Doyle et al.indd

Drug Target Insights 2008:3 13–25 13

ORIGINAL RESEARCH

Correspondence: Robert P. Doyle, Department of Chemistry, Syracuse University, Syracuse, NY 13244-4100,
U.S.A. Tel: +1 315 443 3584; Email: rpdoyle@syr.edu

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Targeting Gallium to Cancer Cells through the Folate Receptor
Nerissa Viola-Villegas, Anthony Vortherms and Robert P. Doyle
Department of Chemistry, Syracuse University, Syracuse, NY 13244-4100, U.S.A.

Abstract: The development of gallium(III) compounds as anti-cancer agents for both treatment and diagnosis is a
rapidly developing fi eld of research. Problems remain in exploring the full potential of gallium(III) as a safe and
successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III) compounds have little
tropism for cancer cells. We have combined the targeting properties of folic acid (FA) with long chain liquid polymer
poly(ethylene glycol) (PEG) ‘spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of
1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) through amide linkages for delivery into target
cells overexpressing the folate receptor (FR). In vitro cytotoxicity assays were conducted against a multi-drug resistant
ovarian cell line (A2780/AD) that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO)
cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer
at pH 7.4.

Keywords: folate receptor, gallium, DOTA, targeting, cytotoxicity

Introduction
The anti-cancer properties of gallium(III) have been extensively investigated since 1971 (Hart et al.
1971). Gallium has numerous ways to induce cell death, including DNA binding and modifi cation
(Hedley et al. 1988), enzyme inhibition (especially ribonucleotide reductase) (Chitambar et al. 1988),
and ion transport disruption (such as calcium effl ux from mitochondria), a known trigger of cellular
apoptosis (Collery et al. 1996).

In general, the poor pharmacokinetic properties of gallium salts investigated have prevented their
widespread use in chemotherapy. Efforts to develop gallium complexes to improve its profi le, by
addressing the problems of hydrolysis, poor absorption, poor solubility, rapid renal excretion and little
tropism for cancer cells are currently underway (Keppler and Jakupec, 2004; Desoize, 2004). Complexes
such as those produced by the groups of Keppler (Keppler et al. 2006), Sharma (Sharma et al. 2007),
Low and Green (Low and Green et al. 1996) have been successful in increasing plasma concentrations
of gallium, providing better antiproliferative effects or improved imaging of cancer cells (when using
gallium radioactive isotopes (67Ga γ, 68Ga β+)) (Greenwood and Earnshaw, 2005). Problems still remain
however especially in regards to renal retention times and cancer cell targeting.

Folic acid (FA) (see Fig. 1) is a vitamin potentially capable of delivering agents specifi cally to folic
acid-receptor (FR) positive tumors (Lee and Sudimack, 2000). FRs are membrane glyco-proteins (Anderson
et al. 1990) overexpressed by a number of tumor cell types such as ovarian, breast, cervical, colorectal,
renal and nasopharyngeal cancers (Antony, 1996). Cells overexpressing the FR bind FA-drug conjugates
tightly (Kd ∼ 0.42 × 10

−9 M) (Shen et al. 1995) and endocytose them inside (Anderson et al. 1988), provided
that chemical modifi cation of the FA upon conjugation does not disrupt recognition by the FR (Liu et al.
2005). Targeting the FR is attractive because in addition to being overexpressed in tumor lines, it is down-
regulated (and inaccessible to blood circulation) in healthy adult cells (Anderson et al. 1988).

We are primarily focused on ovarian tumors since they have been shown to greatly overexpress the
FR (see Table 1). Current treatments for ovarian cancer have a number of serious side effects associated
with their use including kidney damage, hearing loss and even secondary cancers (Sun et al. 2002). In
addition, over 75% of patients are diagnosed when the disease has already progressed to stage III or
IV, with only a 10%–20% 5 year survival rates, respectively (Ries, 1993). New ways to diagnose and/or
treat this illness are therefore urgently needed.

