Supporting information

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1 Supporting information En route to practicality of the polymer grafting technology: Onestep interfacial modification with amphiphilic molecular brushes Nikolay Borodinov 1, Dmitry Gil 2, Mykhailo Savchak 1, Christopher E. Gross 3, Nataraja Sekhar Yadavalli 4, Ruilong Ma 5, Vladimir V. Tsukruk 5, Sergiy Minko 4, Alexey Vertegel 2 and Igor Luzinov 1 * 1 Department of Materials Science and Engineering, Clemson University, Clemson, South Carolina 29634, USA 2 Department of Bioengineering, Clemson University, Clemson, South Carolina 29634, USA 3 Department of Orthopaedics, Medical University of South Carolina, Charleston, SC 29425, USA 4 Nanostructured Materials Laboratory, University of Georgia, Athens, Georgia, USA 5 School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, 30332, USA *To whom correspondence should be addressed: luzinov@clemson.edu S-1

2 CONTENT S1. Calculation of the reactivity ratios and determination of copolymer composition by NMR. S2. Synthesis of GMA/OEGMA/LMA terpolymers. S3. Parameters of the brush-like copolymers. S4. FTIR of P(GMA-OEGMA) and P(GMA-LMA). S5. Measurement of the degree of polymerization and molecular weight. S6. Thermal properties of the copolymers. S7. Estimation of surface energy of the grafted copolymer films. S8. TGA data for graphene oxide modification. S9. AFM of graphene oxide monolayer. S10. MTT assay results. S11. Sequence length distribution for P(G34-O66) and P(G26-L74) copolymers. S12. FTIR analysis of epoxy groups consumption during copolymer grafting. S13. References S-2

3 S1. Calculation of the reactivity ratios and determination of copolymer composition by NMR Parameters X and Y are defined as follows: 1 r X 1 12 Y, (1) 1 1 r21x where Y d[ M d[ M 1 2 ] ] and [ M 1] X [ M ] 2 r k k k, 12, k21 r (2) Constants kij reflect the rate constant for the reaction of monomer j with a growing polymer chain terminated with monomer i, [M1] and d[mi] are molar concentrations of monomer i in the feed and in the polymer respectively. In the case of unknown reactivity ratios, the X and Y values have to be experimentally identified for a series of copolymerization with varying monomer feed ratios. In order to calculate r12 and r21 from this dataset the parameters G and H are defined as follows: G X ( Y 1) X H Y, Y 2 (3) Fineman-Ross method can then be used to find the reactivity ratios using the equation (4): G Hr 12 r 21 (4) Alternatively, inverted Finemann-Ross method (5) can be utilized: G H r 12 1 r H 21 (5) Yet another method of reactivity ratios identification is Kelen-Tüdos method (6): r r / ) /, where (6) ( r21 G H H, H, H H min max (7) Here Hmin and Hmax are minimum and maximum H values for a series of copolymerizations. By linear fitting of the experimental data in the corresponding coordinates (H-G for Finemann- S-3

4 Ross, 1/H-G/H for inverted Fineman-Ross, η-ξ for Kelen-Tüdos) the r12 and r21 values can be calculated. a 3 OEGMA/GMA LMA/GMA LMA/OEGMA b 1 OEGMA/GMA LMA/GMA LMA/OEGMA c OEGMA/GMA LMA/GMA LMA/OEGMA G value 2 1 G/H value 0-1 value d H value r GMA r OEGMA -3 e /H value r GMA -1.0 f e value r LMA r OEGMA Reactivity ratio Reactivity ratio r LMA Reactivity ratio FR ifr KT FR ifr KT FR ifr KT Figure S1. Finemann-Ross (a), inverted Finemann-Ross (b) and Kelen-Tüdos (c) plots reflecting copolymerization of binary systems. The resulting reactivity ratios are presented for OEGMA/GMA (d), LMA/GMA (e) and LMA/OEGMA (f) monomeric pairs. To determine the composition of the molecular brushes we have used NMR-based proton counting. We used three peaks associated with the epoxy ring of the GMA (3.2, 2.9, 2.6 ppm), a group of peaks assigned to CH2 groups of PEG in OEGMA structure ( ppm) and the peak associated with CH2 groups of LMA (1.3 ppm). We have averaged the areas of three peaks corresponding to GMA as each peak corresponds to one proton in its chemical structure. The integrated area of PEG methylene groups was divided by four and then by 21 as one OEGMA units contains 21 units with 4 equivalent protons. The area of the peak at 1.3 ppm was divided by 2 and then by 9 as LMA has 9 equivalent units with 2 protons each contributing to this peak. The resulting numbers were used to calculate molar ratio of GMA, OEGMA and LMA in the molecular brushes (An area of the peak at k ppm, fm the molar ratio of the monomer M in the copolymer). S-4

