Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells

Article abstract of DOI:10.1021/ja5079464

Viruses have evolved specialized mechanisms to efficiently transport nucleic acids and other biomolecules into specific host cells. They achieve this by performing a coordinated series of complex functions, resulting in delivery that is far more efficient than existing synthetic delivery mechanisms. Inspired by these natural systems, we describe a process for synthesizing chemically defined molecular constructs that likewise achieve targeted delivery through a series of coordinated functions. We employ an efficient "click chemistry" technique to synthesize aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different surface markers.

Full text of DOI:10.1021/ja5079464

Article
pubs.acs.org/JACS
Synthetic Aptamer-Polymer Hybrid Constructs for Programmed
Drug Delivery into Specific Target Cells
Seung Soo Oh,†
,‡,§,#
Bongjae F. Lee,
†,∥,⊥,#
Frank A. Leibfarth, Michael Eisenstein, Maxwell J. Robb,
‡,∥
†,‡
‡,∥
Nathaniel A. Lynd,‡,∥ Craig J. Hawker,*
,†,‡,∥
and H. Tom Soh*
,†,‡
†
‡
§
∥
Materials Department, Department of Chemistry and Biochemistry, Department of Mechanical Engineering, Materials Research
Laboratory, University of California, Santa Barbara, California 93106, United States
⊥
Chemical Research Institute, Samsung Cheil Industries, Inc., Seoul, Republic of Korea 140739
S Supporting Information
*
ABSTRACT: Viruses have evolved specialized mechanisms to
efficiently transport nucleic acids and other biomolecules into
specific host cells. They achieve this by performing a coordinated
series of complex functions, resulting in delivery that is far more
efficient than existing synthetic delivery mechanisms. Inspired by
these natural systems, we describe a process for synthesizing
chemically defined molecular constructs that likewise achieve
targeted delivery through a series of coordinated functions. We
employ an efficient “click chemistry” technique to synthesize
aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an
inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the
payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted
killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different
surface markers.
1
5,16
INTRODUCTION
moiety.
For example, the Tan group has shown that
■
aptamer-drug conjugates can facilitate drug uptake by specific
target cells expressing an appropriate surface marker.
Over many millions of years, viruses have evolved elegant and
sophisticated mechanisms for selectively delivering biological
payloads into host cells by performing a sequence of molecular
17,18
Although these constructs that directly couple affinity reagent
to drugs have shown promise thus far, we envision the potential
for further gains in safety and efficacy through the development
of “multifunctional” vehicles thatakin to a virusachieve
targeted cell entry and selective payload release in a stepwise
1
,2
functions in a coordinated and systematic manner. For
example, adeno-associated viruses (AAV) selectively adsorb
onto subsets of host cells that express specific surface
3
,4
markers. Then, the AAV is internalized by these host cells
via endocytosis, after which it makes its escape from the
endosome and releases its protein and DNA contents into the
host cell. Indeed, its excellent delivery efficiency, coupled with
the minimal pathogenicity of the virus, have made AAV a
powerful biotechnological tool for delivering foreign DNA and
1
9−21
fashion.
More specifically, such a delivery mechanism
would entail: (i) prolonged systemic circulation while carrying
therapeutic agents in an inactive state, (ii) efficient tissue
penetration to reach target cells, (iii) specific binding and
internalization into target cells, and (iv) selective activation and
release of therapeutic payload upon internalization. These
features would be particularly valuable in the context of cancer
therapeutics, where the drugs involved are typically highly toxic
and the aim is to selectively eliminate subpopulations of tumor
cells without harming healthy cells in the adjacent tissue.
Toward this end, the integration of aptamers with different
classes of specialized biomaterials into a single, multifunctional
molecular construct offers a compelling strategy for synthetic
delivery vehicles. For example, the Langer and Farokhzad
laboratories have demonstrated the potential utility of aptamers
in this context, working extensively with drug-loaded polymeric
nanoparticles coated with aptamers targeting tumor-specific
5
−7
other biomolecules into host organisms.
The past decade has witnessed considerable effort to devise
synthetic systems that mimic these natural molecular machines
to achieve more effective drug delivery with fewer side effects.
The systems that have made the greatest progress in the clinic
to date are constructed by directly conjugating a targeting
reagent (e.g., an antibody) to a drug.
