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Reprogramming Cellular Signaling Machinery Using SurfaceModified Carbon Nanotubes Yi Zhang, Ling Wu, Cuijuan Jiang, and Bing Yan* School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China ABSTRACT: Nanoparticles, such as carbon nanotubes (CNTs), interact with cells and are easily internalized, causing various perturbations to cell functions. The mechanisms involved in such perturbations are investigated by a systematic approach that utilizes modified CNTs and various chemical−biological assays. Three modes of actions are (1) CNTs bind to different cell surface receptors and perturb different cell signaling pathways; (2) CNTs bind to a receptor with different affinity and, therefore, strengthen or weaken signals; (3) CNTs enter cells and bind to soluble signaling proteins involved in a signaling pathway. Understanding of such mechanisms not only clarifies how CNTs cause cytotoxicity but also demonstrates a useful method to modulate biological/toxicological activities of CNTs for their various industrial, biomedical, and consumer applications.
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CONTENTS
1. Introduction 2. Systematic Modification of Nanoparticle Surfaces through Nanocombinatorial Chemistry 2.1. Combinatorial and High-Throughput Chemistry for Nanosurface Structure−Activity Relationship Research 2.2. Nanocombinatorial Chemistry 3. Reducing Immune Toxicity by Switching the Activation of Cellular Signaling Pathways 3.1. Surface Modifications of MWCNTs Modulate the Immune Response to Differing Extents 3.2. Modifying MWCNT Receptor Binding to Reduce Immunotoxicity 4. Modulation of Autophagy Levels and the Triggering Mechanism by Regulating Cellular Signaling 4.1. Surface-Modified MWCNTs Modulate Autophagy to Differing Extents 4.2. Switching Autophagy Induction from an mTOR-Dependent to an mTOR-Independent Pathway 5. Modulation of Cell Differentiation by Binding to the BMP Receptor with Different Affinities 5.1. MWCNTs Bind to BMPR2 5.2. Down-regulation of BMP Signaling Enhances Myogenic Differentiation 5.3. MWCNTs with Different Surface Modifications Can Fine-Tune Myogenic Differentiation 6. Concluding Remarks Author Information Corresponding Author Funding © XXXX American Chemical Society
Notes Abbreviations References
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1. INTRODUCTION Nanotechnology has led to advancements in various industrial sectors and has improved our daily life. For example, nanotechnology and nanoparticles have been rapidly incorporated into consumer and healthcare products (up to 1700 marketed products),1 which range from antibacterial agents and dental materials to implants and disease diagnostics and treatment. For example, Verigene System from Nanosphere offers a simple testing platform for genetic diseases.2 Compared to traditional reagents, the application of nanomaterials can greatly increase diagnostic and treatment efficiency by the enhanced sensitivity and specificity.3 In addition to direct medical exposures, more and more nanoparticles are being produced, used, and released into the environment, which causes unwanted human exposure. The health effects of human exposure to healthcare products4 and environmental nanoparticles5 have increasingly attracted the attention of the public and researchers. Investigations have repeatedly shown that nanoparticles enter cells and interfere with physiological systems, causing varying degrees of perturbation or damage.5,6 Nanoparticles can bind strongly to biological molecules such as DNA, proteins, and lipids.7 Such interactions likely account for the biological and physiological perturbations caused by nanoparticles. According to our own research and the work
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Special Issue: Chemical Toxicology in China Received: November 23, 2014
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Figure 1. Molecular structures and MWCNT numbering of combinatorial MWCNT library members. Reprinted from ref 25. Copyright 2008 American Chemical Society.
2. SYSTEMATIC MODIFICATION OF NANOPARTICLE SURFACES THROUGH NANOCOMBINATORIAL CHEMISTRY 2.1. Combinatorial and High-Throughput Chemistry for Nanosurface Structure−Activity Relationship Research. Diversity and selection are natural processes for evolution, and they are important driving forces for discovery in many areas, such as drug discovery and the development of novel materials. Combinatorial chemistry can be used to maximize the structural diversity of molecules and to develop high-throughput technologies for synthesis, analysis, screening, and optimization.18 Combinatorial chemistry also has a strong association with computational chemistry in compound and experimental design, big data processing and analysis, quantitative structure−activity relationship studies, and novel lead prediction. 2.2. Nanocombinatorial Chemistry. Alternating molecules on the surfaces of nanoparticles changes the physicochemical properties of the particles and could potentially alter their biological activities. For example, adding carboxyl or amine groups to nanoparticle surfaces produces nanoparticles with distinct properties.19 Therefore, experimental modifications of the surface chemistry of nanoparticles may produce
of others, nanoparticles can alter certain signal transduction pathways by binding to receptor molecules or by affecting the normal interactions between signaling molecules. A unique property of nanoparticles is their extremely large surface area, a property that suggests that surface ligand modifications can change the interactions of nanoparticles with biomolecules and cells.8,9 One-at-a-time surface modifications on nanoparticles, although they can be useful for validation experiments, are not efficient for mechanistic studies or for effective modulation of nano-cell interactions. When properties such as the surface chemistry of nanoparticles are systematically modified, the corresponding biological perturbations are affected. Computational research reveals the nanostructure−activity relationships in such perturbations and generates models to help predict surface ligands that can be attached to nanoparticles to impart desired properties, such as low toxicity or a specific targeting ability.10−12 Carbon nanotubes13 (CNTs) possess unique structures and excellent physical and chemical properties14 that enable their wide applications in industry and medicine. CNTs can enter cells and bind to cytoplasmic proteins causing cytotoxicity. Recent studies have found that the optimized properties and the safety of CNTs can be achieved by both covalent and noncovalent modifications on their surface.15−17 B
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Figure 2. Tuning immune responses in macrophages using the combinatorial MWCNT library. (A) NO generation in THP-1 macrophages in response to MWCNTs from the combinatorial library. (B) MWCNT-COOH binds more to the mannose receptor, and MWCNT 70 preferentially binds to the scavenger receptor. This shift in receptor binding changes the consequent in vivo inflammation response. Reprinted from ref 29. Copyright 2011 American Chemical Society.
nanoparticles with diverse properties. This hypothesis has been proven through random surface modifications of nanoparticles.20−23 High-throughput and combinatorial synthesis of nanoparticle libraries13,23,24 strongly supports the idea that diverse nanoparticle properties are generated, offering greater chances for the discovery of novel agents and materials.25,26 In recent years, we have investigated cellular mechanisms using a nanocombinatorial approach.12,25−29 We have made combinatorial gold nanoparticle libraries and discovered dual-ligand cancer-targeting nanoparticles26 and specific AChE inhibitors.12 We have also developed and investigated a combinatorial multiwalled carbon nanotube (MWCNT) library.25,27−29 Pristine MWCNTs were oxidized to yield carboxylated MWCNTs. A linker molecule with two available reaction sites was then conjugated to the nanotubes. By comparing the predicted properties of a large number of commercially available amines and acylators, we were able to select 10 diverse amines and 8 diverse acylators to synthesize a combinatorial MWCNT library containing 80 members (Figure 1).25 A collection of tools was used to identify and quantify the synthesis products conjugated to the MWCNTs. For example, LC-MS,30 FTIR,25,30 elemental analysis,25 and MAS H1,25 and C13 NMR31,32 have been routinely used in the characterization of modified nanoparticles.
