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Communication
Tumor-Targeted Gene Delivery Using Molecularly Engineered
Hybrid Polymers Functionalized with a Tumor-Homing Peptide
Kris C. Wood, Samira M. Azarin, Wadih Arap, Renata Pasqualini, Robert Langer, and Paula T. Hammond
Bioconjugate Chem., 2008, 19 (2), 403-405 • DOI: 10.1021/bc700408r • Publication Date (Web): 12 January 2008
Downloaded from http://pubs.acs.org on November 16, 2008

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Tumor-Targeted Gene Delivery Using Molecularly Engineered Hybrid

Polymers Functionalized with a Tumor-Homing Peptide
Kris C. Wood,† Samira M. Azarin,† Wadih Arap,§ Renata Pasqualini,§ Robert Langer,*,†,‡ and
Paula T. Hammond*,†
Department of Chemical Engineering, Biological Engineering Division, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, Department of Genitourinary Medical Oncology, University of Texas M.D. Anderson Cancer
Center, Houston, Texas 77030. Received November 3, 2007; Revised Manuscript Received December 12, 2007

Before gene therapy can be used in clinical settings, safe and efficient DNA delivery systems must be developed
to overcome a range of extra- and intracellular transport barriers. As a step toward the development of a modular,
multifunctional gene delivery system to overcome these diverse barriers, we have developed a family of
linear-dendritic “hybrid” polymers which contain functionalities for tissue targeting, minimization of nonspecific
interactions, endosomal buffering, and DNA binding. Here, we demonstrate the rapid three-step, room-temperature,
aqueous synthesis of hybrid polymers, as well as the functionalization of these polymers with a peptide targeting
ligand that specifically binds to glucose-regulated protein-78 kDa (GRP-78), a clinically relevant tumor antigen
identified in human cancer patients. These polymer systems can condense plasmid DNA into small nanoparticle
structures (<210 nm) and transfect cells expressing GRP-78 with efficiencies that exceed that of branched
polyethylenimine (bPEI), one of the best commercially available polymers for in Vitro transfections. The synthetic
approach described here may be useful for the rapid synthesis and optimization of polymer gene delivery systems
bearing a range of diverse functional domains, and the specific GRP-78-targeted systems developed in this study
may potentially have clinical applications in cancer gene therapy.

One approach toward the development of improved nonviral
nucleic acid delivery systems involves the rational design of
integrated, multifunctional systems containing domains which
can be used to impart diverse functions such as DNA binding,
endosomal buffering, and tissue or intracellular targeting (1, 2)
Unfortunately, many early attempts to build such multifunctional
systems have been unsuccessful because the addition of new
domains results in the direct modification of existing domains,
adversely affecting their properties. For example, the conjugation
of poly(ethylene glycol) (PEG) to polyethylenimine (PEI) via
primary and secondary amines, while improving the colloidal
stability of polymer-DNA complexes, also dramatically de-
creases transfection efficiency by directly altering the polymer’s
DNA binding and endosomal buffering properties (3).
In response to these well-established problems, we set out to
design a gene delivery system possessing multiple functional
domains arranged together in a modular fashion, such that new
domains could be added, modified, or removed while only
minimally affecting existing properties. In order to make this
possible, we required that all domains be physically separated
* To whom correspondence should be addressed. 77 Massachusetts
Avenue, Room 66-546, Cambridge, MA 02139. Tel: 617-258-7577;
Fax: 617-258-5766; Email: hammond@mit.edu. 77 Massachusetts
Avenue, Room E25-342, Cambridge, MA 02139; Tel: 617-253-3123;
Fax: 617-258-8827; Email: rlanger@mit.edu.
† statically condense DNA into small nanoparticle structures with
Department of Chemical Engineering, Massachusetts Institute of
Technology.
‡
Biological Engineering Division, Massachusetts Institute of
Technology.
§
University of Texas M.D. Anderson Cancer Center.
