SUPPORTING INFORMATION Functionalization of Cellulose Nanocrystals with PEG-Metal- Chelating Block Copolymers via Controlled Conjugation in Aqueous Media Melinda Guo, 1 Sohyoung Her, 2 Rachel Keunen, 1 Shengmiao Zhang*, 1,3 Christine Allen*, 2, Mitchell A. Winnik* 1 1 Department of Chemistry, University of Toronto, 80 St George St, Toronto ON Canada M5S 3H6 2 Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St, Toronto, Ontario, M5S 3M2 3 Permanent address: School of Material Science and Engineering, East China University of Science and Technology, P.O. Box 289, 130 Meilong Road, Shanghai 200237, China Table of Contents ADDITIONAL RESULTS AND DISCUSSION... S2 SUPPORTING FIGURES... S3 TABLES... S23 REFERENCES... S24 S1
ADDITIONAL RESULTS AND DISCUSSION Calculation of the mean number of polymer molecules per CNC The grafting density of block copolymer to the CNC-4FB sample was calculated from the absorbance value after 20 h reaction from the data Figure 4, main text. Polymer grafting density (σ, polymer molecules / CNC), defined by the number of polymer chains per nanoparticle was calculated by dividing the total content of polymer (in moles) linked through a BAH bond (determined from the absorbance evolution (ΔA)) by the total amount of nanoparticles. σ = DA V 6.02 1023 e 354 l M CNC / p( d (2) 2 )2 L n r CNC where ε354nm is the molar extinction coefficient of the formed BAH bond (ε354nm = 29, 000 M - 1 cm -1 ), 1 l is the absorption path length (l = 1.0 cm), V is the total volume for the measurements (400 μl), MCNC is the total mass of CNC nanoparticles, d and Ln are the width and length of the nanoparticles, respectively and ρcnc is the density of CNC nanoparticles. S2
SUPPORTING FIGURES Figure S1. UV/Vis absorption spectra of FITC labeled CNCs for CNCs reacted with epichlorohydrin for a given time: (a) 1h, (b) 40 min, (c) 2h, (d) 4h, (e) 6h, (f) 20h and (g) an aqueous suspension of unmodified CNCs. Spectra (a) to (f) were taken in NaHCO3 buffer (ph 11), in which the concentration of CNC was the same as the unmodified CNC suspension (0.02 wt%). S3
Figure S2. a) UV/Vis absorption spectra of FITC in ammonium hydroxide (28%, ph 11) between concentrations of 0.0000094 wt% and 0.0003 wt% in presence of CNCs (0.02 wt%) and b) calibration curve of FITC. S4
Figure S3. 1 H NMR spectrum of MeO-PEG-NH2. The degree of polymerization (DP) was calculated by comparing the integration of signal at 3.40 ppm (methyl end group) to that at 3.71 ppm (methylene backbone of z), where DPn = 48. S5
Figure S4. 1 H NMR spectrum of mpeg-pblg18-nh2. The degree of polymerization was calculated by comparing the integration of signal at 3.23 ppm (end group OCH 3) to that at 5.03 ppm (backbone benzyl methylene), where DPn = 18. S6
Figure S5. 1 H NMR spectrum of mpeg-pblg25-nh2. The degree of polymerization was calculated by comparing the integration of signal at 3.26 ppm (end group OCH 3) to that at 5.05 ppm (backbone benzyl methylene), where DPn = 0.5/(0.06/3) = 25. S7
Figure S6. 1 H NMR spectrum of mpeg-pblg18-tboc. The functionality of tboc was calculated by comparing the integration of 1 H NMR signal at 1.52-1.31 ppm (tboc end group) to that at 5.03 ppm (backbone benzyl methylene), where % tboc functionality = 0.20/((0.5/18) 9) 100% = 80%. The degree of polymerization was determined by comparing the integration of peak at 3.23 ppm (end group OCH 3) to that at 5.03 ppm (backbone benzyl methylene), where DPn = 18. This value agrees with that calculated for mpeg-pblg18-nh2. S8
