Heparin Conjugate through Noncovalent

Jan 10, 2011 - Blood compatible graphene/heparin conjugate is simply formulated through noncovalent interaction between chemically reduced graphene an...
12 downloads 27 Views 3MB Size
Download PDF
336

Biomacromolecules 2011, 12, 336–341

Blood Compatible Graphene/Heparin Conjugate through Noncovalent Chemistry Da Young Lee,† Zehedina Khatun,‡ Ji-Hoon Lee,† Yong-kyu Lee,*,‡ and Insik In*,† Departments of Polymer Science and Engineering and Chemical and Biological Engineering, Chungju National University, Daehak-ro 72, Chungju, Chungbuk 380-702, Republic of Korea Received September 2, 2010; Revised Manuscript Received October 25, 2010

Blood compatible graphene/heparin conjugate is simply formulated through noncovalent interaction between chemically reduced graphene and heparin. Charge repulsion of negatively charged heparin on graphene plates renders hydrophobic graphene to be solublized in aqueous media without any precipitation or aggregation even after 6 months. Unfractioned heparin (UFH) with higher molecular weight was effective for graphene solubilization while low molecular weight heparin (LMWH) was not. Noncovalently interacting heparin chains on graphene plates preserve their anticoagulant activity after conjugation with graphene. Graphene/UFH conjugate shows much enhanced anti factor Xa (FXa) activity of 29.6 IU/mL compared with pristine graphene oxide (GO; 1.03 IU/mL).

Introduction Graphene has received increasing interests due to its fascinating electrical, thermal, and mechanical properties in various application.1-7 While most research concentrates on the electronic application of graphene, a recent biomedical application of graphene oxide (GO) or graphene prompts us to study its “compatibility” in biological medium.8-11 Blood compatibility is one of important and critical issues when nanomaterials such as nanoparticles and carbon nanotubes are utilized in various circumstances in vivo or in vitro.12 Heparin is one of glycosaminoglycans that naturally cover the surface of all eukaryotic cells and is reported that heparin acts as anticoagulant, preventing the formation of clot and extension of existing clots within the blood.13 A specific pentasaccharide sequence within the heparin chains binds and activates antithrombin III (AT III). The activated AT III then inactivates thrombin and other serine proteases involved in blood clotting, most notably factor Xa (FXa). The rate of inactivation of these proteases by AT III can increase by up to 1000-fold, owing to the binding of heparin.14 Consequently, it is reasonable to mention that heparinization of nanomaterials can improve blood compatibility of nanomaterials. Actually, heparinization of multiwalled carbon nanotube (MWNT) has been reported to dramatically improve blood compatibility of pristine MWNT.15 Therefore, effective conjugation of graphene with heparin seems to be critical for the preparation of graphene-based nanomaterials or nanodevices with high blood compatibility. To accomplish this goal, two considerations must be clarified. The first is the solubilization of hydrophobic graphene in aqueous biological media. Strong dispersion force between graphene plates makes graphene completely insoluble in aqueous media while it shows limited solubility in organic solvents such as NMP in concentration of less than 10 µg/mL upon sonication.16 Therefore, intense research efforts have been concentrated on developing either covalent or noncovalent chemistry to render enough solubility to reduced graphene in aqueous media. In covalent chemistry, water-soluble polymer-grafted graphene (Figure 1a) or nega* To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. † Departments of Polymer Science and Engineering. ‡ Chemical and Biological Engineering.