8
,9
1
0,11
Two such antibody-
drug conjugates have obtained approval from the U.S. Food
and Drug Administration (FDA), and dozens more are now in
1
2
clinical trials. Although most of the work to date has utilized
monoclonal antibodies as the targeting reagent, nucleic acid
aptamers are an alternative that offer additional advantages,
including smaller size, chemically defined synthesis, and lower
1
3,14
immunogenicity.
Accordingly, several research efforts have
Received: August 8, 2014
Published: October 7, 2014
demonstrated the potential of aptamers as a targeting
©
2014 American Chemical Society
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membrane proteins and have shown that such constructs can
surface marker. After confirming the coordinated molecular
function of the APH based on the successful delivery of the
fluorescent dye coumarin, we show that these APH constructs
can selectively deliver the chemotherapeutic agent doxorubicin
(DOX) into nucleolin-expressing tumor cells, enabling targeted
killing of these cells. Furthermore, we show that our synthetic
approach can be utilized with other aptamer sequences to
achieve flexibility in binding to different surface markers,
demonstrating the generality of our system for targeted drug
delivery.
22−24
enhance selective killing of tumor cells.
We believe block
copolymers constitute an especially promising class of polymers
for the expansion of this strategy because of the diverse
chemical functionalities that can be incorporated; furthermore,
these molecules can be designed to incorporate large numbers
2
5,26
of drug molecules in a chemically defined manner.
In order to realize this potential, we have developed a mild
yet highly effective synthesis strategy based on “click chemistry”
that seamlessly integrates aptamers and block copolymers into a
single, chemically defined molecular construct. Our method-
ology enables efficient and reproducible synthesis of multi-
functional ‘aptamer-polymer hybrids (APHs)’ that combine the
targeting aptamer with a drug-loaded multifunctional block
copolymer, which secures its therapeutic payload in an inactive
state (Figure 1A). Upon binding the target cell, the APH is
RESULTS AND DISCUSSION
■
Design of the APH. We designed our APH with a DNA
27
aptamer that specifically binds to tumor cells that express
2
8,29
nucleolin, a well-known tumor surface marker.
The
nucleolin aptamer sequence is provided in Table S1. Because
nucleolin is known to undergo receptor cycling, it enables
internalization of the APH into the tumor cells through
30,31
endocytosis.
The nucleolin aptamer is covalently con-
jugated to a block copolymer with repeat units of ethylene
32
glycol and ethylene glycol vinyl glycidyl ether (EGVGE)
using a “click-chemistry” scheme. We chose this polymer
scaffold (11.3 kDa, see Scheme 1) because it offers excellent
Scheme 1. Polymerization and Functionalization of
Coumarin Derivative, 4, To Give Aptamer-Polymer Hybrid,
5
Figure 1. Multifunctional APH molecules achieve controlled, targeted
drug delivery. (A) Mechanism of targeted drug delivery. (a) A
PEGylated APH carrying multiple deactivated drug molecules binds a
cancer target cell via aptamer-mediated recognition of nucleolin. (b)
Binding triggers internalization via endocytosis. (c) Esterases within
the endosome induce specific cleavage that (d) releases drug
molecules from the APH scaffold, after which (e) the now-active
small-molecule drugs can diffuse into the cytoplasm. (B) Components
of the modular APH molecule. We conjugated a nucleolin-specific
aptamer modified with Cy5 at its 5′ end to a PEG-based block
copolymer via an efficient, ligand-accelerated CuAAC reaction. This
polymer is loaded with multiple payload molecules, which are released
within target cells via esterase-mediated cleavage.
3
3
biocompatibility, prolonged circulation, and desirable size for
34
efficient tissue penetration (radius ∼10 nm). The cell viabiliy
assay also confirmed that this polymer scaffold is not cytotoxic
to the cells (see Figure S1). Importantly, we designed the
polymer backbone with multiple orthogonal reactive groups to
accommodate multiple DOX molecules that are tethered
through enzyme-cleavable linkers (see Scheme S1). These
linkers are designed to be efficiently cleaved by esterases that
35
internalized via endocytosis, at which point the covalent bonds
linking the payload to the polymer scaffold are cleaved by the
enzymatic milieu of the endosome. This selectively activates
and releases the drug, which then diffuses into the cytoplasm of
the target cell. Our synthesis scheme efficiently conjugates the
two material components with a high yield without
compromising the targeting efficiency of the aptamer or the
programmed drug release functionality of the block copolymer
are endogenous to the endosome of mammalian cells. Once
cleaved, DOX efficiently escapes the endosome due to its small
size and rapidly diffuses in the cytoplasm to reach the nucleus.
