Blood compatibility and biomedical applications of graphene.
Abstract: Graphene discovered as an exciting material for fundamental physics during 2004 is now poised for a wide range of nanotechnology applications. Its biomedical applications particularly on drug delivery and biosensor applications are limited. Although carbon is considered to be highly compatible to blood, nanomaterials' biosafety has caused more and more attention from various scientific and government sectors based on the reported toxicity of carbon nanotubes. Biological application of graphene implies that it should be compatible with blood as it comes in contact with blood in most of the applications including biosensors, cell imaging and drug delivery. An attempt has been made here to study the blood compatibility of graphene. It has been demonstrated that graphene is compatible with blood and do not induce any platelet or complement activation and therefore can be utilized for various in vivo applications.
Article Type: Report
Subject: Graphene (Usage)
Graphene (Health aspects)
Biocompatibility (Measurement)
Hematology (Research)
Authors: Paul, Willi
Sharma, Chandra P.
Pub Date: 07/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: July, 2011 Source Volume: 25 Source Issue: 3
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842720
Full Text: Introduction

The mineral graphite is one of the allotropes of carbon and crystalline "flake" graphite is one of its principal types with plate-like structure. It consists of many sheets stacked together called "graphene", whose structure is one-atom-thick planar sheets of [sp.sup.2]-bonded carbon atoms that are densely packed in a honeycomb crystal lattice (1). Graphene can be visualized as an atomic-scale chicken wire made of carbon atoms and their bonds (figure 1) with a bond length of about 0.142 nanometers (2). It structure relates to the mesh structure commonly seen in real chicken wire, the regular hexagonal (honeycomb-like) patterns found in certain chemical compounds. Six ring carbon atoms have six free bonds which covalently bind to six other carbon atoms forming six other rings of graphene as shown in figure 2. Since its discovery in 2004 (3) graphene is considered to be a super-material in the field of nanotechnology and electronics (1,4), however, its biomedical applications are limited.

Graphene is also the basic structural element of carbon nanotubes and fullerenes. The Nobel Prize in Physics for 2010 was awarded jointly to Andre Geim and Konstantin Novoselov "for groundbreaking experiments regarding the two-dimensional material graphene" (5). Although biological studies on graphene are relatively limited, it could find numerous applications in medical field, like carbon nanotubes and fullerenes. However, some studies have already been reported which is reviewed here. Although carbon is considered to be highly compatible to blood, nanomaterials' biosafety has caused more and more attention from various scientific and government sectors (6) based on the reported toxicity of carbon nanotubes (7). Biological application of graphene implies that it should be compatible with blood as it comes in contact with blood in most of the applications including biosensors, cell imaging and drug delivery. However, blood compatibility of these nanomaterials is still unexamined. Therefore, an attempt has also been made to evaluate the blood compatibility of graphene towards its various in vivo applications.

Limited Biomedical Applications

Graphene has been used initially in sensor related applications where there is no direct interaction with the biological media (8). It has been suggested that the single-stranded DNA was promptly adsorbed onto graphene forming strong molecular interactions, which improved the specificity of its response to complementary DNA. Graphene surface effectively protected ss-DNA from enzymatic cleavage by Dnase I. It also found application in the fabrication of third-generation electrochemical biosensors, where the HRP/ss-DNA/GP/GC electrode performed good electrocatalytic reduction for [H.sub.2][O.sub.2] with good sensitivity and stability with wide linear range (9). In another recent study it has been demonstrated that a biocompatible scaffold developed using graphene accelerates specific differentiation of human mesenchymal stem cells into bone cells without hampering the proliferation. The differentiation rate was comparable to the one achieved with common growth factors, demonstrating graphene's potential for stem cell research (10). Biocompatible nanographene oxides with various physical sizes has been reported which imparted aqueous stability to the NGO in buffer solutions and other biological media by covalently grafting polyethylene glycol starpolymers onto the chemically activated surfaces and edges (11). The photoluminescence from visible to the near-infrared (NIR) range observed with these NGO was used for cellular imaging. Doxorubicin was loaded onto NGO with high capacity, and selectively transported into specific cancer cells by antibody guided targeting which suggests promising applications of graphene materials in biological and medical areas. It has been shown that addition of graphene significantly increased the modulus of chitosan even at a very low content and the composite showed good biocompatibility for L929 cells (12). It has been demonstrated in another paper that macroscopic antibacterial graphene-based paper can be conveniently fabricated with superior inhibition ability to bacteria growth (13). This could be useful in some environmentally friendly applications. However, in a very recent study graphene oxides has been shown to have dose-dependent toxicity to cells and mice, such as inducing cell apoptosis and lung granuloma formation which could not be cleaned by the kidney (14).