Figure 1. Schematic illustration of the preparation methods for soluble graphene from GO through covalent chemistry (a) polymer-grafting and (b) introduction of charge, or through noncovalent chemistry, (c) conjugate with polyelectrolyte, and (d) conjugate with nonpolyelectrolyte.

tively charged graphene (Figure 1b) has been reported to be effective for graphene solubilization in water.17-26 In noncovalent chemistry, various π-rich water-soluble polyelectrolytes such as poly(4-styrene sulfonate) (PSS) or sulfonated polyethynylphenylene (PPE-SO3-) successfully produced stable graphene dispersion via π-π interaction with graphene and subsequent charge repulsion between polyelectrolyte/graphene conjugates (Figure 1c).27-29 Heparin has the highest negative charge density due to the presence of sulfonate groups (-SO3-) among any known biological molecules and relatively hydrophobic cellulose backbones. Recent our studies are revealing that various watersoluble polymers (electrolyte or not) can create efficient conjugation with graphene through hydrophobic interaction rather than π-π interaction, enabling to solubilize their graphene conjugates in aqueous media (Figure 1c,d).30,31 Similarly, hydrophobic interaction between graphene plates and heparin backbones might be also likely while conjugation through π-π interaction is not accessible. The second is developing conjugation method between heparin chains and graphene plates. Heparin is difficult to conjugate on substrates or substances

10.1021/bm101031a  2011 American Chemical Society Published on Web 01/10/2011

Blood Compatible Graphene/Heparin Conjugate

Biomacromolecules, Vol. 12, No. 2, 2011

337

because various organic reactions can be applied to heparin in limited cases.32 Partial esterification of heparin with substrates or substances having hydroxy groups might accomplish heparinization. But optimization of reaction condition is necessary for this covalent heparinization not to lose activity of pentasaccharide sequence within the heparin chains. In both considerations, heparinization of graphene through noncovalent chemistry rather than covalent chemistry seems to be more promising. In this report, direct heparinization of chemically reduced graphene is demonstrated via hydrophobic interaction between chemically reduced graphene plates and heparin chains. Charge repulsion of interacting heparin chains on graphene plates can endow enough solubility and stability for prepared graphene/ heparin conjugates in aqueous biological media. This graphene/ heparin conjugate can not lose its blood compatibility, which is one of the essential features for biomedical application of graphene-based nanomaterials and nanodevices.

Materials and Methods Materials and Instruments. Both unfractioned heparin (UFH, MW: 12000-15000 Da) and low molecular weight heparin (LMWH) were obtained from Dongying Tiandong Biochemical Industry Co. Ltd. (China). The origin of heparin was from porcine intestinal mucosa. All the other chemicals were purchased from Sigma-Aldrich Corporation and used without further purification. UV-vis spectra were obtained from UV-vis spectrometer of Hewlett-Packard. Photoluminescence (PL) spectra were obtained from L550B luminescence spectrometer of Perkin-Elmer with temperature controller. FT-IR spectra were obtained from IR100/IR200 spectrometer of Thermoelectron Corporation. Raman analysis was done from LabRAM high resolution UV/vis/NIR dispersive Raman microscope of Horiba John Yvon. Dynamic light scattering (DLS) data were obtained from particle size analyzer (ELS-Z) of Otsuka Electronics Corporation with temperature controller. FE-SEM images were obtained from JSM-6700 field emission scanning electron microscope of GELO Corporation. AFM images were obtained from XE-100 atomic force microscope of PSIA. Synthesis of Graphene Oxide (GO). GO was prepared from natural graphite by a modified Hummers method.28,33 In the first step, preoxidized graphite powder was synthesized through reaction of natural graphite (3 g), sulfuric acid (12 mL), K2S2O8 (2.5 g), and P2O5 (2.5 g). This preoxidized graphite powder (2 g) was further oxidized by sulfuric acid (120 mL), KMnO4 (15 g), and deionized water (250 mL). After dilution with deionized water (700 mL), decomposition of excess KMnO4 by H2O2 (30 wt %, 20 mL), and complete remove of metal ions by HCl (10 wt %, 1 L), the resulting GO solution was filtered and washed with deionized water several times. The resulting GO powder was shortly dried in air and then transferred into 1 L of water to make bright brown GO solution. The GO solution was dialyzed with deionized water for 1 week. Any insoluble precipitation was removed by centrifuge at 3000 rpm for 5 min. Finally, brown fibrous GO powder was obtained after freeze-drying of centrifuged GO solution under reduced pressure. For experiments, fresh GO solution (1 mg of GO in 10 mL of water) was prepared just before use with another sonication for 30 min. Graphene/Heparin Conjugate. A total of 10 mg of UFH or LMWH in 10 mL of deionized water was simply mixed with aqueous GO solution (1 mg in 10 mL of deionized water). To produce graphene/ UFH solution, hydrazine monohydrate (4 drops) was added into the above GO/UFH solution and then the reaction mixture was maintained at 80 °C for 24 h. The GO/UFH solution with an initially bright brown color darkens in the progress of reduction to graphene/UFH solution. Prepared graphene/UFH solution was stable more than 6 months without any precipitation. Part of the graphene/UFH solution was further dialyzed using MWCO membrane (1000 kDa). But reduction of GO/ LMWH solution in similar conditions produced insoluble precipitation after reaction.

