Cholesterol-tethered platinum II-based supramolecular
nanoparticle increases antitumor efficacy and
Poulomi Senguptaa,b,1, Sudipta Basua,b,1,2,3, Shivani Sonia,b,1,4, Ambarish Pandeya,b,5, Bhaskar Roya,b,5, Michael S. Oha,b,
Kenneth T. Chinc, Abhimanyu S. Paraskara,b, Sasmit Sarangia,b, Yamicia Connora,b,d, Venkata S. Sabbisettib,e,
Jawahar Kopparama,b, Ashish Kulkarnia,b, Katherine Mutoc, Chitra Amarasiriwardenaf,g, Innocent Jayawardenef,g,
Nicola Lupolif,g, Daniela M. Dinulescub,c, Joseph V. Bonventreb,e, Raghunath A. Mashelkara,h,
and Shiladitya Senguptaa,b,d,i,j,3
aLaboratory for Nanomedicine, Division of Biomedical Engineering, Department of Medicine and Center for Regenerative Therapeutics,eRenal Division and
Division of Biomedical Engineering,cDepartment of Pathology, andgChanning Laboratory, Brigham and Women’s Hospital, Cambridge, MA 02139;bHarvard
Medical School, Boston, MA 02115;dHarvard–MIT Division of Health Sciences and Technology, Cambridge, MA 02139;fHarvard School of Public Health,
Boston, MA 02215;hNational Chemical Laboratories, Pune 411021, India;iIndo–United States Joint Center for Nanobiotechnology, Cambridge, MA 02139;
andjDana-Farber Cancer Institute, Brookline, MA 02445
Edited* by Jean-Marie P. Lehn, Universite de Strasbourg, Strasbourg Cedex, France, and approved May 28, 2012 (received for review February 22, 2012)
Nanoscale drug delivery vehicles have been harnessed extensively
as carriers for cancer chemotherapeutics. However, traditional
pharmaceutical approaches for nanoformulation have been a chal-
lenge with molecules that exhibit incompatible physicochemical
properties, such as platinum-based chemotherapeutics. Here we
propose a paradigm based on rational design of active molecules
that facilitate supramolecular assembly in the nanoscale dimen-
sion. Using cisplatin as a template, we describe the synthesis of
a unique platinum (II) tethered to a cholesterol backbone via
a unique monocarboxylato and O→Pt coordination environment
that facilitates nanoparticle assembly with a fixed ratio of phos-
phatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanol-
amine-N-[amino (polyethylene glycol)-2000]. The nanoparticles
formed exhibit lower IC50values compared with carboplatin or
cisplatin in vitro, and are active in cisplatin-resistant conditions.
Additionally, the nanoparticles exhibit significantly enhanced
in vivo antitumor efficacy in murine 4T1 breast cancer and in
K-RasLSL/+/Ptenfl/flovarian cancer models with decreased systemic-
and nephro-toxicity. Our results indicate that integrating rational
drug design and supramolecular nanochemistry can emerge as
a powerful strategy for drug development. Furthermore, given that
platinum-based chemotherapeutics form the frontline therapy for
a broad range of cancers, the increased efficacy and toxicity profile
indicate the constructed nanostructure could translate into a next-
generation platinum-based agent in the clinics.
States and many western countries. In addition, the incidence
is also increasing in less developed and economically tran-
sitioning countries (1). The World Health Organization projects
over 12 million deaths worldwide in 2030 because of cancer, up
from 7.6 million in 2008 (2). To address this growing problem,
there is an urgent need to develop treatment strategies that
are more efficaceous with lesser adverse effects. An increasingly
pursued approach to achieve these goals is the use of nano-
technology to preferentially target anticancer agents to solid
tumors (3). This approach capitalizes on the unique leaky an-
giogenic tumor vasculature coupled with impaired intratumoral
lymphatic drainage, contributing to an enhanced permeation
and retention (EPR) effect (4). Indeed, nanoparticles carrying
a doxorubicin payload or an albumin-paclitaxel nanocomplex
increase intratumoral drug concentration (5, 6) and are currently
in the clinics (7). However, traditional processes for nanofor-
mulation are often incompatible with physicochemical properties
of many chemotherapeutic agents, which limit the entrapment
efficiency or introduce suboptimal release kinetics. To address
ancer remains one of the main causes of death in the United
these challenges, we propose a unique paradigm moving beyond
traditional encapsulation strategies to the rational design of mol-
ecules that facilitate supramolecular assembly in the nanoscale
dimension. In this study, we decided to use cisplatin [cis-dichlor-
odiamineplatinum (II)] as an example to test this hypothesis.
