Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon

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Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging Bilal Demir, Michael Lemberger, Maria Panagiotopoulou, Paulina Medina-Rangel, Suna Timur, Thomas Hirsch, Bernadette Tse Sum Bui, Joachim Wegener, and Karsten Haupt ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16225 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Materials & Interfaces

Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging Bilal Demir†, Michael M. Lemberger‡, Maria Panagiotopoulou§, Paulina X. Medina Rangel§, Suna Timur†,∥, Thomas Hirsch‡, Bernadette Tse Sum Bui§, Joachim Wegener‡,* and Karsten Haupt§,* †

Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey. ‡

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany.

§

Sorbonne Universités, Université de Technologie de Compiègne, CNRS Enzyme and Cell

Engineering Laboratory, Rue Roger Couttolenc, CS 60319, 60203 Compiègne Cedex, France. ∥Central

Research Testing and Analysis Laboratory Research and Application Center, Ege University, 35100 Bornova, Izmir, Turkey.

*e-mail: [email protected] (K. Haupt), [email protected] (J. Wegener)

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ABSTRACT War against cancer constantly requires new affinity tools to selectively detect, localize and quantify biomarkers for diagnosis or prognosis. Herein, carbon nanodots (CDs), an emerging class of fluorescent nanomaterials, coupled with molecularly imprinted polymers (MIPs), are employed as a biocompatible optical imaging tool for probing cancer biomarkers. First, N-doped CDs were prepared by hydrothermal synthesis using starch as carbon source, and L-tryptophan as nitrogen atom provider to achieve a high quantum yield of (25.1 ± 2) %. The CDs have a typical size of ~3.2 nm and produce an intense fluorescence at 450 nm, upon excitation with UV light. A MIP shell for specific recognition of glucuronic acid (GlcA) was then synthesized around the CDs, using the emission of the CDs as an internal light source for photopolymerization. GlcA is a substructure (epitope) of hyaluronan, a biomarker for certain cancers. The biotargeting and bioimaging of hyaluronan on fixated human cervical cancer cells using CD core-MIP shell nanocomposites is demonstrated. Human keratinocytes were used as non-cancerous reference cells and indeed, less staining was observed by the CD-MIP.

KEYWORDS: Cancer cell imaging, hyaluronan, carbon dot, molecularly imprinted polymer, glucuronic acid, synthetic antibody


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INTRODUCTION Recent advances in cancer research and glycobiology have established that accumulation of hyaluronan (HA) is intimately associated with tumor progression in humans.1–3 HA is a linear long chain polymer, ubiquitous to the extracellular matrix and the most abundant glycosaminoglycan in mammalian tissue. In tumors, HA facilitates metastasis by allowing tumor cells to migrate through interaction with cell surface HA receptors such as CD44, RHAMM and ICAM-1. Thus, HA is considered to stimulate growth, survival and angiogenesis within primary tumors or their metastases. Its concentration is elevated for example in colon, breast, prostate, lung, bladder and cervix cancers, and may thus serve as a specific and prognostic biomarker for these illnesses.4 For this reason, advanced cell and tissue molecular imaging tools to detect, localize and quantify HA, are of prime interest. However, HA is non-immunogenic, and production of antibodies for its specific recognition is naturally difficult.5 In this context, molecularly imprinted polymers may be an alternative as synthetic biomimetic receptors to be used instead of antibodies.6-8 Molecular imprinting consists in copolymerizing monomers bearing suitable functional groups with cross-linkers in the presence of a template molecule (the target or a derivative). This generates cavities that are a negative image of the template in terms of size, shape and chemical functionality. The resulting molecularly imprinted polymer (MIP) can bind the target with an affinity and specificity similar to an antibody, and is therefore referred to as antibody mimic. However, in contrast to antibodies, their fabrication is reproducible, rapid and cost-effective, and avoids the use of laboratory animals. Moreover, they are physically, chemically and biochemically more stable. Recently, several groups have reported the targeting and bioimaging of cells via MIP nanoparticles. Most of these MIPs targeted sialic acid9-12 but other sugars like mannose and fucose13 were also addressed and these MIPs were capable of differentiating between normal and cancer cells, with the latter presenting significantly more of these monosaccharides on their surfaces. On our side, we lately synthesized MIPs templated with glucuronic acid (GlcA), a substructure