5 A3.2 A2.9 A GMA, A A LMA, OEGMA (8) f GMA GMA, GMA LMA OEGMA f LMA LMA, GMA LMA OEGMA f OEGMA OEGMA (9) GMA LMA OEGMA In this study, we have identified three pair of reactivity ratios for each of the GMA-OEGMA, LMA-OEGMA and OEGMA-LMA binary systems. For GMA-OEGMA and GMA-LMA systems four copolymers were prepared with 0.2, 0.4, 0.6 and 0.8 GMA molar fraction in the feed. For OEGMA-LMA system LMA molar fraction was ranged from 0.1 to 0.3. The resulting Finemann- Ross, inverted Finemann-Ross and Kelen-Tüdos plots are displayed on Figure S1. S-5

6 S2. Synthesis of GMA/OEGMA/LMA terpolymers Table S1. The composition of the terpolymers prepared in this work. Composition Feed Calculated Experimental (measured by NMR) P(G 15-O 66-L 19) GMA OEGMA LMA P(G 28-O 56-L 16) GMA OEGMA LMA S-6

7 S3. Parameters of the brush-like copolymers To determine parameters for the brush-like polymers we followed the definitions and phase diagrams outlined in the recently published manuscripts. 2-3 The parameters for the copolymers are listed in Table S2. The brush-like macromolecules can be divided in four major classes: (a) loosely grafted combs (LC), (b) densely grafted combs (DC), (c) loosely grafted molecular (bottle) brushes (LM), and (d) densely grafted molecular (bottle) brushes (DM). To differentiate between the classes the following parameters are employed: 2-3 Degree of polymerization of side chains: N SC Degree of polymerization of the backbone spacer: N G Dimensionless parameter: S I = /(bl) 3/2 (10) Dimensionless parameter: S II = /bl 2 (11) where b is the Kuhn length, is the monomeric volume, l is monomeric length. Monomeric length is approximated as l = n d Sin(/2), where n is number of bonds which belong to one monomer in the chain, d is the bond length, and is the angle between the bonds. 4 The Kuhn length is approximated as b = dc, where C, is characteristic ratio of macromolecule. 5 In our estimations we considered that the copolymers are dissolved in theta solvent. Therefore, the root-mean-square size of polymer (Gaussian) chain is: 3 <R 2 > 1/2 = (bln) 1/2 (12) Physical volume of the chain is: 3 <R 2 > 3/2 = (bln) 3/2 = N and, therefore = N/(blN) 3/2 (13) According to phase diagram: 2 For LB: N G > N SC For DB: N G > S I (N SC ) 1/2 and N G < N SC For LM: N G < S I (N SC ) 1/2 and N G > S II For LM: N G < S II In our calculations: d = 0.154nm and = o (as for carbon-carbon bond). The backbone of the graft copolymers, made of the methacrylic monomers, has parameters of methyl methacrylate (MMA): C = 8.2 5, molecular weight of monomeric unit Mo=100 g/mol, n=2, l = nm, b=1.27 nm. The side chain of OEGMA monomer (NSC = 20) has parameters of polyethylene glycol: C = 4.1 5, molecular weight of monomeric unit Mo=44 g/mol, n=3, l = 0.38 nm, b=0.63nm. The side chain of LMA monomer (NSC = 6) has parameters of polyethylene: C = 6.8 5, molecular weight of monomeric unit Mo=28 g/mol, n=2, l = nm, b=1.05nm. Kuhn length and monomer length for the copolymers used in calculations were averaged proportionally to the ratio between NG and NSC. Degree of polymerization of the backbone spacer (for vinyl, MMA monomeric unit) was estimated from geometrical considerations as: NG = 1/(mole fraction of OEGMA or LMA). S-7