Figure 2. (a) Photo images of aqueous GO solution (middle) and chemically reduced graphene solutions in the presence of UFH (left) or LMWH (right).

Anti Factor Xa Activity Assay. Anti FXa activity of the graphene/ UFH conjugate was determined by FXa chromogenic assay with the use of both GO and UFH as control samples. Solution of graphene/ UFH conjugate (25 µL) was mixed with 200 µL of antithrombin III (AT III) solution (0.1 IU/mL), where AT III concentration was in excess to conjugate concentration. This solution was incubated at 37 °C for 2 min, and 200 µL of FXa (4 nkcat/mL) was added. The resulting solution was then incubated for an additional 1 min. The concentration of FXa was also in excess to conjugate concentration. FXa substrate (200 µL, 0.8 µmol/mL) was then added and incubated at 37 °C for 5 min. The reaction was terminated by adding 200 µL of acetic acid (50% solution). The bioactivity in samples was calculated from the absorbance at 405 nm. To obtain the exact anti factor Xa activity of the graphene/heparin conjugate, the absorbance of graphene in the graphene/heparin conjugate at 405 nm was reduced completely.

Results and Discussion Graphene/Heparin Conjugate. To produce graphene/heparin conjugate, freshly as-prepared GO solution (1 mg in 10 mL of water) was mixed with an aqueous solution of UFH or LMWH (10 mg in 10 mL of water). A small amount of hydrazine monohydrate (30 wt %) was dropped into this mixture and then the reaction temperature was adjusted at 80 °C. After reduction for 24 h, the initially brown-colored GO/UFH solution changes to a dark black-colored graphene/UFH solution (Figure 2a, left). In contrast, reduction of GO/LMWH solution produced insoluble black precipitation, which was not soluble again in water even in the presence of UFH (Figure 2a, right). UV-vis spectrum of graphene/UFH solution showed a remarkable decrease in transmittance compared with the initial GO/UFH solution (Figure 2b). This decrease of transmittance originates from the recovery of sp2 carbons of chemically reduced graphene after reduction by hydrazine. In addition, sp2 conjugation is dramatically enhanced after reduction. The initial photoluminescence of GO/heparin was completely disappeared after reduction (Figure 3a). This is because initially incomplete sp2 conjugation of GO is almost fully conjugated after reduction and the prepared chemically reduced graphene becomes conducting. Raman analysis of graphene/HPC conjugate also confirms the formation of reduced graphene after reduction (Figure 3b). While pristine

338

Biomacromolecules, Vol. 12, No. 2, 2011

Lee et al.