Click Chemistry Scheme for Efficient Aptamer-
Polymer Conjugation. APH synthesis requires efficient,
site-specific conjugation of the DNA aptamer to the polymer
scaffold without loss of function or binding affinity. To this end,
we developed an improved synthetic strategy for coupling DNA
aptamers to synthetic polymers based on the novel
tricarboxylate ligand (BimC A) , which stabilizes Cu (I) during
(
Figure 1B). As a model, we demonstrate APH constructs that
specifically target tumor cells that overexpress the nucleolin
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3
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the copper-catalyzed azide−alkyne cycloaddition
As controls, the unconjugated aptamer (lane 2) formed a single
band of the predicted size, whereas the unconjugated polymer
scaffold exhibited no electrophoretic mobility due to its neutral
charge and remained in the loading well (lane 1). Interestingly,
when the polymer was loaded with coumarin dye molecules, it
migrated toward the cathode due to the slightly positive charge
resulting from protonation of coumarin (Figure S2).
3
6−38
(
CuAAC)
(Scheme 1). In comparison to traditional
CuAAC ligands, the use of (BimC A) does not cause
4
3
irreversible damage to the aptamers, leads to higher yield,
and offers superior water solubility compared to tris-
39
(
benzyltriazolylmethyl)amine (TBTA) and other ligands.
The synthesis of our APH involves coupling of the nucleolin
aptamer, which features a single azide at its 3′ terminus (see
Characterization of APH Function. We further confirmed
the retention of aptamer function by comparing the equilibrium
Table S1), with an ω-alkyne-functionalized polyether (Scheme
1
and Methods for details). After purification, dynamic light
scattering of the APHs with concentrations ranging from 0.5 to
0 μM showed that the resulting APH consistently exhibited 10
2 nm in radius (Figure 2A), and we found negligible evidence
dissociation constant (K
the APH. A bead-based binding assay (see Methods) revealed
a K of 8.37 ± 0.75 nM for Cy5-labeled nucleolin aptamer
) of the aptamer in solution to that of
d
40
1
±
d
versus 5.18 ± 0.72 nM for the fully conjugated APH (Figure
2C), confirming that the conjugation scheme described above
does not compromise the binding affinity of the aptamer.
Importantly, the efficiency of quantitative polymerase chain
reaction (qPCR) was comparable for aptamers conjugated to
the polymer scaffold and unconjugated aptamers in solution
of micelle formation or aggregation at the measured
concentrations (Figure 2A).
(
Figure S3). These data offer compelling evidence that our
APH synthesis scheme using (BimC A)3 does not affect
4
aptamer functionality. Finally, we confirmed that the fully
assembled APH retains the affinity of the aptamer by loading
the polymer moiety with coumarin dye. This construct
exhibited a K of 4.01 ± 0.72 nM (Figure 2C, blue) in our
d
bead-based binding assay, essentially equivalent to that of the
aptamer alone or the APH in the absence of a payload. As
expected, the coumarin-loaded polymer scaffold showed
negligible affinity for nucleolin in the absence of aptamer
conjugation (Figure 2C, gray).
Our APH synthetic process can also be generalized to other
aptamers as a means for targeting different cell-surface markers.
To demonstrate this important feature, we used the same
41
chemistry to conjugate DNA aptamers for thrombin and
42
immunoglobulin E (IgE) to the same polymer scaffold. After
verifying that our conjugation strategy preserves the integrity of
the thrombin and IgE aptamers via gel electrophoresis (Figure
S4) and qPCR (Figure S5), we measured the binding affinities
of these APHs (Figures S6 and S7). In both cases, the
difference in binding between the unconjugated aptamer and
the APH was negligible, clearly demonstrating that the
conjugation scheme does not affect the structure or function
of the resulting hybrids. Although the underlying mechanism
behind our ligand is not yet fully understood, we suspect that
the negative charge of the (BimC A) complex electrostatically
Figure 2. Characterizing functional integrity of the APH construct.
