Tr a n s l a t i o n a l O n c o l o g y Volume 4 Number 6 November 2011 pp.

350–354 350
www.transonc.com

Cell Internalization Studies Christopher Scott Berger*, John W. Marks†,

Robert D. Bolskar‡, Michael G. Rosenblum†
of Gadofullerene–(ZME-018) and Lon J. Wilson*
Immunoconjugates into *Department of Chemistry, Smalley Institute for Nanoscale
A375m Melanoma Cells1,2 Science and Technology, and the Center for Biological and
Environmental Nanotechnology, Rice University, Houston,
TX, USA; †Immunopharmacology and Targeted Therapy
Laboratory, Department of Experimental Therapeutics,
MD Anderson Cancer Center, Houston, TX, USA;
‡
TDA Research, Inc, Wheat Ridge, CO, USA

Abstract
Fullerene (C60)–monoclonal antibody (mAb) immunoconjugates have been determined to internalize into target cells
using water-soluble Gd3+ ion–filled metallofullerenes (Gd@C60[OH]x). Two separate conjugations of Gd@C60(OH)x
with the antibody ZME-018 and a murine antibody mixture (MuIgG) were performed in a 1:5 mAb/Gd@C60 ratio.
Characterization of the immunoconjugates was established using inductively coupled plasma mass spectrometry
(ICP-MS) for Gd3+ and UV-Vis spectrometry (for Gd@C60 + C60). Once conjugated, enzyme-linked immunosorbent
assays showed little change in the specific binding of ZME-018. Each immunoconjugate was exposed to two cancer
cell lines, A375m (antigen positive), and T24, bladder carcinoma (antigen negative). Internalization levels of the
immunoconjugate were determined at various time points during 24 hours by harvesting and digesting the cells
with 70% HNO3 for Gd3+ ion analysis by ICP-MS. These results are the first to demonstrate the practicality of a
targeted cancer therapy based on fullerene immunotherapy.

Translational Oncology (2011) 4, 350–354

Introduction
The simple goal of targeted therapies is to increase therapeutic con-
centrations in appropriate biologic areas, thus reducing adverse effects
while allowing higher pharmaceutical doses to be administered sys-
temically. The development of cell-targeted agents for imaging and
therapy in medicine is, therefore, an important area of study.
Peptides, cytokines, growth factors, and monoclonal antibodies
(mAbs) all show promise for their ability to deliver payloads to the cell
surface and into the cytoplasm of targeted cancer cells [1]. Currently,
however, the most versatile and successful class of agents to show tar-
geting capabilities for specific cancers are mAbs.
Using a patient’s own cellular identification system to target cancer
with immunoconjugates is evolving into a potent anticancer therapy
in personalized medicine [2–4]. To date, the US Food and Drug
Administration have approved three immunoconjugates for clinical
use. Two murine mAbs target the B-cell glycoprotein CD20 to treat
non–Hodgkin lymphomas with β-emitting radionuclides. Ibritumomab
tiuxetan is the IgG1-κ mAb radiolabeled with either 111In (γ-emitting
imaging agent) or 90Y (β-emitting therapeutic) [5–7], whereas the sec-
ond agent, tositumomab, is an IgG2a-λ mAb radiolabeled with 131I (γ-
emitting imaging agent and β-emitting therapeutic) [8]. Gemtuzumab
ozogamicin, a third immunoconjugate, is a humanized, anti-CD33
IgG4-κ mAb covalently derivatized with cytotoxic calicheamicin for
use in the treatment of acute myelogenous leukemia [9]. For optimal
therapeutic efficacy, these immunoconjugates must internalize effec-
tively within target cells after binding to the cell surface antigen.
Since the discovery of fullerenes in 1985 [10] and carbon nanotubes
in 1991 [11], one of the most prominent areas of study for such carbon
nanomaterials has been for medical applications [12,13]. Properly
Address all correspondence to: Prof. Lon J. Wilson, 6100 Main St, MS 60, Houston,
TX 77005. E-mail: durango@rice.edu
1
This research was supported by the National Institutes of Health (grant R43CA128277),
the Welch Foundation (grant C-0627), and, in part, the Clayton Foundation for Research.
2
This article refers to supplementary materials, which are designated by Tables W1 to
W3 and Figures W1 and W2 and are available online at www.transonc.com.
Received 5 May 2011; Revised 5 July 2011; Accepted 6 July 2011
Copyright © 2011 Neoplasia Press, Inc. Open access under CC BY-NC-ND license.
1944-7124/11/ DOI 10.1593/tlo.11157
Translational Oncology Vol. 4, No. 6, 2011 Gd@C60-(ZME-018) Internalization into A375m Cells Berger et al. 351

derivatized carbon nanomaterials are nonimmunogenic, biologically and demonstrate impressive cytotoxic effects against established tumors
stable, and are eventually excreted from mammals [14–16]. To date, C60 in orthotopic models [40,41]. The reliable targeting properties of
water-soluble fullerenes have been developed for potential medical uses ZME-018 conjugates and its thorough characterization in various im-
such as neuroprotective agents [17–19], human immunodeficiency virus munoconjugate systems make ZME-018 an ideal antibody platform
type 1 protease inhibitors [20,21], bone-vectoring agents [22], and x-ray for fullerene (C60) conjugate delivery studies.
contrast agents [23]. In addition, these hollow carbon nanomaterials can
be internally loaded, either during initial synthesis or in postproduction
Materials and Methods
steps, with materials, such as Gd3+ ions for magnetic resonance imaging
[24,25], I2 for computed tomography [26], or radionuclides for radio-
Conjugate Preparation
tracer [27] or radiotherapeutic agents [28].
Immunoconjugates of Gd@C60 were prepared using a procedure
The first description of a cell-targeting fullerene (C60)–antibody
similar to previous C60-based immunoconjugates [29], where conjuga-
immunoconjugate was produced in 2005 [29]. Because of the inherently
tion is achieved through supramolecular chemistry rather than by con-
low concentration of antibodies internalizing into cells, detection meth-
ventional covalent attachment. After Gd@C60-mAb conjugation and
ods and sensitivities are key factors that have previously limited deter-
purification, Bio-Rad (Hercules, CA) protein assays were used to deter-
mining the degree of cellular targeting and cell internalization of
mine concentrations of the antibody in the samples. The cell binding
C60-antibody immunoconjugates. However, in the past two decades,
affinity of the Gd@C60-mAb immunoconjugates was evaluated by calcu-
inductively coupled plasma mass spectrometry (ICP-MS) has emerged
lating IP(50) values (the concentration at which 50% of antibody bind-
as an excellent tool in versatility and sensitivity because detection of
ing is achieved) from enzyme-linked immunosorbent assay (ELISA)
many chemical elements on the order of parts per trillion is now regu-
plots. Similar to an earlier C60-immunoconjugate study [29], 96-well
larly achieved [30]. Although carbon is not detectable by ICP-MS, this
plates continuing adherent A375m (antigen-positive) cell plates were
poses a problem for the detection of C60 itself. One solution is to sub-
used. However, to better understand binding efficiencies and nonspecific
stitute C60 fullerene with its endohedral metallofullerene analog
binding, T24 (antigen-negative) cells were also used for comparison with
(M@C60) to determine the amount of C60 internalized into the target
antigen-positive cells. Once the binding efficiency had been determined
cells. Recent innovations in the preparation and purification of water-
to have been retained for the immunoconjugates, samples of the conju-
soluble endohedral gadofullerenes (Gd3+ ion–filled fullerenes) such as
gates (0.667 nM) were digested with 70% HNO3, heated to dryness,
Gd@C60(OH)x (abbreviated hereafter as Gd@C60) now make these
and ICP-MS spectrometry was then used to determine the conjugate
materials available, and given our previous experiences with such
Gd@C60/mAb ratio. In addition, for a second independent determina-
gadofullerenes as magnetic resonance imaging contrast agents, they
tion of the Gd@C60/mAb ratio, the UV-Vis spectra of the naked anti-
provide a well-characterized system for study [25,31,32].
bodies and their Gd@C60 immunoconjugates were obtained.
In this study, Gd@C60 has been conjugated to both a melanoma
antibody (ZME-018) and an irrelevant murine IgG antibody (MuIgG)
as a control [29]. ZME-018 targets the gp240 antigen, found on the Internalization Studies
surface of more than 80% of human melanoma cell lines and biopsy Cell internalization studies for the Gd@C60 immunoconjugates
specimens [33]. Functionalized conjugates of ZME-018 have been were performed to determine the efficiency and rate with which the
used extensively, with studies ranging from in vitro fluorescent studies cell-specific Gd@C60 immunoconjugates internalize into melanoma
of surface antigens [34] to 111In–ZME-018 conjugate targeting as both cells. Antigen-positive (A375m) and -negative (T24) cells were pre-
a laboratory and a clinical in vivo tumor imaging agent [35,36]. ZME- pared on 100-mm2 tissue culture plates at 2 × 106 cells/plate in Dulbecco
018 shows great promise in clinical imaging trials [37] for the delivery modified Eagle medium (10% fetal bovine serum, pH = 7.4). The
of toxins, cytokines, and other therapeutic agents to melanoma cells cells were incubated overnight at 37°C, followed by addition of 1 ×
both in vitro and in vivo [38]. Immunoconjugates containing ZME- 10−6 g/ml (6.67 nM), 10 ml/plate, of the Gd@C60 immunoconjugates
018 reliably and rapidly internalize into melanoma cells [39] and effec- during various time frames. The plates were incubated for 1, 2, 4, and
tively localize into melanoma xenografts after systemic administration 24 hours at 37°C. At the end of the incubation period, the medium was