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Viola-Villegas et al

Drug Target Insights 2008:3

We set out to synthesize, purify and evaluate
in vitro a new FA bioconjugate of gallium and
compare its activity to the unconjugated gallium
analog. We began by initially complexing gallium
(III) to the 1,4,7,10-tetraazacyclo-dodecane-
N,N′,N′′,N′′′-tetraacetic acid (DOTA) ligand
(Doyle et al. 2006). We have previously described
the synthesis and solid state structure of this system
(see Fig. 2). Preceding literature reports have
demonstrated good kinetic and thermodynamic
stability provided by the DOTA ligand in its coor-
dination chemistry and we wished to exploit this
coupled with folate receptor (FR) targeting conju-
gates. In addition to coupling the gallium-DOTA
complex to folic acid we wished to include
poly(ethylene glycol) (PEG) polymer linkers
between the FA and gallium complex since
‘PEGylated’ FA conjugates have been shown to
have greater affi nity for the FR than free FA (Low
and Lee, 1994). Pathways that can break down or
effl ux certain FA conjugates are inhibited by the
polymer-FA conjugate and renal retention times of
certain pharmaceuticals have been improved by
conjugation to PEG units (Anderson et al. 2005).
Hence, the use of FA-PEG conjugates yields syn-
ergistic traits that are of particular interest.

Conjugation of PEG to FA through the glutamate
moiety produces two regioisomer products at the
α- and γ-carboxylic acid functional groups that need
to be separated. FA modifi ed at the α-carboxylic acid
loses its affi nity for the FR, making it unsuitable as
a targeting agent (Yan and Ratnam, 1995). This
separation is diffi cult when using polydisperse PEG
units. Such PEG units are typically all that are com-
mercially available but are approved for use by the
FDA (Qui and Bae, 2006). A facile route to separation
was previously reported by us and this route was used
here to allow access to pure γ-FA-PEG-NH2 for sub-
sequent conjugation to the gallium-(HDOTA)
complex (Doyle et al. 2008).

In vitro cytotoxicity assays were conducted
against adriamycin resistant ovarian cancer cell
line (A2780/AD), which overexpresses the FR, and
contrasted against a non-FR expressing Chinese
hamster ovary (CHO) control line.

Experimental

Chemicals
The following reagents were purchased and used
without further purifi cation: Folic acid (FA) (98%,
Sigma), N,N’-Dicyclohexylcarbodiimide (DCC,
�99%, Fluka), polyethylene glycol bis(amine)
(PEG, MW: 2000) (Fluka), N-Hydroxysuccinimide
(NHS, �97%, Fluka), and 1,4,7,10-tetraazacyclo-
dodecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA,
98%, Strem Chemicals), N-Hydroxysulfosuccin-
imide sodium salt (sulfo-NHS, 98.5%, Fluka), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC, 98%, Alfa Aesar) and trifl uoroacetic acid
(99%, Aldrich). GaCl3 (99.9%) was purchased
from Alfa Aesar and dissolved in 100 mM ammo-
nium acetate (pH 4.8) to make a stock concentra-
tion of 1.325 M. Dimethylsulfoxide (DMSO) (min.
99.9%, Sigma) was dried by running the solvent
through a column of 4 Å molecular sieves (Mallin-
crodt) dried previously overnight at 120 °C. Sol-
vents used for HPLC and growth media are fi ltered
with 0.45 µm fi lter (Fisher). Pyridine (99.9%) was
obtained from Fisher. Triethylamine (99.5%) was
purchased from Sigma Aldrich. 3′-azido-3′-
deoxythymidine (AZT; used as internal control in
cytotoxicity assays) was purchased from Sigma
Aldrich. All other reagents and buffers used were
of reagent grade or higher. Ultra pure water (18.6
MΩ) was used through out the investigation. All
syntheses except for 1 were performed in a

Figure 1. FA with its three major structural components including the
α- and γ-carboxylic acid group of the glutamate moiety indicated.