10.1021/bc700408r CCC: $40.75  2008 American Chemical Society
Published on Web 01/12/2008
from one another, that no domains possess overlapping func-
tions, and that domains be assembled using complementary
(orthogonal) chemistries (Figure S1a). Here, we demonstrate
the rapid synthesis and in Vitro validation of a system which
fulfills all of the above criteria. This system, based on
linear-dendritic “hybrid” polymers consisting of linear PEG
and dendritic poly(amidoamine) (PAMAM), possesses func-
tional domains for electrostatic DNA condensation, endosomal
escape (via the proton sponge mechanism), reduction of
nonspecific tissue interactions, and tissue targeting (Figure S1b)
(4). In order to functionalize hybrid polymers with sensitive
biological functional domains such as peptides, antibodies, and
nucleic acids, we have developed a synthetic scheme that uses
commercially available starting materials and a rapid (three
reaction steps, two days), aqueous chemical conjugation (1, 5).
To examine the utility of this approach, we synthesize hybrid
polymers modified with a peptide ligand (denoted peptide 1,
sequence ) WIFPWIQL) capable of selectively targeting
glucose-regulated protein-78 kDa (GRP-78) (6). GRP-78 is a
functional tumor antigen identified in human cancer patients,
and peptide 1 has been used to target GRP-78 in tumors in Vitro,
in ViVo (in mouse models of breast and prostate cancer), and in
human patient-derived tumor samples (6, 7). We show here that
peptide 1 functionalized hybrid polymer systems can electro-
low, positive zeta potentials and the ability to transfect cells
expressing GRP-78 more efficiently than branched polyethyl-
enimine (bPEI), one of the best commercially available polymers
for in Vitro transfections.
404 Bioconjugate Chem., Vol. 19, No. 2, 2008
Scheme 1. Synthesis of Peptide-Functionalized Hybrid Polymers
The aqueous synthetic approach developed in this work is
shown in Scheme 1. Critical to this approach is the availability
of cystamine-core PAMAM dendrimers (generation 4.0), which
can be reduced to yield dendrons with a single, free thiol in the
interior (8). Briefly, dendrimers were first reduced by addition
to a molar excess of dithiothreitol (DTT) with overnight stirring
under N2 gas, followed by dialysis against pure water to remove
the reducing agent. Ellman’s assay was used to quantify the
fraction of dendrimers reduced prior to the PEG conjugation
(see Supporting Information). In a separate vessel, peptide 1
was added to a bifunctional maleimide-PEG-NHS ester for 90
min, followed by addition of a molar equivalent of reduced
dendrimer at pH 7.4 (phosphate buffered saline) with stirring
for 24–48 h to yield the final conjugate. Note that peptide 1
was dissolved in dimethylsulfoxide (DMSO) prior to the PEG
conjugation reaction only because it is insoluble in water.
Quantitatively, PAMAM reduction proceeded at conversions
of 95–100% (based on total thiol concentration as measured by
Ellman’s method). The peptide-PEG conjugation reaction
proceeded at 78% conversion (Figure S2), and the final
PEG-PAMAM conjugation step went at 74% conversion (based
on the disappearance of thiols; see Table S1). To ensure that
the disappearance of free thiols during the PEG-PAMAM
conjugation step was a result of a reaction with the PEG
maleimide group instead of the reformation of dendritic disul-
fides, a sample of the final product was incubated overnight
with a 10-fold molar excess of DTT, dialyzed, and analyzed
again for thiol content. No additional thiols were observed,
Figure 1. Physical properties of hybrid polymer-DNA complexes. (a) 1% agarose gel electrophoresis demonstrates DNA binding at indicated
mass ratios. (b) Size (nm, bars) and zeta potential (mV, diamonds) of polymer/DNA complexes measured by dynamic light scattering (DLS). Error
bars indicate 1 standard deviation in measured values.
Communications
suggesting that all originally reacted thiols had formed stable
thioether linkages with the maleimide group. Additionally, each
conjugate was also verified using NMR (see Supporting
Information).