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(epitope) of hyaluronan (Figure S1), which were successfully applied for biolabeling and bioimaging of intracellular and extracellular hyaluronan14-16 on human skin tissues and cells. The MIPs were prepared with conventional fluorescent labels, namely, the dye rhodamine or semiconductor quantum dots (QDs). In this study carbon nanodots (CDs), an emerging class of carbon-based materials with remarkable photoluminescent properties,17-19 were examined as core particles for subsequent coating with a MIP layer. CDs are chemically-inert nanomaterials, show improved photostability compared to organic dyes, and, unlike quantum dots, show a stable luminescence without blinking. Besides this, their excitation band is broad due to a superposition of individual bands, making it possible to simultaneously excite multi-colored emission. Moreover, they can be prepared easily and economically, are highly water-dispersible and do not contain heavy metals like QDs, and are thus rather environmentally friendly and less toxic. These properties render carbon dots serious competitors to the traditional QDs as they can be applied in chemical sensing, live-cell and in vivo bioimaging and nanomedicine.20 However, till now they have not been much amalgamated with MIPs, and the fluorescent CD-MIPs described in the literature have mostly been applied for optical sensing.21-25 For additional information on the MIP-CD combination, see reference 26 and references therein.26 In this work, we describe for the first time their combination with MIPs for targeted bioimaging of cells. In this study core-shell CD-MIPs, templated with glucuronic acid were synthesized and applied for biotargeting and bioimaging hyaluronan in human cervix adenocarcinoma cells (HeLa) and human keratinocytes (HaCaT) as models for transformed or healthy cells, respectively. CDs were obtained by hydrothermal synthesis of starch and L-tryptophan and characterized by dynamic light scattering, elemental analysis, fluorescence spectroscopy, X-ray photoelectron spectroscopy and infrared spectoscopy.27 The MIP was synthesized directly as a thin shell on the CDs, using the CDs' luminescence as light source for localized photopolymerization, when excited with a UV lamp (Figure 1). The emitted light, being weak as compared to the excitation light, restricts


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photopolymerization to a volume very close to the CDs' surface. Thus, the CDs have two main functionalities: (i) as an internal light source to trigger photopolymerization and (ii) as a fluorescent probe for bioimaging of cells specifically targeted by the MIP shell. Confocal laser fluorescence microscopy images show that a higher density of MIP core-shell particles was present on HeLa cells than on HaCaT cells, indicating that these MIPs were highly effective for differentiating between tumor and healthy cells.

Figure 1. Internal light emitted from UV-excited carbon dots (CD) generates the photopolymerization by visible lighjt of a thin MIP shell around the CDs. Coumarin 6/triethylamine (TEA) is the photopolymerization initiator system. (4-acrylamidophenyl)(amino) methaniminium acetate (AB) and methacrylamide are functional monomers and ethylene glycol dimethacrylate (EGDMA) is the cross-linker.

EXPERIMENTAL SECTION Reagents and Materials. All chemicals and solvents were of analytical grade and purchased from Sigma-Aldrich (France or Germany) or from VWR International (France), unless otherwise stated. Anhydrous solvents were used for MIP synthesis. Glycine and paraformaldehyde (PFA) were from Applichem (France). Human adult low calcium high temperature (HaCaT) cells were obtained from Cell Lines Service (Eppelheim, Germany). Human cervix adenocarcinoma (HeLa) cells were obtained from ATCC (Virginia, U.S.A.). Glass cover slips, cell culture flasks, 12 and 96 well plates, penicillin/streptomycin, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and 0.25% trypsin/EDTA were from Thermo Scientific (Illkirch, France). Microscope slides for cell specimens were from Roth Sochiel E. U. R. L. (Lauterbourg, France). Water was purified using a Milli-Q system (Millipore, Molsheim, France). ACS Paragon Plus Environment

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Synthesis of N-doped carbon dots. 2 g starch and 350 mg L-tryptophan were suspended in 30 mL water and transferred to a stainless steel autoclave with PTFE liner (~50 mL inner volume). The temperature was increased to 160 °C within about 1 h using a muffle furnace (Nabotherm: LT 15/3) and maintained for 12 h and then cooled to room temperature. The brownish CD raw suspension was centrifuged 10 min at 2500g to remove larger particles, aggregates and other contaminants which precipitate from the suspension. The supernatant was filtered through a 0.22 µm syringe filter with polyether sulfone membrane, resulting in a clear red-brownish suspension. Afterwards, sizeexclusion chromatography using a Sephadex® G-25 fine columnwas applied to remove smaller molecular pyrolysis products using water as eluent. To monitor the progress of the elution, a UV lamp (366 nm, 2 x 4 W) was used to excite the fluorescent CDs in the column. The first fluorescent fraction was collected, lyophilized and used to determine the final CDs concentration gravimetrically after water evaporation. The dried CDs were resuspended in phosphate-buffered saline, pH 7.4 (PBS), and were passed through a 0.22 µm syringe filter to remove potential aggregates from the lyophilization process. These CDs in PBS (Figure S2) are our working stock suspensions for particle characterization and MIP preparation. The suspension was stored at 4 °C until further use. Characterization of CDs. The CDs were characterized using the following techniques:

- Fluorescence spectroscopy. The fluorescence properties of CDs were investigated on a FP-6300 spectrofluorimeter (Jasco Incorporations, U.S.A).

- Dynamic light scattering. The hydrodynamic size distribution of the CDs was analyzed by dynamic light scattering (DLS) on a Zetasizer NanoZS (Malvern Instruments Ltd., U.K.).

- Elemental analysis. Analysis of the elemental composition of dry CDs was carried out by the Central Analytical Services at the Faculty of Chemistry and Pharmacy of the University of Regensburg. 1 – 3 mg of the lyophilized particles were used to determine the mass fraction of carbon, hydrogen and nitrogen. The mass fraction of oxygen was calculated by subtraction.

- Infrared spectroscopy. An IR-Spectrometer 3000 (Varian, California, U.S.A.) was used. For the


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measurement, lyophilized CDs were applied onto the optical bridge.

- X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopy (XPS) measurements were performed on lyophilized CDs on an ESCALAB 250 from Thermo VG Scientific at nanoAnalytics GmbH in Münster (www.nanoanalytics.de), Germany. For the excitation in spectroscopic mode, monochromatic Al Kα X-ray was used (15 kV, 150 W, 500 µm spot size). Overview spectra were recorded with 80 eV pass energy and high-resolution spectra with 30 eV. Synthesis of CD-MIPGlcA. The functional monomer AB was synthesized as previously described (see details in Supporting Information).28 GlcA (0.022 mmol, 4.27 mg) and AB (0.022 mmol, 5.46 mg) were first incubated together for 1 h in 1 mL dimethyl sulfoxide (DMSO). The mixture was then transferred to a 4-mL glass vial containing methacrylamide (MAM) (0.066 mmol, 5.62 mg), ethylene glycol dimethacrylate (EGDMA) (0.423 mmol, 80 µL), triethylamine (TEA) (10 µL) from 72 mM stock solution in DMSO, coumarin 6 (7 µL) from 2.73 mM stock solution in DMSO and CDs (100 µL) from 5 mg/mL stock suspension in PBS. The same procedure was carried out for control CD-NIP by using hexanoic acid as template, instead of not using any template, to help the solubility of AB in DMSO. The vials were fitted with an airtight septum and the mixture was purged with nitrogen for 2 min. Polymerization was initiated by irradiation at 365 nm between two UV lamps (6W each, VILBER LOURMAT), placed at ~ 2 cm from the vials. After 3 h reaction at room temperature, the content was transferred to 2 mL polypropylene microcentrifuge tubes and the particles were sequentially washed 3 times one hour each with DMSO, CH3OH:acetic acid (9:1), 100 mM NH3 in water:CH3OH (7:3), water and CH3OH. The different washing steps allowed to remove the template and unreacted CDs. The CD-polymer nanocomposites were then irradiated with a fluorescent tube (18 W, MAZDAFLUOR, UK) overnight to photobleach the residual fluorescence coming from entrapped coumarin 6. Subsequently, the particles were dried under vacuum. Homogeneous suspensions of 10 mg/mL of CD-MIPGlcA and CD-NIP were prepared in water by sonication. These constituted our working stock suspensions and were stored at 8 °C until further use.