Cisplatin is one of the most widely used chemotherapeutic agents
(8) but poses significant challenges for nanoformulations (9, 10).
For example, SPI-077, a sterically stabilized liposome encapsu-
lating cisplatin, exhibited poor clinical efficacy resulting from
impaired drug release (11, 12).
To achieve supramolecular nanoassembly, we synthesized
a cholesterol-tethered cisplatinum (II) amphiphile. The design of
the tether was inspired by the process of “aquation,” wherein the
chloride leaving groups of cisplatin are rapidly displaced to form
cis-Pt[(NH3)2(OH2)Cl]+and cis-Pt[(NH3)2(OH2)2]2+(8). Self-
assembling cholesterol-succinic acid-cisplatinum II-based nano-
particles (SACNs) exhibited increased potency and efficacy
in vitro and in vivo, respectively. Additionally, the SACNs exceed
the size cutoff for clearance by the kidney (13), and therefore
exhibited limited cisplatin-associated nephrotoxicity (14). We
demonstrate that rational drug design can enable the increase
in the supramolecular dimension from the Angstrom- to the
nano-scale, thereby conferring unique biological properties. Fur-
thermore, only three platinates—cisplatin, carboplatin, and oxa-
liplatin—have been successfully used in the clinics (8). The
increased efficacy with improved therapeutic index of the current
molecule compared with the existing platinates indicates the
Author contributions: P.S., S.B., S. Soni, A.P., B.R., A.S.P., V.S.S., A.K., D.M.D., R.A.M., and
S. Sengupta designed research; P.S., S.B., S. Soni, A.P., B.R., M.S.O., K.T.C., A.S.P., S. Sarangi,
Y.C., V.S.S., J.K., A.K., K.M., C.A., I.J., N.L., and D.M.D. performed research; P.S., S.B.,
S. Soni, A.P., B.R., M.S.O., K.T.C., A.S.P., S. Sarangi, Y.C., V.S.S., D.M.D., J.V.B., and
S. Sengupta analyzed data; and P.S., S.B., S. Soni, A.P., B.R., V.S.S., D.M.D., R.A.M.,
and S. Sengupta wrote the paper.
Conflict of interest statement: P.S., S.B., S. Soni, A.S.P., and S. Sengupta are listed as
inventors on a patent filed by Brigham and Women’s Hospital.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1P.S., S.B., and S. Soni contributed equally to this work.
2Present address: Department of Chemistry, Indian Institute of Science Education and
Research (IISER) Pune, Sutarwadi, Pashan, Pune, Maharashtra, 411021, India.
3To whom correspondence may be addressed. E-mail: email@example.com, or
4Present address: Department of Biological Sciences, Alabama State University, Montgomery,
5A.P. and B.R. contributed equally to this work.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 10, 2012
| vol. 109
| no. 28www.pnas.org/cgi/doi/10.1073/pnas.1203129109
potential for clinical translation as the next-generation platinum-
Synthesis of Cholesterol-Succinic Acid-Pt(II) Molecule. Aquation of
cisplatin results in the rapid formation of active species cis-[Pt
(NH3)2Cl(OH2)]+and cis-[Pt(NH3)2(OH2)2]2+with a rate con-
stant of 8 × 10−5s−1(15). In contrast, the rate constant for
aquation of carboplatin, where the platinum is coordinated with
a stable bidentate 1,1-cyclobutanedicarboxylato ligand, was found
to be 7.2 × 10−7s−1. This difference in their rate of aquation was
matched by their rates of binding to DNA, indicating that the rate
of aquation correlates with potency (16, 17). Indeed, we had
demonstrated that Pt chelated to a polyisobutylene maleic acid
glucosamine copolymer via a monocarboxylato and an O→Pt
coordinate bond release of Pt in a pH-dependent manner, and
more efficiently than when the Pt was chelated using dicarbox-
ylato bonds or via a monocarboxylato and an N→Pt coordinate
bond (18, 19). As a result, we rationalized that the introduction
of a coordination environment where the Pt was chelated via
a monocarboxylato and an O→Pt coordinate bond is critical to
the design of an efficacious platinate. As outlined in the given
scheme (Fig. 1A), we first synthesized cholesterol-ethylenedi-
amine conjugate in near quantitative yield (99.1%) by reacting
cholesteryl chloroformate with excess ethylene diamine. Next, we
introduced monocarboxylato and amide chelating moiety by
reacting cholesterol-ethylenediamine conjugate with succinic
anhydride (at 95% yield). Finally, the conjugate was reacted with
aquated cis-Pt[(NH3)2(OH2)2]2+in 1:1 molar ratio in acidic pH
(pH = 6.4) to obtain cholesterol-cisplatin conjugate, characterized
by monocarboxylato and an O→Pt coordinate bond of an amide,
as indicated by an unique single195Pt NMR peak at −1,621.497
ppm (Fig. S1). All of the other intermediates were characterized
by1H and13C NMR spectroscopy (Figs. S2–S5).
Synthesis and Characterization of SACNs. We engineered the
SACNs from the cholesterol-succinic acid-platinum (II) molecule,
phosphatidylcholine (PC) and 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-
PEG) in 1:2:0.2 weight ratio using a lipid-film hydration self as-
sembly method (20) (Fig. 1A). The ultrastructure analysis using
cryo-transmission electron microscopy (cryo-TEM) (Fig. 1B)
revealed the formation of predominantly uni- and rare multi-
lamellar structures less than 200 nm in diameter, with a membrane
thickness ∼5 nm. Dynamic light scattering further confirmed the
size distribution of SACNs with a mean hydrodynamic diameter of
141.4 ± 1.2 nm (n = 9) (Fig. 1C). To validate the kinetics of cis-
platin release, SACNs were incubated at acidic pH 5.5 over 120 h,
with pH 7 as a reference. As shown in Fig. 1D, SACNs exhibited
a pH-dependent sustained release of cisplatin. Interestingly, the
rate of release was slower than observed earlier using a poly-
meric system, indicating that the cholesterol can incorporate into
the lipid layer in a manner where the Pt moiety is present both
on the outer as well as inner part of the membrane.
In Vitro Efficacy of SACNs. To evaluate the efficacy of the SACNs
in vitro, we performed a cell viability assay using Lewis lung
carcinoma (LLC) and 4T1 breast cancer cell lines. Cell viability
was quantified by using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
salt assay at 48 h postincubation. As shown in Fig. 2 A and B, free
cisplatin induced LLC and 4T1 cell kill with IC50values of 2.91 ±
0.015 μM and 4.72 ± 0.016 μM, respectively. In neither of these
cell lines did carboplatin exert any inhibitory activity at this
concentration range. Interestingly, the SACNs were found to be
more efficacious than cisplatin against both 4T1 and LLC cells,
with IC50 values of 0.44 ± 0.016 μM and 1.16 ± 0.016 μM,
respectively. We next tested the efficacy of the SACNs in a cis-
platin-resistant hepatocellular carcinoma (7404-CP20) cell line.
) d ( ) c (
Size (d. nm)
Scheme for synthesis of cholesterol-cisplatin conju-
gate from cholesteryl chloroformate. Schematic
representation shows synthesis of SACNs by self-as-
sembly from PC, cholesterol-cisplatin conjugate, and
DSPE-PEG. (B) High-resolution cryo-TEM image of
SACNs at lower magnification (Upper) and magni-
fied image (Lower). (Scale bar, Upper, 500 nm).
(C) Distribution of hydrodynamic diameter of SACNs
measured using dynamic light scattering. (D) Graph
shows the pH-dependent release of platinum from
SACNs as quantified over a 120-h period.
Synthesis and characterization of SACNs. (A)
Sengupta et al.PNAS
| July 10, 2012
| vol. 109
| no. 28
Although the IC50value for free cisplatin in this assay was cal-
culated to be 42.84 ± 0.04 μM, consistent with previously
reported values (21), the SACNs were found to be overcome the
resistance with an IC50value of 3.02 ± 0.013 μM (Fig. 2C). To
elucidate the mechanism of cell death, we incubated 4T1 cells
with a sub-IC50concentration of the SACNs, cisplatin or car-
boplatin, for 24 h. The cells were labeled with FITC-Annexin V
that binds to phosphatidylserine, an early marker for apoptosis.