Figure S7. 1 H NMR spectrum of mpeg-pblg25-tboc. The functionality of tboc was calculated by comparing the integration of 1 H NMR signal at 1.48-1.28 ppm (t-boc end group) to that at 5.01 ppm (backbone benzyl methylene), where tboc functionality = 0.17/((0.5/25) 9) 100% = 94%. The degree of polymerization was calculated by comparing the integration of peak at 3.23 ppm (end group OCH 3) to that at 5.01 (backbone benzyl methylene), where DPn = 25. This value agrees with that calculated for mpeg-pblg25-nh2. S9
Figure S8. 1 H NMR spectrum of mpeg-pglu(eda)18-tboc. The functionality of surviving tboc was calculated by comparing the integration of peak at 1.44 ppm (tboc end group) to that at 4.34 ppm (backbone methine), where the surviving % tboc functionality = 0.40 / ((1/18) 9) 100% = 80%. The conversion of aminolysis was calculated by comparing the integration of the residual phenyl group (7.43 ppm) to that of the backbone methine (4.34 ppm), and the conversion of aminolysis = (1-0.14/5) 100% = 97%. S10
Figure S9. 1 H NMR spectrum of mpeg-pglu(eda)25-tboc. The surviving tboc functionality was calculated by comparing integration of peak at 1.43 ppm (tboc end group) to that at 4.33 ppm (backbone methine), where % tboc functionality = 0.26 / (1/25 9) = 73%. The conversion of aminolysis was calculated by comparing the integration of the residual phenyl group (7.33 ppm) to that of the backbone methine (4.33 ppm), giving a conversion of aminolysis of 97%. S11
Figure S10. 1 H NMR spectrum of mpeg-pglu(dtpa)18-tboc. The functionality of DTPA group was calculated by comparing the backbone methine signals to the ethylenediamine associated with DTPA. Each DTPA contains 18 protons from EDA, and the DTPA functionality = (20.76-4-(1/18) 3) / (22-4) 100% = 92%. The tboc functionality was calculated by comparing tboc signal to the backbone methine signal, giving a tboc funcitonality = 74%. S12
Figure S11. 1 H NMR spectrum of mpeg-pglu(dtpa)25-tboc. The functionality of DTPA group was calculated by comparing the integrals of backbone methine to the EDA associated with DTPA. Each DTPA contains 18 protons from EDA, and the functionality of DTPA group = (22.37-4-(1/25) 3) / (22-4) 100% = 100%. The tboc functionality was calculated by comparing the tboc signal (1.43 ppm) to the backbone methine signal (4.33ppm), giving a tboc funcitonality = 73%. S13
Figure S12. 1 H NMR spectrum of mpeg-pglu(dtpa)18-nh2. The disappearance of the peak at 1.44 ppm indicates that the tboc group was completely removed. S14
Figure S13. 1 H NMR spectrum of mpeg-pglu(dtpa)25-nh2. The disappearance of peak at 1.44 ppm indicated that the tboc group was completely removed. S15
Figure S14. 1 H NMR spectrum of mpeg-pglu(dtpa)18-hynic. S16
Figure S15. 1 H NMR spectrum of mpeg-pglu(dtpa)25-hynic. S17
