Effect of nitrogen atomic percentage on N+-bombarded MWCNTs in cytocompatibility and hemocompatibility
© Zhao et al.; licensee Springer. 2014
Received: 14 February 2014
Accepted: 13 March 2014
Published: 25 March 2014
N+-bombarded multi-walled carbon nanotubes (N+-bombarded MWCNTs), with different nitrogen atomic percentages, were achieved by different N ion beam currents using ion beam-assisted deposition (IBAD) on MWCNTs synthesized by chemical vapor deposition (CVD). Characterizations of N+-bombarded MWCNTs were evaluated by X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), Raman spectroscopy, and contact angle. For comparison, the in vitro cytocompatibility of the N+-bombarded MWCNTs with different N atomic percentages was assessed by cellular adhesion investigation using human endothelial cells (EAHY926) and mouse fibroblast cells (L929), respectively. The results showed that the presence of nitrogen in MWCNTs accelerated cell growth and proliferation of cell culture. The higher nitrogen content of N+-bombarded MWCNTs, the better cytocompatibility. In addition, N+-bombarded MWCNTs with higher N atomic percentage displayed lower platelet adhesion rate. No hemolysis can be observed on the surfaces. These results proved that higher N atomic percentage led N+-bombarded MWCNTs to better hemocompatibility.
KeywordsN+-bombarded multi-walled carbon nanotubes Nitrogen atomic percentage Ion beam-assisted deposition Cytocompatibility Hemocompatibility
The last decade has seen a great deal of activity in the use of carbon nanotubes (CNTs) to augment the properties of a variety of materials, including biomaterials . The advantage of carbon nanotubes in biomedicine is their stable conductivity in aqueous physiological environment, thus making them attractive for cellular stimulation . And, the weakness of raw CNTs is their super-hydrophobicity. They can easily aggregate in aqueous media as well as in organic solvents, which strictly restricts their application in biomedical fields because a hydrophilic interface is in favor of enhancing bioactivity . So, in recent years, the enormous progress in nanotechnology and material sciences had stimulated the development and production of engineered carbon nanotubes [4–9]. And, numerous studies in biomaterial development indicated the functionalized water-soluble CNTs to improve cell attachment and growth [5–9]. In our previous work , the improved hemocompatibility and cytocompatibility were also observed in N-doped MWCNTs when compared with pristine MWCNTs using chemical vapor deposition (CVD) method. Recently, many studies on the functionalization of MWCNTs have been reported. Chemical grafting is the main method for CNT functionalization. In previous works, we also synthesized MWCNTs containing N, carboxyl, and hydroxyl groups using CVD and compared the biocompatibility of MWCNTs with and without functional groups [10–12]. A significant improvement in cell and blood behaviors was observed in MWCNTs containing functional groups compared with pure MWCNTs. However, few reports are found to achieve MWCNT functionalization using the ion beam bombardment or ion implantation technique. The advantages of the physical method are its simplicity, small amounts of impurities, and high content of active groups on the surface of MWCNTs. Differing from the traditional chemical grafting, the ion implantation technique was also used to introduce NH2 and COOH groups onto MWCNTs, and graphene which was found to result in favorable effects on their biocompatibility in our previous works [13–16].
To differ from traditional chemical grafting and ion implantation, in this paper, lower-energy N ion beam bombardment method was used to introduce N ions to MWCNTs. Compared with ion implantation, the advantages of low-energy ion beam bombardment are its shallow injection depth and high content of active nitrogen on the surface of MWCNTs. The interaction between cell and substrates primarily occurred on the shallow surface of modified MWCNTs. The larger number of active nitrogen on the surface of MWCNTs which interacted with cells in vitro could increase the number of sites for cell growth. Thus, the modified MWCNT surface should have better bioactivity and biocompatibility.
Due to length limitation, the comparison between pure and N+-bombarded MWCNTs in cytocompatibility and hemocompatibility will be submitted to other journals. This work only focused on the relationships between cell and blood behaviors and N atomic percentages of laboratory-made MWCNTs bombarded at different N+ beam currents (5, 10, and 15 mA), which were evaluated by cell adhesion, hemolysis, and platelet adsorption.
An ion beam-assisted deposition (IBAD) system (FJL560C12, SKY Technology Development Co., Ltd., China) was used to prepare N+-bombarded MWCNTs. This system has two ion sources, one water-cooled sample holder and one water-cooled target holder. In this processing, the chamber was evacuated to a base pressure lower than 3.0 × 10-4 Pa prior to N ion bombardment. Then, the high-purity N2 gas was introduced into low-energy ion source which could perform N ion bombardment to MWCNTs at desired ion bombarding parameters through computer controlling. N ion beams at ion beam currents of 5, 10, and 15 mA and a constant bombarding energy of 200 eV were respectively accelerated to bombard MWCNTs for 30 min to get three N atomic percentages of N+-bombarded MWCNT samples. The working gas pressure was 1.2 × 10-2 Pa.
