A convergent synthetic platform for single-nanoparticle combination cancer therapy: Ratiometric loading and controlled release of cisplatin, doxorubicin, and camptothecin

Article abstract of DOI:10.1021/ja502011g

The synthesis of polymer therapeutics capable of controlled loading and synchronized release of multiple therapeutic agents remains a formidable challenge in drug delivery and synthetic polymer chemistry. Herein, we report the synthesis of polymer nanopar

Full text of DOI:10.1021/ja502011g

Communication
pubs.acs.org/JACS
A Convergent Synthetic Platform for Single-Nanoparticle
Combination Cancer Therapy: Ratiometric Loading and Controlled
Release of Cisplatin, Doxorubicin, and Camptothecin
Longyan Liao,† Jenny Liu,† Erik C. Dreaden,‡ Stephen W. Morton,‡ Kevin E. Shopsowitz,‡
Paula T. Hammond,‡ and Jeremiah A. Johnson*,†
†Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139,
United States
‡Koch Institute for Integrative Cancer Research, Institute for Soldier Nanotechnologies, & Department of Chemical Engineering,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
from cardiotoxicity,20 while those from cisplatin and
camptothecin result from neurotoxicity21 and myelosuppres-
ABSTRACT: The synthesis of polymer therapeutics
sion or hemorrhagic cystitis,22 respectively. Thus, maximum
capable of controlled loading and synchronized release of
multiple therapeutic agents remains a formidable challenge
in drug delivery and synthetic polymer chemistry. Herein,
we report the synthesis of polymer nanoparticles (NPs)
that carry precise molar ratios of doxorubicin, camptothe-
cin, and cisplatin. To our knowledge, this work provides
the first example of orthogonally triggered release of three
drugs from single NPs. The highly convergent synthetic
approach opens the door to new NP-based combination
therapies for cancer.
therapeutic index could be achieved, in principle, via
simultaneous dosing of each drug at or near its maximum
tolerated dose (MTD). We show that three-drug-loaded NPs
with ratios matched to multiples of the MTD of each drug
outperform analogous one- and two-drug-loaded NPs in in vitro
cell viability studies using ovarian cancer (OVCAR3) cells.
Our synthesis relies on the “brush-first” ring-opening
metathesis polymerization (ROMP) method,23,24 which
enables the preparation of nanoscopic brush-arm star polymers
(BASPs). For the purposes of this study, we designed two novel
macromonomers (MMs) and a novel cross-linker (Figure 1A).
CPT-MM and DOX-MM are branched MMs25 that release
unmodified CPT and DOX in response to cell culture media26
and long-wavelength ultraviolet (UV) light,27 respectively. Both
MMs feature a 3 kDa poly(ethylene glycol) (PEG) chain that
confers water solubility and neutral surface charge to the final
NP.28,29
For our cross-linker design, we were drawn to Pt(IV) diester
derivatives, which are widely applied as prodrugs for the
clinically approved chemotherapeutic cisplatin.30−34 Pt(IV)
diesters release cytotoxic Pt(II) species upon glutathione-
induced intracellular reduction. We wondered whether a Pt(IV)
bis-norbornene complex could serve as a cross-linker during
brush-first ROMP. If so, then the resulting BASP core would be
connected via labile Pt−O bonds; reduction would lead to
particle degradation to yield ∼5 nm brush polymers27 and free
cisplatin. To explore the feasibility of this approach, we
designed and synthesized Pt-XL (Figure 1A, see SI for details).
With this pool of novel monomers in hand, we targeted
BASPs with molar ratios of each drug that correspond to 2
times the MTD of CPT,35 2 times the MTD of DOX,36 and 1
times the MTD of cisplatin.37 In the brush-first method, the
final BASP size is determined by the MM to cross-linker ratio.23
A series of stoichiometry screens using a non-drug-loaded MM
(PEG-MM, Figure 1A) and Pt-XL revealed that the most
uniform BASPs formed when the total MM:Pt-XL ratio was
anoparticle (NP)-based combination cancer therapy has
Nthe potential to overcome the toxicity and poorly
controlled dosing of traditional systemic combination thera-
pies.1−3 Though NP-based therapeutics for cancer therapy have
been the subject of numerous investigations over the past
several decades,4−8 ratiometric delivery and synchronized
release of multiple drugs from single NP scaffolds remain
formidable challenges.9−12 Many of the most studied NP
architectures for deliverye.g., liposomes, micelles, and
dendrimersare not readily amenable to incorporation and
release of multiple drugs.
Due to the complex interactions between drugs in living
systems, a NP platform for precise tuning and rapid variation of
drug loading ratios and release kinetics would enable the
discovery of optimal formulations for specific cancer types. We
view this challenge as a synthetic problem: multi-drug-loaded
NP synthesis would be most efficient if serial particle
conjugation and encapsulation reactions were replaced with
highly convergent approaches wherein the key elements of a
desired NP (e.g., drug molecules) are used to build particles
directly.13−17 Herein we present a novel strategy that uses
carefully designed drug conjugates as building blocks for the
parallel construction of a series of multi-drug-loaded NPs; no
extraneous formulation steps are required.
Our NPs carry precise ratios of camptothecin (CPT),
doxorubicin (DOX), and/or cisplatin (Pt). These drugs were
chosen due to their non-overlapping toxicity profiles.18,19 The
most serious dose-limiting side effects from doxorubicin arise
Received: March 3, 2014
Published: April 11, 2014
© 2014 American Chemical Society
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Figure 1. (A) Structures of monomers used in this study. (B) Schematic for synthesis of three-drug-loaded BASP. Drug release occurs in response to
three distinct triggers.
Figure 2. (A) Table of BASP NPs prepared in this study along with the MM stoichiometry used to prepare each particle. BASP diameters as
measured by transmission electron microscopy (DTEM) and dynamic light scattering (DH). aTEM data were obtained from dilute aqueous solutions
cast onto a TEM grid, dried, and imaged without staining. bDH values were measured using 0.1 mg BASP/mL 5% glucose solutions. DLS correlation
functions were fit using the CONTIN algorithm. Values in parentheses correspond to the standard deviation for three particle measurements. (B)
DLS histograms for drug-loaded BASPs. (C) Positively (top) and negatively (bottom) stained TEM images of 3. Scale bars correspond to 100 nm.
7:3. Thus, this ratio was held constant for all drug-loaded
particles; PEG-MM was simply replaced with DOX-MM and/
or CPT-MM. For example, a three-drug-loaded particle (3) was
prepared as follows: CPT-MM (2.07 equiv), DOX-MM (0.83
equiv), and PEG-MM (4.09 equiv) were exposed to Grubbs
third-generation catalyst (cat., 1.00 equiv) for 20 min. Pt-XL
(3.00 equiv) was added, and the mixture was stirred for 6 h at
room temperature. Analogous one- and two-drug-loaded
particles (1, 2a, and 2b) were prepared in parallel following
similar procedures. In this system, the mass fraction of drug
increases with introduction of new drug (3.4% for 1, 6.1% for
2a, 5.1% for 2b, and 7.8% for 3).
solution (Figure S3) showed particle diameters that agree well
with DLS data.
We next studied the cytotoxicity of these BASPs using
OVCAR3 human ovarian cancer cells (Figure 3A). OCVAR3 is
Upon completion of the brush-first ROMP reactions, the
crude reaction mixtures were analyzed by gel permeation
chromatography (Figure S1). In all cases, the conversion of
MM and brush to BASP was >90%. A combination of UV/vis,
1H NMR, and inductively coupled mass spectrometry (ICP-
MS) was used to confirm the drug ratios in 3 (Table S1).
Dynamic light scattering (DLS, Figure 2B) revealed hydro-
dynamic diameters (DH) from 122 to 191 nm for this series
(Figure 2A). These values are larger than we observed for our
previous photocleavable BASPs.23 Regardless, the observed DH
values are suitable for passive tumor targeting via the enhanced
permeation and retention (EPR) effect:38 they are larger than
the ca. 6−8 nm renal clearance threshold39 and smaller than the
200−250 nm splenic clearance cutoff.40 Transmission electron
microscopy (TEM) images of positively (Figure 2C, top) and
negatively (Figure 2C, bottom) stained BASPs showed uniform
NPs (Figure S2). CryoTEM images of the BASPs in aqueous
Figure 3. (A) OVCAR3 cell viability data after 72 h of treatment with
5% glucose (0) and BASPs 1, 2a, 2b, and 3. Data labeled “+hν” were
obtained from cells treated with BASP, irradiated with 365 nm light for
10 min, and then incubated for a total of 72 h. Solid and dashed lines
represent sigmoidal fits for dark and irradiated samples, respectively.