Table 1. Comparison of FR overexpression investigated
in different cancer tissues via immunohistochemistry
(IHC) and reverse transcriptase—polymerase chain
techniques (RT-PCR) (Low and Leamon, 2005).

Tissue IHC (%) RT-PCR (%)
Ovarian 93 100
Endometrial 91 100
Breast 21 80
Lung 33 33
Colorectal 22 20
Kidney 50 100

Targeting gallium to cancer cells through the folate receptor

Drug Target Insights 2008:3

dark-room under a 15 W red light. All reactions were
conducted under nitrogen gas at ambient conditions
unless otherwise stated with sample transfer con-
ducted by cannula (24 inch, 16 gauge).

Physical measurements
and instrumentation
An Agilent 1100 reverse phase high pressure liquid
chromatography (HPLC) with manual injection
and automated fraction collector was fi tted with a
Zorbax C18 analytical column (42 × 10 mm) for
analytical trace analysis with a flow rate of
0.7 ml/min. Purifi cation was achieved using a C18
(9.4 × 250 mm) semi-preparative column using a
fl ow rate of 2 ml/min. Detection was by ultra vio-
let monitoring at 280 nm. The linear gradient used
was: (1) 90% 5 mM Na2HPO4 (pH 7.0) and 10%
acetonitrile over 10 minutes; (2) 40% 5 mM
Na2HPO4 and 60% acetonitrile over 20 minutes.
Ion exchange chromatography (IEC) was conducted

on an Akta Prime Plus with Primeview 5.0 soft-
ware. The ANX (1 ml) and the PD10 Sephadex
G-25M desalting (10 ml) columns were purchased
from GE Healthsciences. 1H nuclear magnetic
resonance (1H NMR) was performed on Bruker
Avance DPX 500 MHz. A Shimadzu LCMS-2010
A mass spectrometer and An Applied Biosystems
Voyager-DE linear Matrix Assisted Laser Desorp-
tion Ionization—Time of Flight mass spectrometer
(MALDI-TOF) were used for mass spectrometry
analysis. Infrared (IR) analyses were performed as
KBr pellets on a Nicolet Magna-IR 850 series II
spectrophotometer. A Perkin Elmer ELAN 6100
was used to conduct inductively coupled plasma
analysis (ICP). Centrifugation was performed
using a Sorvall Legend RT centrifuge typically as
10 minute runs at 4000 rpm at 4 ºC. Optical densi-
ties were measured with a Thermo Multiskan EX
96-well plate reader equipped with Ascent Soft-
ware version 2.6 with 450 nm fi lter.

Chemical synthesis

Synthesis of GaHDOTA (1)
1 was synthesized as reported previously by Doyle
et al. (Doyle et al. 2006).

Synthesis of γ-FA-PEG-NH2 (γ-2)
FA (0.0441 g, 0.100 mmol) was dissolved in 3 ml
of dry DMSO. To this solution, 0.0127 g of NHS
(0.110 mmol) was added. The mixture was stirred
for 5 minutes after which 0.023 g (0.110 mmol) of
DCC was added. The solution was then stirred
overnight. The activated FA was fi ltered through
a 0.45 µM fi lter to remove the dicyclohexylurea
side product. The FA-NHS solution was then added
dropwise to PEG2000 (0.200 g, 0.100 mmol)
previously dissolved in 3 ml DMSO. 100 µL of
pyridine was then added and the reaction stirred
overnight. Approximately 25 ml of chilled
isopropanol (−78 °C) was added forming a light
yellow precipitate. The precipitate was obtained
via centrifugation. The γ- and α-isomers of 2 were
separated via IEC using the following method
[Doyle et al. 2008). 2 was redissolved in water to
give a [20 mg/ml] concentration. This solution was
desalted using a 10 ml sephadex PD10 desalting
column eluting the product in water. 500 µL of this
solution was injected into a 1 ml ANX weak
anion exchange column. The fl ow rate was set
at 0.1 ml/min. The column was then washed with

Figure 2. Crystal structure of Ga(HDOTA) [20].

