Supporting Information. Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging

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1 Supporting Information 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, Bornova, Izmir, Turkey. Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstraße 31, Regensburg, Germany. Sorbonne Universités, Université de Technologie de Compiègne, CNRS Enzyme and Cell Engineering Laboratory, Rue Roger Couttolenc, CS 60319, Compiègne Cedex, France. Central Research Testing and Analysis Laboratory Research and Application Center, Ege University, Bornova, Izmir, Turkey. * karsten.haupt@utc.fr (K. Haupt), joachim.wegener@ur.de (J. Wegener) S- 1

2 Synthesis of 4-acrylamidophenyl)(amino)methaniminium acetate (AB) The functional monomer 4-acrylamidophenyl(amino)methaniminium acetate (AB) was synthesized as previously reported. 1 Briefly, 4-acrylamidophenyl(amino)methaniminium chloride was first synthesized. For this, 34 g (0.25 mol) of sodium acetate trihydrate was dissolved in 200 ml of water and 2 g (9.6 mmole) of 4-aminobenzamidine dihydrochloride was added. The solution was cooled to < 5 C in an ice bath and 4 ml (49 mmol) of acryloyl chloride was added dropwise. The reaction was left to proceed for 1 h. The ph was then adjusted to 4.0 with hydrochloric acid (37%) and precipitation was observed. After filtration, the precipitate was redissolved in 100 ml of water at 40 C. Hydrochloric acid was again added this time to ph 1.0 and the product was left overnight to crystallize at 4 o C. The crystals were collected by filtration and dried in an oven maintained at 50 C. The yield of 4- acrylamidophenyl)(amino)methaniminium chloride was 60%. 1 H NMR (400 MHz, DMSO-d 6 ): (s, 1H), 8.99 (s, 4H), 7.84 (d, 2H), 7.81 (d, 2H), 6.48 (d, 1H), 6.31 (dd, 1H), 5.82 (s, 1H). 4-arylamidophenyl)(amino)methaniminium chloride was then converted to 4- acrylamidophenyl)(amino)methaniminium acetate as the acetate ion is more readily exchangeable with the template s carboxylate. Therefore 1.0 g of 4-acrylamidophenyl)(amino)methaniminium chloride was suspended in 100 ml of saturated sodium acetate solution and stirred overnight. The product was collected by filtration, washed with water to eliminate residual sodium acetate and dried at 50 C. The yield of 4-acrylamidophenyl)(amino)methaniminium acetate, which we term AB in the text, was 60 %. 1 H NMR (400 MHz, DMSO-d 6 ):10.56 (broad s, 5H), 7.84 (d, 2H), 7.78 (d, 2H), 6.48 (dd, 1H), 6.31 (dd, 1H), 5.82 (dd, 1H), 1.70 (s, 3H). Fig. S1. Chemical structure of hyaluronan, repeated disaccharide units of D-glucuronic acid and N-acetyl- D-glucosamine. S- 2

3 Images of CDs Fig. S2. (a) CD suspension diluted to a concentration of 1 mg/ml with PBS buffer and (b) CD suspension under UV excitation in the dark (366 nm, 2 x 4 W). Fig. S3. Mechanism of photoinitiation by coumarin 6-triethylamine. 2 Fig. S4. Calibration curve of glucuronic acid in water. Quantification by the method of Dubois. Absorbance is read at 490 nm. S- 3

4 IR spectroscopy of CDs The spectrum exhibits characteristic peaks between cm -1 (-OH stretching vibration), 2930 and 2890 cm -1 (symmetric and asymmetric C-H stretching vibration), and at 1410 and 1340 cm -1 (C-H deformation vibrations). The peaks at 1660 and 1593 cm -1 are assigned to the amide I (C=O stretching vibration) and II (N-H bending vibration), while those at 1145 and 1012 cm -1 result from C-O stretching vibrations. Fig. S5. IR spectrum of lyophilized CDs with the wavenumbers of the most prominent peaks. XPS spectroscopy of CDs XPS revealed the presence of carbon (C 1s, 285 ev), oxygen (O 1s, 532 ev) and nitrogen (N 1s, 400 ev). The expanded C 1s peak contains signals at ev, ev and ev, which correspond to COOR, C-O/C-N and C-C/C-H moieties, respectively. The expanded N 1s peak indicates two different C- N binding types with signals at ev and ev. Fig. S6. (a) Overview spectrum with assignment of the oxygen, nitrogen and carbon peaks; (b-d) High resolution spectra of the carbon (b), oxygen (c) and nitrogen (d) peaks. Electron binding energy E expressed in ev. S- 4

5 Fig. S7. Emission spectrum (λ ex = 365 nm) of carbon dots (blue) showing an overlapping with the absorbance of coumarin 6 (green). Fig. S8. Polymerization of (A) control sample without the addition of CDs; (B) CD-MIPGlcA and (C) CD-NIP under UV irradiation, followed by further purification comprising washing and photobleaching steps. Fig. S9. Nanoparticle tracking analysis. Size distribution of (A) CD-MIPGlcA and (B) CD-NIP, in water. Quantum yield (QY) of CD-MIPGlcA The emission spectra of suspensions of CDs in water, with absorbance values below 0.1, were recorded and the values of the integrated fluorescence spectra were plotted against the corresponding absorbance. Quinine sulfate in 0.1 M H 2 SO 4 was chosen as reference standard (QY = 54%) [3] and the slope of the linear fit for the plot of the CDs was compared against that of quinine sulfate. Following polymerization, S- 5

6 CD-MIPGlcA particles' quantum yield in water was also calculated using the same method. The quantum yield was calculated using the following equation: Φ x = Φ ST (m x / m ST ) (η 2 x /η 2 ST ) where Φ is the quantum yield, m is the slope, η is the refractive index of the solvent, ST is the standard and X is the sample. The relative quantum yields are shown in Table S1. Table S1. Quantum yields of CDs and CD-MIPGlcA. Sample Quantum Yield (%) Undoped CDs 0.6 ± 0.5 N-doped CDs 25.1 ± 2 CD-MIPGlcA 0.97 ± 0.2 References (1) Nestora, S.; Merlier, F.; Beyazit, S.; Prost, E.; Duma, L.; Baril, B.; Greaves, A.; Haupt, K.; Tse Sum Bui, B. Plastic Antibodies for Cosmetics: Molecularly Imprinted Polymers Scavenge Precursors of Malodors. Angew. Chem. Int. Ed. 2016, 55, (2) Popielarz, R.; Vogt, O. Effect of Coinitiator Type on Initiation Efficiency of Two-component Photoinitiator Systems Based on Eosin. J. Polym. Sci. A: Polym. Chem. 2008, 46, (3) Eaton, D. F. Reference Materials for Fluorescence Measurements. Pure Appl. Chem. 1988, 60, S- 6