Figure 1. ELISA (dry cell) A375m (+) and T-24 (−): 2-hour incubation, dead cell tests of the Gd@C60 immunoconjugates. Results indicate
little change in the specific binding of the ZME-018 immunoconjugate from the original ZME-018 antibody.
352 Gd@C60-(ZME-018) Internalization into A375m Cells Berger et al. Translational Oncology Vol. 4, No. 6, 2011

Table 1. ICP-MS Results for the Gd@C60(OH)x Immunoconjugates and the Calculated Gd@C60 + C60/mAb Ratio.

mAb Conjugate mAb Concentration (nM)* Gd@C60 Concentration (nM) Total Fullerene (Gd@C60 + C60) Concentration (nM)† Total Fullerene/mAb Ratio

Gd@C60–(ZME-018) 0.667 1.30 3.11 4.66

Gd@C60-(MuIgG) 0.667 1.29 3.08 4.62

*Determined by Bio-Rad protein assay.

†
Determined assuming the metallofullerene sample contained 41.9% Gd@C60(OH)x and 58.1% C60(OH)x.

removed, and each cell sample was washed with glycine buffer solution binding over the unmeasurable IP(50) value of the naked ZME-018
(0.05 M, pH 2.5 + 0.1 M NaCl; 10 ml) to remove any remaining cell mAb, the difference between specific and nonspecific binding is still
surface–bound Gd@C60 immunoconjugate. The sample plates were greater by a factor of almost 300. These findings are encouraging for
then washed with a neutralizing buffer (0.15 M Tris, pH 7.4; 10 ml). the future development of fullerene immunotherapy, where C60 con-
Using a trypsin solution (0.25% Trypsin + EDTA [Gibco, Invitrogen, structs containing chemotherapeutic agents are to be targeted to specific
Carlsbad, CA]), the cells were then detached and centrifuged for ICP- cancer cells with the same type of immunoconjugates.
MS analysis. Trypsinization has not been previously shown to be a ICP-MS determined the [Gd3+] of each fullerene immunoconjugate
factor in influencing the outcome of internalization studies. sample, wherein the total fullerene concentration (assuming only
41.9% of the sample is Gd@C60[OH]x) in the sample and the total
fullerene (Gd@C60[OH]x + C60[OH]x)/mAb ratio was calculated, as
Determination of Gd 3+ Ion Concentration
shown in Table 1.
To determine whether Gd@C60 immunoconjugates internalize
In addition, UV-Vis spectra comparing the two immunoconjugates
into target cells and to what extent over time, each sample was placed
to the two naked antibodies exhibited increased absorbance from 250
in an individual 20-ml polytetrafluoroethylene scintillation vial and
to 450 nm, especially at shorter wavelengths (Figure 2). Calculating
digested for ICP-MS analysis using 3 ml of trace metal grade 70%
these differences compared to a Gd@C60(OH)x standard solution
HNO3, while slowly heating the vial to dryness to consume the cells
showed the total fullerene/mAb ratio for Gd@C60–(ZME-018) and
and Gd@C60, leaving behind Gd3+ ions for analysis. After cooling, the
Gd@C60-(MuIgG) to be 5.97 and 5.23, respectively. The discrepancies
resulting residues were dissolved in 3 ml of 2% HNO3, filtered using
between the total fullerene/mAb ratios as measured by ICP-MS (Table 1)
a Millex-GP (Millipore, Billerica, MA) polyethersulfone 0.22-μm
and UV-Vis spectroscopy indicate a small preferential selection by the
syringe filter, heated to dryness again, and then dissolved in 1 ml of
antibody for C60(OH)x over Gd@C60(OH)x.
2% HNO3—the matrix material used for ICP-MS.
For the time-dependent cell internalization experiments, the Gd3+
For each cell internalization study, ICP-MS sampled and mea-
ion concentrations for both the Gd@C60–(ZME-018) and Gd@C60-
sured the [Gd3+] 10 times for each of the Gd@C60–(ZME-018)
(MuIgG) samples were each determined 10 times using ICP-MS for
and Gd@C60-(MuIgG) immunoconjugates. An average of 10 deter-
the experiment run in triplicate (Tables W1, W2, and W3). The
minations per time point was used as the final [Gd3+] with its SD.
Gd@C60–(ZME-018) immunoconjugate exhibited an increase in de-
ICP–optical emission spectroscopy (OES) to determine the Gd3+
livery of Gd@C60 to the A375m cells, peaking in concentration of
concentration of the Gd@C60(OH)x/C60(OH)x sample was per-
7.06 × 1013 Gd@C60 + C60 molecules/cell at the 2-hour time point
formed using a Perkin-Elmer Optima 3000 DV ICP-OES system
(for Table W1 data) and then slowly declining to an average of 4.15 ×
(Waltham, MA). ICP-MS to determine the Gd3+ concentration of
1013 Gd@C60 molecules/cell after 24 hours (Figure 3). The antibody
the Gd@C60–(ZME-018), Gd@C60-(MuIgG), A375m, and T24
samples were performed using a Varian 810 quadruple ICP-MS
system (Bruker, Billerica, MA).

