Formulation, characterization, and antitumor properties of trans- and cis-citral in the 4T1 breast cancer xenograft mouse model

Article abstract of DOI:10.1007/s11095-015-1643-0

Purpose Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Methods Geranial and neral were synthesized. Commerciallypurchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 and 80mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Results Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Conclusions Geranial is significantly more potent than neral and citral at 80 mg/kg (p<0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

Full text of DOI:10.1007/s11095-015-1643-0

Pharm Res
DOI 10.1007/s11095-015-1643-0
RESEARCH PAPER
Formulation, Characterization, and Antitumor Properties
of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft
Mouse Model
San Zeng & Arvinder Kapur & Manish S. Patankar & May P. Xiong
Received: 4 November 2014 /Accepted: 27 January 2015
#
Springer Science+Business Media New York 2015
ABSTRACT
ABBREVIATIONS
Purpose Citral is composed of a random mixture of two geo-
metric stereoisomers geranial (trans-citral) and neral (cis-citral) yet
few studies have directly compared their in vivo antitumor prop-
erties. A micelle formulation was therefore developed.
Methods Geranial and neral were synthesized. Commercially-
purchased citral, geranial, and neral were formulated in PEG-b-
PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro
degradation, drug release, cytotoxicity, flow cytometry, and west-
ern blot studies were conducted. The antitumor properties of
drug formulations (40 and 80 mg/kg based on MTD studies) were
evaluated on the 4T1 xenograft mouse model and tumor tissues
were analyzed by western blot.
BW
Body weight
CMC
DLS
DMEM
EPR
FBS
geranial
i.v.
Critical micelle concentration
Dynamic light scattering
Dulbecco’s modified eagle medium
Enhanced permeability and retention
Fetal bovine serum
trans-citral
Intravenous
Loading efficiency
Maximum tolerated dose
cis-citral
Nanoparticle
LE
MTD
neral
NP
Results Micelles encapsulated drugs with >50% LE at 5–40%
drug to polymer (w/w), displayed sustained release (t1/2 of 8–9 h),
and improved drug stability at pH 5.0. The IC50 of drug formula-
tions against 4T1 cells ranged from 1.4 to 9.9 μM. Western blot
revealed that autophagy was the main cause of cytotoxicity.
Geranial at 80 mg/kg was most effective at inhibiting tumor
growth.
O/W
PBS
PCL
PDI
Oil-in-water
Phosphate buffered saline
Polycaprolactone
Polydispersity index
PEG-b-PCL Poly(ethylene glycol)-block-polycaprolactone
SDS Sodium dodecyl sulfate
Conclusions Geranial is significantly more potent than neral and
citral at 80 mg/kg (p<0.001) and western blot of tumor tissues
confirms that autophagy and not apoptosis is the major mecha-
nism of tumor growth inhibition in p53-null 4T1 cells.
INTRODUCTION
Citral (3,7-dimethyl-2,6-octadienal) is a naturally occurring
bioactive compound found in the essential oils of plants such
as citrus fruits, lemon grass and ginger, and is composed of a
mixture of the two terpenoid geometric stereoisomers geranial
.
.
.
.
KEY WORDS breast cancer citral geranial micelle neral
Electronic supplementary material The online version of this article
doi:10.1007/s11095-015-1643-0) contains supplementary material, which is
(trans-citral) and neral (cis-citral), Fig. 1. Citral has been used as
(
available to authorized users.
a natural additive to food, cosmetics, and beverages due to its
intense lemon aroma and flavor (1, 2). Essential oils containing
citral have been shown to exhibit antimicrobial, antifungal
and antiparasitic properties, (3–7) making citral a natural
preservative.
:
S. Zeng M. P. Xiong (*)
School of Pharmacy, University of Wisconsin–Madison
Madison, Wisconsin 53705-2222, USA
e-mail: mpxiong@pharmacy.wisc.edu
In mammals, the first report of citral as an inducer of cas-
pase 3 activity was reported in 2005 in the leukemic U937 and
HL60 cell lines (8). We have recently demonstrated that the
:
A. Kapur M. S. Patankar
Department of Obstetrics and Gynecology, University of
Wisconsin–Madison, Madison, Wisconsin 53705-2222, USA

Zeng et al.
Fig. 1 Geraniol was reacted with
manganese dioxide to generate
pure geranial (I) with a yield of 95%
yield. Nerol was reacted with
manganese dioxide to generate
pure neral (II) with a yield of 90%.
steam distilled extracts of ginger are actually composed of 30–
to improve citral stability in the context of food, fragrance, cos-
metics or beverage applications (2). However, none of these
reported formulations are ideal for cancer therapeutic applica-
tions since biomaterial safety, micelle size, drug encapsulation
and protection are all critical parameters to control in an i.v.
formulation. NPs such as micelles can typically take advantage
of the EPR effect (15) to successfully extravasate through leaky
blood vessels into the solid tumor.
4
0% citral and that this drug could induce caspase 3 activity,
activate p53 and decrease Bcl-2 expression, resulting in apo-
ptosis in two endometrial cancer cell lines (ECC-1 and
Ishikawa) at IC of 15–25 μM (9). The anticancer mechanism
5
0
and respective potency of citral and its isomers have not yet
been completely elucidated, but citral represents an emerging
class of natural products which demonstrates activity against
p53-expressing ovarian cancer cells (9). In addition, here we
report on evidence of geranial as the more potent isomer of
citral, being capable of inducing autophagy and not apoptosis
as the primary mechanism of cell death both in vitro and in vivo,
in mouse p53-null 4T1 breast cancer cells.
Here, we report on the formulation of citral, geranial, and
neral into biocompatible PEG-b-PCL micelles. PCL is an at-
tractive polymer for drug delivery due to the biocompatible
nature of the degradation products (16) and PCL is currently
approved by the FDA for use in humans. It has been observed
that unstable micelles can fall apart rapidly in plasma (17) and
lead to excessive drug loss. However, recent work has suggested
that PEG-b-PCL micelles can maintain sustained drug release
in the plasma due to enhanced circulation (18, 19) and low
CMCs, indicative of high stability. There are also recent studies
which have confirmed that PEG-b-PCL micelles are indeed
relatively stable in vivo (20, 21). PEG-b-PCL micelles were able
to encapsulate citral, geranial, and neral with high loading
efficiency (>50% between 5 and 40% w/w drug to polymer
ratio), displayed sustained release (t1/2 of 8–9 h), and improved
drug stability at pH 5.0. We also investigated the cytotoxic
properties of the formulations in vitro against the mouse
p53-null 4T1 breast cancer cells. The antitumor properties
of the formulations at two concentrations (40 and 80 mg/kg
based on MTD studies) were evaluated in vivo on the 4T1
xenograft mouse model and tumor tissues were collected at
termination of animal experiments for analysis by western blot.
To properly evaluate the anticancer properties of citral and
its isomers in vivo, a safe intravenous formulation is required.
Citral has a known aqueous solubility of ca. 590 μg/ml at
2
5°C and has previously been shown to degrade at
acidic pH and under oxidative stress into various oxida-
tive products (e.g. p-cymene, p-cresol, p-methylacetophenone,
8-hydroperoxy-p-cymene) dependent on pH, temperature, and
presence of oxygen (10, 11). The majority of citral chemical
stabilization methods reported in the literature have relied on
encapsulating the compound in O/W emulsions with or with-
out the addition of naturally-occurring antioxidants (e.g. ascor-
bic acid, β-carotene, black tea extract) (11). For example, in the
absence of antioxidants, negatively-charged SDS and positively-
charged chitosan were used to create layers of SDS-chitosan
complexes around emulsion droplets (ca. 0.41 μm in diameter)
and this barrier was shown to effectively retard oxidation-
induced citral degradation products, whereas gum arabic stabi-
lized O/W emulsions (ca. 1.1 μm in diameter) seemed more
effective at protecting citral from acid-induced degradation (12).
Citral has also been successfully incorporated into the hydro-
phobic environment of micelles formed from non-ionic
polyoxyethylene-type surfactants, (1, 13) micelles formed from
Tween 80, (14) and reverse micelles formed from polyglycerol
polyricinoleate for enhanced protection against acid-induced
degradations (14). Most of these formulations have been tailored
MATERIALS AND METHODS
Materials
Commercial citral, geraniol, nerol and active manganese (IV)
oxide (MnO ) were purchased from Sigma-Aldrich
2

Antitumor Properties of Trans- and Cis-Citral
1
(
Milwaukee, WI). Citral was analyzed by H-NMR and de-
neral were respectively incubated at 25 or 37°C for up to a
week, and the resulting geometric stability of geranial (Fig. 2a)
and neral (Fig. 2b) were characterized by H-NMR.
termined to have a 2:1 ratio of geranial to neral (see supple-
mental, Fig. 1S). PEG-b-PCL (Mn of PEG=5000 g/mol; Mn
of PCL=10,000 g/mol; Mw/Mn=1.26) was purchased from
Polymer Source (Quebec, Canada). Dialysis tubing
1
Preparation and Characterization of Geranial, Neral, or Citral
in PEG-b-PCL Micelles
(
MWCO=3500) was obtained from SpectraPor. DMEM,
PBS, FBS, trypsin-EDTA (0.05% trypsin, 0.48 mM EDTA
in HBSS) and penicillin-streptomycin were purchased from
Cellgro (MediaTech, Herndon, VA). The 4T1 cells (mouse
breast cancer) were obtained from ATCC and cultured ac-
cording to ATCC protocols. The Annexin V-FITC apoptosis
detection kit I containing propidium iodide to detect for apo-
ptosis and necrosis was purchased from BD Biosciences, San
Jose, CA. For the western blot, primary antibodies against
LC3B and Atg5 proteins were purchased from Cell
Signaling (Danvers, MA). Secondary antibodies against rabbit
were purchased from Jackson Immunoresearch (West Grove,
PA).
The micelles were prepared as previously reported; (23) typi-
cally 0.5 mM of diblock polymer PEG-b-PCL was dissolved in
1
ml of acetone, then 1 ml of ddH O was quickly added to the
2
polymer suspension while vigorously stirring. Geranial-loaded
micelles (geranial/NP), neral-loaded micelles (neral/NP) and
citral-loaded micelles (citral/NP) were prepared by first dis-
solving the drug at various concentrations with the polymer in
acetone and then adding ddH O. Note that citral here was
2
commercially purchased from Sigma-Aldrich and not purified
from ginger as previously done during earlier studies; this
batch of citral contained ca. 2:1 geranial to neral (this ratio
varies depending on the commercial source). The drug
spontaneously loads into the micelle during self-
association of the polymer chains and acetone was removed
by rotary evaporation. Micelles were centrifuged at
1
H-NMR spectra were obtained with a Varian Unity-
Inova 400 MHz NMR spectrometer (Palo Alto, CA), with
temperature regulation of 25°C or otherwise as indicated.
Chemical shifts are reported in ppm with respect to the deu-
terated solvent used.
All aspects of the animal studies were performed in accor-
dance with the guidelines defined by the Animal Research
Committee of the University of Wisconsin. Female BALB/c
mice (6–7 week old) were obtained from Jackson Laboratory
and divided into six groups (5 mice per group) for xenograft
tumor studies. General anesthesia to animals was induced
1
0,000 rpm for 5 min and passed through a 0.2 μm nylon
filter to remove un-encapsulated drug and/or aggregates.
Particle size was characterized at 25°C using a Zeta sizer
Nano-ZS (Malvern Instruments, UK) equipped with a He-Ne
ion laser (λ=633 nm). The hydrodynamic diameters of sam-
ples were obtained by cumulant method and are reported as
Z-average diameters.
The concentration of drugs in the micelles was measured
by reverse-phase HPLC (23, 24) equipped with a Symmetry
Shreld RP18 column from Waters, and monitoring geranial,
neral, or citral by UV detection at 254 nm (the diblock poly-
mer PEG-b-PCL did not absorb at this wavelength). The mo-
bile phase used was a cocktail of 40% water and 60% aceto-
nitrile containing 0.1% acetic acid. The concentration of drug
was increased from 5, 10, 20 to 40% (w/w) while keeping the
concentration of the diblock polymer PEG-b-PCL constant at
with 1.5% isoflurane/oxygen. Tumor volume (volume=
0
2
.5×l×w ) and animal BW were monitored daily.
Results are presented as mean±SEM, n=3–5. To com-
pare between data sets, Graphpad Prism 5 Software was used
to perform one way analysis of variance (ANOVA). A p<0.01
and p<0.001 were considered significant and denoted by * or
*
* respectively to indicate statistical differences.
Methods
0
.5 mM. Drug loaded and LE were calculated using the fol-
Synthesis and Geometric Stability of Geranial and Neral
lowing equations:
Citral isomers, geranial or neral, were prepared from geraniol
or nerol via oxidation of the primary alcohol group to alde-
hyde (Fig. 1) (22). To a flask containing 1.15 g (13.2 mmol) of
weight of drug incorporated
drug loaded ¼
weight of drug þ weight of polymer
activated MnO , a solution of 100 mg (0.65 mmol) of geraniol
2
weight of drug incorporated
loading efficiency ð%Þ ¼
 100
dissolved in 16 ml of hexane was added dropwise at 0°C, while
vigorously stirring. The reaction was allowed to proceed for
weight of drug
6
h at 0°C, followed by filtration. The filtrate was then
concentrated to yield the final product, which was oily,
with 95% yield. Neral was synthesized in the same
manner, using nerol as the primary alcohol, with 90% yield.
The chemical structures of pure geranial and neral were con-
In Vitro Cytotoxicity
4T1 cells were seeded in a 96-well plate at 5000 cells per well
for 24 h. The next day, the medium was replaced with
10 μl of formulations containing either empty NP,
1
firmed by H-NMR (see supplemental, Fig. 1S). Geranial and

Zeng et al.
1
Fig. 2 H-NMR of geranial and
neral in CDCl after incubating for
days at 25 or 37°C. There is no
change in the spectrum of geranial
a) after incubating at 37°C (I) or
5°C (II) for up to 7 days. Similarly,
3
7
(
2
there was no evidence of change
from neral to geranial (b) incubated
at 25°C (II) for up to 7 days;
however two new peaks at ca.
9
.95 ppm characteristic of geranial
begin to appear on day 7 with
incubation at 37°C (I). The ratio of
these two new peaks were
integrated with respect to the neral
peaks at ca. 9.85 ppm and was
determined to be about 1:1
indicative of 50% neral conversion
to geranial by day 7 at 37°C.
citral/NP, neral/NP or geranial/NP; formulations were
added to wells at a final concentration of 0.01–1 mM
drug. The cells were incubated for 72 h and cell viabil-
ity (as measured by metabolic activity) was monitored
with the resazurin assay (25). The IC s were obtained
Flow Cytometry
2
4T1 cells were seeded in 60 cm cell culture dishes and incu-
bated overnight at 37°C/5% CO for 24 h. Cells were then
treated with either 25 μM geranial/NP (9), PBS, or NP as the
negative control for 24 and 48 h. At each time point, both
dead and live cells were collected, washed with PBS, and re-
suspended in 1x binding buffer (10 mM HEPES/NaOH,
2
5
0
by curve fitting with GraphPad Prism 5 Software.
Western Blot
pH 7.4, 140 mM NaCl, 2.5 mM CaCl ) to a final concentra-
2
6
5
tion of 1×10 cells/ml. Next, 1×10 cells were stained with
the Annexin V-FITC apoptosis detection kit I by incubation
at room temperature for 15 min. Afterwards, 400 μl of bind-
ing buffer was added to each sample and the cells were ana-
lyzed on a FACSCalibur flow cytometer. The data was ana-
lyzed with FlowJo® software (version 10.0.6) by gating n=20,
000 cells. Note that gated apoptosis data reported are the sum
of both apoptotic and late apoptotic cells.
