Scavenger Receptor A1 Mediates the Uptake of Carboxylated and Pristine Multi-Walled Carbon Nanotubes Coated with Bovine Serum Albumin.

Previously, we noted that carboxylated multi-walled carbon nanotubes (cMWNTs) coated with Pluronic® F-108 (PF108) bound to and were accumulated by macrophages, but that pristine multi-walled carbon nanotubes (pMWNTs) coated with PF108 were not (Wang et al., Nanotoxicology2018, 12, 677). Subsequent studies with Chinese hamster ovary (CHO) cells that overexpressed scavenger receptor A1 (SR-A1) and with macrophages derived from mice knocked out for SR-A1 provided evidence that SR-A1 was a receptor of PF108-cMWNTs (Wang et al., Nanomaterials (Basel) 2020, 10, 2417). Herein, we replaced the PF108 coat with bovine serum albumin (BSA) to investigate how a BSA corona affected the interaction of multi-walled carbon nanotubes (MWNTs) with cells. Both BSA-coated cMWNTs and pMWNTs bound to and were accumulated by RAW 264.7 macrophages, although the cells bound two times more BSA-coated cMWNT than pMWNTs. RAW 264.7 cells that were deleted for SR-A1 using CRISPR-Cas9 technology had markedly reduced binding and accumulation of both BSA-coated cMWNTs and pMWNTs, suggesting that SR-A1 was responsible for the uptake of both MWNT types. Moreover, CHO cells that ectopically expressed SR-A1 accumulated both MWNT types, whereas wild-type CHO cells did not. One model to explain these results is that SR-A1 can interact with two structural features of BSA-coated cMWNTs, one inherent to the oxidized nanotubes (such as COOH and other oxidized groups) and the other provided by the BSA corona; whereas SR-A1 only interacts with the BSA corona of BSA-pMWNTs.

The Importance of Evaluating the Lot-to-Lot Batch Consistency of Commercial Multi-Walled Carbon Nanotube Products.

The biological response of multi-walled carbon nanotubes (MWNTs) is related to their physicochemical properties and a thorough MWNT characterization should accompany an assessment of their biological activity, including their potential toxicity. Beyond characterizing the physicochemical properties of MWNTs from different sources or manufacturers, it is also important to characterize different production lots of the same MWNT product from the same vendor (i.e., lot-to-lot batch consistency). Herein, we present a comprehensive physicochemical characterization of two lots of commercial pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs) used to study the response of mammalian macrophages to MWNTs. There were many similarities between the physicochemical properties of the two lots of cMWNTs and neither significantly diminished the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 µg cMWNTs/mL). Conversely, several physicochemical properties of the two lots of pMWNTs were different; notably, the newer lot of pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot. Furthermore, a 72-h half maximal inhibitory concentration (IC-50) of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of pMWNTs. These results demonstrate that subtle physicochemical differences can lead to significantly dissimilar cellular responses, and that production-lot consistency must be considered when assessing the toxicity of MWNTs.

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.

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.

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.

Cholera toxin up-regulates endoplasmic reticulum proteins that correlate with sensitivity to the toxin.

Cholera toxin (CT) contains one A chain and five B chains. The A chain is an enzyme that covalently modifies a trimeric G protein in the cytoplasm, resulting in the overproduction of cAMP. The B chain binds the glycosphingolipid G(M1), the cell surface receptor for CT, which initiates receptor-mediated endocytosis of the toxin. After endocytosis, CT enters the endoplasmic reticulum (ER) via retrograde vesicular traffic where the A chain retro-translocates through the ER membrane to reach the cytoplasm. The retro-translocation mechanism is poorly understood, but may involve proteins of the ER stress response, including the ER associated degradation (ERAD) pathway. We report here that treating cells with CT or CTB quickly up-regulates the levels of BiP, Derlin-1, and Derlin-2, known participants in the ER stress response and ERAD. CT did not induce calnexin, another known responder to ER stress, indicating that the CT-mediated induction of ER proteins is selective in this time frame. These data suggest that CT may promote retro-translocation of the A chain to the cytoplasm by rapidly up-regulating a set of ER proteins involved in the retro-translocation process. In support of this idea, a variety of conditions that induced BiP, Derlin-1, and Derlin-2 sensitized cells to CT and conditions that inhibited their induction de-sensitized cells to CT. Moreover, specifically suppressing Derlin-1 with siRNA protected cells from CT. In addition, Derlin-1 co-immunoprecipitated with CTA or CTB from CT-treated cells using anti-CTA or anti-CTB antibodies. Altogether, the results are consistent with the hypothesis that the B chain of CT up-regulates ER proteins that may assist in the retro-translocation of the A chain across the ER membrane.

Single-walled carbon nanotube interactions with HeLa cells.

This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Amphiphilic helical peptide enhances the uptake of single-walled carbon nanotubes by living cells.

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that approximately 6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.

Electrospun linear polyethyleneimine scaffolds for cell growth.

Unique biocompatible scaffolds were produced by electrospinning cross-linked linear polyethyleneimine (PEI) with succinic anhydride and 1,4-butanediol diglycidyl ether. Nonwoven mats of PEI fibers in the range of 1600-687nm were evaluated as interaction scaffolds for normal human fibroblast (NHF) cells. The electrospun scaffolds were characterized by Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy. The growth of the NHF cells was followed by scanning electron microscopy as well as optical and fluorescence microscopies. Cell viability was evaluated by staining with propidium iodide for dead cells and fluorescein diacetate for live cells. Immunofluorescence with fixed cells on the scaffolds was examined by staining the endoplasmic reticulum with rabbit anti-GRP 78/Alexa 488 goat anti-rabbit and staining the nuclei with 4'-6'-diamidino-2-phenylindole. Fluorescence studies confirmed that NHF cells attached and spread throughout the cross-linked linear polyethyleneimine scaffold. The attachment and spreading of cells suggests that electrospun linear polyethyleneimine scaffolds support growth of normal human fibroblasts cells. Thus, these biomaterial scaffolds may be useful in tissue engineering.

Retrograde transport of protein toxins under conditions of COPI dysfunction.

Retrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the epsilon-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport.