Cell-Specific Delivery of a Chemotherapeutic to Lung Cancer Cells
Xin Zhou, Ya-Ching Chang, Tsukasa Oyama, Michael J. McGuire, and Kathlynn C. Brown*
Center for Biomedical InVentions and Department of Internal Medicine,
UniVersity of Texas Southwestern Medical Center, Dallas, Texas 75390-9185
Received September 3, 2004; E-mail: Kathlynn.Brown@UTSouthwestern.edu
The nonspecific toxicity of anticancer drugs toward both cancer-
ous and normal tissues can result in serious side effects, thereby
limiting their clinical applications.1To overcome this obstacle,
methods of delivering anticancer drugs preferentially to cancer cells
have been sought. One approach is to conjugate a drug to a ligand
that recognizes a known cell surface marker preferentially expressed
on malignant cells. Most work in this field has focused on using
antibodies as tumor-homing reagents, and monoclonal antibodies
directed to tumor-associated antigens have been coupled to a variety
of anticancer agents.2
While monoclonal antibodies display high affinity and specificity,
they suffer from clinical limitations. To overcome the short comings
of antibodies, peptides have been employed as targeting ligands.
Peptides can be synthesized in large quantities, are amenable to
derivatization, and are more accessible to solid tumors. A source
of targeting peptides is from known peptidyl ligands that bind to
cell surface receptors overexpressed in neoplastic cells.3When there
is no known peptidic ligand for a desired cellular receptor, peptide
libraries can be screened to isolate tumor-targeting ligands.4Phage
display has been used to identify peptide ligands that bind to well-
characterized tumor-associated cell surface receptors, including RV?3
integrin,5HER2/neu,6transferrin receptor,7ErbB-2,8and ICAM-
We recently reported the isolation of lung cancer-targeting
peptides by panning a phage display library on intact cells.10Our
approach requires no knowledge of the cellular receptor; thus, we
can target tumor cells even when the appropriate tumor antigen is
unknown. These peptides discriminate between cancerous and
normal cells, suggesting that the peptides may have utility as
delivery reagents in vivo. Here we report that the targeting peptides
can be removed from the phage backbone and used to deliver a
chemotherapeutic in a cell-specific fashion.
On the basis of the peptide sequences from our initial experi-
ments, tetrameric peptide constructs were synthesized off a trilysine
core using Fmoc chemistry11(Figure 1). Two peptide sequences
were synthesized: TP H1299.1, which has cell-specific affinity for
a large cell lung carcinoma cell line (H1299), and TP H2009.1,
which binds to two lung adenocarcinoma cell lines (H2009 and
H1648).10,12While the monomeric peptides bind to their target cell
type, the tetrameric scaffold was chosen to increase the affinity of
the ligand for its cell surface receptor by multivalent presentation
of the peptides. Also, it is likely that the peptides must be
multimerized to initiate cellular uptake, which is critical for
intracellular drug delivery. A poly(ethylene glycol) (PEG) segment
was added to each branch of the polymer to increase conformational
flexibility and solubility of the drug conjugates.
To confirm that these peptides bind to their cellular receptor
outside of the phage particle, the ability of these peptides to block
uptake of their cognate phage was assessed (Figure 2). The TP
H2009.1 tetrameric peptide inhibits phage uptake by >97% at 1
µM on both H2009 and H1648 cells, and the peptide exhibits phage
blocking at concentrations as low as 1 nM. Similar results are
observed for the TP H1299.1 tetrameric peptide on H1299 cells,
although less phage blocking is observed at 1 nM. The monomeric
versions of both peptides show significantly reduced ability to block
phage uptake,10supporting the need for multimerization of the
peptide. We previously reported the synthesis of the tetrameric TP
H2009.1 peptide without the PEG linker and its ability to bind
H2009 cells.10Addition of the linker does not alter the affinity of
the peptide scaffold for the cells as determined by this assay.
However, it increases peptide solubility in aqueous solutions. This
aids in purification and ease of handling of the peptides. We were
unable to synthesize and purify the H1299.1 tetrameric peptide
without the PEG linker due to insolubility.
Figure 1. Structure of the tetrameric peptide-doxorubicin conjugates,
1299.1M4-Dox, and 2009.1M4-Dox. The phage-blocking experiments were
performed with the unconjugated peptides in which the cysteine was
protected as an acetamidomethyl group.
Figure 2. Blocking of phage uptake by tetrameric peptides. The blocking
ratio is the ratio of output phage to input phage in the presence of peptide
normalized to the output/input ratio with no peptide added.