4
T1 cells were seeded at 25,000 cells per well in a 6-well plate
for 24 h. Cells were then treated with 25 μM of citral/NP,
neral/NP or geranial/NP for an additional 48 h, using PBS
and NP as control groups. Cell lysates were collected and
loaded onto a 12% SDS-PAGE gel and the gel was run at
1
00 V for 55 min. The protein bands were then transferred to
a PVDF membrane at 100 V for 1 h, blocked with 5% BSA
for 1 h, and the autophagy proteins LC3B and Atg5 were
simultaneously detected by incubating the membrane with
appropriate primary antibodies overnight at 4°C. The mem-
brane was developed through the use of secondary antibodies
conjugated to horseradish peroxidase by incubating at room
temperature for 1 h before imaging.
pH Degradation Studies
For degradation studies, 10 mM of either geranial/NP, free
geranial, neral/NP, free neral, citral/NP or free citral in
0.5 mL final volumes were respectively incubated at pH 5.0

Antitumor Properties of Trans- and Cis-Citral
and pH 7.4 for up to 24 h at room temperature; drug stability
was monitored by HPLC at time t=0, 2, 4, 8, 12, and 24 h.
Empty micelles incubated under these conditions did not show
instability or evidence of PEG-b-PCL polymer degradation
nerol to neral (Fig. 1). The geometric structures of geranial
and neral were confirmed by H-NMR (see supplemental,
1
Fig. 1S). The characteristic peaks of geranial and neral at ca.
10 and ca. 9.9 ppm respectively were used to determine the
ratio of each isomer present in the citral purchased from
Sigma-Aldrich, with results revealing that the ratio of geranial
to neral was 2:1 in this particular batch (see supplemental,
Fig. 1S).
(data not shown). Note that in this assay, we did not identify
degradation products for citral nor its isomers geranial and
neral since it was not the focus of this work; if interested, citral
degradation products have already previously been meticulous-
ly reported (10, 11). Differences between degradation profiles at
pH 5.0 and 7.4 for geranial, neral, and citral were monitored by
HPLC (column temperature of 40°C, mobile phase consisted of
water: acetonitrile at 40:60, v/v, flow rate of 0.5 ml/min) to
investigate drug stability when formulated in micelles.
The geometric stability of free geranial and neral was in-
vestigated by incubation at 25 and 37°C for up to 7 days with
1
H-NMR. For geranial, there was no apparent change to
1
neral found in the H-NMR after incubating at both 37°C
(I) and 25°C (II) for up to 7 days (Fig. 2a). For neral, we
1
observed no apparent change in the H-NMR after incubat-
Animal Studies
ing at 25°C (II) for up to 7 days but there was an obvious peak
shift observed in the H-NMR of neral after incubation
1
For MTD studies, we made an educated guess by starting with
a single 100 mg/kg dose of citral/NP via retro-orbital injections
into normal Balb/c mice (n=3 animals/dose). Generally, about
at 37°C (I) on day 7 (Fig. 2b). More specifically, we
noted the appearance of peaks characteristic of geranial
at ca. 10 ppm; by integrating characteristic neral and
geranial peaks, we obtained a ratio of 1:1 suggesting that
50% of neral had converted to geranial by day 7 when incu-
bated at 37°C.
200 μl of the citral/NP was injected into animals (n=3) which
were then monitored over time. This assay and drug concen-
trations tested were done sequentially, meaning that we didn’t
inject the next dose into animals until we knew whether the
dose tested had been acutely toxic to animals based on changes
in behavior, bleeding, and overall appearance. Final concentra-
Preparation and Characterization of Geranial, Neral,
tions utilized for the MTD studies were 100, 90, and 80 mg/kg. or Citral in PEG-b-PCL Micelles
6
For tumor-bearing studies, approximately 1×10 4T1 cells
were inoculated subcutaneously into the right flanks of anes-
thetized mice. When the tumor size reached about 50 mm ,
Micelles were prepared from PEG-b-PCL (5000:10,000)
3
diblock copolymer by nanoprecipitation. The amount of drug
encapsulated into the micelles was measured by HPLC, and
the LE ranged between 54 and 99% for geranial, 55–97% for
neral, and 51–97% for citral at drug to polymer (w/w) ratio of
5–40% (Table I). Resulting micelle sizes were characterized
with DLS and ranged between 77 and 85 nm for citral/NP,
88–66 nm for geranial/NP, and 74–79 nm for neral/NP at
5–40% w/w drug to polymer ratio (see supplemental,
Table IS).
For the release study, drug-loaded micelles were prepared
at the 5% w/w ratio of drug to polymer to maximize
drug encapsulation within the PCL core rather than at
the interfaces of micelles. The micelle formulations were
diluted with respect to drug concentration to 0.1 mg/ml
2
00 μl of the various formulations (saline, empty NP, C40,
C80, G40, G80, N40, and N80) were injected into the animals
on days 13, 16, 19 and 22. The C, G, or N stands for either
citral, geranial, or neral, and the number represents the con-
centration injected, either 40 or 80 mg/kg; for example, C40
means that 40 mg/kg of citral was injected. The tumor size
and BW of animals were monitored daily. The animals were
sacrificed when tumors in the control group reached about
3
2
000 mm . Tumor tissues were dissected, cut into small pieces
and placed immediately on ice. Next, 250 μl of RIPA buffer
with protease inhibitor was added to 5 mg of minced tissue
and the samples were sonicated on ice to break the tissue
further. The protein lysates from tumor tissues were then load-
ed onto a 12% SDS-PAGE gel for western blot analysis of
autophagy proteins LC3B (14 kDa) and Atg5 (55 kDa).
in ddH O and loaded into a dialysis cassette. The re-
2
lease study was conducted by dialyzing the cassettes
against ddH O at 37°C since citral (and consequently geranial
2
and neral) has a water solubility of ca. 590 μg/mL. At various
time points, 20 μl of suspension was withdrawn from the dial-
ysis cassettes for citral analysis by RP-HPLC to quantify the %
RESULTS
Synthesis and Geometric Stability of Geranial
and Neral
of drug left and 20 μl of ddH O was added to replenish the
2
volumes in the cassettes. For citral/NP we obtained a t1/2 of
ca. 8.5 h to 50% drug release; for geranial/NP we obtained a
t1/2 of ca. 8.3 h and for neral/NP, we obtained a t1/2 of ca.
8.9 h (see supplemental, Fig. 2S).
The two isomers of citral were synthesized by oxidation of
alcohol using MnO , converting geraniol to geranial, and
2

Zeng et al.
Tab le I Characterization of Geranial, Neral, and Citral Loading Levels into 0.5 mM PEG-b-PCL Micelles. On a w/w Ratio of Drug to Polymer, the Drug
Concentration was Then Increased from 5 to 40%
w/w
Initial drug
mg/ml)
Drug solubilized (mg/ml)
Efficiency % loaded
Geranial
(
Geranial
Neral
Citral
Neral
Citral
5
1
2
4
%
0.170
0.341
0.682
1.364
0.168± 0.013
0.289± 0.012
0.380± 0.018
0.739± 0.016
0.166± 0.029
0.202± 0.012
0.383± 0.026
0.748± 0.009
0.165± 0.005
0.260± 0.022
0.397± 0.024
0.692± 0.015
98.8± 1.6
84.7± 1.5
55.7± 3.2
54.2± 1.6
97.4± 3.7
59.3± 2.7
56.2± 1.5
54.8± 1.8
96.7± 2.9
76.2± 6.6
58.3± 3.5
50.7± 1.1
0%
0%
0%
In Vitro Cytotoxicity
breast cancer cells preferably underwent autophagy upon in-
cubation with the drugs.
To compare the cytotoxicity of geranial, neral and citral (the
commercial mixture of geranial and neral at ratio of 2:1 w/w,
Sigma-Aldrich), 4T1 cells were seeded in 96-well plates for
Flow Cytometry
2
4 h. The next day, cells were treated with 0.01 μM–1 mM
Since cytotoxicity and western blot studies demonstrated that
geranial was more potent than neral or citral, only geranial/
NP was investigated for evidence of apoptosis. Flow cytometry
revealed that there were no significant differences between the
control groups and the geranial/NP-treated cells after 24 or
48 h incubation, indicating that the cell death mechanism
induced by geranial was not apoptosis (see supplemental,
Fig. 4S).
drugs and allowed to incubate for 72 h. Cell viability was
measured by the resazurin assay. As shown in Fig. 3a, the
IC50 of geranial/NP at 72 h was found to be 1.4 μM,
neral/NP cytotoxicity was characterized by an IC50 of
9
.9 μM, and citral/NP cytotoxicity was characterized by an
IC50 of 4.5 μM. There was no apparent cytotoxicity to cells
incubated with empty NP (i.e. micelles) compared to cells-only
controls (data not shown).
pH Degradation Studies
Western Blot
Solutions containing neral and neral/NP (Fig. 4a and b),
geranial and geranial/NP (Fig. 4c and d), or citral
and citral/NP (Fig. 4e and f) at final concentrations of
10 mM were incubated at room temperature at pH 5.0
and pH. 7.4 for up to 24 h. The pH degradation of the
drug was monitored via HPLC by UV detection of the
drug at 254 nm. HPLC data for neral, geranial, and
citral incubation at pH 7.4 reveals that all the drugs
4
T1 cells were treated with 25 μM of citral/NP, neral/NP, or
geranial/NP for 48 h and then autophagy proteins LC3B and
Atg5 were detected by western blot (Fig. 3b). The strongest
LC3B and Atg5 band intensities were observed in cells treated
with geranial/NP, followed by citral/NP, and then neral/NP,
in comparison to untreated or NP-only treated cells (see sup-
plemental, Fig. 3S) and confirm that p53-null 4T1 mouse
Fig. 3 Cytotoxicity assay on 4T1 cells after incubating with citral/N P, neral/NP and geranial/NP for 72 h (a). At the highest concentrations of drug tested, empty
NP were not toxic to cells with >90% cell viability, data not shown on graph. The IC50 for citral/N P, neral/NPand geranial/NP was calculated to be 4.5, 9.9, and
1
.4 μM respectively by curve fitting with Graph Pad Prism 5 Software. After 24 h incubation, western blot reveals increased expression of Atg5 (55 kDa) and LC3B
(
14 kDa) proteins in 4T1 cells treated with 25 μM of each formulation, indicative of autophagy (b).

Antitumor Properties of Trans- and Cis-Citral
Fig. 4 The acid-induced
degradation of neral, geranial, and
citral was monitored via HPLC by
incubating drugs at pH 5.0. The
profiles for geranial (a) and geranial/
NP (b) at t=0 show the charac-
teristic peaks for this isomer at ca.
