HyaluronateâPeanut Agglutinin Conjugates for Target-Specific

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Hyaluronate - Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon Cancer Songeun Beack, Minsoo Cho, Young-Eun Kim, G-one Ahn, and Sei Kwang Hahn Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00126 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Bioconjugate Chemistry

Hyaluronate - Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon Cancer

Songeun Beack,† Minsoo Cho,† Young-Eun Kim,‡ G-One Ahn,‡ and Sei Kwang Hahn†,*

†

Department of Materials Science and Engineering, ‡ Division of Integrative Biosciences and

Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongamro, Nam-gu, Pohang 37673, Korea

* CORRESPONDING AUTHOR FOOTNOTE Tel.: +82 54 279 2159. Fax: +82 54 279 2399. E-mail: [email protected] (S.K. Hahn)

ACS Paragon Plus Environment


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Abstract Colon cancer is one of the most common death-related cancers in the world. For treating the colon cancer, it is crucial to detect and remove malignant lesions in the early time. Here, we developed hyaluronate (HA) – peanut agglutinin (PNA) conjugates for bioimaging of colon cancer. The HA-PNA conjugates were successfully synthesized by the coupling reaction between aldehyde modified HA and the N-terminal amine group of PNA. For diagnostic imaging, rhodamine B (RhoB) was chemically conjugated onto PNA in HA-PNA conjugates. After intraluminal injection of HAPNA-RhoB conjugates into tumor-bearing mice, small-sized colon cancers could be effectively visualized by ex vivo IVIS imaging and two-photon microscopy. Taken together, we could confirm the feasibility of HA-PNA-RhoB conjugates as a bioimaging agent for detecting colon cancers. [Keywords] Hyaluronate; Peanut agglutinin; Colon cancer; Two-photon imaging; Diagnostics


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Introduction Colonoscopy has been widely used for the detection of colon cancer in the clinic.1-3 Despite the endoscopic screening, colon cancer remains as one of the most common death-related cancers in the world.4 The colon cancer is easily metastasized to other organs increasing the mortality.5 Most patients of colon cancer have experienced hepatic metastasis.6 Accordingly, early detection is very important to prevent the metastasis of colon cancer. A variety of screening methods have been developed such as optical colonoscopy,7 sigmoidoscopy,8 fecal occult blood test,9 and virtual colonography.10 The conventional screening methods have poor sensitivity and specificity with difficulties in the rapid and accurate detection.11 Colon cancer increases flat and depressed small neoplasms.12 For more accurate detection of colon cancer, fluorescent endomicroscopy has recently been developed including confocal microscopy and two-photon microscopy (TPM).13,14 Fluorescent microscopy has high resolution to visualize the biological processes such as cell trafficking and cell-cell interaction.15 TPM has been extensively investigated for visualizing the morphology of organ tissues such as colon, eye, and skin without labeling.15-18 Intrinsic molecules such as reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and collagen have been successfully exploited for TPM.19 TPM can visualize the morphologies of healthy or diseased colon biopsies like the conventional histological analysis without fixation and staining.20 Peanut agglutinin (PNA) obtained from Arachis hypogea is a homo-tetrameric protein with a molecular weight of 110 kDa.21 PNA has a high binding affinity to β-D-galactosyl-(1-3)-N-acetyl-Dgalactosamine [Gal-β(1-3)GalNAc], which is known as a Thomsen-Friedenreich (TF) antigen expressed on the hyperplastic and malignant epithelial colon cancer cell.22,23 Also, PNA was reported to show antitumor effect by inducing apoptosis and regulating autophagy.21 Meanwhile, hyaluronate (HA), which is a biodegradable, biocompatible, and non-immunogenic polysaccharide, has been widely investigated for target-specific drug delivery to epithelial tumor cells.24,25 Epithelial cancer cells have over-expressed HA receptors like cluster determinant 44 (CD44).26 Especially, the isoform CD44v6 is over-expressed in colon cancers.24 In our previous work, we successfully demonstrated the target-specific delivery of HA derivatives to tumor tissues.18,27 In addition, the isoform of CD44 is



