Selective Uptake of Carboxylated Multi-Walled Carbon Nanotubes by Class A Type 1 Scavenger Receptors and Impaired Phagocytosis in Alveolar Macrophages.

The production and applications of multi-walled carbon nanotubes (MWNTs) have increased despite evidence that MWNTs can be toxic. Recently, we reported that the binding of Pluronic® F-108 (PF108)-coated carboxylated MWNTs (C-MWNTs) to macrophages is inhibited by class A scavenger receptors (SR-As) antagonists (R. Wang et al., 2018. Nanotoxicology 12:677-690). The current study investigates the uptake of PF108-coated MWNTs by macrophages lacking SR-A1 and by CHO cells that ectopically express SR-A1. Macrophages without SR-A1 failed to take up C-MWNTs and CHO cells that expressed SR-A1 did take up C-MWNTs, but not pristine MWNTs (P-MWNTs) or amino-functionalized MWNTs (N-MWNTs). The dependence of C-MWNT uptake on SR-A1 is strong evidence that SR-A1 is a receptor for C-MWNTs. The consequences of SR-A1-dependent C-MWNT accumulation on cell viability and phagocytic activity in macrophages were also studied. C-MWNTs were more toxic than P-MWNTs and N-MWNTs in cell proliferation and colony formation tests. C-MWNTs reduced surface SR-A1 levels in RAW 264.7 cells and impaired phagocytic uptake of three known SR-A1 ligands, polystyrene beads, heat-killed E. coli, and oxLDL. Altogether, results of this study confirmed that SR-A1 receptors are important for the selective uptake of PF108-coated C-MWNTs and that accumulation of the C-MWNTs impairs phagocytic activity and cell viability in macrophages.

Quantitation of cell-associated carbon nanotubes: selective binding and accumulation of carboxylated carbon nanotubes by macrophages.

To understand the influence of carboxylation on the interaction of carbon nanotubes with cells, the amount of pristine multi-walled carbon nanotubes (P-MWNTs) or carboxylated multi-walled carbon nanotubes (C-MWNTs) coated with Pluronic® F-108 that were accumulated by macrophages was measured by quantifying CNTs extracted from cells. Mouse RAW 264.7 macrophages and differentiated human THP-1 (dTHP-1) macrophages accumulated 80-100 times more C-MWNTs than P-MWNTs during a 24-h exposure at 37 °C. The accumulation of C-MWNTs by RAW 264.7 cells was not lethal; however, phagocytosis was impaired as subsequent uptake of polystyrene beads was reduced after a 20-h exposure to C-MWNTs. The selective accumulation of C-MWNTs suggested that there might be receptors on macrophages that bind C-MWNTs. The binding of C-MWNTs to macrophages was measured as a function of concentration at 4 °C in the absence of serum to minimize the potential interference by serum proteins or temperature-dependent uptake processes. The result was that the cells bound 8.7 times more C-MWNTs than P-MWNTs, consistent with the selective accumulation of C-MWNTs at 37 °C. In addition, serum strongly antagonized the binding of C-MWTS to macrophages, suggesting that serum contained inhibitors of binding. Moreover, inhibitors of class A scavenger receptor (SR-As) reduced the binding of C-MWNTs by about 50%, suggesting that SR-As contribute to the binding and endocytosis of C-MWNTs in macrophages but that other receptors may also be involved. Altogether, the evidence supports the hypothesis that macrophages contain binding sites selective for C-MWNTs that facilitate the high accumulation of C-MWNTs compared to P-MWNTs.

Detecting Sonolysis of Polyethylene Glycol Upon Functionalizing Carbon Nanotubes.