6
.1 min (peak 2); the profiles for
neral (c) and neral/NP (d) at t=0
show the characteristic peaks for
this isomer at ca. 5.9 min (peak 1);
the profiles for citral (e) and citral/
NP (f) at t=0 show the character-
istic peaks for this drug at ca. 5.9 min
(
peak 1) and ca. 6.1 min (peak 2)
corresponding to both neral and
geranial respectively. Note that in-
cubation at pH 7.4 for all the drugs
did not show evidence of degrada-
tion up to the 24 h monitored (see
supplemental, Fig. 5S). With in-
creasing incubation time, 0 h (I), 2 h
(
2
II), 4 h (III), 8 h (IV), 12 h (V) and
4 h (VI), we begin to see more
evidence of drug degradation (as
evidenced by the increasing intensi-
ty of peaks observed between 0
and 5 min) compared to the de-
creasing characteristic drug peaks at
t=0 (I).
and micelle formulated drugs were relatively stable at this pH,
with little evidence of drug degradation up to the 24 h mon-
itored (see supplemental, Fig. 5S).
t=0 for this isomer at ca. 5.9 min and with increasing time
(II-VI), we begin to see more evidence of neral degradation
products compared to neral/NP. For example, peak 2 cor-
responding to geranial can be readily observed after 2 h incu-
bation with pure neral (Fig. 4c), but this peak is not observed
with neral/NP until after 8 h incubation (IV) (Fig. 4d). At
pH 5.0 and t=0, citral is characterized by peaks 1 and 2
(Fig. 4e) corresponding to neral and geranial respectively.
Degradation of these peaks occurred much faster for free citral
than for citral/NP over the 0, 2, 4, 8, 12 and 24 h monitored
(I-VI respectively). For example, at t=8 h, there was evidence
At pH 5.0, the degradation of pure geranial (Fig. 4a), peak
2
at approx. 6.1 min, occurred much faster than geranial/NP
(
Fig. 4b) over the 0, 2, 4, 8, 12 and 24 h monitored (I-VI
respectively). For example, at 4 h incubation (III) there is ev-
idence of free geranial conversion to neral (due to appearance
of the neral peak 1 at approx. 5.9 min) and presence of more
degradation products than geranial/NP. Similarly, the HPLC
profile for neral at pH 5.0 shows the characteristic peak 1 at

Zeng et al.
of more degradation products in citral than in citral/NP,
which still revealed two strong peaks for neral and geranial
toxicity in animals based on consistent animal weights
throughout the treatment period compared to controls
(Fig. 5b); a BW loss of± 20% would have indicated acute
toxicities to animals.
(Fig. 4f).
At the end of the study (day 24), animals were all promptly
euthanized and tumors were excised (Fig. 6a, see supplemen-
tal Fig. 6S) for western blot analyses. Western blot analyses of
tumor tissues reveals that autophagy is indeed the major
mechanism of tumor growth inhibition for animals treated
with the formulations (Fig. 6b). At 40 mg/kg treatment con-
centrations, we see evidence of autophagy proteins Atg5 and
LC3B being increasingly expressed across all formulations
(C40, N40 and G40, C80, N80, G80). More specifically, there
was statistically more expression of Atg5 and LC3B compar-
ing G80 to G40, N80 to N40, and C80 to C40, p<0.001;
there was also more expression of Atg5 and LC3B in G80-
treated animals compared to N80-treated animals, p<0.001,
suggesting that geranial is more effective at inducing autoph-
agy (Fig. 6c and d).
Animal Studies
For MTD studies, at dosage of 100 mg/kg we observed that 2
mice died immediately after the injection and 1 mouse died
the next day. When we decreased the dose to 90 mg/kg, we
saw that 1 mouse died the next day and 2 mice survived. At
8
0 mg/kg citral/NP, all three animals survived and looked
normal for up to 2 weeks based on signs of acute toxicities
e.g. changes in behavior, bleeding, and overall appearance).
(
This was deemed the MTD for citral and its respective
isomers.
The xenograft tumor model was established in mice by
subcutaneously injecting 4T1 mouse breast cancer cells into
the right flanks of animals. C40, C80, G40, G80, N40, or N80
was injected i.v. via retro-orbital injections to animals on day
1
3, 16, 19, and 22; the control groups received either empty
NP or saline. The tumor size and BW of animals were mon-
itored daily to assess the therapeutic efficacy and acute toxicity
of the treatments in animals. The empty NP treatments did
not inhibit tumor growth similar to saline (p>0.05), whereas
all the drug formulations at 40 and 80 mg/kg did slow down
growth of this aggressive xenograft tumor model to different
extents when compared to controls (p<0.01). The anti-tumor
efficacy of C40, G40, and N40 formulations were all effective
with a tumor volume reduction of approximately 32–49%
compared to the NP control (Fig. 5a). More significantly, the
results for G80 revealed 92% tumor volume reduction by day
DISCUSSION
We have recently demonstrated that the steam distilled ex-
tracts of ginger composed of 30–40% citral could induce cas-
pase 3 activity, activate p53 and decrease Bcl-2 expression,
resulting in apoptosis in two endometrial cancer cell lines
(ECC-1 and Ishikawa) at IC50 of 15–25 μM (9). The antican-
cer mechanism of citral has not yet been completely elucidat-
ed, but citral represents an emerging class of natural products
which has potential to be developed as a chemotherapeutic
agent against several forms of cancer.
