Binding of pulmonary surfactant proteins to carbon nanotubes; potential for damage to lung immune defense mechanisms

doi:10.1016/j.carbon.2006.10.011

Carbon

Volume 45, Issue 3, March 2007, Pages 607-617

https://doi.org/10.1016/j.carbon.2006.10.011 Get rights and content

Abstract

Potential pulmonary toxicity of carbon nanotubes is a research area that has received considerable attention. Surfactant proteins A and D (SP-A and SP-D) are collectin proteins that are secreted by airway epithelial cells in the lung. They play an important role in first-line defense against infection within the lung. The aim of this study was to investigate the interaction between carbon nanotubes and proteins contained in lung surfactant. By using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, a novel technique of affinity chromatography based on carbon nanotube–Sepharose matrix [1] and electron microscopy data it was shown that SP-A and SP-D selectively bind to carbon nanotubes. The binding was Ca²⁺-ion dependent, and was variable between batches of nanotubes. It was therefore likely to be mediated by surface impurities or chemical modifications of the nanotubes. Chronic level exposure to carbon nanotubes may result in sequestration of SP-D and SP-A. Absence of these proteins in knockout mice leads to susceptibility to lung infection and emphysema.

Introduction

Carbon nanotubes are widely considered to be one of the most versatile families of new materials and will be instrumental in driving forward the nanotechnology industrial revolution in the coming decade. Reported uses in fields as diverse as polymers, electronics or even health care, while promising significant improvements in quality of life, will require production of nanotubes to reach industrial levels. It is therefore essential to investigate thoroughly the effects that carbon nanotubes might have on human health. Three distinct entry routes into the human body are recognised: ingestion, penetration through the skin and inhalation. The last probably constitutes the highest risk as it is notably difficult to deal reliably with suspension in air of extremely small particles. Carbon nanotubes tend to form aggregates, many of which will not penetrate to the lung alveoli when inhaled. However, non-aggregated carbon nanotubes may, because of their small size, penetrate to the alveoli of the lungs, where they may interact with surfactant proteins and lipids.

While in vivo toxicity studies previously reported represent an important first step in highlighting the possible risks associated with exposure to carbon nanotubes, the protocols they rely upon may have produced artefacts that can be difficult to distinguish from effects produced by nanotubes themselves. For example, in instillation studies the aggregation of nanotubes produces mortality by airway obstruction in rats as reported by Warheit et al. [2] and Muller et al. [3]. Further studies are needed to elucidate how carbon nanotubes interact with host pulmonary systems at the molecular level.

The lungs are an important interface between the host and an environment that contains a plethora of potentially harmful microorganisms. The immune defense of the lung involves the pulmonary surfactant, a complex mixture of lipids, phospholipids, and proteins important for normal respiratory function [4]. Two of these surfactant proteins are SP-A and SP-D (Fig. 1), which belong to a family known as the collectins, as they contain a collagenous region and a C-type lectin domain. A major biological role of the collectins is to bind to targets (e.g., microorganisms and allergens) by recognizing patterns of carbohydrate distribution at their surface and to enhance phagocytosis/clearance of the targets. Thus, they play an important role in first-line immune defenses within the lung.

In this communication we report for the first time the selective binding of SP-A and SP-D to carbon nanotubes. These results enable us to shed new light on previously published toxicity studies and also reveal hitherto uncharacterised variability in the nanotubes. The results suggest that long-term exposure to inhalation of nanotubes has the potential to enhance susceptibility to infection and emphysema.

Section snippets

Synthesis of carbon nanotube samples

Catalytic vapor deposition double-walled nanotubes (DWNT) were made as described by Flahaut et al. [5]. After the synthesis the sample was washed with a concentrated aqueous solution of HCl (ca. 12.1 M) to dissolve all the accessible catalyst (including all MgO-based catalyst and non-protected metal particles). The sample was filtered and washed to neutrality. A final rinse was given with ethanol before drying. Subsequently, the carbon nanotube sample was dried at 80 °C in air.

Elemental analysis

Plasma atomic

Results

SDS-PAGE analysis of DWNTs after exposure to BALF (Fig. 3, track 3) shows that in the presence of ethylene diamine tetra acetic acid (EDTA) almost no protein from the BALF supernatant binds to the DWNTs. However in the presence of Ca²⁺ ions several proteins (6–8 bands on SDS-PAGE) bind, and are easily detectable (Fig. 3, track 6). By mass spectrometry analysis the BALF supernatant proteins attached to DWNTs were identified (Fig. 4). Transferrin, Human Serum Albumin (HSA), and IgG were

Discussion

Binding of human plasma proteins including C1q, fibrinogen and high density lipoproteins to carbon nanotubes has been characterised by SDS-PAGE, mass spectrometry and N-terminal sequence analysis [1]. These binding reactions do not require divalent metal ions. In contrast the selective binding of BALF supernatant SP-A and SP-D to carbon nanotubes is Ca²⁺-ion dependent. The binding of SP-A and SP-D to carbon nanotubes can be stopped by using Ca²⁺-ion chelators such as EDTA (Fig. 3, track 3).

Conclusion

In conclusion, this study contributes to the elucidation of a molecular mechanism involved in pulmonary toxicity of carbon nanotubes. In addition, this communication opens up new routes of research in this area, aimed at obtaining a better understanding of the role of the sequestration of SP-A and SP-D by carbon nanotubes. It also suggests that functionalisation while lowering the toxicity in some nanomaterials might also have toxicity implications of its own [32]. More studies are needed to

Acknowledgments

This work was supported partly by the Medical Research Council, UK. C. Salvador-Morales acknowledges the Mexican National Council for Science and Technology (CONACYT) for a Graduate scholarship. We thank Mr. A.C. Willis for the N-terminal sequence analysis measurements and Miss Roona Deb for providing the SP-A protein.

References (34)

C. Salvador-Morales et al.
Complement activation and protein adsorption by carbon nanotubes
Mol Immunol
(2006)
J. Muller et al.
Respiratory toxicity of multi-wall carbon nanotubes
Toxicol Appl Pharmacol
(2005)
N. Palaniyar et al.
Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin
J Biol Chem
(2004)
A. Suwabe et al.
Calcium dependent association of surfactant protein A with pulmonary surfactant: application to simple surfactant protein A purification
Arch Biochem Biophys
(1996)
P. Strong et al.
A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid
J Immunol Methods
(1998)
H.J. Hoppe et al.
Trimeric C-type lectin domain in host defence
Structure
(1994)
K. Hakansson et al.
Crystal structure of the trimeric α-helical coiled-coil and the three lectin domains of human lung surfactant protein D
Structure
(1999)
J.F. Head et al.
Crystal structure of trimeric carbohydrate recognition and neck domains of surfactant protein A
J Biol Chem
(2003)
T.R. Korfhagen et al.
Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo
J Biol Chem
(1998)
T.R. Korfhagen et al.
Surfactant protein A (SP-A) gene targeted mice
Biochim Biophys Acta
(1998)

J. Muller et al.

Respiratory toxicity of carbon nanotubes: how worried should we be?

Carbon

(2006)

D.B. Warheit et al.

Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats

Toxicol Sci

(2004)

T.P. Hickling et al.

Collectins and their role in lung immunity

J Leukoc Biol

(2004)

E. Flahaut et al.

Gram-scale CCVD synthesis of double-walled carbon nanotubes

Chem Commun

(2003)

J.U. Kim et al.

Raman and IR spectroscopy of chemically processed single-walled carbon nanotubes

J Am Chem Soc

(2005)

E. Murray et al.

Expression of surfactant protein D in the human gastric mucosa and during helicobacter pyroli infection

Infect Immun

(2002)

M.M. Bradford

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing in the principle of protein-dye binding

Anal Biochem

(1978)

Cited by (96)