Viola-Villegas et al

Drug Target Insights 2008:3

5 column volumes of water. After the fi rst peak
was eluted, the column was then washed following
a gradient (solvent A, water; solvent B, 100 mM
ammonium acetate, pH 10) of 10% B for 17 column
volumes, 50% B for 17 column volumes, 80% B
for 15 column volumes. A column volume of 5 ml
of 0.5 M NaCl was used to fl ush the column. 1H
NMR (D2O): δ 8.62 (s, 1H), δ 7.83 (t, 2H), δ 6.63
(d, 2H), δ 3.50–3.80 (m, PEG). Yield: 60% based
on PEG.

Only the isolated γ -isomer (γ -2) was used for
subsequent coupling.

Synthesis of γ-FA-PEG-H3DOTA ( γ-3)
The sulfo-succinamide ester of DOTA was prepared
by activating 0.121 g (0.300 mmol) of the ligand
with 26.5 µL (0.150 mmol) EDC and 0.0260 g
(0.120 mmol) sulfo-NHS in 2 ml water. γ-2 (0.0726 g,
0.03 mmol) was dissolved in 2 ml water and cooled
to 4 °C. To the DOTA solution, γ-2 was added
dropwise and the pH was adjusted to 8.5. The
reaction was left to stand overnight. γ-3 was
obtained via HPLC with a retention time, of Tr =
20.4 minutes. 1H NMR (D2O): δ 8.76 (s, 1H), δ
7.62 (d, 2H), δ 6.73 (d, 2H), δ 4.61 (s, 2H), δ 4.48
(m, 2H), δ 3.90 – 3.29 (m, PEG), δ 2.87 (d, 2H).
Yield: 64.4% based on γ-2.

Synthesis of γ-FA-PEG-Ga(HDOTA) ( γ-4)
1 (3.42 mg, 0.00726 mmol) was dissolved in 1 ml
of 20:80 water:DMSO solution. A volume of 1 ml
containing dissolved NHS (0.800 mg, 0.00695
mmol) and DCC (1.5 mg, 0.00727 mmol) was
added to the solution of 1. This mixture was
stirred for 30 minutes. γ-2 (17.6 mg, 0.00726
mmol) was subsequently dissolved in 1 ml of
DMSO. A volume of 100 µl of triethylamine was
added to this solution and was also stirred for 30
minutes. The solution of γ-2 was then added
dropwise to the solution of 1. The mixture was
left to react overnight. The resulting solution was
filtered with a 0.45 µm filter and the crude
product precipitated with 25 ml of chilled
isopropanol (−78 °C). A yellow solid was isolated
by centrifugation and redissolved with 1 ml water
and purifi ed by HPLC. 1H NMR (D2O): δ 8.64
(s, 1H), δ 7.68 (d, 2H), δ 6.85 (d, 2H), δ 4.61 (s,
2H), δ 3.83 – 3.20 (m, PEG), δ 2.32 (d, 12H), δ
1.18 – 1.13 (t, 11H). MALDI-TOF: 2715.00 m/z
(M+H+) calculated 2876.25 for γ-4. Yield: 82.9%
based on γ-2.

Cell lines and culture conditions
Adriamycin resistant ovarian cancer cell line
(A2780/AD) and Chinese hamster ovary (CHO) cell
line were cultured as adherent monolayers in RPMI
1640 (Invitrogen) growth media containing L-glu-
tamine and FA supplemented with 10,000 units
penicillin and 10 mg/ml streptomycin (Sigma), 10%
(v/v) fetal bovine serum (Sigma). CHO cells were
obtained from the ATCC. The A2780/AD cell line
used for testing was provided by the Fox Chase
Cancer Centre, Philadelphia. Cells were incubated
and grown in a VWR mammalian incubator at 5%
CO2 and 95% humidity. The presence of the FR in
the A2780/AD line (and indeed absence in CHO
cells) was followed by RT-PCR and confocal micros-
copy (Doyle et al, unpublished results). All prepara-
tions for cell culture and assays were conducted in
a sterile environment under a Labconco Purifi er I
Laminar fl ow hood. Cells were cultured in Millipore
250 mL culture bottles with vented lids.