These cells recognize foreign matter by way of genetically encoded membrane receptors, such as Toll-like receptors, the mannose receptor, and the scavenger receptor. 33 The recognition and binding of foreign matter by these receptors activate related signaling pathways leading to immune responses. For example, the activation of the mannose receptor pathway results in inflammation, but the activation of the scavenger receptor pathway does not.34,35 Both single- and multiwalled CNTs (SWCNTs and MWCNTs) have been reported to cause perturbations in the immune system in animal models.36 We13 and others19 have shown that CNTs disturb the cell signaling pathways that govern immune responses. We have also demonstrated that surface-modified CNTs reprogram immune functions by selectively binding to different cellular receptors and activating different signaling pathways.13 3.1. Surface Modifications of MWCNTs Modulate the Immune Response to Differing Extents. Macrophages in the major reticuloendothelial system (RES) use specialized membrane receptors, such as the mannose receptor and the scavenger receptor, to recognize pathogens or foreign matter and elicit phagocytosis. For example, the mannose receptor binds microorganisms through the recognition of terminal mannose or fucose residues of the glycoproteins on the surface of microorganisms. Binding and activation of the mannose receptor typically activate downstream NFκB-mediated inflammatory responses.37 The scavenger receptors are known to recognize oxidized or negatively charged lipoproteins, and they do not induce inflammation.38 Recent studies have suggested that the cellular effects of CNTs are associated with their recognition of membrane receptors on immune cells21 and the activation of downstream signaling pathways.39,40 We hypothesize that surface chemistry modification may modulate immune responses and other signaling perturbations. Although carboxylation of MWCNTs enhances their dispersibility and reduces the toxicity caused by
3. REDUCING IMMUNE TOXICITY BY SWITCHING THE ACTIVATION OF CELLULAR SIGNALING PATHWAYS The human immune system uses innate immunity and adaptive immunity to prevent the invasion of external pathogens. Whereas innate immunity uses myeloid progenitor-derived cells to recognize and eliminate pathogens, adaptive immunity relies on B and T cells to specifically recognize foreign matter. The myeloid progenitor-derived cells include neutrophils, eosinophils, basophils, mast cells, dendritic cells, and macrophages. C
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Figure 3. Autophagy induction by MWCNT library members in LC3-GFP U87 cells. (A) Surface-modified MWCNTs (100 μg/mL) induce cell autophagy at different levels at 24 h. The scale bar represents 10 μm, and this scale applies to all panels. (B) The autophagy induction capabilities of MWCNTs are ranked into four categories. Reprinted from ref 27 Copyright 2014 American Chemical Society.
MWCNT agglomeration,41 studies have shown that carboxylated MWCNTs induce inflammation and immunotoxicity,42,43 which suggests that further modifications are needed for safe application. To screen the surface-diversified combinatorial MWCNT library, a simple and rapid high-throughput assay is needed. By detecting the generation of nitric oxide (NO), a well-known immune function marker, we measured the immune responses of macrophages to MWCNTs by high-throughput screening.29 The results show that MWCNTs with various surface modifications are able to tune macrophage activity to different levels (Figure 2A). These results show that surface modifications on MWCNTs effectively modulate their immune
toxicity, which demonstrates the power of nanocombinatorial chemistry. 3.2. Modifying MWCNT Receptor Binding to Reduce Immunotoxicity. The preferences of modified MWCNTs for specific cell surface receptors were tested by using receptorspecific inhibitors for two selected MWCNTs, the more toxic MWCNT-COOH and less toxic MWCNT 70. Comparing the effects of the receptor-specific inhibitors reveals that the cell uptake of MWCNT-COOH is more susceptible to mannose receptor inhibition, whereas MWCNT 70 is more susceptible to scavenger receptor inhibition. Thus, these two modified MWCNTs have different receptor-binding preferences. Switching from mannose receptor activation to scavenger receptor D
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4.1. Surface-Modified MWCNTs Modulate Autophagy to Differing Extents. To test whether surface modifications affect MWCNT interactions with autophagy pathways, we established a high-throughput screen using the autophagyreporting cell line LC3-GFP U87, taking advantage of the fact that LC3 proteins are recruited into autophagosomal membranes during autophagy. In this cell line, autophagy can be visualized by fluorescence, and the extent of autophagy can be measured by counting the number of green fluorescent puncta.62 The screening of the MWCNT library shows that MWCNTs with different surface modifications exhibit different levels of autophagy induction (Figure 3). Free ligands, in contrast, do not induce autophagy.27 MWCNTs may cause strong or weak autophagy due to different perturbations of mTOR signaling. However, analysis of MWCNTs that generate high levels of autophagy shows that they either release mTOR inhibition on autophagy or induce autophagy through an mTOR-independent mechanism. 4.2. Switching Autophagy Induction from an mTORDependent to an mTOR-Independent Pathway. To elucidate the mechanisms that lead to different levels of autophagy activation, we examined the effects of MWCNTCOOH and MWCNT 41 on 84 autophagy-related genes using a microarray. The results indicate that MWCNT-COOH downregulates the level of IGF1, whereas MWCNT 41 downregulates IFNA2.27 Cells use different transmembrane receptors to sense extracellular stress signals by regulating ligand expression after the recognition of foreign matter. IGF1 and IFNA2 are such ligands, and changes in their concentrations may lead to cell autophagy.63,64 Because MWCNT surface modifications may change binding preferences for cell membrane receptors, the altered chemistry on the surface of MWCNT 41 shifts its binding preference from the IGF1 receptor to the IFNA2 receptor (Figure 4). Previous findings show that inhibition of cell receptors down-regulates their respective ligands.65 The significance of these findings is 2-fold. First, nanoparticles, including CNTs, may induce cell autophagy by preventing receptors from sensing extracellular stress signals. Second, surface modifications may not only tune cell autophagy to
activation reduces the activation of the NFκB pathway. As a result, MWCNT-COOH-induced pulmonary inflammation in the lung is alleviated by using MWCNT 70 (Figure 2B).29 The above findings show that surface chemical modifications of MWCNTs shift the receptor binding on the macrophage surface from the mannose receptor to the scavenger receptor and alter the resulting inflammation in mice. CNTs are easily internalized by cells via both energy-dependent and -independent pathways.18,24 Inside the cell, CNTs are located in different subcellular organelles and interact with cytoplasmic proteins.18,44 Studies from other researchers also suggest that chemical modifications may change the binding of SWCNTs or MWCNTs to intracellular immune proteins, such as complement proteins.45−47 Altered immune toxicity correlates with changes in the levels of complement system activation. The physicochemical properties of modified SWCNTs or MWCNTs, such as surface charge, hydrophobicity, molecular volume and shape, may partially account for the selectivity of protein bindings. Such a switching of receptor binding is also observed in the regulation of another cellular function: cell autophagy.