Contact angle, XPS, SEM, TEM, and Raman analysis
Water contact angles were measured using a face contact angle meter (CAM KSV021733, Nunc, Finland). The detailed measurement process can be found in our previous work [17–19]. Characterization by X-ray photoelectron spectroscopy (XPS) (PHI5000 VersaProbe system, Physical Electronics, Chanhassen, MN, USA) was used to prove the existence of the main functional groups in the three samples. The morphology of N+-bombarded MWCNTs was examined with a field emission scanning electron microscope (FESEM; 18SI, FEI, Hillsboro, OR, USA) operated at 10.0 kV and a field emission scanning electron microscope (SU8020, HITACHI, Tokyo, Japan) operated at 1.0 kV. The detailed morphologies and chemical bonding states of the samples were characterized using a JOEL JEM 2100 transmission electron microscope (TEM; Tokyo, Japan) and Renishaw micro-Raman 2000 system (Wotton-under-Edge, UK) and a 514-nm laser line excitation.
Cell adhesion assays
The human endothelial cell line EAHY926 and mouse fibroblast cells (L929) were used to investigate the cytocompatibility of N+-bombarded MWCNTs. The processes of cell culture and cell vaccination can be found in our previous work [13–16]. Endothelial cells were harvested from the cultures and replaced into 24-well plate (5 × 104 cells/ml) in four groups (three kinds of N+-bombarded MWCNTs and blank control group). The inoculum density of fibroblast cells is 2.5 × 104 cells/ml. After 1 to 7 days in an incubator (culture intervals of 0.5, 1, 2, 3, 5, and 7 days), the medium was removed, and the cell monolayer was washed several times with PBS and then isolated by trypsin for enumeration.
Immunofluorescence staining was done as described with mouse monoclonal anti-α-tubulin (clone B-5-1-2, 1:1,000 dilution; Sigma, St. Louis, MO, USA), followed by 1:200 dilution of various fluorochrome-conjugated secondary antibodies. Finally, DNA was stained with DAPI (1 μg/ml) for 5 min. For immunostaining, mouse fibroblast cells were grown on three kinds of N+-bombarded MWCNTs at 2.5 × 104 cells/ml for 24 h. Confocal scanning laser microscopy (CSLM) (Nikon Eclipse 90, Shinjuku, Tokyo, Japan) was employed to observe cell morphology and stretching on the three samples. The scanning electron microscope (SEM) (FEI QUANTA 200) was employed to observe endothelial cells' and mouse fibroblast cells' morphology and stretching on three materials.
where A is the total number of platelets and B is the number of platelets remaining in the blood after the platelet adhesion test . The morphology of adherent platelets was assessed using SEM.
Anticoagulant blood solution was obtained by adding normal saline to anticoagulant blood which was prepared from healthy rabbit blood plus 2% potassium oxalate. The samples were placed in each Erlenmeyer flask and added with 5 ml normal saline. The same numbers of Erlenmeyer flasks with either 5 ml normal saline or distilled water were used as negative and positive control groups, respectively. The detailed process can be found in our previous work [10, 17, 18].
Results and discussions
Wettability, evaluated through the measurement of the contact angle of a liquid on a surface, is a sensitive way to detect surface modifications . Furthermore, it is a measurement of the hydrophilic/hydrophobic character of a material, a relevant property regarding biocompatibility, since it has a major influence on protein adsorption and interaction with cells . In this work, the wettability of the three samples was evaluated by water contact angle measurements, as shown in Figure 2f,g,h. The values of N+-bombarded MWCNTs at nitrogen concentrations of 7.81%, 8.67%, and 9.28% are 61.89°, 17.16°, and 45.48°, respectively. It is worth noting that the increase of contact angle is not related to the increase of nitrogen concentration and ion beam current. The results show a slight decrease in contact angle with the decrease of the sp2 C-O content.
Endothelial cells have been shown to be more sensitive than mouse fibroblast cells to the same sample. The numbers of endothelial cells on N+-bombarded MWCNTs still increase rapidly after the 5-day incubation. And, it far exceeds the control group on the seventh day (Figure 5d). The highest nitrogen concentration displays the highest cell numbers. Thus, the high nitrogen concentration stimulates cell growth and proliferation of cell culture, revealing superior cytocompatibility.