A range of techniques was used to measure the physical
properties of polymer-DNA complexes. In Figure 1a, gel
electrophoresis demonstrates binding and charge neutralization
of plasmid DNA (pCMV-Luciferase) by hybrid polymers at
polymer/DNA mass ratios greater than 1:1 (N:P 0.8:1), with
complete charge neutralization occurring at mass ratios equal
to or greater than 5:1 (N:P 3.9:1). Zeta potential measurements
indicate that complexes formed at polymer/DNA ratios of up
to 40:1 (N:P 31:1) have low, positive zeta potentials, and
dynamic light scattering shows that small particles (<210 nm)
are formed with diameters equal to or less than the reported
cutoff of ∼200 nm for efficient cellular uptake in ViVo (Figure
1b) (9). Particle size showed some sensitivity to the polymer/
DNA mass ratio, with small ratios yielding smaller (∼140–150
nm) particles and larger ratios yielding slightly larger particles
(180–210 nm), likely due to increased steric or charge repulsion
in complexes formed at high mass ratios.
To test for the ability of these peptide-functionalized hybrid
polymers to target and deliver plasmid DNA to cells expressing
GRP-78, we transfected DU145 prostate carcinoma cells, which
have been shown to bind and internalize peptide 1 in a GRP-
78-dependent manner (6). As shown in Figure 2, peptide-
functionalized hybrid polymers can transfect DU145 cells at
levels nearly 10-fold higher than an optimized formulation of
Communications
Figure 2. Transfection of DU145 prostate carcinoma cells expressing
GRP-78 using peptide-modified hybrid polymers. Polymers with and
without the peptide ligand, and peptide-modified polymers transfected
in the presence of polyclonal anti-GRP-78 antiserum to block receptor
binding, are shown (6). * indicates p < 0.1. Results normalized to an
optimized formulation of PEI (2:1 PEI/DNA ratio (m:m)). All results
are given as average +/- standard error.
branched PEI (25 kDa). Moreover, this process is receptor-
mediated, as evidenced by the fact that cells transfected using
polymers lacking a peptide ligand, as well as cells transfected
with peptide-functionalized polymers in the presence of poly-
clonal anti-GRP-78 antiserum (to block receptor–ligand bind-
ing), yielded significantly lower levels of transfection (Figure
2). (For more information on serum stability and cytotoxicity
of hybrid polymer systems, see ref (4).)
In conclusion, we have developed a rapid and simple aqueous
synthesis to construct multidomain, biomolecule-functionalized
polymers for nonviral gene delivery applications. Unlike previ-
ous efforts to functionalize spherical dendrimers with PEG,
targeting groups, or other moieties through DNA binding
peripheral amines, this approach allows for the synthesis of
asymmetric polymers with well-controlled architectures whose
DNA binding and endosomal buffering properties are not
directly altered in the functionalization process (10–12). As a
demonstration of this design and its utility, we synthesized
hybrid polymers functionalized with short peptides which
efficiently target and transfect cells expressing GRP-78, a
clinically relevant tumor antigen. Preliminary in ViVo biolumi-
nescence imaging experiments performed using mice bearing
EF43-FGF4 tumors demonstrate that a single, intravenous
injection of these GRP-78-targeted systems can also lead to
transient, tumor-specific transgene expression (data not shown).
In the future, the peptide-functionalized hybrid polymers
developed in this study may potentially find use in clinical
applications involving the targeted delivery of “suicide” trans-
genes to solid tumors (13). More broadly, the general synthetic
approach described here could be a useful tool for the rapid
synthesis and optimization of next-generation gene delivery
systems bearing biological functional domains such as nuclear
localization sequences, adjuvants, and protein transduction
domains (2).
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
(R01-EB00244 and R21-EB004122) and the Division of Ma-
terials Research of the National Science Foundation (DMR
9903380). K.C.W. thanks the National Cancer Institute and the
Bioconjugate Chem., Vol. 19, No. 2, 2008 405
MIT Center for Cancer Research for a Ludwig Research
Fellowship. We thank Drs. Gregory Zugates and Amin Hajitou
for helpful discussions and Drs. Donald Tomalia and Ryan
Hayes (Dendritic Nanotechnologies, Inc.) for assistance in
obtaining cystamine-core PAMAM dendrimers.
Supporting Information Available: Schematic of hybrid
polymers, conjugation of peptide 1 to Maleimide-PEG-NHS as
monitored by the fluoraldehyde assay, table of reaction conver-
sions, detailed experimental methods, and NMR characterization.
This information is available free of charge via the Internet at
http://pubs.acs.org/BC.
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