MDA-MB-231, BT474, U87, A549 and M2-10B4 cells were seeded into 6-well plates and incubated with 20 µM of Ac4ManNAz, Ac4GalNAz and Ac4GlcNAz containing medium for 72 h at 37 oC CO2 incubator, respectively. In addition, U87 and MCF7 cells were treated with 0 to 50 µM of Ac4ManNAz for 48 h at 37 oC in a CO2 incubator for dose-dependent azide generation. Then, the cells were washed twice with cold DPBS and were lysed using RIPA buffer (Thermo Fisher Scientific Inc., USA) with 1 % protease inhibitor cocktail. Lysates were centrifuged at 14,000 rpm for 20 min at 4 oC to remove cell debris. The total protein of each sample was quantified by BCA assay (Pierce® BCA Protein Assay Kit, Thermo Scientific Inc., USA) and the lysates were incubated with 500 nM of phosphine–PEG3-biotin (Pierce, Rockford, IL, USA) for 6 h at 37oC. The proteins from each sample were mixed with 1 x sodium dodecyl sulfate (SDS) gel-loading buffer (125 mol/L Tris, pH 6.8, 5 % glycerol, 2 % SDS, 1 % βmercaptoethanol and 0.006% bromophenol blue) and boiled for 5 min. Then, 50 µg of 9



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proteins were separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature in 0.5% bovine serum albumin (BSA) containing 1 x TBST solution (10 mol/L Tris, pH 7.4, 100 mol/L NaCl and 0.1 % Tween 20). Then, the membranes were incubated with streptavidinHRP (Pierce, Rockford, IL, USA) containing 1 x TBST solution (Pierce, Rockford, IL, USA) for 16 h at 4 oC. Finally, the membranes were washed three times using 1 x TBST and protein band was detected with an ECL system.

2.5. In vitro tumor cell binding analysis. To compare in vitro binding efficiency of azide reporter-targeting with biological ligand-targeting, we used targeting ligands cRGD and BCN to target integrin αvβ3 (CD61) and azide groups, respectively. Firstly, to observe binding effect of BCN-CNPs and cRGD-CNPs in U87 cells, which express integrin αvβ3, 1 x 105 U87 cells were seeded into a 35-mm cover glass-bottom dish. Then, the cells were incubated in 20 µM of Ac4ManNAz-containing medium for 48 h at 37 oC CO2 for azide generation. For visualization of CNPs on the cell surface, the cells were washed twice with DPBS and further incubated in fresh medium containing 0 - 10 mg/ml of Cy5.5-labeled cRGD-CNPs or BCNCNPs for 1 h at 37 oC. Then, the cells were washed twice with DPBS and fixed with formaldehyde-glutaraldehyde combined fixative for 10 min in dark condition. After fixation, the cells were washed twice with DPBS and mounted with DAPI. The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Germany) with 405 diode (405 nm), Ar (488, 514 nm) and He-Ne (633nm) lasers. Fluorescence intensity of each image was analyzed by Image J software (NIH, USA). In addition, we compared targeting effect between integrin αvβ3 positive and negative cell line. U87 cell line 10


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was selected as integrin αvβ3 high expression group whereas MCF7 cell line was selected as integrin αvβ3 low expression group.26 In brief, 1 x 105 cells of U87 and MCF7 cells were seeded into 35mm cover glass bottom dish and the cells were incubated with 20 µM of Ac4ManNAz for 48 h at 37 oC CO2 incubator for the azide generation. To observe BCNCNPs and cRGD-CNPs on the cell surface, the cells were washed twice with DPBS and further incubated with 1mg/ml of fluorescein isothiocyanate (FITC) labeled cRGD-CNPs or BCN-CNPs contained fresh medium for 1 h at 37 oC. Then, the cells were washed twice with DPBS and fixed with formaldehyde-glutaraldehyde combined fixative for 10 min in dark condition. After fixation, the cells were washed twice with DPBS and mounted with DAPI. The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Germany) with 405 diode (405 nm), Ar (488, 514 nm) and He-Ne (633nm) lasers. Z-scanning analysis was performed using Leica Application Suite Advanced Fluorescence (LAS AF 4.3, Leica Microsystems CMS GmbH, Germany). For quantification of fluorescence on U87 and MCF7 cells, they were analyzed by flow cytometer (Guava easy CyteTM, Merck Millipore, Germany).

