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Proc Natl Acad Sci U S A. Jun 24, 2008; 105(25): 8697–8702.
Published online Jun 16, 2008. doi:  10.1073/pnas.0803557105
PMCID: PMC2438394
Medical Sciences

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; CD22CD25+ 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.

Keywords: immunoconjugates, lymphoma cells, monoclonal antibodies, nanotechnology, near infrared light

Despite the success of current treatments for several types of cancer, all known treatments have major limitations. Conventional chemotherapy or radiotherapy damage many cells, and both have significant side effects. In addition, tumor cells develop resistance to many chemotherapeutic agents (1), and most chemotherapeutic drugs kill dividing cancer cells and not dormant ones. To decrease nonspecific toxic effects and kill nondividing cells, targeted therapies are being developed and some have already been approved by the Food and Drug Administration for use in humans. These include both small molecules that target specific intracellular pathways in tumor cells and mAbs that target molecules on their surface. Some of these targeted agents are cytostatic and not cytotoxic, and they are often given in combination with chemotherapy in an effort to both lower the dose of chemotherapy required and hence reduce side effects and achieve additive or synergistic effects. With regard to mAbs, strategies include increasing cytotoxicity by coupling them to drugs, radionuclides, toxins, drugs, or prodrugs (2, 3). These agents (collectively called immunoconjugates) are potent, and three have been approved for human use (4, 5). However, they also have side effects because they carry toxic payloads. We and others have successfully tested the antitumor activity of different agents, including signaling antibodies and immunotoxins, alone or in combination with pharmacological agents, in disseminated or solid human tumors grown in immunocompromised mice (59). We have also tested four different immunotoxins in humans (1015). To optimize the use of mAbs in cancer therapy, it is important to explore their use with new types of payloads and carriers, including carbon nanotubes (CNTs). The ability of CNTs to convert near-infrared (NIR) light into heat provides an opportunity to create a new generation of immunoconjugates for cancer photo-therapy with high performance and efficacy. Moreover, hyperthermia has been clinically used in the management of solid tumors because it can synergistically enhance tumor cytotoxicity when combined with chemotherapy or radiotherapy (16, 17). Hyperthermia also preferentially increases the permeability of tumor vasculature compared with normal vasculature, which can enhance the delivery of drugs into tumors. Therefore, the thermal effects generated by targeted CNTs may have important advantages. Recent pharmacokinetic studies have reported that CNTs dispersed by different procedures lack nonspecific toxic effects in mice (1820).

The use of NIR-resonant nanostructures, including gold nanoshells and CNTs, to thermally ablate cancer cells is being explored by several groups (2126). The use of NIR light in the 700- to 1,100-nm range for the induction of hyperthermia is particularly attractive because living tissues do not strongly absorb in this range (27). Hence, an external NIR light source should effectively and safely penetrate normal tissue and ablate any cells to which the CNTs are attached. The critical aspect for selective CNT-mediated thermal ablation of cells is to stably attach targeting moieties that will not interfere with the optical properties of the CNTs and yet retain targeting specificity. The targeting of CNTs to tumor cells can be accomplished by coating them with cell-binding ligands such as peptides or mAbs (25, 26, 2830). Several studies have reported that the targeting of such CNTs is “specific” (25, 26, 29, 30), but no study has used both a control ligand and a control cell to convincingly demonstrate ligand-specific thermal ablation of tumors cells with CNTs. Specificity is critical because nonspecific binding to antigen-negative cells in vivo could cause major side effects, which has been a confounding issue in the cancer targeting field for >25 years.

The aim of this study was to design and prepare an anti-CD22-targeted CNT construct to ablate human Burkitt's lymphoma cells in vitro. Herein, we describe the physical properties of these CNT constructs, their selective binding to tumor cells, and the NIR-induced thermal ablation of the targeted tumor cells. Importantly, both a control CNT construct and a control cell were used to definitely prove specificity.


Dispersion of CNTs.