As seen in Fig. 2D, treatment with platinates induced both ap-
optosis and necrosis of the tumor cells, with both cisplatin and
SACNs being more efficacious than carboplatin. Studies using
FITC-labeled SACNs revealed that the nanoparticles were in-
ternalized and localized in the endolysosomal compartment in
a temporal manner (Fig. 2E). This finding is further validated by
incubating the tumor cells (4T1 and 7404-CP20) with FITC-
labeled SACNs at 37 °C and 4 °C, wherein the internalization of
SACNs into the cells was decreased in the latter condition
(Fig.2F). To dissect the mechanism underlying the efficacy of
SACNs in the cisplatin-resistant 7404-CP20 cell line, we quan-
tified the intracellular concentration of Pt in the cells following
incubation with cisplatin or SACNs containing equivalent levels of
Pt. As shown in Fig. 2G, the SACNs resulted in significantly ele-
vated intracellular levels of Pt compared with cisplatin. Incubating
cells with SACNs at 4 °C, which inhibits energy-dependent en-
docytosis, reduced the intracellular Pt concentration to cisplatin-
treated levels, validating that the SACNs can enter these cells
Efficacy of SACNs in an in Vivo 4T1 Breast Cancer Model. Motivated
by the sustained release of Pt and enhanced in vitro efficacy of
SACNs, we evaluated its antitumor efficacy in vivo. As the first
step, we established the maximum tolerated dose (MTD) for the
SACNs in BALB/c mice to be 16 mg/kg compared with 9 mg/kg
of cisplatin (Fig. 3A). We next dosed syngeneic BALB/c mice
bearing 4T1 breast tumors (mean tumor volume ∼100 mm3) with
a single dose of cisplatin (8 mg/kg). Other groups of animals
received vehicle, carboplatin, or SACNs, (the latter two received
a Pt dose equivalent to 8 mg/kg dose of cisplatin). As shown in
Fig. 3B, although all of the platinates resulted in significant tu-
mor inhibition compared with the vehicle-treatment, the SACNs
exerted the maximal tumor inhibition (P < 0.01 vs. control)
followed by cisplatin and carboplatin. Furthermore, although
treatment with carboplatin or cisplatin exerted only minor in-
crease in survival over vehicle-treated controls, the SACNs sig-
nificantly increased overall survival trend (Fig. 3C). We next
tested the effects of multiple low-dose treatment with cisplatin,
carboplatin, or the SACNs, with the highest platinum dose in
each case adding up to the levels of Pt delivered at the MTD of
cisplatin. Two additional groups were included that were treated
with a lower dose of cisplatin or SACNs (equivalent of 1 mg/kg
dose of platinum). As shown in Fig. 3 D and E, treatment with
cisplatin resulted in a dose-dependent inhibition of tumor
growth. Interestingly, although at the highest doses the tumor
inhibition with the SACNs or cisplatin were identical, at the
lower doses the SACNs exerted a superior antitumor effect
compared with free cisplatin (P < 0.05, ANOVA). Furthermore,
cisplatin resulted in a significant reduction in mean body weight
(P < 0.05, ANOVA) compared with the SACN-treated groups
(Fig. 3F), indicating that the latter can reduce the systemic tox-
icity associated with cisplatin chemotherapy. Interestingly, even
at the lower dose both the SACNs and cisplatin exerted greater
tumor inhibition as opposed to the higher dose of carboplatin
(Fig. 3 E and F). At the higher dose, both cisplatin and SACNs
were found to increase survival, although the latter was superior
(Fig. 3G). To elucidate the mechanism underlying of increased
in vivo efficacy, the tumors were excised posttreatment and
processed for TUNEL as a marker for apoptosis. As shown in
Fig. 3D, SACNs induced significantly greater apoptosis than
cisplatin, but at the higher doses both the SACNs and cisplatin
induced similar apoptosis, consistent with the tumor inhibition
results. However, at the latter dose level, cisplatin but not SACNs
resulted in significant nephrotoxicity as evident by reduced kidney
weight and up-regulation of kidney injury molecule-1 (KIM1)
expression (Fig. 4 A and B). Additionally, TUNEL of the excised
(15 min)(30 min)
(4 h) (18 h)
4T1 breast cancer cellsCP20 hepatocellular carcinoma
37 Co37 Co 4 C
cell viability of (A) LLC, (B) 4T1, and (C) 7404-CP20 cell lines,
respectively, after 48-h incubation with increasing concen-
trations of cisplatin, carboplatin, and SACNs. (D) Treatment
with SACNs induces cell death by apoptosis. Representative
FACS distribution of 4T1 cells treated with carboplatin, cis-
platin, and SACNs at 1 μM Pt concentration. The cells were
incubated for 24 h, following which they were labeled with
Annexin-V FITC and counter-stained with propidium iodide.
Each quadrant represents the percentage of cells in early
apoptosis (Lower Right), late apoptosis (Upper Right), necrosis
(Upper Left), and healthy cells (Lower Left). Data shown are
mean ± SE from n = 3 independent experiments. (E) Repre-
sentative epifluorescence imaging of 4T1 tumor cells for
monitoring internalization of SACNs. SACNs were labeled with
FITC (green), the endolysosomal compartment was labeled by
LysoTracker red, and the nucleus was labeled with DAPI blue.