Drug cytotoxicity
The proliferation of the exponential phase cultures
of A2780/AD and CHO cells was assessed by
colorimetric assay. WSK-8 (Dojindo) was per-
formed according to manufacturer’s instructions.

Adherent cell cultures were harvested by strip-
ping of culture fl asks by a non-enzymatic cell
stripper (Mediatech) after a 30 minute incubation
period. The cells were then collected. The cell
densities were adjusted using FA-free RPMI 1640
media to 3.0 × 104 cells/ml to guarantee exponen-
tial growth for the period of drug exposure. To each
well, aliquots of 100 µL were inoculated. After a
24 hour incubation time to facilitate adherence, the
FA free RPMI media was removed and replaced
with 200 µl of fresh media containing different
concentrations of 1, γ-2, γ-3, γ-4, DOTA and a
control of AZT. The cells were then incubated for
72 hours. Optical densities were measured at
450 nm using a plate reader. The percentage of cell
viability was determined relative to untreated con-
trol microcultures.

Stability studies
2 mM solutions of γ-4 were prepared from 25 mM
HEPES (pH 7.4) and RPMI 1640 FA-free media.
These solutions were incubated over 72 hours at
37 °C. Solutions made from the media were fi ltered
by centrifugation using a centrifugal fi lter (Pall
Life Sciences, MW: 1000 g/mol) at 4,000 rpm over

Targeting gallium to cancer cells through the folate receptor

Drug Target Insights 2008:3

15 minutes. Analytical C18 reverse phase HPLC
analysis was conducted at 0, 1, 24, 48 and 72 hours.
At 72 hours, fractions were analyzed for the
presence of gallium via inductively coupled
plasma (ICP).

Results and discussion

Chemical synthesis
The synthesis of 1 (Scheme 1) was prepared by
direct addition of stoichiometric equivalents of
gallium to DOTA under acidic (pH 4.8) conditions.
Crystals were grown after concentrating the solu-
tion to its saturation point. Five volume equivalents
of acetone was then added and the suspension
fi ltered. The clear, colorless solution was then
placed at 4 °C. Colorless needle-shaped crystals
formed after 24 hours.

FA was activated by a reaction with DCC/NHS
to couple it to PEG. α/γ-2 was subsequently
separated via IEC using a weak anion exchange
column as shown in Scheme 2. The fi rst and second
peak eluted both isomers. Upon increasing the
conductivity to 3 mS/cm, a third peak eluted to
give γ-2. Analytical HPLC runs of fractions
collected from IEC confi rmed the identity of the
isomers. Previous work separating both isomers
established the α-2 eluting at a later time than the
γ-isomer with reverse phase HPLC (Doyle et al.
2008). The identities of the peaks from fractions
collected from the IEC were confi rmed with γ-2
eluting at Tr = 23.64 minutes and α-2 subsequently
eluting at Tr = 26.34 minutes.

γ-3 was made with sulfo-NHS/EDC water
soluble cross linkers to give the metal-free
compound. EDC formed an O-acylisourea
intermediate with DOTA. This converts to the
sulfo-hydroxysuccinimide DOTA ester in the
presence of the amine reactive sulfo-NHS. Amidation
proceeds upon addition of the amine group of γ-2
(see Scheme 3).

Coupling of γ-2 and 1 proceeded using DCC/
NHS coupling agents (see Scheme 4). TEA was
added to γ-2 improving the nucleophilicity of the
amine end of the conjugate. Since 1 is only soluble
in water, it was dissolved in a minimal amount of
water followed by addition of DMSO. The water
to DMSO ratio was 20:80.

