- Email: [email protected]
Comparative in vivo biocompatibility study of single- and multi-wall carbon nanotubes
Available online at www.sciencedirect.com
Acta Biomaterialia 4 (2008) 1593–1602 www.elsevier.com/locate/actabiomat
Comparative in vivo biocompatibility study of singleand multi-wall carbon nanotubes Aneta Fraczek a,*, Elzbieta Menaszek b, Czeslawa Paluszkiewicz a, Marta Blazewicz a a
AGH – University of Science and Technology, Faculty of Materials Science and Ceramics, 30-059 Krakow, al Mickiewicza 30, Poland b Jagiellonian University, Collegium Medicum, Department of Cytobiology and Histochemistry, 30-068 Krakow, Medyczna 9, Poland Received 24 October 2007; received in revised form 17 April 2008; accepted 9 May 2008 Available online 18 June 2008
Abstract Carbon nanotubes are expected to be of use in both genetic engineering and biomaterials engineering. In each of these potential areas of application, nanoparticles are introduced into a living organism either in the form of active biomolecule carriers or as a result of the degradation process of an implant. In the present study we focus on the in vivo behavior of two types of carbon nanotubes (single- and multi-wall nanotubes). Raman and Fourier transform infrared spectroscopy, thermogravimetric analysis and differential scanning calorimetry techniques are used to characterize the materials before introducing them into the living system. The nanotubes were implanted into the skeletal rat muscle. A comparative analysis of the tissue reaction to the presence of the two types of carbon nanotubes was made. It was observed that multi-wall carbon nanotubes were found to form large aggregates within the living tissue, while distinctly smaller particles consisting of single-wall nanotubes were easily phagocytosed by macrophages and transported to local lymph nodes. Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Carbon nanotubes; Biocompatibility; In vivo investigation
1. Introduction Carbon nanotubes (CNTs) are the objects of research in an increasing number of works. Due to their unique mechanical, physical and chemical properties, it is expected that they will find use in a range of medical techniques [1–5]. CNTs comprise a carbon fibrous form consisting of one (single-wall carbon nanotubes – SWNTs) to tens of concentric tubes (multi-wall carbon nanotubes – MWNTs) of carbon elements with adjacent graphene sheets separated by 0.34 nm. Their diameters range from two to several nanometers. Due to their unique form and set of physical, chemical and biological properties, they are becoming more and more attractive for use in medical applications. At present, CNTs are used as drug carriers [6,7], in gene therapy [8–10], as membrane elements, as materials for tissue engineering [11,12] as scaffolds for cells [13,14] and in neuronal *
Corresponding author. Tel.: +48 12 617 37 59. E-mail address: [email protected] (A. Fraczek).
growth [15–17] using the effect of electrostimulation, specifically in compositions with polymers. In vitro experiments show that carbon nanotubes can be used to mimic neural fibers for neuronal growth. The surface of carbon nanotubes can additionally be subjected to chemical processing to create electrically charged sites, capable of fixing various biomolecules [18–20]. On the other hand, CNTs are very mobile within the living body and easily migrate through biological membranes, skin, hair follicles and the respiratory and alimentary tracts, and diffuse through biological tissue and cellular membranes [21,22]. These features make CNTs useful materials as drug and gene carriers. Their mobility within living systems is a highly advantageous feature in diagnostics, gene transport and drug delivery devices. Attempts have been made to design CNTs with specific shapes and properties in order to produce a new class of biocompatible composite implants [12,23,24]. Polymeric matrices modified with CNTs present greater biocompatibility in contact with cell cultures than do pure polymers. However, a few studies have reported reactions between living tissue
1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2008.05.018
1594
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
and CNTs. Taking into consideration the significant increase in the industrial use of nanomaterials, deeper knowledge about such possible reactions in living systems is required. An increasing number of companies are involved in manufacturing and developing various nanoparticulates and nanotechnologies. Thus, more questions relating to the safety and potential hazards due to their application are arising. In this respect, studies on what happens with nanotubes after delivering the drug or as a result of implant degradation in the living body, and determination of how CNTs react with tissue, are needed [12,25–29]. An essential aspect of the use of CNTs is the functionalization of their surface [30–32]. Previous studies on carbon fibrous biomaterials have revealed that the chemical state of the surface of CNTs may strongly influence tissue response [30,33]. Chemically treating the surface of CNTs can alter their susceptibility to form agglomerates or to disperse in an environment, as well as to evoke an interaction with the cells responsible for inflammation. AS well as the chemical state of the material surface, the shape and size of particles may also affect cells’ response to a foreign body [34]. The influence of catalytic particles, like Fe, Ni, V and Y, applied during the synthesis of CNTs (catalytic residues) on the toxicity of CNTs has also been reported [12,29]. However, the amount of impurities necessary to influence the pathogenic activity of CNTs has not still been determined. Some results have shown that CNTs containing from 1 to 5 lg ml 1 metallic impurities evoked no cytotoxic effect on a mesothelioma cell line, although the results have often been divergent or controversial [34]. In our study we analyse the in vivo biocompatibility of two types of CNTs differing in their surface chemical state and structure.
Both types of nanotubes were characterized by Fourier transform infrared (FTIR) and FT Raman spectroscopy. The FTIR spectra of the samples were recorded by means of a Bio-Rad FTS60v spectrometer. The transmission FTIR spectra were registered in the range of 4000– 400 cm 1 using KBr pellets. The FT Raman spectra were collected in the range of 1700–1100 cm 1 on a BioRad FT Raman accessory spectrometer (FTS 6000) with a Ge detector. The samples were excited at 1064 nm with a diode pumped Nd-YAG Spectra Physics laser. The spectra were measured at a resolution of 4 cm 1. After spectra normalization, the areas of two bands of CNTs existing in the Raman spectra were fitted to a Lorenzian line shape and the area ratio of the G and D bands was calculated. An STA (simultaneous thermal analysis) type SDT 2960 thermogravimetric analyser (TA Instruments Co.) was used to determine the differences in both types of nanotubes. The samples (5 mg) were placed in a platinum crucible and heated in air at 10 °C min 1 to 1000 °C. Differential scanning calorimetry (DSC) with a type DSC 2010 calorimeter (TA Instruments Co.) was used in order to determine the differences between single- and multi-wall CNTs. The samples (3 mg) were placed in a aluminum crucible and heated in air at 5 °C min 1 to 500 °C. Measurements of the particles size (agglomerates) before implantation were conducted in water by the dynamic light scattering (DLS) technique (Malvern Zetasizer Nano ZS) in the range from 0.6 to 6 lm, with the laser light source of wave length k = 520 nm. Larger particles formed during agglomeration were determined by stereoscopic microscopy (Alpha, Vision Engineering Ltd.) aided with a PixelFox computer image analyser.
2. Materials and methods
2.1. Animal experiments
The CNTs examined in this study were from NanoCraft, Inc. of Renton (USA). Multi- and single-wall CNTs were synthesized using arc-discharge evaporation of graphite rods. The SWNTs were 2–3 nm in diameter and 30– 50 nm in length, with a 19° closed end, and called a carbon nanohorn. They were grown in the presence of iron as catalyst. The concentration of Fe catalyst determined by atomic absorption spectrometry (ASA) using an electrothermal technique (spectrometer Model 3110, Perkin– Elmer Co.) was about 1.8 wt.%. MWNTs were 5–20 nm in diameter and 300–2000 nm long. The structure of these nanotubes contains 99.5% carbon elements in sp2 form. The residual metal particles were not detected in this material. The surface areas of the MWNTs and SWNTs were about 20 and 220 m2 g 1, respectively. Both types of nanotubes were used in the experiments without any additional purification or treatment. SWNTs in the form of nanohorns, rather than normal nanotubes, are expected to become a low-cost raw material for practical use in medicine. The details of these samples in terms of their structure and functionality are presented in the next section.
The experiments were performed according to the EU ISO 10993-6 guidelinesand the study protocol was approved by the I Local Bioethics Committee in Krakow, Poland (No. 25/2007). 2.1.1. Implantation Before implantation, the specimens of nanotubes were sterilized at 200 °C for 1 h. The nanotubes were implanted under sterile conditions into the gluteal muscle of adult hooded Oxford (HO/Krf) inbred rats. Animals were anesthetized and the skin at the site of surgery was shaved and disinfected with iodine (Cefarm Lublin, Poland). A small incision was made in the skin and the underlying muscle to create a 4 mm deep pouch. Equal portions (4 mg) of SWNTs or MWNTs were inserted into the bottom of the pouch. The muscle and skin wounds were closed with 5/0 PDS II (polydioxanone) monofilament absorbable sutures (Ethicon Ltd., UK). All animals survived the surgery. No wound healing complications were observed after the surgery or during the whole experiment. Before and after surgery the animals were maintained under standard conditions with free access to food and water.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
2.1.2. Retrieval of specimens At 7, 30 and 90 days after implant surgery, four animals at each time point were sacrificed with an overdose of Vetbutal (Biowet Pulawy Ltd., Poland) and tissue specimens containing the implanted material were excised. The samples were immediately frozen in liquid nitrogen and next cut into 8 lm thick slides in a cryostatic microtome. To estimate the processes of tissue regeneration, histological and histochemical reactions were carried out on the obtained slides. Additionally, the lymph nodes neighbouring the place of implantation were excised and examined. After fixation in 4% buffered paraformaldehyde, the nodes were embedded in Paraplast and cut with a microtome into 4 lm thick slides. Histological staining: to estimate the morphology of tissues, tissue slides were stained by the May–Gru¨nwald– Giemsa (MGG) method. Slides of Paraplast-embedded lymph nodes were processed according to the Masson–Goldner method and examined for the presence of carbon particles. 2.1.3. Histochemical reactions The presence and thickness of fibrous capsule were verified on the slides stained by the van Gieson method. The presence of cytochrome C oxidase in muscle fibers was examined by Pearse’s method [35]. 3. Results and discussion 3.1. Spectroscopic, thermogravimetric analysis (TG) and DSC studies The Raman spectroscopy allows to displays structural features of the carbonaceous skeleton, whereas FTIR
1595
analysis provides information about the presence of covalent functionalities in carbon materials. Raman spectroscopy is a sensitive technique for characterization of carbon materials, included molecular allotropes of carbon. Raman spectra of carbon nanoforms allow the differences in their structural features to be distinguished, as well as those between the fullerenes and tubular structures corresponding to CNTs. The Raman spectrum of carbon structure contains two main bands: the G-band (1570–1610 cm 1), which is assigned to the E2g C–C (sp2-bonded) stretching mode of a well-ordered graphitic structure; and the D-band (1280–1360 cm 1), attributed to the A1g (sp3-bonded) stretching mode resulting from the presence of a disordered structure or lattice defects in the graphite structure (substitutional heteroatoms, vacancies or chemically bonded heteroatoms). The nature of the spectra of the nanotubes shown in the Fig. 1 indicates that their structural ordering is relatively low. The differences are associated with their integral intensity of bands; the D-line of SWNTs is distinctly broadened in comparison to the MWNTs spectrum. The D-band of SWNTs is located at about 1280 cm 1, whereas in MWNTs spectrum at 1300 cm 1. Raman investigation also reveals the differences in the frequencies of the Dband in the spectra of both nanotubes. The spectra show that the structures of the two types of nanotubes differ significantly from the viewpoint of their lattice defects in the graphite sheet and the quantity and nature of the lattice defects in the graphite sheet. A comparison of Gbands of SWNTs and MWNTs at 1580 cm 1, originating from the atomic vibration in sp2 hybridized carbon, indicates that these bands have similar intensity and frequency. However, a distinct difference is observed for the D-bands of both materials. The intensity ratio of
Fig. 1. Raman spectra of SWNTs and MWNTs.
1596
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 2. IR spectra of SWNTs (c: initial; a: after baseline correction) and MWNTs (d: initial; b: after baseline correction).
the G-line (IG) to the D-line (ID) in Raman spectra is a useful parameter to evaluate the quality of carbon materials, including carbon nanoforms. A high ID/IG ratio means that there are more defects inside the carbon layers. This parameter equals 1.3 and 0.8 for SWNTs and MWNTs, respectively. The higher value obtained for the SWNTs means that the degree of crystalline perfection for this type of nanotubes is distinctly lower. Fig. 2 presents two IR spectra (initial and after baseline correction) for SWNTs and MWNTs. The initial IR spectra (Fig. 2c and d) exhibit almost straight lines without any bands. After the baseline correction, these spectra reveal several bands useful for analysing the chemical state of CNTs (Fig. 2a and b). The major differences of the spectra are related to the bands attributed to the carbon–oxygen and oxygen–hydrogen bonds existing in the structure of CNTs. A broad envelope of band in the range of 800–1200 cm 1 is assigned to C–O–H stretching vibration. As is indicated from this figure, such a very broad band is observed for SWNTs only. The presence of hydroxyl groups in this carbon sample is also confirmed by an intensive stretching band at 3300–3500 cm 1. This band is considerably stronger in the SWNTs spectrum. It seems reasonable to conclude that SWNTs contain chemical functionalities such as –OH and –COOH carboxyl groups. Such groups make the SWNTs surface more hydrophilic in character, while MWNTs seem to be hydrophobic. Evidence of further chemical groups existing in SWNTs is demonstrated in the TG and DSC curves. Fig. 3A and B shows the TG and DSC curves for both analysed samples. The observed differences are related to the lower temperature range of mass losses for SWNTs. The majority of mass loss of SWNTs starts at 677.9 °C, and at 774.4 °C for MWNTs. Moreover, the TG curve of SWNTs shows 3% mass loss, which occurs in the range between 150 and 300 °C, not observed for MWNTs. In this
temperature range the DSC curve corresponding to SWNTs shows a peak that is not observed for MWNTs. This mass loss can be associated with the presence of the surface chemical groups in SWNTs, which, due to their low stability, are removed at lower temperature. The hydrophilic and hydrophobic nature of single- and multi-wall CNTs can be simply observed after their immersion in water (Fig. 4). The SWNTs are uniformly dispersed in the whole volume of the water, while the MWNTs accumulate near the surface. To determine the initial (as-received) distribution of nanotubes particles, the samples were subjected to short sonication in water. The degree of dispersion of CNTs was measured by the DLS method. The mean value of MWNT agglomerates was about 0.955 ± 0.064 lm, whereas for SWNTs a bimodal distribution system was observed, with a lower mean value of 0.044 ± 0.003 lm and a higher one of 1.106 ± 0.074 lm. 3.2. In vivo assessment The in vivo study indicated that both types of CNTs induce a rapid initial tissue regeneration process. Enzymatic activity in the muscle tissue surrounding the implanted nanotubes was observed, and numerous regenerating muscle fibres appeared both in proximity to the implantation site and inside the area of the implant. Muscle fibres that appeared in the middle of the implantation site consisted of newly formed fibres. Regenerating immature muscle fibres are smaller in diameter and have centrally located nuclei. Sometimes, it is possible to confuse regenerating and degenerating fibres. To estimate the metabolic processes in the regenerating fibres, we performed histoenzymatic staining on the specimens. The results of reactions detecting the activity of oxidative enzymes (OCC) confirmed our histological observations (Fig. 5A;
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1597
Fig. 3. (A) Thermogravimetry analysis (TG) of multi- and single-wall CNTs; and (B) differential scanning calorimetry (DSC) of multi- and single-wall CNTs.