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Characterization

The major features of the Raman spectra of graphene are the G band at 1584 [cm.sup.-1] and the G2 ?band at 2700 [cm.sup.-1]. The G band is due to the E2g vibrational mode, and the G2 ?band is a second-order two-phonon mode. A third feature, the D band at 1350 [cm.sup.-1], is not Raman active for pristine graphene but can be observed where symmetry is broken by edges or in samples with a high density of defects. The figure 3 shows the Raman spectra of the graphene sample studied. Since the D band is visible this sample could be having high density of defects. The high resolution AFM micrograph as shown in figure 4 clearly shows the honeycomb-like structure of the graphene. It reveales the hidden atom in graphene showing all atoms within the hexagonal cell with an image size 2 x 2 [nm.sup.2].

Blood Compatibility Studies

The initial event when a material comes in contact with blood is the adsorption of proteins. The nature of protein and amount of protein adsorbed will directly influence the compatibility of the particles with the blood. One of the negative effects of the clinical application of various blood-contacting materials is the activation of the platelets and complement system induced by the foreign surface. The response of blood in contact with the material depends on phisico-chemical features such as surface area, surface charge, hydrophobicity/hydrophilicity etc. The response depends directly on the surface area. Adsorption of C3 triggers complement activation (15). The aggregation of blood cells and activation of platelets and complement on exposure of graphene to blood were evaluated by a reported procedure (16). Graphene (QGraphene[R], Graphene 50) was obtained from Quantum Materials Corporation, Bangalore, India with the following specifications. Flakes of single layered carbon sheets with platelet morphology; Mean particle size = 80nm; Specific surface area > 50[m.sup.2]/g; >90% purity.

The aggregations of the blood cells on interaction with the nanoparticles are shown in figure 5 for RBC, WBC and platelets. It revealed no aggregation of blood cells on incubation of graphene a higher interaction ratio of 10mg/ml. Polyethyeleneimine (PEI) which was used as positive control showed aggregation whereas saline used as negative control did not show any aggregation. The same was visible with the haemolytic property of the nanoparticles. The hemolysis induced by grapheme was only 0.1 % which was well within the acceptable limits of 1% (17).

Measuring C3a or C5a in blood or serum after contact with a material has been the most usual way of assessing complement activation. It has been claimed that a surface is biocompatible if these markers are not increased in the fluid phase (18). Since C3 is cleaved to C3a and C3b by the contact of the surface with blood, irrespective of whether the activation occurs via classical or alternative pathways, and also C3a could be adsorbed on to the material surface just like any other proteins, C3 depletion in the medium can be taken as an indirect measure of complement activation. The amount of C3 in blood (pre-incubation) was 125 [+ or -] 5 mg%. After incubation with graphene it was 124 [+ or -] 3 mg% indicating no significant changes in the complement protein level.

Platelet factor 4 (PF4) is as a platelet-specific protein secreted when platelet is activated and belongs to the C-X-C chemokine family. Measurements of plasma levels of PF 4 have been shown to be the marker of platelet degranulation and increased level of PF4 is used to detect platelet activation of the circulating pool of platelets (19). On incubation with graphene the level of platelet factor 4 in plasma did not change appreciably compared to control plasma. The PF4 level in control plasma was 9.5 [+ or -] 0.3 IU/ml and after incubation with graphene for 15 min it was 9.6 [+ or -] 0.5 IU/ml. It has been demonstrated in this study that the activation of platelets, adsorption of C3 or aggregation of blood cells on grapheme incubation is insignificant indicating that these preparations do not induce any complement activation or platelet activation on contact with blood. Thus the graphene sample tested seems to be blood compatible.

Conclusion

Graphene discovered as an exciting material for fundamental physics is now poised for a wide range of nanotechnology applications and also finds application in medical applications. Although it is being widely studied for electronic nanotechnology applications, its biomedical applications particularly on drug delivery and biosensor applications are limited. It can be presumed that graphemes ability for chemical modification can help tremendously towards its biological applications. This is also supported by the present study indicating its blood compatibility.

The accumulation of graphene in various organs like liver, spleen, kidney and brain, is an important factor which needs to be evaluated with a long term study on suitable animal models. The discovery of a method for isolating graphene sheets led to an explosion in graphene research for advanced computing applications, digital displays and other types of flexible electronics, and advanced composite materials. With the new potential applications in biomedical field coming up, graphene's usefulness is only just beginning to be discovered and has a bright future.

Acknowledgement

We are grateful to the Director and the Head BMT Wing of SCTIMST for providing facilities for the completion of this work. This work was supported by the Department of Science & Technology, Govt. of India through the project 'Facility for nano/microparticle based biomaterials--advanced drug delivery systems' #8013, under the Drugs & Pharmaceuticals Research Programme.

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Willi Paul and Chandra P. Sharma *

Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram 695012, India

* corresponding author sharmacp@sctimst.ac.in

Received 3 April 2011; Accepted 1 May 2011; Available online 29 May 2011
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