4. MODULATION OF AUTOPHAGY LEVELS AND THE TRIGGERING MECHANISM BY REGULATING CELLULAR SIGNALING Autophagy is an evolutionarily conserved catabolic recycling process by which lysosomes degrade soluble macromolecules or organelles.48 Through this process, damaged cell components, such as misfolded proteins and aged or damaged organelles, become reusable building blocks for new biomolecules. Autophagy can help eukaryotic cells survive various stress conditions.49 During autophagy, the cytoplasmic contents are engulfed by pieces of membrane that form double-membrane structures known as autophagosomes. The autophagosomes later fuse with lysosomes, which leads to the degradation of the autophagosomal contents by lysosomal enzymes.50 The mTOR (mammalian target of rapamycin)-dependent pathway is a known signaling pathway that regulates autophagy. mTOR is a serine/threonine protein kinase. Under nutrientrich or nonstress conditions, this pathway suppresses autophagy by inhibiting another serine/threonine kinase, Ulk1, such that autophagy is limited to a basal level. Stresses, such as starvation or rapamycin treatment, relieve mTOR’s suppression of Ulk1, and the latter forms complexes with autophagy-related (Atg) proteins, leading to the formation of autophagosomes.51 In addition to the mTOR-dependent pathway, mTOR-independent signaling pathways have also been reported. Currently, four mTOR-independent signaling pathways have been identified:52 the inositol signaling pathway; the Ca2+/calpain pathway; the cAMP/Epac/Ins(1,4,5)P3 pathway; and the JNK1/Beclin-1/ PI3KC3 pathway. Dysregulation of autophagy can lead to diseases including neurological disorders53 and cancers.54,55 Therefore, proper modulation of autophagy has been considered as a novel therapeutic strategy. Recent studies have found that different nanoparticles, such as Nd2O3 nanoparticles,56 quantum dots,57,58 MnO nanocrystals,59 and gold nanoparticles,60 induce autophagy in different cells. Although the underlying mechanisms are still not known, autophagy induction is a common feature of many nanoparticles. This suggests that autophagy induction may be a source of nanotoxicity. At the same time, nanoparticles may be promising novel autophagy regulators.61
Figure 4. Surface modifications alter the receptor binding preferences of MWCNTs, leading to different levels of autophagy pathway activation. MWCNT-COOH induces mTOR-dependent autophagy by binding to IGF1R, and MWCNT 41 induces mTOR-independent autophagy by preferentially binding to IFNA2R. Reprinted from ref 27. Copyright 2014 American Chemical Society. E
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different levels but also change the underlying pathways, which could provide a feasible way to maximize the biomedical benefits of CNTs.
5. MODULATION OF CELL DIFFERENTIATION BY BINDING TO THE BMP RECEPTOR WITH DIFFERENT AFFINITIES Cell differentiation is a process by which less specialized cells, such as stem or progenitor cells, give rise to more specialized cell types under the tight regulation of signaling networks. Recently, mesenchymal stem cells have shown great promise in tissue engineering for regenerative disease treatment.66 The proliferation of mesenchymal stem cells requires various growth factors.67 Bone morphogenetic proteins (BMPs) or factors from differentiated muscle cells can induce myogenic68 or osteogenic69 differentiation in mesenchymal stem cells. 5.1. MWCNTs Bind to BMPR2. The hydrophobic nature of the CNT surface leads to preferential binding of protein domains that are rich in hydrophobic amino acids. The binding of proteins to CNTs also depends on the CNT diameter or size.70,71 Studies from our group28,72,73 and others74 have consistently found that carboxylated SWCNTs and MWCNTs down-regulate the BMP signaling pathway. MWCNTs do not affect BMP signaling initiated by constitutively active receptors (receptors remain active without the need for ligand binding and receptor heterodimer formation), which suggests that MWCNTs do not affect intracellular signal transduction steps. Using a proximity ligation assay, we found that MWCNTCOOH preferentially binds to BMPR2, not BMPR1.28 As a result of this binding, the association between receptor 2 and receptor 1 was partially inhibited. This inhibition may be caused by two possible mechanisms (Figure 5): (1) MWCNT binds to a remote site on BMPR2 and changes the conformation of the BMPR1-binding domain; and (2) MWCNT binds directly to the BMPR1-binding domain on BMPR2. 5.2. Down-regulation of BMP Signaling Enhances Myogenic Differentiation. Activation of BMP signaling triggers the expression of target genes, including the Id (inhibitor of differentiation/DNA binding) family of genes: Id1, Id2, Id3, and Id4.75 The Id genes are ubiquitously expressed in a wide variety of cell types, and they play important roles in development. In stem cells, Id proteins help maintain self-renewal and inhibit premature differentiation.76 In other cells, they strongly promote cell proliferation.77 Id proteins contain a helix−loop−helix (HLH) domain but lack a DNA-binding domain. Therefore, they primarily act as negative regulators of transcription factors with a basic HLH (bHLH) structure. In mesenchymal stem cells, myogenic differentiation is under the control of a family of transcription factors with bHLH structures, including MyoD, myf4, myf5, and myogenin.78 These proteins form heterodimers with E2A proteins that activate the expression of myogenesis-specific genes, such as MYH and desmin. The Id proteins competitively dimerize with E2A, which blocks their binding with transcription factors and inhibits the transcription of the myogenesis-specific genes.79 BMP signaling blocks myogenic differentiation by inducing the expression of Id proteins, and this process is down-regulated by MWCNTs, which increases myogenic differentiation (Figure 6). 5.3. MWCNTs with Different Surface Modifications Can Fine-Tune Myogenic Differentiation. Because inter-
Figure 5. Alternative models explaining how MWCNTs bind to BMP receptor 2 (BMPR2) and affect cell differentiation signaling. MWCNT may bind to a remote site on BMPR2 and change its conformation so that the binding of BMPR1 is disfavored (mechanism 1). Alternatively, MWCNT may compete with BMPR1 for the binding site on BMPR2 (mechanism 2). In both models, the dimerization of two receptors has been partially blocked, and the subsequent signaling steps have been affected.