red arrows1 indicate new muscle fibers). Regenerating muscle fibres arise due to satellite cells. These cells begin to proliferate and fuse with each other into novel myotubes, or
1 For interpretation of the references to color in this figure, the reader is referred to the web version of this article.
they fuse with the damaged muscle fibers. The presence of myotubes is evident and indicates there generative process (Fig. 5B, red arrows). The inflammation response was observed around the site of the nanotubes, while 7 days after surgery a chronic phase with few neutrophiles and abundant macrophage influx were observed [36]. Microphotographs of the implantation
1598
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 4. MWNTs (A) and SWNTs (B) immersed in water.
sites show that the nanotubes were not encapsulated within a continuous fibrous capsule, or in some places a very thin capsule occurred and disappeared in an early stage after implantation (Fig. 6A–D). They were often found to be in direct contact with the muscle tissue (Fig. 6C and D). Each muscle fibre is surrounded by a thin connective tissue layer (endomysium), and groups of the fibres are divided by a thicker layer (perimysium). The fragment visible in the Fig. 6D is not thicker than perymysium. Moreover, in some implantation sites a non-continuous thin capsule is seen. The lack of a fibrous capsule in the implant’s vicinity (or its presence in a non-continuous form) and the relatively fast regeneration process would suggest that both forms of carbon are highly biocompatible. There are, however, other features indicating negative phenomena associated with the presence of nanotubes in living tissue. These phenomena may be associated with the susceptibility of the nanotubes grouping together to
form secondary particles (agglomerates). The nanotubes create particles in the tissue that differ in size and shape. Our investigations suggest that, depending on the agglomeration process, both types of CNT may evoke different host responses. SWNTs formed small aggregates uniformly dispersed in the tissue and were well phagocytosed (Fig. 7B), while MWNTs cumulated into large size aggregates (Fig. 7A). These aggregates were accompanied by abundant foreign body giant cells (Fig. 7E). The average values of the agglomerate sizes were assessed from histological microphotographs of the implant sites. Analysis of the microphotographs showed that the ‘‘diameter” of MWNT agglomerates increased with the time of implantation, while SWNTs maintained almost the same size during the entire experiment. After 7 days the biggest size of MWNT agglomerates (the longest distance between two points of the object in microscopic image) was 75 lm, and the smallest was 5 lm, respectively (Fig. 6A). In contrast, for SWNTs the longest distance across the agglomerate was 35 lm, and the smallest dimension was 5 lm (Fig. 6B). After 90 days the elongated forms of MWNT agglomerates (Fig. 7C), and the more regular small particles of SWNTs were still visible (Fig. 7D). The typical image of the implant site after follow-up for 90 days is this: the size of MWNT agglomerated particles varied from a maximum of 300 lm (Figs. 6C and 7C) to a minimum, across an elongated region, of 5 lm. In the case of SWNTs, the size of agglomerates varied from 5 to 30 lm (Figs. 6D and 7D), although bigger agglomerates (rarely) occurred up to a maximum of 100 lm (Fig. 6D). A distinctly narrower range of agglomerate sizes was observed for SWNTs. Fig. 7C and E shows MWNTs in the form of large particles with irregular shapes. SWNTs agglomerated in the form of round particles (Fig. 7D).
Fig. 5. Cross-section area of tissue with implants stained by Pearse’s method. The regeneration muscle fibers containing of cytochrome C oxidase (OCC) inside the area of the implant are visible (A). New muscle fibers containing myotubule inside the implantation site are visible (B). Magnification 20 and 100.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1599
Fig. 6. Cross-sections of the muscle with implanted nanotubes stained by van Gieson’s method. No uniform fibrous capsule separating the implant from the muscle tissue (after 7 and 90 days of implantation) is observed. Magnification 10 and 20.
MWNT particles were surrounded by numerous giant cells, indicating that phagocytosis of that material was hampered (Fig. 7E). The phagocytosis of SWNT particles was carried out by macrophages from the place of implantation (Figs. 7F and 8A). Noticeable differences were revealed by analysis of the lymph nodes draining the muscle region containing the implanted materials. Lymph nodes neighbouring the SWNT implants were grey, indicating the accumulation of that material inside the nodes. This was also confirmed by microscopic analyses (Fig. 8B). On the contrary, MWNT particles were not found in the local lymph nodes. Although both materials differ in their shape and size, as well as in their structural ordering, examination of the tissue response to the presence of these materials during the early stages of implantation revealed no significant differences. Both materials induced a rapid initial regenerative process of muscles in the vicinity of the implantation site, a short acute state, a proper level of enzymatic activity in muscle fibers, and the lack or presence of a non-continuous thin capsule around the implant. These features are characteristic of biocompatible materials.
One difference between the materials was that MWNTs formed bigger aggregates. It seems that this process is rather more dependent upon the surface state of nanotubes than upon other factors. Both forms of nanotube are known to have high specific surface energy, and they have a natural tendency to form agglomerates. The agglomerates were observed in the case of multi- and single-wall CNTs. However, the effect of agglomeration for SWNTs was considerably lower, which can be attributed to the presence of chemical functional groups on their surface. A probable explanation for the presence of oxygen-containing SWNTs is that they show a pronounced Raman spectrum defect mode (D) (higher than MWNTs), caused by amorphous carbon, and when exposed to ambient conditions (humidity, room temperature) and sterilization they can behave as activated carbon. The hydrophilic nature of the surface of SWNTs decreased their surface energy and favoured their better dispersion in muscle tissue. The easily phagocytosed small particles of SWNTs were transported by macrophages to the local lymph nodes. In contrast, MWNTs created flattened agglomerates of larger size after being implanted. The latter fact may be interpreted as being caused by the lack of func-
1600
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 7. Cross-sections of muscle with implanted SWNTs and MWNTs. Histological staining: MGG. (A, C) An irregular shape of MWNT agglomerates is seen. (B, D) SWNTs in the form of small round particles. (E) Elongated MWNT agglomerates with attached multinucleated giant cells. (F) SWNTs attached to single cell membranes or existing in cell cytoplasm. Magnification 20, 40 and 100.
tional groups in this type of nanotube. As a consequence of the relatively large size of the agglomerates, the phagocytosis process was inhibited. The absence of a connective tissue capsule around the implantation site of CNTs and the fast tissue regeneration are characteristic symptoms of biocompatible materials. On the other hand, the presence of abundant multinucle-
ated cells attached to MWNT agglomerations and the translocation of SWNTs from implant sites to lymph nodes may suggest undesirable effects related to cytotoxicity. No evidence of the influence of the iron residue in SWNTs on their toxicity was noted. However, this must remain a possibility, and further experiments are being performed to study this problem.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1601
Fig. 8. Cross-section of tissue with implants stained by MGG. Cells containing carbon materials (SWNTs) between muscle fibres are visible (A). Crosssections of the lymph nodes neighboring the SWNT implantation site, Masson–Goldner staining. Carbon particles are seen in the lymph nodes (B). Magnification 100.
