Redox-Sensitive Citronellol-Cabazitaxel Conjugate: Maintained in Vitro Cytotoxicity and Self-Assembled as Multifunctional Nanomedicine

Article abstract of DOI:10.1021/acs.bioconjchem.6b00155

Citronellol-cabazitaxel (CIT-ss-CTX) conjugate self-assembled nanoparticles (CSNPs) were designed and prepared by conjugating cabazitaxel with citronellol via the disulfide bond that is redox-sensitive to the high concentration of glutathione within tumor cells. Notably, the CSNPs maintained in the cell cytotoxicity. Moreover, the AUC_0-t of CSNPs was 6.5-fold higher than that of cabazitaxel solutions and the t_1/2 was prolonged 2.3 times. Furthermore, we found that CSNPs could be employed as an efficient carrier for other hydrophobic drugs or imaging agents. Thus, the in vivo targeting study was implemented via using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)-loaded CSNPs as imaging agent, which showed CSNPs could effectively accumulate at the tumor site. Curcumin, a hydrophobic anticancer drug, was successfully loaded in CSNPs which exhibits good stability and synergistic antitumor effects. The citronellol-cabazitaxel conjugate therefore has a promising perspective as a multifunctional nanomedicine for combination therapy and theranostics attributed to its long-circulation property, redox-sensitive mechanism, and high drug coloading capability.

Full text of DOI:10.1021/acs.bioconjchem.6b00155

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Article
Redox-Sensitive Citronellol-Cabazitaxel Conjugate: Maintained in Vitro
Cytotoxicity and Self-Assembled as Multifunctional Nanomedicine
Peng Xue, Dan Liu, Jing Wang, Na Zhang, Jiahua Zhou, Lin Li,
Weiling Guo, Mengchi Sun, Xiangfei Han, and Yongjun Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00155 • Publication Date (Web): 12 Apr 2016
Downloaded from http://pubs.acs.org on April 16, 2016
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Redox-Sensitive Citronellol-Cabazitaxel Conjugate: Maintained in
Vitro Cytotoxicity and Self-Assembled as Multifunctional
Nanomedicine
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Peng Xue,† Dan Liu,‡ Jing Wang,† Na Zhang,‡ Jiahua Zhou,† Lin Li,† Weiling Guo,†
Mengchi Sun,† Xiangfei Han,† and Yongjun Wang*,†
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† School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang,
110016, China
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Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
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Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China
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ABSTRACT GRAPHIC
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ABSTRACT
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Citronellolꢀcabazitaxel (CITꢀssꢀCTX) conjugate selfꢀassembled nanoparticles
(CSNPs) were designed and prepared by conjugating cabazitaxel with citronellol via
the disulfide bond that is redoxꢀsensitive to the high concentration of glutathione
within tumor cells. Notably, the CSNPs maintained in the cell cytotoxicity. Moreover,
the AUC0ꢀt of CSNPs was 6.5ꢀfold higher than that of cabazitaxel solutions and the t1/2
was prolonged 2.3 times. Furthermore, we found that CSNPs could be employed as an
efficient carrier for other hydrophobic drugs or imaging agents. Thus the in vivo
targeting
study
was
implemented
via
using
1,1ꢀdioctadecylꢀ3,3,3,3ꢀ
tetramethylindotricarbocyanine iodide (DiR)ꢀloaded CSNPs as imaging agent, which
showed CSNPs could effectively accumulate at tumor site. And curcumin, a
hydrophobic anticancer drug, was successfully loaded in CSNPs which exhibits good
stability and synergistic antitumor effects. The citronellolꢀcabazitaxel conjugate
therefore has a promising perspective as a multifunctional nanomedicine for
combination therapy and theranostics attribute to its long circulation property, redoxꢀ
sensitive mechanism and high drug coꢀloading capability.
KEYWORDS: cabazitaxel, disulfide bond, conjugate selfꢀassembled nanoparticles,
redoxꢀsensitive, multifunctional nanomedicine
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48 INTRODUCTION
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Cabazitaxel (CTX) is an anticancer drug of taxanes which can be used for the
treatment of metastatic castrationꢀresistant prostate cancer (mCRPC).1,2 It has the
similar mechanism with docetaxel that act by binding to microtubules, thereby
promoting the assembly of tubulin into stable microtubules and simultaneously
inhibiting disassembly, which results in the blockage of mitosis at the G2/M phase
and leads to cell death.3,4 Cabazitaxel (Jevtana, Sanofi–Aventis) was approved by the
US Food and Drug Administration (FDA) in June 2010.5 Jevtana used in clinic
requires two times of dilutions before intravenous infusion, and it has a significant
advantage that can overcome multidrug resistance (MDR) due to its poor affinity to Pꢀ
glycoprotein (Pꢀgp) — a member of the MDR protein family that pumps the
intracellular drug out of cells — compared with docetaxel and paclitaxel.6 But there
also exists some problems: the drug solutions must be prepared immediately before
use because of the instability of cabazitaxel and it has adverse side effects especially
to neutropenia and leucopenia.7 In the past years, biocompatible nanosystems such as
polymeric micelles and polymeric nanoparticles have been designed for the delivery
of cabazitaxel8ꢀ12 and have improved safety and the ability of targeting tumor site due
to the enhanced permeability and retention (EPR) effect.13 Nevertheless, the
nanosystems suffer low drug loading capacity and poor stability in vivo. An
appropriate drug delivery system should be developed to improve the drug loading
capacity and control the release of drug in tumor site.
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Tumor microenvironment, referring to cellular components such as endothelial
cells, tumorꢀassociated macrophages, cancerꢀassociated fibroblasts, inflammatory
cells and nonꢀcellular components such as matrix metalloproteinases, integrins,
cytokines and their receptors, is suitable for tumor growth, invasion and metastasis.14ꢀ
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These cellular and nonꢀcellular components give tumor microenvironment special
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features including hypoxia, acidic, interstitial hypertension and chronic inflammatory
response.17 And the tumor intracellular microenvironment is quite particular because
of its strong redox status caused by the high concentration of glutathione (GSH, 1ꢀ11
mM) compared with GSH in blood circulatory system (4ꢀ8 ꢁM).18,19 Thus, some
studies have designed redoxꢀsensitive drug delivery system such as redoxꢀsensitive
micelles for improving the targeting effect on tumor cells. These sensitive micelles
usually contain disulfide linkage that is stable in blood circulation and can be rapidly
cleaved through thiolꢀdisulfide exchange reactions with tumor intracellular GSH.19ꢀ21
In our previous study, the disulfideꢀlinked prodrugs such as paclitaxelꢀssꢀvetamin E
were synthesized and used selfꢀassembled into nanoparticles.22 DSPEꢀPEG2000 was
used as stabilizer which the hydrophobic part was inserted into the nanoparticles and
the PEG chains were exposed to the surface to give the nanoparticles a feature of long
circulation that the nanoparticles could not be recognized and cleared by the
reticuloendothelial system.16 The nanoparticles have good stability and GSHꢀtriggered
drug release, demonstrating that selfꢀassembled nanoparticles can be employed for
effective delivering drugs to tumor intracellular microenvironment.
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Citronellol (CIT) is a monoterpene alcohol which has antioxidant and
antiinflammatory properties.23,24 It has been reported that citronellol has moderate
anticancer activities, and it may sensitize the activities of other anticancer drugs.25 It
also was reported that citronellol could improve the immune function of cancer
patients and their ability to fight off the cancer.23 For these reasons, we synthesized
the novel cabazitaxel prodrug by conjugating cabazitaxel with citronellol via disulfide
bond. Then the conjugate selfꢀassembled nanoparticles (CSNPs) were prepared and
the physical and chemical properties of the CSNPs were characterized. In vitro, the
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cell viability assay, cell uptake, cell cycle analysis and cell apoptosis on A549 cells
and PC3 cells of the CSNPs were studied. In vivo, the pharmacokinetics and
biodistribution of the CSNPs injected via tail vein were evaluated in rats. Furthermore,
we put forward an innovative idea that CSNPs can be utilized as an effective carrier
for other hydrophobic drugs or imaging agents. So fluorescence dyes 6ꢀcoumarin and
1,1ꢀdioctadecylꢀ3,3,3,3ꢀtetramethylindotricarbocyanine iodide (DiR) were chosen to
prepare the 6ꢀcoumarinꢀloaded CSNPs and DiRꢀloaded CSNPs for the study of cell
uptake and in vivo biodistribution of CSNPs, respectively. And curcumin with diverse
effects including antiproliferative, antiꢀinflammatory, antiangiogenic and antioxidant
activities26ꢀ28 was selected as a model drug to load in CSNPs, then the stability and
synergistic antitumor effects of curcuminꢀloaded CSNPs was investigated.
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123 RESULTS AND DISCUSSION
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Synthesis of Citronellol-cabazitaxel Conjugate. Cabazitaxel was conjugated with
citronellol via disulfide bond (Scheme 1). Firstly, dithiodiglycolic acid (DTDA) was
converted to the corresponding anhydride with acetic anhydride as dehydration agent.
Then, it reacted with citronellol to obtain CITꢀDTDA with 4ꢀdimethylaminopyridine
(DMAP) as catalyst. Finally, CITꢀDTDA was reacted with cabazitaxel under 1ꢀethylꢀ
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3ꢀ(3ꢀdimethyllaminopropyl)carbodiimide
hydrochloride
(EDCI),
1ꢀ
Hydroxybenzotriazole (HOBt) and DMAP to gain the target compound citronellolꢀ
cabazitaxel conjugate (CITꢀssꢀCTX).
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Scheme 1. Synthesis of citronellolꢀcabazitaxel conjugatea
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aConditions : (a) Ac2O, 25°C; (b) citronellol, DMAP, DCM, r.t.; (c) cabazitaxel, EDCI, HOBt,
DMAP, DCM, 0ꢀr.t.
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The chemical structure of citronellolꢀcabazitaxel conjugate was confirmed by H
NMR (Figure 1). The 1H NMR spectra of citronellolꢀcabazitaxel conjugate (400 MHz,
CDCl3) was analyzed as following: δ8.10 (2H, d, 3″7″), 7.61 (1H, m, 5″), 7.50 (2H, m,
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4″6″), 7.40ꢀ7.34 (4H, m, 5′6′8′9′), 7.32 (1H, m, 7′), 6.24 (1H, t, H13), 5.64 (1H, d,
H2), 5.49 (1H, br.s, H3′), 5.34 (1H, br.s, H2′), 5.08 (1H, t, CH(CH3)2=CH), 4.98 (1H,
d, H5), 4.82 (1H, s, H10), 4.30(1H, d, H20), 4.18 (2H, m, ꢀCO2CH2), 4.16 (1H, d,
H20), 3.90 (1H, m, H7), 3.84 (1H, d, H3), 3.62 (2H, s, ꢀSCH2CO2ꢀCTX), 3.54 (2H, s,
ꢀSCH2CO2ꢀCIT), 3.43 (3H, s, 10ꢀOCH3), 3.30 (3H, s, 7ꢀOCH3), 2.70 (1H, m, H6),
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2.43 (3H, s, 4ꢀOCOCH3), 2.27 (1H, m, H14), 2.00 (2H, s, =CHCH2ꢀCH2), 1.99 (3H, s,
18ꢀCH3), 1.79ꢀ1.75 (2H, m, H14, H6), 1.71 (3H, s, 19ꢀCH3), 1.68 (3H, s, =CH(CH3)2),
1.60 (3H, s, =CH(CH3)2), 1.55ꢀ1.48 (4H, m, ꢀOCH2CH2CH(CH3)CH2), 1.35 (9H, s,
(OCH3)3), 1.21 (3H, s, 16ꢀCH3), 1.19 (3H, s, 17ꢀCH3), 0.92 (3H, d, ꢀOCH2CH(CH3)).