Figure 4. DLS spectra of (a) GO/UFH and (b) graphene/UFH solutions (1:100). Figure 3. (a) PL spectra of GO/UFH and graphene/UFH solutions with λext of 260 nm and (b) Raman spectra of GO powder, graphene/ UFH conjugate (1:100), and natural graphite powder with a He-Ne laser with a wavelength of 660 nm.

graphite shows only G peak at 1585 cm-1, graphene/heparin conjugate shows both G and D peaks at 1588 cm-1 and 1331 cm-1, respectively. GO also shows both G and D peaks at 1592 cm-1 and 1338 cm-1 but the intensity ratio, I(D)/I(G) is much larger in graphene/heparin compared with GO. The presence of both G and D peaks and increased I(D)/I(G) of graphene/ heparin conjugate supports the formation of chemically reduced graphene in conjugate.34 The as-prepared graphene/UFH solution is optically clear and stable in aqueous media for more than six months. All these results support that conjugation between hydrophobic graphene plates and UFH is quite effective for producing stable dispersion of chemically reduced graphene in aqueous media. Hydrophobic interaction between hydrophobic graphene plates and heparin backbones might contribute for effective graphene/heparin conjugation. Hydrophobic interaction strongly depends on both electron density and geometry between molecules. Use of LMWH instead UFH can decrease this hydrophobic interaction because electron density of LMWH is much smaller than UFH. Formation of graphite-like precipitation after reduction supports that conjugation between graphene and LMWH is not effective. Once graphene/heparin conjugate forms in the case of UFH, graphene/heparin conjugate strongly repels each other due to the presence of plentiful negative charges in the pendent sulfonate groups of heparin. The high stability of prepared graphene/UFH solution comes from these highly charged characteristics of heparin. Graphene/heparin conjugation resembles that of graphene/PSS or graphene/PPE-SO3-.27,29 While charge repulsion contributes to the stability of graphene dispersion in both systems (Figure 1c), the interacting parameter between graphene and polymer backbone is different. Graphene/ heparin conjugates use hydrophobic interaction rather than π-π interaction of graphene/PSS or graphene/PPE-SO3- systems for

conjugation. It is quite interesting to examine the structural rearrangement of graphene/heparin conjugate in solution. DLS can provide indirect information on the solution structure of graphene/heparin conjugate. GO/UFH solution showed particle diameter distribution centered on 415 nm (Figure 4a) and graphene/UFH solution showed shifted distribution centered on 440 nm (Figure 4b). Because the interaction between GO and heparin is expected to be much weaker than the hydrophobic interaction between graphene and heparin, an increase in the particle size in DLS analysis in the graphene/heparin conjugate might originate from the presence of interacting heparin layers on the graphene plate. Any aggregation of graphene plates will show particle site distribution in bigger particle sizes but both GO and graphene solutions showed a similar distribution profile in similar dimensions. Aggregation of chemically reduced graphene is believed to be minimized in the presence of interacting heparin chains. Next, prepared graphene/UFH solution was purified by dialysis using a MWCO membrane (1000000 Da). Any free heparin chains in aqueous media (not interacting on graphene surface) can be removed by dialysis from a graphene/heparin conjugate because the size of the graphene/heparin conjugate is much bigger than the pore size of MWCO, while the molecular size of heparin (MW 12000-15000 Da) is much smaller. Interestingly, partial precipitation of reduced graphene was observed after dialysis. DLS analysis shows the formation of much bigger aggregates (typically bigger than 10 µm) after dialysis. Probably, the graphene/heparin conjugate is in an equilibrium state with free heparin in solution. Removal of free heparin by dialysis shifts this equilibrium to the dissociation of the graphene/heparin conjugate and finally graphene/graphene aggregation starts to be formed. Similar structural disruption of graphene/heparin conjugate was also observed in the process of vacuum filtration of graphene/UFH solution using anodized aluminum oxide (AAO) membrane (0.2 µm pore). After vacuum filtration and extensive washing with water, prepared films were not soluble

Blood Compatible Graphene/Heparin Conjugate

Biomacromolecules, Vol. 12, No. 2, 2011

339

Figure 6. Anti FXa activity of the graphene/UFH conjugate (1:100), GO, and UFH.