Figure 6. Molecular interactions involved in MWCNT-enhanced cell differentiation. MWCNT binds to BMP receptor 2 (BMPR2) and inhibits heterodimerization between BMPR1 and BMPR2, which blocks the phosphorylation of BMPR1. This inhibition attenuates the expression of Id proteins, which are members of the negative HLH family of proteins. Lower level of Id proteins reduces the ability of these proteins to inhibit complex formation between ubiquitous Eprotein (HEB) and tissue-specific E-proteins (MyoD and myogenin). These positive HLH protein complexes activate the expression of differentiation-specific genes (myogenin and MYH) by binding to the E-box motifs in their promoter regions. Reprinted with permission from ref 28. Copyright 2012 Nature Publishing Group.
actions between MWCNTs and BMPR are governed by MWCNT surface properties, chemically modified MWCNTs F
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Figure 7. Surface-modified MWCNTs fine-tune cell differentiation. (a) The chemical structures of MWCNTs that regulate the expression of Id1 at different levels. Various levels of activation of Id1 (b) and myogenin (c) induced by MWCNTs (25 mg/mL). Effects of MWCNTs on the terminal myogenic differentiation of C2C12 cells, evaluated by the nuclear fusion index (d). (e) Western blots of proteins involved in BMP signaling and cell differentiation, showing that almost all signaling molecules are tuned to various levels. In panels b−e, −COOH indicates MWCNT-COOH. (f) Representative images showing various levels of myotube formation after treatment with different MWCNTs (25 mg/mL) for 72 h (scale bar: 100 mm). Reprinted with permission from ref 28. Copyright 2012 Nature Publishing Group.
possible to modulate myogenic differentiation to any desired level.
can have different effects on cell differentiation. This hypothesis was tested in C2C12 cells, a myogenic progenitor cell model. Different MWCNTs exhibit different inhibitory effects on BMP signaling, which is demonstrated by the altered phosphorylation levels of BMP receptor 1 and Smad1/5/8, as well as the expression of Id proteins and differentiation-regulating proteins. Consequently, various signaling activities directly induce different levels of cell differentiation28 (Figure 7). In summary, the interaction of MWCNTs and BMP receptor 2 down-regulates BMP signaling activity. As a result, the expression of the targeted Id genes is inhibited. The relief of Id protein inhibition results in the promotion of myogenic differentiation in C2C12 cells. Because the binding affinity of MWCNTs for BMP receptor 2 is controllable by MWCNT surface chemistry, a combinatorial MWCNT library makes it
6. CONCLUDING REMARKS Cellular physiological homeostasis is maintained by complex signaling networks. Considerable experimental evidence has demonstrated that surface chemistry modifications of CNTs alter the interactions between CNTs and biomolecules on the surfaces and interiors of cells. When the binding partner is a cell surface receptor, modified CNTs directly affect the cell signaling pathways. CNTs can change the binding preferences for various types of cell receptors and, therefore, can activate different signaling pathways (Figure 8, MWCNT a vs b). CNTs may also bind to the same receptor with different affinities to adjust a particular signaling pathway to particular levels of G
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Funding
This work was supported by the Natural Science Foundation of China (21137002) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030401). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS AChE, acetylcholinesterase; bHLH, basic helix−loop−helix; BMP, bone morphogenetic proteins; BMPR, bone morphogenetic protein receptor; C13 NMR, C13 nuclear magnetic resonance; cAMP, cyclic adenosine monophosphate; CNT, carbon nanotube; MWCNT, multiwalled carbon nanotube; SWCNT, singlewalled carbon nanotube; Epac, exchange protein directly activated by cAMP; FTIR, Fourier transform infrared spectroscopy; GFP, green fluorescent protein; Id, inhibitor of differentiation/DNA binding; IFNA2, interferon alpha 2; IGF-1, insulin-like growth factor 1; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; JNK1, c-Jun N-terminal kinase 1; LC-MS, liquid chromatography−mass spectrometry; LC3, microtubule-associated protein 1A/1B-light chain 3; MAS H1, magic-angle spinning H1 nuclear magnetic resonance; mTOR, mammalian target of rapamycin; NMR, nuclear magnetic resonance; NO, nitric oxide; PI3KC3, Class III phosphatidylinositol 3-kinase; RES, reticuloendothelial system; ULK1, unc51 like autophagy activating kinase 1
Figure 8. Three modes by which surface-modified MWCNTs modulate cell fate by reprogramming cell signaling pathways. Mode A: Modifications of the MWCNT determine the selectivity of receptor binding. MWCNT a binds to receptor 1 and activates signaling pathway A; MWCNT b binds to receptor 2 and activates pathway B. Mode B: Modifications of the MWCNT determine the binding affinity to a specific membrane receptor. MWCNT c exhibits a greater binding affinity for receptor 2 than MWCNT b; therefore, MWCNT c induces a stronger signal in pathway B. Mode C: A modified MWCNT may exhibit altered binding to intracellular signaling proteins45,47 and affect signaling pathway C.
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signaling (Figure 8, MWCNT b vs c). Surface-modified CNTs may also have altered affinities for intracellular signaling proteins (Figure 8, MWCNT d). These CNT−cell interactions significantly affect cellular signaling machinery and cellular functions. The physiochemical properties of the modified CNTs modulate cell functions probably by modulating their interactions with cellular signaling proteins. The dimension, hydrophobicity, and stereochemistry of the nanosurface may determine the binding domains of the target proteins. This preference may account for the selectivity of the target receptor and binding affinity. A nanocombinatorial library approach helps us modulate the interaction of nanoparticles with cells and elucidate the quantitative nanostructure−activity relationship.10−12 However, cell signaling machinery is so complex that our current understanding is only a beginning. In the future, we will further substantiate the nanocombinatorial chemistry approach with more diverse modifications of nanoparticles, and we will incorporate more nanoparticle varieties. Furthermore, various biological and chemical methods will be applied to help us understand complex nanocell interactions.