1H NMR spectrum displayed new peaks at δ5.08/4.18/0.92 and δ3.62/3.54, which can
be attributed to the protons of CIT and methylene protons of the disulfide linker,
respectively. Furthermore, the chemical shift of H2’ in CTX moved to the low field to
5.34, indicating that CITꢀDTDA was successfully conjugated to CTX via an ester
bond at C2’.
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The final product was also characterized by Bruker micrOTOFꢀQ time of flight
mass spectrometer which was a powerful method to detect the mass of the compound.
The accurate m/z 1160.4682 ions was determined as sodium adduct
([C59H79NO17S2+Na]+ ions) for citronellolꢀcabazitaxel conjugate (Figure 2). The
predicted molecular formula is matched with actual molecular formula.
The results above indicated that cabazitaxel was successfully conjugated to
citronellol via disulfide bond.
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Figure 1. 1H NMR spectra of citronellolꢀcabazitaxel conjugate (400 MHz, CDCl3).
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Figure 2. MS spectra of citronellolꢀcabazitaxel conjugate.
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Preparation and Characterization of CSNPs. It was found that selfꢀassembled
nanoparticles could be prepared by nanoꢀprecipitation method in our previous work.22
In this study, the citronellolꢀcabazitaxel conjugate selfꢀassembled nanoparticles were
prepared by the same way. DLS analysis revealed that the CSNPs had a size of 153.3
± 0.17 nm, PDI of 0.081 ± 0.009 and Zeta potential of ꢀ19.0 ± 2.55 mV (Table S1),
suggesting that the CSNPs could accumulate abundantly by EPR effect because the
particle size of the CSNPs was between the endothelial junctions of normal vessels
(less than 10 nm) and the fenestrations of tumor vasculatures (more than 200 nm).29
And TEM micrograph (Figure 3B) indicated that CSNPs were spherical and the
particle size was nearly 150 nm that close to the results detected by DLS. The loading
content of cabazitaxel was 61.21 wt%, which was significantly higher than other
nanomedicine such as polymeric micelles,30 liposomes,31 nanoemulsions,32 and
nanoparticles for delivery of taxane drug.33 In addition, the stability of CSNPs stored
at 4 °C was detected by DLS for two months (Figure S1C). And there were no
significant changes in size and PDI of CSNPs. The particle size only slightly
increased in the initial days which might caused by the internal hydrophobic
interaction and the PDI was always below 0.2, indicting that the CSNPs were stable
during storage at 4 °C. All these results proved the successful formation of stable
spherical CSNPs with monomodal particle size distribution and low polydispersities.
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And we thought that electrostatic repulsion of the surface charge of nanoparticles and
the feature of conjugate that inhibits crystallization made the main contribution to the
stable selfꢀassembly of citronellolꢀcabazitaxel conjugate (Figure S2).
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Figure 3. Size distribution of CSNPs determined by DLS (A) and TEM image of CSNPs (bar =
200 nm) (B).
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In Vitro Drug Release Study. The in vitro drug release of CSNPs was studied at
37 °C under four GSH conditions, including GSH 10 mM, 1 mM, 10 ꢁM and, 1 ꢁM
respectively. The results showed that degradation of citronellolꢀcabazitaxel conjugate
and the release of cabazitaxel were significantly accelerated under high concentration
of GSH (Figure 4). For instance, 35.0%, 47.3%, 17.5%, 13.3% of cabazitaxel were
released from the CSNPs and 98.5%, 89.4%, 73.9%, 72.8% of citronellolꢀcabazitaxel
conjugate were degraded at 37 °C in 24h in PBS (pH 7.4) with 10 mM GSH, 1 mM
GSH, 10 ꢁM GSH and 1 ꢁM GSH, respectively. The breakage of disulfide bond of
citronellolꢀcabazitaxel conjugate caused by the high concentration of GSH can not
directly generate cabazitaxel, it also need extra reaction to cleave the ester bond for
the release of cabazitaxel which was a slow step. So the release rate of cabazitaxel
was slow than the degradation rate of citronellolꢀcabazitaxel conjugate. And the
release of cabazitaxel in the condition of 10 mM GSH was less than that of 1 mM
GSH which might due to the slightly instability of cabazitaxel at high concentration of
10 mM GSH (Figure S3). Notably, we observed that the degradation rate of
citronellolꢀcabazitaxel conjugate was: 10 mM GSH > 1 mM GSH > 10 ꢁM GSH > 1
ꢁM GSH. And it was obvious that the high concentration of GSH (1ꢀ10 mM)
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corresponding to the intracellular GSH concentration in tumor cells18,
could
promote stronger cleavage of disulfide bonds of citronellolꢀcabazitaxel conjugate to
release cabazitaxel (47.3% and 35.0%) compared with low concentration of GSH (1ꢀ
10 ꢁM) which was set as the redox condition in blood plasma(13.3% and 17.5%) in
24 h, which indicated that the CSNPs could reduce systemic toxicity by slow release
of cabazitaxel at the blood circulation and rapid release within the tumor intracellular
microenvironment.
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Figure 4. In vitro release studies. The GSHꢀsensitive release of cabazitaxel (A) and degradation of
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citronellolꢀcabazitaxel conjugate (B) from CSNPs were studied at 37 °C under four different
conditions, i.e, 10 mM, 1 mM, 10 ꢁM and 1 ꢁM GSH, respectively (n = 3).
Rat Plasma Stability. The addition of an esterase inhibitor is a usual method to
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stabilize compounds which can be hydrolyzed by esterase in serum, and
phenylmethanesulfonyl fluoride (PMSF) has been tested and used as an inhibitor for
esterase.34ꢀ36 So the stability of CSNPs was studied at 37 °C in two media which were
rat plasma containing 2.5 mM PMSF and without PMSF, respectively. As shown in
Figure S4, although PMSF could stabilize the ester bonds in citronellolꢀcabazitaxel
conjugate. The conjugate had the similar degraded behavior in two media, which
suggested that the ester bonds have slightly influence on the degradation of conjugate.
And we found that the degraded rate of conjugate in rat plasma just like that in PBS
with 1~10ꢁM GSH that revealed the GSH level in plasma (Figure 4B). This indicated
that the major factor on the unstability of conjugate was the breakage of disulfide
bond in terms of GSH. In addition, a reference about paclitaxel prodrug proved that
the disulfide bond was cleaved through thiolꢀdisulfide exchange reactions prior to the
breakage of ester bonds in blood circulation.37
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Cell Viability Assay. The in vitro antitumor activity of cabazitaxel solutions,
CSNPs and citronellol solutions was investigated by MTT assays. Figure 5 showed
that CSNPs maintained the cell cytotoxicity to both PC3 and A549 cells. Notably,
CSNPs induced similar antitumor drug efficacy to cabazitaxel solutions in both PC3
and A549 cells. The IC50 of PC3 cells incubated with cabazitaxel solutions and
CSNPs were 0.12 ± 0.01 nM and 0.70 ± 0.04 nM, respectively. And the IC50 of A549
cells incubated with cabazitaxel solutions and CSNPs were 4.26 ± 0.17 nM and 4.33 ±
0.27 nM, respectively (Table S2). Cabazitaxel solutions and CSNPs had almost the
same antitumor activity on PC3 cells at various concentrations, however, antitumor
activity of CSNPs became higher than cabazitaxel solutions on A549 cells at drug
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concentrations less than 1 nM. Usually the cytotoxic activity of anticancer drug would
decrease in dozens to hundreds of times after conjugation,38,39 so it is noteworthy that
the cytotoxic activity of CSNPs keeps almost unchanged. The similar antitumor
activity of CSNPs and cabazitaxel solutions might be due to their fast drug release
within cells resulting from the redoxꢀresponsive characteristic of citronellolꢀ
cabazitaxel conjugate, as well as the potential anticancer sensitizing effect of
citronellol to cabazitaxel (Figure S5). The cell inhibition rate (%) of PC3 cells
incubated with citronellol solutions was less than 15% at concentrations varying from
0.001 nM to 10 nM, suggesting that free citronellol had little antitumor effect to PC3
cells, but citronellol solutions had no antitumor effect to A549 cells.
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Figure 5. MTT assays of cabazitaxel solutions, CSNPs and citronellol solutions on PC3 cells (A)
and A549 cells (B). The cells were incubated with formulations for 48 h at concentrations varying
from 0.001 nM to 10 nM. Data are presented as mean ± SD (n = 3).
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Cell Uptake Study. CSNPs had no fluorescence signal, hence we attempted to
prepare the coumarinꢀ6 loaded CSNPs to investigate the cell uptake of CSNPs in PC3
and A549 cells. The coumarinꢀ6 loaded CSNPs were successfully prepared with light
yellow green opalescence (Figure S6). And the weight of citronellolꢀcabazitaxel
conjugate was 10 times of that of coumarinꢀ6 and the cells were treated with
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formulations for only 1 h and 3 h, so the effect of citronellolꢀcabazitaxel conjugate on
cells could be ignored.
The CLSM images were presented in Figure 6. It clearly showed that compared
with the treatment with CSNPs loaded 1 ꢁg/mL coumarinꢀ6 for 1 h, the cells treated
with CSNPs loaded 3 ꢁg/mL coumarinꢀ6 for 1 h and 1 ꢁg/mL coumarinꢀ6 for 3 h
showed stronger fluorescence, which indicated that more coumarinꢀ6 was uptaken,
that is to say, the uptake of CSNPs was timeꢀdependent and concentrationꢀdependent.
This result was consentient with the uptake behavior studies performed up to date.40
Moreover, it indicated that concentration contributed more to the uptake of CSNPs by
PC3 cells than time, which was just opposite to A549 cells, suggesting that different
types of cells owned different uptake behavior.
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The fluorescence was examined by FACS and the data was also presented in Figure
6. Flow cytometry profile presented the relative intensity of fluorescence signal in X
axis and the relative number of cells in Y axis. The results were respectively
consistent with those of the CLSM images.
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Figure 6. Cellar uptake of coumarinꢀ6ꢀloaded CSNPs in PC3 and A549 cells (n = 3). The CLSM
images of PC3 cells (A) and A549 cells (B) treated with coumarinꢀ6ꢀloaded CSNPs at different
times and concentrations: 1 h (1 ꢁg/mL, a), 3 h (1 ꢁg/mL, b), 1 h (3 ꢁg/mL, c). Cells were
counterstained with DAPI (for nuclei). Flow cytometry profile of PC3 cells (C) and A549 cells (D)
presented the relative intensity of fluorescence signal in X axis and the relative number of cells in
Y axis. Black line: control, red line: CSNPs containing 1ꢁg/mL coumarinꢀ6 for 1 h, blue line:
CSNPs containing 1 ꢁg/mL coumarinꢀ6 for 3 h, green line: CSNPs containing 3 ꢁg/mL coumarinꢀ
6 for 1 h.
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Cell Cycle Analysis. Cabazitaxel has been reported to target tubulin and inhibit
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depolymerization of microtubules, which results in cellꢀcycle arrest.4 Hence, cellꢀ
cycle arrest effects of cabazitaxel solutions, CSNPs and citronellol solutions were
investigated. Cell cycle tests were performed using a KeyGEN Cell Cycle Detection
Kit and tested by FACS, and the results were presented in Figure 7. Compared with
control group, it was obvious that more PC3 and A549 cells treated with cabazitaxel
solutions and CSNPs were accumulated in G2/M phases and fewer in G0/G1 phases
by a concentration dependent manner. In particular, compared with cabazitaxel
solutions, CSNPs induced slightly fewer cells arrest in the G2/M phase and both
cabazitaxel solutions and CSNPs had more cellꢀarrest effects in PC3 cells than A549
cells. And citronellol solutions induced very little cells arrest in the G2/M phase in
both PC3 and A549 cells. All these results are concordant with the data acquired by
MTT analysis and suggesting that the CSNPs can effectively block the mitosis at
G2/M phase.
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Figure 7. Effects on the cell cycle of PC3 cells (A) and A549 cells (B) treated with cabazitaxel
solutions, CSNPs and citronellol solutions (n = 3). The test groups were incubated with equivalent
cabazitaxel of 10 nM and 20 nM for 24 h. And the control groups were incubated with only fresh
media for 24 h.
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Cell Apoptosis Study. Cell apoptosis test was performed using a BD Annexin Vꢀ
Fluorescein Isothiocyanate Apotpsis Detection Kit and tested by FACS, and the
results were presented in Figure 8. To measure the quantitative apoptosis effects of
cabazitaxel solutions, CSNPs and citronellol solutions, AnnexinVꢀFITC/PI was used
to coꢀstain the cells, which could distinguish living cells from dead cells or early
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apoptosis cells from late apoptosis ones. In the flow cytometry profiles, the lower left,
upper left, lower right and upper right quadrants denoted live, necrotic, early apoptotic
and late apoptotic regions, respectively.
In Figure 8A, most PC3 cells treated with formulations were live and a large part
was on the stage of early apoptosis with increased concentration. Markedly, citronellol
solutions induced many cells apoptosis, although fewer than that induced by
cabazitaxel solutions and CSNPs, which might be the reason why cabazitaxel
solutions and CSNPs caused almost the same number of apoptosis including early and
late apoptosis. In Figure 8B, it showed that cabazitaxel solutions and CSNPs were
more effective to A549 cells than to PC3 cells, which may be because A549 cells can
take in more drugs than PC cells. And most A549 cells treated with cabazitaxel
solutions and CSNPs were on early apoptosis, which was also concentrationꢀ
dependent. However, citronellol solutions induced scarcely cell apoptosis.
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Figure 8. Induction of apoptosis on PC3 cells (A) and A549 cells (B) by cabazitaxel solutions,
CSNPs and citronellol solutions (n = 3). Apoptosis was evaluated after cells treated with
cabazitaxel solutions, CSNPs or citronellol solutions containing 1 ꢁM and 10 ꢁM drugs for 48 h.
Flow cytometry profile showed PI in Y axis and AnnexinꢀVꢀFITC in X axis. The normal cells were
showed in the lower left quadrant, the late apoptotic cells were showed in the upper right quadrant
and the early apoptotic cells were showed in the lower right quadrant.
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In Vivo Pharmacokinetics and Biodistribution. The main pharmacokinetic
parameters of cabazitaxel in rats after intravenous administration of cabazitaxel
solutions and CSNPs were summarized in Table S3. And the plasma concentration–
time curves of cabazitaxel were shown in Figure 9A. The results indicated that there
was an apparent difference for main pharmacokinetic parameters, AUC0ꢀt and t1/2,
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between cabazitaxel solutions and CSNPs. The AUC0ꢀt for CSNPs was 2717.6 ± 989.1
ng·h/mL which was 6.5ꢀfold higher than that of cabazitaxel solution (420.3 ± 83.9
ng·h/mL), demonstrating that the selfꢀassembled nanoparticles was stable and
pegylation can reduce the clearance of CSNPs from the body. Compared with
cabazitaxel solutions, the halfꢀtime of CSNPs was also increased to 6.7 ± 1.8 h (2.3ꢀ
fold increase) implying that the blood circulation time of cabazitaxel was prolonged.
To explore whether CSNPs can effectively improve the accumulation in tumor, the
in vivo targeting property of the CSNPs was investigated in tumorꢀbearing A549 nude
mice. The common used near infrared fluorescence dye DiR loaded by CSNPs was
selected to reflect the distribution of CSNPs in the body after intravenous
administration. The DiR solutions and DiRꢀloaded CSNPs were injected into mice
with a dose of 2 mg/kg, respectively. At desired time points, three mice were imaged
in vivo in each group, and other two mice were sacrificed and the major organs and
tumor were harvested for ex vivo imaging at 4 h, 24 h postꢀinjection in each group.
And the results of the fluorescence imaging at predetermined time intervals of only
one live mouse for each group were exhibited in Figure 9B. Apparently, the
fluorescence of mice treated with DiR solutions was mainly detected at liver and had
increased in lung with time pasted. For DiRꢀloaded CSNPs, although there was still
high level of fluorescence in the liver, strong fluorescence can be found at the tumor
site at desired time intervals. The same result was observed in Figure 9C, for control
group, the fluorescence was major in the liver at 4 h postꢀtreatment and not detected
in tumor. Even 24 h postꢀadministration, there was still no fluorescence can be found
in tumor and there were high levels of fluorescence in the liver and lung. The results
indicated most of DiR were captured by RES and accumulated at liver and lung sites.
Other researches were also reported the RES is the major obstacle for many
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antitumorꢀdrugs due to its nonꢀspecific defense mechanisms.41ꢀ43 As for the DiRꢀ
loaded CSNPs group, the fluorescence in the tumor can be apparently observed.
Although liver has strong fluorescence, low levels of fluorescence were detected in
heart, spleen, lung and kidney. These results demonstrated that CSNPs have effect on
reducing the RES uptake and improving tumor biodistribution.
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Figure 9. Plasma concentrationꢀtime profiles of cabazitaxel after intravenous administration of
cabazitaxel solutions and CSNPs of the equivalent cabazitaxel dose (3 mg/kg) in rats (mean ± SD,
n = 5) (A). In vivo fluorescence imaging of tumorꢀbearing A549 nude mice at predetermined time
intervals postꢀinjection of DIRꢀsolutions and DIRꢀloaded CSNPs (B). Ex vivo fluorescence
imaging of tumor and major organs taken from tumorꢀbearing A549 nude mice at 4 h and 24 h
time points (C).
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Preparation and Characterization of Curcumin-loaded CSNPs. The CSNPs can
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be used as a redoxꢀresponsive nanocarrier for other anticancer drugs for rapidly
release, providing an attractive approach for releasing drug within tumor cells and
combination therapy. In this study, curcumin, a hydrophobic drug that has diverse
effects including cytotoxicity26,27 was loaded by CSNPs. The mixed ethanol solutions
of curcumin and citronellolꢀcabazitaxel conjugate were added into water and stirred
for 1 min to form the curcuminꢀloaded CSNPs. The particle size, size distribution and
Zeta potential analysis of curcuminꢀloaded CSNPs were carried out by DLS, showing
that the CSNPs had small particle size of 129.7 ± 3.04 nm, a fair PDI of 0.076 ± 0.064
and mean Zeta potential of ꢀ24.5 ± 0.17 (Table S1), and TEM revealed that curcuminꢀ
loaded CSNPs were spherical and similar with CSNPs (Figure S1B). The curcuminꢀ
loaded CSNPs with drug loading content of 7.98 ± 0.07 wt% (curcumin) and 56.33 ±
0.04 wt% (cabazitaxel) and entrapment efficiency of 83.22 ± 0.77%, implied that the
CSNPs can successfully encapsulate curcumin and have high entrapment efficiency,
which was not reported before. Compared with other strategies for codeliver drugs,
such as using PLGA nanoparticles coꢀloaded with paclitaxel and Stat3 siRNA,44 lipidꢀ
albumin nanoassemblies codeliver paclitaxel and borneol,45 coreꢀmatched
nanoemulsions codeliver paclitaxel and 5ꢀfluoroucacil,46 the multifunctional CSNPs
showed apparent advantage that can increase the capability of loading drugs.
Moreover, the long term stability study of curcuminꢀloaded CSNPs (Figure S1C) was
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investigated at 4 °C. The results determined by DLS suggested there were no obvious
changes in particle size and PDI of the nanoparticles after several weeks. Therefore,
the structure of curcuminꢀloaded CSNPs was stable.
In Vitro Anti-tumor Effect of Combined Therapy. Figure 10 showed that
curcumin had no cell inhibitionꢀeffect on both PC3 and A549 cells at concentration
varying from 0.001 nM to 10 nM. However, the employment of curcumin in
curcuminꢀloaded CSNPs could effectively enhance the antiꢀtumor effect of
citronellolꢀcabazitaxel conjugate. Curcuminꢀloaded CSNPs showed slight synergistic
antiꢀtumor effects on both PC3 and A549 cells at concentration varing from 0.001 nM
to 1 nM. Notablely, at concentration from 1 nM to 10 nM curcuminꢀloaded CSNPs
presented strong synergistic effects for inhibiting PC3 and A549 cells, and the cell
inhibition reached 91.8% and 90.5% at 10 nM on PC3 and A549 cells, respectively
compared with CSNPs (53.8% and 44.7%, respectively). The IC50 of curcuminꢀloaded
CSNPs on PC3 and A549 cells were 0.02 ± 0.01 nM and 0.28 ± 0.03 nM, respectively
compared with CSNPs (0.70 ± 0.04 nM and 4.33 ± 0.27 nM, respectively) (Table S4).
In fact, the quantity of both curcumin (≤ 3.9 nM) and citronellolꢀcabazitaxel conjugate
(≤ 10 nM) was very small. Hence, the synergistic antiꢀtumor effects of curcumin and
citronellolꢀcabazitaxel conjugate in curcuminꢀloaded CSNPs could be significantly
employed in enhancing the antiꢀtumor effect, reducing the amount of drugs, thereby
reducing the incidence of side effects.
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Figure 10. The koinonia by MTT assays of curcumin, CSNPs and curcuminꢀloaded CSNPs on
PC3 cells (A) and A549 cells (B). The cells were incubated with preparations for 48 h at
concentrations varying from 0.001 nM to 10 nM. Data are presented as mean ± SD (n = 3). **:
compared with curcumin solutions, P < 0.01; #: compared with CSNPs, P < 0.05; ##: compared
with CSNPs, P < 0.01.
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In conclusion, we have successfully synthesized a novel disulfideꢀlinked
cabazitaxel prodrug by conjugating cabazitaxel with citronellol via the disulfide bond,
and the citronellolꢀcabazitaxel conjugate selfꢀassembled nanoparticles were prepared.
Notably, the CSNPs have several unique features: (a) The preparation method is
simple and the CSNPs are stable spherical nanoparticles with uniform size distribution.
(b) The loading content of cabazitaxel in CSNPs is significantly higher than other
biocompatible nanosystems such as polymeric micelles and polymeric nanoparticles.
(c) The disulfide bond is redoxꢀresponsive so that the CSNPs can reduce the general
toxicity and rapidly release cabazitaxel within tumor cells. (d) The cell uptake of
CSNPs is both timeꢀdependent and concentrationꢀdependent. And CSNPs can block
the mitosis at G2/M phase and induce the apoptosis of PC3 and A549 cells that
produce higher antitumor effect. (e) The in vivo pharmacokinetics of CSNPs is
apparently improved, and the CSNPs have target effect for accumulating at tumor site.
Furthermore, CSNPs exhibit an excellent property that can load other hydrophobic
drugs or imaging agents. In this article, fluorescence dyes 6ꢀcoumarin and DiR were
successfully loaded in CSNPs for the experiment of cell uptake and in vivo
biodistribution of CSNPs, respectively. And curcuminꢀloaded CSNPs were also
prepared and expressed strong synergistic anticancer effects. All these results indicate
that the redoxꢀsensitive citronellolꢀcabazitaxel conjugate can be utilized as a smart
multifunctional nanomedicine for combination cancer therapy and theranostics.
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Materials. Cabazitaxel (CTX) was purchased from Beijing Maisuo Chemical
Technology Co Ltd (Beijing, China). Citronellol (CIT) was obtained from Chengdu
Gracia Chemical Technology Co. Ltd. (Chengdu, China). Dithiodiglycolic acid
(DTDA), 4ꢀDimethylaminopyridine (DMAP) and phenylmethanesulfonyl fluoride
(PMSF) were bought from Aladdin Industrial Corporation (Shanghai, China). 1ꢀ
Ethylꢀ3ꢀ(3ꢀdimethyllaminopropyl)carbodiimide hydrochloride (EDCI) and 1ꢀ
Hydroxybenzotriazole (HOBt) were purchased from Energy Chemical Co. Ltd.
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(Shanghai,
China).
2ꢀdistearoylꢀsnglyceroꢀ3ꢀphosphoethanolamineꢀNꢀ
[methoxy(polyethylene glycol)ꢀ2000] (DSPEꢀPEG2000) was obtained from Shanghai
Advanced Vehicle. The human lung cancer cells (A549) and human prostatic
carcinoma cells (PC3) were purchased from the cell bank of Chinese Academy of
Sciences (Beiijng, China). Roswell Park Memorial Institute (RPMIꢀ1640), Dulbecco’s
Modified Eagle Medium (DMEM, high glucose), trypsin and 3ꢀ(4,5ꢀdimethylthiazolꢀ
2ꢀyl)ꢀ2,5ꢀdiphenyltetrazoliumbromide (MTT) were obtained from Gibco (Beijing,
China). Nutrient Mixtuee Fꢀ12 Ham Kaghn’s Modification (F12K) and Coumarinꢀ6
were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and CS
were purchased from Hyclone (Beijing, China) and KeyGEN (Nanjing, China),
respectively. Dimethylsulfoxide (DMSO) was purchased from Kemeng (Tianjin,
China). 1,1ꢀdioctadecylꢀ3,3,3,3ꢀtetramethylindotricarbocyanine iodide (DiR) and 4′,6ꢀ
diamidinoꢀ2ꢀphenylindole (DAPI) were brought from AAT Bioquest (Beiijng, China)
and Beijing Founction biotechnology company (Beiijng, China), respectively.
Isoflurane was bought from RWD life science Co. Ltd. (Shenzhen, China). Curcumin
was purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China)All other
reagents and solvents used in this article were of analytical grade.