Figure 5. (a) FT-IR spectra of filtered graphene film, natural graphite, and GO and (b) FE-SEM images of filtered graphene film (left, top view; right, side view).

again in water. Even the addition of UFH did not solubilize these films, and therefore, it is regarded that most of the interacting heparin chains on graphene plates were removed during the filtration process. FT-IR analysis of prepared films showed no presence of heparin chains after filtration. Instead, the FT-IR spectrum of the prepared film was mostly matching with chemically reduced graphene, which was formed by the reduction of GO without UFH. Carbonyl stretching (around 1730 cm-1) of GO is almost disappearing in the filtered film, while aromatic carbon double bond stretching (around 1620 cm-1) is preserved in both spectra (Figure 5a). Therefore, it is concluded that the filtered film is mainly chemically reduced graphene. FE-SEM images of this film show planar surface structure with some wrinkles in the top view and typical layered structures in the side view (Figure 5b).10 Monitoring of individual graphene plates is not available here because dense and layered film structures are formed due to 2D planar structures of graphene plates. The sheet resistance of graphene film is dependent on the film thickness (typically, 105∼106 ohm/square). This high sheet resistance value of as-prepared chemically reduced graphene film can be dramatically decreased by subsequent thermal treatment.10 Thermal annealing at 400 °C allowed graphene film to show a sheet resistance of 104∼105 ohm/square. Blood Compatibility of Graphene/Heparin Conjugate. Highly heparinated structural features of graphene/UFH conjugate prompts us to test its blood compatibility. FXa plays a critical role in blood coagulation. Regulation of thrombin generation is the primary physiologic function of FXa. FXa is, therefore, an attractive and potentially specific target for new anticoagulant agents. Therefore, anti FXa activity of the graphene/UFH conjugate was analyzed by using an anti FXa chromogenic assay. For comparison, anti FXa activity of UFH or GO itself (instead of graphene) was also measured in the same experimental conditions because nonconjugated graphene itself is not soluble in water. The concentration of GO or graphene was adjusted to 50 µg/mL in both GO and graphene/ UFH solutions and concentration of UFH is adjusted to 500 µg/mL in both graphene/UFH and UFH solutions. The observed anti FXa activity from the assay was summarized in Figure 6. While GO shows anti FXa activity of 1.03 ( 0.04 IU/mL, graphene/UFH conjugate shows enhanced anti FXa activity of

29.6 IU/mL ( 0.03. In general, chondroitin or nonanticoagulant heparin does not show any anti factor Xa because of the relation between anticoagulant activity and Factor Xa activity. This almost 30-fold increase of bioactivity after heparinization demonstrates that conjugation between graphene/UFH preserves inherent high anti FXa activity of UFH (85.6 ( 0.04 IU/mL). The reason for the decrease of heparin activity after conjugation with graphene is unclear. It is regarded that binding with FXa requires only a pentasaccharide sequence of heparin. Fondaparinux (MW: 1727 Da) is synthetic pentasaccharide for FXa inhibition.35 From the fact that the molecular weight of UFH is between 12000 and 15000 Da, UFH is estimated to have 35-43 saccharide units along the heparin chain. Because the conjugation between graphene and heparin is based on totally noncovalent chemistry, it is uncertain whether some part of the pentasaccharide sequence of UFH is interacting on the graphene plate or not. For comparison, LMWH failed to produce soluble graphene conjugate. LMWH is estimated to have less than 23 saccharide units. Therefore, it is regarded that significant parts (probably more than 23 saccharide units) of UFH chains might interact on graphene plates for solubilization of chemically reduced graphene. In this case, some of the above pentasaccharide of UFH can lose its activity for binding with FXa and an observed decrease of anti FXa activity of graphene/UFH conjugate compared with pure UFH is reasonable. To elucidate the detailed structural features of graphene/UFH conjugate, AFM images of graphene/UFH conjugate were monitored. Highly diluted graphene/UFH assembly or GO solution was dropped on silicon wafer and the samples were completely dried for AFM analysis. While the AFM image of GO shows typical singlelayered plates (height of ∼1 nm) with varying shapes and sizes (Figure 7a), the AFM image of the graphene/UFH conjugate shows plates with heights between 1 and 1.3 nm (Figure 7b). The most significant difference is the shape of plates of graphene/UFH conjugate. The plates of graphene/UFH conjugate have rounded plate edges, while plates of GO have sharp edges. In addition, there are some fiber-like connected structures among conjugate plates. All these differences in the graphene/UFH conjugate compared with GO might result from the presence of interacting UFH chains on graphene plates and further aggregation of UFH chains (free or interacting on graphene plate) between neighboring graphene plates on drying. Actually, the fact that even dialyzed graphene/UFH solution showed a similar AFM image excludes the possibility of the aggregation of free UFH chains. Probably, graphene/UFH conjugates rearrange their structures on sample preparation and finally aggregate each other. But we can not completely disregard the possibility that some of the interacting UFH chains are detached from the conjugate structure and then these free UFH chains