Published on Web 11/10/2004
15656 9 J. AM. CHEM. SOC. 2004, 126, 15656-15657
10.1021/ja0446496 CCC: $27.50 © 2004 American Chemical Society
As the cell-targeting tetrameric peptides bind their cellular target,
we sought to determine if they could deliver an active therapeutic
in a cell-specific fashion. The chemotherapeutic doxorubicin was
coupled to the tetrameric peptides via a ?-maleimidopropinoic linker
at a unique cysteine placed before the branch point of the polymer
(Figure 1).13The hydrazone linkage between the peptide linker and
doxorubicin was chosen so that the drug could be released from
the peptide under the acidic environment of the endosome.14To
confirm that the drug could be released from the targeting moiety,
the stability of the conjugate was assessed at acidic pH. The
peptide-doxorubicin conjugate was found to release >50% doxo-
rubicin at 37 °C within 24 h at pH 4, while <7% release was
observed at neutral pH.
To evaluate the ability of the peptide conjugates to deliver active
doxorubicin in a cell-specific manner, a cytotoxicity screen was
conducted for four cell lines: IMR 90, H460, H1299, and H1648.
IMR 90, a normal lung fibroblast cell line, and H460, a large cell
lung carcinoma cell line, serve as negative controls as neither
peptide displays significant affinity for these cells (Supporting
Information). The H2009 cells were not tested in this assay as they
are resistant to doxorubicin, even at high concentrations. Among
the four cell lines, cell viability in the presence of free doxorubicin
is <20%, and the normal lung fibroblasts are affected as well as
the cancer cell lines (Figure 3). In contrast, cell viability of H1299
in the presence of 1299.1M4-Dox was 32 ( 4.5%, while no cell
death was observed for H460 and IMR 90 cells. The cell viability
of H1648 cells was slightly reduced (69 ( 3.6%) when exposed to
1299.1M4-Dox. This is not unexpected as TP H1299.1 peptide has
a moderate affinity for H1648 cells. A similar pattern of cell
viability was observed for 2009.1M4-Dox, with cell viability being
reduced to 43 ( 4.5% for H1648 cells, but with little effect observed
on IMR 90, H460, and H1299 cells. Treatment with unconjugated
tetrameric peptides did not affect the growth of the cells, indicating
that the decrease in cell viability is not due to the addition of the
targeting peptide. Furthermore, addition of unconjugated tetrameric
peptides had no effect on the toxicity of free doxorubicin.
The IC50values of doxorubicin, 1299.1M4-Dox, and 2009.1M4-
Dox on the four cell lines were determined (Table 1). The IC50
value of 1299.1M4-Dox toward H1299 cells is similar to that of
free doxorubicin, but the peptide-drug conjugate is less toxic to
the other three cell lines than free doxorubicin. The 2009.1M4-
Dox is also more toxic to H1648 than to the other three cell lines.
However, the H2009.1M4-Dox conjugate is 13-fold less potent than
free doxorubicin on H1648 cells. Similar decreases in potency have
been observed in other drug conjugates.15This may stem from low
drug uptake, inefficient drug release from the peptide carrier, or
incorrect cellular trafficking. These problems can be overcome by
increasing the drug load of the conjugate or by changing the linkage
of the drug to the targeting agent. Nonetheless, the increase in the
therapeutic window observed suggests that higher concentrations
of the peptide-drug conjugate may be employed in order to achieve
the same drug efficacy while decreasing cell toxicity to normal
Our data indicate that these targeting peptides can deliver an
active anticancer agent in a cell-specific fashion. Conjugation of
cell-permeable drugs to the targeting peptides renders them cell-
impermeable, except to the target cells. This results in an increase
of the therapeutic index of the targeted drug compared to systemic
delivery. The efficacy of the peptide conjugate correlates to the
phage binding for a particular cell line. Thus, cell-specific targeted
drugs can be synthesized, even when the cell surface target is
unknown. Efforts to increase the drug load of the peptide conjugates
and to test these targeted drugs in animals are underway.
Acknowledgment. This work was supported by Texas Ad-
vanced Technology Program Grant 010019-0049-2001 (K.C.B.),
and The University of Texas SPORE P50CA70907. We thank Dr.
John D. Minna for helpful discussions.
Supporting Information Available: Experimental methods for
peptide and conjugate synthesis, cell viability assays, peptide selectivity
assays, and IC50determinations. This material is available free of charge
via the Internet at http://pubs.acs.org.
(1) Langerak, A. D. Chemotherapy Regimens and Cancer Care; Landes
Bioscience: Georgetown, TX, 2001; p 209.
