A nanoparticle formulation that selectively transfects
metastatic tumors in mice
Jian Yanga,b,c,1, William Hendricksa,b,c,1, Guosheng Liuc,d, J. Michael McCafferye, Kenneth W. Kinzlera,b,c,
David L. Husoc,d, Bert Vogelsteina,b,c,2, and Shibin Zhoua,b,c,2
aLudwig Center for Cancer Genetics and Therapeutics,bHoward Hughes Medical Institute, and
Hopkins, Baltimore, MD 21231;dDepartment of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD
21287; andeDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218
cSidney Kimmel Comprehensive Cancer Center at Johns
Contributed by Bert Vogelstein, July 19, 2013 (sent for review April 18, 2013)
Nanoparticle gene therapy holds great promise for the treatment
of malignant disease in light of the large number of potent, tumor-
specific therapeutic payloads potentially available for delivery. To
be effective, gene therapy vehicles must be able to deliver their
therapeutic payloads to metastatic lesions after systemic adminis-
tration. Here we describe nanoparticles comprised of a core of high
molecular weight linear polyethylenimine (LPEI) complexed with
DNA and surrounded by a shell of polyethyleneglycol-modified
(PEGylated) low molecular weight LPEI. Compared with a state-
of-the-art commercially available in vivo gene delivery formulation,
more than a 16,000-fold increase in the ratio of tumor to nontumor
transfection. The vast majority of examined liver and lung metasta-
ses derived from a colorectal cancer cell line showed transgene ex-
pression after i.v. CPS injection in an animal model of metastasis.
Histological examination of tissues from transfected mice revealed
that the CPS nanoparticles selectively transfected neoplastic cells
rather than stromal cells within primary and metastatic tumors.
However, only a small fraction of neoplastic cells (<1%) expressed
the transgene, and the extent of delivery varied with the tumor cell
line, tumor site, and host mouse strain used. Our results demon-
strate that these CPS nanoparticles offer substantial advantages
over previously described formulations for in vivo nanoparticle
gene therapeutics. At the same time, they illustrate that major
increases in the effectiveness of such approaches are needed for
utility in patients with metastatic cancer.
transduction pathways whose component genes are altered in
human tumors (1). Extraordinary efforts have been devoted to
developing therapeutics that selectively target these altered path-
ways. These efforts have been remarkably successful, resulting in
a new generation of agents that can shrink tumors without causing
unacceptable side effects (reviewed in refs. 2–4). The effects of
these targeted agents are generally short-lived, however, as
a result of the inevitable outgrowth of cells with mutations that
confer resistance (5–7). This phenomenon has spurred efforts to
identify complementary approaches that are less likely to be
subverted by mutations present in the tumor cells before therapy.
One of the most promising approaches to achieving this ob-
jective involves the exploitation of the abnormal vasculature in
tumors. To maintain their high relative growth rate, tumors must
recruit blood vessels, both afferent and efferent, to supply oxygen
and nutrients. But given the abnormal organization of cancer cells
with respect to the underlying stroma, the recruited vasculature is
quite unlike that present in normal tissues (8). This tumor vascu-
lature is characterized by chaotic organization, leakiness, and poor
lymphatic drainage; thus, agents that target the abnormal vascu-
lature should be particularly powerful weapons against cancers.
Nanoparticles represent one class of such weapons. When
administered systemically, nanoparticles with unique size and
charge characteristics may selectively accumulate in solid tumors,
because they can more easily extravasate through the leaky tumor
esearch on cancer genomes combined with functional and
biochemical studies has led to the identification of signal
endothelium and less easily vacate through the tumor’s poorly
developed lymphatic system (9, 10). This accumulation is known
as the enhanced permeability and retention (EPR) effect (11).
Several types of nanoparticles have been developed to exploit
the EPR effect in experimental model systems, some of which
have demonstrated clinical value (12, 13). Nanoparticles consist-
ing of nucleic acids complexed with cationic polymers also have
been extensively evaluated for therapeutic purposes (reviewed in
ref. 14). DNA-containing nanoparticles offer a wealth of thera-
peutic possibilities because they can encode highly toxic genes
whose tumor-specific expression can be tailored via engineering of
the nanoparticle itself or the molecular payload.
Although this potential has long been recognized, and much
work has been done to optimize nanoparticle gene delivery for-
mulations in vitro, relatively few studies have attempted to sys-
temically deliver genes to experimental tumors in vivo (see refs.
15–17 for examples of in vivo work). In vivo studies have often
used s.c. rather than internal tumors, global measures of trans-
fection that can be achieved with whole-body imaging or whole-
tissue lysates, and intratumoral rather than systemic delivery.
However, for clinical relevance, it is critical not only to test
systemic delivery of nanoparticle gene therapy formulations to
internal tumors, but also to evaluate the nature and number of
cells within tumors that express transfected reporters. Ultimately,
to be clinically viable, gene delivery vehicles must be able to po-
tently and selectively transfect metastatic tumor cells. Given that
primary tumors often can be surgically excised, the treatment of
metastatic disease is the most important clinical setting for nano-
In the present study, we attempted to improve the efficiency
and specificity of nanoparticle gene therapeutics, focusing on
To be effective, gene therapy vehicles must be able to deliver
their therapeutic payloads to widely dispersed tumor lesions
after systemic administration. We describe novel nanoparticles
that provided a >16,000-fold increase in the ratio of tumor to
nontumor cell delivery over conventional formulations. How-
ever, only a small fraction of neoplastic cells expressed the
transgene, and the extent of delivery varied with the tumor
cell line, tumor site, and host mouse strain used. Although our
nanoparticles represent a technical advance, they also illustrate
the challenges that remain before nonviral gene therapy can
be applied to cancer patients.
Author contributions: J.Y., W.H., K.W.K., B.V., and S.Z. designed research; J.Y. and W.H.
performed research; G.L. and J.M.M. contributed new reagents/analytic tools; J.Y., W.H.,
K.W.K., D.L.H., B.V., and S.Z. analyzed data; and J.Y., W.H., B.V., and S.Z. wrote the paper.
The authors declare no conflict of interest.
1J.Y. and W.H. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or sbzhou@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 3, 2013
| vol. 110
| no. 36
difficult to transfect compared with identical tumors in immu-
nodeficient mice (Fig. S6). This finding suggests that the innate
immune system plays a role in limiting gene transfection, which is
unfortunate, given that humans with cancer are generally
The variable transfection efficiency with different experimen-
tal tumor systems that we observed has important implications
not only for gene therapy, but also for any cancer therapy that
uses nanoparticles or targets the vasculature. An abnormal vas-
culature is always found in tumors and, coupled with advances in
nanoparticle-based technologies, offers extraordinary therapeu-
tic potential. However, this study emphasizes that much more
basic research into the factors governing the interface between
tumor cells and this vasculature is needed before this potential is
likely to be realized in practice.
Materials and Methods
Materials and methods used in polymer synthesis and characterization,
plasmid preparation, formulation and analysis of nanoparticles, and in vitro
and in vivo transfections are detailed in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Tim Phelps for the artwork; Hai-Quan
Mao, Yong Ren, George Sgouros, and Hong Song for sharing their laboratory
instruments; E. Latice Watson and Qiang Liu for technical support; and Yuan
Qiao, Surojit Sur, and Ian Cheong for inspiring discussions. This project was
supported by the Virginia and D. K. Ludwig Fund for Cancer Research and
National Institutes of Health Grants CA062924 and CA043460.
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