(A) Dynamic light scattering measurements revealed a narrow radius
distribution of ∼10 nm with negligible aggregation in phosphate-
buffered saline buffer. (B) Gel electrophoresis shows that APH
conjugation via (BimC A)3 ligand-accelerated CuAAC greatly
4
minimizes DNA damage. Lanes: 1, unconjugated polymer; 2,
unconjugated aptamer (45 nt); 3, APH synthesized with (Bim) ; 4,
3
APH synthesized with (BimC A) ; 5, 20-bp ladder. (C) Affinity
4
3
measurements from a bead-based nucleolin-binding assay show that
aptamer target affinity is preserved within the APH construct. The
unconjugated aptamer exhibits a K of 8.37 ± 0.75 nM (black), while
d
4
3
APH molecules consisting of Cy5-labeled aptamer and coumarin-
repels the negatively charged phosphate backbone of DNA and
thereby prevents Cu (I) from inflicting oxidative damage on the
aptamer.
labeled polymer scaffold display similar K s of 5.18 ± 0.72 and 4.01 ±
d
0.72 nM based on Cy5 (red) and coumarin (blue) intensities,
respectively. In contrast, unconjugated polymer (gray) exhibits
negligible nucleolin affinity. (D) Time-dependent fluorescence
measurements confirm selective payload release. Untreated APHs
emit a red-shifted, self-quenched signal (red), but esterase treatment
shifts the peak fluorescence wavelength to that of free coumarin
Finally, we verified that the therapeutic payload remains
sequestered in an inactive state within the APH until it is
cleaved by the enzymes in the endosome. To assess the
efficiency of this cleavage reaction, we measured the release of
coumarin by porcine liver esterase (PLE) as a function of
(
blue), with a signal that increases over time as more coumarin is
released (dashed lines).
35
time. Without PLE, we observe minimal fluorescence from
the coumarin because it is tightly sequestered within the APH
(Figure 2D), even after 3 h in either buffer or undiluted human
serum (Figure S8). This latter result demonstrates that drug
payloads should remain stably sequestered in the physiological
environment prior to internalization by target cells.
However, in the presence of PLE, we observed a dramatic
increase in fluorescence at 467 nm, the expected peak for free
coumarin. Quantification of the data showed that 50% of the
payload is released within 30 min at 15 U/mL PLE (Figure S9).
As expected, the rate of coumarin release increased propor-
We confirmed that CuAAC coupling causes minimal damage
to the aptamer. This was clearly evident in the electrophoresis
shown in Figure 2B. Specifically, APHs synthesized using
conventional conjugation schemes with TBTA or the TBTA
3
9
analogue (Bim)3 caused significant damage to the aptamer,
which is evident in the heavy smearing of the band due to the
fragmentation of DNA (Figure 2B, lane 3). In contrast, APH
synthesis with (BimC A) gave rise to a sharply defined single
4
3
band, indicating minimal damage to the DNA aptamer (lane 4).
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tionally with the enzyme concentration (Figure S10). We
confirmed that this process was enzyme dependent rather than
chemically driven, as exposure to the acidic pH typically found
in the endosome was insufficient to promote coumarin release
does not occur without nucleolin-mediated APH binding and
internalization (Figure 3, bottom).
The power of this modular coupling was clearly demon-
strated through a series of control experiments that show how
each component of the APH makes a critical contribution to its
targeted delivery function. The deletion of the aptamer
component rendered the polymer scaffold incapable of target
recognition, and we observed no internalization by MCF-7 cells
(Figure 4A). Similarly, a mixture of unconjugated aptamers and
(
Figure S11). Taken together, these results confirm that our
APH retains the binding affinity of the parent aptamer and
requires the enzymatic activity of endosomal esterases to
release their therapeutic payload.
Verification of APH Functionality and Mechanism. We
used confocal fluorescence microscopy to verify targeted APH
binding, internalization and payload release in live cells. As
target cells, we used tumorigenic human breast epithelial cells
(
MCF-7) that overexpress nucleolin on their membrane, with
nontumorigenic human breast epithelial cells (MCF-10A) that
43
do not express nucleolin as negative control. In order to
fluorescently track the payload and APH independently inside
cells, we used coumarin as the model payload (blue
fluorescence) and labeled the aptamer component of the
APH with Cy5 (red fluorescence).