2.6. In vivo and ex vivo distribution and tumor-targeting analysis by near-infrared fluorescence (NIRF) imaging. All experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Korea Institute of Science and Technology (KIST) and institutional committees have approved the experiments. Athymic nude mice (5-weeks old, 18 - 20 g, male) were purchased from Orient Bio Inc. (Gyeonggi-do, Korea). To prepare tumor-bearing mice models, a suspension of 2ⅹ106 U87 cells in RPMI1640 (60 µl) was subcutaneously injected into left flanks of mice. When tumors 11



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grew to approximately 350 – 400 mm3 in volume, 50 mM of Ac4ManNAz (20 µl) was injected into tumor via intratumoral injection once in a day for 4 days. Then, 200 µl PBS (pH 7.4) solution containing Cy5.5-labeled cRGD-CNPs or BCN-CNPs (1 mg/ml) was injected into the mice via tail vein. The time-dependent biodistribution and accumulation profiles of nanoparticles were observed by using an eXplore Optix system with a 670-nm-pulsed laser diode (Advanced Research Technologies Inc., Montreal, Canada). To observe NIRF in major organs, each mouse was sacrificed 48 h post-injection. Then, major organs with tumors were excised, and NIRF was measured using the IVIS Lumina Series III (PerkinElmer, Massachusetts, USA). Fluorescence intensities in organs were analyzed using the Living Image® software (PerkinElmer, Massachusetts, USA). For the high-resolution tissue fluorescence analysis, the integrin αvβ3 in tumor tissue was immunofluorescence-stained using FITC-conjugated mouse monoclonal anti-integrin αvβ3 antibody. In brief, dissected tumor tissues were retrieved from U87 tumor-bearing mice, fixed in OCT compound, and frozen for 24 h in a deep freezer. Then, the tissue blocks were sectioned at 10 µm thickness. For integrin αvβ3 staining, the tissue slide was washed with PBS (pH 7.4) twice and treated with blocking solution (0.3 % BSA contained PBS (pH7.4)) for 1 h at room temperature. Then, the tissue slide was washed with PBS twice and incubated with mouse anti-human integrin αvβ3 antibody (1 µg/ml, 0.3 % BSA contained PBS (pH7.4)) for 2 h at room temperature. After washing the tissue slide with PBS twice, the tissue slide was mounted using cover glass and fluorescence in the tumor tissue was observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Germany) with 405 diode (405 nm), Ar (458, 488, 514 nm) and He-Ne (633nm) lasers.

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2.7. Ex vivo tumor tissue analysis after simultaneous injection of cRGD-CNPs and BCNCNPs. For the observation of distribution of cRGD-CNPs and BCN-CNPs in tumor tissues, a suspension of 2ⅹ106 U87 cells in RPMI1640 (60 µl) was subcutaneously injected into left flanks of mice to prepare tumor-bearing mice. When tumors grew to approximately 350 – 400 mm3 in volume, 50 mM of Ac4ManNAz (20 µl) was injected daily into the tumor via intratumoral injection for 4 days. Then, 300 µl PBS (pH 7.4) solution containing IR800CW (λEx = 774 nm, λEm = 789 nm)-labeled cRGD-CNPs (0.2 mg) and Cy5.5 (λEx = 675 nm, λEm = 720 nm)-labeled BCN-CNPs (0.2 mg) was injected into the mice via tail vein. Tumor tissue was dissected from mice at 48 h post injection and the fluorescence signals were measured using in vivo Fluorescence Molecular Imaging Systems (OV-100, Olympus, USA)

