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.

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.

Specific thermal ablation of tumor cells using single-walled carbon nanotubes targeted by covalently-coupled monoclonal antibodies.

CD22 is broadly expressed on human B cell lymphomas. Monoclonal anti-CD22 antibodies alone, or coupled to toxins, have been used to selectively target these tumors both in SCID mice with xenografted human lymphoma cell lines and in patients with B cell lymphomas. Single-walled carbon nanotubes (CNTs) attached to antibodies or peptides represent another approach to targeting cancer cells. CNTs convert absorbed near-infrared (NIR) light to heat, which can thermally ablate cells that have bound the CNTs. We have previously demonstrated that monoclonal antibodies (MAbs) noncovalently coupled to CNTs can specifically target and kill cells in vitro. Here, we describe the preparation of conjugates in which the MAbs are covalently conjugated to the CNTs. The specificity of both the binding and NIR-mediated killing of the tumor cells by the MAb-CNTs is demonstrated by using CD22+CD25- Daudi cells, CD22-CD25+ phytohemagglutinin-activated normal human peripheral blood mononuclear cells, and CNTs covalently modified with either anti-CD22 or anti-CD25. We further demonstrate that the stability and specificity of the MAb-CNT conjugates are preserved following incubation in either sodium dodecyl sulfate or mouse serum, indicating that they should be stable for in vivo use.

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.

Thermal ablation of tumor cells with antibody-functionalized single-walled carbon nanotubes.

Single-walled carbon nanotubes (CNTs) emit heat when they absorb energy from near-infrared (NIR) light. Tissue is relatively transparent to NIR, which suggests that targeting CNTs to tumor cells, followed by noninvasive exposure to NIR light, will ablate tumors within the range of NIR. In this study, we demonstrate the specific binding of antibody-coupled CNTs to tumor cells in vitro, followed by their highly specific ablation with NIR light. Biotinylated polar lipids were used to prepare stable, biocompatible, noncytotoxic CNT dispersions that were then attached to one of two different neutralite avidin-derivatized mAbs directed against either human CD22 or CD25. CD22(+)CD25(-) Daudi cells bound only CNTs coupled to the anti-CD22 mAb; CD22(-)CD25(+) activated peripheral blood mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to NIR light.

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.

Importance of aromatic content for peptide/single-walled carbon nanotube interactions.

We have previously demonstrated that a designed amphiphilic peptide helix, denoted nano-1, coats and debundles single-walled carbon nanotubes (SWNTs) and promotes the assembly of these coated SWNTs into novel hierarchical structures via peptide-peptide interactions. The purpose of this study is to better understand how aromatic content impacts interactions between peptides and SWNTs. We have designed a series of peptides, based on the nano-1 sequence, in which the aromatic content is systematically varied. Atomic force microscopy measurements and optical absorption spectroscopy reveal that the ability to disperse individual SWNTs increases with increasing aromatic residues in the peptide. Altogether, the results indicate that pi-stacking interactions play an important role in peptide dispersion of SWNTs.

Diameter-selective solubilization of single-walled carbon nanotubes by reversible cyclic peptides.

We have utilized reversible cyclic peptides (RCPs)-peptides containing alternating l- and d-amino acids with N- and C-termini derivatized with thiol-containing groups allowing reversible peptide cyclization-to solubilize and noncovalently functionalize carbon single-walled nanotubes (SWNTs) in aqueous solution. Solubilization occurs through wrapping of RCPs around the circumference of a SWNT, followed by the formation of head-to-tail covalent bonds, yielding closed rings on the nanotubes. By controlling the length of the RCPs, we have demonstrated limited diameter-selective solubilization of the SWNTs as revealed by UV/vis/NIR and Raman spectroscopies, as well as atomic force microscopy.

p97 Is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm.

Certain protein toxins, including cholera toxin, ricin, and Pseudomonas aeruginosa exotoxin A, are transported to the lumen of the endoplasmic reticulum where they retro-translocate across the endoplasmic reticulum membrane to enter the cytoplasm. The mechanism of retrotranslocation is poorly understood but may involve the endoplasmic reticulum-associated degradation pathway. The AAA ATPase p97 (also called valosin-containing protein) participates in the retro-translocation of cellular endoplasmic reticulum-associated degradation substrates and is therefore a candidate to participate in the retrotranslocation of protein toxins. To investigate whether p97 functions in toxin delivery to the cytoplasm, we measured the sensitivity to toxins of cells expressing either wild-type p97 or a dominant ATPase-defective p97 mutant under control of a tetracycline-inducible promoter. The rate at which cholera toxin and related toxins entered the cytoplasm was reduced in cells expressing the ATPase-defective p97, suggesting that the toxins might interact with p97. To detect interaction, the cholera toxin A chain was immunoprecipitated from cholera toxin-treated Vero cells, and co-immunoprecipitation of p97 was assessed by immunoblotting. The immunoprecipitates contained both cholera toxin A chain and p97, evidence that the two proteins are in a complex. Altogether, these results provide functional and structural evidence that p97 participates in the transport of cholera toxin to the cytoplasm.

Preparation and characterization of individual peptide-wrapped single-walled carbon nanotubes.

Two challenges for effectively exploiting the remarkable properties of single-walled carbon nanotubes (SWNTs) are the isolation of intact individual nanotubes from the raw material and the assembly of these isolated SWNTs into useful structures. In this study, we present atomic force microscopy (AFM) evidence that we can isolate individual peptide-wrapped SWNTs, possibly connected end-to-end into long fibrillar structures, using an amphiphilic alpha-helical peptide, termed nano-1. Transmission electron microscopy (TEM) and well-resolved absorption spectral features further corroborate nano-1's ability to debundle SWNTs in aqueous solution. Peptide-assisted assembly of SWNT structures, specifically in the form of Y-, X-, and intraloop junctions, was observed in the AFM and TEM images.

Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI.

Cholera toxin, Shiga toxin and ricin are examples of protein toxins that require retrograde transport from the Golgi complex into the endoplasmic reticulum (ER) to express their cytotoxic activities and different toxins appear to use different pathways of retrograde transport. Cholera toxin contains the mammalian retrograde targeting signal KDEL and is believed to exploit the coat protein I (COPI) and KDEL receptor-dependent pathway to go from the Golgi complex to the ER. Shiga toxin, however, has no KDEL sequence to specify its inclusion in COPI-coated retrograde vesicles and is believed to use a recently discovered COPI-independent and Rab6A-dependent retrograde pathway to enter the ER. Ricin, like Shiga toxin, does not contain a KDEL sequence and is therefore a candidate to use the COPI-independent and Rab6A-dependent pathway of retrograde transport to access the ER. We measured the effect of the GDP-restricted mutant of Rab6A (Rab6A-T27N) on the cytotoxic activity of ricin and found that expressing Rab6A-T27N in cells did not inhibit the cytotoxicity of ricin, suggesting that ricin enters the cytoplasm by a retrograde pathway that does not involve Rab6A. Moreover, ricin still intoxicated cells when Rab6A and COPI were simultaneously inhibited, implying that ricin requires neither Rab6A nor COPI to intoxicate cells.