Our APHs exhibited highly selective binding to cells that
overexpress nucleolin. With MCF-7 cells, we observed APH
binding at the cell surface immediately after incubation, as
indicated by strong red fluorescence along the cellular contour
(
Figures 3, top, and S12). In contrast, negligible binding was
Figure 4. Control experiments demonstrate that each component of
the APH contributes essentially to targeted delivery. (A) In the
absence of a nucleolin-targeting aptamer, the polymer scaffold is no
longer bound or internalized by MCF-7 cells. (B) MCF-7 cells
incubated with an unconjugated mixture of nucleolin aptamers and
polymer scaffolds internalized the aptamer but not the polymer. (C)
APHs that target IgE are not bound or internalized by MCF-7 cells,
which do not express this protein. (D) Nucleolin-targeting APHs bind
MCF-7 cells but are no longer internalized at temperatures that inhibit
endocytosis. (E) Coumarin release is greatly reduced when the
payload is linked to the polymer scaffold via esterase-resistant bonds.
Scale bar = 40 μm.
Figure 3. Live-cell imaging confirms APH-mediated, targeted
coumarin delivery to nucleolin-expressing cells. We incubated APHs
that couple a Cy5-labeled nucleolin aptamer (red) with a polymer
scaffold loaded with coumarin (blue) with MCF-7 (top) and MCF-
10A (bottom) cells, which express high and low levels of nucleolin,
respectively. After 10 min, we washed the cells thoroughly; over the
next 4 h, we observed nucleolin-mediated cell-surface binding, APH
internalization and cytoplasmic payload release in MCF-7 but not
MCF-10A cells. Scale bar = 40 μm.
observed with MCF-10A cells (Figure 3, bottom), confirming
that nucleolin expression is a prerequisite for specific binding.
After 10 min, APH is internalized into endosomes, based on the
localization of red fluorescence within the MCF-7 cells, and
within 1 h, we observed evidence of cleavage of the ester
linkages and endosomal escape of coumarin based on the
permeation of blue fluorescence into the cytoplasm. After 4 h,
efficient endosomal escape of coumarin was observed, with
widespread diffusion throughout the cytoplasm. In contrast, the
red fluorescence remained localized within the endosome at
this late time-point, indicating that the polymer scaffold
remained trapped, presumably due to its larger size. These
data show that endosomal internalization efficiently triggers
payload cleavage and release into the cellular interior. Critically,
we observed a complete lack of blue fluorescence within MCF-
polymer scaffolds only showed localized red fluorescence within
endosomes due to aptamer uptake but no blue fluorescence
when incubated with MCF-7 cells (Figure 4B). Replacing the
nucleolin aptamer in the APH with a Cy5-labeled IgE aptamer
also prevented uptake by MCF-7 cells due to the lack of IgE
receptors on their cell surface (Figure 4C).
We confirmed that APH molecules are actively internalized
via endocytosis rather than diffusion or another passive
mechanism by incubating MCF-7 cells with nucleolin-targeting
APHs at 4 °C, a temperature at which endocytotic pathways are
44
inactive. Even after 4 h, red fluorescence remained entirely
localized at the cellular surface, indicating that the APHs could
successfully bind nucleolin but were no longer being
internalized at this temperature (Figure 4D). Finally, we
verified that payload release requires esterase-mediated cleavage
10A cells even after 4 h, demonstrating that payload delivery
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in the endosome through the synthesis of an alternate APH
wherein coumarin was coupled to the polymer scaffold through
amide linkages (Scheme S2) that cannot be cleaved by
esterases. In this case, only minimal blue fluorescence could
be detected after 4 h at 37 °C (Figure 4E) with the magnitude
of the signal being dramatically reduced in comparison to APHs
with esterase-cleavable linkages (Figure 3, top row).
APHs Selectively Kill Target Cells. Finally, we con-
structed APH with the chemotherapeutic agent DOX as the
payload and used it to selectively kill nucleolin-expressing target
cells. To synthesize the APH, we conjugated the hydroxyl
group of DOX to the polymer scaffold via the esterase-cleavable
linker described above (Scheme S1). We found that DOX-
loaded APH molecules efficiently killed MCF-7 cells at a
concentration of 500 nM. We used a live/dead cell viability
assay (see Methods) to measure the cytotoxicity of the DOX-
loaded APHs (Figure 5) and determined that this treatment
CONCLUSION
■
In this work, we describe the design and synthesis of
multifunctional drug-delivery vehicles that combine the
advantages of aptamers and functional polymers to enable
controlled, targeted therapy. The resulting APH constructs
incorporate an aptamer component and a block copolymer
component. The aptamer serves to selectively bind cell-surface
markers specific to the target cell, such that the APH is
subsequently internalized via an active endocytotic mechanism.