Colocalization of the signals in the merged images reveals
internalization of FITC-SACNs in the endolysosomal compart-
ments within 4 h. (F). Epifluorescence imaging of 4T1 and
CP20 tumor cells to monitor the internalization of FITC tagged
SACN at 37 °C and 4 °C. Images were captured using a Nikon Ti
epifluorescence microscope at 40× magnification. 4T1 images
were captured at 1,000 × 700 pixel resolution and the 7404-
CP20 images are 900 × 600 pixels. (G) Graph shows Pt levels in
7404-CP20 cells treated with cisplatin or SACNs (20 μM Pt
concentration). Cells incubated with similar concentration of
SACNs at 4 °C to inhibit energy-dependent endocytosis exhibit
lower intracellular Pt concentrations (*P < 0.05, **P < 0.01,
ANOVA, Newman–Keuls post hoc test).
In vitro characterization of SACNs. (A–C) Graphs show
| www.pnas.org/cgi/doi/10.1073/pnas.1203129109 Sengupta et al.
kidney sections (Fig. 4C) indicated significant apoptosis in cis-
platin-treated mice, whereas the SACNs demonstrated negligible
apoptosis even at the higher dose.
SACNs Home Preferentially to the Tumors and Bypass Kidney. To
elucidate the mechanism underlying the enhanced apoptosis in
the tumor and reduced nephrotoxicity evident with the SACNs,
we probed the tumor and reticuloendothelial system (RES)
organs for the platinum biodistribution. Tumor-bearing animals
were dosed with cisplatin or SACNs at doses equivalent to 1 and
3 mg/kg of platinum. As shown in Fig. 4D, there was a dose-
dependent accumulation of platinum (as quantified per gram of
tissue using inductive-coupled plasma atomic absorption spectra)
in the RES tissues. Interestingly, the SACNs (3 mg/kg Pt dose)
resulted in significantly higher concentration in the tumor than
achieved following dosing of an equivalent amount of cisplatin.
Furthermore, at this dose, cisplatin resulted in significantly
higher platinum build-up in the kidney, which could account for
cisplatin-associated nephrotoxicity, compared with the SACN-
Efficacy of SACNs in an in Vivo K-RasLSL/+/Ptenfl/flOvarian Cancer
Model. In recent years, it has been well established that fre-
quent somatic PTEN and K-Ras mutations are implicated in
wide spectrum of human cancers, including endometrioid ovar-
ian cancer (22–24). As shown in Fig. S6 A and B, the animals
bearing medium and large K-rasLSL/+/Ptenfl/flovarian cancer
treated with SACNs (Pt dose equivalent to 3 mg/kg of cisplatin)
resulted in greater regression compared with cisplatin treatment.
TUNEL revealed apoptosis in both SACN- and cisplatin-treated
tumor. However, although cisplatin induced apoptosis of neph-
rons, negligible cell death was evident in the kidney with SACNs
(Fig. S6C), which correlated with elevated levels of Pt in the
tumor with reduced concentrations in the kidney following
SACN treatment compared with cisplatin-treatment (Fig. S6D).
Supramolecular chemistry, the development of complex chem-
ical systems from molecular building blocks that interact via
noncovalent intermolecular forces (25), has emerged as a field
that explains and impacts many biological and physical concepts.
In an elegant perspective, Jean-Marie Lehn had envisioned a
unique paradigm called supramolecular nanochemistry (26).
Indeed, gadolinium (III)-encapsulated supramolecular nanopar-
ticles were recently shown to enhance relaxivity with increased
sensitivity, and serve as a tool for diagnosis of cancer metastasis
(27). In another study, camptothecin was encapsulated in a su-
pramolecular nanoparticle (28). However, although these
emerging studies have focused on using supramolecular inter-
actions to encapsulate molecules for targeting cancer, we report
here the rational redesign of a cancer chemotherapeutic drug to
enable supramolecular assembly into a nanostructure.
Although cisplatin [cis-dichlorodiamineplatinum (II)] is the
drug of choice as a first or second line chemotherapy for most
cancers, its clinical efficacy is dose-limited because of nephro-
toxicity, resulting from a peritubular uptake in both proximal and
distal tubules mediated by a organic cation transporter 2 (29). As
nanoparticles > 5.5 nm can bypass glomerular filtration (13),
cisplatin made an excellent candidate for rational engineering
into a supramolecular nanostructure to potentially overcome
nephrotoxicity. As the first step, we converted cis-platinum (II)
into an amphiphile via conjugation to a cholesterol succinic acid
conjugate, which facilitated the supramolecular assembly of this
platinate into SACNs with PC and DSPE-PEG arising from
hydrophilic-hydrophobic interactions (30, 31). Cholesterol and
PC were selected as both are components of biological cellular
membranes, and the 3β-OH group of cholesterol is easily ame-
nable to conjugation, and alter pharmacodynamic/pharmacoki-
netic profile as well as cellular uptake of the active agent (32).
