Functionalized Carbon Nanotubes Are Non-Cytotoxic and Preserve

We discovered that f-CNT 1, which is highly water soluble, did not influence the ..... ACS Nano 0 (proofing), ... Bioconjugate Chemistry 0 (proofing),...
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Functionalized Carbon Nanotubes Are Non-Cytotoxic and Preserve the Functionality of Primary Immune Cells

2006 Vol. 6, No. 7 1522-1528

He´le`ne Dumortier,*,† Ste´phanie Lacotte,† Giorgia Pastorin,† Riccardo Marega,‡ Wei Wu,† Davide Bonifazi,‡ Jean-Paul Briand,† Maurizio Prato,*,‡ Sylviane Muller,† and Alberto Bianco*,† CNRS UPR 9021, Institute of Molecular and Cellular Biology, 67084 Strasbourg, France, and Department of Pharmaceutical Sciences, UniVersity of Trieste, Trieste 34127, Italy. Received May 21, 2006; Revised Manuscript Received June 9, 2006

ABSTRACT Carbon nanotubes are emerging as innovative tools in nanobiotechnology. However, their toxic effects on environment and health have become an issue of strong concern. In the present study, we address the impact of functionalized carbon nanotubes (f-CNTs) on cells of the immune system. We have prepared two types of f-CNTs, following the 1,3-dipolar cycloaddition reaction (f-CNTs 1 and 2) and the oxidation/ amidation treatment (f-CNTs 3 and 4), respectively. We have found that both types of f-CNTs are uptaken by B and T lymphocytes as well as macrophages in vitro, without affecting cell viability. Subsequently, the functionality of the different cells was analyzed carefully. We discovered that f-CNT 1, which is highly water soluble, did not influence the functional activity of immunoregulatory cells. f-CNT 3, which instead possesses reduced solubility and forms mainly stable water suspensions, preserved lymphocytes’ functionality while provoking secretion of proinflammatory cytokines by macrophages.

Introduction. Carbon nanotubes (CNTs), with their unique and fascinating one-dimensional nanostructure, are currently under careful scrutiny as new tools for biomedical applications.1-9 The incorporation of CNTs in living systems has, however, raised many concerns because this type of nanomaterial is practically insoluble and could accumulate into cells, organs, and tissues with dangerous effects.10 The problem of solubility has now been solved because a wide variety of noncovalent and covalent methods for CNT functionalization have been developed, which make their manipulation easier.11-16 As a consequence, the biocompatibility of f-CNTs is clearly improved.17-19 This property already became evident when f-CNTs were proposed as a novel type of carrier system for therapeutic agents.7,8,20-23 In addition, recent biodistribution and clearance studies have shown that f-CNTs are rapidly excreted through the kidneys and visualized in urine.24 This feature renders f-CNTs worth of consideration for various therapeutic applications.25 In a * Corresponding authors. He´le`ne Dumortier and Alberto Bianco, UPR 9021 CNRS Immunologie et Chimie The´rapeutiques, IBMC, 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France. E-mail: h.dumortier@ ibmc.u-strasbg.fr; [email protected]. Maurizio Prato, Dipartimento di Scienze Farmaceutiche, Universita` di Trieste, Piazzale Europa 1, Trieste 34127, Italy. E-mail: [email protected]. † CNRS UPR 9021, Institute of Molecular and Cellular Biology. ‡ University of Trieste. 10.1021/nl061160x CCC: $33.50 Published on Web 06/23/2006