4. Conclusions The biocompatibility of two types of CNTs was studied in vivo. Both types of nanotubes differed significantly with respect to their size, shape, length and chemical surface state. The results suggest that the reaction of tissue to the presence of carbon materials is dependent on their tendency to group together to create secondary particles. SWNTs created smaller particles, which were easily dispersed in the tissue environment and were subsequently transport to the local lymph nodes by macrophages. In contrast, MWNTs created aggregates of relatively large size in the vicinity of the implantation site and such aggregates increased with time. No connective tissue capsules were observed in the vicinity of the implantation site or, in some cases, capsules were present but were non-continuous and thin. A fast muscle regeneration process was observed in the vicinity of the implanted materials. The presence of abundant multinucleated cells attached to the MWNT agglomerations and the transport of SWNTs from the implant sites to the lymph nodes may suggest undesirable effects related to cytotoxicity. The results of our in vivo experiments suggest the need for further careful study to better understand the fundamental processes involved in the interaction between this specific type of material and the living body. Acknowledgements This work was financially supported by the Polish Ministry of Science and Higher Education Grant No. 3763/ T02/2006/31. References [1] Iijima S. Carbon nanotubes: past, present, and future. Physica B 2002;323:1–5. [2] Bonard JM, Croci M, Klinke C, Kurt R, Noury O, Weiss N. Carbon nanotube films as electron field emitters. Carbon 2002;40:1715–28.
[3] Zhu W, Bower C, Zhou O, Kochanski G, Jin S. Large current density from carbon nanotube field emitters. Appl Phys Lett 1999;75:873–5. [4] Xie S, Li W, Pan Z, Chang B, Sun L. Mechanical and physical properties of carbon nanotube. J Phys Chem Solid 2000;61:1153–8. [5] Dresselhaus MS, Dresselhaus G. Carbon nanotubes. Berlin: Springer; 2001. [6] Bogunia-Kubik K, Sugisaka M. From molecular biology to nanotechnology and nanomedicine. BioSystem 2002;265:123–38. [7] Bianco A, Kostarelos K, Prato M. Applications of CNTs in drug delivery. Curr Opin Chem Biol 2005;9:674–9. [8] Ciu D, Ozkan CS, Ravindran S, Kong Y, Gao H. Encapsulation of Pt-labelled DNA molecules inside CNTs. Mech Chem Biosyst 2004;1:113–21. [9] Gao H, Kong Y, Cui D, Ozkan CS. Spontaneous insertion of DNA oligonucleotides into CNTs. Nanotechnol Lett 2003;3:471–3. [10] Wang SG, Wang R, Sellin PJ, Zhang Q. DNA biosensors based on self-assembled carbon nanotubes. Biochem Biophys Res Commun 2004;325:1433–7. [11] Wood J. Nanotube scaffolds for tissue engineering. Mater Today 2004;7:10. [12] Harrison BS, Atala A. Carbon nanotube applications for tissue engineering. Biomaterials 2007;28:344–53. [13] MacDonald RA, Laurenzi BF, Viswanathan G, Ajayan PM, Stegemann JP. Collagen–carbon nanotube composite materials as scaffolds in tissue engineering. J Biomed Mater Res 2005;74A:489–96. [14] Correa-Duarte MA, Wagner N, Rojas-Chapana J, Morsczeck C, Thie M, Giersig M. Fabrication and biocompatibility of carbon nanotube-based 3D networks as scaffolds for cell seeding and growth. Nano Lett 2004;4:2233–6. [15] Hu H, Ni YC, Montana V, Haddon RC, Parpura V. Chemically functionalized carbon nanotubes as substrates for neuronal growth. Nano Lett 2004;4:507–11. [16] Zang X, Prased S, Niyogi S, Morgan A, Ozkan M, Ozkan CS. Guide neurite growth on patterned CNTs. Sensor Actuat B 2005;106:843–50. [17] McKenzie JL, Waid MC, Shi ME, Webster TJ. Decreased functions of astrocytes on carbon nanofiber materials. Biomaterials 2004;25:1309–17. [18] Huang TS, Tzeng Y, Liu YK, Chen YC, Walker KR, Guntopalli R, et al. Immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications. Diam Relat Mater 2004;13:1098–102. [19] White BJ, Harmon HJ. Novel optical solid – state glucose sensor using immobilized glucose oxidase. Biochem Biophys Res Commun 2002;296:1069–71. [20] Chen RJ, Bangsaruntip S, Drouvalakis KA, Kam NWS, Shim M, Li Y, et al. Non-covalent functionalization of CNTs for highly specific electronic biosensors. Proc Natl Acad Sci USA 2003;100:4984–9.
1602
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
[21] Smart SK, Cassady AI, Lu GQ, Martin DJ. The biocompatibility of CNTs. Carbon 2006;44:1034–47. [22] Pulskamp K, Diabate S, Krug HF. Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminations. Toxicol Lett 2007;168:58–74. [23] Krul LP, Volozhyn AI, Belov DA, Poloiko NA, Artushkevich AS, Zhdanok SA , et al. Nanocomposites based on poly-D,L-lactide and multiwall CNTs. Biomol Eng 2007;24:93–5. [24] Elias KL, Price RL, Webster TJ. Enhanced functions of osteoblasts on nanometer diameter carbon fibers. Biomaterials 2002;23:3279–87. [25] Muller J, Huaux F, Moreau N, Misson P, Heilier J, Delos M, et al. Respiratory toxicity of multi-wall carbon nanotubes. Toxicol Appl Pharmacol 2005;207:221–31. [26] Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol Lett 2005;155:377–84. [27] Yokoyama A, Sato Y, Nodasaka Y, Yamamoto S, Kawasaki T, Shindoh M, et al. Biological behavior of hat-stacked carbon nanofibers in the subcutaneous tissue in rats. Nano Lett 2005;5(1):157–61. [28] Huczko A, Lange H. Carbon nanotubes: experimental evidence for a null risk of skin irritation and allergy. Fullerene Sci Technol 2001;9:247–50.
[29] Grubek-Jaworska H, Nejman P, Czubinska K, Przybylowski T, Huczko A, Lange H, et al. Preliminary results on the pathogenic effects of intratracheal exposure to one-dimensional nanocarbons. Carbon 2006;44:1057–63. [30] Kumar C. Biofuncionalization of nanomaterials. Weinheim: WileyVCH; 2005. [31] Lee WH, Kim SJ, Lee JG, Haddon RC, Reucroft PJ. X-ray photoelectron spectroscopic studies of surface modified single-walled carbon nanotubes material. Appl Surf Sci 2001;181:121–7. [32] Kyotani T, Nakazaki S, Xu WH, Tomita A. Chemical modification of the inner walls of carbon nanotubes by HNO3 oxidation. Carbon 2001;39:771–85. [33] Bianco A, Kostarelos K, Partidos CD, Prato M. Biomedical applications of functionalized carbon nanotubes. Chem Commun 2004. [34] Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich F, Roth S, et al. The degree and kind of agglomeration affect carbon nanotubes cytotoxicity. Toxicity Lett 2007;168:121–31. [35] Stoward PJ, Pearse AGE. Histochemistry theoretical and applied. Churchill; 1991, [chapter 4]. [36] Menaszek E, Zolnierek M, Fraczek A. Blazewicz M. Histochemical and immunohistochemical studies of tissue reaction to carbon nanotubes. In: XLI Symposium PTHC; 2006. p. 72.