Well dispersed single-walled CNTs were prepared by sonicating CNTs in the presence of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol) 2000] [DSPE-PEG(2000)-biotin], followed by centrifugation to recover the biotinylated CNTs (B-CNTs). The resulting B-CNT suspension contained 0.06 mg CNT/ml and ≤3 parts per million metals, as determined by thermal gravimetric analysis (TGA) and inductively coupled plasma mass spectrometry (MS) (data not shown). The dispersions were stable and did not aggregate at room temperature for >120 days. Atomic force microscopy (AFM) analysis demonstrated that the suspension was free of nontubular carbon structures and the CNTs were either individually dispersed or in small bundles. The lengths of the CNTs ranged from 0.2 to 1.4 μm with an average of 0.59 μm (Fig. 1a). Analysis by transmission electron microscopy (TEM) of the B-CNT samples probed with gold-labeled goat antibiotin demonstrated that biotin was distributed along the entire surface of the B-CNT (Fig. 1b). The biotin content of the B-CNT dispersion was determined by using a competitive ELISA and adding dilutions of the B-CNT dispersion to biotin-HRP and plating them onto neutralite avidin (NA)-coated plates. The amount of HRP-labeled biotin was detected by the development of color in the presence of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. Using this assay, we found that the content of biotin was 0.02 mmol/g of B-CNT. The UV-visible (Vis)-NIR spectra of the B-CNTs confirmed the quality of these dispersions with the presence of electronic transitions between van Hove singularities, suggesting that the optical properties of the CNTs were maintained after the adsorption of DSPE-PEG-biotin (Fig. 1c). The Raman spectra of the B-CNTs showed a number of well characterized CNT resonances such as the radial breathing mode region between 100 and 300 cm−1 (data not shown) and the tangential (G-band) peak at 1,590 cm−1, confirming the presence of CNTs in the sample (Fig. 1d).

Fig. 1.
Water-soluble CNTs functionalized with biotinylated polar lipids. (a) AFM image of B-CNTs shows CNTs coated by the biotinylated polar lipid, DSPE-PEG-biotin. (b) TEM images of individual B-CNTs show uniform coverage of biotin after immunodetection with ...

To determine whether B-CNTs were inherently cytotoxic (in the absence of NIR), cells from the IgM+ CD22+CD25 Burkitt's lymphoma cell line Daudi were incubated for 24 h with up to the highest amount of B-CNTs used in the binding and killing assays (3.6 μg). No toxicity was observed using a [3H]thymidine incorporation assay (data not shown).

Preparation of mAb-NA Targeting Moieties.

To prepare the targeting agents, we coupled NA to mAbs. To this end, mouse IgG anti-human CD22 (RFB4) or mouse IgG anti-human CD25 (RFT5) were thiolated with 2-iminothiolane (Traut's reagent). NA was activated with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Then, the purified thiolated IgG was mixed with MBS-activated NA at a 2:1 ratio as shown by preliminary experiments to give the best yields as determined by the elution profile of the resulting conjugate on a Sephacryl S-300 HR column (Fig. 2a). The concentration of the mAb-NA conjugates was determined by using the bicinchoninic acid assay (BCA). Both mAb-NA conjugates were free of impurities as judged by Western blot analysis (Fig. 2a). We next determined whether these conjugates were cytotoxic to Daudi cells after incubation of cells for 24 h with up to 10 μg/ml of RFB4-NA; cytotoxicity was determined by [3H]thymidine incorporation. Similar concentrations of unconjugated RFB4 and NA were used as negative controls, and goat anti-IgM (which induces apoptosis of Daudi cells) (31) was used as the positive control. We found that the RFB4-NA conjugates were not cytotoxic, whereas (as predicted) the goat anti-IgM reduced [3H]thymidine incorporation by >50% (Fig. 2b).

Fig. 2.
Analysis of mAb-NA conjugates. (a) A typical chromatographic separation of RFB4-NA from unconjugated RFB4 and NA using a Sephacryl S-300 HR column. Fractions of the first peak containing the RFB4-NA conjugate were pooled and concentrated. (Inset) Purified ...