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As a further control experiment, a MIP pre-polymerization mixture without CDs (source of internal light for photopolymerization) was run in parallel under the same conditions as the MIP and the NIP, to verify that the polymerization was due to the internally emitted light of the CDs and not from the UV excitation light. The mechanism of photoinitiation by coumarin 6 and TEA is shown in Figure S3. Binding assay of CD-MIPGlcA. The recognition properties of CD-MIPGlcA particles for GlcA were evaluated using the method of Dubois.29 Dubois’s method is a versatile approach to quantify free sugars, glycosides, oligosaccharides and polysaccharides. This colorimetric method, also known as phenol-sulfuric acid method is based on the pre-conversion of sugars into furfural derivatives upon heating with strong acids, followed by the formation of a colored complex with phenol. The reaction mixture is composed of sample/standard solution of 50 µL sample solution containing GlcA , 50 µL phenol solution (99 % in water) and 150 µL of sulphuric acid (95 %), in a 96 well plate and incubated in an oil-bath for 20 min at 90 oC. After cooling to room temperature, the absorbance was read at 490 nm on an MRX microplate reader (DYNEX Technologies, U.S.A). Prior to the binding assay, a calibration curve was generated with standard solutions of GlcA (0.1 – 5 mM) in water (Figure S4). To evaluate the binding properties of CD-MIPGlcA, different concentrations of the composite polymers (0.1 – 2.5 mg/mL) in 2-mL polypropylene microcentrifuge tubes, were incubated with a fixed concentration of GlcA (1 mM) in water for 17 h at room temperature under agitation. Samples were centrifuged at 42000g for 1 h. Unbound GlcA was determined in the supernatant by the Dubois method and the amount of bound GlcA was calculated by subtracting the amount of unbound analyte from the total amount of the analyte added to the mixture. Physico-chemical characterization of CD-MIPGlcA. The size distribution of the particles was measured using nanoparticle tracking analysis on a NanoSight NS500 (Malvern Instruments Ltd., France). The measurement was performed recording videos of 10 seconds duration at 25 °C. The zeta potential of CD-MIPGlcA was recorded in a disposable capillary cell (DTS1061) using


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dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments Ltd., France) at 25 oC. The morphology of CD-MIPGlcA was examined by transmission electron microscopy (TEM) imaging. TEM images were captured using a JEM-2100F (JEOL, Japan). The TEM grid was a 300 mesh carbon-coated copper grid from AGAR Scientific (Stansted, U.K.). Fluorescence measurements of polymer coated CDs were performed on a FluoroLog-3 spectrofluorimeter (Horiba Jobin Yvon, Longjumeau, France). Cell Culture. HaCaT and HeLa cells were cultured in DMEM supplemented with 10% (v/v) FBS, 2 mM glutamin, 100 µg/mL penicillin/streptomycin, in 75 cm2 flasks at 37 °C, 5% CO2 and 100% humidity, until reaching 80% confluency. The cells were passaged using 0.25% (w/v) trypsin/EDTA in PBS buffer. Sample Preparation for Cell Imaging. After trypsinization, the cells were harvested and centrifuged for 3 min at 400g. Cells were resuspended in 10 mL culture medium and an aliquot was diluted in a 1:4 ratio with 0.4% (w/v) trypan blue solution in a microcentrifuge tube for subsequent cell counting on a disposable hemocytometer.30 Live cells that did not internalize trypan blue were scored as life cells. Cells were cultured in 12-well plates (well diameter 22.1 mm) equipped with round glass cover slips (diameter 12 mm). 105 cells suspended in 100 µL culture medium were pipetted onto each cover slip. After a 3 h incubation to allow for cell settling, 2 mL of medium was added to the cells. Subsequently, cells were allowed to grow to confluence for 48 h. To prepare the specimen, each cover slip of HaCaT and HeLa cells was washed 3 times with 2 mL PBS and fixed at room temperature for 10 min in 600 µL PFA (3 % w/v) in PBS. To terminate the fixation process and block non-specific binding, each cell sample was incubated three times with 1 mL 20 mM glycine in PBS for 20 min at room temperature and finally the samples were washed three times with 2 mL PBS. Afterwards, the cells were washed with 1 mL CH3OH:water (1:30) three times and then incubated with 1 mL of a freshly sonicated polymer suspension (100 µg/mL) or CDs only (100

µg/mL) in CH3OH:water solution (1:30) at 37 oC for 90 min.15-16 As a next step, each fixed cell layer was washed three times with 1 mL CH3OH:water (1:30). Staining of the cell nuclei was


