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Fluorescent Neomannosyl Bovine Serum Albumin as Efficient Probe for Mannose Receptor Imaging and MCF-7 Cancer Cell Targeting Yang Yang, Tian-Wei Jia, Fei Xu, Wei Li, Shun Tao, Li-Qiang Chu, Yun He, Yu Li, Suri S. Iyer, and Peng Yu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00134 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 26, 2018
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Fluorescent Neomannosyl Bovine Serum Albumin as Efficient Probe for Mannose Receptor Imaging and MCF-7 Cancer Cell Targeting
Yang Yang,a Tian-Wei Jia,a Fei Xu,a Wei Li,a Shun Tao,b Li-Qiang Chu,b Yun He,c Yu Li,a *
Suri S. Iyerd * and Peng Yua *
a
China International Science and Technology Cooperation Base of Food
Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
b
College of Chemical Engineering and Materials Science, Tianjin University of Science
and Technology, Tianjin 300457, China.
c
Research Institute of Tsinghua University in Shenzhen, Nanshan District, Shenzhen
518057, China.
d
Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State
University, Atlanta, GA 30302, USA.
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E-mail: liyu@tust.edu.cn (Y. Li)
siyer@gsu.edu (S. Iyer)
yupeng@tust.edu.cn (P. Yu)
KEYWORDS Glycoprotein, Mannose, Surface Plasmon Resonance, Microarray, Fluorescent imaging.
ABSTRACT
Robust carbohydrate conjugated fluorescent bovine serum albumin (BSA) as useful tool to study carbohydrate-receptor interactions and in vivo targeting is reported. Amine terminated α-mannoside was attached to fluorescein labelled BSA via diethyl squarate strategy. The surface functionalization and lectin binding specific to fluorescent neomannosyl
glycoprotein
were
confirmed
using
matrix-assisted
laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface plasmon resonance (SPR) and microarray imaging. The glycoconjugate was further used to image Concanavalin A (ConA), pili of E. coli K12 and lysosomes in MCF-7 cancerous cells successfully, suggesting that this neomannosyl glycoprotein can be used as suitable probe to elucidate carbohydrate-protein interactions, image cancers and target drug specifically towards tumors.
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1. INTRODUCTION
A high density of carbohydrates presented as glycolipids and glycoproteins covers the mammalian cell surface 1-5. Their presence on the cell surface underscores their role in a number of physiological and pathological processes, e.g. protein folding,6,7 celladhesion,8,9 cellular signalling,10,11 inflammation12,13 and infections induced by viral14,15 and bacterial16-18 agents. These processes are highly dependent on molecular recognition involving carbohydrate ligand-specific protein receptor interactions19; the study of these complex interactions is not well understood, except for limited systems. There is a real need to develop biochemical tools to investigate carbohydrate-receptor interactions. Several methods that produce sensitive and detectable signals have been used to analyze carbohydrate-receptor interactions;
20,21
using fluorescent labels is a common
and widely accepted strategy.22 Fluorescent parameters such as fluorescence intensity, emission and excitation spectrum, stokes shift, fluorescence quantum yield and fluorescence lifetime could be used for monitoring carbohydrate-protein interactions.23 Furthermore, fluorescence microscopy can pinpoint the locations of the carbohydrate receptors.24,25 However, attaching fluorescent molecules directly to a unimolecular carbohydrate is limited in scope, because monomeric carbohydrate–protein binding interactions are not very strong with dissociation constants in the mM range26-30. This problem can be overcome by taking advantage of the glycoside cluster effect31,32; multiple glycan residues are usually attached to a carrier, such as luminescent materials 3 Environment ACS Paragon Plus
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33,34
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, nanoparticles 35,36, etc. This approach has also an added advantage of mimicking the
multivalent displaying of glycolipids and glycoproteins to achieve specific cellular responses.