The specific binding of RFB4-NA and RFT5-NA conjugates to CD22+CD25 Daudi cells and CD22CD25+ phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs), respectively, was demonstrated by flow cytometry, using either FITC-labeled goat anti-mouse Ig (GAMIg) or FITC-biotin (data not shown for FITC-biotin). The latter was confirmed by the ability of the cell-bound mAb-NA to bind to B-CNT. Daudi cells were precoated with a saturating concentration of RFB4-NA, washed, and incubated with increasing amounts of B-CNTs. The RFB4-NA, but not the RFT5-NA conjugate could target an average of 0.237 pg of B-CNTs per cell (Fig. 2c).

Preparation and Testing of mAb-CNT Complexes.

We next prepared the mAb-CNT conjugates by coupling the B-CNTs to either RFB4-NA or RFT5-NA for 35 min at room temperature. After the removal of the supernatant containing the unreacted mAb-NA, the optical properties of the freshly prepared mAb-CNT were tested. The UV-Vis-NIR spectra of the mAb-CNT conjugates displayed the same metallic and semiconducting CNT types as observed for the B-CNTs, indicating that the optical properties of the CNTs were not affected by the coupling (Fig. 3a), and the characteristic CNT resonances displayed in the Raman spectra of the mAb-CNTs again confirmed the presence of CNTs in the sample (Fig. 3b).

Fig. 3.
Optical properties of CNTs following coupling with mAbs (mAb-CNT). (a) UV-Vis-NIR spectrum of RFB4-CNTs show the same metallic and semiconducting CNT types as observed for the B-CNTs, indicating the retention of the optical properties of CNTs after the ...

The ability of the mAb-CNT conjugates to bind to antigen-positive but not antigen-negative target cells was assessed by flow cytometry. The components of the cell-bound mAb-CNT were detected by using FITC-GAMIg (which binds to mouse mAb) and phycoerythrin-streptavidin (PE-SA) (which binds to biotin), respectively. We found that RFB4-CNT and RFB4 (positive control) bound equally well to Daudi cells, whereas RFT5-CNT (negative control) bound poorly (P < 0.001) (Fig. 4a). Conversely, RFT5-CNT and RFT5 bound equally well to CD22CD25+ PHA-activated PBMCs (95% CD25+ cells), whereas the negative control conjugate, RFB4-CNT, did not (P < 0.002) (Fig. 4b). These results demonstrate that the coupling of the mAbs to CNTs does not alter their mAb-binding activity and that the mAb-CNTs bind to antigen-expressing cells as specifically as the uncoupled mAbs.

Fig. 4.
Binding of mAb-CNTs to target cells. One million cells were incubated with saturating concentrations of RFB4-CNTs or RFT5-CNTs and then incubated either with FITC-GAMIg to detect the mAbs or with PE-SA to detect the B-CNTs and analyzed on a FACScan. ( ...

Having demonstrated that the mAb-CNT conjugates retained the binding activity of the mAb and the optical properties of the CNTs, we next determined whether cells targeted by the mAb-CNTs could be thermally ablated after exposure to NIR light. Cells were incubated with the mAb-CNTs in PBS, washed three times with PBS, and then dispensed into 96-well plates in cell culture media. The cells in the plate were exposed to an 808-nm laser (5 W/cm2) for 7 min and pulsed for the next 12 h with 1 μCi [3H]thymidine to assess cell viability. As shown in Fig. 5a, as compared with treatment with the nonbinding RFT5-CNTs, the viability of the RFB4-CNT-treated Daudi cells was significantly reduced after exposure to NIR light (P < 0.0001). Conversely, when activated PBMCs were used as target cells, RFT5-CNT, but not RFB4-CNT, killed the cells after exposure to NIR light (P < 0.0001) (Fig. 5b). These experiments demonstrate that the binding of the mAb-CNTs to their respective antigen-positive target cells leads to their specific ablation after exposure to NIR light.

Fig. 5.
Ablation of mAb-CNT-coated cells with NIR. One million cells were incubated with saturating concentrations of RFB4-CNTs or RFT5-CNTs. Cells were dispensed into 96-well plates, exposed for 7 min to 808-nm NIR light (5 W/cm2), pulsed with 1 μCi ...

