Targeted Quantum Dot Conjugates for siRNA Delivery
Austin M. Derfus,†,‡Alice A. Chen,§,3Dal-Hee Min,§Erkki Ruoslahti,‡,#and Sangeeta N. Bhatia*,†,§
Department of Bioengineering, University of California at San Diego, La Jolla, California 92093, Burnham Institute for Medical
Research, La Jolla, California 92037, Health Sciences and Technology/Electrical Engineering and Computer Science,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and School of Engineering and Applied Sciences,
Harvard University, Cambridge, MA 02138. Received November 25, 2006; Revised Manuscript Received April 3, 2007
Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic
modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle
promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to
improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and
disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA)
therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using
a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to
functional groups on the particle’s surface. We found that the homing peptide was required for targeted
internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the
peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached
to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible
thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the
tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation.
Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment
led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target
(e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image
The development of multifunctional nanoparticles for treat-
ment of focal disease is attractive for several reasons: they ex-
hibit unique pharmacokinetics including minimal renal filtration,
they have high surface to volume ratios enabling modification
with surface functional groups that can be used to specifically
target the delivery of therapeutic agents to sites of disease, and
they can serve as vehicles for integration of diagnostic imaging
and therapeutic drug delivery, a potentially transformative
clinical paradigm. Use of a nanoparticle imaging core that is
decorated with functional moieties provides a strategy that is
particularly amenable to modular design of a multifunctional
nanoparticle where features may be interchanged or combined
to tailor formulations for a plethora of applications. In an attempt
to move toward this goal, we have previously combined peptides
derived from phage display- a powerful biological screening
technique- with fluorescent semiconductor quantum dots to
target multivalent nanoparticles to tumors (1). In this report,
we further explore the feasibility of incorporating an oligo-
nucleotide-based therapeutic cargo, siRNA. Short, double-
stranded small-interfering-RNAs (siRNA) are one manifestation
of a phenomenon known as RNA interference whereby transla-
tion of a target protein is inhibited. This type of therapeutic
cargo is of particular interest in recent years because it has the
potential to modulate so-called ‘nondruggable’ targets (2, 3).
The untargeted, systemic delivery of siRNA has been
explored by conglomeration of duplexes into nanosized com-
plexes that reduce their renal filtration rates, extending the
circulation half-life well beyond the ∼6 min observed for
unmodified siRNA (4). For example, cholesterol-siRNA con-
jugates bind serum albumin after intravenous injection, forming
long-circulating “natural” nanoparticles (4). Similarly, siRNA-
carrier complexes can be formed ex vivo, prior to injection, by
condensing the nucleic acid with a cationic protein (e.g.,
protamine (5)) or polymer (e.g., poly(ethylene imine) (PEI) (6),
cyclodextrin-containing polycations (7), or PEG-based block
catiomer (8)). In addition, targeted delivery of such agents has
the potential to limit collateral toxicity and ‘off-target’ effects.
Targeting has been explored through use of through the
attachment of antibodies (5), small molecules (e.g., transferrin
(7)), aptamers (9, 10), or well-established peptide ligands (6));
however, these approaches are typically not modular nor
multifunctional (i.e., do not incorporate imaging moieties).
Addition of a nanoparticle-based imaging agent to siRNA
delivery strategies may be particularly advantageous as protein
knockdown by RNAi is delayed (>48 h or more after
administration), and many fluorescent dyes are not stable for
monitoring delivery over extended periods of time in vivo. In
vitro, co-delivery of fluorescent reporter plasmid along with the
siRNA is often utilized; however, this is unlikely to be used
clinically given the potential risks associated with integration
into host DNA.
Quantum dots offer the potential to serve as photostable
beacons to track siRNA delivery. We have previously explored
their utility for monitoring siRNA delivery in vitro by co-
complexing QDs with cationic liposomes and siRNA (11, 12);
however, this approach is not amenable to either systemic
delivery, because of their relatively large size and rapid uptake
* Author to whom correspondence should be addressed. Phone:
(617) 324-0221, fax: (617) 324-0740, e-mail: email@example.com.
†University of California at San Diego.
‡Burnham Institute for Medical Research.
#Burnham Institute for Medical Research, University of California,
Santa Barbara, Santa Barbara, CA 93106-9610.
Bioconjugate Chem. 2007, 18, 1391−1396
10.1021/bc060367e CCC: $37.00© 2007 American Chemical Society
Published on Web 07/14/2007
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