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REFERENCES
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AUTHOR INFORMATION
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*E-mail: [email protected]. H
DOI: 10.1021/tx500480d Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Reprogramming Cellular Signaling Machinery Using SurfaceModified Carbon Nanotubes Yi Zhang, Ling Wu, Cuijuan Jiang, and Bing Yan* School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China ABSTRACT: Nanoparticles, such as carbon nanotubes (CNTs), interact with cells and are easily internalized, causing various perturbations to cell functions. The mechanisms involved in such perturbations are investigated by a systematic approach that utilizes modified CNTs and various chemical−biological assays. Three modes of actions are (1) CNTs bind to different cell surface receptors and perturb different cell signaling pathways; (2) CNTs bind to a receptor with different affinity and, therefore, strengthen or weaken signals; (3) CNTs enter cells and bind to soluble signaling proteins involved in a signaling pathway. Understanding of such mechanisms not only clarifies how CNTs cause cytotoxicity but also demonstrates a useful method to modulate biological/toxicological activities of CNTs for their various industrial, biomedical, and consumer applications.
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CONTENTS
1. Introduction 2. Systematic Modification of Nanoparticle Surfaces through Nanocombinatorial Chemistry 2.1. Combinatorial and High-Throughput Chemistry for Nanosurface Structure−Activity Relationship Research 2.2. Nanocombinatorial Chemistry 3. Reducing Immune Toxicity by Switching the Activation of Cellular Signaling Pathways 3.1. Surface Modifications of MWCNTs Modulate the Immune Response to Differing Extents 3.2. Modifying MWCNT Receptor Binding to Reduce Immunotoxicity 4. Modulation of Autophagy Levels and the Triggering Mechanism by Regulating Cellular Signaling 4.1. Surface-Modified MWCNTs Modulate Autophagy to Differing Extents 4.2. Switching Autophagy Induction from an mTOR-Dependent to an mTOR-Independent Pathway 5. Modulation of Cell Differentiation by Binding to the BMP Receptor with Different Affinities 5.1. MWCNTs Bind to BMPR2 5.2. Down-regulation of BMP Signaling Enhances Myogenic Differentiation 5.3. MWCNTs with Different Surface Modifications Can Fine-Tune Myogenic Differentiation 6. Concluding Remarks Author Information Corresponding Author Funding © XXXX American Chemical Society
Notes Abbreviations References
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1. INTRODUCTION Nanotechnology has led to advancements in various industrial sectors and has improved our daily life. For example, nanotechnology and nanoparticles have been rapidly incorporated into consumer and healthcare products (up to 1700 marketed products),1 which range from antibacterial agents and dental materials to implants and disease diagnostics and treatment. For example, Verigene System from Nanosphere offers a simple testing platform for genetic diseases.2 Compared to traditional reagents, the application of nanomaterials can greatly increase diagnostic and treatment efficiency by the enhanced sensitivity and specificity.3 In addition to direct medical exposures, more and more nanoparticles are being produced, used, and released into the environment, which causes unwanted human exposure. The health effects of human exposure to healthcare products4 and environmental nanoparticles5 have increasingly attracted the attention of the public and researchers. Investigations have repeatedly shown that nanoparticles enter cells and interfere with physiological systems, causing varying degrees of perturbation or damage.5,6 Nanoparticles can bind strongly to biological molecules such as DNA, proteins, and lipids.7 Such interactions likely account for the biological and physiological perturbations caused by nanoparticles. According to our own research and the work
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Special Issue: Chemical Toxicology in China Received: November 23, 2014
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Figure 1. Molecular structures and MWCNT numbering of combinatorial MWCNT library members. Reprinted from ref 25. Copyright 2008 American Chemical Society.
2. SYSTEMATIC MODIFICATION OF NANOPARTICLE SURFACES THROUGH NANOCOMBINATORIAL CHEMISTRY 2.1. Combinatorial and High-Throughput Chemistry for Nanosurface Structure−Activity Relationship Research. Diversity and selection are natural processes for evolution, and they are important driving forces for discovery in many areas, such as drug discovery and the development of novel materials. Combinatorial chemistry can be used to maximize the structural diversity of molecules and to develop high-throughput technologies for synthesis, analysis, screening, and optimization.18 Combinatorial chemistry also has a strong association with computational chemistry in compound and experimental design, big data processing and analysis, quantitative structure−activity relationship studies, and novel lead prediction. 2.2. Nanocombinatorial Chemistry. Alternating molecules on the surfaces of nanoparticles changes the physicochemical properties of the particles and could potentially alter their biological activities. For example, adding carboxyl or amine groups to nanoparticle surfaces produces nanoparticles with distinct properties.19 Therefore, experimental modifications of the surface chemistry of nanoparticles may produce
of others, nanoparticles can alter certain signal transduction pathways by binding to receptor molecules or by affecting the normal interactions between signaling molecules. A unique property of nanoparticles is their extremely large surface area, a property that suggests that surface ligand modifications can change the interactions of nanoparticles with biomolecules and cells.8,9 One-at-a-time surface modifications on nanoparticles, although they can be useful for validation experiments, are not efficient for mechanistic studies or for effective modulation of nano-cell interactions. When properties such as the surface chemistry of nanoparticles are systematically modified, the corresponding biological perturbations are affected. Computational research reveals the nanostructure−activity relationships in such perturbations and generates models to help predict surface ligands that can be attached to nanoparticles to impart desired properties, such as low toxicity or a specific targeting ability.10−12 Carbon nanotubes13 (CNTs) possess unique structures and excellent physical and chemical properties14 that enable their wide applications in industry and medicine. CNTs can enter cells and bind to cytoplasmic proteins causing cytotoxicity. Recent studies have found that the optimized properties and the safety of CNTs can be achieved by both covalent and noncovalent modifications on their surface.15−17 B
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Figure 2. Tuning immune responses in macrophages using the combinatorial MWCNT library. (A) NO generation in THP-1 macrophages in response to MWCNTs from the combinatorial library. (B) MWCNT-COOH binds more to the mannose receptor, and MWCNT 70 preferentially binds to the scavenger receptor. This shift in receptor binding changes the consequent in vivo inflammation response. Reprinted from ref 29. Copyright 2011 American Chemical Society.
nanoparticles with diverse properties. This hypothesis has been proven through random surface modifications of nanoparticles.20−23 High-throughput and combinatorial synthesis of nanoparticle libraries13,23,24 strongly supports the idea that diverse nanoparticle properties are generated, offering greater chances for the discovery of novel agents and materials.25,26 In recent years, we have investigated cellular mechanisms using a nanocombinatorial approach.12,25−29 We have made combinatorial gold nanoparticle libraries and discovered dual-ligand cancer-targeting nanoparticles26 and specific AChE inhibitors.12 We have also developed and investigated a combinatorial multiwalled carbon nanotube (MWCNT) library.25,27−29 Pristine MWCNTs were oxidized to yield carboxylated MWCNTs. A linker molecule with two available reaction sites was then conjugated to the nanotubes. By comparing the predicted properties of a large number of commercially available amines and acylators, we were able to select 10 diverse amines and 8 diverse acylators to synthesize a combinatorial MWCNT library containing 80 members (Figure 1).25 A collection of tools was used to identify and quantify the synthesis products conjugated to the MWCNTs. For example, LC-MS,30 FTIR,25,30 elemental analysis,25 and MAS H1,25 and C13 NMR31,32 have been routinely used in the characterization of modified nanoparticles.