Polyethylene glycol (PEG) and related polymers are often used in the solubilization and noncovalent functionalization of carbon nanomaterials by sonication. For example, carbon nanotubes are frequently sonicated with PEG-containing surfactants of the Pluronic® series or phospholipid-PEG polymers to noncovalently functionalize the nanotubes. However, PEG is very sensitive to degradation upon sonication and the degradation products can be toxic to mammalian cells and to organisms such as zebrafish embryos. It is therefore useful to have a simple and inexpensive method to determine the extent of potential PEG sonolysis, as described in this chapter. Intact PEG polymers and degraded fragments are resolved on sodium dodecyl sulfate polyacrylamide gels by electrophoresis and visualized by staining with barium iodine (BaI2). Digitized images of gels are acquired using a flatbed photo scanner and the intensities of BaI2-stained PEG bands are quantified using ImageJ software. Degradation of PEG polymers after sonication is readily detected by the reduction of band intensities in gels compared to those of non-sonicated, intact PEG polymers. In addition, the approach can be used to rapidly screen various sonication conditions to identify those that might minimize PEG degradation to acceptable levels.

The impact of subcellular location on the near infrared-mediated thermal ablation of cells by targeted carbon nanotubes.

Single-walled carbon nanotubes (SWNTs) are used in the near infrared (NIR)-mediated thermal ablation of tumor cells because they efficiently convert absorbed NIR light into heat. Despite the therapeutic potential of SWNTs, there have been no published studies that directly quantify how many SWNTs need be associated with a cell to achieve a desired efficiency of killing, or what is the most efficient subcellular location of SWNTs for killing cells. Herein we measured dose response curves for the efficiency of killing correlated to the measured amounts of folate-targeted SWNTs that were either on the surface or within the vacuolar compartment of normal rat kidney cells. Folate-targeted SWNTs on the cell surface were measured after different concentrations of SWNTs in medium were incubated with cells for 30 min at 4 °C. Folate-targeted SWNTs within the vacuolar compartments were measured after cells were incubated with different concentrations of SWNTs in medium for 6 h at 37 °C. It was observed that a SWNT load of ∼13 pg/cell when internalized was sufficient to kill 90% of the cells under standardized conditions of NIR light irradiation. When ∼3.5 pg/cell of SWNTs were internalized within the endosomal/lysosomal compartments, ∼50% of the cells were killed, but when ∼3.5 pg/cell of SWNTs were confined to the cell surface only ∼5% of the cells were killed under the same NIR irradiation conditions. The SWNT subcellular locations were verified using Raman imaging of SWNTs merged with fluorescence images of known subcellular markers. To our knowledge, this is the first time that SWNT amounts at known subcellular locations have been correlated with a dose-normalized efficacy of thermal ablation and the results support the idea that SWNTs confined to the plasma membrane are not as effective in NIR-mediated cell killing as an equivalent amount of SWNTs when internalized within the endosomal/lysosomal vesicles.

Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108.

Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity.

Rapid detection of polyethylene glycol sonolysis upon functionalization of carbon nanomaterials.

Polyethylene glycol (PEG) and related polymers are often used in the functionalization of carbon nanomaterials in procedures that involve sonication. However, PEG is very sensitive to sonolytic degradation and PEG degradation products can be toxic to mammalian cells. Thus, it is imperative to assess potential PEG degradation to ensure that the final material does not contain undocumented contaminants that can introduce artifacts into experimental results. Described here is a simple and inexpensive polyacrylamide gel electrophoresis method to detect the sonolytic degradation of PEG. The method was used to monitor the integrity of PEG phospholipid constructs and branched chain PEGs after different sonication times. This approach not only helps detect degraded PEG, but should also facilitate rapid screening of sonication parameters to find optimal conditions that minimize PEG damage.

A carbon nanotube-based Raman-imaging immunoassay for evaluating tumor targeting ligands.