Figure S16. Following the bis-aryl hydrazone bond formation with UV/Vis-spectroscopy at = 354 nm during the conjugation of 4FB-acid to (a) mpeg-pglu(dpta)18-hynic and (b) mpeg-pglu(dpta)25- HyNic. Experiments were carried out in sodium acetate buffer ph 5.0 at room temperature. [mpeg-p(glu- EDA-DPTA)18-HyNic] = 34 μm, [mpeg-p(glu-eda-dpta)25-hynic] = 19 μm and [4FB-acid] = 4.5 mm. The red curve represents a fit to a stretched-exponential function and the green curve represents a fit to a sum of two exponential terms. The stretched-exponential fitted parameters are: ΔA0 = 0.71, β = 0.49 and k = 0.30 min -1 for mpeg-pglu(dpta)18-hynic, and ΔA0 = 0.30, β = 0.43 and k = 0.25 min -1 for mpeg- PGlu(DPTA)25-HyNic. A best fit (R 2 = 0.99) was found for fitting to a sum of two exponential terms: ΔA = ΔA1 (1-exp(-k1t)) + ΔA2 (1-exp(-k2t)). For mpeg-pglu(dpta)18-hynic: ΔA1 = 0.24, k1 = 0.036 min -1 ; ΔA2 = 0.76, k2 = 0.48 min -1. For mpeg-pglu(dpta)25-hynic: ΔA1 = 0.29, k1 = 0.031 min -1 ; ΔA2 = 0.71, k2 = 0.52 min -1. S18
Figure S17. UV/Vis spectra and time-evolution of hydrazone absorbance in the reaction of mpeg- PGlu(DTPA)25-HyNic to A) and B)Rh 552 -CNC, C) and D) A 488 -CNC. E) represents the photograph of an aqueous suspension of Rh 552 -CNC- PGlu(DTPA)25-PEG and F) an aqueous suspension of (1) A 488 -CNC- PGlu(DTPA)25-PEG and (2) unlabeled CNCs. S19
Figure S18. Kinetics study of 4FB-HyNic conjugations between CNC-4FB and a) 2-hydrazinopyridine and b) mpeg-pglu(dtpa)18-hynic (top curve), and mpeg-pglu(dtpa)25-hynic (bottom curve). Experiments were performed in sodium acetate buffer ph 5.0 and the data were collected every 30 s. The concentration of CNC-4FB was 0.0625 wt% for these experiments. Solid lines are fits to a stretchedexponential decay model using the software Origin. S20
Figure S19. Cell viability of HEYA8 cells following 24 hours of treatment with (a) Rh 552 -CNC-4FB- HyNic-PGlu(DTPA)25-mPEG or (b) A 488 -CNC-4FB-HyNic-PGlu(DTPA)25-mPEG. Data represents mean ± SD (n = 3). Figure S20. Following UV/Vis absorbance with time for the quantification of 4FB groups on CNC-4FB. S21
Figure S21. Following UV/Vis absorbance with time at λ = 354 nm for the conjugation of mpeg- PGlu(DPTA)18-HyNic to CNC-4FB. Figure S22. Following UV/Vis absorbance with time at λ = 354 nm for the conjugation of mpeg- PGlu(DPTA)25-HyNic to CNC-4FB. S22
Figure S23 Linkage between CNCs and NHS-dye molecule. SUPPORTING TABLES Table S1. Reaction of 4-formylbenzoic acid with HyNic-diblock copolymers: concentrations used and fitted stretched-exponential parameters ΔA0, β, and k. All experiments were performed at room temperature. Sample Conc.(mM) ΔA0 β k (min -1 ) mpeg-pglu(dtpa)18-hynic 0.034 0.71 0.49 0.30 mpeg-pglu(dtpa)25-hynic 0.019 0.30 0.43 0.25 Table S2. Reactions of mpeg-pglu(dtpa)25-hynic with labeled CNC-4FB and unlabeled CNC-4FB: summarization of grafting densities and fitted stretched-exponential parameters ΔA0, β, and k. All experiments were performed at room temperature. Unlabeled CNC Rh 552 -CNC-4FB A 488 -CNC-4FB ΔA0 0.12 0.13 0.16 β 0.68 0.42 0.50 k (min -1 ) 4.0 10-3 5.1 10-2 2.9 10-2 μmol polymers / g CNCs 6.9 7.2 8.6 Polymer molecules / g CNCs 185 193 230 S23
REFERENCES (1) Grotzky, A.; Nauser, T.; Erdogan, H.; Schluter, A. D.; Walde, P. A Fluorescently Labeled Dendronized Polymer-Enzyme Conjugate Carrying Multiple Copies of Two Different Types of Active Enzymes. J. Am. Chem. Soc. 2012, 134, 11392 11395. S24