340

Biomacromolecules, Vol. 12, No. 2, 2011

Lee et al.

References and Notes

Figure 7. AFM topographic images of (a) GO and (b) graphene/UFH conjugate (1:100).

induce fiber-like aggregation. But, it is definitely clear that UFH chains are interacting on chemically reduced graphene and retain significantly their anti FXa activity even after formation of the graphene/UFH conjugate.

Conclusions The blood compatible graphene/heparin conjugate is simply formulated through noncovalent interaction between chemically reduced graphene and heparin. The chain length of heparin is critical for the successful conjugation and subsequent solubilization of chemically reduced graphene in water. Conjugation with UFH dramatically enhances the anti FXa activity of graphene (29.60 IU/mL) compared with GO (1.03 IU/mL). While the graphene/UFH conjugate shows some decrease of anti FXa activity compared with UFH itself (85.61 IU/mL), it is important to elucidate that biomolecules such as heparin noncovalently interacting on the graphene plate preserve their bioactivity after conjugation with graphene. This designing of the graphene/biomolecule conjugate is very simple and more versatile than covalent chemistry because it is based on totally noncovalent chemistry, which does not require any chemical bonding and purification. Further multicomponent designing of graphene/biomolecule conjugate is under study to endow multiple bioactivities such as biocompatibility, targeting, delivery, and cell viability to the graphene-based biomaterials or devices. Acknowledgment. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2010-0004806) and financially supported by the Ministry of Education, Science Technology (MEST) and Korea Institute for Advancement of Technology (KIAT) through the Human Resource Training Project for Regional Innovation. We thank Ms. Hee Won Seo in Research Support Team (KAIST, Central Research Instrumental Facility) for kind help with the Raman experiment.