Mass spectra
A MALDI-TOF mass spectrum of the commercial
PEG displayed a mass range of 1900–2100 m/z due
to the polydispersity of the polymer. The observed
mass of γ-2 is centered at 2422 m/z in agreement
with the calculated theoretical mass of ~ 2400 m/z
for the polydisperse PEG containing system. The
calculated theoretical mass of γ-4 is ~ 2900 m/z.
A central range at 2846 m/z was observed from
MALDI-TOF mass spectrometry analysis. The
44 m/z spacing is indicative of one unit of ethylene
glycol (MW: 44 g/mol). Figure 3a–b displays the
mass spectra obtained for γ-2 and γ-4 respectively.

In vitro biological activity
IC50 concentrations were calculated using an expo-
nential fi t. Table 2 shows the potency of 1, γ-2, γ-3,

Scheme 1. Synthesis of 1 involving chelation of Ga(III) to DOTA in ammonium acetate buffer (pH 4.8).

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Drug Target Insights 2008:3

Scheme 2. Synthesis and separation, via IEC of the regioisomers of the α- and γ-isomers of 2.

Scheme 3. Synthesis of γ-3 using EDC/sulfo-NHS as coupling agents.

Targeting gallium to cancer cells through the folate receptor

Drug Target Insights 2008:3

γ-4 and DOTA against both the A2780/AD and
CHO cell lines. AZT was used as an internal con-
trol (data not shown). In all cases toxicity was
greater in the A2780/AD line over the CHO line.
1, γ-3, γ-4 and DOTA displayed between [0.18 and
1.85 mM] activity against A2780/AD cells and
between [0.8 and 2.93 mM] in CHO cells. γ-2
provided no IC50 concentration over 72 hours at
concentrations up to [100 mM]. The fact that the
FA-PEG moiety is not toxic indicates that the activ-
ity of the completed conjugates stems from the
gallium metal or DOTA ligand itself.

Interestingly gallium compounds were noted as
less toxic than free DOTA containing controls. This
was the case in both cell lines. The IC50 concentra-
tions for both compounds containing gallium
(namely 1 and γ-4) indicate that gallium is in fact
reducing the toxicity of the DOTA moiety, presum-
ably by chelation, hence preventing the scavenging
by DOTA of other essential metals.

Toxicity of the FA-PEG containing DOTA
compound (γ-3) against CHO cells can then also
be explained as metal scavenging outside the cell,
with uptake not necessary (and not possible in
CHO without the FR). DOTA and γ-3 displayed

IC50 concentrations of 800 µM and 1.35 mM
against CHO and 580 µM and 180 µM against
A2780/AD cell lines respectively. The presence of
the FR receptor in A2780/AD and more rapid divi-
sion in A2780/AD over CHO helps explain the
greater toxicity.

Toxicity of γ-4 in CHO cells was noted as
2.93 mM and 1.85 mM in A2780/AD cells. The
reduced toxicity of the DOTA compounds previously
complexed with gallium is consistent with the
observed toxicity of free DOTA and supports the
idea that the toxicity lies with metal scavenging by
DOTA and supercedes any gallium related toxicity.
The toxicity in CHO cells can only be explained then
if gallium is leaching from the DOTA macrocyle,
since this would produce a gallium salt or complex
and would leave DOTA now uncomplexed and free
to chelate other metals.

Gallium decomplexation from a ligand such as
DOTA that renders such thermodynamic and
kinetic stability is possible even with a reported
stability constant of log K ~ 21.33 (Clark and
Martell, 1991) (compared to say to open-chain
multidentate ligands like ethylenediamine
(log K ∼ 17.2) (Harris and Martell, 1976)). A key

Scheme 4. Synthesis of γ-4 illustrates the coupling of γ-2 and 1 using DCC and NHS as coupling agents in dry DMSO.

Viola-Villegas et al

Drug Target Insights 2008:3

factor in this release is an increase in DOTA’s
electron density due to inductive effects contributed
by the ethylene bridges (Hancock and Martell,
1995).