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accomplished by adding 0.6 mL propidium iodide (PI) (1:3000 diluted solution from 1.5 mM stock in PBS) and incubating the cells for 10 min at room temperature, followed by washing 3 times with PBS buffer. Each cover slip was mounted for bioimaging on a microscope slide with 5 µL mounting medium consisting of 0.5 mL water, 0.5 mL 1 M Tris-HCl buffer pH 8 and 9 mL glycerol. Bioimaging was carried out using a confocal fluorescence microscope (Zeiss LSM 710, AxioObserver, Jena, Germany) equipped with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Lasers with emission lines at 405 nm and 488 nm were used for all images. Cytotoxicity of CD-MIPGlcA. Cell viability in presence of the particles was determined using the MTT assay.31 The MTT colorimetric assay assesses the metabolic activity of living cells by reading the concentration of NADH or NADPH as an indicator for the number of metabolically active, viable cells. Cellular dehydrogenases reduce the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to form insoluble formazan, which has a purple color. HaCaT and HeLa cells were grown to 80% confluency and trypsinized as described above. The cells were then suspended in cell culture medium such that 15,000 cells were seeded in each well of a 96-well plate in a volume of 200 µL. After 24 h, cells were incubated with CDs, CD-MIPGlcA and CD-NIP (25 - 500 µg/mL) for 24 h in cell culture medium. After adding the MTT solution (0.5 mg/mL in PBS:DMEM (1:9)) to the wells (200 µL per well) and incubation for 4 h, the MTT was removed and 200 µL of DMSO was added to each well to dissolve the formazan formed in the cells due to their metabolic activity. Following 10 min incubation, 25 µL of Sorenson's buffer (containing 100 mM glycine and NaCl, pH 10) was added to each well and the absorbance of each well was read with a spectrophotometric plate reader at 570 nm and 630 nm as the reference wavelength. Cell viability results were calculated by dividing the absorbance obtained for treated cells by that of the untreated controls.

RESULTS AND DISCUSSION


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Characterization of N-doped CDs. Fluorescent N-doped CDs were obtained by hydrothermal synthesis, using starch and L-tryptophan (as nitrogen source). Doping with nitrogen was used to achieve higher quantum yields for the particles’ photoluminescence.32-34 The quantum yield of the CDs was found to be (25.1 ± 2) % (n = 5) as compared to a reference standard of quinine sulfate in 0.1 M H2SO4. Without nitrogen doping (350 mg per 2 g of starch), the quantum yield of pure CDs from starch was only 0.6 ± 0.5 %. Several techniques were applied to characterize the CDs in terms of optical properties, size and chemical composition. General photoluminescence properties were studied by measuring the absorption, excitation and emission spectra of CDs in PBS buffer. As shown in Figure 2A, a concentration of 1 mg/mL of CDs exhibits the highest fluorescence intensity at an emission maximum of 452 nm and an excitation of 371 nm. The emission spectrum is broad, covering the whole visible spectrum, indicating that CDs, as used here, are a mixture of particles with slightly different photochemical properties or that individual particles host more than one emissive state or moieties. Therefore, the emission spectra of an aqueous suspension of CDs was recorded for different excitation wavelengths between 300 and 500 nm in 20 nm steps (Figure 2B-C). Irradiation with wavelengths between 300 and 400 nm produces a maximum emission at 450 nm, with the highest intensity being reached at an excitation wavelength of 360 nm. At higher excitation wavelengths, the emission maxima are increasingly red shifted but fluorescence intensity decreases.

Figure 2. (A) Absorption, excitation and emission spectra of CDs with Ex/Em 371/452 nm; (B) Native emission spectra of CDs, excitation wavelength was varied between 300 and 500 nm in 20 nm steps and (C) Emission spectra normalized to their respective maximum. CDs: 1 mg/mL in PBS.


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The hydrodynamic diameter of the CDs was measured by DLS analysis and found to be (3.2 ± 0.9) nm (Figure 3).

Figure 3. Size distribution of CDs in PBS (solid line), CD-MIPGlcA (dashed line) and CD-NIP (dotted line), in water, as analyzed by dynamic light scattering at 25 °C.

The chemical composition of the CDs was determined by elemental analysis which shows a C, O, H and N content (w/w) of 44.9 %, 47.9 %, 6.1 % and 1.1 %, respectively. Chemical moieties on the particles surface were identified by IR spectroscopy. The IR data shows hydroxyl, carbonyl, and in particular amide groups on the surface (Figure S5). In addition, XPS measurements were performed to retrieve more detailed information about the chemical binding of the elements within the CDs. The spectra are shown in Figure S6. In agreement with IR data, the XPS analysis confirms the presence of these surface functionalities: C-OH, C=O, C-N, C=N. The carbon-nitrogen bonds are considered to enable the photoluminescence as the electron donating groups of nitrogen (lone pair) influence the emitters’ band gap. Synthesis of core-shell CD-MIPGlcA. Reports of CDs associated with MIPs are still in their infancy, and have been mostly applied for optical sensing purposes. The synthetic methods to obtain these CD-MIP materials are mainly based on encapsulating CDs with molecularly imprinted silica via sol-gel polymerization, using silica-modified CDs as starting material.26 The main rationale advanced in these reports is that photoluminescence properties are maintained in silica as it is a transparent and inert material. CDs combined with organic MIPs have scarcely been reported and the strategy employed to graft MIPs on CDs, which we report in this study, is very versatile,