Although
several
glycan
functionalized
fluorescent
semiconductor
nanomaterials have been developed and explored for the study of carbohydratereceptor interactions, the potential application of these nanosized glycoprobes is still in the early stage because of its complicated preparation, surface modification steps and potential toxicity.37,38 To overcome these limitations, we decided to combine small organic fluorophores, the multivalency of carbohydrate presentation and a biomacromolecule backbone for the preparation of glycan fluorescein-labeled conjugate. Herein, Bovine Serum Albumin (BSA) was chosen as the backbone since BSA has excellent nonspecific absorption and biocompatibility characteristics39-41. To establish proof of principle, multiple copies of mannose (Man) as functional ligands were attached to fluorescent BSA. This new fluorescent
neoglycoprotein
was
characterized
by
matrix-assisted
laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface plasmon resonance (SPR) and lectin microarray techniques. Additionally, this fluorescent mannosyl neoglycoprotein was further used to label lectins, E. coli K12 and cancerous cells.
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2. EXPERIMENTAL METHODS
2.1 Materials and methods All materials and solvents were obtained from commercial suppliers and were used as received. Thin-layer chromatography (TLC) plate was purchased from EMD Co. Ltd. Flash column chromatography was performed on silica gel 200-300 mesh size. The compounds were detected using UV light and/or were stained with 5% H2SO4 in ethanol. The final neomannosyl glycoconjugates were purified using SephadexTM G-75 size exclusion chromatography (GE Healthcare). NMR spectra were recorded on a 400 MHz Bruker AVANCE III instruments. Chemical shifts (δ) were reported in parts per million downfield from the internal standard, TMS and J values were given in Hz. The molecular weights of the conjugates and mannosides were confirmed using ultrafleXtreme Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (Bruker Daltonics MALDI-TOF/TOF, Bremen, Germany) or Electrospray Ionization Mass Spectra (ESI-MS) on a hybrid Ion Trap-Time of Flight Mass Spectrometer (Shimadzu LCMS-IT-TOF, Kyoto, Japan). Fourier Transform-Infrared Spectra were recorded on a Thermo Scientific FT-IR spectrometer (Nicolet™ iS™ 50). Photoluminescence (PL) spectra were obtained on RF-5301PC spectrofluorimeter (Shimadzu, Kyoto, Japan). Micrographs were taken using a SU1510 scanning electron microscope, SEM (Hitachi, Tokyo, Japan). Fluorescence microscopy images were captured by Ti-DH (Nikon, Japan) or Olympus FV1000 confocal laser scanning 5 Environment ACS Paragon Plus
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microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) instrument. Surface plasmon resonance (SPR) experiments were performed using a custom-built combination based on standard Kretschmann configuration42. SPR spectra were fitted using Winspall 2.0 software (developed by Max-Planck-Institute for Polymer Research, Mainz, Germany). Bovine Serum Albumin, (BSA, Lot No. B2064, ≥98%), Concanavalin A from Canavalia ensiformis (Jack bean) (Con A, Lot No. L7647, Affinity-purified), Lectin from Arachis hypogaea (peanut), (PNA, Lot No. L0881, Affinity-purified) and fluorescein isothiocyanate isomer I (FITC, Molecular Weight: 389.4, Lot No. F7250, ≥90%) were obtained from Sigma (Shanghai, China). E. coli K12 wild-type strains and Bacillus natto obtained from the Center of Industrial Culture Collection, College of Biotechnology, Tianjin University of Science and Technology were grown in sterile Luria-Bertani (LB) medium. Cultures were grown overnight until the value of A600 reached 0.6. The mixture was centrifuged (6000 rpm, 10 min) and washed twice with PBS buffer. Influenza A/Chicken/Beijing/AT609/2014 (H9N2) strains were provided by the National Institute for Viral Disease Control and Prevention (China CDC). Strains were propagated in embryonated chicken eggs. After propagation, the allantoic fluid was collected and centrifuged at 3000 rpm for 10 min. Next the supernatant was separated and stored at -80 ℃. Virus was inactivated by β-propiolactone (β-PL) and used for all experiments.