A plausible explanation for the possibility of
gallium release concerns the formation of the FA
conjugate. The stability of γ-4 may be affected by
the conjugation of one of the pendant carboxylate
arms of DOTA. This phenomenon has been
observed by several investigations that involve
modifi cation of the DOTA side arms. Sherry et al.
in their work involving gadolinium-DOTA conju-
gated to a propylamide group via one carboxylate
arm has reported a stability constant that is con-
siderably lower (105 fold) than the DOTA complex

owing to the decrease in basicity of the amine
macrocycles (Sherry et al. 1989). A recent
investigation reported that substitution with a
p-NO2-benzyl group at either one of the DOTA
arms resulted in a reduction in the cooperative
binding of the ligand and a lower thermodynamic
stability constant compared to unmodifi ed metal-
DOTA complex (Sherry et al. 2004). Of course
thermodynamic stability does not necessarily
translate into in vivo stability with kinetic inertness
often being of greater importance. This may also
have a role to play in gallum’s release.

Structural studies by Csajbok et al. via 1H NMR
reveal the occurrence of ring inversion and fl ux-
ionality in DOTA with an increase in temperature

(a)

(b)

Figure 3. MALDI-TOF mass spectrometry analysis of a) γ-2 and b) γ-4 showing a central peak at ca. 2400 m/z and 2846 m/z respectively.
ICP also confi rmed the presence of gallium in γ-4.

Targeting gallium to cancer cells through the folate receptor

Drug Target Insights 2008:3

(Csajbok et al. 2004). Similarly, proton exchange
can occur between ring amine groups and the car-
boxylate pendant arms, which may trigger decom-
plexation (Goddard et al. 2001). With the ligand’s
dynamic exchange process occurring in solution
decomplexation can occur. A whole series of
gallium compounds have been screened for
in vitro cytotoxicity. A series of gallium compounds

with signifi cant toxicity are shown for comparision
in Table 3.

To prove that gallium has indeed been “freed”
from its macrocyclic cage, stability studies in
HEPES buffer and RPMI 1640 media over 72 hours
coupled with HPLC and ICP techniques were con-
ducted. New peaks were observed between 48 and
72 hours indicative of gallium release (see supple-
mental material). These peaks were analyzed via
ICP and gallium was noted. In addition, a slight
precipitate could be removed by fi ltration (0.22 µM
fi lters) and ICP confi rmed the presence of gallium
in the collected solid fraction. Attempts to identify
the new species were unsuccessful by electrospray
mass spectrometry and 1H NMR and attempts to
obtain crystals for X-ray structural analysis also
proved unsuccessful. It is likely that both soluble
and insoluble gallium salts (such as gallium hydrox-
ides) are forming and/or gallium is complexing with
compounds found in the RPMI media. The compa-
rable observed cytotoxicities on both cell lines can
then be ascribed to gallium leaching from DOTA
over 72 hours.

Table 2. IC50 concentrations for 1, γ-2, γ-3, γ-4 and
DOTA against A2780/AD ovarian cells and Chinese
hamster ovary (CHO) cells. (−) indicates no IC50 was
recorded. AZT was used as a control and returned an
IC50 concentration of ∼ 6–8 mM consistent with literature
values (Doyle et al. 2008).

Compound IC50 (mM) (72hrs)
CHO A2780/AD
1 1.61 0.77
γ-2 (−) (−)
γ-3 1.35 0.18
γ-4 2.93 1.85
DOTA 0.80 0.58

Table 3. Ga compounds of various ligands (L) tested on cell lines showing signifi cant antiproliferative activity.
IC50 concentrations were obtained at 72 hours unless otherwise noted. PIH is Pyridoxal Isonicotinoyl Hydrazone.