(2) (a) Green, M. C.; Murray, J. L.; Hortobagyi, G. N. Cancer Treat. ReV.
2000, 26, 269-286. (b) Dubowchik, G. M.; Walker, M. A. Pharmacol.
Ther. 1999, 83, 67-123. (c) Trail, P.; Bianchi, A. B. Curr. Opin. Immunol.
1999, 11, 584-588.
(3) (a) Schally, A. V.; Nagy, A. Eur. J. Endocrinol. 1999, 141, 1-14. (b)
Schally, A. V.; Nagy, J. A. Life Sci. 2003, 72, 2305-2320.
(4) (a) Aina, O. H.; Sroka, T. C.; Chen, M.-L.; Lam, K. S. Biopolymers 2002,
66, 184-199. (b) Brown, K. C. Curr. Opin. Chem. Biol. 2000, 4, 16-21.
(5) (a) Pasqualini, R.; Koivunen, E.; Ruoslahti, E. Nat. Biotechnol. 1997, 15,
542-546. (b) Arap, W.; Pasqualini, R.; Ruoslahti, E. Science 1998, 279,
(6) Urbaelli, L.; Ronchini, C.; Fontana, L.; Menard, S.; Orlandi, R.; Monaci,
P. J. Mol. Biol. 2001, 313, 965-976.
(7) Schatzlein, A. G.; Rutherford, C.; Corrihons, F.; Moore, B. D. J. Controlled
Release 2001, 74, 357-362.
(8) Karasseva, N. G.; Glinsky, V. V.; Chen, N. X.; Komatireddy, R.; Quinn,
T. P. J. Protein Chem. 2002, 21, 287-296.
(9) Belizaire, A. K.; Tchistiakova, L.; St-Pierre, Y.; Alakhov, V. Biochem.
Biophys. Res. Commun. 2003, 309, 625-630.
(10) Oyama, T.; Sykes, K. F.; Samli, K. N.; Minna, J. D.; Johnston, S. A.;
Brown, K. C. Cancer Lett. 2003, 202, 219-230.
(11) Tam, J. P. J. Immunol. Methods 1996, 196, 17-32.
(12) Elayadi, A. N.; Samli, K. N.; Oyama, T.; Brown, K. C. Submitted.
(13) Kruger, M.; Beyer, U.; Shumacher, P.; Unger, C.; Zahn, H.; Kratz, F.
Chem. Pharm. Bull. 1997, 45, 399-401.
(14) Kaneko, T.; Wilner, D.; Monkovic, I.; Knipe, J. O.; Braslawsky, G. R.;
Greenfiled, R. S.; Vyas, D. M. Bioconjugate Chem. 1991, 2, 133-141.
(15) (a) Kratz, F.; Beyer, U.; Roth, T.; Tarasova, N.; Collery, P.; Lechenault,
F.; Cazabat, A.; Schumacher, P.; Unger, C.; Falken, U. J. Pharm. Sci.
1998, 87, 338-346. (b) Rodrigues, P. C.; Beyer, U.; Schumacher, P.;
Roth, T.; Fiebig, H. H.; Unger, C.; Messori, L.; Orioli, P.; Paper, D. H.;
Mulhaupt, R.; Kratz, F. Bioorg. Med. Chem. 1999, 7, 2517-2524. (c)
Luo, Y.; Bernshaw, N. J.; Lu, Z.-R.; Kopecek, J.; Prestwich, G. D. Pharm.
Res. 2002, 19, 396-402.
Figure 3. Viability of cells treated with 10 µM doxorubicin, 1299.1M4-
Dox, and 2009.1M4-Dox. Cells were exposed to the drug for 2 days
followed by a 3 day recovery. Cell viability was determined by quantitation
of ATP, a measure of viable, metabolically active cells. Cell viability is
normalized against the untreated cells. Similar results are observed when
the cells are exposed to the drug conjugates for 5 days.
Table 1. Cytotoxicity of Doxorubicin and Peptide-Doxorubicin
Conjugates against IMR 90, H460, H1299, and H1648 Cells (IC50,
cell line doxorubicin
1.0 ( 0.06
0.096 ( 0.007
2.3 ( 0.02
0.56 ( 0.005
4.2 ( 0.03
7.2 ( 0.03
aLess than 10% growth inhibition at 35 µM; IC50 not determined.
bExtrapolated from the growth inhibitory data. The IC50 values were
obtained from polynomial-fitted curves of cell viability versus concentration.
C O M M U N I C A T I O N S
J. AM. CHEM. SOC. 9 VOL. 126, NO. 48, 2004 15657