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because it allows coating CDs with a MIP shell or any other type of polymer shell to improve the CDs’ biocompatibility and stability, in a straightforward manner and in one single step, at room temperature. Thereby, the association of CDs with the polymer is solely by physical entrapment, thus no chemical modification of the CD surface or chemical coupling are necessary. MIPGlcA was synthesized as a thin shell around CDs by photopolymerization. The shell was grafted by using the fluorescent light from the CDs excited by in UV (365 nm). This was done using a suitable initiator system having an absorption wavelength that overlaps the emission of the CDs35 (coumarin 6/TEA) (Figure S7). Light emitted by CDs is weak compared to the excitation light and loses intensity from the emitter to the bulk phase due to absorption of the initiators, polymerization is confined to the CD surface, yielding core-shell nanoparticles. The MIP polymerization mixture in DMSO as porogen, was composed of GlcA, AB, MAM and EGDMA in a 1:1:3:20 ratio. This composition had been optimized in an earlier publication.14 AB contains a basic amidine moiety and this so-called stoichiometric monomer36 forms strong ionic interactions with the carboxylate group of glucuronic acid, with a high association constant (Ka(DMSO-d6)) of 7.1 x 103 M-1.15 It was essential to add MAM to form H-bonds with GlcA and to confer hydrophilicity to the MIP to prevent aggregation in the aqueous medium during cell imaging. Control polymer particles (CD-NIP) were prepared in the same way, using hexanoic acid as the imprinting template instead of GlcA. In fact, non-imprinted polymers (NIPs) usually show a different general morphology than MIPs, thus, this is not a good control. Therefore, we have synthesized a different MIP, imprinted with hexanoic acid, and used it as a NIP-like control. A further control composed of a polymerization MIP mixture without CDs was run in parallel to make sure that the polymerization was initiated by the CDs’ emission and not by the UV excitation light (Figure S8). Indeed, no polymer was observed in the latter vial. Finally, the resulting polymers were photobleached for 8 hours to eliminate entrapped residual coumarin 6 fluorescence so that it does not interfere with subsequent fluorescence measurements.


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Characterization of core-shell CD-MIPGlcA. Size determination. The hydrodynamic particle diameter of CD-MIPGlcA and CD-NIP was determined by nanosight tracking analysis (NTA) on Nanosight NS500. Compared to other techniques to estimate particle size, this method directly observes the thermal motion of individual particles, producing more reliable results in terms of particle size distribution and concentration.37,38 Figure S9A shows the size distribution of CDMIPGlcA; an intense peak at 48 nm with 3 smaller peaks at 123, 173 and 208 nm. Size distribution of CD-NIP (Figure S9B) shows that the biggest population has a diameter of 82 nm. The other particle populations have diameters of 163, 238 and 348 nm. For comparison, size measurements were also done using dynamic light scattering on a Zetasizer NanoZS. CD-MIPGlcA’s size was 184 ± 5 nm (n = 3, PDI: 0.184) and CD-NIP’s size was 219 ± 14 nm (n = 3, PDI: 0.204) (Figure 3). NTA gave smaller mean particle diameters as compared to DLS because DLS is biased towards large particles which scatter light more intensely than small particles.

Zeta potential measurements. The zeta potential of CD-MIPGlcA and CD-NIP was determined to be +(24 ± 2) mV and +(19 ± 2) mV (n = 3), respectively. The positive surface charge is due to the presence of the amidinium moiety of the AB monomer.

TEM imaging. The morphological structure of the core-shell particles was examined by TEM imaging. Even though the contrast in TEM is weak because of the carbon-based nature of the CDs, the size of bare CDs was estimated as ~ 3-10 nm (Figure 4A), similar to the size obtained by DLS analysis (Figure 3). Figure 4B-C (inset) shows that a thin polymer shell (7-10 nm) has been grafted on CD-MIPGlcA and CD-NIP. The data clearly shows that the core CDs in the nanocomposites are larger than the ones found in Figure 4A, indicating that CD particles have aggregated during polymerization, which took place in DMSO. However, the mean particle sizes of the final core-shell particles, (43 ± 22) nm for the MIP and (80 ± 14) nm for the NIP are similar to the sizes obtained by nanoparticle tracking analysis (Figure S9). In order to assess the colloidal stability of the CDMIPGlcA composite particles, we have characterized by DLS and NTA particles that had been kept


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in the refrigerator for several months, and we have not detected significant changes in particle size and size distribution, confirming the stability of these highly cross-linked polymer particles. Fluorescence measurements. The quantum yield of the CD-MIPGlcA composites was lower by a factor of ~25 compared to naked CDs, which is probably due to some quenching by the acrylamidebased polymer shell (see SI, Table S1). Figure 4B-C represent the fluorescence emission spectra of 500 µg/mL CD-MIPGlcA and CD-NIP in water, using excitation wavelengths of 365, 400, 420 and 445 nm. The spectra show that CDs were still highly fluorescent with the MIP and NIP coating, with a maximum emission at ~500 nm. These results together with the TEM image strongly indicate the presence of CDs within the polymers. Due to the optical characteristics of the CD-MIPGlcA composites, bioimaging studies were carried out using blue and green fluorescence. The fluorescence of the CDs was not affected by the 8-h photobleaching step used to eliminate residual fluorescence by entrapped coumarin 6 (see above), indicating an excellent optical stability.

Figure 4. (A) Representative TEM image of bare CDs; Emission spectra in water of 500 µg/mL (B) CD-MIPGlcA and (C) CD-NIP, with different excitation wavelengths in the range of 365 - 445 nm, slit 2.5 nm. The small peak around 420 nm at excitation 365 nm is due to the water Raman peak. Insets are corresponding TEM images.

Binding assay. The recognition properties of CD-MIPGlcA and CD-NIP were evaluated by equilibrium binding assays in water. Dubois’s method, which was used to quantify GlcA, allows to determine sugars colorimetrically. Figure 5 shows that CD-MIPGlcA bind specifically to GlcA as the binding to the hexanoic acid-imprinted control (NIP) was significantly lower. These results are similar to those obtained with quantum dot-MIP composites (QD-MIPGlcA), prepared by a similar strategy.15,16 The significant binding to the NIP control is due to the presence in these particles of amidinium groups from the functional monomer AB. Some of them may be accessible to GlcA, ACS Paragon Plus Environment

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although in general binding to the NIP does not represent the non-specific part of the binding to the MIP, since in the latter these groups are located in the imprinted cavities. Previous competitive binding experiments with glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and Nacetylneuraminic acid (sialic acid), other monosaccharides that are present on the cell surface, indicated that MIPGlcA was very selective towards GlcA as only < 1% cross-reactivity was observed.15 Therefore, we can confidently apply the CD-MIPGlcA for selective hyaluronan bioimaging.

Figure 5. Equilibrium binding isotherm of GlcA (1 mM) with CD-MIPGlcA and CD-NIP in water. Data are means of three independent experiments. The error bars represent standard deviations.

Application of CD-MIPGlcA for quantitative cell imaging. A standard fluorescence immunoimaging protocol14-16 was used with fluorescent MIPs instead of antibodies for cell imaging, to detect hyaluronic acid on keratinocytes and HeLa cells. This protocol was previously optimized for the use with MIPs.14,15 Cells were fixed with paraformaldehyde, which has a low background fluorescence. Blocking was then performed with glycine to stop the fixation and reduce nonspecific binding. Finally, cells were incubated with the MIP and the control NIP nanocomposites and bare CDs, for 90 min before imaging. The spatial distribution and localization of the particles was analyzed by confocal fluorescence microscopy. Color images of the CDs, emitting both blue and green fluorescence globally shows that they were present in higher quantities in samples incubated with CD-MIPGlcA as compared to CD-NIP or bare CD (Figure 6). Blue and green fluorescent particles on the cells were quantified after background subtraction by fluorescent-image ACS Paragon Plus Environment

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analysis using ImageJ software. Figure 7 shows that there were ~ 4 times more CD-MIPGlcA on HeLa cells than bound to HaCaT cells, indicating that our CD-MIPGlcA is capable of differentiating between cells with different hyaluronan content, such as healthy and tumor cells. This is clearly visible on a representative example of a zoom image of the particles on one single cell (Figure 7A-B).