Results
The Gd@C60(OH)x sample used for antibody conjugation contained
both Gd@C60(OH)x and C60(OH)x species [31]; therefore, ICP–
optical emission spectroscopy (ICP-OES), with better than nanogram
sensitivity, was used to determine the percent of Gd@C60(OH)x
contained in the sample (41.9%).
The Gd@C60–(ZME-018) and Gd@C60-(MuIgG) immunocon-
jugate ELISA binding curves and IP(50) values were determined
for both cell lines (Figure 1). The IP(50) values reflecting binding
efficiencies to A375m cells for the Gd@C60–(ZME-018) immuno-
conjugate was 44.8 ng/ml. This is practically identical to nonconjugated
ZME-018, which demonstrated a IP(50) value of 43.9 ng/ml. When
compared with negative nonspecific MuIgG, which showed an IP(50)
value of 1228 ng/ml for the Gd@C60-(MuIgG) immunoconjugate
(nearly 800 times less efficient), clearly the Gd@C60–(ZME-018) Figure 2. UV-Vis spectra of Gd@C60(OH)x/C60(OH)x sample, the mAbs,
immunoconjugate retained cell specificity. The IP(50) value of the and their respective immunoconjugates. Increased absorption for
Gd@C60–(ZME-018) immunoconjugate binding to T24 cells was the immunoconjugates over the mAbs reflects the presence of
13,320 ng/ml. Although this represents an increase in nonspecific Gd@C60(OH)x (and C60(OH)x) in the immunoconjugates.
Translational Oncology Vol. 4, No. 6, 2011 Gd@C60-(ZME-018) Internalization into A375m Cells
Figure 3. Internalization of Gd@C60-mAb immunoconjugates into cells over time (experiment 1). Data indicate an increase in Gd@C60
specific delivery to the A375m cells, peaking at the 2-hour time point, and then slowly declining to the 24-hour end time point. Error bars
are SDs for 10 individual [Gd3+] determinations for each time point.
internalization experiment was repeated twice more (Tables W2 and
W3 and Figures W1 and W2) with similar results.
Discussion
Although it cannot be conclusively demonstrated that the immuno-
conjugates internalize into the cells, it is reasonable to infer from these
results that the immunoconjugates remain localized with the cell after
any remaining immunoconjugate is washed away, either internalized
within the cell or tightly bound to the plasma membrane surface.
These internalization experiments demonstrate the feasibility of
using ICP-MS for determining [Gd3+] at extremely low concentrations
(parts per trillion) after cell internalization of Gd@C60 immuno-
conjugates into A375m cells and are the first such studies to quantita-
tively determine antibody internalization concentration using metal ion
labeling experiments. For comparison, attempts to internalize the
Gd@C60 immunoconjugates into T24 antigen-negative cells were also
performed. These experiments showed significantly lower [Gd3+] in the
T24 cells (Tables W1, W2, and W3), demonstrating that the Gd@C60
immunoconjugates retained their cell-specific targeting properties for
A375m cells as well as verifying successful internalization into the cells.
These results suggest that Gd@C60-based immunoconjugates do, in-
deed, internalize into target cells, which is encouraging for the future
development of fullerene immunotherapy.
Acknowledgments
The authors thank Yongjun Gao and Minako Righter at the Univer-
sity of Houston-Main Campus for their help with the collection of
ICP-MS data.
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Table W1. Cell Internalization Results Over Time for the Gd@C60–(ZME-018) and Gd@C60-(MuIgG) Immunoconjugate Internalization (Experiment 1).