Acta Biomaterialia 4 (2008) 1593–1602 www.elsevier.com/locate/actabiomat
Comparative in vivo biocompatibility study of singleand multi-wall carbon nanotubes Aneta Fraczek a,*, Elzbieta Menaszek b, Czeslawa Paluszkiewicz a, Marta Blazewicz a a
AGH – University of Science and Technology, Faculty of Materials Science and Ceramics, 30-059 Krakow, al Mickiewicza 30, Poland b Jagiellonian University, Collegium Medicum, Department of Cytobiology and Histochemistry, 30-068 Krakow, Medyczna 9, Poland Received 24 October 2007; received in revised form 17 April 2008; accepted 9 May 2008 Available online 18 June 2008
Abstract Carbon nanotubes are expected to be of use in both genetic engineering and biomaterials engineering. In each of these potential areas of application, nanoparticles are introduced into a living organism either in the form of active biomolecule carriers or as a result of the degradation process of an implant. In the present study we focus on the in vivo behavior of two types of carbon nanotubes (single- and multi-wall nanotubes). Raman and Fourier transform infrared spectroscopy, thermogravimetric analysis and differential scanning calorimetry techniques are used to characterize the materials before introducing them into the living system. The nanotubes were implanted into the skeletal rat muscle. A comparative analysis of the tissue reaction to the presence of the two types of carbon nanotubes was made. It was observed that multi-wall carbon nanotubes were found to form large aggregates within the living tissue, while distinctly smaller particles consisting of single-wall nanotubes were easily phagocytosed by macrophages and transported to local lymph nodes. Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Carbon nanotubes; Biocompatibility; In vivo investigation
1. Introduction Carbon nanotubes (CNTs) are the objects of research in an increasing number of works. Due to their unique mechanical, physical and chemical properties, it is expected that they will find use in a range of medical techniques [1–5]. CNTs comprise a carbon fibrous form consisting of one (single-wall carbon nanotubes – SWNTs) to tens of concentric tubes (multi-wall carbon nanotubes – MWNTs) of carbon elements with adjacent graphene sheets separated by 0.34 nm. Their diameters range from two to several nanometers. Due to their unique form and set of physical, chemical and biological properties, they are becoming more and more attractive for use in medical applications. At present, CNTs are used as drug carriers [6,7], in gene therapy [8–10], as membrane elements, as materials for tissue engineering [11,12] as scaffolds for cells [13,14] and in neuronal *
Corresponding author. Tel.: +48 12 617 37 59. E-mail address: [email protected] (A. Fraczek).
growth [15–17] using the effect of electrostimulation, specifically in compositions with polymers. In vitro experiments show that carbon nanotubes can be used to mimic neural fibers for neuronal growth. The surface of carbon nanotubes can additionally be subjected to chemical processing to create electrically charged sites, capable of fixing various biomolecules [18–20]. On the other hand, CNTs are very mobile within the living body and easily migrate through biological membranes, skin, hair follicles and the respiratory and alimentary tracts, and diffuse through biological tissue and cellular membranes [21,22]. These features make CNTs useful materials as drug and gene carriers. Their mobility within living systems is a highly advantageous feature in diagnostics, gene transport and drug delivery devices. Attempts have been made to design CNTs with specific shapes and properties in order to produce a new class of biocompatible composite implants [12,23,24]. Polymeric matrices modified with CNTs present greater biocompatibility in contact with cell cultures than do pure polymers. However, a few studies have reported reactions between living tissue
1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2008.05.018
1594
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
and CNTs. Taking into consideration the significant increase in the industrial use of nanomaterials, deeper knowledge about such possible reactions in living systems is required. An increasing number of companies are involved in manufacturing and developing various nanoparticulates and nanotechnologies. Thus, more questions relating to the safety and potential hazards due to their application are arising. In this respect, studies on what happens with nanotubes after delivering the drug or as a result of implant degradation in the living body, and determination of how CNTs react with tissue, are needed [12,25–29]. An essential aspect of the use of CNTs is the functionalization of their surface [30–32]. Previous studies on carbon fibrous biomaterials have revealed that the chemical state of the surface of CNTs may strongly influence tissue response [30,33]. Chemically treating the surface of CNTs can alter their susceptibility to form agglomerates or to disperse in an environment, as well as to evoke an interaction with the cells responsible for inflammation. AS well as the chemical state of the material surface, the shape and size of particles may also affect cells’ response to a foreign body [34]. The influence of catalytic particles, like Fe, Ni, V and Y, applied during the synthesis of CNTs (catalytic residues) on the toxicity of CNTs has also been reported [12,29]. However, the amount of impurities necessary to influence the pathogenic activity of CNTs has not still been determined. Some results have shown that CNTs containing from 1 to 5 lg ml 1 metallic impurities evoked no cytotoxic effect on a mesothelioma cell line, although the results have often been divergent or controversial [34]. In our study we analyse the in vivo biocompatibility of two types of CNTs differing in their surface chemical state and structure.
Both types of nanotubes were characterized by Fourier transform infrared (FTIR) and FT Raman spectroscopy. The FTIR spectra of the samples were recorded by means of a Bio-Rad FTS60v spectrometer. The transmission FTIR spectra were registered in the range of 4000– 400 cm 1 using KBr pellets. The FT Raman spectra were collected in the range of 1700–1100 cm 1 on a BioRad FT Raman accessory spectrometer (FTS 6000) with a Ge detector. The samples were excited at 1064 nm with a diode pumped Nd-YAG Spectra Physics laser. The spectra were measured at a resolution of 4 cm 1. After spectra normalization, the areas of two bands of CNTs existing in the Raman spectra were fitted to a Lorenzian line shape and the area ratio of the G and D bands was calculated. An STA (simultaneous thermal analysis) type SDT 2960 thermogravimetric analyser (TA Instruments Co.) was used to determine the differences in both types of nanotubes. The samples (5 mg) were placed in a platinum crucible and heated in air at 10 °C min 1 to 1000 °C. Differential scanning calorimetry (DSC) with a type DSC 2010 calorimeter (TA Instruments Co.) was used in order to determine the differences between single- and multi-wall CNTs. The samples (3 mg) were placed in a aluminum crucible and heated in air at 5 °C min 1 to 500 °C. Measurements of the particles size (agglomerates) before implantation were conducted in water by the dynamic light scattering (DLS) technique (Malvern Zetasizer Nano ZS) in the range from 0.6 to 6 lm, with the laser light source of wave length k = 520 nm. Larger particles formed during agglomeration were determined by stereoscopic microscopy (Alpha, Vision Engineering Ltd.) aided with a PixelFox computer image analyser.
2. Materials and methods
2.1. Animal experiments
The CNTs examined in this study were from NanoCraft, Inc. of Renton (USA). Multi- and single-wall CNTs were synthesized using arc-discharge evaporation of graphite rods. The SWNTs were 2–3 nm in diameter and 30– 50 nm in length, with a 19° closed end, and called a carbon nanohorn. They were grown in the presence of iron as catalyst. The concentration of Fe catalyst determined by atomic absorption spectrometry (ASA) using an electrothermal technique (spectrometer Model 3110, Perkin– Elmer Co.) was about 1.8 wt.%. MWNTs were 5–20 nm in diameter and 300–2000 nm long. The structure of these nanotubes contains 99.5% carbon elements in sp2 form. The residual metal particles were not detected in this material. The surface areas of the MWNTs and SWNTs were about 20 and 220 m2 g 1, respectively. Both types of nanotubes were used in the experiments without any additional purification or treatment. SWNTs in the form of nanohorns, rather than normal nanotubes, are expected to become a low-cost raw material for practical use in medicine. The details of these samples in terms of their structure and functionality are presented in the next section.