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known to be the major PNA-binding protein.28 Furthermore, HA has been used for novel endoscopic resection of a large colorectal polyp.29 In this work, we developed a facile diagnostic probes using PNA conjugated HA for colon cancer. HA-PNA conjugates were synthesized by coupling reaction of aldehyde modified HA (HA-ALD) with N-terminal primary amine group of PNA. Although HA or PNA has been applied for the targetspecific delivery to colon cancer,11,21 to our knowledge, this is the first report on diagnostic application of HA-PNA conjugates to the colon cancer model of azoxymethane and dextran sulfate sodium (AOM-DSS) induced colitis. AOM-DSS induced colitis is disrupted by inflammation, which has been recognized as a model of human ulcerative colitis.30 The intestinal barrier in colon epithelia revealed the opening of epithelial gaps. HA-PNA conjugates were expected to accumulate in these epithelial gaps of polyps. After labeling with a red fluorescence dye of Rhodamine B (RhoB), we carried out TPM of intraluminally administered HA-PNA conjugate to the healthy and diseased colon tissues. These findings are discussed for further development of a novel target-specific imaging probes of colon cancer using endoscopy.


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Results and Discussion Synthesis and characterization of HA-PNA conjugate Figure 1 shows a schematic representation for the synthesis of HA-PNA conjugate. HA-ALD was prepared by the treatment with sodium periodate, as reported elsewhere.31 HA-PNA conjugates were synthesized via the coupling reaction between aldehyde groups of HA-ALD and N-terminal amine groups of PNA. To reduce the adverse effect of unreacted aldehyde groups in HA-PNA conjugate, ethyl carbazate was used to block the remaining aldehyde group. The successful synthesis of HA-PNA conjugate was confirmed by GPC (Figure 2A) and SDS-PAGE (Figure 2B). The retention time of native PNA with a molecular weight of 110 kDa was ca. 58 min, which was shortened to ca. 53 min after conjugation to 20 mol% aldehyde modified HA with a molecular weight of 100 kDa. The bioconjugation efficiency was determined to be ca. 46% from the reacted PNA peak area before purification. Figure 2B shows the SDS-PAGE of HA, PNA, and HA-PNA conjugates. PNA showed a clear band at around 27 kDa, indicating that PNA has a tetramer structure composed of four identical subunits.21 Sodium dodecyl sulfate dissociates proteins into polypeptide subunits.32 In the case of HA alone, the band was not detected by coomassie staining in SDS-PAGE. As similar with that of poly(ethylene glycol) and poly(L-glutamic acid) in SDS-PAGE,33 a smeared band was observed in the lane of HA-PNA conjugate, confirming the successful synthesis of HA-PNA conjugate. The retardation effect might occur due to the polydispersity of HA. The secondary structure of PNA after conjugation was analyzed by CD (Figure 2C). The CD spectrum of HA-PNA conjugate was almost identical with that of PNA. The negative peak at 220 nm reflects that the secondary structure of PNA was maintained stably as a β-sheet form after conjugation to HA. The immunobiological activity of PNA in HA-PNA conjugates was also confirmed from the ratios of PNA concentrations in HA-PNA conjugates determined by ELISA and Bradford assay (Figure 2D).



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A Blood vessel Lymph node Colon cancer

Intraluminal injection of HA-PNA conjugates

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Figure 1. Schematic illustration for (A) the target-specific treatment of colon cancers after intraluminal administration of HA-PNA conjugates and (B) the synthesis of HA-PNA conjugates.


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Absorbance at 280 nm (a.u.)

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Figure 2. (A) Gel permeation chromatograms (GPCs) of PNA (blue) and HA-PNA conjugates (pink). (B) Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) of HA, PNA, and HA-PNA conjugates. (C) Circular dichroism (CD) spectra of HA-ALD, PNA, the mixture of HA and PNA, and HA-PNA conjugates. (D) The ratios of PNA concentrations in HAPNA conjugates determined by ELISA and Bradford assay (n=3).



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In vitro cellular uptake and cytotoxicity test of HA-PNA conjugates After labelling PNA and HA-PNA conjugate with RhoB, confocal microscopy was performed to visualize the cellular uptake of PNA and HA-PNA conjugates. Confocal images showed more effective intracellular delivery of HA-PNA conjugates than that of PNA and RhoB (Figures 3A and S1). Cancer cells are known to have many HA receptors such as CD44.26 Also, colon cancer cells are known to express many TF antigen.21 To check the cellular uptake mechanism of HA-PNA conjugates, the cells were pre-incubated with excess HA or PNA for the competitive binding tests. Although it was not possible to increase the PNA concentration above 3 mg/mL for pre-incubation due to the low solubility of PNA in PBS, the cells pre-incubated with excess HA or PNA showed less red fluorescence than cells treated without excess HA or PNA (Figure 3A and S2). These results indicate that HA-PNA conjugates were uptaken to the cells via HA or PNA-receptor mediated endocytosis. In vitro cytotoxicity of HA-PNA conjugates was assessed by MTT assay. Figures 3B and C show the viability of normal colon cells and colon cancer cells after treatment with PNA and HA-PNA conjugates. PNA and HA-PNA conjugates showed similar cell viability in normal colon cells, but PNA and HA-PNA conjugates caused more significant cytotoxicity in colon cancer cells at the concentration of 100 µg/mL. In particular, HA-PNA conjugates showed more toxic effect in colon cancer cells than PNA, which might be ascribed to dual targeting effect via HA-receptor mediated endocytosis and PNA-TF antigen binding. The results are consistent with confocal images showing more effective intracellular delivery of HA-PNA conjugates than PNA and RhoB (Figures 3A).


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Figure 3. (A) Confocal microscopy of HCT116 cells after treatment with PBS, RhoB, PNA, or HA-PNA conjugate (scale bar = 50 µm) without and with HA pre-incubation or PNA preincubation (blue: DAPI and red: RhoB). (B) CCD841CoN cell viability after treatment with PNA or HA-PNA conjugate. (C) HCT 116 Cell viability after treatment with PNA or HA-PNA conjugate.



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Bioimaging of AOM-DSS induced colitis using HA-PNA-RhoB conjugates Ex vivo imaging with an IVIS was carried out to visualize AOM-DSS induced colitis. The AOMDSS induced colitis closely resembles human ulcerative colitis due to the similarly advanced cancer stage like adenoma-to-carcinoma sequence.30 As shown in Figure 4, the fluorescent signals were observed at the adeno-carcinomas after intraluminal administration of HA-PNA-RhoB conjugates. The RhoB signals from each sample were obtained at the excitation and emission wavelengths of 570 nm and 620 nm. Although the polyps were very small, the strong fluorescence signals of RhoB after administration of HA-PNA-RhoB conjugates were distinguished from the background tissues and other treated groups, enabling the excellent colon cancer detection. These results demonstrate the target-specificity of HA-PNA conjugate to detect small colon cancers at early stages without timeconsuming histological analysis. Two-photon microscopy of AOM-DSS induced colon cancer After intraluminal injection of HA-PNA-RhoB conjugates, TPM was performed for the dissected fresh colon tissues (Figure 5 and S3). The TPM at an excitation wavelength of 780 nm could visualize the morphology of colon tissues like individual cells and subcellular structures by intrinsic contrast.16 The blue fluorescent signals reflect the autofluorescence from NADH, FAD, and so on. Colon cancer tissues showed the irregular crypt architecture and the intercellular distance in the crypt was wider than that of normal colon tissues.30 The yellow fluorescent signals represent immune cells of colon cancer and RhoB. When DSS induced colitis, inflammatory cell infiltration was observed from epithelia to lamina propria. Lamina propria contains antigen-presenting cells, lymphocytes, and fibroblasts. Lysosomes in antigen-presenting cells show yellow fluorescence at the excitation wavelength of 780 nm.34,35 Ex vivo TPM images clearly showed that HA-PNA-RhoB conjugates were widely distributed compared to the PNA-RhoB conjugate and RhoB. The RhoB signals at the wavelength of 1000 nm showed the different contrasts between the tumor tissues treated with HA-PNA-RhoB conjugates and those treated with HA-RhoB, PNA-RhoB, RhoB, and the control. The tumorigenesis of colon tissues are known to upregulate TF antigen and CD44 receptors in the epithelia surface.21,36 Considering the