(1) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Electric Field Effect in Atomically Thin Carbon Films. Science 2007, 306 (5696), 666–669. (2) Dikin, D. A.; Stankovich, S.; Zimney, E. J.; Piner, R. D.; Dommett, G. H. B.; Evmenenko, G.; Nguyen, S. T.; Ruoff, R. S. Preparation and Characterization of Graphene Oxide Paper. Nature 2007, 448 (26), 457–460. (3) Geim, A. K.; Novoselov, K. S. The Rise of Graphene. Nat. Mater. 2007, 6, 183–191. (4) Wu, J.; Pisula, W.; Mllen, K. Graphenes as Potential Material for Electronics. Chem. ReV. 2007, 107 (3), 718–747. (5) Freitag, M. Graphene: Nanoelectronics Goes Flat Out. Nat. Mater. 2008, 3, 455–457. (6) Eda, G.; Fanchini, G.; Chhowalla, M. Large-Area Ultrathin Films of Reduced Graphene Oxide as a Transparent and Flexible Electronic Material. Nat. Nanotechnol. 2008, 3, 270–274. (7) Kim, K. S.; Zhao, Y.; Jang, H.; Lee, S. Y.; Kim, J. M.; Kim, K. S.; Ahn, J.-H.; Kim, P.; Choi, J.-Y.; Hong, B. H. Large-Scale Pattern Growth of Graphene Films for Stretchable Transparent Electrodes. Nature 2009, 457 (5), 706–710. (8) Watcharotone, S.; Dikin, D. A.; Stankovich, S.; Piner, R.; Jung, I.; Dommett, G. H. B.; Evmenenko, G.; Wu, S.-E.; Chen, S.-F.; Liu, C.P.; Nguyen, S. T.; Ruoff, R. S. Graphene-Silica Composite Thin Films as Transparent Conductors. Nano Lett. 2007, 7 (7), 1888–1892. (9) Gilje, S.; Han, S.; Wang, M.; Wang, K. L.; Kaner, R. B. A Chemical Route to Graphene for Device Applications. Nano Lett. 2007, 7 (11), 3394–3398. (10) Becerril, H. A.; Mao, J.; Liu, Z.; Stoltenberg, R. M.; Bao, Z.; Chen, Y. Evaluation of Solution-Processed Reduced Graphene Oxide Films as Transparent Conductors. ACS Nano 2008, 2 (3), 463–470. (11) Eda, G.; Chhowalla, M.; Goki, E. Graphene-Based Composite Thin Films for Electronics. Nano Lett. 2009, 9 (2), 814–818. (12) Dobrovolskaia, M. A.; Clogston, J. D.; Neun, B. W.; Hall, J. B.; Patri, A. K.; McNeil, S. E. Method for Analysis of Nanoparticle Hemolytic Properties in Vitro. Nano Lett. 2008, 8 (8), 2180–2187. (13) Kidane, A. G.; Salacinski, H.; Tiwari, A.; Bruckdorfer, K. R.; Seifalian, A. M. Anticoagulant and Antiplatelet Agents: Their Clinical and Device Application(s) Together with Usages to Engineer Surfaces. Biomacromolecules 2004, 5 (3), 798–813. (14) Luppi, E.; Cesaretti, M.; Volpi, N. Purification and Characterization of Heparin from the Italian Clam Callista chione. Biomacromolecules 2005, 6 (3), 1672–1678. (15) Murugesan, S.; Park, T.-J.; Yang, H.; Mousa, S.; Linhardt, R. J. Blood Compatible Carbon Nanotubes: Nano-Based Neoproteoglycans. Langmuir 2006, 22 (8), 3461–3463. (16) Hernandez, Y.; Nicolosi, V.; Lotya, M.; Blighe, F. M.; Sun, Z.; De, S.; Mcgovern, I. T.; Holland, B.; Byrne, M.; Gun’ko, Y. K.; Boland, J. J.; Niraj, P.; Duesberg, G.; Krishnamurthy, S.; Goodhue, R.; Hutchison, J.; Scardaci, V.; Ferrari, A. C.; Coleman, J. N. High-Yield Production of Graphene by Liquid-Phase Exfoliation of Graphite. Nat. Nanotechnol. 2008, 3, 563–568. (17) Stankovich, S.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Synthesis and Exfoliation of Isocyanate-Treated Graphene Oxide Nanoplatelets. Carbon 2006, 44 (15), 3342–3347. (18) Sun, X.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. Nano-graphene Oxide for Cellular Imaging and Drug Delivery. Nanoscale Res. 2008, 1 (3), 203–212. (19) Mohanty, N.; Berry, V. Graphene-Based Single-Bacterium Resolution Biodevice and DNA Transistor: Interfacing Graphene Derivatives with Nanoscale and Microscale Biocomponents. Nano Lett. 2008, 8 (12), 4469–4476. (20) Li, D.; Mu¨ller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G. Processable Aqueous Dispersions of Graphene Nanosheets. Nat. Nanotechnol. 2008, 3, 101–105. (21) Shan, C.; Yang, H.; Han, D.; Zhang, Q.; Ivaska, A.; Niu, Li. WaterSoluble Graphene Covalently Functionalized by Biocompatible PolyL-lysine. Langmuir 2009, 25 (20), 12030–12033. (22) Salavagione, H. J.; Go´mez, M. A.; Martı´nez, G. Polymeric Modification of Graphene through Esterification of Graphite Oxide and Poly(vinyl alcohol). Macromolecules 2009, 42 (17), 6331–6334. (23) Wang, G.; Shen, X.; Wang, B.; Yao, J.; Park, J. Synthesis and Characterisation of Hydrophilic and Organophilic Graphene Nanosheets. Carbon 2009, 47 (5), 1359–1364.