(B) Bar chart of IC50 values along with statistical comparisons. Error
represents standard error of the mean of four technical replicates.
an established model cell line derived from a patient with
platinum-refractory41 disease that exhibits genotypic similarity
with the high-grade serous subtype.42 Given the widespread
clinical use of anthracyclines and topoisomerase I inhibitors in
second-line therapies for recurrent ovarian carcinoma,
OVCAR3 is a suitable model for BASP combination chemo-
therapy.43,44 Exposure of OVCAR3 cells to 365 nm UV light for
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Figure 4. Phototriggered release of DOX in OVCAR3 cells as monitored by live-cell confocal fluorescence imaging. Cells were loaded with 2b for 30
min and concurrently exposed to 405 nm UV irradiation during imaging of doxorubicin (red; λex/λem = 561/595 nm) and nuclei (acridine orange,
green; λex/λem = 488/525 nm). Scale bar is 5 μm.
10 min (black circles) induced no observable toxicity. A non-
drug-loaded BASP23 displayed toxicity only at very high
concentrations (>650 μg/mL) in the presence and absence of
UV light (Figure S4). Among the drug-loaded BASPs, 1 (purple
curve) had the largest IC50 value: 192 46 μg BASP/mL (23
5 μM drug).
alternative MMs, drug linkers, and cross-linkers, libraries of
multi-drug-loaded BASPs can be readily synthesized in parallel
for efficacy optimization. These studies along with in vivo
analysis of the current BASP systems are currently ongoing in
our laboratories.
ASSOCIATED CONTENT
* Supporting Information
BASP 2a (green curve) showed a much lower IC50: 44 15
μg BASP/mL (8 2 μM drug). BASP 2b had an IC50 of 217
■
S
Supplemental figures, experimental procedures, and spectral
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
23 μg BASP/mL (32
3 μM drug) in the absence of
irradiation (red trace), which is not significantly different from
that of 1; exposure to UV for 20 min led to a 2.3 0.3-fold
decrease in IC50 to 93 11 μg BASP/mL (14 1 μM drug).
No significant decrease in viability was observed following
photoexposure of 1 and 2a (P = 0.078 and 0.018, respectively).
These results suggest that therapeutically active cisplatin and
CPT are released from these BASPs without an external trigger;
DOX release is only significant upon irradiation.
AUTHOR INFORMATION
Corresponding Author
jaj2109@mit.edu
■
Notes
The authors declare no competing financial interest.
When cells were treated with three-drug-loaded BASP 3
without UV irradiation (blue curve), the IC50 was 42 6 μg
BASP/mL (9.2 0.8 μM drug). This result can be rationalized
via extrapolation of the results for 1, 2a, and 2b: in the absence
of light, 3 only released CPT and cisplatin, i.e., it behaved
similarly to 2a (P = 0.81). After UV irradiation for 10 min, the
IC50 for 3 dropped 2.3 0.4-fold to 18 2 μg BASP/mL (4.0
0.3 μM total drug); the three-drug-loaded NP outperformed
the one- and two-drug-loaded systems.
To examine cellular internalization of BASPs, we conducted a
series of confocal fluorescence imaging experiments on live cells
using the inherent fluorescence of DOX. After 30 min of
incubation with 2b in the dark, cells were briefly irradiated with
405 nm laser light once per minute and imaged immediately
afterward for 25 min (DOX λex/λem = 561/595 nm). Figure 4
shows images collected at various times (see Figure S5 for full
series). Initially, punctate, extranuclear DOX fluorescence was
observed to colocalize with acridine orange in the endo/
lysosomes (Figure 4, far left); photoinduced DOX release led
to rapid redistribution of fluorescence throughout the
cytoplasm and nucleus and a 2.7-fold fluorescence intensity
increase (Figure S6). To ensure that these results were due to
DOX release, an experiment was conducted wherein cells were
pulsed with 561 nm light rather than 405 nm. In this case, the
particles remained in the endosomes (Figure S7), and no
increase in mean fluorescence intensity was observed.
ACKNOWLEDGMENTS
■
We acknowledge support from the MIT Research Support
Committee, the MIT Lincoln Laboratories Advanced Concepts
Committee, the Department of Defense Ovarian Cancer
Research Program Teal Innovator Award, the National
Institutes of Health (E.C.D., Ruth L. Kirschstein NRSA
1F32EB017614-01), the National Sciences and Engineering
Research Council (postdoctoral fellowship for K.E.S. and
graduate fellowship for J.L.), and the Koch Institute Support
(core) Grant P30-CA14051 from the National Cancer Institute.
We thank the Koch Institute Swanson Biotechnology Center
for technical support.
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