© 2006 American Chemical Society

previous work, we have demonstrated that f-CNTs can be used as support for a peptide antigen vaccine to elicit specific antibodies.26,27 In the study presented here, we have explored in more detail the biocompatibility of different types of f-CNTs toward cells that are key players in setting up immune responses against potentially harmful external substances and objects that penetrate the body. The impact of CNTs, functionalized via the 1,3-dipolar cycloaddition reaction and/or via the oxidation/amidation protocol, on murine primary immune cells was analyzed in terms of cytotoxicity and cell functionality. Results and Discussion. Preparation of f-CNTs. For this study, two types of f-CNTs were used. We have modified pristine single-walled carbon nanotubes (SWNTs) via the 1,3dipolar cycloaddion reaction,28,29 or via the oxidation/ amidation methodology,21,30-32 obtaining ammonium f-CNTs 1 and 3, respectively (Figure 1). (See the Supporting Information for details.) The first type of nanotube (1) is highly soluble in physiological conditions. Oxidized nanotubes (3), derivatized with poly(ethylene glycol) (PEG1500) chains at the carboxylic functions, form instead homogeneous suspensions.30-32 Both f-CNTs 1 and 3 were further modified with fluorescein isothiocyanate (FITC) to generate fluorescent nanotubes 2 and 4, respectively, for the immune cell uptake study. The

Figure 2. Penetration of f-CNTs into splenocytes. Total spleen cells (2 × 106/well in a 24-well plate) were incubated for 24 h with 10 µg/mL of f-CNT 2 and analyzed by confocal microscopy. Images correspond to consecutive slices of two splenocytes (from the top to the bottom of the cells; left to right) and show the presence of f-CNTs (green fluorescence) in the cytoplasm of one of them.

Figure 1. Molecular structures of f-CNTs used in this study.

excess of FITC was removed carefully by repeated reprecipitation or dialysis of f-CNTs (see the Supporting Information). Uptake of f-CNTs by Primary Immune Cells. We and others have previously described the capacity of f-CNTs to penetrate transformed cell lines from different origins including 3T3 (mouse fibroblasts),20 HeLa (human cervical carcinoma cells),23 MCF7 (human breast cancer cells),33 and Jurkat cells (human T-cell leukemia).7,21,34 In the present study, we have focused our attention on primary immune cells isolated from mouse lymphoid organs. These cells are involved in the mechanisms allowing protection of the body against pathogens and foreign particles. First, the capacity of fluorescently labeled f-CNTs to be internalized in mouse spleen cells was assessed by confocal microscopy. For this purpose, total spleen cells isolated from healthy BALB/c mice (see the Supporting Information) were incubated with different concentrations of f-CNT 2 (1-10 µg/mL) for different periods of time (4-24 h) in 24-well culture plates (2 × 106 cells/well). After extensive washings with culture medium, the cells were embedded into an antifading medium before being mounted on glass slides and examined with a confocal microscope. The best results in terms of number of stained cells and fluorescence intensity were obtained when the cells were cultured with 10 µg/mL of f-CNT 2 for 24 h.35 Representative images corresponding to consecutive slices of two spleen cells are shown in Figure 2. One cell has clearly internalized f-CNT 2, as shown by the intracellular green fluorescence. In addition, f-CNT 2 can be observed in the cytoplasm but not in the nucleus, as suggested by the central round shape corresponding to the nucleus, which remains nonfluorescent. The preferential cytoplasmic localization of f-CNT 2 might be attributed to the nature of the functional groups around the tubes. Indeed, it has been Nano Lett., Vol. 6, No. 7, 2006

shown, for example, that CNTs functionalized with nucleic acids can reach the nucleus.23,33,36 Because we observed that not all spleen cells were fluorescently labeled, we next examined whether the different cell types present in the spleen and other lymphoid organs were equally susceptible to f-CNT penetration. The major immune cell types, that is, B lymphocytes, T lymphocytes, and macrophages, were isolated from the spleen, lymph nodes, and peritoneal cavity of BALB/c mice, respectively. B and T lymphocytes were purified using classical negative selection-based procedures allowing depletion of unwanted cells to obtain up to more than 95% of a pure single cell population (see the Supporting Information for experimental protocols). The macrophages, present in high numbers among peritoneal cells, were harvested by successive washes of the peritoneal cavity with culture medium. The isolated cell populations were then incubated for 24 h with FITC-labeled nanotubes 2 or 4 at 10 µg/mL, washed and stained with fluorescently labeled antibodies, specific for surface markers, before being examined by confocal microscopy. As shown in Figure 3, both types of solubilized f-CNTs (visualized as green fluorescence) are found in the cytoplasm of the three cell types, suggesting that they have been actively captured by the cells or that they have diffused through the cell membrane. Precise penetration pathways responsible for the presence of these f-CNTs inside lymphocytes and macrophages are under investigation. The images show that not all cells are stained with fluorescent f-CNTs. This might be attributed to different factors such as the dose of nanotubes used or varied intrinsic cell metabolism levels. Moreover, in the case of f-CNT 4, we found that these tubes formed a suspension in water with the presence of some aggregates, which settled in the cell culture. As a consequence, f-CNT 4 can be detected as big bundles in the medium around B and T lymphocytes (Figure 3D and E) but inside the macrophages (Figure 3F). The latter observation supports the hypothesis that an active uptake mechanism is likely involved in the entry of f-CNTs into the macrophages, widely 1523