2
4 compared to the NP vehicle (p<0.001) and G40 displayed
a 49% tumor volume reduction by day 24 compared to the
NP vehicle (p<0.001). Comparing G80 to C80 and N80,
there was a statistically significant difference in tumor volume
reduction between G80 and C80 (p<0.001), and G80 and
N80 (p<0.001). The formulations did not generate acute
Although citral is known to be composed of a mixture of
geranial (trans-citral) and neral (cis-citral), little work has been
done to investigate the anticancer properties of these respec-
tive isomers. For example, the potent anticancer agent cisplat-
in can kill cancer cells by binding to DNA and interfering with
Fig. 5 Mice (n=5) received four retro-orbital injections on days 13, 16, 19, and 22 of either saline, empty N P, C40, N40, G40, C80, N80, or G80. The saline
and NP treatments did not inhibit tumor growth (p>0.05), whereas C40, N40 and G40 were relatively all effective at reducing tumor burden 32–49%
compared to NP controls by day 24. The most significant tumor regression was observed with G80. There was significant difference in tumor volume reduction
between G80 and C80 (p<0.001), and G80 and N80 (p<0.001) (a) and suggest that geranial is more potent than neral at 80 mg/kg. The formulations did not
generate acute toxicity in animals based on consistent animal weights throughout the treatment period (b). **p<0.001.

Antitumor Properties of Trans- and Cis-Citral
Fig. 6 Animals were euthanized and tumors were excised (a) for western blot analysis of autophagy proteins Atg5 and LC3B (b). There was more expression of
Atg5 and LC3B in G80 tumors compared to G40 tumors, and there was also more expression of Atg5 (c) and LC3B (d) in G80 tumors compared to N80
tumors, suggesting that geranial is more effective than neral or citral at inducing autophagy at the same concentrations. The degree of tumor inhibition appears to
correlate with increasing induction of autophagy in tumor tissues. *p<0.01 and **p<0.001.
cell repair mechanisms but its trans isomeric form transplatin
binds differently to DNA and is therefore inactive (26, 27).
Before formulation of citral was investigated, we set about
synthesizing pure geranial and neral in the lab (Fig. 1) to
compare each isomer’s relative cytotoxicity with respect to
commercially purchased citral from Sigma, containing a ratio
EPR effect for extravasation through leaky blood vessels into
the solid tumor tissue. Clearly, the geometric conformation of
geranial and neral does not appear to play a critical role in LE
of the isomers into PEG-b-PCL micelles. Citral was previously
successfully encapsulated into micelles formed from non-ionic
polyoxyethylene alkylether surfactants and was also found to
preferentially solubilize citral within the alkylether core of
C H -PEO micelles, (1) but to the best of our knowledge
this is the first report of drug loading properties comparing
geranial to neral. The in vitro release study of geranial, neral,
and citral from PEG-b-PCL micelles was performed by the
dialysis cassette method at 37°C against water for 48 h and
demonstrated comparable sustained release properties for all
the drugs, characterized by a half-life of ca. 8.5 h for citral, 8.3 h
for geranial, and 8.9 h for neral (see supplemental, Fig. 2S).
When we evaluated the cytotoxicity of geranial/NP,
neral/NP, and citral/NP on 4T1 mouse breast cancer
cells, the IC50 of geranial was calculated to be 1.4 μM,
9.9 μM for neral, and 4.5 μM for citral. Empty NP (i.e.
PEG-b-PCL micelles) did not display evidence of cytotoxicity
to cells at the highest concentration of drugs tested (>90% cell
viability) so we did not investigate cytotoxicity of the polymer
for lower concentrations (data could not be plotted in Fig. 3 so
it is not shown). It should also be noted that PCL is well-known
1
of approximately 2:1 geranial to neral based on H-NMR (see
supplemental, Fig. 1S). The yield for syntheses of geranial and
neral were 95 and 90% respectively. We incubated each iso-
mer for up to 7 days at temperatures of 25 and 37°C, and
found that the geranial (trans-citral) isomer was stable with no
detectible conversion to neral (cis-citral) for either temperature
12
25
4
(Fig. 2a). Similarly, neral (cis-citral) did not display evidence of
change to geranial after incubating at 25°C up to 7 days;
however, we did observe 50% conversion of cis-citral to trans-
citral by day 7 at higher incubation temperature of 37°C
(Fig. 2b).
We successfully encapsulated citral and its isomers geranial
and neral into PEG-b-PCL micelles. The drug encapsulation
levels for geranial, neral, and citral were similar, with all drug
loading above 50% at 5–40% (w/w) drug to polymer ratio
(Table I). Drug loaded micelles were also similarly sized, rang-
ing between 66 and 88 nm in diameter (see supplemental,
Table IS) and are small enough to take advantage of the

Zeng et al.
to be biocompatible (16), being currently approved by the
FDA for use in humans (e.g. biodegradable sutures). When
reduction between G80 and C80 (p<0.001), and G80 and
N80 (p<0.001) (Fig. 5a). In vivo results suggest that geranial is
more potent than neral at 80 mg/kg, with a 92% reduction in
tumor volume by day 24 compared to NP controls. Since C80
contains approximately 2:1 geranial to neral in the formula-
tion, it makes sense that its antitumor properties would be
slightly less effective than G80 but better than N80. In addi-
tion, the formulations did not generate acute toxicity in ani-
mals based on consistent animal weights throughout the treat-
ment period (Fig. 5b). In vivo results suggest that geranial is
therefore more potent than neral at 80 mg/kg.
At the end of the study (day 24), all animals were eutha-
nized and tumors were excised (Fig. 6a, see supplemental
Fig. 6S) for western blot analysis of autophagy proteins Atg5
and LC3B. Confirming the in vitro western blot data (Fig. 3b),
western of tumor tissues revealed that autophagy was indeed
the major mechanism of tumor growth inhibition for animals
treated with the formulations (Fig. 6b). Interestingly,
there was statistically more expression of Atg5 and
LC3B for G80-treated animals compared to G40,
p<0.001, and there was also more expression of Atg5 and
LC3B in G80-treated animals compared to N80-treated ani-
mals, p<0.001, suggesting again that geranial is more effective
than neral or citral at inducing autophagy at the same con-
centrations (Fig. 6c and d). The degree of tumor inhibition
appears to correlate with increasing induction of autophagy
in tumor tissues.
4
T1 cells were treated with 25 μM citral/NP, neral/NP, or
geranial/NP, western blot detected up-regulation of LC3B
and Atg5 proteins (Fig. 3b), which are known to be involved
in the regulation of autophagy (28). Autophagy is a self-
digestion mechanism that is activated to ensure cell sur-
vival under stress and there is strong evidence of a link
between cancer and regulation of autophagy as either a
tumor suppressor or a tumor promoter (29). One possi-
ble role for autophagy-induced recycling in established
cancers is to maintain a functional pool of mitochondria
after mitochondrial damage, (28, 29) which may explain
the up-regulation of autophagy proteins and decrease in
metabolism (cell viability) detected in 4T1 cells treated with
the drugs. Although we had found in previous studies that the
mechanism of cell death by citral in two endometrial cancer
cell lines was mainly due to apoptosis via activation of p53, (9)
this seemed unlikely to be the case for 4T1 cells since they do
not express p53 (p53-null) (30). This was verified by treating
4
T1 cells with 25 μM of geranial/NP for up to 48 h and
assaying cells for evidence of apoptosis by flow cytometry
see supplemental, Fig. 4S); data indeed confirmed that the
(
mechanism of cell death was not apoptosis.
The acid-induced degradation of neral, geranial, and citral
formulated in micelles was investigated at pH 5.0 by incubat-
ing samples at room temperature for up to 24 h (Fig. 4). It’s
important to note that none of the drugs, in free form or when
formulated in micelles, showed evidence of degradation when
incubated at room temperature at pH 7.4 (see supplemental,
Fig. 5S). This data does agree with our previous NMR stabil-
ity studies conducted at 25°C which had revealed that both
geranial and neral remained stable geometric stereoisomers at
this temperature (Fig. 2). At pH 5.0, all the drugs were indeed
better protected from acid-induced degradation compared to
the free drugs (Fig. 4), with degradation correlating to drug
release from micelles. Empty micelles incubated under these
conditions did not show evidence of micelle instability or poly-
mer degradations during the course of the study (data not
shown). Protection from acid-degradation is most likely a re-
sult of the drug preferentially partitioning into the hydropho-
bic core of PEG-b-PCL micelles.
For the animal studies, mice (5 per group) received four
retro-orbital injections on days 13, 16, 19, and 22 of either
saline, empty NP, C40, N40, G40, C80, N80, or G80. The
tumor size and BW of animals were monitored daily to assess
the therapeutic efficacy and acute toxicity of the treatments in
animals. The saline and NP treatments did not inhibit tumor
growth (p>0.05), whereas C40, N40 and G40 were relatively
all effective at reducing tumor burden 32–49% compared to
NP controls by day 24. The most significant tumor regression
was observed with G80. Comparing G80 to C80 and N80,
there was a statistically significant difference in tumor volume
CONCLUSIONS
Citral is a naturally occurring bioactive compound found in
the essential oils of plants such as citrus fruits, lemon grass and
ginger, and is composed of a mixture of the two terpenoid
isomers geranial (trans-citral) and neral (cis-citral). PEG-b-
PCL micelles were able to encapsulate citral, geranial and
neral with >50% loading efficiency at 5–40% drug/polymer
(w/w) and displayed similar patterns of sustained drug release
(t1/2 of 8–9 h). The IC50 of geranial, citral and neral formu-
lation against p53-null 4T1 cells ranged from 1.4 to 4.5 to
9.9 μM respectively; western blot revealed in vitro evidence of
autophagy as the primary mechanism of cell growth inhibition
in 4T1 cells. Flow cytometry revealed that apoptosis/necrosis
did not play a role in inhibiting cell growth. The rate of drug
degradation decreased at pH 5.0 up to 24 h when formulated
in micelles compared to free drugs; there was no evidence of
drug degradation (free or formulated into NP) at pH 7.4 up to
24 h. In vivo studies on mice bearing 4T1 xenograft tumors
revealed that geranial is the more potent isomer of citral and
western blot of tumor tissues confirmed that autophagy was
the major mechanism of tumor growth inhibition in p53-null
4T1 cells. None of the formulations tested exhibited evidence
of adverse acute toxicity to animals based on body weights.

Antitumor Properties of Trans- and Cis-Citral
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ACKNOWLEDGMENTS AND DISCLOSURES
This research was supported by NIH grant R01DK099596
and startup funds from the University of Wisconsin-Madison,
School of Pharmacy.
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