2.8. Tumor cell isolation from tumor tissues and cell analysis. For isolated tumor cell analysis, athymic nude mice (5-weeks old, 18 - 20 g, male) were purchased from Orient Bio INC. (Gyeonggi-do, Korea). To prepare tumor-bearing mice models, a suspension of 2ⅹ106 U87 cells in saline (60 µl) was subcutaneously injected into left flanks of mice. After 30 days, tumors grew to approximately 350 – 400 mm3 in volume, 50 mM of Ac4ManNAz (20 µl) was injected daily into left tumor via intratumoral injection for 4 days. After that, tumor tissues were extracted from the mouse then isolated cancer cells using a Cancer Cell Isolation Kit (Panomics Inc., CA, USA). In brief, the tumor tissue was placed into a cell culture dish containing 10 ml RPMI-1640 medium (1% Antibiotics) and non-tumor tissues and necrotic tumor tissues were picked out. Then, tumor tissue was transferred to a new dish and chopped up into small pieces. They were suspended with 20 ml of RPMI-1640 medium and

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centrifugation at 1200 rpm for 6 min at room temperature. The supernatant was discarded and the pelleted material was resuspended and incubated with 10 ml of tumor cell digestion solution for 3 h at 37 oC. After incubation, 10 ml of tumor-cell suspension solution was added into the tumor-cell digestion solution and filtrated by 100 mm cell strainer. Then, the solution was centrifuged at 1200 rpm for 8 min and the supernatant removed. The pellet was resuspended with 20 ml of tumor-cell suspension solution for the further purification. For purification of the cell suspension solution, 20 ml of tumor-cell purification solution was centrifuged at 1200 rpm for 2 min at room temperature. Then, cell suspension solution was transferred slowly to tumor-cell purification solution and stood in a vertical position for 6 min at room temperature. To collect tumor cells, 6 ml of solution from the bottom was transferred to a new tube and centrifuged at 1200 rpm for 8 min and the supernatant removed. Isolated cells were incubated with 1mg/ml of cRGD-CNPs or BCN-CNPs containing fresh medium for 1 h at 37 oC. The cells were washed twice with DPBS and collected in 2 % FBS containing DPBS. The fluorescence analysis of the cRGD-CNPs or BCN-CNPs treated cells was performed using flow cytometer (Guava easyCyteTM, Merck Millipore, Germany).

2.9. In vivo azide generation analysis. The U87 tumor-bearing nude mice model was established as described above, and 50 mM of Ac4ManNAz (20 µl) was injected daily into tumor via intratumoral injection for 4 days. To quantify generated azide groups in glycoproteins in tumor tissue, tumor tissue was dissected from mouse and washed twice with cold DPBS then lysed using RIPA buffer (Thermo Fisher Scientific Inc., USA) with 1 % protease inhibitor cocktail. Lysates were centrifuged at 14,000 rpm for 20 min at 4 oC to removed cell debris. The total protein of each sample was quantified by BCA assay (Pierce® 14


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BCA Protein Assay Kit, Thermo Scientific Inc., USA) and the lysates were incubated with 500 nM of phosphine–PEG3-biotin (Pierce, Rockford, IL, USA) for 6 h at 37 oc. The proteins from each sample were mixed with 1 x sodium dodecyl sulfate (SDS) gel-loading buffer (125 mol/L Tris, pH 6.8, 5 % glycerol, 2 % SDS, 1 % β-mercaptoethanol and 0.006% bromophenol blue), then boiled for 5 min. Then, 50 µg of proteins were separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature in 0.5% bovine serum albumin (BSA) containing 1 x TBST solution (10 mol/L Tris, pH 7.4, 100 mol/L NaCl and 0.1 % Tween 20). Then, the membranes were incubated with streptavidin-HRP (Pierce, Rockford, IL, USA) containing 1 x TBST solution (Pierce, Rockford, IL, USA) for 16 h at 4 oC. Finally, the membranes were washed three times using 1 x TBST, and protein band was detected with an ECL system.

2.10. Statistical analysis. In this study, the differences between experimental and control groups were analyzed using one-way ANOVA and considered statistically significant (marked with an asterisk (*) in figures) if p < 0.05.