Figure 4. f-CNTs have no effect on immune cell viability. B and T lymphocytes (4 × 105/well in a 96-flat bottomed well plate) and macrophages (2 × 105/well) were either left untreated or incubated with 10 µg/mL of f-CNT 1 or 3. Twenty-four hours later, cell death was assessed by flow cytometry (annexin V staining combined with propidium iodide incorporation). Cell viability is expressed as the percentage of viability measured for untreated cells (control). Each bar/value corresponds to one sample resulting from the mix of at least two culture wells. Data are derived from one representative experiment out of 3. White bar, control; black bar, f-CNT 1; gray bar, f-CNT 3.

Figure 3. f-CNTs localize inside B and T lymphocytes as well as macrophages. Three major immune cell populations, i.e., B lymphocytes (A, D), T lymphocytes (B, E), and macrophages (C, F), were isolated from the spleen, lymph nodes, and peritoneal cavity of BALB/c mice, respectively. They were then incubated for 22-24 h with 10 µg/mL of either f-CNT 2 (A-C) or 4 (D-F) (2 × 106 cells/well in a 24-well plate; green fluorescence). Plasma membrane staining (orange fluorescence) was then performed using antibodies specific either for MHC (major histocompatibility complex) class II molecules in the case of lymphocytes (expressed on B cells but not on T cells) or for the pan-marker F4-80 in the case of macrophages. Cells were analyzed by confocal microscopy. The white arrows indicate bundles of f-CNT 4 that are located outside lymphocytes (D, E) but inside the cytoplasm of macrophages (F). Upper left quadrant, intracellular nanotubes (green fluorescence); upper right quadrant, visible light image; lower left quadrant, membrane staining (orange fluorescence); lower right quadrant, combined light and fluorescence images.

known as highly phagocytic cells. In addition, the important role of macrophages as cleaning cells from external bodies including bundles of less-soluble nanotubes is confirmed by our results. Effect of f-CNTs on Primary Immune Cell Viability. Several studies have pointed out the fact that insoluble, pristine CNTs induce cell death in vitro.37-44 Concerning the nanotubes that underwent a chemical modification to improve their solubility, only two reports were published recently.17,18 These works describe a direct correlation between the degree of functionalization of CNTs and the observed cytotoxic 1524

effects. In the context of our study, we have analyzed the impact of the two types of f-CNTs on primary immune cell viability. For this purpose, 4 × 105 purified B and T lymphocytes and 2 × 105 macrophages were cultured in the presence of ammonium-functionalized CNTs 1 and 3. Upon incubation and subsequent washing steps to remove the excess f-CNTs, cell viability was assessed by flow cytometry upon cell staining with two markers of cell death, corresponding to fluorescently labeled annexin V and propidium iodide (PI), respectively. Annexin V is a protein that binds to membrane phosphatidyl serine residues, which are externalized and accessible only in dying cells. PI is an intercalating DNA dye that incorporates in dead cells. As displayed in Figure 4, we do not observe any significant loss of cell viability upon incubation of the three cell types with 10 µg/mL of f-CNTs 1 or 3 for 24 h, as compared to untreated cells. The same results were obtained when cells were incubated with higher amounts of f-CNTs 1 or 3 (50 µg/mL) and/or for a longer period of time (48 h) (data not shown). Therefore, our results clearly indicate that f-CNTs do not induce primary immune cell death. Taken together, the results on cell uptake and viability indicate that both types of f-CNTs can enter primary immune cells without exerting any cytotoxicity in terms of cell viability. However, cell function is another key parameter that might be altered upon interaction with f-CNTs and their subsequent internalization. Because any modification in the functional activity of immune cells might have drastic consequences on the body homeostasis and on the balance between healthy and diseased status, we decided to investigate two aspects of cell functionality: (i) the potential activation of immune cells by f-CNTs and (ii) the capacity of immune cells that have previously encountered f-CNTs to respond to a physiological stimulus. Influence of f-CNTs on Lymphocyte Functionality. One of the functional characteristics of lymphocytes is their ability to proliferate when they are activated. B and T cells were isolated and cultured (4 × 105 cells/well) in the presence of Nano Lett., Vol. 6, No. 7, 2006

10 µg/mL or 50 µg/mL of f-CNTs 1 or 3 for 48 h in order to assess the direct stimulatory effect of f-CNTs on lymphocytes. The bacterial endotoxin lipopolysaccharide (LPS) was used as a positive control for B-lymphocyte activation. In the case of T lymphocytes, we used a combination of stimulatory monoclonal antibodies, which are specific for surface molecules mediating T-cell activation (i.e., CD3 and CD28). Because f-CNT 3 is modified by PEG1500 chains, we decided to use PEG1500-diamine as a control, in an amount equivalent to that covalently linked to the f-CNT 3 samples (1.7 or 8.8 µg/mL for 10 or 50 µg/mL of f-CNT 3, respectively). Cultures were then pulsed with tritiated thymidine (1 µCi/well; 6.7 mCi/mmol) and the DNA-incorporated radioactivity, reflecting active cell proliferation, was measured 18 h later using a direct beta counter. Proliferative responses are presented in Figure 5 and are expressed as stimulation index (SI; cpm treated cells/cpm untreated cells). As shown in Figure 5A, B lymphocytes do not proliferate in response to f-CNTs 1 and 3, or to PEG. Indeed, the calculated SI remain below 2, which is the minimum value usually considered as biologically significant. As expected, LPS induces an intense proliferation of B cells as visualized by a high SI value (>20). In the same manner, we could not measure any proliferation of T lymphocytes in response to f-CNT 1, f-CNT 3, or PEG, whereas SI obtained with stimulatory anti-CD3/anti-CD28 antibodies is very high (>300). The secretion of cytokines reflecting T-cell activation (such as IL2 and IFNγ) is not induced either, as measured by ELISA (data not shown). In parallel, we also tested whether incubation of lymphocytes with f-CNTs would affect their capacity to further respond to a physiological stimulus. For this purpose, we incubated purified B and T lymphocytes with f-CNT 1, 3, or PEG in the same conditions reported above. After 24 h incubation, LPS or anti-CD3/anti-CD28 antibodies were added to the cultures at suboptimal concentrations (0.1 µg/ mL and 0.2 µg/mL/0.1 µg/mL, respectively) to get sensitive test conditions and to be able to visualize any modification of cell reactivity that may occur following interaction with f-CNTs. Proliferation was measured 65-70 h later. Figure 5B shows that B- and T-cell proliferation is not significantly affected upon incubation with f-CNTs 1 or 3 because the calculated SI are comparable in the presence or absence of nanotubes in the cell cultures. This was confirmed by the statistical analysis (student’s t test) because all p values are higher than 0.05 (in comparison to untreated cells). These results are in accord with preliminary data suggesting that the function of mouse splenocytes is not affected upon treatment with different doses of cationic f-CNTs.45 Influence of f-CNTs on Macrophage BehaVior. We next analyzed the impact of f-CNTs on macrophages. Peritoneal harvested cells (2 × 105/well) were cultured together with 10 µg/mL or 50 µg/mL of f-CNTs 1 or 3 for 24 h. Alternatively, the cells were cultured with LPS as a positive control or PEG. As a key sign of macrophage activation, the levels of secreted proinflammatory cytokines TNFR and IL6 were measured in culture supernatants using a standard double-sandwich ELISA (see the Supporting Information). Nano Lett., Vol. 6, No. 7, 2006

