Mechanically Stiff Nanocomposite Hydrogels at
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1 Supporting Information Mechanically Stiff Nanocomposite Hydrogels at Ultralow Nanoparticle Content Manish K. Jaiswal #, Janet R. Xavier #, James K. Carrow #, Prachi Desai #, Daniel Alge #,$, and Akhilesh K. Gaharwar #,$,, * # Department of Biomedical Engineering, Texas A&M University, College Station, TX (USA) $ Department of Materials Science and Engineering, Texas A&M University, College Station, TX (USA) Center for Remote Health Technologies and Systems, Texas A&M University, College Station, TX 77843, USA *Corresponding author: gaharwar@tamu.edu (AKG) 1
2 Figure S1: Fe 3 O 4 nanocrystals synthesis in two steps. Step 1 explains the precipitation of ferric oleate complex due to sodium oleate and ferric chloride at 70 C which when heated in octadecane bp 325 C) (step 2) at a controlled rate of 3.3 C/min, will form spherical nanocrystals of Fe 3 O 4. The size of these nanocrystals can be tuned by reaction time. Figure S2: Ligand exchange by nitro-dopamine- PEG using DCC-NHS chemistry in organic media. 2
3 Stress sweep of nanocomposite: G', G" (Pa) G', G'' (Pa) Figure S3: Rheological analysis of nanocomposites by stress sweep showing storage modulus, G (symbol O) and loss modulus, G (symbol ) measured at 1 Hz. The addition of nanoparticles enhances both the storage modulus as well as crossover (intersection of G and G indicating network breakdown) due to the formation of stiffer network. The clear shift in crossover can be seen using red dashed line for control. For 12 nm the crossover did not happen in the measurement range indicating a very strong network formation. Frequency sweep of nanocomposite: Figure S4: Representative data of rheological analysis of nanocomposites by frequency sweep analysis showing storage modulus, G (symbol O) and loss modulus, G (symbol ) measured at 1 Pa. The addition of nanoparticles (12 nm) enhanced G from approx Pa (control) to about 2600 Pa (MNPs) due to stiff network formation. 3
4 Figure S5: (a) The semicrystalline property of Gel was utilized to understand the MNPs crosslinking with its layered polymeric configuration using X-ray diffraction pattern of gelatin and nanocomposite. A forward angle shift in the characteristic gelatin peaks in the nanocomposites due to MNPs crosslinking which otherwise indicate the decrement in interchains spacing, hence confirming the strong chemical crosslinking of MNPs to the gelatin polymer chains. (b) Presence of Fe 3 O 4 in prepolymer matrix was confirmed by electron diffraction pattern as well. The indexed diffraction rings indicated magnetite as the major phase of the sample. Figure S6: Physical characterization of nanocomposites (a) Swelling degree and (b) degradation profile of nanocomposites. No significant change in hydration degree or degradation profile of the nanocomposites was observed. In contrast with 96% of pristine GelMA, nanocomposites were about 94% hydrated, a marginal decline by about 2% only. 4
5 10 Modulus (kpa) ns Figure S7: The effect of addition of MNPs to PEG diacrylate (Mw~10kDa). (ns, no significant difference) Concentration of MNP(µg/mL) 3-D cellular spreading of MC 3T3: Figure S8: 3-Dimensional cell encapsulation of preosteoblast (MC 3T3 cells) in nanocomposites. (a) Shows the cell spreading after 24 h of encapsulation in the gel nanocomposites. (b). The cell further proliferated and remained fairly viable at 72 h inside the gel matrix. The presence of nanoparticles helped cell spreading as shown in fig. 5 (c). (b) Shows the cellular perimeter and circularity parameters calculated for presoteoblast at 72 h using image J software. There was significant increase in perimeter with larger size, 12 nm due to enhanced stiffness of the matrix. Due to spreading the circularity parameters significantly reduced in the nanocomposites in 72 h of observation. 5
6 3-D cellular viability & spreading of hmsc on nanocomposite hydrogels: 3 h 24 h 72 h 100 Cell spreading after 72 hours Viability (%) h 24 h 72 h Figure S9: 3-dimensional cell encapsulation of human mesenchymal stem cell (hmsc) within nanocomposites (Gel/MNPs 12 nm) show high cell viability. After 72 hours, significant cell spreading was observed. Green cells represent live cells and red cells represent dead cells. Table S1: XPS references N 1s O 1s C 1s Peak assignment -NH + 3, primary amine, amide Nitro Carbonyl, anhydride, carboxylate Nitro C=O, C-C, C-N, O-C=O, C=C, amide, C-OH References [1], [2], [3] [4] [5] [6] [7], [8] 6
7 REFERENCES: 1. Shieh, D.-B.; Cheng, F.-Y.; Su, C.-H.; Yeh, C.-S.; Wu, M.-T.; Wu, Y.-N.; Tsai, C.-Y.; Wu, C.-L.; Chen, D.-H.; Chou, C.-H. Aqueous Dispersions of Magnetite Nanoparticles with + NH 3 Surfaces for Magnetic Manipulations of Biomolecules and MRI Contrast Agents. Biomaterials 2005, 26, Zhu, Y.; Gao, C.; Shen, J. Surface Modification of Polycaprolactone with Poly(methacrylic acid) and Gelatin Covalent Immobilization for Promoting its Cytocompatibility. Biomaterials 2002, 23, Cheng, F.-Y.; Su, C.-H.; Yang, Y.-S.; Yeh, C.-S.; Tsai, C.-Y.; Wu, C.-L.; Wu, M.-T.; Shieh, D.-B. Characterization of Aqueous Dispersions of Fe 3 O 4 Nanoparticles and Their Biomedical Applications. Biomaterials 2005, 26, Schmitz, P. J.; Baird, R. J. NO and NO 2 Adsorption on Barium Oxide: Model Study of the Trapping Stage of NO x Conversion via Lean NO x Traps. J. Phys. Chem. B 2002, 106, Zhou, J.-H.; Sui, Z.-J.; Zhu, J.; Li, P.; Chen, D.; Dai, Y.-C.; Yuan, W.-K. Characterization of Surface Oxygen Complexes on Carbon Nanofibers by TPD, XPS and FT-IR. Carbon 2007, 45, Sarma, D. D.; Sreedhar, K.; Ganguly, P.; Rao, C. N. R. Photoemission Study of YBa 2 Cu 3 O 7 Through the Superconducting Transition: Evidence for Oxygen Dimerization. Phys. Rev. B 1987, 36, Ruiz-Hitzky, E.; Darder, M.; Fernandes, F. M.; Zatile, E.; Palomares, F. J.; Aranda, P. Supported Graphene from Natural Resources: Easy Preparation and Applications. Adv. Mater. 2011, 23, Xu, L. Q.; Yang, W. J.; Neoh, K.-G.; Kang, E.-T.; Fu, G. D. Dopamine-Induced Reduction and Functionalization of Graphene Oxide Nanosheets. Macromolecules 2010, 43,
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