Carbohydrate dependent targeting of cancer cells by bleomycin-microbubble conjugates

Full text of DOI:10.1021/ja8091104

Published on Web 02/02/2009
Carbohydrate Dependent Targeting of Cancer Cells by
Bleomycin-Microbubble Conjugates
†
†,‡
†,§
§
Jean-Charles Chapuis, Ryan M. Schmaltz, Krystal S. Tsosie, Marek Belohlavek, and
,
†,‡
Sidney M. Hecht*
Center for BioEnergetics and Department of Chemistry, Arizona State UniVersity, Tempe, Arizona 85287,
Departments of Chemistry and Biology, UniVersity of Virginia, CharlottesVille, Virginia 22904, and College of
Medicine, Department of Research, Mayo Clinic, Scottsdale, Arizona 85259
Received November 20, 2008; E-mail: sid.hecht@asu.edu
5
The bleomycins (BLM A , 1) are clinically used antitumor agents
efficacious in the treatment of squamous cell carcinomas and
1
malignant lymphomas. Their antitumor activity is believed to result
from their ability to mediate the selective oxidative cleavage of
2
3
DNA and possibly also RNA.
One property of bleomycin that has been recognized for decades
is its ability to target tumors; this has been documented in numerous
4,5
tumor imaging studies that employed BLMs bound to radionuclides.
The importance of tumor targeting to the therapeutic efficacy of
BLM is underscored by the therapeutic dose of BLM (∼5 µmol),
which is quite low relative to the doses of many clinically used
agents. Understanding the molecular nature of tumor targeting by
BLM would facilitate the synthesis of analogues with improved
properties and might also enable the selective delivery of other
probes and drugs to tumor cells.
Microbubbles, consisting of a shell enclosing a gas core, are used
in ultrasonography as contrast agents and intravascular blood flow
tracers. The shell is usually composed of albumin, carbohydrates,
6
or lipids. The gas core is usually air, nitrogen, or a perfluorocarbon.
While individual microbubbles may exist in varying sizes, they are
usually smaller than red blood cells and their mean diameter is
typically within the range 1-4 µm. This small size permits free
flow of the microbubbles through the (micro)circulation. In recent
years, microbubbles have been modified with ligands that bind
specific receptors expressed by cell types of interest, including
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inflamed and cancer cells. The majority of ligands to date have
been monoclonal antibodies. Presently we describe the conjugation
Figure 1. Synthesis of biotinylated derivatives of bleomycin A
deglycobleomycin A
5
and
5
.
of BLM A
selectively to cancer cells, and report that the carbohydrate moiety
of BLM A is required for tumor cell targeting.
The C-terminus of BLM A (1) was acylated with biotin, as
shown in Figure 1, to afford BLM A
preparation of BLM A -conjugated microbubbles was then ac-
5
to microbubbles, demonstrate that the conjugate adheres
5
5
8
5
-biotin conjugate 2a. The
5
complished by admixture of BLM-biotin 2a to commercially
9
available microbubbles derivatized with streptavidin. A schematic
representation of the resulting targeted microbubble is shown in
Figure 2.
MCF-7 human breast carcinoma cells were grown on sterile glass
coverslips (40 mm) and incubated at 37 °C until they reached
Figure 2. Constitution of the bleomycin A
5
-microbubble conjugate.
4
0-60% confluency in a 5% fetal bovine serum/RPMI 1640
medium. They were then assembled into a parallel plate flow
chamber with a constant temperature maintained at 37 °C. The
suspension containing the BLM-microbubble conjugate was
introduced into the flow chamber at a controlled rate of 0.01 mL/
min, and possible attachment of the microbubbles to the cultured
MCF-7 cells was imaged using an inverted microscope fitted with
a camera. As shown in Figure 3, the BLM-microbubble conjugate
adhered to the cultured MCF-7 cells, but microbubbles lacking
attached BLM 2a failed to adhere to the cultured MCF-7 cells.