8 Table S2. Molecular parameters for the copolymers synthesized in this work. Copolymer/parameter NG NSC b, nm l, nm, nm 3 SI (NSC) 1/2 SII Copolymer type average average average P(GMA-OEGMA) P(G85-O15) LM P(G73-O27) DM P(G61-O39) DM P(G34-O66) DM P(GMA-LMA) P(G83-L17) DC/LM P(G65-L35) DM P(G46-L54) DM P(G26-L74) DM P(GMA-OEGMA-LMA) Only OEGMA units were considered in calculations P(G15-O66-L19) DM P(G28-O56-L16) DM S-8

9 S4. FTIR of P(GMA-OEGMA) and P(GMA-LMA). a b OEGMA fraction Absorbance [a.u.] LMA fraction Absorbance [a.u.] Wavenumber [cm -1 ] Wavenumber [cm -1 ] Figure S2. Fourier-transform infrared spectra of P(GMA-LMA) (a) and P(GMA-OEGMA) (b) molecular brushes. Table S3. The areas of the key peaks in FTIR spectra. GMA/LMA GMA/OEGMA LMA content C-H stretch (~3000 cm -1 ) Epoxy (900 cm -1 ) Ester (1730 cm -1 ) OEGMA content C-H stretch (~3000 cm -1 ) Ester (1730 cm -1 ) Ether (1100 cm -1 ) S-9

10 S5. Measurement of the degree of polymerization and molecular weight A detailed investigation of the degrees of polymerization is essential for the usage of poly(gma- -OEGMA-LMA) copolymers in practical applications. Even though expansion factor for polystyrene and poly(gma-ran-oegma) in methyl ethyl ketone may by different, the calibration can be used to estimate the range of poly(gma-ran-oegma) molecular weight by dynamic light scattering (DLS). It yields the hydrodynamic diameter, which is proportional to the square root of the number of the monomeric units in the chain. (Nn). 6 Figure S3a displays the degrees of polymerization of the copolymers in three binary systems calculated using this relationship. It is evident that products of LMA/GMA and OEGMA/GMA polymerizations have about monomeric units while decreasing of GMA content leads to increase of degrees of polymerization up to LMA/OEGMA system is generally characterized by higher values reaching 7000 which decreases when more LMA is presented in the copolymer. These trends are stemming from the steric hindrance of the termination step during the macromonomer radical copolymerization. 7 Using the NMR data regarding copolymer composition, degrees of polymerization can be converted to molecular weights (Figure S3b). The resulting values can be finely tuned during the reaction using chain transfer agents (CTAs) that can effectively decrease the obtained degrees of polymerization of the product. Carbon tetrabromide was found to be an efficient CTA that decreased the molecular weight of poly(gma-ran-oegma) with 0.66 OEGMA fraction (poly(g34-o66) copolymer) by a factor of 10 while the composition remained the same. S-10

11 Degree of polymerization a high OEGMA high GMA high LMA Monomer fraction GMA/OEGMA GMA/LMA OEGMA/LMA high LMA high OEGMA Molecular weight [Da] b high OEGMA high GMA high LMA Monomer fraction GMA/OEGMA GMA/LMA OEGMA/LMA high LMA high OEGMA Figure S3. Degrees of polymerization (a) and molecular weights (b) of P(GMA-OEGMA), P(GMA-LMA) and P(LMA-OEGMA) copolymers. Lines are only guide for eyes. S-11

12 S6. Thermal properties of the copolymers Temperature [ o C] (a) T g LMA transition LMA molar fraction Temperature [ o C] 80 (b) T g T m OEGMA molar fraction Figure S4. Temperature transitions observed in P(GMA-LMA) (a) and P(GMA-OEGMA) (b) copolymers. Lines are only guide for eyes. S-12

13 S7. Estimation of surface energy of polymer films. The surface energy of was calculated according to Owens-Wendt method (equation 13). 8 γ l1 1 + cosθ 1 = 2 (γ s d γ l1 d + 2 (γ s p γ l1 p γ l2 1 + cosθ 2 = 2 (γ s d γ l2 d + 2 (γ s p γ l2 p γ s = γ s d + γ s p (14) where γs and γl are the surface tensions of the solid and liquid, respectively. The subscripts d and p correspond to dispersion and polar components of the surface tension, respectively. Surface free energy (γs) and its polar (γ p s ) and dispersion (γ d s ) components of the surfaces were determined using two sets of contact angle measurements of water and hexadecane. The γ p d l and γ l components of liquids shown in Table S3 were used in the calculations. Table S4. γ l p and γ l d components of CA probe liquids. 9 γ l d γ l p γ l hexadecane water S-13

14 S8. TGA data for graphene oxide modification Weight [%] P(G 34 -O 66 ) P(G 15 -O 66 -L 19 ) Pristine GO (a) Temperature [ C] Weight [%] h 2.5 h 1 h 20 min GO/P(G 34 -O 66 ) Weight [%] h 2.5 h 1 h 20 min GO/P(G 15 -O 66 -L 19 ) 0.2 (b) Temperature [ C] 0.2 (c) Temperature [ C] Figure S7. Thermogravimetric analysis of: (a) P(G34-O66), P(G15-O66-L19) polymers and pristine GO; (b) GO grafted with P(G34-O66) as a function of grafting time; (c) GO grafted with P(G15-O66- L19) as a function of grafting time. S-14

15 S9. AFM of modified graphene oxide monolayer. (a) 10x10 μm 2 AFM cross-sections, h indicates thickness of GO sheet h = (0.9 ± 0.2) 30x30 (b) 10x10 μm 2 h = (2.3 ± 0.2) 30x30 h = (2.8 ± 0.2) (c) 7x7 μm 2 30x30 Figure S8. AFM images and cross-sectional analysis showing (a) pristine GO (vertical scale-15 and 5 nm for 30x30 and 10x10, respectively) layer and GO sheets modified with (b) P(G34-O66) and (c) P(G15-O66-L19) molecular brushes (vertical scale-10 and 5 nm for 30x30 and 10x10/7x7, respectively). S-15

16 S10. MTT assay results. The standard MTT assay was conducted in order to evaluate the effect of the PGMA/POEGMA (P(G34-O66)) coatings on the osteoblast proliferation rate. The protocol for this experiment was adapted from the literature. 10 Osteoblasts were passaged after reaching 80% confluency, and aliquots containing ~40,000 cells were transferred into a sterile 24-well plate. Samples were incubated in the presence of cells for 2, 4 and 7 days at 37 C in 5% CO2. Following the incubation, the samples were removed from the wells, and osteoblasts were exposed to the 5 mg/ml MTT reagent for 4 hours. Then, DMSO was added to completely dissolve formazan crystals, and the optical density of the resulting solution was measured at 570 nm using a microplate reader (Bio- Tek Synergy HT, Winooski, VT). The results (Figure S9) were compared with plain tissue-grade polystyrene. Absorbance at 570 nm reflects cell count and as evident from the analysis, the difference between the control and studied samples was not found to be statistically different (p-value>0.05, for n=6 samples). Hence, multiple evidences suggest that polymer coatings are non-cytotoxic, which confirms the findings observed with LIVE/DEAD assay. PGMA P(G34-O66) PS control Figure S9. The results of MTT assay for PGMA and P(G34-O66). S-16