3. Results and discussions 3.1. Preparation and characterization of glycol chitosan nanoparticles (CNPs, BCNCNPs, cRGD-CNPs). In this study, we prepared the CNPs modified with BCN or cRGD molecules as active-targeting ligands. Artificial azide reporter-targetable ligand, BCN, was directly conjugated to glycol chitosan polymers using amide bond linkages. Integrin αvβ3 15



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targeting ligand, cRGD, was directly conjugated to BCN molecules of BCN-CNPs via copper-free azide-alkyne cyclic addition reaction (Figure 1a). CNPs, BCN-CNPs, and cRGDCNPs can form self-assembled nanoparticle structure in aqueous condition due to their amphiphilicity.27 In addition, they showed narrow size distribution in dynamic laser scattering (DLS) results (Figure 1b). CNPs, BCN-CNPs, and cRGD-CNPs showed 258.5 ± 14.14, 266.8 ± 11.96, and 285.8 ± 3.96 nm in size, respectively. The surface charges (ζ) of CNPs, BCNCNPs, and cRGD-CNPs were measured as 13.8 ± 0.17, 5.08 ± 1.22, and 15.3 ± 0.30 mV, respectively. Transmission electron microscopy (TEM) images of CNPs, BCN-CNPs, and cRGD-CNPs showed that they formed spherical nanostructures (Figure 1c). The conjugation ratio of BCN molecules was measured as about 49.73 ± 0.25 nmol/mg in BCN-CNP by fluorescence quantification method after incubation with azide-quenched fluorescence dye, 3azido-7-hydroxycoumarin.28 To control conjugation ratio of cRGD with BCN equally, we conjugated cRGD-N3 molecules to BCN-CNPs via copper-free click reaction. Conjugation ratio of cRGD molecules was calculated as 47.75 ± 0.91 nmol/mg in cRGD-CNPs by UV absorption at 280 nm from BioPhotometer Plus (Eppendorf, Hamburg, Germany). As expected, they have similar physicochemical properties, as summarized in Table 1.

3.2. In vitro azide-reporter generation analysis. Since the azide-reporters can be generated using various metabolites, such as Ac4ManNAz, Ac4GalNAz and Ac4GlcNAz, the azidereporters generation analysis must be performed to find optimized metabolite for making tumor cells more uniform. Ac4ManNAz, Ac4GalNAz and Ac4GlcNAz were used as precursors for making azide-reporter on tumor cell surface via glycan biosynthetic pathway.18, 29

Various type of cancer cells, such as breast cancer (MCF7, MDA-MB-231, BT-474), brain 16


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cancer (U87), and lung cancer (A549), were incubated with 20 µM of Ac4ManNAz, Ac4GalNAz and Ac4GlcNAz for 48 h, and generated azide reporters were analyzed by DBCO-Cy5. As shown in Figure 2a, Ac4ManNAz-treated cells showed higher and moreuniform fluorescence intensity of DBCO-Cy5 on tumor cells surface than those of Ac4GalNAz- or Ac4GlcNAz-treated cells. This is because N-acetylmannosamine is a biological precursor of sialic acid on the tumor cell surface, and most tumor cells show increased expression of sialic acid on the surface.30-31 In addition, strong fluorescence intensity was observed in Ac4ManNAz- or Ac4GalNAz-treated murine bone marrow stroma cells (M2-10B4). To further quantification of azide-reporter in the cells, the mean fluorescence intensity (MFI) of DBCO-Cy5 labeled cells was evaluated by flow cytometer (Figure S1). Ac4ManNAz- or Ac4GalNAz-treated cells showed higher MFI than those of Ac4GlcNAz- or DBCO-Cy5-treated cells. Western blot results also showed higher azidereporter generation in Ac4ManNAz-treated cells than those of Ac4GalNAz or Ac4GlcNAztreated cells (Figure 2b). Strong band intensities in Ac4ManNAz- or Ac4GalNAz-treated M210B4 cells further supported that azide-reporters can be generated onto stroma cells as well as cancer cells. Although the amount of azide expression varied on the type of tumor cells due to their genetic properties and the tumor cell surface glycans were composed of various combinations of different monosaccharides and linkages, the outer site and most exposed saccharide of glycans is sialic acid, converted from D-mannosamine.32-33 Therefore, we expected that azide modified N-acetyl D-mannosamine (Ac4ManNAz) is an appropriate metabolite to generate targetable azide-reporters onto tumor cell surface.