These cells recognize foreign matter by way of genetically encoded membrane receptors, such as Toll-like receptors, the mannose receptor, and the scavenger receptor. 33 The recognition and binding of foreign matter by these receptors activate related signaling pathways leading to immune responses. For example, the activation of the mannose receptor pathway results in inflammation, but the activation of the scavenger receptor pathway does not.34,35 Both single- and multiwalled CNTs (SWCNTs and MWCNTs) have been reported to cause perturbations in the immune system in animal models.36 We13 and others19 have shown that CNTs disturb the cell signaling pathways that govern immune responses. We have also demonstrated that surface-modified CNTs reprogram immune functions by selectively binding to different cellular receptors and activating different signaling pathways.13 3.1. Surface Modifications of MWCNTs Modulate the Immune Response to Differing Extents. Macrophages in the major reticuloendothelial system (RES) use specialized membrane receptors, such as the mannose receptor and the scavenger receptor, to recognize pathogens or foreign matter and elicit phagocytosis. For example, the mannose receptor binds microorganisms through the recognition of terminal mannose or fucose residues of the glycoproteins on the surface of microorganisms. Binding and activation of the mannose receptor typically activate downstream NFκB-mediated inflammatory responses.37 The scavenger receptors are known to recognize oxidized or negatively charged lipoproteins, and they do not induce inflammation.38 Recent studies have suggested that the cellular effects of CNTs are associated with their recognition of membrane receptors on immune cells21 and the activation of downstream signaling pathways.39,40 We hypothesize that surface chemistry modification may modulate immune responses and other signaling perturbations. Although carboxylation of MWCNTs enhances their dispersibility and reduces the toxicity caused by
3. REDUCING IMMUNE TOXICITY BY SWITCHING THE ACTIVATION OF CELLULAR SIGNALING PATHWAYS The human immune system uses innate immunity and adaptive immunity to prevent the invasion of external pathogens. Whereas innate immunity uses myeloid progenitor-derived cells to recognize and eliminate pathogens, adaptive immunity relies on B and T cells to specifically recognize foreign matter. The myeloid progenitor-derived cells include neutrophils, eosinophils, basophils, mast cells, dendritic cells, and macrophages. C
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Figure 3. Autophagy induction by MWCNT library members in LC3-GFP U87 cells. (A) Surface-modified MWCNTs (100 μg/mL) induce cell autophagy at different levels at 24 h. The scale bar represents 10 μm, and this scale applies to all panels. (B) The autophagy induction capabilities of MWCNTs are ranked into four categories. Reprinted from ref 27 Copyright 2014 American Chemical Society.
MWCNT agglomeration,41 studies have shown that carboxylated MWCNTs induce inflammation and immunotoxicity,42,43 which suggests that further modifications are needed for safe application. To screen the surface-diversified combinatorial MWCNT library, a simple and rapid high-throughput assay is needed. By detecting the generation of nitric oxide (NO), a well-known immune function marker, we measured the immune responses of macrophages to MWCNTs by high-throughput screening.29 The results show that MWCNTs with various surface modifications are able to tune macrophage activity to different levels (Figure 2A). These results show that surface modifications on MWCNTs effectively modulate their immune
toxicity, which demonstrates the power of nanocombinatorial chemistry. 3.2. Modifying MWCNT Receptor Binding to Reduce Immunotoxicity. The preferences of modified MWCNTs for specific cell surface receptors were tested by using receptorspecific inhibitors for two selected MWCNTs, the more toxic MWCNT-COOH and less toxic MWCNT 70. Comparing the effects of the receptor-specific inhibitors reveals that the cell uptake of MWCNT-COOH is more susceptible to mannose receptor inhibition, whereas MWCNT 70 is more susceptible to scavenger receptor inhibition. Thus, these two modified MWCNTs have different receptor-binding preferences. Switching from mannose receptor activation to scavenger receptor D
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4.1. Surface-Modified MWCNTs Modulate Autophagy to Differing Extents. To test whether surface modifications affect MWCNT interactions with autophagy pathways, we established a high-throughput screen using the autophagyreporting cell line LC3-GFP U87, taking advantage of the fact that LC3 proteins are recruited into autophagosomal membranes during autophagy. In this cell line, autophagy can be visualized by fluorescence, and the extent of autophagy can be measured by counting the number of green fluorescent puncta.62 The screening of the MWCNT library shows that MWCNTs with different surface modifications exhibit different levels of autophagy induction (Figure 3). Free ligands, in contrast, do not induce autophagy.27 MWCNTs may cause strong or weak autophagy due to different perturbations of mTOR signaling. However, analysis of MWCNTs that generate high levels of autophagy shows that they either release mTOR inhibition on autophagy or induce autophagy through an mTOR-independent mechanism. 4.2. Switching Autophagy Induction from an mTORDependent to an mTOR-Independent Pathway. To elucidate the mechanisms that lead to different levels of autophagy activation, we examined the effects of MWCNTCOOH and MWCNT 41 on 84 autophagy-related genes using a microarray. The results indicate that MWCNT-COOH downregulates the level of IGF1, whereas MWCNT 41 downregulates IFNA2.27 Cells use different transmembrane receptors to sense extracellular stress signals by regulating ligand expression after the recognition of foreign matter. IGF1 and IFNA2 are such ligands, and changes in their concentrations may lead to cell autophagy.63,64 Because MWCNT surface modifications may change binding preferences for cell membrane receptors, the altered chemistry on the surface of MWCNT 41 shifts its binding preference from the IGF1 receptor to the IFNA2 receptor (Figure 4). Previous findings show that inhibition of cell receptors down-regulates their respective ligands.65 The significance of these findings is 2-fold. First, nanoparticles, including CNTs, may induce cell autophagy by preventing receptors from sensing extracellular stress signals. Second, surface modifications may not only tune cell autophagy to
activation reduces the activation of the NFκB pathway. As a result, MWCNT-COOH-induced pulmonary inflammation in the lung is alleviated by using MWCNT 70 (Figure 2B).29 The above findings show that surface chemical modifications of MWCNTs shift the receptor binding on the macrophage surface from the mannose receptor to the scavenger receptor and alter the resulting inflammation in mice. CNTs are easily internalized by cells via both energy-dependent and -independent pathways.18,24 Inside the cell, CNTs are located in different subcellular organelles and interact with cytoplasmic proteins.18,44 Studies from other researchers also suggest that chemical modifications may change the binding of SWCNTs or MWCNTs to intracellular immune proteins, such as complement proteins.45−47 Altered immune toxicity correlates with changes in the levels of complement system activation. The physicochemical properties of modified SWCNTs or MWCNTs, such as surface charge, hydrophobicity, molecular volume and shape, may partially account for the selectivity of protein bindings. Such a switching of receptor binding is also observed in the regulation of another cellular function: cell autophagy.
4. MODULATION OF AUTOPHAGY LEVELS AND THE TRIGGERING MECHANISM BY REGULATING CELLULAR SIGNALING Autophagy is an evolutionarily conserved catabolic recycling process by which lysosomes degrade soluble macromolecules or organelles.48 Through this process, damaged cell components, such as misfolded proteins and aged or damaged organelles, become reusable building blocks for new biomolecules. Autophagy can help eukaryotic cells survive various stress conditions.49 During autophagy, the cytoplasmic contents are engulfed by pieces of membrane that form double-membrane structures known as autophagosomes. The autophagosomes later fuse with lysosomes, which leads to the degradation of the autophagosomal contents by lysosomal enzymes.50 The mTOR (mammalian target of rapamycin)-dependent pathway is a known signaling pathway that regulates autophagy. mTOR is a serine/threonine protein kinase. Under nutrientrich or nonstress conditions, this pathway suppresses autophagy by inhibiting another serine/threonine kinase, Ulk1, such that autophagy is limited to a basal level. Stresses, such as starvation or rapamycin treatment, relieve mTOR’s suppression of Ulk1, and the latter forms complexes with autophagy-related (Atg) proteins, leading to the formation of autophagosomes.51 In addition to the mTOR-dependent pathway, mTOR-independent signaling pathways have also been reported. Currently, four mTOR-independent signaling pathways have been identified:52 the inositol signaling pathway; the Ca2+/calpain pathway; the cAMP/Epac/Ins(1,4,5)P3 pathway; and the JNK1/Beclin-1/ PI3KC3 pathway. Dysregulation of autophagy can lead to diseases including neurological disorders53 and cancers.54,55 Therefore, proper modulation of autophagy has been considered as a novel therapeutic strategy. Recent studies have found that different nanoparticles, such as Nd2O3 nanoparticles,56 quantum dots,57,58 MnO nanocrystals,59 and gold nanoparticles,60 induce autophagy in different cells. Although the underlying mechanisms are still not known, autophagy induction is a common feature of many nanoparticles. This suggests that autophagy induction may be a source of nanotoxicity. At the same time, nanoparticles may be promising novel autophagy regulators.61
Figure 4. Surface modifications alter the receptor binding preferences of MWCNTs, leading to different levels of autophagy pathway activation. MWCNT-COOH induces mTOR-dependent autophagy by binding to IGF1R, and MWCNT 41 induces mTOR-independent autophagy by preferentially binding to IFNA2R. Reprinted from ref 27. Copyright 2014 American Chemical Society. E
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different levels but also change the underlying pathways, which could provide a feasible way to maximize the biomedical benefits of CNTs.
5. MODULATION OF CELL DIFFERENTIATION BY BINDING TO THE BMP RECEPTOR WITH DIFFERENT AFFINITIES Cell differentiation is a process by which less specialized cells, such as stem or progenitor cells, give rise to more specialized cell types under the tight regulation of signaling networks. Recently, mesenchymal stem cells have shown great promise in tissue engineering for regenerative disease treatment.66 The proliferation of mesenchymal stem cells requires various growth factors.67 Bone morphogenetic proteins (BMPs) or factors from differentiated muscle cells can induce myogenic68 or osteogenic69 differentiation in mesenchymal stem cells. 5.1. MWCNTs Bind to BMPR2. The hydrophobic nature of the CNT surface leads to preferential binding of protein domains that are rich in hydrophobic amino acids. The binding of proteins to CNTs also depends on the CNT diameter or size.70,71 Studies from our group28,72,73 and others74 have consistently found that carboxylated SWCNTs and MWCNTs down-regulate the BMP signaling pathway. MWCNTs do not affect BMP signaling initiated by constitutively active receptors (receptors remain active without the need for ligand binding and receptor heterodimer formation), which suggests that MWCNTs do not affect intracellular signal transduction steps. Using a proximity ligation assay, we found that MWCNTCOOH preferentially binds to BMPR2, not BMPR1.28 As a result of this binding, the association between receptor 2 and receptor 1 was partially inhibited. This inhibition may be caused by two possible mechanisms (Figure 5): (1) MWCNT binds to a remote site on BMPR2 and changes the conformation of the BMPR1-binding domain; and (2) MWCNT binds directly to the BMPR1-binding domain on BMPR2. 5.2. Down-regulation of BMP Signaling Enhances Myogenic Differentiation. Activation of BMP signaling triggers the expression of target genes, including the Id (inhibitor of differentiation/DNA binding) family of genes: Id1, Id2, Id3, and Id4.75 The Id genes are ubiquitously expressed in a wide variety of cell types, and they play important roles in development. In stem cells, Id proteins help maintain self-renewal and inhibit premature differentiation.76 In other cells, they strongly promote cell proliferation.77 Id proteins contain a helix−loop−helix (HLH) domain but lack a DNA-binding domain. Therefore, they primarily act as negative regulators of transcription factors with a basic HLH (bHLH) structure. In mesenchymal stem cells, myogenic differentiation is under the control of a family of transcription factors with bHLH structures, including MyoD, myf4, myf5, and myogenin.78 These proteins form heterodimers with E2A proteins that activate the expression of myogenesis-specific genes, such as MYH and desmin. The Id proteins competitively dimerize with E2A, which blocks their binding with transcription factors and inhibits the transcription of the myogenesis-specific genes.79 BMP signaling blocks myogenic differentiation by inducing the expression of Id proteins, and this process is down-regulated by MWCNTs, which increases myogenic differentiation (Figure 6). 5.3. MWCNTs with Different Surface Modifications Can Fine-Tune Myogenic Differentiation. Because inter-
Figure 5. Alternative models explaining how MWCNTs bind to BMP receptor 2 (BMPR2) and affect cell differentiation signaling. MWCNT may bind to a remote site on BMPR2 and change its conformation so that the binding of BMPR1 is disfavored (mechanism 1). Alternatively, MWCNT may compete with BMPR1 for the binding site on BMPR2 (mechanism 2). In both models, the dimerization of two receptors has been partially blocked, and the subsequent signaling steps have been affected.
Figure 6. Molecular interactions involved in MWCNT-enhanced cell differentiation. MWCNT binds to BMP receptor 2 (BMPR2) and inhibits heterodimerization between BMPR1 and BMPR2, which blocks the phosphorylation of BMPR1. This inhibition attenuates the expression of Id proteins, which are members of the negative HLH family of proteins. Lower level of Id proteins reduces the ability of these proteins to inhibit complex formation between ubiquitous Eprotein (HEB) and tissue-specific E-proteins (MyoD and myogenin). These positive HLH protein complexes activate the expression of differentiation-specific genes (myogenin and MYH) by binding to the E-box motifs in their promoter regions. Reprinted with permission from ref 28. Copyright 2012 Nature Publishing Group.
actions between MWCNTs and BMPR are governed by MWCNT surface properties, chemically modified MWCNTs F
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Figure 7. Surface-modified MWCNTs fine-tune cell differentiation. (a) The chemical structures of MWCNTs that regulate the expression of Id1 at different levels. Various levels of activation of Id1 (b) and myogenin (c) induced by MWCNTs (25 mg/mL). Effects of MWCNTs on the terminal myogenic differentiation of C2C12 cells, evaluated by the nuclear fusion index (d). (e) Western blots of proteins involved in BMP signaling and cell differentiation, showing that almost all signaling molecules are tuned to various levels. In panels b−e, −COOH indicates MWCNT-COOH. (f) Representative images showing various levels of myotube formation after treatment with different MWCNTs (25 mg/mL) for 72 h (scale bar: 100 mm). Reprinted with permission from ref 28. Copyright 2012 Nature Publishing Group.
possible to modulate myogenic differentiation to any desired level.
can have different effects on cell differentiation. This hypothesis was tested in C2C12 cells, a myogenic progenitor cell model. Different MWCNTs exhibit different inhibitory effects on BMP signaling, which is demonstrated by the altered phosphorylation levels of BMP receptor 1 and Smad1/5/8, as well as the expression of Id proteins and differentiation-regulating proteins. Consequently, various signaling activities directly induce different levels of cell differentiation28 (Figure 7). In summary, the interaction of MWCNTs and BMP receptor 2 down-regulates BMP signaling activity. As a result, the expression of the targeted Id genes is inhibited. The relief of Id protein inhibition results in the promotion of myogenic differentiation in C2C12 cells. Because the binding affinity of MWCNTs for BMP receptor 2 is controllable by MWCNT surface chemistry, a combinatorial MWCNT library makes it
6. CONCLUDING REMARKS Cellular physiological homeostasis is maintained by complex signaling networks. Considerable experimental evidence has demonstrated that surface chemistry modifications of CNTs alter the interactions between CNTs and biomolecules on the surfaces and interiors of cells. When the binding partner is a cell surface receptor, modified CNTs directly affect the cell signaling pathways. CNTs can change the binding preferences for various types of cell receptors and, therefore, can activate different signaling pathways (Figure 8, MWCNT a vs b). CNTs may also bind to the same receptor with different affinities to adjust a particular signaling pathway to particular levels of G
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Funding
This work was supported by the Natural Science Foundation of China (21137002) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030401). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS AChE, acetylcholinesterase; bHLH, basic helix−loop−helix; BMP, bone morphogenetic proteins; BMPR, bone morphogenetic protein receptor; C13 NMR, C13 nuclear magnetic resonance; cAMP, cyclic adenosine monophosphate; CNT, carbon nanotube; MWCNT, multiwalled carbon nanotube; SWCNT, singlewalled carbon nanotube; Epac, exchange protein directly activated by cAMP; FTIR, Fourier transform infrared spectroscopy; GFP, green fluorescent protein; Id, inhibitor of differentiation/DNA binding; IFNA2, interferon alpha 2; IGF-1, insulin-like growth factor 1; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; JNK1, c-Jun N-terminal kinase 1; LC-MS, liquid chromatography−mass spectrometry; LC3, microtubule-associated protein 1A/1B-light chain 3; MAS H1, magic-angle spinning H1 nuclear magnetic resonance; mTOR, mammalian target of rapamycin; NMR, nuclear magnetic resonance; NO, nitric oxide; PI3KC3, Class III phosphatidylinositol 3-kinase; RES, reticuloendothelial system; ULK1, unc51 like autophagy activating kinase 1
Figure 8. Three modes by which surface-modified MWCNTs modulate cell fate by reprogramming cell signaling pathways. Mode A: Modifications of the MWCNT determine the selectivity of receptor binding. MWCNT a binds to receptor 1 and activates signaling pathway A; MWCNT b binds to receptor 2 and activates pathway B. Mode B: Modifications of the MWCNT determine the binding affinity to a specific membrane receptor. MWCNT c exhibits a greater binding affinity for receptor 2 than MWCNT b; therefore, MWCNT c induces a stronger signal in pathway B. Mode C: A modified MWCNT may exhibit altered binding to intracellular signaling proteins45,47 and affect signaling pathway C.
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signaling (Figure 8, MWCNT b vs c). Surface-modified CNTs may also have altered affinities for intracellular signaling proteins (Figure 8, MWCNT d). These CNT−cell interactions significantly affect cellular signaling machinery and cellular functions. The physiochemical properties of the modified CNTs modulate cell functions probably by modulating their interactions with cellular signaling proteins. The dimension, hydrophobicity, and stereochemistry of the nanosurface may determine the binding domains of the target proteins. This preference may account for the selectivity of the target receptor and binding affinity. A nanocombinatorial library approach helps us modulate the interaction of nanoparticles with cells and elucidate the quantitative nanostructure−activity relationship.10−12 However, cell signaling machinery is so complex that our current understanding is only a beginning. In the future, we will further substantiate the nanocombinatorial chemistry approach with more diverse modifications of nanoparticles, and we will incorporate more nanoparticle varieties. Furthermore, various biological and chemical methods will be applied to help us understand complex nanocell interactions.
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DOI: 10.1021/tx500480d Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