Because we anticipate using these mAb-CNTs in vivo, it was important to demonstrate that they retained activity in serum at 37°C. Therefore, the mAb-CNTs were incubated in mouse serum at 37°C for 0, 24, 48, and 72 h. At each time point, the mAb-CNTs were washed with PBS, incubated with Daudi cells, and irradiated with NIR light in a procedure similar to the thermal ablation described above. No loss in their ability to thermally ablate Daudi cells was observed, even after 72 h in mouse serum at 37°C (Fig. 5c).


The first critical challenge in the field of targeted CNTs is to create soluble and stable CNTs that retain both the specificity of the targeting moiety and the thermal activity of the CNTs even in serum at physiological temperatures. In this article, we demonstrate that this can be accomplished. Our strategy involved the generation of targeting moieties consisting of mAb-NAs attached to dispersed biotinylated CNTs. The use of B-CNTs and mAb-NAs gives us the flexibility to “assemble” the targeted CNTs by using any cell-binding mAb. Second, the one-step strategy of generating dispersed CNTs by using biotinylated polar lipids has the advantage of preventing subsequent chemical treatments that remove the polar lipids and/or destroy their optical properties. Of equal importance is the specificity of the targeting strategy. Thus, previous studies have demonstrated that folic acid-coated CNTs could be targeted to folate receptor (FR)-positive cells and that NIR light killed the cells (25). Although FR-negative cells were used as a control, CNTs coated with an irrelevant ligand were not. In other studies, rArg-Gly-Asp (RGD)-CNTs were used to deliver adsorbed doxorubicin (29). These CNTs were also evaluated for in vivo biodistribution (19), but control peptide-CNTs were not used to demonstrate specificity. Another approach for targeting CNTs to cells is to noncovalently attach mAbs that can be used in photothermal therapy (26) or imaging (30). However, attachment of mAbs by direct adsorption on CNTs involves a potential loss of the targeting function of the mAbs and, indeed in the study cited, specificity controls were not reported, and cell viability studies showed 50% collateral damage by the irrelevant mAb-CNT control after exposure to NIR light (26). In another very elegant study, mAbs were covalently attached to CNTs to deliver radionuclides to cells (28). These studies achieved their goal of killing target cells by radiotherapy and showed both linkage stability and specific targeting. However, because the objective of these studies was not to ablate cells with NIR light, we do not know whether the optical properties of the CNTs were preserved.

Having demonstrated excellent specificity of both targeting and thermal ablation in vitro, the next step is to evaluate the pharmacokinetics, biodistribution, toxicity, and activity of these mAb-CNT constructs in vivo.

Materials and Methods


Purified CNTs (HiPco) were purchased from Carbon Nanotechnologies. The polar lipid DSPE-PEG(2000) biotin was purchased from Avanti Polar Lipids. Mouse IgG1 anti-human CD22 (RFB4) and mouse IgG anti-human CD25 (RFT5) were prepared and purified in our laboratory at UT Southwestern Medical Center. Traut's reagent and MBS were purchased from Pierce/Endogen. NA was purchased from Accurate Chemical and Scientific.

Cell Culture.

Daudi cells (American Type Culture Collection) were cultured in RPMI 1640 medium (Sigma) containing 1% antibiotic-antimycotic mixture (penicillin/streptomycin/Amphotericin B) (Sigma), 10% heat-inactivated FCS (HyClone), and 2 mM l-glutamine (Sigma) (complete medium). PBMCs from normal healthy donors were isolated from the fresh heparinized blood by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. Normal activated CD25+ cells were generated by culturing the PBMCs for 72 h at 1 × 106 cells/ml in complete medium supplemented with 5 μg/ml PHA (Sigma).

CNT Solubilization by Biotinylated Polar Lipids and Characterization.