3.3. In vitro tumor cell binding analysis. BCN and cRGD molecules were selected as 17



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active-targeting ligands of CNPs, which can bind azide-reporters or integrin αvβ3, respectively. We selected a U87 cell line with high expression of integrin αvβ3 group34-37 and MCF7 cell line with lower expression.37-38 Firstly, we observed dose-dependent azidereporter generation in U87 and MCF7 cells using western blot analysis. Figure 3a and 3b showed that Ac4ManNAz-treated U87 and MCF7 cells successfully generated azide reporter in a dose-dependent manner. Furthermore, artificially generated azide-reporters could have successfully targeted using BCN-CNPs both U87 and MCF7 cell lines, resulting in expression of strong fluorescence on the cell membrane (Figure 3c). In Figure 3c, BCNCNPs showed stronger and more-uniform fluorescence intensities in both azide-reportergenerated U87 and MCF7 cells than that of those treated with cRGD-CNPs. However, cRGD-CNPs showed different fluorescence intensities between U87 and MCF7 cells, showing strong fluorescence intensity in U87 cells only. The mean fluorescence intensity (MFI) of Ac4ManNAz/BCN-CNPs-treated U87 cells showed 14.8 and 2 fold higher than nontreated and cRGD-CNPs-treated U87 cells, respectively (Figure 3d). In addition, MFI of Ac4ManNAz/BCN-CNPs-treated MCF7 cells showed 18.6 and 15.7 fold higher than nontreated and cRGD-CNPs-treated MCF7 cells, respectively (Figure 3e). Z-axis fluorescence analysis results further supported that BCN-CNPs can more uniformly bind onto both U87 and MCF7 cells after azide-reporter generation than cRGD-CNPs can (Figure 4a). This is because that MCF7 cells have lower expression of integrin αvβ3 than U87 cells, resulting in saturation of cRGD-CNPs binding. Moreover, saturated binding of cRGD-CNPs also could be observed in high magnification fluorescence images of U87 cells (Figure 4b). cRGDCNPs-treated U87 cells showed heterogeneous distribution of fluorescence and lower intensity than BCN-CNPs-treated U87 cells. Next, we observed concentration-dependent

18


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targeting properties of BCN-CNPs and cRGD-CNPs. As shown Figure 5, when we increase the concentration of particles to 10 mg/ml, fluorescence intensities from Ac4ManNAz/BCNCNP-treated U87 cells dramatically increased up to 67.8 fold by dose-dependent manner (Figure 5a and 5b). However, fluorescence intensities of cRGD-CNPs-treated U87 cells increased up to only 25.1 fold and saturated (Figure 5c and 5d). This is because azide-reporter generation can be controlled by dose of Ac4ManNAz (Figure 3a and 3b) and the amount of generated azide reporters is sufficient to prevent saturation by binding of BCN-CNPs. Therefore, based on the Ac4ManNAz dose-dependent azide-reporter generation and concentration-dependent targeting analysis, we expect that azide-reporter can be utilized for tumor targeting strategy in vivo as well as in vitro.