The experiments were performed according to the EU ISO 10993-6 guidelinesand the study protocol was approved by the I Local Bioethics Committee in Krakow, Poland (No. 25/2007). 2.1.1. Implantation Before implantation, the specimens of nanotubes were sterilized at 200 °C for 1 h. The nanotubes were implanted under sterile conditions into the gluteal muscle of adult hooded Oxford (HO/Krf) inbred rats. Animals were anesthetized and the skin at the site of surgery was shaved and disinfected with iodine (Cefarm Lublin, Poland). A small incision was made in the skin and the underlying muscle to create a 4 mm deep pouch. Equal portions (4 mg) of SWNTs or MWNTs were inserted into the bottom of the pouch. The muscle and skin wounds were closed with 5/0 PDS II (polydioxanone) monofilament absorbable sutures (Ethicon Ltd., UK). All animals survived the surgery. No wound healing complications were observed after the surgery or during the whole experiment. Before and after surgery the animals were maintained under standard conditions with free access to food and water.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
2.1.2. Retrieval of specimens At 7, 30 and 90 days after implant surgery, four animals at each time point were sacrificed with an overdose of Vetbutal (Biowet Pulawy Ltd., Poland) and tissue specimens containing the implanted material were excised. The samples were immediately frozen in liquid nitrogen and next cut into 8 lm thick slides in a cryostatic microtome. To estimate the processes of tissue regeneration, histological and histochemical reactions were carried out on the obtained slides. Additionally, the lymph nodes neighbouring the place of implantation were excised and examined. After fixation in 4% buffered paraformaldehyde, the nodes were embedded in Paraplast and cut with a microtome into 4 lm thick slides. Histological staining: to estimate the morphology of tissues, tissue slides were stained by the May–Gru¨nwald– Giemsa (MGG) method. Slides of Paraplast-embedded lymph nodes were processed according to the Masson–Goldner method and examined for the presence of carbon particles. 2.1.3. Histochemical reactions The presence and thickness of fibrous capsule were verified on the slides stained by the van Gieson method. The presence of cytochrome C oxidase in muscle fibers was examined by Pearse’s method [35]. 3. Results and discussion 3.1. Spectroscopic, thermogravimetric analysis (TG) and DSC studies The Raman spectroscopy allows to displays structural features of the carbonaceous skeleton, whereas FTIR
1595
analysis provides information about the presence of covalent functionalities in carbon materials. Raman spectroscopy is a sensitive technique for characterization of carbon materials, included molecular allotropes of carbon. Raman spectra of carbon nanoforms allow the differences in their structural features to be distinguished, as well as those between the fullerenes and tubular structures corresponding to CNTs. The Raman spectrum of carbon structure contains two main bands: the G-band (1570–1610 cm 1), which is assigned to the E2g C–C (sp2-bonded) stretching mode of a well-ordered graphitic structure; and the D-band (1280–1360 cm 1), attributed to the A1g (sp3-bonded) stretching mode resulting from the presence of a disordered structure or lattice defects in the graphite structure (substitutional heteroatoms, vacancies or chemically bonded heteroatoms). The nature of the spectra of the nanotubes shown in the Fig. 1 indicates that their structural ordering is relatively low. The differences are associated with their integral intensity of bands; the D-line of SWNTs is distinctly broadened in comparison to the MWNTs spectrum. The D-band of SWNTs is located at about 1280 cm 1, whereas in MWNTs spectrum at 1300 cm 1. Raman investigation also reveals the differences in the frequencies of the Dband in the spectra of both nanotubes. The spectra show that the structures of the two types of nanotubes differ significantly from the viewpoint of their lattice defects in the graphite sheet and the quantity and nature of the lattice defects in the graphite sheet. A comparison of Gbands of SWNTs and MWNTs at 1580 cm 1, originating from the atomic vibration in sp2 hybridized carbon, indicates that these bands have similar intensity and frequency. However, a distinct difference is observed for the D-bands of both materials. The intensity ratio of
Fig. 1. Raman spectra of SWNTs and MWNTs.
1596
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 2. IR spectra of SWNTs (c: initial; a: after baseline correction) and MWNTs (d: initial; b: after baseline correction).
the G-line (IG) to the D-line (ID) in Raman spectra is a useful parameter to evaluate the quality of carbon materials, including carbon nanoforms. A high ID/IG ratio means that there are more defects inside the carbon layers. This parameter equals 1.3 and 0.8 for SWNTs and MWNTs, respectively. The higher value obtained for the SWNTs means that the degree of crystalline perfection for this type of nanotubes is distinctly lower. Fig. 2 presents two IR spectra (initial and after baseline correction) for SWNTs and MWNTs. The initial IR spectra (Fig. 2c and d) exhibit almost straight lines without any bands. After the baseline correction, these spectra reveal several bands useful for analysing the chemical state of CNTs (Fig. 2a and b). The major differences of the spectra are related to the bands attributed to the carbon–oxygen and oxygen–hydrogen bonds existing in the structure of CNTs. A broad envelope of band in the range of 800–1200 cm 1 is assigned to C–O–H stretching vibration. As is indicated from this figure, such a very broad band is observed for SWNTs only. The presence of hydroxyl groups in this carbon sample is also confirmed by an intensive stretching band at 3300–3500 cm 1. This band is considerably stronger in the SWNTs spectrum. It seems reasonable to conclude that SWNTs contain chemical functionalities such as –OH and –COOH carboxyl groups. Such groups make the SWNTs surface more hydrophilic in character, while MWNTs seem to be hydrophobic. Evidence of further chemical groups existing in SWNTs is demonstrated in the TG and DSC curves. Fig. 3A and B shows the TG and DSC curves for both analysed samples. The observed differences are related to the lower temperature range of mass losses for SWNTs. The majority of mass loss of SWNTs starts at 677.9 °C, and at 774.4 °C for MWNTs. Moreover, the TG curve of SWNTs shows 3% mass loss, which occurs in the range between 150 and 300 °C, not observed for MWNTs. In this
temperature range the DSC curve corresponding to SWNTs shows a peak that is not observed for MWNTs. This mass loss can be associated with the presence of the surface chemical groups in SWNTs, which, due to their low stability, are removed at lower temperature. The hydrophilic and hydrophobic nature of single- and multi-wall CNTs can be simply observed after their immersion in water (Fig. 4). The SWNTs are uniformly dispersed in the whole volume of the water, while the MWNTs accumulate near the surface. To determine the initial (as-received) distribution of nanotubes particles, the samples were subjected to short sonication in water. The degree of dispersion of CNTs was measured by the DLS method. The mean value of MWNT agglomerates was about 0.955 ± 0.064 lm, whereas for SWNTs a bimodal distribution system was observed, with a lower mean value of 0.044 ± 0.003 lm and a higher one of 1.106 ± 0.074 lm. 3.2. In vivo assessment The in vivo study indicated that both types of CNTs induce a rapid initial tissue regeneration process. Enzymatic activity in the muscle tissue surrounding the implanted nanotubes was observed, and numerous regenerating muscle fibres appeared both in proximity to the implantation site and inside the area of the implant. Muscle fibres that appeared in the middle of the implantation site consisted of newly formed fibres. Regenerating immature muscle fibres are smaller in diameter and have centrally located nuclei. Sometimes, it is possible to confuse regenerating and degenerating fibres. To estimate the metabolic processes in the regenerating fibres, we performed histoenzymatic staining on the specimens. The results of reactions detecting the activity of oxidative enzymes (OCC) confirmed our histological observations (Fig. 5A;
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1597
Fig. 3. (A) Thermogravimetry analysis (TG) of multi- and single-wall CNTs; and (B) differential scanning calorimetry (DSC) of multi- and single-wall CNTs.