DSPE-PEG was incorporated to impart “stealth” property to
SACNs as surface modification of nanoparticles with PEG has
been reported to decrease interaction with opsonin (33), and
thereby reduce clearance by the RES. Indeed, consistent with the
above hypothesis, our biodistribution studies revealed that the
SACNs could bypass glomerular filtration in the kidney, evident
by the significantly lower Pt concentration in the kidney com-
pared with cisplatin. This finding was further validated by low
expression of KIM1, an early marker for kidney injury (34), with
cancer model. (A) Graph shows body weight loss of animals
with increasing doses of cisplatin or SACNs (Cisplatin NP).
Maximum tolerated dose is calculated at 20% body weight
loss. (B) Graph shows change in tumor volume in different
treatment groups in 4T1 murine breast cancer model fol-
lowing a single dose of platinum chemotherapy at the MTD
platinum dose of cisplatin. (C) Kaplan–Meier curve shows ef-
fect of different treatments on survival at MTD platinum dose
of cisplatin (P = 0.0189 Logrank test for trend). (D) Multiple-
dose effects of treatment on 4T1 breast cancer growth. Cells
were implanted subcutaneously on day 0. Mice were treated
with PBS, carboplatin (3 mg/kg), cisplatin (3 mg/kg and
1 mg/kg), and SACNs (3 mg/kg and 1 mg/kg) (n = 4, doses are
Pt equivalent) on days 9, 11, and 13 posttumor implantation.
Upper row shows representative images of excised tumors,
and Lower row shows tumor cross sections processed for
TUNEL as marker for apoptosis. Images were captured using
a Nikon Ti epifluorescence microscope at 20× magnification
to capture a large view field. (E) Growth curves show the
effect of the different multiple-dose treatments on tumor
volume. (F) Graph shows change in body weight of animals in
different treatment groups. (G) Kaplan–Meier curve shows
effect of different treatments on survival (P = 0.0022, Logrank
In vivo antitumor activity of SACNs in 4T1 breast
Sengupta et al.PNAS
| July 10, 2012
| vol. 109
| no. 28
concomitant decrease in kidney apoptosis observed following
SACNs treatment. Furthermore, the SACNs preferentially ac-
cumulated in the tumor are consistent with previous reports where
such stealth nanosystems were reported to home into the tumors
via the EPR effect (5).
Although the SACNs enable enhanced intratumoral concen-
trations, a critical driver of efficacy is the efficient release of
active cis-[Pt(NH3)2]2+moiety. For example, the stable cyclo-
butanedicarboxylate chelating ligand lowers the rate of aquation
of carboplatin by two-to-four orders of magnitude than cisplatin,
and to obtain cytotoxicity comparable to cisplatin a 4- to 20-fold
higher dose of carboplatin is required (16). Similarly, AP5280,
a N-(2-hydroxypropyl) methacrylamide copolymer-bound plati-
num was found to exert minimal nephrotoxicity in clinical studies
(35), but was less potent than carboplatin because the platinum
is held to an aminomalonic acid chelating agent coupled to
the COOH terminal glycine of a tetra-peptide spacer (36). The
criticality in the amphiphile design in this study was therefore the
introduction of the monocarboxylato and O→Pt coordination
environment between the platinum and the leaving group, in this
case the cholesterol succinic acid conjugate. We have previously
demonstrated that this coordination environment was more ef-
ficient in releasing activated Pt in a pH-dependent manner than
when coordination is via more stable dicarboxylato linkages or
monocarboxylato and N→Pt linkage (18, 19). This finding is
consistent with the increased potency of SACNs compared with
carboplatin as observed in vitro and validated by increased tumor
cell apoptosis and necrosis. Interestingly, the SACNs also
exhibited slightly improved efficacy compared with cisplatin in
the breast cancer (4T1) and LLC cells, and was significantly
superior to cisplatin in the hepatocellular carcinoma (7404-
CP20), which could be explained by the SACNs harnessing ad-
ditional mechanisms of uptake into the 7404-CP20 cells besides
traditional platinum transporters that are mutated in these cells.
Indeed our studies using fluorescently tagged SACNs revealed
internalization via endocytosis in a temporal manner. Inhibition
of endocytosis decreased the intracellular levels of Pt following
SACN treatment to that achieved by treatment with cisplatin.