Herein, we describe a versatile immunoassay that uses biotinylated single-walled carbon nanotubes (SWNTs) as a Raman label, avidin-biotin chemistry to link targeting ligands to the label, and confocal Raman microscopy to image whole cells. Using a breast tumor cell model, we demonstrate the usefulness of the method to assess membrane receptor/ligand systems by evaluating a monoclonal antibody, Her-66, known to target the Her2 receptors that are overexpressed on these cells. We present two-dimensional Raman images of the cellular distribution of the SWNT labels corresponding to the distribution of the Her2 receptors in different focal planes through the cell with validation of the method using immunofluorescence microscopy, demonstrating that the Her-66-SWNT complexes were targeted to Her2 cell receptors.

Use of gel electrophoresis and Raman spectroscopy to characterize the effect of the electronic structure of single-walled carbon nanotubes on cellular uptake.

It is well-known that the uptake of single-walled carbon nanotubes (SWNTs) by living cells depends on factors such as SWNT length and surface chemistry. Surprisingly, little is known about whether the electronic structure of a SWNT influences uptake. One reason for this has been the lack of methods to measure the uptake of SWNTs by cell populations. Previously, we developed a rapid, sensitive, and label-free sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) method for measuring the amount of SWNTs in lysates prepared from cultured cells ( Wang et al. Anal. Chem. 2009 , 81 , 2944 ). Herein, we describe the use of SDS-PAGE and microprobe Raman spectroscopy to detect and distinguish the electronic structure of SWNTs internalized by mammalian cells. Using normal rat kidney (NRK) cells and SWNTs dispersed with bovine serum albumin (BSA), we demonstrate that the method can detect both metallic and semiconducting SWNTs in lysates of cells that had internalized BSA-SWNTs and that the uptake of BSA-SWNTs by NRK cells is not influenced by SWNT electronic structure.

Generation of toxic degradation products by sonication of Pluronic® dispersants: implications for nanotoxicity testing.

Poloxamers (known by the trade name Pluronic®) are triblock copolymer surfactants that contain two polyethylene glycol blocks and one polypropylene glycol block of various sizes. Poloxamers are widely used as nanoparticle dispersants for nanotoxicity studies wherein nanoparticles are sonicated with a dispersant to prepare suspensions. It is known that poloxamers can be degraded during sonication and that reactive oxygen species contribute to the degradation process. However, the possibility that poloxamer degradation products are toxic to mammalian cells has not been well studied. We report here that aqueous solutions of poloxamer 188 (Pluronic® F-68) and poloxamer 407 (Pluronic® F-127) sonicated in the presence or absence of multi-walled carbon nanotubes (MWNTs) can became highly toxic to cultured cells. Moreover, toxicity correlated with the sonolytic degradation of the polymers. These findings suggest that caution should be used in interpreting the results of nanotoxicity studies where the potential sonolytic degradation of dispersants was not controlled.

STUDY OF THE NEAR INFRARED-MEDIATED HEATING OF DISPERSIONS OF PROTEIN-COATED PRISTINE AND CARBOXYLATED SINGLE-WALLED CARBON NANOTUBES.

Previously, we demonstrated the selective NIR-mediated ablation of tumor cells in vitro using pristine single-walled carbon nanotubes (SWNTs) with adsorbed tumor-targeting ligands and carboxylated SWNTs with covalently-attached ligands. The covalent approach is advantageous in ensuring that protein ligands remain associated with the NIR-absorbing SWNTs in biological matrices and the noncovalent approach has the advantage of enabling SWNT functionalization without perturbation of the SWNT lattice and photothermal properties. Herein, we compare the ability of moderately-carboxylated (~4 at.% carboxylic acid groups) and pristine SWNT materials to absorb 808 nm radiation and convert it to heat. Under conditions of a constant 808 nm laser power density, the approach involved measuring the temperature of aqueous dispersions of protein-coated SWNTs as a function of the irradiation time. Nearly identical temperature profiles were observed for dispersions of moderately-carboxylated and pristine SWNTs possessing matched 808 nm optical densities and equivalent concentrations of carbonaceous species (i.e., SWNTs and amorphous carbon impurities). The results indicate that the amount of carbonaceous species in purified dispersions of protein-coated SWNTs is more important for converting absorbed 808 nm radiation into heat than whether or not the SWNTs were moderately carboxylated, and that moderately-carboxylated SWNTs could be the SWNT-material of choice for the targeted photothermal ablation of tumor cells.