red arrows1 indicate new muscle fibers). Regenerating muscle fibres arise due to satellite cells. These cells begin to proliferate and fuse with each other into novel myotubes, or
1 For interpretation of the references to color in this figure, the reader is referred to the web version of this article.
they fuse with the damaged muscle fibers. The presence of myotubes is evident and indicates there generative process (Fig. 5B, red arrows). The inflammation response was observed around the site of the nanotubes, while 7 days after surgery a chronic phase with few neutrophiles and abundant macrophage influx were observed [36]. Microphotographs of the implantation
1598
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 4. MWNTs (A) and SWNTs (B) immersed in water.
sites show that the nanotubes were not encapsulated within a continuous fibrous capsule, or in some places a very thin capsule occurred and disappeared in an early stage after implantation (Fig. 6A–D). They were often found to be in direct contact with the muscle tissue (Fig. 6C and D). Each muscle fibre is surrounded by a thin connective tissue layer (endomysium), and groups of the fibres are divided by a thicker layer (perimysium). The fragment visible in the Fig. 6D is not thicker than perymysium. Moreover, in some implantation sites a non-continuous thin capsule is seen. The lack of a fibrous capsule in the implant’s vicinity (or its presence in a non-continuous form) and the relatively fast regeneration process would suggest that both forms of carbon are highly biocompatible. There are, however, other features indicating negative phenomena associated with the presence of nanotubes in living tissue. These phenomena may be associated with the susceptibility of the nanotubes grouping together to
form secondary particles (agglomerates). The nanotubes create particles in the tissue that differ in size and shape. Our investigations suggest that, depending on the agglomeration process, both types of CNT may evoke different host responses. SWNTs formed small aggregates uniformly dispersed in the tissue and were well phagocytosed (Fig. 7B), while MWNTs cumulated into large size aggregates (Fig. 7A). These aggregates were accompanied by abundant foreign body giant cells (Fig. 7E). The average values of the agglomerate sizes were assessed from histological microphotographs of the implant sites. Analysis of the microphotographs showed that the ‘‘diameter” of MWNT agglomerates increased with the time of implantation, while SWNTs maintained almost the same size during the entire experiment. After 7 days the biggest size of MWNT agglomerates (the longest distance between two points of the object in microscopic image) was 75 lm, and the smallest was 5 lm, respectively (Fig. 6A). In contrast, for SWNTs the longest distance across the agglomerate was 35 lm, and the smallest dimension was 5 lm (Fig. 6B). After 90 days the elongated forms of MWNT agglomerates (Fig. 7C), and the more regular small particles of SWNTs were still visible (Fig. 7D). The typical image of the implant site after follow-up for 90 days is this: the size of MWNT agglomerated particles varied from a maximum of 300 lm (Figs. 6C and 7C) to a minimum, across an elongated region, of 5 lm. In the case of SWNTs, the size of agglomerates varied from 5 to 30 lm (Figs. 6D and 7D), although bigger agglomerates (rarely) occurred up to a maximum of 100 lm (Fig. 6D). A distinctly narrower range of agglomerate sizes was observed for SWNTs. Fig. 7C and E shows MWNTs in the form of large particles with irregular shapes. SWNTs agglomerated in the form of round particles (Fig. 7D).
Fig. 5. Cross-section area of tissue with implants stained by Pearse’s method. The regeneration muscle fibers containing of cytochrome C oxidase (OCC) inside the area of the implant are visible (A). New muscle fibers containing myotubule inside the implantation site are visible (B). Magnification 20 and 100.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1599
Fig. 6. Cross-sections of the muscle with implanted nanotubes stained by van Gieson’s method. No uniform fibrous capsule separating the implant from the muscle tissue (after 7 and 90 days of implantation) is observed. Magnification 10 and 20.
MWNT particles were surrounded by numerous giant cells, indicating that phagocytosis of that material was hampered (Fig. 7E). The phagocytosis of SWNT particles was carried out by macrophages from the place of implantation (Figs. 7F and 8A). Noticeable differences were revealed by analysis of the lymph nodes draining the muscle region containing the implanted materials. Lymph nodes neighbouring the SWNT implants were grey, indicating the accumulation of that material inside the nodes. This was also confirmed by microscopic analyses (Fig. 8B). On the contrary, MWNT particles were not found in the local lymph nodes. Although both materials differ in their shape and size, as well as in their structural ordering, examination of the tissue response to the presence of these materials during the early stages of implantation revealed no significant differences. Both materials induced a rapid initial regenerative process of muscles in the vicinity of the implantation site, a short acute state, a proper level of enzymatic activity in muscle fibers, and the lack or presence of a non-continuous thin capsule around the implant. These features are characteristic of biocompatible materials.
One difference between the materials was that MWNTs formed bigger aggregates. It seems that this process is rather more dependent upon the surface state of nanotubes than upon other factors. Both forms of nanotube are known to have high specific surface energy, and they have a natural tendency to form agglomerates. The agglomerates were observed in the case of multi- and single-wall CNTs. However, the effect of agglomeration for SWNTs was considerably lower, which can be attributed to the presence of chemical functional groups on their surface. A probable explanation for the presence of oxygen-containing SWNTs is that they show a pronounced Raman spectrum defect mode (D) (higher than MWNTs), caused by amorphous carbon, and when exposed to ambient conditions (humidity, room temperature) and sterilization they can behave as activated carbon. The hydrophilic nature of the surface of SWNTs decreased their surface energy and favoured their better dispersion in muscle tissue. The easily phagocytosed small particles of SWNTs were transported by macrophages to the local lymph nodes. In contrast, MWNTs created flattened agglomerates of larger size after being implanted. The latter fact may be interpreted as being caused by the lack of func-
1600
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
Fig. 7. Cross-sections of muscle with implanted SWNTs and MWNTs. Histological staining: MGG. (A, C) An irregular shape of MWNT agglomerates is seen. (B, D) SWNTs in the form of small round particles. (E) Elongated MWNT agglomerates with attached multinucleated giant cells. (F) SWNTs attached to single cell membranes or existing in cell cytoplasm. Magnification 20, 40 and 100.
tional groups in this type of nanotube. As a consequence of the relatively large size of the agglomerates, the phagocytosis process was inhibited. The absence of a connective tissue capsule around the implantation site of CNTs and the fast tissue regeneration are characteristic symptoms of biocompatible materials. On the other hand, the presence of abundant multinucle-
ated cells attached to MWNT agglomerations and the translocation of SWNTs from implant sites to lymph nodes may suggest undesirable effects related to cytotoxicity. No evidence of the influence of the iron residue in SWNTs on their toxicity was noted. However, this must remain a possibility, and further experiments are being performed to study this problem.
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
1601
Fig. 8. Cross-section of tissue with implants stained by MGG. Cells containing carbon materials (SWNTs) between muscle fibres are visible (A). Crosssections of the lymph nodes neighboring the SWNT implantation site, Masson–Goldner staining. Carbon particles are seen in the lymph nodes (B). Magnification 100.