Figure 5. f-CNTs do not affect lymphocyte functionality. B and T lymphocytes (4 × 105/well in a 96-flat bottomed well plate) were left untreated or incubated with 10 µg/mL (dark gray bar) or 50 µg/mL (light gray bar) of f-CNT 1 or 3, or incubated with PEG (1.7 or 8.8 µg/mL). Cell activation was assessed 65-70 h later by measuring proliferation (expressed as a stimulation index, SI: cpm treated cells/cpm untreated cells). LPS (0.1 µg/mL) and agonistic anti-CD3/anti-CD28 antibodies (0.2 µg/mL/0.1 µg/mL) were used as positive controls for B and T lymphocytes activation, respectively (A). In some experiments, the capacity of cells pretreated with f-CNTs for 24 h to subsequently respond to an activation signal was assessed. For this purpose, proliferation was measured following secondary stimulation of B and T lymphocytes with LPS and anti-CD3/anti-CD28 antibodies, respectively (B). Results are expressed as the mean SI ((sem) and are derived from two independent experiments. B and T cell responses are considered biologically significant when the SI values are higher than 2. 1525

Figure 6. Impact of f-CNTs 1 and 3 on macrophage function. Macrophages (2 × 105/well) were either left untreated or incubated with 10 µg/mL (dark gray bar) or 50 µg/mL (light gray bar) of f-CNT 1 or 3, or incubated with PEG (1.7 or 8.8 µg/mL). Secretion of TNFR and IL6 in the supernatants was measured 24 h later by ELISA. LPS (8 ng/mL) was used as a positive control (A). In some experiments, the capacity of macrophages pretreated with f-CNTs for 24 h to subsequently respond to an activation signal was assessed. For this purpose, TNFR and IL6 levels were measured following secondary exposure of macrophages to LPS for 24 h (B). Results shown consist of the mean concentrations of secreted TNFR and IL6 in supernatants ((sem) and are derived from two independent experiments. Statistical significance (student’s t test) is indicated by: *, p < 0.05 (compared to untreated cells). 1526

The results are presented in Figure 6A and indicate that f-CNT 1 is not stimulatory because it does not induce macrophages to secrete either TNFR or IL6. In contrast, macrophages cultured with f-CNT 3 produce significant amounts of both cytokines, as compared with untreated cells. It should be noted, however, that the secreted amounts are remarkably lower than those measured when macrophages are stimulated by LPS (less than 1 ng/mL versus more than 6 ng/mL for TNFR, and 5 ng/mL versus 100 ng/mL for IL6, respectively). Macrophage activation is not related to the presence of PEG molecules on f-CNT 3, as shown by the absence of cytokine secretion when PEG is added to the cells (Figure 6A). The observed response is likely due to the presence of f-CNT 3 aggregates in the culture medium because this material is not fully soluble (in contrast to f-CNT 1) but rather forms a suspension (see Figure 3). Interestingly, no upregulation of classical surface activation markers such as CD86 could be observed by flow cytometry on macrophages upon incubation with both f-CNTs 1 and 3 (data not shown). In parallel to these direct activation assays, and in the same manner as described above for lymphocytes, we assessed whether macrophages, which had been first incubated with f-CNTs, were still able to behave normally in response to a physiological signal (i.e., LPS). For this purpose, isolated peritoneal cells were cultured in the presence of f-CNTs in the conditions described before. After 24 h incubation, a suboptimal dose of LPS (8 ng/mL) was added to the cells and culture supernatants were harvested 24 h later in order to measure cytokine production. As shown in Figure 6B, the response of macrophages to LPS is not significantly affected by f-CNT 1 because secretion levels of both TNFR and IL6 are not statistically different from those measured in the case of untreated cells. However, the suspended f-CNT 3 has an effect on macrophage function as shown by the unexpected diminished concentrations of the same proinflammatory cytokines as compared with cells incubated without f-CNT 3. We also noticed a decrease in cytokine secretion when macrophages were first cultured in PEG-containing medium (not significantly different from the decrease observed with f-CNT 3), suggesting that PEG molecules surrounding the f-CNT 3 might be responsible for this change. Published data concerning the capacity of PEG to disturb adhesion properties of a macrophage cell line might bring a possible explanation to our observation.46 Summary and Conclusions. We have here reported the first thorough study on the impact of f-CNTs on primary cells belonging to the immune system. Interestingly, we show that water-soluble f-CNTs, functionalized according to the 1,3-dipolar cycloaddition reaction, are not cytotoxic. Indeed, they induce neither cell death nor activation of lymphocytes and macrophages, and they do not disturb cell functions. In contrast, PEG-functionalized CNTs, obtained using an oxidation/amidation procedure and that form water suspensions, have an effect on primary immune cells and especially on macrophages because they activate them (as visualized by proinflammatory cytokines secretion) and modify their subsequent capacity to respond to a physiological stimulus. Nano Lett., Vol. 6, No. 7, 2006

This potentially harmful consequence of the interactions between cells and suspended f-CNTs fits with other reports, which have described multiple cytotoxic effects triggered by nonfunctionalized and therefore insoluble SWNTs and/or multiwalled carbon nanotubes (MWNTs). In fact, cell cycle arrest and apoptosis in a human embryo kidney cell line38 or in human untransformed skin fibroblasts42 and oxidative stress in human keratinocytes40,41 were evidenced. Concerning immune-related cells, only a few studies have been reported.45 High concentrations of pristine and oxidized MWNTs have been shown to generate massive loss of viability of the transformed human Jurkat T cell line and human peripheral blood lymphocytes.39 In contrast, pristine SWNTs embedded into Pluronic surfactant, which reduces hydrophobic interactions and ameliorates solubility, did not modify adhesion and growth of mouse phagocytic cells.47 Production of TNFR by total mouse spleen cells and the human monocytic cell line THP-1 was induced upon incubation with nonfunctionalized SWNTs.43 In the same manner, nonfunctionalized SWNTs and MWNTs were responsible for profound viability and functional defects of guinea pig pulmonary macrophages in culture.37 On the contrary, we and others have shown that stable aqueous solutions of f-CNTs do not exert toxic effects on immortalized cell lines from different origins (e.g., murine fibroblasts,20 and human promyelocytic leukemia cells21). Similarly, Sayes et al. have very recently reported a direct correlation between increasing functionalization density and decreasing cytotoxicity of SWNTs toward human dermal fibroblasts.17 The new data presented in this paper support these observations because f-CNT 1, which is fully soluble in aqueous culture medium, does not modify primary immune cells’ viability and functionality in vitro. In conclusion, our results strengthen the promising possibility of using soluble f-CNTs as carriers of biological and therapeutic molecules without affecting the immune system. Acknowledgment. This work was supported by CNRS, MRNT (Program BioIng 2003, Re´seau GenHomme), University of Trieste, and MIUR (PRIN 2004, prot. 2004035502). H.D. and G.P. were recipients of a postdoctoral fellowship from MNRT. TEM and confocal images were collected at the Microscopy Facility Plate-form of the Esplanade Campus (Strasbourg, France). We are also grateful to Je´roˆme Mutterer for his help with confocal microscopy measurements. H.D. was also supported by a fellowship from Fondation pour la Recherche Me´dicale. Note Added after Print Publication. On p 1527, a sentence in the acknowledgment section and an update for reference 30 were omitted in the version posted on the Web June 23, 2006 (ASAP), and published in the July 2006 issue (Vol. 6, No. 7, pp 1522-1528); the correct electronic version of the paper was published on December 21, 2006, and an Addition and Correction appears in the December 2006 issue (Vol. 6, No. 12). Supporting Information Available: Materials, full experimental details of CNT functionalization, description of the procedures for cell isolation, and ELISA for cytokine Nano Lett., Vol. 6, No. 7, 2006

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Nano Lett., Vol. 6, No. 7, 2006