Blood Compatible Graphene/Heparin Conjugate (24) Park, S.; Dikin, D. A.; Nguyen, S. T.; Ruoff, R. S. Graphene Oxide Sheets Chemically Cross-Linked by Polyallylamine. J. Phys. Chem. C 2009, 113 (36), 15801–15804. (25) Tung, V. C.; Allen, M. J.; Yang, Y.; Kaner, R. B. High-Throughput Solution Processing of Large-Scale Graphene. Nat. Nanotechnol. 2009, 4, 25–29. (26) Fan, X.; Peng, W.; Li, Y.; Li, X.; Wang, S.; Zhang, G.; Zhang, F. Deoxygenation of Exfoliated Graphite Oxide under Alkaline Conditions: A Green Route to Graphene Preparation. AdV. Mater. 2008, 20 (23), 4490–4493. (27) Stankovich, S.; Piner, R. D.; Chen, X.; Wu, N.; Nguyen, S. T.; Ruoff, R. S. Stable Aqueous Dispersions of Graphitic Nanoplatelets via the Reduction of Exfoliated Graphite Oxide in the Presence of Poly(sodium 4-styrenesulfonate). J. Mater. Chem. 2006, 16, 155–158. (28) Xu, Y.; Bai, H.; Lu, G.; Li, C.; Shi, G. Flexible Graphene Films via the Filtration of Water-Soluble Noncovalent Functionalized Graphene Sheets. J. Am. Chem. Soc. 2008, 130 (18), 5856–5857. (29) Yang, H.; Zhang, Q.; Shan, C.; Li, F.; Han, D.; Niu, Li. Stable, Conductive Supramolecular Composite of Graphene Sheets with Conjugated Polyelectrolyte. Langmuir 2010, 26 (9), 6708–6712.

Biomacromolecules, Vol. 12, No. 2, 2011

341

(30) Yoon, S.; In, I. Solubilization of Reduced Graphene in Water through Noncovalent Interaction with Dendrimers. Chem. Lett. 2010, 39 (11), 1160–1161. (31) Park, Y.-J.; Park, S. Y.; In, I.; Preparation of Water Soluble Graphene Using Polyethylene Glycol: Comparison of Covalent Approach and Noncovalent Approach. J. Ind. Eng. Chem. 2011, in press. (32) Murugesan, S.; Xie, J.; Linhardt, R. J. Immobilization of Heparin: Approaches and Applications. Curr. Top. Med. Chem. 2008, 8 (2), 80–100. (33) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80 (6), 1339. (34) Stankovich, S.; Dikin, D. A.; Piner, R. D.; Kohlhaas, K. A.; Kleinhammes, A.; Jia, Y.; Wu, Y.; Nguyen, S. T.; Ruoff, R. S. Synthesis of Graphene-Based Nanosheets via Chemical Reduction of Exfoliated Graphite Oxide. Carbon 2007, 45 (7), 1558–1565. (35) Alferiev, I. S.; Connolly, J. M.; Stachelek, S. J.; Ottey, A.; Rauova, L.; Levy, R. J. Surface Heparinization of Polyurethane via Bromoalkylation of Hard Segment Nitrogens. Biomacromolecules 2006, 7 (1), 317–322.

BM101031A