4. Conclusions The biocompatibility of two types of CNTs was studied in vivo. Both types of nanotubes differed significantly with respect to their size, shape, length and chemical surface state. The results suggest that the reaction of tissue to the presence of carbon materials is dependent on their tendency to group together to create secondary particles. SWNTs created smaller particles, which were easily dispersed in the tissue environment and were subsequently transport to the local lymph nodes by macrophages. In contrast, MWNTs created aggregates of relatively large size in the vicinity of the implantation site and such aggregates increased with time. No connective tissue capsules were observed in the vicinity of the implantation site or, in some cases, capsules were present but were non-continuous and thin. A fast muscle regeneration process was observed in the vicinity of the implanted materials. The presence of abundant multinucleated cells attached to the MWNT agglomerations and the transport of SWNTs from the implant sites to the lymph nodes may suggest undesirable effects related to cytotoxicity. The results of our in vivo experiments suggest the need for further careful study to better understand the fundamental processes involved in the interaction between this specific type of material and the living body. Acknowledgements This work was financially supported by the Polish Ministry of Science and Higher Education Grant No. 3763/ T02/2006/31. References [1] Iijima S. Carbon nanotubes: past, present, and future. Physica B 2002;323:1–5. [2] Bonard JM, Croci M, Klinke C, Kurt R, Noury O, Weiss N. Carbon nanotube films as electron field emitters. Carbon 2002;40:1715–28.
[3] Zhu W, Bower C, Zhou O, Kochanski G, Jin S. Large current density from carbon nanotube field emitters. Appl Phys Lett 1999;75:873–5. [4] Xie S, Li W, Pan Z, Chang B, Sun L. Mechanical and physical properties of carbon nanotube. J Phys Chem Solid 2000;61:1153–8. [5] Dresselhaus MS, Dresselhaus G. Carbon nanotubes. Berlin: Springer; 2001. [6] Bogunia-Kubik K, Sugisaka M. From molecular biology to nanotechnology and nanomedicine. BioSystem 2002;265:123–38. [7] Bianco A, Kostarelos K, Prato M. Applications of CNTs in drug delivery. Curr Opin Chem Biol 2005;9:674–9. [8] Ciu D, Ozkan CS, Ravindran S, Kong Y, Gao H. Encapsulation of Pt-labelled DNA molecules inside CNTs. Mech Chem Biosyst 2004;1:113–21. [9] Gao H, Kong Y, Cui D, Ozkan CS. Spontaneous insertion of DNA oligonucleotides into CNTs. Nanotechnol Lett 2003;3:471–3. [10] Wang SG, Wang R, Sellin PJ, Zhang Q. DNA biosensors based on self-assembled carbon nanotubes. Biochem Biophys Res Commun 2004;325:1433–7. [11] Wood J. Nanotube scaffolds for tissue engineering. Mater Today 2004;7:10. [12] Harrison BS, Atala A. Carbon nanotube applications for tissue engineering. Biomaterials 2007;28:344–53. [13] MacDonald RA, Laurenzi BF, Viswanathan G, Ajayan PM, Stegemann JP. Collagen–carbon nanotube composite materials as scaffolds in tissue engineering. J Biomed Mater Res 2005;74A:489–96. [14] Correa-Duarte MA, Wagner N, Rojas-Chapana J, Morsczeck C, Thie M, Giersig M. Fabrication and biocompatibility of carbon nanotube-based 3D networks as scaffolds for cell seeding and growth. Nano Lett 2004;4:2233–6. [15] Hu H, Ni YC, Montana V, Haddon RC, Parpura V. Chemically functionalized carbon nanotubes as substrates for neuronal growth. Nano Lett 2004;4:507–11. [16] Zang X, Prased S, Niyogi S, Morgan A, Ozkan M, Ozkan CS. Guide neurite growth on patterned CNTs. Sensor Actuat B 2005;106:843–50. [17] McKenzie JL, Waid MC, Shi ME, Webster TJ. Decreased functions of astrocytes on carbon nanofiber materials. Biomaterials 2004;25:1309–17. [18] Huang TS, Tzeng Y, Liu YK, Chen YC, Walker KR, Guntopalli R, et al. Immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications. Diam Relat Mater 2004;13:1098–102. [19] White BJ, Harmon HJ. Novel optical solid – state glucose sensor using immobilized glucose oxidase. Biochem Biophys Res Commun 2002;296:1069–71. [20] Chen RJ, Bangsaruntip S, Drouvalakis KA, Kam NWS, Shim M, Li Y, et al. Non-covalent functionalization of CNTs for highly specific electronic biosensors. Proc Natl Acad Sci USA 2003;100:4984–9.
1602
A. Fraczek et al. / Acta Biomaterialia 4 (2008) 1593–1602
[21] Smart SK, Cassady AI, Lu GQ, Martin DJ. The biocompatibility of CNTs. Carbon 2006;44:1034–47. [22] Pulskamp K, Diabate S, Krug HF. Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminations. Toxicol Lett 2007;168:58–74. [23] Krul LP, Volozhyn AI, Belov DA, Poloiko NA, Artushkevich AS, Zhdanok SA , et al. Nanocomposites based on poly-D,L-lactide and multiwall CNTs. Biomol Eng 2007;24:93–5. [24] Elias KL, Price RL, Webster TJ. Enhanced functions of osteoblasts on nanometer diameter carbon fibers. Biomaterials 2002;23:3279–87. [25] Muller J, Huaux F, Moreau N, Misson P, Heilier J, Delos M, et al. Respiratory toxicity of multi-wall carbon nanotubes. Toxicol Appl Pharmacol 2005;207:221–31. [26] Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol Lett 2005;155:377–84. [27] Yokoyama A, Sato Y, Nodasaka Y, Yamamoto S, Kawasaki T, Shindoh M, et al. Biological behavior of hat-stacked carbon nanofibers in the subcutaneous tissue in rats. Nano Lett 2005;5(1):157–61. [28] Huczko A, Lange H. Carbon nanotubes: experimental evidence for a null risk of skin irritation and allergy. Fullerene Sci Technol 2001;9:247–50.
[29] Grubek-Jaworska H, Nejman P, Czubinska K, Przybylowski T, Huczko A, Lange H, et al. Preliminary results on the pathogenic effects of intratracheal exposure to one-dimensional nanocarbons. Carbon 2006;44:1057–63. [30] Kumar C. Biofuncionalization of nanomaterials. Weinheim: WileyVCH; 2005. [31] Lee WH, Kim SJ, Lee JG, Haddon RC, Reucroft PJ. X-ray photoelectron spectroscopic studies of surface modified single-walled carbon nanotubes material. Appl Surf Sci 2001;181:121–7. [32] Kyotani T, Nakazaki S, Xu WH, Tomita A. Chemical modification of the inner walls of carbon nanotubes by HNO3 oxidation. Carbon 2001;39:771–85. [33] Bianco A, Kostarelos K, Partidos CD, Prato M. Biomedical applications of functionalized carbon nanotubes. Chem Commun 2004. [34] Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich F, Roth S, et al. The degree and kind of agglomeration affect carbon nanotubes cytotoxicity. Toxicity Lett 2007;168:121–31. [35] Stoward PJ, Pearse AGE. Histochemistry theoretical and applied. Churchill; 1991, [chapter 4]. [36] Menaszek E, Zolnierek M, Fraczek A. Blazewicz M. Histochemical and immunohistochemical studies of tissue reaction to carbon nanotubes. In: XLI Symposium PTHC; 2006. p. 72.