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one-time incubation of HA-PNA-RhoB conjugate for only 10 min, the successful staining of colon cancer might result from the high binding affinity to TF antigen and CD44, showing a great promise for diagnostics. Figure 6 showed the 3D TPM images of AOM-DSS induced colitis after treatment with each sample. The images of colons treated with HA-PNA-RhoB conjugates showed that HA-PNA-RhoB conjugates were deeply uptaken into the polyp. The normal colon tissues showed tightly packed colonic crypts without the disruption of tight junctions at the wavelength of 780 nm (Figure S2). Remarkably, yellow fluorescent signals from RhoB at the excitation wavelength of 1000 nm were not observed in normal colon tissues, reflecting that PNA and HA-PNA conjugates have no binding affinity to healthy colon tissues. The quantitative analysis of RhoB signals from the TPM images demonstrated that HA-PNA-RhoB conjugates were highly accumulated in the colon cancer (Figure 7).



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l tro hoB hoB hoB hoB n R -R -R -R Co H A PN A P N A HA Figure 4. (A) Ex vivo merged pseudocolor IVIS images of colons in AOM-DSS induced colon cancer model mice after treatment with PBS, RhoB, HA-RhoB, PNA-RhoB, or HA-PNA-RhoB conjugate, respectively. (B) Quantitative analysis of RhoB fluorescence intensities in tumor tissues (*P < 0.05, HA-PNA-RhoB conjugate vs PNA-RhoB, ***P < 0.001, PNA-RhoB vs RhoB).


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Figure 5. Ex vivo two-photon microscopic images of AOM-DSS induced colon cancer tissues at 10 min post-intraluminal administration of (A) PBS, (B) RhoB, (C) HA-RhoB, (D) PNA-RhoB, or (E) HA-PNA-RhoB conjugate. First row shows the autofluorescence images by 780 nm excitation and second row represents those images by 1000 nm excitation (objective = 25×, scale bar = 100 µm).


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Figure 6. 3D two-photon microscopic images of excised tumors after intraluminal administration of (A) PBS, (B) RhoB, (C) HA-RhoB, (D) PNA-RhoB, or (E) HA-PNA-RhoB conjugate. First row images were obtained at the excitation wavelength of 780 nm and the second row images were by 1000 nm excitation. Left column shows the XYZ direction of colons from the colonic epithelial surface to 100 µm in depth. Right column shows the XY plane of colons from the depth of 100 µm to the colonic epithelial surface.

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Figure 7. Quantitative analysis of ex vivo two-photon microscopic images of normal colon tissues and colon cancer tissues at 10 min post-intraluminal administration of PBS, RhoB, HA-RhoB, PNA-RhoB, or HA-PNA-RhoB conjugate (*P < 0.05, HA-PNA-RhoB conjugate vs PNA-RhoB and PNA-RhoB vs RhoB in colon cancer tissues).


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Conclusion HA-PNA conjugates were synthesized by coupling reaction between aldehyde group of HA-ALD and N-terminal amine group of PNA. GPC, SDS-PAGE, CD, and ELISA confirmed the successful synthesis of HA-PNA conjugate. According to in vitro test of HA-PNA conjugates in colon epithelial cells and colon cancer cells, HA-PNA conjugates were higher targeting efficiency to cancer cells than that of free PNA. IVIS imaging, and TPM imaging clearly visualized the effective target-specific delivery of HA-PNA conjugates to colon cancer tissues. Our results suggest that HA-PNA conjugates have promising potential as target-specific delivery probes for effective colon cancer detection agent using endoscopy.