Degassed ultrapure deionized (DI) water was used for all solutions. CNTs (0.3 mg) were suspended in 1 ml of 166 μM DSPE-PEG(2000)-biotin. The mixture was sonicated with a 2-mm probe tip connected to a Branson Sonifier 250 (VWR) for 10 min at a power level of 10 W, with the sample immersed in an ice water bath. To remove excess DSPE-PEG-biotin, samples were washed twice in DI water by centrifugation for 15 min at 90,000 × g at 4°C. The supernatant was discarded, the pellet was resuspended in 1 ml of DI water, and the procedure was repeated. The samples were then centrifuged two times for 10 min at 16,000 × g at room temperature, and the upper 50% of the supernatant containing the B-CNT was recovered. To obtain more concentrated samples, the B-CNT suspension was centrifuged for 60 min at 16,000 × g at 4°C, the supernatant was discarded, and the pellet was resuspended in 0.2 ml of DI water.

Sample concentration was detected by TGA using a Pyris-1 thermal gravimetric analyzer (PerkinElmer) equipped with a high-temperature furnace and sample thermocouple. AFM was performed in air under ambient conditions by using a Digital Instruments Nanoscope III Multimode scanning probe microscope (Veeco Metrology). Images were acquired in the TappingMode by using cantilevers with 0.9 Nm−1 force constants as described (32). A dual-beam Lambda 900 UV-Vis-NIR spectrophotometer (PerkinElmer) with a scan speed of 25 nm/min and a 0.4-s integration time was used for absorption spectra. Raman spectroscopy at 633-nm excitation was performed with a LabRAM high-resolution confocal Raman microscope system (Jobin Yvon). Wave number calibration was performed by using the 520.5-cm−1 line of a silicon wafer; the spectral resolution was ≈1 cm−1 as described (33). TEM was performed with a JEOL JEM-1200EX II electron microscope. The B-CNT dispersion was probed with 5-nm gold beads labeled with goat antibiotin (Kirkegaard & Perry Laboratories), and then imaged.

Preparation of mAb-NA Conjugates.

To couple the B-CNTs to mAbs, we used a modified protocol (34). Briefly, 10 mg of RFB4 or RFT5 in 1 ml of 0.15 M borate buffer, 0.1 mM EDTA, pH 8.5 were thiolated by incubation for 1 h at room temperature with a 20:1 molar excess of Traut's reagent. After incubation, the reaction was quenched with 0.1 M glycine. In parallel, 10 mg of NA dissolved in 1 ml of 0.01 M PBS, 0.1 mM EDTA, pH 7.4, was activated by 30-min incubation at room temperature by using a 6:1 molar excess of MBS. The unreacted Traut's reagent and MBS were removed by gel filtration on Sephadex G-25 columns in 0.01 M PBS, 0.1 mM EDTA, pH 7.4. The thiolated mAb was conjugated to the activated NA at a molar ratio of 1:2 for 2 h at room temperature with gentle shaking. The resultant conjugate was purified by gel filtration on a Sephacryl S-300 HR column (GE Healthcare) by using 0.1 M PBS, 0.05% Tween-20, pH 7.4. The protein concentration in the purified conjugate was quantified by using the BCA assay (Pierce/Endogen). The size and integrity of the conjugate was analyzed by Western blot. The samples were electrophoresed on a 7.5% nondenaturing polyacrylamide gel and transferred to PVDF membranes (Bio-Rad), probed with HRP-labeled sheep anti-mouse IgG, and visualized by using an enhanced chemiluminescence system (GE Healthcare).

Competition ELISA.

NA-coated 96-well plates were blocked with 1% BSA in 0.01 M PBS, 0.05% Tween-20 (PBST) for 1 h. B-CNTs were added to each well together with biotin-labeled HRP and incubated for 1 h. After washing five times with PBST, the substrate ABTS was added, and absorbance was measured at 405 nm. The amount of biotin bound to the CNTs (biotin mmol/μg B-CNT) was calculated by using a standard curve constructed by plotting OD against the biotin concentration (ng/ml) prepared by coincubating increasing amounts of biotin in the presence of a constant amount of HRP-biotin. A similar procedure was used to detect the amount of NA in mAb-NA conjugates.

Preparation of mAb-CNT Conjugate.