†
Arizona State University.
University of Virginia.
‡
To judge the ability of the BLM A
5
-microbubble conjugate to
§
Mayo Clinic Scottsdale.
bind selectively to tumor cells, the experiment was repeated using
2
438 9 J. AM. CHEM. SOC. 2009, 131, 2438–2439
10.1021/ja8091104 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
As shown in Figure 4 for the cultured MCF-7 cells, no adhesion
of the deglyco BLM A
was observed.
5
-microbubble conjugate to either cell line
The foregoing experiments enable direct, visual observation of
the binding of a BLM conjugate to cultured tumor cells and
demonstrate that BLM adheres selectively to tumor cells, as
compared with the same “normal” cells. Also established is the
requirement for the carbohydrate moiety of BLM to support tumor
cell targeting. Not yet resolved by these experiments is the related
question of the sufficiency of the BLM carbohydrate moiety to
support tumor cell targeting, a finding that could enable novel
strategies for selective drug delivery and antitumor therapy.
Acknowledgment. K.S.T. was supported by R25GM071798
from the National Institute of General Medical Sciences.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 3. Monolayers of cultured MCF-7 breast cancer cells treated with
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(
8) Cu(II) ·BLM A
as outlined in Figure 1, in analogy with attachment of BLM A
support (Abraham, A. T.; Zhou, X.; Hecht, S. M. J. Am. Chem. Soc. 2001,
5
was treated with the N-hydroxysuccinmide ester of biotin,
5
to a solid
Figure 4. Monolayers of cultured MCF-10A breast cells treated with the
BLM A
with the deglyco BLM A
5
-microbubble conjugate (top) and of cultured MCF-7 cells treated
2+
1
23, 5167). Following the removal of Cu by treatment with EDTA, the
5
-microbubble conjugate (bottom).
BLM A
-biotin conjugate (2a) was purified by C18 reversed phase HPLC.
5
B
(
9) Targestar Ultrasound Contrast Agent, containing streptavidin attached
through a PEG spacer, was used for attachment of BLM 2a and deglyco
BLM 2b. These microbubbles have a consistent mean diameter of ∼2.5
the “normal” breast cell line MCF-10A. As shown in Figure 4,
there was no adhesion of the BLM A microbubble-conjugate to
the cultured MCF-10A cells. Thus the BLM A -microbubble
conjugate adhered selectively to the cancer cell line.
The realization of a system for monitoring the selective inter-
action of BLM A with tumor cells provides a vehicle for evaluating
those structural elements of BLM that contribute to tumor cell
targeting. Accordingly, biotinylated deglyco BLM A (2b) was
5
6
µm with ∼1.5 × 10 streptavidin molecules/microbubble. The density of
5
streptavidin was about 30 times greater than that of antibody in antibody-
derivatized microbubbles (Talkalkar, A. M.; Klibanov, A. L.; Rychak, J. J.;
Lindner, J. R.; Ley, K. J. Controlled Release 2004, 96, 473). A 1-mL
1
0
8
aqueous suspension of 1.5 × 10 streptavidin-derivatized microbubbles was
treated with 50 µL of aq. 500 µM BLM 2a or 2b. The combined solution
was agitated gently for 20 min, then free BLM was removed by repeated
centrifugation (400 × g, 3 min, 10 °C) and washing with buffer.
(10) Analogous experiments were carried out using other sets of cultured cancer
5
5
5
and normal cells. Selective adhesion of the BLM A -microbubble conjugate
prepared to permit a direct assessment of the role of the carbohy-
drate moiety of BLM in tumor cell targeting. BLM 2b was
conjugated to the same microbubbles and employed in experiments
designed to test adhesion to cultured MCF-7 and MCF-10A cells.
was observed using SW480 colon carcinoma cells (but not CRL-1541 colon
cells), DU145 prostate carcinoma cells (but not CRL-2221 prostate cells),
and A549 lung carcinoma cells (but not CCL-75 lung cells).
JA8091104
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