Materials and Methods Materials Sodium hyaluronate (HA) with a molecular weight (MW) of 100 kDa was purchased from Lifecore Co. (Chaska, MN). Peanut agglutinin (PNA), sodium periodate (NaIO4), ethylene glycol (EG), and sodium cyanoborohydride, ethyl carbazate, tert-butyl carbazate, human serum, and 3,3’,5,5’-tetramethylbenzidine (TMB), azoxymethane (AOM) were obtained from Sigma-Aldrich (St. Louis, MO). Coomassie plus protein assay reagent was purchased from Thermo Scientific (Rockford, IL). Dextran sodium sulfate was obtained from MP Biomedicals (Santa Ana, CA). Affinity purified chicken anti-peanut protein and HRP conjugated affinity purified chicken anti-peanut protein were purchased from Gallus Immunotech (Fergus, Ontario, Canada). Lissamine rhodamine B sulfonyl chloride (RhoB), Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), antibiotics, and phosphate buffered saline (PBS) were purchased from Invitrogen (Carlsbad, CA). All reagents were used without purification. Synthesis of HA-PNA conjugate HA-aldehyde (HA-ALD) with an aldehyde content of 20 mol% was synthesized as reported elsewhere.31 Briefly, 10 mg/mL of HA (MW = 100 kDa, 100 mg) aqueous solution was reacted with 1



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molar excess of NaIO4. After reaction with stirring in dark place for 24 h, excess amount of EG (100 µL) was added to the mixture for the termination of the reaction. The resulting solution was poured into the cellulose ester dialysis membrane bag (MWCO of 7000) and dialyzed against deionized (DI) water for 3 days. The degree of aldehyde content in HA-ALD was analyzed by 1H NMR (DPX500, Bruker, Germany). After that, PNA and HA-ALD was dissolved in pH 5.5 sodium acetate buffer and 5 molar excess of sodium cyanoborohydride to aldehyde group was added into the mixed solution. The number of PNA molecules per HA chain in the feed was 4 to synthesize HA-PNA conjugates. An excess molar of ethyl carbazate was added to the reaction solution for blocking the unreacted aldehyde groups in HA-PNA conjugates. Finally, HA-PNA conjugate was dialyzed against a large excess amount of PBS for a day. Characterization of HA-PNA conjugate The synthesized HA-PNA conjugates was analyzed by gel permeation chromatography (GPC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). GPC analysis was carried out to check the change of retention time before and after conjugation of PNA to HA using the following systems: Waters 717 plus autosampler, Waters 1525 binary HPLC pump, Waters 2847 dual λ absorbance detector, and UltrahydrogelTM 1000 connected with UltrahydrogelTM 500 column. The eluent was PBS at pH 7.4 and the flow rate was 0.3 mL/min. The detection wavelengths were 210 nm for HA and 280 nm for PNA. SDS-PAGE was performed in 12 % PA gel. A MW reference marker (MW 10-250 kDa), HA, PNA, or HA-PNA conjugate solution (final concentration at 0.5 mg/mL PNA) were loaded onto PA gel, and run at 50 V for 30 min and 100 V for 1 h. After electrophoresis, the gel was stained with Coomassie Blue R-250 for 2 h. The secondary structure of HA-PNA conjugate was analyzed by circular dichroism (CD). The CD spectra for PNA and HA-PNA conjugate in PBS (pH 7.4) were obtained with a UV spectrophotometer (JASCO J-715, Essex, UK) at 25 °C over the range of 190-250 nm under a nitrogen atmosphere. The data were acquired at 0.2 nm intervals. Each spectrum was subtracted from that of buffer and the residual ellipticity was calculated as an average of two scans. The PNA content in the conjugate was determined by Bradford assay. The affinity of PNA and HA-PNA conjugates to anti-PNA antibody was assessed by PNA ELISA


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measuring the absorbance at 450 nm with a microplate reader (SpectraFluor Plus, TECAN, Mannedorf, Switzerland). In vitro cytotoxicity effect of HA-PNA conjugate A human colon cancer cell line of HCT116 was cultured in DMEM supplemented with 10 vol% FBS and 1 wt% antibiotics in a humidified 5% CO2 incubator at 37 °C. A human normal colon cell line of CCD841CoN was cultured in EMEM supplemented with 10 vol% FBS and 1% antibiotics in a humidified 5% CO2 incubator at 37 °C. Each cell at a density of 1 × 104 was seeded on 96-well plates. The cells were incubated with different concentrations of PNA or HA-PNA conjugate (0, 0.1, 1, 10, 100 µg/mL) at 37 °C for 24 h, respectively. After washing with PBS, serum free medium was added to the cells. The cellular toxicity of HA-PNA conjugate was determined by MTT assay (n = 4). The absorbance was measured at 540 nm. Labeling of PNA and HA-PNA conjugate with RhoB PNA and HA-PNA conjugate were labeled with RhoB following the protocol to visualize their target-specific delivery into colon cancer cells and tissues. The RhoB solution in sodium bicarbonate buffer (1 M, pH 9) was added to the protein solutions of PNA and HA-PNA conjugate at a molar ratio to protein molecule of 10. HA-PNA-RhoB conjugates were prepared by the chemical conjugation between sulfonyl chloride group of Lissamine RhoB sulfonyl chloride and amine group of PNA. The reaction was performed in dark place with mild stirring for 12 h. Then, the conjugate was dialyzed against a large excess amount of PBS for a day. The degree of substitution was calculated by measuring the absorbance at 280 and 540 nm. Confocal microscopy for cellular uptake of HA-PNA conjugates CCD841CoN cells and HCT116 cells were seeded on each eight chamber confocal slide at a density of 2 × 104 cells per well and incubated for a day. Then, RhoB, PNA-RhoB, and HA-PNARhoB conjugates in 200 μL of DMEM were added to the wells. To confirm PNA and HA receptormediated endocytosis of HA-PNA conjugate, the cells were pre-incubated with an excess amount of PNA and HA for 2 h before HA-PNA conjugate treatment. After 2 h incubation, the cells were washed



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with PBS and fixed with 4% paraformaldehyde solution for 3 min. The cells were mounted with Vectashield mounting medium containing 4’,6’-diamidino-2-phenylindole (DAPI). The cellular uptake of HA-PNA conjugates was monitored by confocal microscopy. AOM-DSS induced colon cancer model mice To prepare colorectal cancer model mice, AOM (10 mg/kg) was intraperitoneally injected to male C57BL/6 mice (8 weeks old, 20-25 g), which were maintained with feeding normal water for 7 days. Then, the mice were treated with 2% DSS in the drinking water for 7 days followed by regular access to water for 14 days. This cycle for administration of DSS was repeated once again. Ex vivo bioimaging of colon cancer using IVIS imaging systems Ex vivo bioimaging was carried out with IVIS imaging systems at the excitation and emission wavelengths of 570 and 620 nm. All of the data were quantified using the region of interest (ROI) in the captured images and presented as a mean ± standard deviation for the groups of four animals. We have complied with the POSTECH institutional ethical protocols for using animals. Ex vivo bioimaging of colon cancer by two-photon fluorescence microscopy HA-PNA-RhoB conjugates were intraluminally injected to AOM-DSS induced colon cancer model mice to investigate the targeted delivery of HA-PNA conjugates (n = 4). PBS, RhoB, HARhoB, or PNA-RhoB was also injected to the mice for comparison. After 10 min, the mice were sacrificed and the colon tissues were surgically excised, longitudinally cut to locate the mucosal layer upward, and washed with PBS. The colon tissues were unrolled on the glass slide and overlaid with a cover glass. The images of fresh colon tissues were obtained with a two-photon microscope (TCS SP5 II, Leica) using a Ti-Sapphire laser. The 3D imaging was performed by using 25× objective lenses (HCX IRAPO L25×, NA 0.95 W, Leica) with the stepwise increment of 2 µm in the depth direction. The excitation laser was tuned to 780 and 1000 nm for autofluorescence and the fluorescence of RhoB, respectively. Statistical analysis The data were expressed as mean values ± S.D from several separate experiments. Statistical


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analysis was carried out via the t-test using the software of SigmaPlot 12.0. The value for *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Acknowledgement This research was supported by a grant of the Bio & Medical Technology Development Program (No. 2012M3A9C6049791) and Mid-career Researcher Program (No. 2015R1A2A1A15053779) of the National Research Foundation (NRF) funded by the Korea government (MEST).

Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI:



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– For Table of Contents Use Only –

Hyaluronate – Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon cancer

Songeun Beack,† Minsoo Cho,† Young-Eun Kim,‡ G-One Ahn,‡ and Sei Kwang Hahn†,*

Normal colon

Polyp

Colon cancer

Colon HA-PNA conjugate


HyaluronateâPeanut Agglutinin Conjugates for Target-Specific

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