Fresh mAb-CNTs were prepared immediately before use by mixing B-CNT with mAb-NA in a 1:2 (wt/wt) ratio. The mixture was placed on a rocker for 35 min at room temperature and vortexed gently every 5 min. After coupling, the mixture was centrifuged for 5 min at 16,000 × g at 4°C, the supernatant containing unreacted mAb-NA was discarded, and the pellet was resuspended in 40 μl of PBS for every 3.6 μg of B-CNT and used immediately.

Binding of mAb-CNTs to Target Cells.

One million Daudi cells or PHA-activated PBMCs (>95% CD25+ cells) were incubated with the mAb-CNTs for 20 min at 4°C in PBS. Cells were washed two times with ice-cold PBS and then incubated with either PE-SA (Jackson ImmunoResearch) or FITC-GAMIg (Kirkegaard & Perry Laboratories) for 20 min at 4°C. The cells were washed two times with ice-cold PBS and resuspended in 0.5 ml of PBS, and the bound fluorescence was analyzed on a FACScan (Becton Dickinson).

Determination of the Amount of B-CNTs Bound per Cell.

One million Daudi cells were incubated with saturating amounts of RFB4-NA or RFT5-NA for 15 min at 4°C in PBS. Cells were washed two times with ice-cold PBS, incubated with incremental amounts of B-CNT for 20 min at 4°C in PBS, and then washed two times with ice-cold PBS. The amount of B-CNT bound to cells was determined by measuring the absorbance at 808 nm of the B-CNT suspension before and after incubation with Daudi cells. The amount of B-CNT bound per cell was determined by using the extinction coefficient [ε0.1% = 25 (mg/ml)−1] calculated from the linear fit (Beer-Lambert law) of absorbance at 808 nm versus the B-CNT concentration.

Ablation of mAb-CNT-Coated Cells with NIR Light.

One million cells were incubated with 40 μl of the mAb-CNTs in PBS for 20 min at 4°C. Cells were washed three times with ice-cold PBS, and then 105 cells were dispensed in triplicate wells in a 96-well plate in 200 μl of complete medium. The cells were exposed to continuous NIR light by using a FAP-Sys-30W 805- to 811-nm laser system (Coherent) for 7 min at 5 W/cm2. Cell death was assessed by pulsing the cells for the next 12 h with 1 μCi [3H]thymidine per well, and the incorporated radioactivity was measured by liquid scintillation counting. The incorporated radioactivity for each sample was calculated relative to the corresponding nonirradiated samples. For functional stability, mAb-CNTs, prepared as described above, were suspended in 0.1 ml of mouse serum (Sigma) and incubated at 37°C for 0–72 h. At each time point, the suspension was washed with ice-cold PBS, and the pellet was resuspended in 40 μl of PBS. The ablation of mAb-CNT-coated Daudi cells with NIR light was tested as described above.


Data were analyzed by using Student's t test. Values are given as mean ± SD. P < 0.05 was considered statistically significant.


We thank Dr. Gregg R. Dieckmann (University of Texas, Dallas) for use of the sonicator and centrifuge; Hadi N. Yehia for help during the initial stages of the project; Vicky Poenitzsch for assistance with the AFM images; Kate Walker, Meredith Daigrepont, and Jonathan Belew for assistance with elemental analyses; and Drs. Peter Wilk, Robert Stirbl, Jimmy Xu, and Malcolm Snead for unwavering support, ideas, and interactions. This work was supported by The Cancer Immunobiology Center, University of Texas Southwestern Medical Center (E.S.V., R.M., and P.C.), Robert A. Welch Foundation Grants AT-1364 (to P.P.) AT-1326 (to I.H.M.), the Department of Defense (to E.S.V. and R.K.D.) and the Center for Applied Biology at the University of Texas, Dallas. N.S.Z. and A.D.-E.S. were summer students supported by the Summer Undergraduate Research Fellowship program at the University of Texas Southwestern Medical Center.


Conflict of interest statement: E.S.V., R.K.D., P.P., and I.H.M. are affiliated with Medical Nanotechnologies, Inc. E.S.V. is a coinventor on an issued patent encompassing this work.


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