Cytotoxicity screening of single-walled carbon nanotubes: detection and removal of cytotoxic contaminants from carboxylated carbon nanotubes.

This study compares the cytotoxicity to cultured mammalian cells of nine different single-walled carbon nanotube (SWNT) products synthesized by a variety of methods and obtained from a cross section of vendors. A standard procedure involving sonication and centrifugation in buffered bovine serum albumin was developed to disperse all the SWNTs in a biocompatible solution to facilitate comparisons. The effect of the SWNTs on the proliferative ability of a standard cell line was then assessed. Of the nine different SWNT materials tested, only two were significantly toxic, and both were functionalized by carboxylation from different vendors. This was unexpected because carboxylation makes SWNTs more water-soluble, which would presumably correlate with better biocompatibility. However, additional purification work demonstrated that the toxic material in the carboxylated SWNT preparations could be separated from the SWNTs by filtration. The filtrate that contained the toxic activity also contained abundant small carbon fragments that had Raman signatures characteristic of amorphous carbon species, suggesting a correlation between toxicity and oxidized carbon fragments. The removal of a toxic contaminant associated with carboxylated SWNTs is important in the development of carboxylated SWNTs for pharmacological applications.

The importance of cellular internalization of antibody-targeted carbon nanotubes in the photothermal ablation of breast cancer cells.

Single-walled carbon nanotubes (CNTs) convert absorbed near infrared (NIR) light into heat. The use of CNTs in the NIR-mediated photothermal ablation of tumor cells is attractive because the penetration of NIR light through normal tissues is optimal and the side effects are minimal. Targeted thermal ablation with minimal collateral damage can be achieved by using CNTs attached to tumor-specific monoclonal antibodies (MAbs). However, the role that the cellular internalization of CNTs plays in the subsequent sensitivity of the target cells to NIR-mediated photothermal ablation remains undefined. To address this issue, we used CNTs covalently coupled to an anti-Her2 or a control MAb and tested their ability to bind, internalize, and photothermally ablate Her2(+) but not Her2(-) breast cancer cell lines. Using flow cytometry, immunofluorescence, and confocal Raman microscopy, we observed the gradual time-dependent receptor-mediated endocytosis of anti-Her2-CNTs whereas a control MAb-CNT conjugate did not bind to the cells. Most importantly, the Her2(+) cells that internalized the MAb-CNTs were more sensitive to NIR-mediated photothermal damage than cells that could bind to, but not internalize the MAb-CNTs. These results suggest that both the targeting and internalization of MAb-CNTs might result in the most effective thermal ablation of tumor cells following their exposure to NIR light.

Gel electrophoresis method to measure the concentration of single-walled carbon nanotubes extracted from biological tissue.

A rapid and sensitive method to detect single-walled carbon nanotubes (SWNTs) in biological samples is presented. The method uses polyacrylamide gel electrophoresis (PAGE) followed by quantification of SWNT bands. SWNTs dispersed in bovine serum albumin (BSA) were used to develop the method. When BSA-SWNT dispersions were subjected to sodium dodecyl sulfate (SDS)-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected. The SWNTs, however, accumulated in a sharp band at the interface between the loading well and the stacking gel. The intensities from digitized images of these bands were proportional to the amount of SWNTs loaded onto the gel with a detection limit of 5 ng of SWNTs. To test the method, normal rat kidney (NRK) cells in culture were allowed to take up SWNTs upon exposure to medium containing various concentrations of BSA-SWNTs for different times and temperatures. The SDS-PAGE analyses of cell lysate samples suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis at a rate of 30 fg/day/cell upon exposure to medium containing 98 microg/mL SWNTs.