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2.2. Synthesis 2.2.1. 6-Aminohexyl-α-D-mannopyranoside (2) Sodium methoxide (29 mg, 0.5 eq) was added to a solution of 6-azidohexyl-2, 3, 4, 6-tetra-O-acetyl-α-D-mannopyranoside 1 (500 mg, 1 eq) in dry MeOH (10 mL). The reaction mixture was kept at room temperature (rt) and stirring rate was set at 1000 rpm for 3 h. Dowex 50W X 8 (H+) resin was used to neutralize the solution to pH 7. After filtration to remove the resin, the solution was evaporated to dryness. The resultant syrup was dissolved in dry methanol, 10% palladium-carbon (20 mg) added and the mixture was subjected to 1 atm hydrogen gas. After 12 h, the mixture was filtered and evaporated to dryness to give the title compound 2 as a colorless syrup (250 mg, 85%). 1
H NMR (400 MHz, D2O) δ 4.83 (s, 1H, H-1), 3.92 – 3.81 (m, 2H, H-6a, H-2), 3.79 – 3.65
(m, 3H, –OCHHCH2, H-3, H-6b), 3.65 – 3.56 (m, 2H, H-4, H-5), 3.56 – 3.47 (m, 1H, – OCHHCH2), 2.94 (t, J = 7.9 Hz, 2H, –CH2NH2), 1.61 (br, 4H), 1.37 (br, 4H); 13C NMR (100 MHz, D2O) δ 99.68 (C-1), 72.74 (C-5), 70.65 (C-3), 70.11 (C-2), 67.76 (C-4), 66.81 (– OCH2(CH2)5NH2), 60.95 (C-6), 39.59 (–O(CH2)5CH2NH2), 28.30, 26.96, 25.43, 24.96. IR (KBr): 3415 – 3289 (s, OH), 2932 – 2857 (m, –CH2 in pyranose), 1142 – 1049 (s, C–O–C). HRMS (ESI): m/z calculated for C12H26NO6 [M+H]+: 280.1755, found: 280.1747. 2.2.2. 6-O-(2-ethoxy-3, 4-dioxocyclobut-1-en-1-amino) hexyl -α-D-mannopyranoside (3)
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To a solution of 2 (300 mg, 1 eq) in phosphate buffer (pH 7.0, 10 mL), diethyl squarate (365 mg, 2 eq) was added and the solution was stirred at rt for 14 h. The solvent was removed in vacuo and the residue was subjected to flash column chromatography to yield the desired product as a colorless oil (325 mg, 75%). 1H NMR (400 MHz, D2O) δ 4.78(s, 1H, H-1), 4.66 (q, J = 7.3 Hz, 2H, –OCH2CH3), 3.85 – 3.9 (m, 2H, H-6a, H-2), 3.72 – 3.65 (m, 3H, –OCHHCH2, H-3, H-6b), 3.60 – 3.52 (m, 2H, H-4, H-5), 3.48 – 3.41 (m, 3H, –OCHHCH2, –CH2NHC=C) 1.55 – 1.54 (m, 4H), 1.41 – 1.27 (m, 7H); 13
C NMR (100 MHz, D2O) δ 176.79 (C=O), 99.65 (C-1), 72.72 (C-5), 70.65 (C-3), 70.53 (–
OCH2CH3), 70.09 (C-2), 67.71 (C-4), 66.73 (–OCH2(CH2)5NH), 60.90 (C-6), 44.47(– O(CH2)5CH2NHC=C), 29.50, 28.36, 25.29, 24.94, 15.09 (–OCH2CH3). IR (KBr): 3523 – 3404 (s, OH), 2934 – 2865 (m, –CH2 in pyranose), 2099 (s, C=O), 1166 – 1052 (s, C–O–C). HRMS (ESI): m/z calculated for C18H29NO9Na [M+Na]+: 426.1735, found: 426.1749. 2.2.3. FITC Labelling of BSA (BF) To a stirring solution of BSA (1000 μL, 0.01 mg/mL) in 100 mM sodium carbonate buffer (pH 9.0), fluorescein isothiocyanate isomer I, FITC (50 μL, 0.01 mg/mL) was added in anhydrous dimethyl sulfoxide. The reaction was stirred at 4 ℃ for 16 h. This solution was purified by Sephadex® G75, followed by lyophilization to afford the FITClabelled BSA, BF.
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2.2.4. Synthesis and characterization of fluorescent neomanosylglycoprotein (BFM) BF (55 mg, 1 eq.) was dissolved in sodium carbonate buffer (10.0 mL, pH 9.0). Next, 3 (16.7 mg, 50 eq.) was added and the solution was stirred for 12 h at rt. The reaction mixture was desalted using an Amicon ultrafiltration device (MWCO 10 kDa, Millipore). The solution was concentrated to 2 mL and purified by Sephadex® G-75, followed by lyophilization to afford the fluorescent neomannosyl glycoprotein, BFM. MALDI-TOF-MS in positive linear ion mode was used to determine the molecular weight of the protein conjugates using 2, 5-dihydroxybenzoic acid (DHB) as matrix. FlexAnalysisTM was used for data analysis. 2.3. Surface plasma resonance analysis toward ConA. Freshly-deposited gold substrate was attached to a flow cell and PBS buffer was injected into the cell using a peristaltic pump. After baseline stabilization, BFM was covalently immobilized onto the gold chip by introducing the BFM solution (1 mg/mL in PBS buffer, pH 7.0) using a flow rate of 700 μL/min. To reduce nonspecific binding, BSA (1 mg/mL) was used to block the unreacted sites. Next, the analyte in PBS running buffer were injected over the immobilized BFM at a flow rate of 700 μL/min for 30 min. The adsorption kinetic was obtained by monitoring the reflectivity at a fixed angle. The SPR spectra were recorded before and after the adsorption and analyzed using Winspall
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2.0 software. Regeneration was performed using 0.01 M NaOH in PBS buffer immediately after the disassociation phase until the reflectivity reached baseline values. 2.4. Microarray studies. Microarrays
were
fabricated
using
the
OmniGrid
Accent
microarrayer
(GeneMachines, San Carlos, CA) onto NHS-activated glass slides (NEXTERION® Slide H, Schott Technical Glass Solutions GmbH, Jena, Germany) according to protocols provided by the manufacturer. Various concentrations (0.5, 1 and 1.5 mg/mL) of proteins (ConA and PNA) in print buffer (150 mM phosphate buffer, pH 8.5, 0.01 % Tween® 20) were printed on to the glass in replicates of seven. Printed slides were allowed to react in an atmosphere of 50% humidity and then placed in an atmosphere of 75% humidity for 12 h. The unreacted NHS groups were blocked using blocking buffer (100 mM phosphate buffer, 25 mM ethanol amine, 0.01 % Tween® 20, pH 8.5) for 1 h, washed with wash buffer thrice, dried and stored at 4 ℃ before use. Lectin functionalized slide was incubated with BFM for 2 h. After incubation, the glass slide was washed sequentially with PBST (PBS with 0.01 % Tween® 20), PBS and deionized H2O for 5 min each and dried. Fluorescence intensities were detected by using an InnoScan® 710 microarray scanner (Arrayit Corporation, Sunnyvale, CA, USA). 2.5. Fluorescent neomannosylprotein − Concanavalin A interactions 2.5.1. Luminescent aggregates assays toward ConA
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A series of concentrations of ConA (1, 5, 10 and 25 nM, 200 μL) and PNA (25 nM, 200 μL) in PBS buffer (1 mM, pH 7.0) were incubated with BFM (0.5 μM, 800 μL) at rt for 30 min under gentle shaking. Subsequently, the solution was centrifuged (13000 rpm, 20 min). The pellet was visualized using UV light. The supernatant was transferred into a 1 mL quartz cuvette and the fluorescence spectra were recorded at an excitation wavelength of 495 nm. 2.5.2. Competitive inhibition assays toward ConA Aliquots of ConA (25 nM, 200 μL) and BFM (0.5 μM, 800 μL) in PBS were equilibrated at 25 ℃
for 30 min. Next, the mixtures were centrifuged (13000 rpm, 20
min) and different carbohydrates (10 mM mannose, 100 mM lactose, 100 mM galactose and 75 mM glucose) were added to the BFM-ConA solution and shaken throughly. All solutions were transferred into 1 mL quartz cuvettes to record the fluorescence spectra at an excitation wavelength of 495 nm. 2.6. Fluorescent neomannosylprotein − Escherichia coli interactions Different concentrations of E. coli K12 (0.2 mL of a suspension), Bacillus natto and Influenza A/Chicken/Beijing/AT609/2014 (H9N2) were incubated with BFM (0.5 μM, 800 μL) in PBS buffer for 30 min at rt with gentle shaking. The solutions were transferred into 1 mL quartz cuvettes and the fluorescence spectra were recorded at an excitation wavelength of 495 nm. For fluorescence microscopy studies, a drop of the
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solution was spotted on a microscope slide and covered using a cover slide. For Scanning Electron Microscope (SEM) studies, a drop of the samples was spotted on muscovite mica substrate and air dried for 2 h before observation. 2.7. BFM labelling MCF-7 cancer cells Cells from the human breast cancer cell line MCF-7 were grown at 37 0C under a 5% CO2 atmosphere for 12 h in Dulbecco's Modified Eagle Medium (DMEM) enhanced with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 1× nonessential amino acids and 1× penicillin/streptomycin. After 12 h, the media was replaced with fresh media and BFM was added at a concentration of 50 μg/mL. After 48 h, the supernatant was removed and cells were washed three times with PBS buffer. The cells were mounted onto a glass slide and imaged using a Nikon Ti-DH fluorescence microscope. Lysosomes were stained with Lysotracker-Red (Beyotime Biotechnology Inc. Nantong, China) according to protocols provided by the manufacturer. 2.8. In vitro analysis of cytotoxicity on HUVEC cells HUVEC cells were seeded at 5×104 cells/well in a 96-well plate and grown overnight in DMEM media containing 10% fetal bovine serum. Cells were incubated with different concentrations of BFM for 24 h at 37 °C under 5% CO2. 20 μL of MTT [3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] was added to each well and incubated for 4 h. Next the supernatants were discarded and 200 μL DMSO was
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added to each well. A microplate reader was used to measure the absorbance of each well at 492 nm. Percent viability was calculated using the equation, (Asample /Acontrol) ×100%, where Asample and Acontrol are the absorbance of a well containing BFM and only media (DMEM) as control, respectively. The experiments were performed in triplicate. 3. RESULTS AND DISCUSSION 3.1. Synthesis of fluorescent mannosylprotein probe The fluorescent protein scaffold BF was prepared by reacting the isothiocyanate group of FITC with the primary amines of BSA.43 The number of FITC labels on the protein was determined by MALDI-TOF-MS44-46 (Supporting Information). Mass analysis of BF and BSA revealed that, on average, one FITC was conjugated to one BSA molecule (Table 1). Coupling of mannosyl moiety to BF was carried out by the squaric acid diethyl esters method as described previously.47-50 Briefly, fully protected mannosyl azide, 1 was synthesized in good yield using trichloroacetimidate method.51,52 After deacylation with NaOCH3 in CH3OH and hydrogenation with Pd(OH)2/C under 1 atm hydrogen gas, the amine 2 was reacted with one of squaric acid diethyl esters in potassium phosphate buffer (pH 7.0) to give 3 (Scheme 1). The ester group of 3 was conjugated to the amines present on the side chains of lysine in borate buffer (pH 9.0) to obtain the fluorescent neomanosylprotein BFM. The difference in the molecular weights of BF and BFM indicated that an average number of 10 mannose residues were attached to a single BF molecule (Table 1).
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Scheme 1. Synthesis of fluorescent neomannosyl glycoprotein BFM. Reagents and conditions: (a): (i) CH3ONa/CH3OH; (ii) H2, Pd(OH)2/C; (b): squaric acid diethyl esters, pH 7.0, 68% over two steps; (c) BF, pH 9.0.
Table 1. The conjugation of FITC and mannosyl ligand with BSA. Loading
a
Entry
Molecular Weight (Da)a
FITC
Mannose
BSA
66574
0
0
BF
66948
1
0
BFM
70844
1
10
Molecular weight was determined by MALDI-TOF-MS
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3.2 Specific binding of Concanavalin A to BFM 3.2.1 SPR analysis With the fluorescent mannosylprotein BFM in hand, the specific binding of the BFM to Concanavalin A (ConA), a lectin that selectively binds αmannopyranosyl and α-glucopyranosyl residues,53 was investigated by SPR. This sensitive and label-free technique is widely accepted for in situ monitoring interactions between biomolecules. In this study, an angle-modulated SPR setup was developed42 to study the association and disassociation process of the BFMlectin interaction (Figure 1). After covalently immobilizing BFM onto the gold chip via Au-thiol coupling,48 ConA or the interfering lectin PNA, a lectin from Arachis hypogaea (Peanut), which specifically bind oligosaccharides bearing a terminal galactose moiety54 in PBS buffer was injected and the data was analyzed. A dose-dependent SPR sensorgram was obtained with increasing the concentration of ConA. However, when compared with the SPR curves of ConA, the binding of PNA with BFM is negligible despite increasing the concentration of PNA to 10 μM. These studies indicate that the glycoprobe BFM retains its lectin binding preference and therefore, can be used to investigated mannose mediated binding processes.
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Figure 1. Kinetic binding response of ConA and PNA to BFM immobilized on SPR sensor chip. 3.2.2 Microarray analysis Next, BFM was employed to label ConA on a small lectin microarray system.55 The microarray was fabricated on a NHS-activated glass slides (Nexterion Slide H) following the protocols provided by the manufacturer (Schott Technical Glass, Germany). ConA and PNA in printing buffer were printed on the slides using a microarray printer. After blocking with ethanolamine, the slide was incubated with BFM for 2 h. After washing, the fluorescence intensities were measured to obtain an image of microarray (Figure 2). As anticipated, the fluorescence of BFM was visible on all ConA spots. At a 1 mg/mL printing concentration, the relative fluorescence intensity on ConA was ~3 times higher than those on PNA. These
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data also showed that the BFM can be used for fluorescent labelling and identification of ConA as mannose receptor at the molecular level.
Figure 2. Fluorescence images and intensities of lectin microarray incubated with BFM. Data are mean of seven independent spots. 3.2.3 ConA labelling Since mannose retains its selective lectin recognized properties after fluorescent BSA conjugation, another direct ConA labelling assay was conducted using fluorescence spectroscopy (Scheme 2)56.
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Scheme 2. Graphical representation of agglutination of BFM by Con A or E. coli K12 in PBS. In this assay, BFM was first dissolved in PBS buffer and different protein solutions were added. The decrease in the fluorescence intensity of BFM solutions in the presence of BSA and PNA with high concentration up to 100 nM were not significant. In contrast, there was a clear decrease in fluorescence density when different concentrations of ConA were added, presumably due to the complex formed by multiple mannose moieties present in BFM with the mannose binding lectin (Figure 3). We further titrated ConA into BFM solution in PBS and the measured fluorescence intensities of the supernatants were inversely proportional to ConA concentrations. Moreover, when we
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increased the concentration to 100 nM, a florescent pellet of a ConA-BFM complex was observed under UV light.
Figure 3. Relative fluorescence intensities of BFM in the presence of different lectins. Inset: visualization of BFM in the absence and presence of ConA.
Next, competitive inhibition assays based on increasing the fluorescence density of ConA-BFM complex in the presence of different carbohydrates (Mannose, Man; Glucose, Glc; Lactose, Lac and Galactose, Gal) were developed to further study the interactions between ConA and our fluorescent mannosyl probe. BFM was incubated with Man, Glc, Lac and Gal as negative control. After the addition of one of the carbohydrates to the BFM-ConA complex and vigorous shaking, an increase in fluorescence was observed when mannose and glucose were present at 10 and 75 mM,
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respectively. This indicates that these two carbohydrates can compete for the mannose binding sites of ConA and release the fluorescent BFM leading to the inhibition of the luminescent aggregation.
The fluorescence spectra also showed that the binding
affinity between ConA and mannose is much higher than that with glucose. In contrast, no increasing fluorescence was measured when galactose and lactose were added even at 100 mM (Figure 4). These results are in good agreement with previous reports.53
Figure 4. Fluorescence spectra of BFM and BFM-ConA complex in the presence of different carbohydrates. 3.3. Escherichia coli K12 binding studies. Encouraged by the positive results of our fluorescent mannosyl probe using lectins, we further investigated the capability of BFM binding to mannose-specific type 1 pili in
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E. coli.57 Type 1 pili are composed of FimA, FimF, FimG, and FimH proteins, which is responsible for the mannose-specific adhesion.58 We chose E. coli K12 expresses wildtype
1
pili
for
our
studies.
For
negative
controls,
we
chose
influenza
A/Chicken/Beijing/AT609/2014 (H9N2) and Bacillus natto. These pathogens do not have mannose receptor on their cell surface. After incubation of the bacterial suspensions (106-108 cells/mL) with the BFM and centrifugation, a decrease in the fluorescence intensity was observed indicative of strong binding between BFM and E. coli K12. No fluorescent intensity change was measured when influenza virus or Bacillus natto was added. Similar fluorescence intensity increases (Scheme 2) was also observed when mannose was added to the mixture of BFM-E. coli K12 complex verifying a competitive interaction (Figure 5).
Figure 5. Fluorescence intensities of BFM used as probe to investigate the interactions with E. coli K12 (106−108 cells/mL), Influenza A/Chicken/Beijing/AT609/2014 (H9N2) and Bacillus natto.
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The interactions between E. coli K12 and BFM were also studied by fluorescence microscopy and scanning electron microscopy, SEM (Figure 6). Fluorescence microscopy images showed that E. coli K12 are homogeneously dispersed in PBS solution in absence of BFM. However, green fluorescent aggregates 59 of E. coli K12 were seen after incubation with BFM indicating the interactions between the terminal mannose residues present on BFM and type 1 pili located on the bacteria. The SEM results60,61 were similar to the images obtained from fluorescence microscopy52.
(a)
10 µm
(d)
(c)
(b)
50 µm
50 µm
(e) E. coli K12
(f)
aggregates
Figure 6. Fluorescence microscopy images of a) E. coli K12 in bright field b) E. coli K12 incubate with BFM in bright field c) E. coli K12 incubate with BFM under green excitation. SEM images of d) E. coli K12 before and e), f) after incubate with BFM.
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3.4. Lysosomes in MCF-7 cancer cell labelling Next, the utility of BFM labelling technique was investigated by applying BFM to MCF-7 cancer cell imaging. It has been shown that, mannose capped fluorescent silicon nanoparticles (Man-SiNPs) can accumulate in cancerous cells MCF-7.62 Encouraged by this report, we studied the targeting of MCF-7 using the BFM probe. Lysotracker-Red which can stain lysosomes and BF (with no mannose) were chosen as positive and negative controls, respectively. The selective uptake and intracellular accumulation of BFM in MCF-7 cells is shown in Figure 7a-d. As a control, MCF-7 cells with incubation of BF, which do not have mannosyl residues and Lysotracker-Red are shown in Figure 7e-h. The merged results indicated that the BFM is endocytosed by MCF-7 cells, in accordance with previous results using Man-SiNPs62. The lack of the green fluorescence accumulation in Figure 7e-h illustrates that some mannose receptors may be located on the surface of MCF-7, which could mediate the selective endocytosis. These results also demonstrated the feasibility of mannose modified conjugates as potential drug delivery agents. 3.5. Cytotoxicity of fluorescent neomannosylprotein probe Since one of the potential applications is targeted drug delivery, we wanted to demonstrate that the new construct is not cytotoxic. To this end, we examined cytotoxicity of BFM in Human Umbilical Vein Endothelial Cells (HUVEC) using tetrazolium dye (MTT) based assay63,64 (Figure 8). Camptothecin (CPT) a widely used
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anticancer agent65 was chosen as positive control. Cell viability studies showed that toxicity was not observed with BFM concentrations up to 1 μM. This indicates that the non-toxic neomannosyl glycoprotein could potentially be used for targeted drug delivery. (a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 7. Fluorescence labelling of MCF-7 cancerous cells with BFM and LysotrackerRed: (a) bright field; (b) green fluorescence from BFM inside the cells after 48 h
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incubation with green wavelength excitation; (c) red fluorescence from Lysotracker-Red inside the cells after 48 h incubation with red wavelength excitation; (d) merged image. Pictures were taken on live cells using a Nikon Ti-DH fluorescence microscope. Fluorescence labelling of MCF-7 cancerous cells with BF and Lysotracker-Red were applied as a control: (e) bright field; (f) after 48 h incubation with green wavelength excitation; (g) after 48 h incubation with red wavelength excitation; (h) merged image. Pictures were taken on live cells using an Olympus FV1000 confocal laser scanning microscope.
Figure 8. Cytotoxicity study of BFM using HUVEC cells.
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4. Conclusions An easy method to generate mannose tailored fluorescein labelled BSA was developed. MALDI-TOF spectra confirmed that the protein is modified with multiple copies of mannose. Probing carbohydrate–lectin interactions on SPR and microarray platforms demonstrated the feasibility of using this fluorescent neomannosyl glycoprotein as a potential bioimaging agent. ConA and bacteria labelling experiments further demonstrated the applications in bioanalysis, diagnostics and biosensor applications. Additionally, MCF-7 cancer cell targeting imaging showed potential tumor-targeting therapy applications. In further, fluorescent neoglycoproteins with different carbohydrates could be prepared using this general method and used to understand carbohydrate-protein interactions and functions. This study also indicates that this approach could be used in theranostic applications. Supporting Information.
1
H, 13C NMR and IR spectra of key intermediates, MALDI-TOF mass spectra of protein
conjugates associated with this article can be found at ACS. org.
Funding Sources The National Natural Science Foundation of China (No. 21402140), Youth Innovation Research Foundation of Tianjin University of Science and Technology (No. 2016LG08) and Shenzhen Peacock Plan (No. KQTD2016053114253158) supported this research.
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ACKNOWLEDGMENT
The authors appreciate personnel in charge of NMR, IR, HRMS and MALDI-TOF mass spectroscopy facilities in the Research Centre of Modern Analytical Technology at Tianjin University of Science and Technology and National Institute for Viral Disease Control and Prevention, China CDC for the influenza virus strains. We also thank Dr. Ning Liu and Dr. Jian-Song Cheng, Nankai University for kind use of the microarray printing facilities.
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