Cells, mAbs, Incubation Period Gd Concentration in Sample (ppt)* No. Cells Gd@C60 + C60 per Cell mAbs per Cell

A375m, No mAb, 0 h 0.0 (0.00) 2.67e + 06 0.00e + 00 0.00e + 00

A375m, ZME-018, 1 h 4.0 (0.03) 9.00e + 05 4.05e + 13 8.41e + 12
A375m, ZME-018, 2 h 13.5 (0.13) 1.75e + 06 7.06e + 13 1.47e + 13
A375m, ZME-018, 4 h 10.2 (0.07) 1.63e + 06 5.71e + 13 1.19e + 13
A375m, ZME-018, 24 h 13.8 (0.09) 3.03e + 06 4.15e + 13 8.61e + 12
A375m, MuIgG, 1 h 4.2 (0.03) 1.43e + 06 2.68e + 13 5.61e + 12
A375m, MuIgG, 2 h 7.1 (0.06) 1.40e + 06 4.66e + 13 9.77e + 12
A375m, MuIgG, 4 h 8.0 (0.18) 1.94e + 06 3.77e + 13 7.91e + 12
A375m, MuIgG, 24 h 13.6 (0.11) 4.88e + 06 2.55e + 13 5.35e + 12
T24, No mAb, 0 h 0.0 (0.00) 1.15e + 06 0.00e + 00 0.00e + 00
T24, ZME-018, 1 h 1.1 (0.01) 1.24e + 06 8.18e + 12 1.70e + 12
T24, ZME-018, 2 h 2.3 (0.01) 5.40e + 05 3.86e + 13 8.01e + 12
T24, ZME-018, 4 h 2.8 (0.02) 8.00e + 05 3.19e + 13 6.63e + 12
T24, ZME-018, 24 h 4.5 (0.03) 2.14e + 06 1.93e + 13 4.01e + 12
T24, MuIgG, 1 h 4.1 (0.03) 1.00e + 06 3.78e + 13 7.93e + 12
T24, MuIgG, 2 h 2.4 (0.01) 5.80e + 05 3.82e + 13 8.01e + 12
T24, MuIgG, 4 h 1.8 (0.02) 5.00e + 05 3.22e + 13 6.74e + 12
T24, MuIgG, 24 h 8.3 (0.03) 2.07e + 06 3.66e + 13 7.66e + 12

*Gd3+ ion concentration in parts per trillion from the average of 10 individual ICP-MS measurements per sample with the SD in parentheses.

Table W2. Cell Internalization Results Over Time for the Gd@C60–(ZME-018) and Gd@C60-(MuIgG) Immunoconjugate Internalization (Experiment 2).

Cells, mAbs, Incubation Period Gd Concentration in Sample (ppt)* No. Cells Gd@C60 + C60 per Cell mAbs per Cell