Figure 6. Confocal microscope images of fixed HaCaT and HeLa cells treated with CDs, CD-NIP and CD-MIPGlcA. The three types of particles exhibit blue and green fluorescence under proper filter sets of confocal microscopy. Nuclei stained with PI (red). Scale bar: 50 µm.


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Figure 7. Confocal micrographs showing labeling of GlcA on a single (A) HeLa and (B) HaCaT cell, by CD-MIPGlcA (green) and nuclear staining with PI (red); (C, D) Phase-contrast images of HaCaT and HeLa cells, respectively; (E) Analysis of labeled cells with CD-MIPGlcA, CD-NIP and CD as obtained from ImageJ by measuring the normalized fluorescence of each single cell area from 5 different images. Error bars represent S.D, n (number of cells in analysis) = 20. *** shows the statistical analysis with a value of p < 0.001.

Cytotoxicity of CD-MIPGlcA. For potential applications of these particles in vivo, it was interesting to verify cell viability in their presence. Cell viability of HaCaT and HeLa cells in the presence of CDs, CD-MIPGlcA and CD-NIP was determined using an MTT assay after 24 h of particle exposure (Figure 8). CDs, at all concentrations tested (25-500 µg/mL), demonstrated no cytotoxicity when applied to HaCaT cells. These findings are in agreement with previous studies reporting no significant toxicity of CDs prepared from cabbage, up to 500 µg/mL, on HaCaT cells.39 In the case of HeLa cells, cell viability was not affected up to 250 µg/mL of CDs (95 ± 7%). Similar results were reported for N-doped CDs on HeLa cells after 24 h treatment using similar concentrations.40 On the other hand, both CD-MIPGlcA and CD-NIP showed a minor cytotoxic effect at concentrations above 100 µg/mL in both, HaCaT and HeLa cells. This is probably due to the positive charge on the MIP and NIP particles, which can interact with the negatively charged membrane and get easily internalized.


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Figure 8. Cell viability (MTT) assay of (A) HaCaT and (B) HeLa cells in the presence of CDs, CDMIPGlcA and CD-NIP, for 24 h. Data are means from 5 different experiments.

CONCLUSION Since they were discovered in 2004, much work has been done on fluorescent carbon dots for their potential as biocompatible, non-toxic and eco-friendly nanomaterials to compete with conventional semiconductor quantum dots. In this study, we have exploited their fluorescent emission as an internal light source for the photopolymerization of a thin MIP shell around them. Previous studies in our group have used a similar strategy to coat quantum dots and upconverting nanoparticles, but these particles have been reported to be rather cytotoxic. The possibility of offering safer alternatives, coupled with selective biotargeting capabilities due to the recognition cavities present on the MIP, renders these nanocomposites powerful tools for imaging of tumor biomarkers. This versatile strategy can be used to coat any type of polymer shell around the CD for protection, stability, biocompatibility or drug delivery, paving the way to new potential applications in theranostics.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI : Details of the synthesis of the monomer, 4-acrylamidophenyl (amino)methaniminium acetate (AB), chemical structure of hyaluronan, mechanism of photoinitiation by coumarin 6-triethylamine, calibration curve of glucuronic acid, FTIR and XPS spectra of CDs, size distribution by nanoparticle tracking analysis, quantum yield determination of CD-MIPGlcA, optical images of CD and CD-MIP suspensions (PDF).

ACKNOWLEDGMENTS The Scientific and Technological Research Council of Turkey (TUBITAK)-BIDEB 2214/A International Fellowship program (PhD scholarship of B.D.) is acknowledged. B.D and S.T also thank Ege University Scientific Research Project (Project Grant No: 2017 FEN 007) for partial funding. P.X.M.R. thanks the Mexican National Council for Science and Technology (CONACYT) and the Instituto para el Desarrollo de la Sociedad del Conocimiento del Estado de Aguascalientes (IDSCEA) for PhD scholarship. C. Boulnois is thanked for TEM and confocal laser fluorescence microscopy analysis. This work was supported by the European Regional Development Fund and the Region of Picardy (CPER2007–2013 and project Polysense). Funding by the European Commission (FP7 Marie Curie Actions: SAMOSS, PITN-2013-607590) is also acknowledged. M.M.L. was supported by the DFG Research Training Group “Electronic Properties of Carbon Based Nanostructures” (GRK 1570).

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Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon

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