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Synthesis of Citronellol-cabazitaxel Conjugate. DTDA (0.5 g, 2.74 mmol) and 2
mL acetic anhydride were added to a 100 mL roundꢀbottom flask equipped with a
magnetic stirrer. The mixture was stirred for 2 hours at 25 °C under nitrogen
atmosphere. Thereafter, 20 mL methylbenzene was repeatedly added and then dried in
vacuo (< 30 °C) for three times. The crude product was subjected to the subsequent
step without further purification. The anhydride was dissolved in 20 mL anhydrous
dichloromethane, and then citronellol (0.17 mL, 0.928 mmol) and DMAP (13 mg,
0.106 mmol) were added. The reaction was stirred at 25 °C for 1 h and the completion
of reaction was monitored by TLC (Hex/EA = 1:1). The crude product was collected
by filtration and dried in vacuo (< 30 °C) as a yellow oily solid (0.627 g). It was
purified by silica gel column chromatography (Hex/EA = 5:1，1 ‰ HOAc) to give a
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light yellow solid compound (CITꢀDTDA, 0.26 g, 87.9%). CITꢀDTDA(56 mg, 0.175
mmol)，EDCI (40 mg, 0.209 mmol)，HOBt (28 mg, 0.209 mmol and DMAP (6 mg,
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0.049 mmol) in 4 mL anhydrous dichloromethane were stirred at ꢀ5~ꢀ8 °C for 1 h
under nitrogen atmosphere, then cabazitaxel (150 mg, 0.179 mmol) was added to the
reaction liquid, and the mixture was further stirred at 25 °C for 4 h under nitrogen
atmosphere. The completion of reaction was tested by TLC (Hex/EA = 1:1). The
reaction liquid was extracted by water (20 mL × 3) and the organic phase was dried by
anhydrous sodium sulfate. The crude product was purified by HPLC to obtain
citronellolꢀcabazitaxel conjugate (169 mg, 82.8%) as a white solid.
1H NMR spectra was recorded on a Bruker ARXꢀ400 NMR spectrometer operating
at 400 MHz and using deuterated chloroform (CDCl3) as a solvent. The chemical
shifts were corrected against residual solvent signals. The accurate mass of sample
was performed using a Bruker micrOTOFꢀQ time of flight mass spectrometer, mass
range of m/z was 50 ~ 3000, ion type is [M + Na]+.
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Preparation and Characterization of CSNPs. CSNPs were prepared via the
existing nanoꢀprecipitation method.22 Briefly, The ethanol solution containing 10
mg/mL citronellolꢀcabazitaxel conjugate was slowly added into water that was stirred
(800 rpm) at room temperature. After 1 min, the selfꢀassembled nanoparticles
(containing 2 mg/mL citronellolꢀcabazitaxel conjugate) were formed and 10 mg/mL
DSPE ꢀ PEG2000 (20%, w/w) was then slowly added to the solution for pegylation.
The CSNPs were stored at 4 °C.
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The size and Zeta potential of the CSNPs were measured by DLS. The
measurements that performed in triplicate were carried out via a Zetasizer (Nano ZS,
Malvern. UK and the results were evaluated by mean ± standard deviation (SD). The
morphology of CSNPs was observed through TEM (JEM2100, JEOL. Japan) operated
at an accelerating voltage of 200 kV. The samples were prepared by dropping 10 ꢁL
of nanoparticle solution on a carbonꢀcoated copper grid for 3 min and then dried by
filter paper. Finally, 1% uranyl acetate (5 µL) was dropped for 30 seconds and air
dried.
The Mechanism of the Conjugate to Self-assemble into Nanoparticles. A simple
study to explore the mechanism of the conjugate to selfꢀassemble into nanoparticles
was conducted by imaging the dynamics of crystal growth. Ethanol containing
cabazitaxel and citronellolꢀcabazitaxel conjugate was placed onto a glass slide,
respectively. After drying and desiccating, add a drop of water to each sample.
Pictures were taken after the slides were kept in a humidified chamber at room
temperature for 6 h.
To investigate selfꢀassembly interaction among molecules, computational
simulation was also used. The citronellolꢀcabazitaxel conjugate structures were
constructed by package and optimized with the material studio 8.0 software. All
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molecular dynamics (MD) simulations were performed by using the Amorphous Cell
and Forcitemodule of the Material studio simulation package with COMPASS. In the
MD simulation, firstly, amorphous cell module was employed to construct a box,
which was inundated with 200 water molecules and 4 citronellolꢀcabazitaxel
conjugate molecules. Then, 100 ns MD simulations were performed under the
constant temperature of 200 K. The results were displayed in accompanying
Supporting Information.
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In Vitro Drug Release Study. The release of cabazitaxel from CSNPs and the
degradation of citronellolꢀcabazitaxel conjugate were studied under shaking (100 rpm)
at 37 °C in four different media which were the phosphate buffer saline (PBS, pH 7.4)
containing 0.5% Tween 80 with 10 mM, 1 mM, 10 ꢁM and 1 ꢁM GSH, respectively.
Typically, 1 mL of CSNPs dispersions (2 mg/mL) was added to 25 mL of release
media. The release studies were conducted at different time intervals: 1, 2, 3, 6, 12
and 24 h. At desired time intervals, 1 mL samples was withdrawn and replenished
with the equal volume of fresh media. The content of cabazitaxel and citronellolꢀ
cabazitaxel conjugate was determined by highꢀperformance liquid chromatography
(HPLC) with Waters e2695 Separations Module and Waters 2489 UV/Visible
Detector on a reverse ODS CosmosilꢀC18 column (250 mm × 4.6 mm, 5 ꢁm)
thermostated at T = 40 °C with UV detection at 227 nm using a mixture of
acetonitrile/water (80:20, v/v) as a mobile phase with a flow rate of 1 mL/min. Each
experiment was repeated in triplicate and the results were expressed by mean ± SD.
Rat Plasma Stability. The stability of CSNPs in rat plasma was analyzed by
incubating CSNPs in rat plasma without or with polymethylsulfonyl flouride (PMSF,
2.5 mM). In each group, CSNPs were mixed with rat plasma to reach the
concentration of 50 ꢁg/mL and stored at 37 °C. At desired time intervals, 100ꢁL
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