A375m, No mAb, 0 h 0.0 (0.00) 6.75e + 06 0.00e + 00 0.00e + 00

A375m, ZME-018, 1 h 35.7 (0.98) 7.30e + 06 4.46e + 13 9.27e + 12
A375m, ZME-018, 2 h 97.5 (2.08) 9.50e + 06 9.38e + 13 1.95e + 13
A375m, ZME-018, 4 h 55.6 (2.63) 7.60e + 06 6.68e + 13 1.39e + 13
A375m, ZME-018, 24 h 62.3 (1.32) 1.20e + 07 4.75e + 13 9.85e + 12
A375m, MuIgG, 1 h 20.6 (0.86) 7.50e + 06 2.51e + 13 5.25e + 12
A375m, MuIgG, 2 h 39.3 (1.45) 8.10e + 06 4.43e + 13 9.29e + 12
A375m, MuIgG, 4 h 43.7 (1.29) 1.08e + 07 3.70e + 13 7.75e + 12
A375m, MuIgG, 24 h 19.7 (0.28) 9.30e + 06 1.93e + 13 4.05e + 12
T24, No mAb, 0 h 0.0 (0.00) 5.70e + 06 0.00e + 00 0.00e + 00
T24, ZME-018, 1 h 5.1 (0.12) 6.05e + 06 7.66e + 12 1.59e + 12
T24, ZME-018, 2 h 33.3 (1.20) 9.10e + 06 3.34e + 13 6.94e + 12
T24, ZME-018, 4 h 24.0 (0.71) 9.80e + 06 2.24e + 13 4.64e + 12
T24, ZME-018, 24 h 13.9 (0.20) 9.80e + 06 1.30e + 13 2.69e + 12
T24, MuIgG, 1 h 17.9 (0.73) 6.40e + 06 2.56e + 13 5.36e + 12
T24, MuIgG, 2 h 24.1 (1.35) 6.20e + 06 3.55e + 13 7.45e + 12
T24, MuIgG, 4 h 34.3 (1.74) 9.90e + 06 3.17e + 13 6.65e + 12
T24, MuIgG, 24 h 28.2 (1.08) 1.14e + 07 2.26e + 13 4.74e + 12

*Gd3+ ion concentration in parts per trillion from the average of 10 individual ICP-MS measurements per sample with the SD in parentheses.

Table W3. Cell Internalization Results Over Time for the Gd@C60–(ZME-018) and Gd@C60-(MuIgG) Immunoconjugate Internalization (Experiment 3).

Cells, mAbs, Incubation Period Gd Concentration in Sample (ppt)* No. Cells Gd@C60 + C60 per Cell mAbs per Cell

A375m, No mAb, 0 h 0.0 (0.00) 6.65e + 06 0.00e + 00 0.00e + 00

A375m, ZME-018, 1 h 19.8 (0.38) 4.30e + 06 4.19e + 13 8.70e + 12
A375m, ZME-018, 2 h 36.9 (1.73) 3.60e + 06 9.34e + 13 1.94e + 13
A375m, ZME-018, 4 h 33.5 (1.32) 4.50e + 06 6.79e + 13 1.41e + 13
A375m, ZME-018, 24 h 27.8 (0.90) 5.90e + 06 4.31e + 13 8.93e + 12
A375m, MuIgG, 1 h 18.6 (0.62) 6.55e + 06 2.60e + 13 5.38e + 12
A375m, MuIgG, 2 h 17.2 (0.39) 3.35e + 06 4.69e + 13 9.72e + 12
A375m, MuIgG, 4 h 23.9 (0.60) 5.85e + 06 3.72e + 13 7.72e + 12
A375m, MuIgG, 24 h 17.8 (0.89) 7.40e + 06 2.20e + 13 4.56e + 12
T24, No mAb, 0 h 0.0 (0.00) 1.43e + 06 0.00e + 00 0.00e + 00
T24, ZME-018, 1 h 1.9 (0.11) 2.12e + 06 8.14e + 12 1.69e + 12
T24, ZME-018, 2 h 14.5 (0.49) 3.30e + 06 4.00e + 13 8.30e + 12
T24, ZME-018, 4 h 11.6 (0.24) 3.38e + 06 3.14e + 13 6.50e + 12
T24, ZME-018, 24 h 3.6 (0.05) 2.32e + 06 1.43e + 13 2.96e + 12
T24, MuIgG, 1 h 10.2 (0.36) 3.15e + 06 2.96e + 13 6.14e + 12
T24, MuIgG, 2 h 11.1 (0.51) 2.55e + 06 3.96e + 13 8.22e + 12
T24, MuIgG, 4 h 9.1 (0.23) 2.75e + 06 3.03e + 13 6.29e + 12
T24, MuIgG, 24 h 8.6 (0.19) 3.25e + 06 2.42e + 13 5.03e + 00

*Gd3+ ion concentration in parts per trillion from the average of 10 individual ICP-MS measurements per sample with the SD in parentheses.
Figure W1. Internalization of Gd@C60-mAb immunoconjugates into cells over time (experiment 2). Error bars are SDs for 10 individual
[Gd3+] determinations for each time point.

Figure W2. Internalization of Gd@C60-mAb immunoconjugates into cells over time (experiment 3). Error bars are SDs for 10 individual
[Gd3+] determinations for each time point.