Ligand (L) IC50 Cell line References
2-acetylpyridine 4N-
dimethylthiosemicarbazone

1.33 +/− 0.43 nM – 96 hr
2.10 +/− 0.90 nM – 96 hr
0.18 +/− 0.02 nM – 96 hr

Ovarian: 41M
Mammary: SK-BR3

Colon: SW480

Kenpaullone �1 µM – 48 hr Lung: CCRF-CEM; 27
K-562; MLT-4

�5 µM – 48 hr Colon: HCT-116; HCT-15;
HT29; SW-620

�1 µM – 48 hr Melanoma: SK-MEL-28; SK-MEL-5
�10 µM – 48 hr Ovarian: OVCAR-3
�10 µM -48 hr Breast: MCF7

PIH 50 µM Lung: CCRF-CEM 28
Chloride 175 µM – 48 hr Leukemia: L1210 29

16 µM – 96 hr
Transferrin 1.1 +/- 0.2 µM Leukemia: HL60 30
Nitrate 120 µM Lung: CCRF-CEM 2

80 µM S-phase arrest Lung
1 1.61 mM Ovarian: CHO This work

0.77 mM Ovarian: A2789/AD
γ-3 1.35 mM Ovarian: CHO This work

180 µM Ovarian: A2789/AD
γ-4 2.93 mM Ovarian: CHO This work

1.85 mM Ovarian: A2789/AD

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Drug Target Insights 2008:3

Conclusion
We have successfully synthesized, characterized,
and investigated the in vitro cytotoxicity studies
of DOTA based gallium complexes and conducted
controls to track the source of the toxicity. These
results demonstrate that while a ligand of extraor-
dinary kinetic and thermodynamic stability gallium
can ‘leach’ from DOTA over a 72-hour period.
What is also clear is that DOTA itself has between
[500–800 µM] toxicity, an interesting note in and
of itself. Toxicity in both lines could be explained
by the uptake, by diffusion, of free DOTA or
gallium-DOTA, or the presence of uncomplexed
DOTA and/or the release of gallium from the con-
jugate as applicable in the FA-PEG containing
systems. Clearly the fact that free DOTA has
greater toxicity than the gallium complexed forms
described herein, make them unsuitable as anti-
cancer agents themselves. There is however a
signifi cant difference on FR containing cells over
non-FR containing cells in terms of selectivity, as
well as suffi cient stability, to suggest that coupling
the γ-emitting 67Ga isotope or the β-emitting 68Ga
isotope to the FA-PEG conjugate unit may provide
a suitable route to targeting radioisotopes of gal-
lium to cell lines for use as diagnostic agents. This
work is currently being investigated in the group.

Acknowledgments
The authors wish to thank Syracuse University and
the iLEARN program for funding. We also thank
Karen L. Howard (State University of New York,
ESF) and Chris Incarvito (Yale University) for
assistance obtaining MALDI-TOF mass spectra
and Colin Fuss (CESE, SU) for conducting ICP.

Supporting Material
HPLC stability traces and IC50 graphs showing
exponential plots.

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Drug Target Insights 2008:3

Supplementary Data

(a) (b)

Figure S1. HPLC traces of γ-4 displaying peaks after incubation at 37 °C in 25 mM HEPES buffer (pH 7.4) at a) 0 hr b) 24 hrs c) 48 hrs
d) 72 hrs. ICP confi rmed presence of gallium at the new peaks growing after 72 hrs at a retention time of Tr = 15.6–16.3 min.

Targeting gallium to cancer cells through the folate receptor

Drug Target Insights 2008:3

Figure S2. Cytotoxic effects of 1 (♦), γ-3 (▲), γ-4 ( ) and DOTA ( ) against CHO cancer cells. Error bars represent the standard deviation
of the mean of three experiments (where n = 3 for each experiment) calculated for each concentration. Lines are exponential fi ts with R2
values of 0.8333, 0.9982, 0.9942 and 0.9652 for 1, γ-3, γ-4 and DOTA respectively.

Figure S3. Cytotoxic effects of 1 (♦), γ-3 (▲), γ-4 ( ) and DOTA ( ) against A2780/AD cells. Error bars represent the standard deviation of
the mean of three experiments (where n = 3 for each experiment) calculated for each concentration. Lines are exponential fi ts with R2 values
of 0.9905, 0.7274, 0.9444, and 0.9645 for 1, γ-3, γ-4 and DOTA.

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/NOR <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>
/SVE <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>
/ENU <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>
>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice