Maximizing Synergistic Activity When Combining RNAi and Platinum-Based Anticancer Agents

Article abstract of DOI:10.1021/jacs.6b12108

RNAi approaches have been widely combined with platinum-based anticancer agents to elucidate cellular responses and to target gene products that mediate acquired resistance. Recent work has demonstrated that platination of siRNA prior to transfection may negatively influence RNAi efficiency based on the position and sequence of its guanosine nucleosides. Here, we used detailed spectroscopic characterization to demonstrate rapid formation of Pt-guanosine adducts within 30 min after coincubation of oxaliplatin [OxaPt(II)] or cisplatin [CisPt(II)] with either guanosine monophosphate or B-cell lymphoma 2 (BCL-2) siRNA. After 3 h of exposure to these platinum(II) agents, >50% of BCL-2 siRNA transcripts were platinated and unable to effectively suppress mRNA levels. Platinum(IV) analogues [OxaPt(IV) or CisPt(IV)] did not form Pt-siRNA adducts but did display decreased in vitro uptake and reduced potency. To overcome these challenges, we utilized biodegradable methoxyl-poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly(l-lysine) (mPEG-b-PCL-b-PLL) to generate self-assembled micelles that covalently conjugated OxaPt(IV) and/or electrostatically complexed siRNA. We then compared multiple strategies by which to combine BCL-2 siRNA with either OxaPt(II) or OxaPt(IV). Overall, we determined that the concentrations of siRNA (nM) and platinum(II)-based anticancer agents (μM) that are typically used for in vitro experiments led to rapid Pt-siRNA adduct formation and ineffective RNAi. Coincorporation of BCL-2 siRNA and platinum(IV) analogues in a single micelle enabled maximal suppression of BCL-2 mRNA levels (to <10% of baseline), augmented the intracellular levels of platinum (by ～4×) and the numbers of resultant Pt-DNA adducts (by >5×), increased the cellular fractions that underwent apoptosis (by ～4×), and enhanced the in vitro antiproliferative activity of the corresponding platinum(II) agent (by 10-100×, depending on the cancer cell line). When combining RNAi and platinum-based anticancer agents, this generalizable strategy may be adopted to maximize synergy during screening or for therapeutic delivery.

Full text of DOI:10.1021/jacs.6b12108

Subscriber access provided by Georgetown University | Lauinger and Blommer Libraries
Article
Maximizing Synergistic Activity When Combining
RNAi and Platinum-based Anticancer Agents
Haihua Xiao, Ruogu Qi, Ting Li, Samuel G. Awuah, Yaorong Zheng, Wei
Wei, Xiang Kang, Haiqin Song, Yongheng Wang, Yingjie Yu, Molly A. Bird,
Xiabin Jing, Michael B. Yaffe, Michael J. Birrer, and P. Peter Ghoroghchian
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b12108 • Publication Date (Web): 06 Feb 2017
Downloaded from http://pubs.acs.org on February 7, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Maximizing Synergistic Activity When Combining RNAi and
Platinum-based Anticancer Agents
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Haihua Xiao,† Ruogu Qi,† Ting Li,† Samuel G. Awuah,†,‡ Yaorong Zheng,†,‡ Wei Wei,§ Xiang Kang†, Haiqin
Song,† Yongheng Wang,† Yingjie Yu,† Molly A. Bird,† Xiabin Jing,⊥ Michael B. Yaffe,† Michael J. Birrer,§
, *
∥
and P. Peter Ghoroghchian†,
†Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, United
States
‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States
§Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United
States
⊥State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun 130022, People's Republic of China
∥Dana-Farber Cancer Institute, Boston, MA, 02115, United States
KEYWORDS: breast cancer, cisplatin, drug delivery, nanoparticle, ovarian cancer, oxaliplatin, platinum(IV), polymer micelle,
siRNA.
ABSTRACT: RNAi approaches have been widely combined with platinum-based anticancer agents to elucidate cellular responses
and to target gene products that mediate acquired resistance. Recent work has demonstrated that platination of siRNA prior to
transfection may negatively influence RNAi efficiency based on the position and sequence of its guanosine nucleosides. Here, we
used detailed spectroscopic characterization to demonstrate rapid formation of Pt-guanosine adducts within 30 min after co-
incubation of oxaliplatin [OxaPt(II)] or cisplatin [CisPt(II)] with either guanosine monophosphate or BCL-2 siRNA. After 3 h of expo-
sure to these platinum(II) agents, >50% of BCL-2 siRNA transcripts were platinated and unable to effectively suppress mRNA lev-
els. Platinum(IV) analogs [OxaPt(IV) or CisPt(IV)] did not form Pt-siRNA adducts but did display decreased in vitro uptake and re-
duced potency. To overcome these challenges, we utilized biodegradable methoxyl-poly(ethylene glycol)-block-poly-(ε-
caprolactone)-block-poly(L-lysine) (mPEG-b-PCL-b-PLL) to generate self-assembled micelles that covalently conjugated OxaPt(IV)
and/or electrostatically complexed siRNA. We then compared multiple strategies by which to combine BCL-2 siRNA with either
OxaPt(II) or OxaPt(IV). Overall, we determined that the concentrations of siRNA (nM) and platinum(II)-based anticancer agents
(µM) that are typically used for in vitro experiments led to rapid Pt-siRNA adduct formation and ineffective RNAi. Co-incorporation
of BCL-2 siRNA and platinum(IV) analogs in a single micelle enabled maximal suppression of BCL-2 mRNA levels (to < 10% of base-
line), augmented the intracellular levels of platinum (by ~4x) and the numbers of resultant Pt-DNA adducts (by >5x), increased the
cellular fractions that underwent apoptosis (by ~4x), and enhanced the in vitro anti-proliferative activity of the corresponding
platinum(II) agent (by 10-100x depending on the cancer cell line). When combining RNAi and platinum-based anticancer agents,
this generalizable strategy may be adopted to maximize synergy during screening or for therapeutic delivery.
INTRODUCTION
Since the late 1970’s, platinum(II)-based anticancer
proliferative advantages are areas of very active investiga-
tion.5-11 RNAi approaches have been used to screen for gene
products that mediate these tumorigenic properties,12-16 to
validate putative drug targets,17-19 and to develop therapeutic
constructs.20-22 Many of these experiments employ double-
stranded small interfering RNAs (siRNAs) or microRNAs (miR-
NAs) that are synthesized with near-perfect complementarity
to specific messenger RNA (mRNA) sequences. The siRNA (or
agents, including cisplatin and oxaliplatin, have become the
most common class of chemotherapies used worldwide.1-2
Whereas initial clinical response rates to platinum-containing
regimens are high, the duration of activity in patients with
metastatic cancers is generally short.3-4 The various mecha-
nisms by which these tumors develop resistance and acquire
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 21
miRNA) is delivered into the cell via a lipid or polymer-based num(IV) analogs are activated by reduction to their corre-
1
2
3
4
5
6
7
8
transfection reagent, whereby it binds to the RNA-induced
silencing complex (RISC) and leads to cleavage of its target
mRNA.23 The duration and level of mRNA knockdown are de-
pendent on the cell type and the concentration of the siRNA
that is delivered.24 When combining siRNA and platinum(II)-
based anticancer agents, both species must be introduced
into the cellular environment within a narrow temporal win-
dow in order to capture synergistic treatment effects.12-19 The
success of such endeavors relies implicitly on the assumption
that the siRNA remains active, even for a few hours, when
utilized in combination with platinum-based therapies.
sponding platinum(II) species only after intracellular uptake
within cancer cells, which contain supraphysiologic levels of
glutathione and ascorbic acid (~100 to ~1000 fold excess over
normal cells).39, 41-45 Employing platinum(IV)-containing agents
offers an attractive means by which to overcome challenges
associated with combining RNAi and platinum(II)-based ther-
apies for discovery research and cancer treatment. The MCF-
7 breast cancer and the OVCAR4 ovarian cancer cell lines
were mainly employed as the in vitro model systems in our
studies; and, the anti-apoptosis protein BCL-2 was selected as
the target for RNAi. BCL-2 upregulation contributes to the
development of acquired CisPt(II) resistance in various solid
tumor malignancies.5, 46 Here, we used a BCL-2 siRNA that
contained multiple GG and GA regions but only a single GA
sequence outside of the seed portion of the antisense strand.
Platination at this latter site was expected to result in maxi-
mal thermal destabilization with decreased mRNA silencing36-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
It has long been appreciated that platinum(II)-based an-
ticancer agents interact with DNA nucleobases, forming intra-
and interstrand cross-links through the generation of adducts
at the N7 positions of both guanine (G) and/or adenine (A).25-
27
The most commonly formed cross-links include d(pGpG)
(i.e. the 1,2-guaninosine-guanosine intrastrand cross-link) and
d(pApG) (i.e. the 1,2-adenosine-guanosine intrastrand cross-
link), which account for 47-50% and 23-28% of adducts, re-
spectively.25, 28 With increasing quantities of intracellular Pt-
DNA adducts, cell cycle arrest and eventually apoptosis en-
sue.29-32 Given the base homology in RNA and DNA, recent
investigations have sought to determine the presence, as well
as to ascertain the biological consequences, of Pt-RNA ad-
ducts that may be similarly generated.
37
(Scheme 1A). A micelle-based transfection reagent com-
prised of the triblock copolymer of methoxyl-poly(ethylene
glycol)-block-poly-(ε-caprolactone)-block-poly(L-lysine)
(mPEG-b-PCL-b-PLL) was used to deliver OxaPt(IV) and/or
siRNA (Scheme 1B). This vehicle was also employed to vali-
date an optimal strategy for combining RNAi and platinum-
based anticancer agents (Scheme 1C).
Cytoplasmic RNA is a major binding pool for platinum(II)-
based anticancer agents; and, the concentrations of Pt-RNA
adducts that are formed are ~4-20 times greater than those
of Pt-DNA adducts, following intracellular uptake of
cisplatin.33-35 Further, platinum(II)-containing agents readily
form bidentate GG, GA/AG, and GXG (X≠G) adducts with siR-
NA and miRNA.36-37 Whereas platination of neither the sense
strand nor the seed portions of the antisense strand seems to
interfere with silencing activity, Pt-RNA adducts that are
formed in non-seed regions of the antisense strand induce
thermal destabilization of the duplex of siRNA (or miRNA),
which leads to decreased silencing of the complementary
target mRNA.36-37 The results of these investigations raise
important questions as to whether the concentrations of siR-
NA and platinum(II)-based anticancer agents that are admin-
istered in combination treatments are sufficient to form Pt-
siRNA adducts in the cellular environment. siRNA inactivation
through Pt-siRNA adduct formation could have implications
for the results of numerous preclinical studies that have uti-
lized such approaches either for discovery or for therapeutic
intervention.12-22 Moreover, different strategies by which to
combine these two reactive species have not, hitherto, been
rigorously compared.
EXPERIMENTAL SECTION
Materials. mPEG-b-PCL-b-PLL was synthesized as previ-
ously described.47 Its structure was verified by H-NMR spec-
1
troscopy and consisted of mPEG114-b-PCL25-b-PLL25, where the
subscript denotes the degree of polymerization of each indi-
vidual monomer in a given block (Figure S1). This triblock
copolymer
is
hereafter
abbreviated
“P”.
N-
hydroxysuccinimide (NHS), 1-(3-dimethyl aminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC HCl), guanosine 5’-
monophosphate disodium salt (5’-GMP), and sodium ascor-
bate were purchased from Sigma-Aldrich. OxaPt(II) and
CisPt(II) were purchased from ChemiChem International De-
velopment Co., Ltd (Shenzhen, China). OxaPt(IV) analogs were
synthesized and characterized by nuclear magnetic resonance
(NMR) spectroscopy and mass spectrometry (MS), following
procedures described below and in our previous studies48
(Figures S2-S6). The CisPt(IV) derivatives were similarly syn-
thesized and characterized as described.38, 40 All other chemi-
cals and solvents were used without further purification. Con-
trol (c-) siRNA that targeted the sequence 5’-
GGGUAAGUGUCCUACU-GAAGU-3’, BCL-2 siRNA that targeted
the sequence 5’-UGUGGAUGACUGAGUACC UGA-3’, and
Alexa488-labeled BCL-2 siRNA (Alexa488 siRNA) were pur-
chased from Integrated DNA Technologies (Coralville, Iowa);
the c-siRNA did not match any known sequence in the human
genome. Luciferase GL3 siRNA was purchased from
GenePharma Co. Ltd. (Shanghai, China).
Here, we sought to determine a generalizable method by
which to combine RNAi and platinum-containing therapies in
order to prevent premature siRNA inactivation and to maxim-
ize synergistic activity. We utilized oxaliplatin [OxaPt(II)] and
cisplatin [CisPt(II)] as model platinum(II)-based anticancer
agents. To advance our design, we also synthesized chemical-
ly inert platinum(IV) derivatives, denoted by OxaPt(IV) and
CisPt(IV),38-40 respectively (Schemes 1A, S1, and S2). Plati-
General measurements. NMR, MS, graphite furnace
atomic absorption spectroscopy (GFAAS), and UV-VIS spec-
troscopy were used to study the reactions of various platinum
2
ACS Paragon Plus Environment

Page 3 of 21
Journal of the American Chemical Society
species with either 5’-GMP or BCL-2 siRNA (see Supplemental Formation and Characterization of Polymeric Micelles. P-
1
2
3
4
5
6
7
8
Materials and Methods in the Supporting Information). In
based micelles (M(P)) were prepared by an established nano-
precipitation method.47 In brief, P (50 mg) was dissolved in
DMF (10 mL); water (50 mL) was then added drop-wise into
the flask under continuous agitation, forming M(P) in suspen-
sion. The suspension was then dialyzed against water to re-
move the organic solvent and subsequently freeze-dried for
storage. The critical micelle concentrations (CMCs) of M(P)
and other micellar formulations (vide infra) were measured as
previously reported,49 using Nile red as the fluorescent probe.
Particle sizes and zeta potentials were measured using a Nano
ZS90 Zetasizer equipped with a vertically polarized He-Ne
laser (Malvern Instruments, Malvern, PA). Images of various
micelle suspensions were obtained using a JEOL-1100 trans-
mission electron microscope (JEOL USA, Peabody, MA).
1
brief, H, 13C, and 195Pt NMR spectra at RT were measured at
400, 100, and 86 MHz, respectively, using a Bruker NMR spec-
trometer (Bruker Corporation, Billerica, MA). MS measure-
ments were performed on a Quattro Premier XE system
equipped with an electrospray interface (ESI-MS; Waters,
Milford, MA) as well as on a matrix-assisted laser-desorption
ionization and time-of-flight MS (MALDI/TOF-MS) instrument
(Bruker model MicroFlex). GFAAS and UV-Vis measurements
were conducted using an AAnalyst 600 GFAAS instrument
(Perkin Elmer, Waltham, MA) and an Infinite® M200 Pro mi-
croplate reader (Tecan Systems, Inc., CA, USA), respectively.
GFAAS was also used to determine the Pt content in all mi-
celle formulations. Inductively coupled plasma MS (ICP-MS;
ICP-MS 7900, Agilent Technologies, CA, USA) was used for
quantitative determination of trace levels of Pt in cancer cells
and to quantify the numbers of intracellular Pt-DNA adducts
that were formed after treatment with various experimental
groups.
Cell Culture. MCF-7 (human breast cancer), LUC+ MCF-7
(luciferase expressing MCF7), OVCAR4 (human ovarian can-
cer), A2780 (CisPt(II)-sensitive), and A2780DDP (CisPt(II)-
resistant) cell lines were obtained from ATCC and cultured at
37 °C, 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM,
Gibco, Carlsbad, CA; MCF-7) or RPMI 1640 (LUC+ MCF-7,
OVCAR4, A2780, and A2780DDP) supplemented with 10%
fetal bovine serum (FBS, Gibco).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conjugation of OxaPt(IV) to M(P). EDC/NHS chemistry
was employed to conjugate OxaPt(IV) to P via an amide bond.
Briefly, EDC·HCl (0.191 g, 1 mmol) and NHS (0.115 g, 1 mmol)
were dissolved in deionized H2O under stirring. OxaPt(IV)
(0.42 g, 0.8 mmol) was then added to the solution. After the
suspension mixture became clear, P (0.5 g; containing 1.25
mmol NH2 groups) was added to the reaction mixture (100
mL) and stirred at RT for 24 h; the suspension was then dia-
lyzed against water for an additional 12 h and lyophilized to
isolate OxaPt(IV)-conjugated micelles (M(OxaPt(IV))). The Pt
content of the micelles was measured by GFAAS.
Complexation of siRNA with Polymeric Micelles. Suspen-
sions of M(P) or M(OxaPt(IV)) were diluted with Opti-MEM
media® (Invitrogen) to different concentrations, varying the
final numbers of amino groups in solution. Equal volume solu-
tions containing BCL-2 or c-siRNA where then added to the
micelles, varying the initial ratios of free amines to phos-
phates (N/P) in suspension. siRNA-complexed micelles were
then formed by gentle pipetting and allowed to equilibrate
for 30 min at RT. For electrophoresis-retardation analyses,
complexes formed from either M(P) or M(OxaPt(IV)) and siR-
NA at different initial N/P ratios, ranging from 0 to 4, were
prepared for a fixed concentration of siRNA (1 mM). The
samples were run on a 1% agarose gel in 0.5 mM tris-borate-
EDTA buffer (TBE buffer; 89 mM Tris, 90 mM boric acid, 2 mM
EDTA, pH 8.3) at 80 V and for 30 min. Bands containing free
and micellar-bound siRNA were visualized using a UV illumi-
nator (Tanon 3000, Biotanon, Shanghai, China) after ethidium
bromide staining.
Quantitative Reverse Transcription (RT-q)PCR. BCL-2
siRNA was first pre-incubated with platinum(II) or plati-
num(IV) species for various time periods and subsequently
transfected into MCF-7 and OVCAR4 cells in 6-well plates,
using an RNAiMAX Kit and by following the manufacturer’s
instructions (Invitrogen, Thermo-Fisher, USA). Briefly, 48 h
after transfection, total RNA was isolated, using an RNeasy
mini-kit (Qiagen, Germantown, MD), and quantified by
NanoDropTM (NanoDrop 2000, Thermo Scientific). A total of
300 ng of mRNA was subsequently subjected to RT-qPCR
analysis, utilizing primers that targeted BCL-2 and glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH), the SYBR Premix
Ex (Clontech, Mountain View, CA), and an Applied Biosystems
StepOne Real-Time PCR System (Thermo Scientific). Relative
gene expression values were determined by the ΔΔCT meth-
od using StepOne Software v2.1 (Applied Biosystems, Foster
City, CA). Data are presented as fold differences in mRNA
expression for BCL-2 normalized to the housekeeping gene
GAPDH and were obtained by employing a standard curve;
results are reported relative to untreated (control) cells. The
sequence of the primers used for BCL-2 and GAPDH are as
follows: BCL-2 forward – 5’-CTGCACCTGACGCCCTTCACC-3’;
To determine the maximal loading of siRNA and the effi-
ciency of micellar complexation, Alexa488-labeled BCL-2 siR-
NA was complexed with M(P) or M(OxaPt(IV)) at different
initial N/P ratios, varying from 0 to 16, and subsequently
quantified by both UV-VIS and fluorescence spectroscopy. To
determine the amounts of micellar-complexed siRNA neces-
sary for effective RNAi, Luciferase GL3 siRNA was complexed
with M(P) or M(OxaPt(IV)) at an initial N/P ratio of 8; and, the
percentage of initial luciferase mRNA expression (relative to
controls) was quantified as a function of different concentra-
tions of Luciferase GL3 siRNA that were used for transfection,
ranging from 25-150 nM. Detailed information on micelle
preparation and various siRNA-complexed formulations may
BCL-2 reverse
GAPDH forward
–
5’-CACATGACCCCACCGAACTCAAAGA-3’;
– 5’-TTCACCACCATGGAGAAGGC-3’; and
GAPDH reverse – 5’-GGCATGGACTGTGGTCA TGA-3’ (Integrat-
ed DNA Technologies). The specificity of each set of primers
was verified by melting curve analysis. Additional details may
be found in Supplemental Materials and Methods in the Sup-
porting Information.
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 21
be found in Supplemental Materials and Methods in the Sup- A2780, A2780DDP; 100 L) supplemented with 10% FBS was
1
2
3
4
5
6
7
8
porting Information.
added to each well. The cells were then incubated at 37 °C for
24 h prior to the addition of OxaPt(II), OxaPt(IV), or
M(OxaPt(IV)) with or without micellar-complexed BCL-2 or c-
siRNA. For siRNA-containing treatments, an equivalent con-
centration of siRNA (100 nM) was added to each well. The
cells were subsequently allowed to incubate at 37 °C for 72 h
prior to evaluation of cellular viability, using the colorimetric
MTT assay. In brief, MTT reagent in in PBS buffer (5 mg/mL;
20 μL) was added to each well and allowed to incubate for 4
h. DMSO (150 μL) was then added to replace the MTT-
containing media. After gentle agitation (5 min), the absorb-
ance of each well at 570 nm was recorded on a Bio-Rad Plate
Reader (Hercules, CA). ABS at 650 nm was set as background
and used for subtraction. All experiments were conducted in
triplicate.
Intracellular Uptake of Fluorophore-labeled Micelles. For
confocal microscopy experiments, MCF-7 and OVCAR4 cells
were plated on coverslips in 6-well plates (4105 cells/well)
and cultured with RPMI 1640 supplemented with 10% FBS for
24 h. The cells were then incubated either for 30 min or for 4
h with micelles that were electrostatically complexed with
Alexa488-labeled BCL-2 siRNA (50 nM) and that were cova-
lently conjugated to Cy5.5 with and without OxaPt(IV), form-
ing M(Cy5.5/Alexa488 siRNA) and M(Cy5.5/Alexa488 siR-
NA/OxaPt(IV)), respectively. After removal of the media, the
cells were then washed twice with cold phosphate buffered
saline (PBS, pH 7.4, 0.01 M) and fixed with 4% formaldehyde
(Sigma-Aldrich, St. Louis, USA). To label the cell nuclei, sam-
ples were incubated with 1 µL of DAPI (1 mg/mL; Sigma-
Aldrich, St. Louis, USA) for 15 min in PBS and were subse-
quently rinsed extensively with PBS. Slides were mounted on
coverslips and imaged, using a confocal laser scanning mi-
croscopy system (Olympus FV1000, Olympus Corporation,
Bridgeport, CT).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cellular Apoptosis Assays. MCF-7 cells were cultured in 6-
well plates (5x105 cells/well) and incubated with various
treatments at a fixed Pt concentration (20 M). After 36 h,
apoptotic cells were detected by flow cytometry, using the
Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San
Jose, CA); the results were analyzed using FlowJo software
Version 7.6.2.
For flow cytometry measurements, MCF-7 and OVAR4
cells were similarly cultured in 6-well plates (4105 cells/well)
prior to incubation with M(Cy5.5/Alexa488 siRNA) or
M(Cy5.5/Alexa488 siRNA/OxaPt(IV)) either for 30 min or for 4
h. The cells were washed twice with cold PBS, lysed with tryp-
sin–EDTA solution, collected by centrifugation (1500 rpm, 5
min), and, finally, analyzed for fluorescence content, using a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
Cells with fluorescence above the threshold intensity for un-
treated cells (i.e. blank) were quantified and compared. The
percentage of these cells in the total population was consid-
ered as a measure of uptake efficiency. Data were analyzed
using FlowJo software Version 7.6.2 (Tree Star, Ashland, OR).
RESULTS AND DISCUSSION
Monitoring the Reaction of siRNA with Various Platinum-
based Species. OxaPt(II) and CisPt(II) bind preferentially to
guanine and adenine bases in both DNA and RNA.25-27, 33-35 To
compare the relative reactivity of OxaPt(IV) and CisPt(IV) to
their platinum(II) counterparts, we first incubated each com-
pound for different time intervals with 5’-GMP (i.e. 30 min, 1
h, 3 h, 6 h or 12 h) and in conditions that would mimic a typi-
cal in vitro RNAi experiment (i.e. co-incubation of the two
species at 37 oC). Formation of Pt-(5’-GMP)2 adducts was
readily detectable by ESI-MS when 5’-GMP was incubated
with OxaPt(II) but not with OxaPt(IV) (Figures 1A, 1B and S7).
These findings were further confirmed by MALDI/TOF-MS
measurements of the same suspensions, showing that Pt-(5’-
GMP)2 adducts were detected within 1 h in solutions contain-
ing OxaPt(II) or CisPt(II) but not OxaPt(IV) or CisPt(IV) (Figures
S8 and S9). To further quantify differences in the kinetics of
Pt-(5’-GMP)2 adduct formation, 1H NMR spectra were ob-
tained for 5’-GMP solutions at various time intervals after the
addition of different platinum(II) or platinum(IV) species and
under identical conditions. The chemical shifts of the H8 pro-
ton on the guanosine base (8.5 ppm in the (diamino-1,2-
cyclohexane)-Pt(5’-GMP)2, DACHPt(5’-GMP)2) adduct vs. 8.09
ppm in unreacted 5’-GMP) were monitored at several time
points after co-incubation of 5’-GMP (10 mM) with either
OxaPt(II) or OxaPt(IV) (5 mM) (Figure S10). By integrating the
ratio of the two peaks at each time point, the relative
amounts of Pt-bound 5’-GMP to free 5’-GMP were deter-
mined in solutions containing OxaPt(II) (green) or OxaPt(IV)
(red) and plotted as a function of time (Figure 1C). The results
showed that approximately 10% of the 5’-GMP pool consisted
of DACHPt(5’-GMP)2 adducts after 30 min of incubation with
Quantification of Intracellular Uptake of Platinum Spe-
cies. MCF-7 cells were seeded in 12-well plates (1x105
cells/well) and incubated overnight in DMEM containing 10%
FBS (1 mL). Various platinum-containing agents (OxaPt(II),
OxaPt(II) + M(c-siRNA), OxaPt(II) + M(BCL-2), M(OxaPt(IV)/c-
siRNA), or M(OxaPt(IV)/BCL-2)) were then added to each well
(100 µL aliquots and to final Pt concentration of 10 µM). For
siRNA-containing treatments, the final siRNA concentration in
each well was 100 nM. After either 1 or 4 h of incubation,
media were removed and the cells were thoroughly washed
x5 with cold PBS. The cells were then lysed with nitric acid
prior to ICP-MS measurements. For quantification of Pt-DNA
adducts, cells were similarly incubated with each platinum-
containing agent but for 24 h. The cells were then washed
and the DNA was extracted using a Genome DNA extraction
kit, following the manufacturer’s instructions (Thermo Scien-
tific, USA). DNA concentrations were determined by
NanoDropTM; and, the quantity of DNA-bound Pt was deter-
mined by ICP-MS.
In vitro Cytotoxicity. MCF-7, OVCAR4, A2780, and
A2780DDP cells were seeded in 96-well plates (5103
cells/well). DMEM (MCF-7; 100 L) or RPMI-1640 (OVCAR4,
4
ACS Paragon Plus Environment

Page 5 of 21
Journal of the American Chemical Society
OxaPt(II), that the number of adducts that were formed in- dependent platination of ds-siRNA, which has been shown to
1
2
3
4
5
6
7
8
creased over the first 6 h, reaching 40% of the total pool, and
that they continued to increase over time. Similar early for-
mation and time dependent increases in the numbers of
Pt(NH3)2(5’-GMP)2 adducts were seen when 5’-GMP was in-
cubated with CisPt(II) but not with CisPt(IV) (Figure S11). Tak-
en together, these model reactions of 5’-GMP with various
platinum species support a rationale for combining RNAi ap-
proaches with platinum(IV)- as opposed to platinum(II)-based
anticancer agents, which would otherwise react with similar
nucleosides in RNA macromolecules and which would lead to
inactivation of the siRNA or miRNA species.
convert it to ss-siRNA, via monitoring of the hyperchromic
(i.e. ΔA-value at λ = 260 nm) and bathochromic shifts (from a
maximum at λ ca. 258 nm to a maximum at λ ca. 260 nm) that
follow nucleic acid duplex melting.37b By first varying the ini-
tial concentrations of ds-siRNA (from 0.5-2 µM) for a fixed
and excess (> 10-fold) amount of CisPt(II) (20 µM), we verified
that ss-siRNA was generated via a pseudo-first order reaction
and that the apparent second-order rate constant (k2,app
)
9
could be derived from the linear relationship between the
observed rate constant (kobs) and the initial concentration of
the platinum species (see Figure S15 and Table S1):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We next sought to ascertain the kinetics of Pt-siRNA ad-
duct formation by using the anti-sense strand of BCL-2 siRNA.
This single-stranded (ss-)siRNA (50 M) was incubated with
different platinum(II)- and platinum(IV)-based anticancer
agents (500 M), which were present in a 10-fold excess in
order to account for the relative concentrations of RNA to
platinum species that are typically employed in RNAi and cy-
totoxicity experiments, respectively.12-19 MALDI/TOF-MS spec-
tra were taken at various time points after their co-incubation
[ss-siRNA]t = [ds-siRNA]o * (1- e(-kobs*t)
)
k2,app= kobs/[platinum species]o,
UV-Vis spectroscopy was similarly used to observe the
reactions of other platinum species with ds-siRNA, verifying
that the k2,app for OxaPt(II), CisPt(IV), and OxaPt(IV) were ap-
proximately 40%, 25% and 2% that of CisPt(II) (Table 1).
Moreover, when ds-siRNA was incubated with CisPt(II) at
concentrations that are typically employed for in vitro cyto-
toxicity experiments (i.e. > 10 µM), approximately 50% of the
ds-siRNA transcripts were converted to ss-siRNA by 1-3 h.