Figure 5b,c,d,e,f shows clearly the difference at the amount and morphology of the adhered cells on N+-bombarded MWCNTs with N 8.61% and 9.28%. As we can see from the SEM images with low magnification, the cell concentration with N 8.67% (Figure 5b,e) is significantly less than that with N 9.28% (Figure 5c,f), which is consistent with the results given by Figure 4 and Figure 5a,d. And, the adhered cells all spread flat with richer pseudopod and microvilli, as shown at a high magnification. These results add to growing evidence that the increase of nitrogen content promoted cell adherence and growth.
The ability of substrates to promote adhesion of cells depends on how well they adsorb proteins from the culture medium that interact with receptors on the cell surface . Adsorption of proteins in an active conformation, in turn, is likely to be affected by the functional groups of the substrate. All proteins have NH2 and COOH groups at the ends, where the NH tends to be positively charged and the COOH negatively charged . Thus, a surface with an organized arrangement of functional groups can act as a site for cell growth. The formation of functional sp2 C-N and sp3 C-N bonds on the N+-bombarded MWCNTs by N ion beam bombardment induces polarization at the surface due to the difference in electronegativity between carbon and nitrogen . In addition, from the XPS results (Figure 1d,e,f), it is clear that with the increase of nitrogen concentration, the ratio of the sp2 C-N bond decreases and the sp3 C-N bond increases while the unsaturated degree of the N bond increases. Therefore, the number of protein attached on the material's surface increases with increasing unsaturated degree of the N bond, and adhesion of cells are promoted.
The morphological change of the adherent platelets is a common qualitative criterion to assess activation of adherent platelet on the materials' surface. Baurschmidt  reported that the formation of thrombus on the biomaterial surface is correlated with charge transferring from fibrinogen to the material surface. Fibrinogen can transform to fibrin monomer and fibrinopeptides when it losses charge. The crosslink of fibrin monomer causes an irreversible thrombus. Thus, the suitable density of charge will promote the hemocompatibility [37, 38]. A suitable ratio of sp3 C-N to sp2 C-N can provide the optimum density of charge to promote hemocompatibility. The possible reason for the decrease of platelet adhesion rates is the significant change in the electronic characteristics due to the increase of sp3 C-N bond.
The hemolysis ratio was calculated by the formula , where A, B, and C are the absorbance values of the specimens, negative control group (physiological salt water), and the positive control group (H2O), respectively [17, 18]. The average OD values of the N+-bombarded MWCNTs with 7.81%, 8.67%, and 9.28% are 0.027, 0.029, and 0.026, respectively. The hemolytic rates of all the N+-bombarded MWCNTs are all 0%. According to the YY/T0127.1 standard, a hemolytic rate below 5% is acceptable [38–40]. These results indicate that the three materials all have good hemocompatibility.
In this paper, the cytocompatibility and hemocompatibility of the N+-bombarded MWCNTs with three N atomic percentages are investigated and compared. The cell adhesion assays indicate clearly that with the increase of nitrogen concentration, the ratio of the sp2 C-N bond decreases and the sp3 C-N bond increases while the unsaturated degree of the N bond increases. It may increase the number of protein which attached on the material's surface; so, the adhesion of cells is promoted. Thus, the cytocompatibility of N+-bombarded MWCNTs are promoted with the increase of nitrogen concentration. The blood experiments also show that N+-bombarded MWCNTs with higher nitrogen content displayed lower platelet adhesion rates and lower hemolytic rate values. In conclusion, bombarding N ions into MWCNTs by IBAD is a great feature and desirable for biomaterial industry.
MZ is an Assistant Experimentalist in the College of Physics and Materials Science, Tianjin Normal University, Tianjin, China. YC and XL are Masters degree candidates of College of Physics and Materials Science, Tianjin Normal University, Tianjin, China. JD is a Lecturer in the College of Physics and Materials Science, Tianjin Normal University, Tianjin, China. DL is a Professor in the College of Physics and Materials Science, Tianjin Normal University, Tianjin, China. HG is a Professor in Tianjin Institute of Urological Surgery, Tianjin Medical University, Tianjin and in School of Medicine, Ninth People's Hospital, Shanghai Jiao Tong University, Shanghai, China.
multi-walled carbon nanotubes
- N+-bombarded MWCNTs:
N+ ion-bombarded multi-walled carbon nanotubes
red blood cell.
This work was supported by the National Natural Science Foundation of China (51272176) and National Basic Research Program of China (973 Program, 2012CB933600). The Key Project of Tianjin Municipal Natural Science Foundation of China (13JCZDJC33900), National Natural Science Foundation of China for Youth Science Funds (51302187), and the Youth Foundation of Tianjin Normal University (52XQ1204) also supported this work.
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