In parallel, the polymer component incorporates multiple
payload molecules, which are enzymatically cleaved from the
scaffold and subsequently diffuse into the cytoplasm only after
uptake of the APH in an endosomal compartment. As a result,
drug-mediated cytotoxicity is restricted to target cells that
express the surface marker of interest, and control experiments
verified that there is minimal drug release in the absence of
cellular binding and endosomal internalization.
Importantly, APH synthesis is achieved via a click chemistry
scheme that offers reproducibility and high yield under mild
conditions that ensure the structural and functional integrity of
both the aptamer and polymer components. We showed that
our synthesis strategy is modular and can be used with a variety
of aptamers to target other cell surface markers. Finally, given
that our scheme could be readily adopted for other polymer
4
7
systems and functional nucleic acids such as DNAzymes,
RNA riboswitches, and structure-switching aptamers,
48
49,50
we
believe our strategy of integrating aptamer technology with
functional, biocompatible synthetic polymers into a single
chemical platform may enable the development of other useful
functional materials for biomedical applications.
ASSOCIATED CONTENT
Figure 5. APH-mediated DOX delivery efficiently targets and kills
cancer cells. MCF-7 cells incubated with DOX-loaded APHs for 4 days
at 37 °C exhibited 36% viability, while the cells with free DOX yielded
■
*
S
Supporting Information
Synthetic procedures, experimental details, and other support-
ing data. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
8% viability relative to untreated controls. Similar treatment with
either unconjugated aptamers or DOX-loaded polymers alone resulted
in minimal cytotoxicity.
AUTHOR INFORMATION
■
Corresponding Authors
hawker@mrl.ucsb.edu
tsoh@engr.ucsb.edu
reduced cell viability by 64%. The potency of the APH was also
readily apparent in microscopic images of treated cells, in which
viability could be visually ascertained based on changes in cell
morphology and adhesion characteristics (Figure S13A).
However, the cytotoxicity of the APH is lower than equivalent,
free DOX at a concentration of 5 μM, indicating that some
ester linkages between the APH and the DOX would be still
intact within the endosome.
Author Contributions
These authors contributed equally.
#
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
As demonstrated above with coumarin-loaded APHs, we
found that each component of the APH plays an integral role in
the targeted delivery of chemotherapy to nucleolin-expressing
cancer cells. Equal concentrations of unconjugated nucleolin
aptamers (Figure S13B) or DOX-loaded polymers (Figure
S13C) alone had negligible effect on MCF-7 cell viability, even
after 4 days of incubation, with levels of cell death that were
essentially indistinguishable from untreated cells in culture
media (Figure S13D). Based on our cell viability assay, cells
treated with unconjugated aptamers or DOX-loaded polymers
respectively exhibited ∼100% and 91% viability relative to
untreated cells (Figure 5); the latter result confirms that the
polymer keeps the drug safely sequestered in an inactive state,
which is an important feature when delivering toxic
■
This work was supported by the Institute for Collaborative
Biotechnologies through the U.S. Army Research Office
(W911NF-09-D-0001) (S.S.O., B.F.L., H.T.S., C.J.H.) and
W911NF-10-2-0114 (H.T.S., S.S.O.), the Garland Intitiative
(S.S.O., H.T.S.), the MRSEC Program of the National Science
Foundation (NSF) under Award DMR-1121053 (F.A.L.,
M.J.R., C.J.H.) and the National Institutes of Health (NIH)
as a Program of Excellence in Nanotechnology
(HHSN268201000046C) (N.A.L., C.J.H.). Partial support
from the NIH U54 DK093467 (H.T.S., S.S.O.), U01
HL099773-1 (H.T.S., S.S.O.), and the NRI-MCDB Microscopy
Facility and the Spectral Laser Scanning Confocal supported by
the Office of The Director, the NIH under Award
S10OD010610 is gratefully acknowledged. We thank Prof.
4
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therapeutics.
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James A. Thomson, Prof. Patrick S. Daugherty, and Dr. Jack
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