3.4. In vivo and ex vivo tumor-targeting analysis by near-infrared fluorescence (NIRF) imaging. The in vivo biodistribution and tumor-targeting efficacy of BCN-CNPs and cRGDCNPs in azide-reporter generating tumor tissue were evaluated by monitoring the whole body NIRF intensity in U87 tumor mice model. To evaluate the in vivo biodistribution and their tumor-targeting efficacy, Cy5.5-labeled BCN-CNPs (5 mg/kg) and cRGD-CNPs (5 mg/kg) were intravenously administered to the mice with/without intratumoral pretreatment of Ac4ManNAz. Then, the fluorescence of BCN-CNPs and cRGD-CNPs were non-invasively observed as a function of time for up to 48 h (Figure 6a). The fluorescence intensity at the tumor tissue was slightly increased for 12 h and decreased at 48 h post injection of BCNCNPs in mice without Ac4ManNAz treatment. This might originate from the enhanced permeation and retention (EPR) effect in the tumor tissue and showed that BCN-CNPs could provide long circulation in body. cRGD-CNPs showed higher accumulation at tumor tissue 19



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up to 48 h via active-targeting effect. The fluorescence intensity of BCN-CNPs in azidereporter-generated tumor tissue (N3/BCN-CNPs) was gradually increased for 12 h and maintained up to 48 h post-injection. This showed tumor targeting effect23 via bioorthogonal click reaction between BCN molecule and azide-reporter as well as EPR effect in tumor tissue. At 48 h post-injection, major organs were enucleated from mouse and fluorescence distribution was measured using IVIS Lumina series (PerkinElmer, USA). Ex vivo fluorescence image showed strong fluorescence intensity in cRGD-CNPs and N3/BCNCNPs-treated tumor tissues (Figure 6b). Quantified fluorescence intensities showed 1.28- and 1.6-fold higher in tumor tissues from cRGD-CNP or N3/BCN-CNPs-injected mice, respectively, compared to the case of BCN-CNPs injection without Ac4ManNAz treatment (Figure 6c). The strong NIRF signals of N3/BCN-CNPs (red color) in tumor tissues were also observed using fluorescence microscopy after immunofluorescence (IF) staining, wherein the expression of integrin αvβ3 (green color) was observed using FITC-labeled anti-integrin αvβ3 antibody (Figure 6d).

3.5. Ex vivo tumor tissue distribution analysis after simultaneous injection of cRGDCNPs and Ac4ManNAz/BCN-CNPs. To compare distribution of cRGD-CNPs and BCNCNPs in tumor tissue precisely, Cy5.5-labeled BCN-CNPs and IR800CW-labeled cRGDCNPs were co-injected intravenously into U87 tumor-bearing mice model after pretreatment of Ac4ManNAz. Then, the tumor tissue (360 ± 20 mm3 in volume) was enucleated from mice, and fluorescence in tumor tissue was observed using in vivo fluorescence molecular imaging systems. As shown in Figure 7a, the fluorescence of both cRGD-CNPs and N3/BCN-CNPs was similarly concentrated in the perimicrovessel tissue. To further observe the binding of 20


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cRGD-CNPs and BCN-CNPs to tumor cells after formation of tumor tissue, we performed binding analysis using freshly isolated tumor cells from azide-generated U87 tumor tissues (Figure S2). Isolated tumor cells were incubated with 1mg/ml of Cy5.5-labeled cRGD-CNPs or BCN-CNPs for 1 h at 37 oC. The fluorescence analysis of the cRGD-CNPs- or BCNCNPs-bound cells was performed using flow cytometer. Figure 7b showed that 50.9 % and 92.9 % of U87 tumor cells were bound with cRGD-CNPs and BCN-CNPs, respectively. The MFI of BCN-CNPs-treated U87 tumor cells was 3.42-fold higher than cRGD-CNPs-treated cells (Figure 7c). This result showed that U87 tumor cells formed different phenotype subpopulations during tumor progression and that our method can target the heterogeneous tumor cells more effectively.39

4. Conclusion In this study, we have demonstrated the artificial azide-reporter-targeting strategy using metabolic engineering and bioorthogonal click chemistry to improve the tumor-targeting ability of nanoparticles and overcome tumor heterogeneity. Based on the metabolic engineering, azide-containing chemical reporters were successfully generated onto the various tumor cells in vitro. In addition, we observed inter-heterogeneity between U87 and MCF7 cells and heterogeneous distribution of integrin αvβ3-targeted cRGD-CNPs in vitro. Artificially targetable azide reporters also could be generated in U87 tumor tissues using intratumoral injection of Ac4ManNAz, and tumor targeting efficiency of BCN-CNPs in these mice was higher than that of cRGD-CNPs. As shown in the isolated U87 tumor cells from tumor tissue, azide reporters could be more uniformly targeted with BCN-CNPs via bioorthogonal click reaction compared to integrin αvβ3-targeted cRGD-CNPs. Therefore, our 21



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artificial azide-reporter-targeting strategy can be utilized as an effective heterogeneous tumor cell targeting method via bioorthogonal click reaction in nanoparticle delivery and may provide an alternative method for tumor targeting. However, direct injection of Ac4ManNAz into the tumor tissues is hardly utilized to the clinical field. Because it requires the information of the location of the tumor tissue and surgical treatment may be preferable. Therefore, tumor-specific delivery system of the azido precursors in in-vivo should be developed together for further clinical applications of artificial azide-reporter-targeting strategy.

Acknowledgment This work was supported by the GiRC (NRF-2012K1A1A2A01055811), the GRL project (NRF-2013K1A1A2A02050115) and the Intramural Research Program (CATS) of KIST.

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Scheme 1. Schematic illustration of artificial azide-reporter targeting strategy. (a) Conventional single-molecule targeting strategy in heterogeneous tumor cells using activetargeting ligands such as aptamer, antibody, glucose, and peptide. (b) Artificial azide-reporter generation using azide-containing mannosamine derivative with metabolic engineering of tumor cells. (c) Artificial azide-reporter targeting using bioorthogonal click reaction between azide molecules on tumor cell surface and bicyclononyne (BCN) on glycolchitosan nanoparticles.

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Figure 1. In vitro physicochemical properties of BCN-CNPs and cRGD-CNPs. (a) Schematic diagram of fluorescence-labeled glycolchitosan nanoparticle with structure of bicyclononyne (BCN, R1) and azide-functionalized cyclic RGD (cRGD, R2). (b) Hydrodynamic size distribution of CNPs, BCN-CNPs, and cRGD-CNPs. (c) Transmission electron microscope (TEM) images of CNPs, BCN-CNPs, and cRGD-CNPs. Scale bar indicates 200 nm.

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Figure 2. In vitro azide-reporter generation in various cancer cell lines and stromal cell. (a) Confocal microscopy images of Ac4ManNAz-, Ac4GalNAz-, and Ac4GlcNAz-treated breast cancer cell line (MCF7, MDA-MB231, BT-474), brain cancer cell line (U87), lung cancer cell line (A549) and stromal cell (M2-10B4), respectively. Azide-reporters were labeled and visualized with DBCO-Cy5 via bioorthogonal click reaction. Scale bar indicates 50 µm. (b) Western blot analysis of azide-reporters in glycoproteins in vitro. The coomassie staining shows the total amount of protein.

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Figure 3. In vitro Ac4ManNAz dose-dependent azide-reporter generation and binding properties in U87 (integrin αvβ3 high) and MCF7 (integrin αvβ3 low) cells. Western blot analysis of azide-reporters in glycoproteins, collected from (a) U87 and (b) MCF7 cell lines. The coomassie staining shows the total amount of protein. (c) Confocal microscope images of fluoreceinisothiocyante (FITC)-labeled BCN-CNPs- or cRGD-CNPs-treated U87 and MCF cells. Azide-reporter was introduced onto the surface of U87 and MCF7 cells by treatment of 20 µM of Ac4ManNAz. Blue channel: DAPI, green channel: CNPs. Scale bar indicates 100 µm. Quantification of mean fluorescence intensity (MFI) from non-treated, cRGD-CNPs-, and BCN-CNPs-treated U87 (b) and MCF7 (c) cells, respectively. *Indicates difference at the p < 0.001 significance level. 31



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Figure 4. Z-axis analysis of azide-reporter-generating U87 and MCF7 cell lines treated FITC-labeled cRGD-CNPs or BCN-CNPs. (a) Confocal microscope images were obtained with 10 µm thickness. Max projection images were merged with fluorescence image stacks. (b) High magnified confocal microscope images of cRGD-CNPs or BCN-CNPs treated U87 cell line. Scale bar indicates 25 µm.