Although previous studies have reported that cholesterol has
been shown to facilitate cellular uptake through caveolin-medi-
ated endocytosis (37), we observed that pretreatment of the cells
with chlorpromazine, an inhibitor of clathrin-mediated endocy-
tosis, nystatin, a caveolae-mediated endocytosis inhibitor, or cy-
tochalasin D, an inhibitor of macropinocytosis/phagocytosis,
could not fully abolish internalization of the SACNs (Fig. S7),
which could suggest a redundancy in the mechanisms of SACN
internalization. However, it should be noted that recent reports
have questioned the specificity of endocytosis inhibitors (38), and
in our study we did observe changes in cellular morphology, even
at lower doses and short incubation times. Although the SACNs
were also internalized in a similar manner by endothelial cells
and fibroblasts in vitro (Fig. S7), we anticipate that in vivo the
preferential EPR-mediated intratumoral accumulation, and the
tendency of SACNs to internalize within the low pH endolyso-
somal compartment together with the predisposition of the
SACNs to release activated cis-[Pt(NH3)2]2+in an acidic envi-
ronment, may further contribute to preferential tumor targeting.
Although the SACNs exhibit increased potency, we also ob-
served an increase in the MTD in vivo compared with cisplatin,
suggesting that it may be possible to overcome the dosing limits
associated with cisplatin in the clinics. We selected the 4T1
breast cancer and the genetically engineered K-rasLSL/+/Ptenfl/fl
ovarian adenocarcinoma mouse models for our in vivo studies
because these closely mimic human tumor progression. In-
terestingly, even at a single sub-MTD platinum dose comparable
to the MTD of cisplatin, the SACNs exerted superior antitumor
efficacy, both in terms of tumor inhibition and survival, which
could be attributed to the preferential accumulation and in-
creased potency. Furthermore, there may be a metronomic
dosing effect involved in the therapeutic outcome potentially
arising from the sustained release because, even at the lower
doses, SACNs were more efficacious than cisplatin. Interestingly,
recent clinical reports have indicated that metronomic dosing of
cisplatin exerts an antiangiogenic effect (39). Interestingly, we
observed that administration of lower multiple doses of cisplatin
was more effective in increasing survival compared with a single
MTD. This finding indicates that therapeutic efficacy of SACNs
can be optimized by tailoring the dosing regimen.
In conclusion, our results support the hypothesis that in-
tegrating rational drug design and supramolecular nanochemistry
can emerge as a powerful strategy for drug development. Fur-
thermore, because platinum-based chemotherapeutics form the
frontline therapy for a broad range of cancers, including testicular,
ovarian, cervical, endometrial, bladder, head and neck, lung, and
gastro-esophageal cancers, the increased efficacy and improved
toxicity profile, resulting from an increase in the molecular di-
mension through supramolecular assembly, indicates that the
constructed nanostructure could translate into the next-generation
platinum-based agent in the clinics.
Materials and Methods
Synthesis and Characterization of SACNs. The synthesis and characterization of
cholesterol-cisplatin conjugate isdescribed in SIMaterialsand Methods. Briefly,
a thin and uniform lipid-drug film of PC, cholesterol-cisplatin conjugate,
and DSPE-PEG was coated using a rotary evaporator, then hydrated for 1 h at
60 °C, passed although Sephadex G-25 column, and extruded at 65 °C to ob-
tain sub-200 nm particles. Nanoparticles were analyzed using a nanozetasizer
and using cryo-TEM. For release kinetics, drug loaded nanoparticles were
suspended in buffer (pH = 5.5 or 7) and sealed in a dialysis membrane (mo-
lecular weight cutoff = 500 Da). The dialysis bags were incubated in 30 mL PBS
buffer at room temperature with gentle shaking. An aliquot was collected
from the incubation medium at predetermined time intervals, and the released
drug was quantified.
Cell Viability/Apoptosis Assay. The LLC cells, breast cancer cell line (4T1), and
hepatocellular carcinoma cells (CP20) were seeded into 96-well flat bottomed
plates (4 × 103cells per well). Drugs or SACNs were added at equivalent Pt
concentrations and incubated for 48 h. Viability was quantified using the
Cispla?n (3 mg/kg)
Cispla?n (1 mg/kg)
SACNs (3 mg/kg)SACNs (1 mg/kg)
Cispla?n (3 mg/kg)Cispla?n (1 mg/kg)
SACNs (3 mg/kg)SACNs (1 mg/kg)
and exert reduced nephrotoxicity. Mice were treated with PBS, carboplatin
(3 mg/kg), cisplatin (3 mg/kg and 1 mg/kg), and SACNs (cisplatin NP, 3 mg/kg
and 1 mg/kg) (n = 4, doses are Pt equivalent) on days 9, 11, and 13 posttumor
implantation. (A) Bar graph shows weight of excised kidney in different
treatment groups. (B) Representative images of cross sections of kidney
stained for KIM1 expression. Images were captured using a Nikon Eclipse 90i
microscope (Left). (C) Representative images of kidney cross-sections pro-
cessed for TUNEL as marker apoptosis (Right). Images were captured using
a Nikon Ti epifluorescence microscope at 20× magnification to capture
a large view field; 1,000 × 700 pixels. (D) Tissue distribution of platinum in
different treatment groups as determined by inductively coupled plasma-
MS. *P < 0.05, **P < 0.01 vs. cisplatin (3 mg/kg)-treated group (ANOVA
followed by Newman–Keuls post hoc test).