17 S11. Sequence length distribution for P(G34-O66) and P(G26-L74) copolymers Sequence length distribution for the copolymer can be found if the reactivity ratios are known. The probabilities or mole fractions (N1)x and (N2)x of forming M1 and M2 sequences of the length x is given as: 1 ( N ) ( p ) p ( x1) 1 x ( N ) ( p ) p p p p p ( x1) 2 x r1 r [ M ] / [ M ] [ M 2] r [ M ] [ M ] [ M1] 1 r [ M ] [ M ] r1 r [ M ] [ M ] (15) where [M1] and [M2] are molar concentrations of respective monomers and r1 and r2 are their reactivity ratios. For OEGMA/GMA copolymerization to obtain P(G34-O66), [MGMA] = 0.2, and [MOEGMA] = 0.8, r1 = 1.4, r2 = 0.3. As evident from the Figure S10, the resulting product does not have a tendency to have long blocks GMA OEGMA Fraction Sequence length, units Figure S10. Sequence length distribution for GMA/OEGMA copolymerization. S-17

18 For LMA/GMA copolymerization to obtain P(G26-L74), [MGMA] = 0.2, and [MLMA] = 0.8, r1 = 1.2, r2 = 0.7. As evident from the Figure S11, the resulting product does not have a tendency to have long blocks as well GMA LMA Fraction Sequence length, units Figure S11. Sequence length distribution for GMA/LMA copolymerization. S-18

19 S12. FTIR analysis of epoxy groups consumption during copolymer grafting P(G66-O34) and P(G15-O66-L19) copolymers were deposited on Si wafers, annealed at 80 C for 1.5 hours and rinsed in MEK as described in the experimental section. FTIR transmission spectra were acquired to estimate consumption of epoxy groups during the grafting. As evident from Figure S12 the change of the epoxy peak (900 cm -1 ) area is on the level of 10%. The result indicates that the grafting/cross-linking of the anchored layer involves only small fraction of the epoxy groups. Epoxy peak area, a.u Polymer layer prior grafting \ P(G 66 -O 34 ) \ P(G 15 -O 66 -L 19 ) Grafted polymer layer Figure S12. Epoxy peak (900 cm -1 ) areas for the 50 nm copolymer films before and after the grafting. S-19

20 S 13: References 1. Odian, G. G. Principles of Polymerization, Wiley-Interscience: Hoboken, N.J., Daniel, W. F. M.; Burdynska, J.; Vatankhah-Varnoosfaderani, M.; Matyjaszewski, K.; Paturej, J.; Rubinstein, M.; Dobrynin, A. V.; Sheiko, S. S. Solvent-free, Supersoft and Superelastic Bottlebrush Melts and Networks. Nat. Mater. 2016, 15, Paturej, J.; Sheiko, S. S.; Panyukov, S.; Rubinstein, M. Molecular Structure of Bottlebrush Polymers in Melts. Sci. Adv. 2016, 2, e Rubinstein, M.; Colby, R. H. Polymer Physics, Oxford University Press: Oxford 2003; p Sperling, L. H. Introduction to Physical Polymer Science, Chapter 5, Fourth ed; Wiley- Interscience Hoboken, New Jersey, 2006; p Sperling, L. H. Introduction to Physical Polymer Science, Chapter 3, Fourth ed; Wiley- Interscience Hoboken, New Jersey, 2006; p Ito, K.; Tanaka, K.; Tanaka, H.; Imai, G.; Kawaguchi, S.; Itsuno, S. Poly(ethylene oxide) Macromonomers. 7. Micellar Polymerization in Water. Macromolecules 1991, 24, Owens, D. K.; Wendt, R. C. Estimation of the Surface Free Energy of Polymers. J. Appl. Polym. Sci. 1969, 13, Janczuk, B.; Wojcik, W.; Zdziennicka, A.; Bruque, J. M. Components of the Surface Free Energy of Low Rank Coals in the Presence of N-Alkanes. Powder Technol. 1996, 86, Wu, F.; Meng, G.; He, J.; Wu, Y.; Wu, F.; Gu, Z. Antibiotic-Loaded Chitosan Hydrogel with Superior Dual Functions: Antibacterial Efficacy and Osteoblastic Cell Responses. ACS Appl. Mater. Interfaces 2014, 6, S-20