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Figure 5. Dose-dependent binding properties in U87 (integrin αvβ3 high) cells after treatment with BCN-CNPs or cRGD-CNPs. (a) Confocal microscope images of Cy5.5 labeled BCNCNPs (0.5 - 10 mg/ml)-treated U87 cells. Blue channel: DAPI, red channel: BCN-CNPs. Scale bar indicates 100 µm. (b) Quantification of fluorescence intensities (pixel/cell) of BCNCNPs (0 – 10 mg/ml) using Image J software. (c) Confocal microscope images of Cy5.5labeled cRGD-CNPs (0.5 - 10 mg/ml)-treated U87 cells. Blue channel: DAPI, red channel: cRGD-CNPs. Scale bar indicates 100 µm. (d) Quantification of fluorescence intensities (pixel/cell) of cRGD-CNPs (0 – 10 mg/ml) using Image J software.

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Figure 6. In vivo biodistribution of Cy5.5-labeled BCN-CNPs, cRGD-CNPs and azide reporter-targeted BCN-CNPs in U87 tumor-bearing mice. (a) Whole-body near-infrared fluorescence (NIRF) images of the mice. White circles indicate tumor sites. (b) Ex vivo NIRF images of tumors and organs 48 h post-injection of Cy5.5-labeled BCN-CNPs and cRGDCNPs. (c) Relative NIRF intensities in the tumor tissues in (b). *Indicates difference at the p < 0.05 significance level. (d) Confocal microscope images of tumor tissues from (b). Integrin αvβ3 in tumor tissue was visualized by immunofluorescence stain. Blue channel: DAPI, red channel: CNPs, green channel: integrin αvβ3. Scale bar indicates 25 µm. 34


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Figure 7. NIRF distribution of Cy5.5-labeled BCN-CNPs and IR800CW-labeled cRGDCNPs in U87 tumor tissue. 5 mg/kg of BCN-CNPs and cRGD-CNPs were co-administered via tail vein of U87 tumor-bearing mice. Scale bar indicates 3 mm. (b) Flow cytometry analysis of isolated azide-reporter-generating U87 cells, incubated with Cy5.5 labeled BCNCNPs or cRGD-CNPs. (c) Mean fluorescence intensities (MFI) of Cy5.5-labeled BCN-CNPsor cRGD-CNPs-bound U87 cells in (b). *Indicates difference at the p < 0.001 significance level.

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Diameter (nm)

ξ-potential (mV)

BCN (nmol/mg)

cRGDyK (nmol/mg)

CNPs

258.5 ± 14.14

13.8 ± 0.17

-

-

BCN-CNPs

266.8 ± 11.96

5.08 ± 1.22

49.73 ± 0.25

-

cRGD-CNPs

285.8 ± 3.96

15.3 ± 0.30

-

47.75 ± 0.91

Table 1. In vitro characterization of CNPs, BCN-CNPs and cRGD-CNPs.

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30

MFI (A.U.)

Ac4ManNAz,

Ac4GalNAz

Ac4GlcNAz,

DBCO-Cy5

20

10

M 210 B4

A5 49

U8 7

BT -4 74

M DA -M B

23 1

0 M CF 7

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Figure S1. In vitro azide-reporter generation in various cancer cell lines and stromal cell. Mean fluorescence intensity (MFI) of Ac4ManNAz-, Ac4GalNAz- and Ac4GlcNAz-treated breast cancer cell line (MCF7, MDA-MB231, BT-474), brain cancer cell line (U87), lung cancer cell line (A549) and stromal cell (M2-10B4) was quantified by flow cytometer after labeling with DBCO-Cy5 via bioorthogonal click reaction.

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Figure S2. Schematic illustration of U87 tumor cells from tumor tissue. U87 tumor cells were collected from U87 tumor tissue at 30 days (362 mm2) after inoculation.

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TOC

39


Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal

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