SACNs preferentially accumulate in the tumor bypassing the kidney,
| www.pnas.org/cgi/doi/10.1073/pnas.1203129109Sengupta et al.
Cell-Titer 96 Aqueous One Solution reagent. Cellular apoptosis was quanti-
fied using Annexin-V-Alexa Fluor 488 conjugate and propidium iodide
staining followed by FACS.
SACN Internalization Study. The 4T1 cells were seeded on glass cover-slips and
treated with FITC-encapsulated SACNs for a time-course ranging from 30 min
to 18 h. At the indicated times, cells were washed twice in PBS and incubated
in LysoTracker red for 30 min at 37 °C. Images taken in three random fields
were captured at using an inverted epifluorescence deconvolution micro-
scope (Nikon). To study the role of endocytosis in SACN internalization,
the cells were incubated at 4 °C or pretreated with endocytosis inhibitors
(described in detail in SI Materials and Methods).
In Vivo Murine 4T1 Breast Cancer Model. The 4T1 breast cancer cells (3 × 105)
were implanted subcutaneously in the flanks of 4-wk-old BALB/c mice. The
drug therapy consisted of intravenous administration of SACNs (1 mg/kg and
3 mg/kg equivalent Pt dose), cisplatin (1 mg/kg and 3 mg/kg equivalent Pt
dose), and carboplatin (3 mg/kg equivalent Pt dose). PBS (100 μL) by tail-vain
injection was used as a control for drug treatment. Treatment was started on
day 9 postimplantation, and administered every alternate day till day 13. The
tumor volumes and body weights were monitored on a daily basis. The tumor
volume was calculated by using the formula, L × B2. All animal procedures were
approved by the Harvard Institutional Use and Care of Animals Committee.
Transgenic Ovarian Cancer Tumor Model. Ovarian adenocarcinomas were in-
duced in genetically engineered K-rasLSL/+/Ptenfl/flmice via intrabursal delivery
of adenovirus carrying Cre recombinase. Tumor cells were engineered to ex-
press luciferase once activated by Adeno-Cre, to make tumor imaging feasible
before and after drug treatment. The drug therapy consisted of tail vein ad-
ministration of SACNs (3 mg/kg equivalent Pt dose), cisplatin (3 mg/kg equiv-
alent Pt dose), or PBS (100 μL). Each animal was dosed three times over the
course of treatment given every alternate day. Treatment efficacy was
quantified by examining the fold increase in bioluminescence of the post-
treatment signal compared with baseline. A detailed description is available
in SI Materials and Methods.
Biodistribution of SACNs. Tumor-bearing animals were treated as described
earlier. Organs were harvested, weighed, and dissolved in concentrated
HNO3. To these mixtures 30% (vol/vol) H2O2was added; the resulting sol-
utions were stirred for 24 h at room temperature and then heated for an-
other 12 h to evaporate the liquids. All solid residues were redissolved in 1
mL water and then amount of platinum was measured by inductive-coupled
plasma-atomic absorption spectrometry/MS.
Histopathology. The tissues were fixed in 10% formalin, paraffin-embedded,
and sectioned at the Harvard Medical School Core Facility. Tumor and kidney
paraffin sections were deparaffinized and stained with a standard TMR red
fluorescent TUNEL kit following the manufacturer’s protocol (In Situ Cell
Death Detection Kit, TMR-Red; Roche). The kidney sections were also
immunolabeled for KIM1 expression. Images were obtained using a Nikon
Eclipse TE2000 fluorescence microscope equipped with red filter. For further
details see SI Materials and Methods.
ACKNOWLEDGMENTS. This work is supported by US Department of Defense
Breast Cancer Research Program Era of Hope Scholar Award W81XWH-07-1-
0482; a Department of Defense Collaborative Innovator Grant; National
Institutes of Health Grant R01 CA135242-01A2; a Charles A. King Trust
Fellowship; a Department of Defense Breast Cancer Research Program
Postdoctoral Fellowship Award; the Burroughs-Wellcome Foundation; a Har-
vard Ovarian Cancer Spore Award; the Canary Fund; the Mary Kay Ash
Foundation; and a V Foundation for Cancer Research Scholar Award.
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| July 10, 2012
| vol. 109
| no. 28