Development of PNC-27 targeted codelivery system for survivin-shRNA and SN38 against colon adenocarcinoma in vitro and in vivo
2022, Journal of Drug Delivery Science and Technology
Combination therapy, as a treatment modality with simultaneous use of two or more therapeutic agents, could provide more attention in the field of cancer therapy. In this regard, the combination of anti-cancer agents could increase the drug efficacy due to reduction of tumor growth and metastasis as well as apoptosis induction while reducing the drug resistance. In the current study, a single-walled carbon nanotube (SWCNT) nanoparticulate carrier, modified with polyethylene glycol and branched polyethylenimine (PEI 10 kDa), was synthesized for the simultaneous delivery of plasmid encoding survivin (psur) shRNA and SN38. The aforementioned nanoparticle was tagged with a targeting peptide, PNC27, for enhancing the internalization of therapeutic agents to cancerous cells. Competition assay using flow cytometry (FCM) experiment revealed the higher uptake of the targeted platform into C26 cells, treated with targeted carrier whereas cells treated with either untargeted NP or pretreated with an excess amount of free PNC27 peptide exhibited lower internalization efficiency. Furthermore, compared with either irinotecan at high dose or SWCNT-PEG10%PEI(10Br)/SN38/pEGFP at an equivalent dose, SWCNT-PEG10%PEI(10Br)/SN38/psur shRNA and SWCNT-PEG10%PEI(10Br)/SN38/psur shRNA/PNC27 significantly reduced the tumor volume in C26 tumorized female BALB/c mice, demonstrating the synergistic effects of SN38 and psur shRNA. In addition, the carrier conjugated with PNC27 facilitated the accumulation of psur shRNA and SN38 in cancer tissue causing a substantial inhibitory effect compared to that of untargeted groups. It could be concluded that the novel modified SWCNT loaded with SN38 and psur shRNA could enhance the therapeutic efficacy in vitro and in vivo, thereby providing an innovative approach for cancer treatment.
Far-reaching advances in the role of carbon nanotubes in cancer therapy
2020, Life Sciences
Cancer includes a group of diseases involving unregulated cell growth with the potential to invade or expand to other parts of the body, resulting in an estimate of 9.6 million deaths worldwide in 2018. Manifold studies have been conducted to design more efficacious techniques for cancer therapy due to the inadequacy of conventional treatments including chemotherapy, surgery, and radiation therapy. With the advances in the biomedical applications of nanotechnology-based systems, nanomaterials have gained increasing attention as promising vehicles for targeted cancer therapy and optimizing treatment outcomes. Owing to their outstanding thermal, electrical, optical and chemical properties, carbon nanotubes (CNTs) have been profoundly studied to explore the various perspectives of their application in cancer treatment. The current study aims to review the role of CNTs whether as a carrier or mediator in cancer treatment for enhancing the efficacy as well as the specificity of therapy and reducing adverse side effects. This comprehensive review indicates that CNTs have the capability to be the next generation nanomaterials to actualize noninvasive targeted eradication of tumors. However, further studies are needed to evaluate the consequences of their biomedical application before the transition into clinical trials, since possible adverse effects of CNTs on biological systems have not been clearly understood.
Qualitative and quantitative analysis of the biophysical interaction of inhaled nanoparticles with pulmonary surfactant by using quartz crystal microbalance with dissipation monitoring
2019, Journal of Colloid and Interface Science
Understanding the interaction between inhaled nanoparticles and pulmonary surfactant is a prerequisite for predicting the fate of inhaled nanoparticles. Here, we introduce a quartz crystal microbalance with dissipation monitoring (QCM-D)-based methodology to reveal the extent and nature of the biophysical interactions of polymer- and lipid-based nanoparticles with pulmonary surfactant. By fitting the QCM-D data to the Langmuir adsorption equation, we determined the kinetics and equilibrium parameters [i.e., maximal adsorption (Δm_max), equilibrium constant (K_a), adsorption rate constant (k_a) and desorption rate constant (k_d)] of polymeric nanoparticles adsorption onto the pulmonary surfactant (e.g., an artificial lipid mixture and an extract of porcine lung surfactant). Furthermore, our results revealed that the nature of the interactions between lipid-based nanoparticles (e.g., liposomes) and pulmonary surfactant was governed by the liposomal composition, i.e., incorporation of cholesterol and PEGylated phospholipid (DSPE-PEG₂₀₀₀) into DOPC-based liposomes led to the adsorption of intact liposomes onto the pulmonary surfactant layer and the mass exchange between the liposomes and pulmonary surfactant layer, respectively. In conclusion, we demonstrate the applicability of the QCM-D technique for qualitative and quantitative analysis of the biophysical interaction of inhaled nanoparticles with pulmonary surfactant, which is vital for rational design and optimization of inhalable nanomedicines.
High-throughput virtual screening to rationally design protein - Carbon nanotube interactions. Identification and preparation of stable water dispersions of protein - Carbon nanotube hybrids and efficient design of new functional materials
2019, Carbon
Carbon nanotubes, CNTs, and proteins could not be more chemically and physically distant. CNTs are hardly soluble and strongly hydrophobic. They are smooth wires, mechanically strong, with electronic properties that make them unique. Many proteins are water soluble and hydrophilic. They are highly corrugated and elastic. They can recognize a target molecule with high selectivity and sensitivity and can have catalytic activity. Integration of the remarkably different features of CNTs and proteins would create a new class of multifunctional materials. The conundrum to solve lies in how to best match proteins and CNTs. High-throughput virtual screening is used to predict the ability of 1207 proteins to recognize carbon tubes with a well-defined 1.3 nm diameter. The propensity for formation of protein-CNT hybrids is ranked. Experiments carried out in this work validated the computational results and show that the identified proteins are able to bind and disperse in water the selected CNTs. The highest scoring proteins are further examined in detail to identify general rules for binding and discussed for a variety of practical applications.
Interaction of nano carbon particles and anthracene with pulmonary surfactant: The potential hazards of inhaled nanoparticles
2019, Chemosphere
Understanding the alteration of the air-liquid interfacial properties of pulmonary surfactant (PS) in the presence of nanoparticles (NPs) and polycyclic aromatic hydrocarbons (PAHs) is particularly important for pulmonary risk assessment. Here, we investigated the interaction of natural PS (extracted from pig's lungs) with nano carbon particles (NCPs) and anthracene as a representative PAH. Our results showed that PS exhibited a significant solubilization effect on anthracene. Solubilization experiment for the substructures of PS demonstrated that the mixed phospholipid components of PS played the primary role in the solubilization of PS for anthracene. Adsorption experiment indicated that in the mixed system of PS, NCPs, and anthracene, PS can inhibit the adsorption of anthracene on NCPs due to the solubilization, agglomeration, and competitive adsorption. In addition, the surface tension, phase behavior, and foaming ability of PS were obviously altered in the presence of NCPs. These findings indicate that the solubilization effect of PS on anthracene, the inhibitive effect of PS for the adsorption of anthracene on NCPs, and the alternation of air-liquid interfacial properties of PS containing NCPs may increase the pulmonary risk in the exposure of atmospheric environment containing both PAHs and NCPs.
The asbestos-carbon nanotube analogy: An update
2018, Toxicology and Applied Pharmacology
Nanotechnology is an emerging industry based on commercialization of materials with one or more dimensions of 100 nm or less. Engineered nanomaterials are currently incorporated into thin films, porous materials, liquid suspensions, or filler/matrix nanocomposites with future applications predicted in energy and catalysis, microelectronics, environmental sensing and remediation, and nanomedicine. Carbon nanotubes are one-dimensional fibrous nanomaterials that physically resemble asbestos fibers. Toxicologic studies in rodents demonstrated that some types of carbon nanotubes can induce mesothelioma, and the World Health Organization evaluated long, rigid multiwall carbon nanotubes as possibly carcinogenic for humans in 2014. This review summarizes key physicochemical similarities and differences between asbestos fibers and carbon nanotubes. The “fiber pathogenicity paradigm” has been extended to include carbon nanotubes as well as other high-aspect-ratio fibrous nanomaterials including metallic nanowires. This paradigm identifies width, length, and biopersistence of high-aspect-ratio fibrous nanomaterials as critical determinants of lung disease, including mesothelioma, following inhalation. Based on recent theoretical modeling studies, a fourth factor, mechanical bending stiffness, will be considered as predictive of potential carcinogenicity. Novel three-dimensional lung tissue platforms provide an opportunity for in vitro screening of a wide range of high aspect ratio fibrous nanomaterials for potential lung toxicity prior to commercialization.

View all citing articles on Scopus

View full text

Binding of pulmonary surfactant proteins to carbon nanotubes; potential for damage to lung immune defense mechanisms

Abstract

Introduction

Section snippets

Synthesis of carbon nanotube samples

Elemental analysis

Results

Discussion

Conclusion

Acknowledgments

Mol Immunol

Toxicol Appl Pharmacol

J Biol Chem

Arch Biochem Biophys

J Immunol Methods

Structure

Structure

J Biol Chem

J Biol Chem

Biochim Biophys Acta

Carbon

Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats

Toxicol Sci

Collectins and their role in lung immunity

J Leukoc Biol

Gram-scale CCVD synthesis of double-walled carbon nanotubes

Chem Commun

Raman and IR spectroscopy of chemically processed single-walled carbon nanotubes

J Am Chem Soc

Expression of surfactant protein D in the human gastric mucosa and during helicobacter pyroli infection

Infect Immun

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing in the principle of protein-dye binding

Anal Biochem