Notably, other investigators have previously measured higher
reaction rate constants for CisPt(II) with ds-RNA, whereby
similar results would be expected in less than 1 h of co-
incubation (Table 1 and 33-35). Given the decreased reactivity
of OxaPt(II) when compared to CisPt(II), analogous levels of
ds-siRNA deactivation (from conversion to ss-siRNA via plati-
nation under identical reaction conditions) could be expected
between 3-6 h of co-incubation. Notably, as both CisPt(IV)
and OxaPt(IV) were even less active than OxaPt(II), neither
significant ds-siRNA platination nor deactivation were ex-
pected to proceed in their presence and under identical in
vitro reaction conditions.
o
at 37 C. These experiments demonstrated that Pt-siRNA ad-
ducts could be detected as early as 1 h after the incubation of
ss-siRNA with OxaPt(II) (Figures 1D and S12). Three major
peaks were detected and attributed to unreacted BCL-2 ss-
siRNA (m/z=7227.888), BCL-2 ss-siRNA with one DACHPt2+
adduct (m/z=7535.592), and BCL-2 ss-siRNA with two
DACHPt2+ adducts (m/z=7841.982). Note, the molecular
weight of DACHPt was 309.08 and the Δm/z of ss-siRNA upon
formation of the siRNA-DACHPt2+ mono adduct was 307.704;
ss-siRNA lost two H+ after binding with OxaPt(II) and added
another H+, keeping it at the +1 charge state. The unmodified
BCL-2 ss-siRNA had a molecular weight of 7224.401 and dis-
played a +1 charged mass peak at m/z=7226.122 (Figure 1E,
inset). The margin of error for the MALDI/TOF-MS was +/-
0.1%; thus, for free ss-siRNA a variation of 0.722 Dalton units
was within the range of precision for the MALDI/TOF-MS in-
strument. Compared to OxaPt(II), CisPt(II) is known to under-
go more facile aquation of its planar ligands, decreasing its
relative stability and increasing its reactivity towards ss-siRNA.
When BCL-2 ss-siRNA was incubated with CisPt(II), numerous
Pt-siRNA adducts were detected as early as 30 min after
CisPt(II) addition (Figure S13). Notably, no Pt-siRNA adducts
were detected in solutions of BCL-2 ss-siRNA containing ei-
ther OxaPt(IV) or CisPt(IV) and even after 24 h of co-
incubation under similar physiological conditions (pH 7.4;
Figure 1E, S12 and S13).
To determine the exact sites of platination on a nucleic
acid transcript, we next conducted MALDI/TOF-MS experi-
ments using a model ss-oligonucleotide of DNA that con-
tained a single GG site, verifying the formation of mono- and
bis-Pt-DNA adducts in the presence of OxaPt(II) but not
OxaPt(IV) (Figure S16). MALDI/TOF-MS was then performed
after enzymatic cleavage of the antisense strand of BCL-2
siRNA by RNase A, either before (Figure 1G) or after incuba-
tion with OxaPt(II). These experiments demonstrated two
distinct sites on the ss-siRNA transcript that were platinated
with either one or two DACHPt moieties, including the 5’-
AGGU-3’ segment in the seed portion and the 5’-AGU-3’ site
in the non-seed portion of the anti-sense strand (Figures 1H
and S17).
GFAAS measurements of ds-siRNA that had been isolated
after co-incubation with various platinum species were also
conducted to quantify the amounts of siRNA-bound Pt over
time. These results indicate that a total of ~4 Pt atoms bound
to ds-siRNA within 3-6 h after incubation with OxaPt(II) (Fig-
ure S14), which corresponded to platination of ~40% of the G
nucleotides of the ds-siRNA (Figure 1F). Notably, no Pt was
detected by GFAAS even in ds-siRNA solutions that had been
incubated with OxaPt(IV) for up to 12 h. These results were
further corroborated by UV-Vis measurements of the time-
Platination of the non-seed portion of the anti-sense
strand was expected to affect RNAi efficacy.36-37 To test this
hypothesis, we studied changes in the relative silencing activi-
ty of BCL-2 ds-siRNA that was pre-incubated with different
platinum(II) and platinum(IV) agents prior to cellular expo-
sure. Using a fixed concentration of siRNA (7.5 nM), different
concentrations of platinum-based species (either 0.75 or 7.5
µM), and various incubation times (ranging from 30 min to 24
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 21
h), we effectively varied the numbers of Pt-siRNA adducts micelle, the siRNA was protected from interacting with the
1
2
3
4
5
6
7
8
that were present on the siRNA transcript prior to transfec-
tion. The siRNA was subsequently diluted (to a final concen-
tration of 1.5 nM) and combined with the commercially avail-
able transfection reagent Lipofectamine® (RNAiMAX) for de-
livery to both MCF-7 and OVCAR4 cells. RT-qPCR of BCL-2
mRNA isolated from these cells at 48 h after transfection
showed ineffective BCL-2 silencing (i.e. greater than 30% of
baseline mRNA expression) by siRNA (1.5 nM) that had been
pre-incubated with a 1000-fold excess of OxaPt(II) for > 3 h
(Figure 2 and S18). CisPt(II) was more reactive than OxaPt(II)
and ineffective BCL-2 mRNA suppression was seen even after
30 min of co-incubation of BCL-2 siRNA with CisPt(II) under
identical conditions. The activity of the siRNA was unper-
turbed by > 24 h of exposure to similar concentrations of ei-
ther OxaPt(IV) or CisPt(IV). Note, both the concentrations and
the relative ratios of siRNA to platinum-based species that
were used for these studies were typical of those employed
for in vitro RNAi and cytotoxicity experiments, respectively.12-
platinum species both by physical separation in different lay-
ers of the nanocomplex as well as by utilization of an unreac-
tive platinum(IV) analog (e.g. OxaPt(IV)). Each of these micel-
lar constructs was generated to examine its relative ability to
protect siRNA from platination, thereby preserving the effec-
tiveness of RNAi. When taken up into cancer cells, the Pt in
M(Oxa(IV)) rapidly reacted with intracellular reducing agents,
including glutathione and ascorbic acid, liberating free
OxaPt(II) that was capable of binding nuclear DNA (Scheme
1C).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
M(P) had a mean diameter of 70.8 ± 0.5 nm, as deter-
mined by DLS, and a zeta potential of +46.1 ± 4.2 mV (Figure
S19 and Table S2). To generate M(OxaPt(IV)), OxaPt(IV) was
allowed to react with P at an initial molar ratio of 0.6:1 Pt to
NH2 groups (on the PLL block). Once coupled to PLL, the
OxaPt(IV) conjugate became less water soluble, driving its
segregation within the PCL core and leaving unreacted PLL
chains in the outer layer of M(OxaPt(IV)). This assertion is
supported by experimental evidence that demonstrated a
decrease in the CMC from 37.2 to 24 µg/mL (Figure S20), an
increase in the mean particle diameter to 92.6 ± 2.6 nm (DLS),
and a reduction in the zeta potential to +34.8 ± 1.9 for
M(OxaPt(IV)) as compared to M(P) (Table S2). Overall,
M(OxaPt(IV)) was comprised of 10 wt% Pt (by GFAAS), corre-
sponding to a final OxaPt(IV) content of 20.4 wt%. This degree
of loading was expected to yield 72% unreacted amino groups
on the PLL corona of M(OxaPt(IV)), which were available for
additional complexation of siRNA through electrostatic inter-
actions.
19
Taken together, these experiments support a generalizable
phenomenon whereby siRNA rapidly reacts with platinum(II)
species that are present at > 1 µM concentrations, leading to
irreversible Pt-siRNA adduct formation and ineffective RNAi
within as little as 1-3 h of co-incubation. These results further
highlight the value of combining RNAi approaches with chem-
ically-inert platinum(IV) prodrugs to circumvent these limita-
tions.
Nanoparticle Characterization. There are many examples
of utilizing nanoparticles for the delivery of siRNA along with
different anticancer agents.50-53 Whereas several studies have
attempted nanoparticle incorporation of both siRNA and
CisPt(II),54-56 the results presented here reveal the advantages
of utilizing a platinum(IV) agent to prevent siRNA platination
and premature inactivation. To date, however, there have
been only a few examples that utilize this latter approach.57-59
Further investigations were, therefore, necessary to identify
the optimal strategy by which to combine RNAi and plati-
num(IV) species to maximize their individual activities and to
harness their synergy. Accordingly, we devised a transfection
reagent capable of intracellular delivery of both platinum(IV)
prodrugs and siRNA. For this work, we chose mPEG-b-PCL-b-
PLL (P), which self-assembled into biodegradable micelles
with a hydrophobic PCL core and a corona consisting of hy-
drophilic mPEG and positively-charged PLL segments (M(P),
where “M” stands for micelles; Scheme 1B). OxaPt(IV) was
coupled to the PLL polymer through formation of an amide
bond between the carboxyl group of its auxiliary ligand and
the free amino group of lysine, forming micelles that con-
tained OxaPt(IV) (M(OxaPt(IV)). Similarly, siRNA was com-
plexed with unreacted PLL chains through electrostatic inter-
actions, forming micelles that contained BCL-2 (M(BCL-2)) or
c-siRNA (M(c-siRNA)) (Scheme S3). Both OxaPt(IV) and siRNA
species were also combined in a single micellar construct
(M(OxaPt(IV)/BCL-2) or M(OxaPt(IV)/c-siRNA), respectively),
consisting of a hydrophilic mPEG corona, a middle layer com-
prised of PLL/siRNA, and a hydrophobic core of PCL and PLL-
OxaPt(IV) conjugates. When co-incorporated within a single
To prove that M(OxaPt(IV)) was able to complex with
BCL-2 siRNA, agarose gel electrophoresis experiments were
conducted on siRNA after co-incubation with either M(P) or
M(OxaPt(IV)) (Figure S21A and S21B). The results showed
that the mobility of the siRNA was completely inhibited when
electrostatically complexed with M(P) above a ratio of nitro-
gen atoms in the PLL block of the polymer to phosphate units
of the siRNA (N/P) that was greater than 2. Additionally, the
mobility of the siRNA was reduced when complexed with
M(OxaPt(IV)) above an N/P ratio of 4. By using Alexa488-
labeled BCL-2 siRNA, the loading efficiency was determined to
be approximately 99% and the final micelle composition con-
sisted of 30% siRNA by weight (Figure S21C-E). Representa-
tive micelles, containing both BCL-2 siRNA and OxaPt(IV)
formed at an N/P ratio of 8 (i.e. M(OxaPt(IV)/BCL-2)), were
spherical with a mean particle diameter of 78.9 ± 10.4 nm and
hydrodynamic size of 104.9 ± 2.1 nm, as determined by TEM
and DLS, respectively (Figure 3A and 3B). M(OxaPt(IV)/BCL-2)
had a reduced zeta potential of +26.6 ± 1.5 mV as compared
to either M(P) or M(OxaPt(IV)) (Table S2), further supporting
the shielding of free amines on the PLL corona of the micelles
by siRNA. BCL-2 siRNA was also directly complexed to M(P) to
generate M(BCL-2) with a mean particle diameter of 55.4 ±
0.7 nm (DLS) and a zeta potential of +35.5 ± 0.6 mV (Table
S2). M(OxaPt(IV)/BCL-2) rapidly released OxaPt(II) under high
reducing conditions in situ (Figure 3C), modeling the intracel-
lular environment of tumor cells. However, it exhibited a
6
ACS Paragon Plus Environment

Page 7 of 21
Journal of the American Chemical Society
slower and pH-dependent release of Alexa488-labeled siRNA
individual and synergistic activities of these otherwise reac-
tive species.
Optimum Strategy for Combining RNAi and Platinum-
1
2
3
4
5
6
7
8
(Figure 3D). Together, these data support a mechanism of
rapid release of free OxaPt(II) followed by controlled release
of siRNA after the intracellular uptake of M(OxaPt(IV)/BCL-2).
based Anticancer Agents. After having validated effective
intracellular delivery with our transfection reagent, we next
compared various strategies for combining BCL-2 siRNA and
platinum-based anticancer agents in order to achieve maxi-
mal synergistic activity. For all siRNA-containing groups, the
concentration of siRNA (100 nM) was kept constant and was
transfected using various polymeric micelles formed at an
N/P ratio of 8. These micelles had no inherent effects on cel-
lular viability in the absence of the platinum-based species
(Figure S25). We next conducted a series of experiments in
which MCF-7 cells were treated with increasing concentra-
tions of different platinum formulations (i.e. free OxaPt(II),
free OxaPt(IV), or M(OxaPt(IV))), either individually or in
combination with M(BCL-2) or M(c-siRNA). Cells were also
treated with siRNA and platinum-based species that were co-
delivered via a single micellar construct (i.e. M(OxaPt(IV)/BCL-
2) or M(OxaPt(IV)/c-siRNA)). In all cases, BCL-2 mRNA levels
were measured by RT-qPCR, intracellular Pt concentrations
and the numbers of Pt-DNA adducts were quantified by ICP-
MS, and cellular viability was determined by the colorimetric
MTT assay after each treatment.
Cellular Uptake of Micelles. In order to determine the op-
timal conditions for siRNA delivery, we next used different
concentrations of Luciferase GL3 and BCL-2 siRNA to achieve
mRNA knockdown of their respective targets in a LUC+ MCF-7
cell line. For both M(P) and M(OxaPt(IV)), an N/P ratio of 8
was employed for siRNA complexation. For effective RNAi of
luciferase expression, 100 nM of Luciferase GL3 siRNA had to
be utilized with either micellar construct (Figure S22). Alt-
hough this was a higher concentration value for siRNA than
was required with the commercially available RNAiMAX trans-
fection kit (typically 1-2 nM), it was within the range of siRNA
concentrations that have been utilized with other polymer-
based transfection reagents.60-62 To better study the kinetics
and the mechanisms of micelle uptake and in vitro siRNA re-
lease, a Cy5.5-conjugated mPEG-b-PCL-b-PLL polymer was
synthesized (Scheme S4). Alexa488-labeled siRNA was then
complexed to micelles formed from this Cy5.5-conjugated
polymer, generating M(Cy5.5/Alexa488 siRNA). OxaPt(IV) was
also chemically bound to the Cy5.5-conjugated polymer to
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
yield
dual
fluorophore-labeled
micelles
(i.e.
M(Cy5.5/Alexa488 siRNA/OxaPt(IV))), which enabled simul-
taneous but independent in vitro tracking of both polymer
and siRNA species (Scheme S5). MCF-7 cells were subse-
quently incubated with M(Cy5.5/Alexa488 siRNA/OxaPt(IV))
either for 30 min or for 4 h and were visualized by confocal
laser scanning microscopy (Figure 3E). After 30 min of incuba-
tion, there was a co-localization of Cy5.5 (red) and Alexa488
fluorescence (green) in a punctate distribution within cells
(depicted in orange within the magnified overlay image), con-
sistent with the intracellular localization of intact micelles
within endosomes. After 4 h, however, Alexa488 emission
extended beyond the areas of corresponding Cy5.5 fluores-
cence and was apparent throughout the cytoplasm. Flow
cytometry was used to quantify the relative intracellular lev-
els of Cy5.5-labeled polymer and Alexa488-labeled siRNA
both in MCF-7 (Figure 3F and 3G) and in OVCAR4 cells over
time (Figure S23). Notably, cells that were treated with either
M(Cy5.5/Alexa488 siRNA) or M(Cy5.5/Alexa488 siR-
Decreased amounts of intracellular Pt were detected af-
ter both 1 and 4 h of exposure to free OxaPt(IV) as compared
to free OxaPt(II) (Figure 4A and Table S3). Intracellular Pt
levels were markedly enhanced (by ~3-8 fold) when treated
with M(OxaPt(IV)) as opposed to various free drug formula-
tions of either OxaPt(II) or OxaPt(IV) that were administered
at identical Pt concentrations. The intracellular levels of Pt
also increased 2-fold between 1 and 4 h after administration
of M(OxaPt(IV)), which, again, supported a time-dependent
mechanism of micelle uptake via active endocytosis.
Co-administration of M(OxaPt(IV)) and a separate micel-
lar construct containing only siRNA (e.g. M(BCL-2)), however,
resulted in a relative 2-fold decrease in the intracellular con-
tent of Pt as compared to cells that were treated with either
M(OxaPt(IV)) alone or M(OxaPt(IV)/BCL-2) at equivalent Pt
concentrations. These results indicate that micelle uptake via
endocytosis was not only time-dependent but also saturable.
These observations were further supported by analogous
measurements of the numbers of intracellular Pt-DNA ad-
ducts that could be detected after 24 h of treatment (Figure
4B and Table S3). A markedly enhanced number of Pt-DNA
adducts (~10 fold increase) were measured after treatment
with M(OxaPt(IV)) as opposed to either free OxaPt(II) or free
OxaPt(IV) administered at equivalent Pt concentrations and
for similar incubation periods. Co-administration of two sepa-
rate micellar constructs of M(OxaPt(IV)) and M(BCL-2) result-
ed in the generation of half the numbers of Pt-DNA adducts
as compared to treatment with M(OxaPt(IV)) alone or with
M(OxaPt(IV)/BCL-2), which, again, supported a saturable limit
for micelle uptake by MCF-7 cells. To promote the highest
intracellular levels of Pt and the maximal numbers of Pt-DNA
adducts, combined delivery of OxaPt(IV) and BCL-2 siRNA via
a single micellar construct was found to be optimal.
NA/OxaPt(IV)) exhibited
a 10-fold increase in Cy5.5-
conjugated polymer and 40-fold increase in Alexa488-labeled
siRNA after 4 h (Figure 3F), supporting active intracellular
uptake of each species. Utilization of different small molecule
inhibitors implicated multiple cellular uptake pathways but
with a predominance of clathrin-dependent endocytosis (Fig-
ure S24). Together these results support a mechanism of ac-
tive endocytosis of Cy5.5-conjugated micelles, rapid conver-
sion of M(OxaPt(IV)) to free OxaPt(II)), and a slower cyto-
plasmic redistribution of Alexa488-labeled siRNA, which was
consistent with controlled release and endosomal escape.
This relative temporal and spatial separation between
OxaPt(II) and siRNA release was expected to limit the for-
mation of intracellular Pt-siRNA adducts and to promote the
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 21
Thereafter, we examined the relative levels of BCL-2 cytometry to quantify the populations of MCF-7 cells that
1
2
3
4
5
6
7
8
mRNA within cells after treatment with each experimental
group (Figure 4C and S26). Whereas free OxaPt(II) decreased
BCL-2 mRNA levels to 65% that of untreated (control) cells,
consistent with its known pro-apoptotic activity,63 effective
mRNA suppression was only achieved with BCL-2 siRNA
treatment (i.e. suppression to less than 30% of baseline
mRNA levels). Co-administration of free OxaPt(II), free
OxaPt(IV), or M(OxaPt(IV)) together with M(BCL-2) did not
further inhibit mRNA levels beyond that which was obtained
with M(BCL-2) treatment alone. These results indicate that
when siRNA was electrostatically complexed with polymeric
micelles prior to platinum(II) exposure, it was effectively pro-
tected from platination. Notably, co-incorporation of BCL-2
siRNA and OxaPt(IV) in the same micellar construct (i.e.
M(OxaPt(IV)/BCL-2)) did achieve a statistically significant im-
provement in BCL-2 mRNA suppression (to less than 10% of
baseline levels). As such, delivery of BCL-2 siRNA and
OxaPt(IV) with a single micelle not only ensured siRNA tran-
script stability but also promoted maximal suppression of
mRNA levels beyond that which was achievable with either
agent only or when they were combined by alternative
means.
were apoptotic after 36 h of exposure to each experimental
group (Figure 4F and S28). When compared to treatment with
free OxaPt(II) (at equivalent Pt concentrations and for identi-
cal durations of time), treatment with M(OxaPt(IV)/BCL-2)
resulted in a 4-fold increase in the total number of cells that
had underwent apoptosis. Notably, this level of pro-apoptotic
activity was twice that of M(OxaPt(IV)) alone and 1.5-fold
greater than that of the combination of M(OxaPt(IV)) and
M(BCL-2). These experiments confirmed that the delivery of
BCL-2 siRNA and OxaPt(IV) via a single micellar construct re-
sulted in the highest numbers of apoptotic events as com-
pared to all other strategies for combining BCL-2 siRNA with
different platinum-based species.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSIONS
RNAi approaches are extensively used to study molecular
responses that mediate acquired cellular resistance to plati-
num-containing regimens. The success of these endeavors
relies on the preserved activity of the exogenous RNAi tran-
script when combined with platinum-based anticancer
agents. Here, we demonstrated rapid and extensive Pt-siRNA
adduct formation when siRNA was incubated with plati-
num(II)-based agents at concentrations and for time intervals
that are typically used for in vitro RNAi experiments. Notably,
siRNA was protected from inactivation by combining it with a
platinum(IV) analog, but this combination resulted in reduced
cytotoxicity as compared to using the active platinum(II)
agent alone. In order to promote effective RNAi and to max-
imize the individual and synergistic activities of siRNA and
platinum-based anticancer agents, we found that both spe-
cies had to be co-incorporated within a single micelle delivery
vehicle. A generalized adoption of this strategy may lead to
the development of novel therapeutics and may be used to
identify drug targets that would otherwise be missed in con-
ventional screens that typically combine RNAi with plati-
num(II)-based anticancer agents.
Ultimately, the goals of combining RNAi with platinum-
based therapies are to explore changes in treatment respons-
es and biological activities that are mediated by the suppres-
sion of specific gene products. We, therefore, interrogated
changes in the in vitro viability of MCF-7 cells that were treat-
ed with various experimental combinations of platinum-
based species and BCL-2 siRNA. We measured changes in
cellular viability as a function of increasing quantities of free
OxaPt(II), free OxaPt(IV), or M(OxaPt(IV)), either as single
treatments or in combinations with either M(BCL-2) or M(c-
siRNA) (Figure 4D and S27). Cells were also treated with
M(OxaPt(IV)/BCL-2) or M(OxaPt(IV)/c-siRNA) at equivalent Pt
concentrations for comparative purposes. Synergistic treat-
ment responses from any combination of platinum-based
species and siRNA were defined as a decrease in the IC50 val-
ue as compared to cells that were exposed to the same plati-
num-based therapy alone. Although the combination of free
OxaPt(IV) with M(BCL-2) did show a synergistic treatment
effect, which was not obvious in the combination of free
OxaPt(II) and M(BCL-2), free OxaPt(IV)-containing treatments
were approximately 10-fold less potent than free OxaPt(II) at
inducing cellular toxicity (Figure 4E). By comparison,
M(OxaPt(IV)) was 100x more potent than free OxaPt(IV) and
displayed 10-fold greater cytotoxicity than free OxaPt(II).
Whereas the combination of M(BCL-2) and M(OxaPt(IV)) did
result in a statistically significant improvement in anti-
proliferative activity, M(OxaPt(IV)/BCL-2) displayed the high-
est in vitro potency, yielding an IC50 value of 60 nM as com-
pared to 23 +/-0.25 µM for free OxaPt(II) alone. This dramati-
cally enhanced efficacy for M(OxaPt(IV)/BCL-2) was further
corroborated by testing each of the various treatment combi-
nations on additional cell lines (i.e. OVCAR4, A2780 and
A2780DDP; Table 2). To investigate the biological mecha-
nisms underlying this improved activity, we performed flow
ASSOCIATED CONTENT
Supporting Information
"Supporting Information.PDF” is available free of charge via the
Internet at http://pubs.acs.org and contains the Supplemental
Materials and Methods, Schemes S1 thru S5, Figures S1 thru S28,
and Tables S1 thru S3.
AUTHOR INFORMATION
Corresponding Author
* P. Peter Ghoroghchian, M.D., Ph.D. (ppg@mit.edu).
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version.
Funding Sources
This research was funded by the Charles W. and Jennifer C. John-
son Clinical Investigator Fund from the Koch Institute at MIT as
8
ACS Paragon Plus Environment

Page 9 of 21
Journal of the American Chemical Society
well as by a 2015 Amgen Research Grant from the Foundation for
Women’s Cancer.
(17) Kwok, J. M. M.; Peck, B.; Monteiro, L. J.; Schwenen, H.
D. C.; Millour, J.; Coombes, R. C.; Myatt, S. S.; Lam, E. W. F. Mol
Cancer Res 2010, 8, 24.
1
2
3
4
5
6
7
8
Notes
(18) McAuliffe, S. M.; Morgan, S. L.; Wyant, G. A.; Tran, L. T.;
Muto, K. W.; Chen, Y. S.; Chin, K. T.; Partridge, J. C.; Poole, B. B.;
Cheng, K.-H.; Daggett, J.; Cullen, K.; Kantoff, E.; Hasselbatt, K.;
Berkowitz, J.; Muto, M. G.; Berkowitz, R. S.; Aster, J. C.;
Matulonis, U. A.; Dinulescu, D. M. Proc Natl Acad Sci U S A 2012,
109, E2939.
(19) Moreno-Smith, M.; Halder, J. B.; Meltzer, P. S.; Gonda,
T. A.; Mangala, L. S.; Rupaimoole, R.; Lu, C.; Nagaraja, A. S.;
Gharpure, K. M.; Kang, Y.; Rodriguez-Aguayo, C.; Vivas-Mejia, P.
E.; Zand, B.; Schmandt, R.; Wang, H.; Langley, R. R.; Jennings, N.
B.; Ivan, C.; Coffin, J. E.; Armaiz, G. N.; Bottsford-Miller, J.; Kim, S.
B.; Halleck, M. S.; Hendrix, M. J. C.; Bornman, W.; Bar-Eli, M.; Lee,
J.-S.; Siddik, Z. H.; Lopez-Berestein, G.; Sood, A. K. J Clin Invest
2013, 123, 2119.
(20) Xue, W.; Dahlman, J. E.; Tammela, T.; Khan, O. F.; Sood,
S.; Dave, A.; Cai, W.; Chirino, L. M.; Yang, G. R.; Bronson, R.;
Crowley, D. G.; Sahay, G.; Schroeder, A.; Langer, R.; Anderson, D.
G.; Jacks, T. Proc Natl Acad Sci U S A 2014, 111, E3553.
(21) Reyes-González, J. M.; Armaiz-Peña, G. N.; Mangala, L.
S.; Valiyeva, F.; Ivan, C.; Pradeep, S.; Echevarría-Vargas, I. M.;
Rivera-Reyes, A.; Sood, A. K.; Vivas-Mejía, P. E. Mol Cancer Ther
2015, 14, 2260.
(22) Nascimento, A. V.; Gattacceca, F.; Singh, A.; Bousbaa,
H.; Ferreira, D.; Sarmento, B.; Amiji, M. M. Nanomedicine (Lond)
2016, 11, 767.
(23) Rana, T. M. Nat Rev Mol Cell Biol 2007, 8, 23.
(24) Dykxhoorn, D. M.; Novina, C. D.; Sharp, P. A. Nat Rev
Mol Cell Biol 2003, 4, 457.
(25) Jamieson, E. R.; Lippard, S. J. Chem Rev 1999, 99, 2467.
(26) Reedijk, J. Chem Rev 1999, 99, 2499.
(27) Baik, M.-H.; Friesner, R. A.; Lippard, S. J. J Am Chem Soc
2003, 125, 14082.
The Authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to acknowledge Professor Stephen J. Lip-
pard for helpful discussions as well as Dr. Fang Wang for assis-
tance with NMR spectroscopy. PPG acknowledges support from
the Kathryn Fox Samway Foundation. Research support was also
provided from the Flow Cytometry, Microscopy, and Nano-
materials Core Facilities in the Swanson Biotechnology Center of
the Koch Institute at MIT.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ABBREVIATIONS
BCL-2, B-cell lymphoma 2; CisPt(II), cisplatin; CisPt(IV), cispla-
tin(IV); mRNA, messenger ribonucleic acid; miRNA, micro-
ribonucleic acid; OxaPt(II), oxaliplatin; OxaPt(IV), oxaliplatin(IV);
Pt-RNA, platinum-ribonucleic acid adduct; siRNA, small interfer-
ing ribonucleic acid.
REFERENCES
(1)
Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V.
H. Nature 1969, 222, 385.
(2)
Alderden, R. A.; Hall, M. D.; Hambley, T. W. J Chem
Educ 2006, 83, 728.
(3)
Fennell, D. A.; Summers, Y.; Cadranel, J.; Benepal, T.;
Christoph, D. C.; Lal, R.; Das, M.; Maxwell, F.; Visseren-Grul, C.;
Ferry, D. Cancer Treat Rev 2016, 44, 42.
(4)
Ho, G. Y.; Woodward, N.; Coward, J. I. G. Crit Rev Oncol
Hematol 2016, 102, 37.
(5)
(6)
Agarwal, R.; Kaye, S. B. Nat Rev Cancer 2003, 3, 502.
Stordal, B.; Pavlakis, N.; Davey, R. Cancer Treat Rev
2007, 33, 688.
(28) Fichtinger-Schepman, A. M.; van der Veer, J. L.; den
Hartog, J. H.; Lohman, P. H.; Reedijk, J. Biochemistry 1985, 24,
707.
(29) Sorenson, C. M.; Barry, M. A.; Eastman, A. J Natl Cancer
Inst 1990, 82, 749.
(30) Gonzalez, V. M.; Fuertes, M. A.; Alonso, C.; Perez, J. M.
Mol Pharmacol 2001, 59, 657.
(31) Gatti, L.; Supino, R.; Perego, P.; Pavesi, R.; Caserini, C.;
Carenini, N.; Righetti, S. C.; Zuco, V.; Zunino, F. Cell Death Differ
2002, 9, 1352.
(7)
Shen, D.-W.; Pouliot, L. M.; Hall, M. D.; Gottesman, M.
M. Pharmacol Rev 2012, 64, 706.
(8)
Chien, J.; Kuang, R.; Landen, C.; Shridhar, V. Front Oncol
2013, 3, 251.
(9)
Davis, A.; Tinker, A. V.; Friedlander, M. Gynecol Oncol
2014, 133, 624.
(10) O'Grady, S.; Finn, S. P.; Cuffe, S.; Richard, D. J.; O'Byrne,
K. J.; Barr, M. P. Cancer Treat Rev 2014, 40, 1161.
(11) Konstantinopoulos, P. A.; Ceccaldi, R.; Shapiro, G. I.;
D'Andrea, A. D. Cancer Discov 2015, 5, 1137.
(12) Bartz, S. R.; Zhang, Z.; Burchard, J.; Imakura, M.;
Martin, M.; Palmieri, A.; Needham, R.; Guo, J.; Gordon, M.;
Chung, N.; Warrener, P.; Jackson, A. L.; Carleton, M.; Oatley, M.;
Locco, L.; Santini, F.; Smith, T.; Kunapuli, P.; Ferrer, M.; Strulovici,
B.; Friend, S. H.; Linsley, P. S. Mol Cell Biol 2006, 26, 9377.
(13) Arora, S.; Bisanz, K. M.; Peralta, L. A.; Basu, G. D.;
Choudhary, A.; Tibes, R.; Azorsa, D. O. Gynecol Oncol 2010, 118,
220.
(32) Qin, L. F.; Ng, I. O. L. Cancer Lett 2002, 175, 27.
(33)
a) Hostetter, A. A.; Chapman, E. G.; DeRose, V. J. J Am
Chem Soc 2009, 131, 9250; b) Osborn MF.; White JD.; Haley MM.;
DeRose VJ. ACS Chem. Biol 2014, 9 , 2404; c) Alberti E.;
Zampakou M.; Donghi D. J Inorg Biochem 2016,163,278.
(34) Chapman, E. G.; DeRose, V. J. J Am Chem Soc 2010, 132,
1946.
(35)
Hostetter, A. A.; Osborn, M. F.; DeRose, V. J. ACS Chem
Biol 2012, 7, 218.
(36) Hedman, H. K.; Kirpekar, F.; Elmroth, S. K. C. J Am Chem
Soc 2011, 133, 11977.
(37) a) Polonyi, C.; Elmroth, S. K. C. Dalton Trans 2013, 42,
14959; b) Polonyi, C.; Alshiekh, A.; Sarsam, LA.; Clausén
,M.;Elmroth, S. K. C. Dalton Trans 2014, 43, 11941.
(38) Xiao, H.; Qi, R.; Liu, S.; Hu, X.; Duan, T.; Zheng, Y.;
Huang, Y.; Jing, X. Biomaterials 2011, 32, 7732.
(14) Diehl, P.; Tedesco, D.; Chenchik, A. Drug Discov Today
Technol 2014, 11, 11.
(15) Mackay, C.; Carroll, E.; Ibrahim, A. F. M.; Garg, A.;
Inman, G. J.; Hay, R. T.; Alpi, A. F. Cancer Res 2014, 74, 2246.
(16) Leung, A. W. Y.; Dragowska, W. H.; Ricaurte, D.; Kwok,
B.; Mathew, V.; Roosendaal, J.; Ahluwalia, A.; Warburton, C.;
Laskin, J. J.; Stirling, P. C.; Qadir, M. A.; Bally, M. B. Oncotarget
2015, 6, 17161.
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 21
(39) Xiao, H.; Song, H.; Yang, Q.; Cai, H.; Qi, R.; Yan, L.; Liu,
S.; Zheng, Y.; Huang, Y.; Liu, T.; Jing, X. Biomaterials 2012, 33,
6507.
(40) Zheng, Y.-R.; Suntharalingam, K.; Johnstone, T. C.; Yoo,
H.; Lin, W.; Brooks, J. G.; Lippard, S. J. J Am Chem Soc 2014, 136,
8790.
(41) Reiber, H.; Ruff, M.; Uhr, M. Clin Chim Acta 1993, 217,
163.
(42) Jones, D. P.; Carlson, J. L.; Mody, V. C.; Cai, J.; Lynn, M.
J.; Sternberg, P. Free Radic Biol Med 2000, 28, 625.
(43) Khorsand, B.; Lapointe, G.; Brett, C.; Oh, J. K.
Biomacromolecules 2013, 14, 2103.
(44) Mikirova, N.; Casciari, J.; Riordan, N.; Hunninghake, R. J
Transl Med 2013, 11, 191.
(45) Zhang, A.; Zhang, Z.; Shi, F.; Xiao, C.; Ding, J.; Zhuang,
X.; He, C.; Chen, L.; Chen, X. Macromol Biosci 2013, 13, 1249.
(46) Cho, H. J.; Kim, J. K.; Kim, K. D.; Yoon, H. K.; Cho, M.-Y.;
Park, Y. P.; Jeon, J. H.; Lee, E. S.; Byun, S.-S.; Lim, H. M.; Song, E.
Y.; Lim, J.-S.; Yoon, D.-Y.; Lee, H. G.; Choe, Y.-K. Cancer Lett 2006,
237, 56.
(52) Saraswathy, M.; Gong, S. Materials Today 2014, 17,
298.
(53) Jones, S. K.; Lizzio, V.; Merkel, O. M.
Biomacromolecules 2016, 17, 76.
(54) Ganesh, S.; Iyer, A. K.; Weiler, J.; Morrissey, D. V.; Amiji,
M. M. Mol Ther Nucleic Acids 2013, 2, e110.
(55) Meng, H.; Mai, W. X.; Zhang, H.; Xue, M.; Xia, T.; Lin, S.;
Wang, X.; Zhao, Y.; Ji, Z.; Zink, J. I.; Nel, A. E. ACS Nano 2013, 7,
994.
(56) Babu, A.; Wang, Q.; Muralidharan, R.; Shanker, M.;
Munshi, A.; Ramesh, R. Mol Pharm 2014, 11, 2720.
(57) Xu, X.; Xie, K.; Zhang, X.-Q.; Pridgen, E. M.; Park, G. Y.;
Cui, D. S.; Shi, J.; Wu, J.; Kantoff, P. W.; Lippard, S. J.; Langer, R.;
Walker, G. C.; Farokhzad, O. C. Proc Natl Acad Sci U S A 2013,
110, 18638.
(58) Shen, S.; Sun, C.-Y.; Du, X.-J.; Li, H.-J.; Liu, Y.; Xia, J.-X.;
Zhu, Y.-H.; Wang, J. Biomaterials 2015, 70, 71.
(59) Yu, H.; Guo, C.; Feng, B.; Liu, J.; Chen, X.; Wang, D.;
Teng, L.; Li, Y.; Yin, Q.; Zhang, Z.; Li, Y. Theranostics 2016, 6, 14.
(60) Gabrielson, N. P.; Lu, H.; Yin, L.; Kim, K. H.; Cheng, J.
Mol Ther 2012, 20, 1599.
(61) Kozielski, K. L.; Tzeng, S. Y.; Green, J. J. Wiley Interdiscip
Rev Nanomed Nanobiotechnol 2013, 5, 449.
(62) Sun, C.-Y.; Shen, S.; Xu, C.-F.; Li, H.-J.; Liu, Y.; Cao, Z.-T.;
Yang, X.-Z.; Xia, J.-X.; Wang, J. J Am Chem Soc 2015, 137, 15217.
(63) Hayward, R. L.; Macpherson, J. S.; Cummings, J.; Monia,
B. P.; Smyth, J. F.; Jodrell, D. I. Mol Cancer Ther 2004, 3, 169.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(47) Qi, R.; Hu, X.; Yan, L.; Chen, X.; Huang, Y.; Jing, X. J
Control Release 2011, 152 Suppl 1, e167.
(48) Xiao, H.; Li, W.; Qi, R.; Yan, L.; Wang, R.; Liu, S.; Zheng,
Y.; Xie, Z.; Huang, Y.; Jing, X. J Control Release 2012, 163, 304.
(49) Goodwin, A. P.; Mynar, J. L.; Ma, Y.; Fleming, G. R.;
Fréchet, J. M. J. J Am Chem Soc 2005, 127, 9952.
(50) Deng, Z. J.; Morton, S. W.; Ben-Akiva, E.; Dreaden, E. C.;
Shopsowitz, K. E.; Hammond, P. T. ACS Nano 2013, 7, 9571.
(51) Gandhi, N. S.; Tekade, R. K.; Chougule, M. B. J Control
Release 2014, 194, 238.
10
ACS Paragon Plus Environment

Page 11 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 21
Scheme 1. OxaPt(II) but not OxaPt(IV) deactivates siRNA and a strategy for combining RNAi with an OxaPt(IV) prodrug. A) Planar
OxaPt(II) rapidly forms Pt-RNA adducts with BCL-2 siRNA, resulting in transcript inactivation; kinetically inert octahedral OxaPt(IV) spe-
cies, however, do not. As predicted from the sequence of the BCL-2 siRNA, platination of a single GA region in the non-seed portion of
the anti-sense strand (red/underlined) would be expected to affect silencing efficacy; the sense and anti-sense strands also contain other
potential sites for further platination (red/italics). B) Illustration of mPEG-b-PCL-b-PLL micelles (M(P)), incorporating OxaPt(IV)
(M(OxaPt(IV)) or both OxaPt(IV) and BCL-2 siRNA in a single construct (M(OxaPt(IV)/BCL-2)); note, OxaPt(IV) is linked through an amide
bond while BCL-2 siRNA is bound through electrostatic complexation to amino groups on the PLL chains of the micelles. C) Intracellular
uptake and the ultimate fate of M(OxaPt(IV)/BCL-2). The micelles are taken up by endocytosis, where high intracellular concentrations of
ascorbic acid (Asc) and glutathione (GSH) rapidly convert OxaPt(IV) to OxaPt(II); free OxaPt(II) subsequently binds genomic DNA, resulting
in Pt-DNA adduct formation. BCL-2 siRNA is liberated more slowly from the micelles, escaping from the endosome to down regulate BCL-
2 mRNA expression in the cytoplasm.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
12
ACS Paragon Plus Environment

Page 13 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 21
Figure 1: OxaPt(II) rapidly binds 5’-GMP as well as siRNA, forming Pt-RNA adducts, while OxaPt(IV) does not. Representative ESI-MS
spectra of solutions of 5’-GMP (10 mM) at 12 h after incubation with either A) OxaPt(II) (5 mM) or B) OxaPt(IV) (5 mM) at 37 C. The
o
1
2
3
4
5
6
7
8
presence of the DACHPt(5’-GMP)2 adduct was monitored over time by measuring changes in the relative height of the peak at
m/z=1035.1778; the structure and molecular weight of bis-adducts are shown in A, inset. Note: other peaks in the ESI spectra correspond
to adducts formed with Na+ and K+; see also Figure S7 (Supporting Information) for spectra at other time points. C) Kinetics of the binding
of various Pt-based agents to 5’-GMP; results are depicted as the molar percentage of the total 5’-GMP pool in solution and were deter-
mined by 1H NMR spectroscopy. Representative MALDI/TOF-MS spectra of the products formed after 6 h of incubation of BCL-2 ss-siRNA
(50 µM) with either D) OxaPt(II) (500 µM) or E) OxaPt(IV) (500 µM) at 37 oC. The spectrum of unreacted ss-siRNA is shown in E, inset. F)
Binding kinetics of OxaPt(II) and OxaPt(IV) (50 µM) to BCL-2 ds-siRNA (5 µM) as measured by GFAAS. Data are expressed as the percent-
age of platinum-bound G nucleotides within the ds-siRNA suspension. MALDI/TOF-MS was also conducted after enzymatic cleavage of
the antisense strand of BCL-2 siRNA (50 µM) by RNase A either G) before or H) after incubation with OxaPt(II) (500 µM) to demonstrate
sites of platination. 5’-AC-3’, 5’-AGU-3’, and 5’-AGGU-3’ are the cleavage products of the native BCL-2 ss-siRNA. Platination of the G
and/or A sites in the BCL-2 ss-siRNA resulted in the addition of one or more DACHPt moieties, yielding 5’-A(Pt)GU-3’(*), 5’-A(Pt)GGU-
3’(**), and 5’-A(Pt)G(Pt)GU-3’(**); note, here Pt denotes DACHPt.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14
ACS Paragon Plus Environment

Page 15 of 21
Journal of the American Chemical Society
Table 1: Summary of different ds-RNA macromolecules, various platinum-containing anticancer agents, their reaction rate constants
(k2,app), and the percentages of ds-siRNA that are expected to be deactivated due to platination as a function of reaction time and ini-
tial concentration of the platinum-containing species.
1
2
3
4
5
6
7
8
% ds-siRNA deactivated due to platination (i.e. % ss-siRNA)
30 min
10
µM µM µM µM µM µM
1 h
3 h
6 h
24 h
k2,app
Std
1
1
10
1
10
1
µM
10
1
10
REF
RNA
Length
# Gs
Species
(M-1s-1) Dev
µM µM
µM
9
37a
RNA-1
RNA-2
miR-146a
RNA-1
RNA-1-1-S
RNA-1-2
RNA-1-3
RNA-1-4
BCL-2
15
15
21
15
13
13
15/16
17
6
6
9
6
5
6
6
6
CisPt(II)
CisPt(II)
CisPt(II)
CisPt(II)
CisPt(II)
CisPt(II)
CisPt(II)
CisPt(II)
22.1
20.1
20.4
7.7
10.5
23.6
29.7
>30.0
1.7
1.1
0.6
0.5
0.6
1.0
1.1
3.9
3.6
3.6
1.4
1.9
4.2
5.2
33
30
31
13
17
35
41
42
7.6
7.0
7.1
2.7
3.7
8.1
10
55
52
52
24
32
57
65
66
21
20
20
8.0
11
23
27
27
91
89
89
57
68
92
96
96
38
35
36
15
20
40
47
48
99
99
99
81
90
99
100
100
85
82
83
49
60
87
92
93
100
100
100
100
100
100
100
100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
37b
N/A 5.3
10
This
work
siRNA
20
10
CisPt(II)
OxaPt(II)
12.7
5.3
1.2
2.3
2.3
0.9
20
9
4.5
1.9
37
17
13
75
44
24
11
94
68
67
37
100
99
5.6
CisPt(IV)
OxaPt(IV)
3.0
0.2
1.2
0.5
5
1.1
0.1
10
3.2
0.2
28
6.2
0.4
47
23
92
16
N/A 0.0
0.4
0.7
2.2
4.3
1.7
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2: Incubation of siRNA with OxaPt(II) or CisPt(II) results in a time-dependent decrease in RNAi efficacy. Relative BCL-2 mRNA
levels in A) MCF-7 breast cancer cells and B) OVCAR4 ovarian cancer cells at 37 oC and at 48 h after Lipofectamine®-based transfection of
BCL-2 siRNA. BCL-2 siRNA (1.5 nM) was utilized directly or was first pre-incubated with either different platinum(II)-based anticancer
agents (i.e. OxaPt(II) or CisPt(II)) (1.5 µM) or with their platinum(IV) analogs (i.e. OxaPt(IV) or CisPt(IV)) (1.5 µM) for different time peri-
ods, ranging from 0.5 to 24 h prior to transfection. BCL-2 mRNA levels were quantified by NanoDropTM and the data are presented as
relative to that of untreated cells, which served as the negative control (NC) treatment. Data are depicted as mean values ± SEM with n =
3 samples per condition; note, 3 experimental replicates were similarly conducted for each cell line and treatment, yielding analogous
results. Significance is defined as ** p < 0.01; *** p<0.001.
16
ACS Paragon Plus Environment

Page 17 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Figure 3: Micelles that covalently conjugate OxaPt(IV) and that are electrostatically complexed with BCL-2 siRNA demonstrate con-
trolled release of both species. A) TEM and B) DLS results depicting the average size distributions for M(OxaPt(IV)/BCL-2) that were
formed at an amine (per monomer unit, N) to phosphate (per nucleic acid, P) ratio (N/P ratio) of 8. The resultant micelles had a mean
particle diameter of 78.9 +/- 10.4 nm by TEM and 104.9+/-2.1 nm by DLS. C) Rapid in situ release of free OxaPt(II) was observed in high
reducing environments (10 mM sodium ascorbate, NaSC) as compared to under physiologic (pH 7.4) or acidic (pH 5.0) in situ conditions.
Individual data points are expressed as mean values ± SD for an n = 3 technical replicates per condition. A statistically significant separa-
tion in the curves was observed at all time points after 12 h (p < 0.05 via ANOVA). D) The release of Alexa488-labeled siRNA from
M(OxaPt(IV)/Alexa488 siRNA) was enhanced in low pH environments (pH 5.0 vs. pH 7.4). Individual data points are again expressed as
mean values ± SD for an n = 3 technical replicates per condition. A statistically significant separation in the curves was observed at all
time points after 1 h (p < 0.05 via two-tailed t-test). E) Confocal laser scanning microscopy images were taken to visualize cellular uptake
and cytoplasmic delivery of M(Cy5.5/Alexa488 siRNA/OxaPt(IV)). Images were obtained at 30 min (upper panel) and at 4 h (lower panel)
after the addition of micelles to the MCF-7 cells. Note, cell nuclei were stained with DAPI (blue); Cy5.5-conjugated mPEG-b-PCL-b-PLL
polymer (red) and Alexa488-labeled BCL-2 siRNA (green) were utilized to simultaneously and independently track the polymer and RNAi
species, respectively (scale bar = 100 μm). Flow cytometry was employed to quantify the relative uptake of F) Cy5.5-conjugated polymer
and G) Alexa488-labeled BCL-2 siRNA at various time points after the addition of M(Cy5.5/Alexa488 siRNA/OxaPt(IV)) to MCF-7 cells.
Signals generated from the untreated (blank) cells are included for reference.
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
ACS Paragon Plus Environment

Page 19 of 21
Journal of the American Chemical Society
Figure 4: Co-encapsulation of OxaPt(IV) and BCL-2 siRNA within a single micellar transfection reagent (i.e. M(OxaPt(IV)/BCL-2)) quanti-
tatively maximizes the intracellular uptake of Pt, the numbers of Pt-DNA adducts that are formed, the suppression of BCL-2 mRNA
levels, and the cytotoxicity to MCF-7 cells through augmentation of cellular apoptosis. A) Pt uptake into MCF-7 cells after 1 h (red) and
4 h (green) of cellular exposure to various treatments as determined by ICP-MS. Equal concentrations of Pt (10 µM) and/or BCL-2 siRNA
(100 nM) were used in each group and were respresentative of the concentrations of each species that are typically employed for in vitro
cytotoxicity and RNAi experiments, respectively. B) Total numbers of Pt-DNA adducts as assessed after 24 h of incubation with each ex-
perimental group. DNA was isolated after cellular lysis and the content of DNA-bound Pt was determined by ICP-MS. C) Relative BCL-2
mRNA levels as determined by RT-qPCR conducted at 48 h after treatment with each experimental group. D) Dose dependent cell viabil-
ity plots and E) IC50 values for cellular toxicity as determined by the MTT cellular viability assay, which was performed at 72 h after incu-
bation with each experimental group. Experiments in A-D were performed in triplicate. IC50 values were derived from three independent
experiments that each had three technical replicates per condition. Data are displayed as the mean values ± the standard deviation of
the mean. F) Total percentages of MCF-7 cells that underwent apoptosis as determined by flow cytometry at 36 h after treatment with
various experimental groups. Equivalent concentrations of Pt (10 µM) +/- BCL-2 siRNA (100 nM) were used for each condition. Data are
depicted as mean values ± SEM with n = 3 samples per condition. In all cases, significance is defined as *p<0.05, **p<0.01, and ***
p<0.001. # denotes p>0.05.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 21
Table 2: Summary of IC50 values (µM) for MCF-7, OVCAR4, A2780 and A2780DDP cells treated with various com-
binations of platinum-containing anticancer agents and/or siRNA species.
1
2
3
4
5
6
7
8
9
Treatment groups
OxaPt(II)
MCF-7
23.05 ± 0.25
28.51 ±0.34
19.20 ± 0.42
>100
OVCAR4
15.45 ± 0.50
21.60 ± 0.71
9.24 ± 1.50
>100
A2780
A2780DDP
18.80 ± 0.99
22.25 ± 1.63
10.55 ± 1.34
>100
2.90 ± 0.28
3.75 ± 0.91
1.60 ± 0.14
15.85 ± 2.76
12.65 ± 1.20
8.60 ± 1.13
0.87 ± 0.13
0.72 ± 0.09
0.51 ± 0.03
0.59 ± 0.06
0.39 ± 0.02
OxaPt(II)+M(c-siRNA)
OxaPt(II)+M(BCL-2)
OxaPt(IV)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OxaPt(IV)+M(c-siRNA)
OxaPt(IV)+M(BCL-2)
M(OxaPt(IV))
>100
>100
>100
>100
>100
>100
0.54 ± 0.03
0.43 ± 0.01
0.17 ± 0.02
0.43 ± 0.02
0.06 ± 0.01
1.79 ± 0.10
1.37 ± 0.12
0.43 ± 0.01
1.80 ± 0.25
0.12 ± 0.01
1.45 ± 0.07
1.06 ± 0.20
0.60 ± 0.08
0.72 ± 0.05
0.42 ± 0.08
M(OxaPt(IV))+M(c-siRNA)
M(OxaPt(IV))+M(BCL-2)
M(OxaPt(IV)/c-siRNA)
M(OxaPt(IV)/BCL-2)
20
ACS Paragon Plus Environment

Page 21 of 21
Journal of the American Chemical Society
Table of contents (TOC) graphic
1
2
3
4
5
6
7
8
9
Pt(II)
Pt(IV)
A
N
R
i
s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Tumor
Cells
Screening/Therapy
siRNA
Pt(IV)
M(P)
M(Pt(IV))
M(Pt(IV)/siRNA)
21
ACS Paragon Plus Environment