Amphiphilic nanoparticles of resveratrol-norcantharidin to enhance the toxicity in zebrafish embryo

Article abstract of DOI:10.1016/j.bmcl.2015.12.099

Direct coupling of a hydrophobic drug and a hydrophilic natural product via an ester bond produced an amphiphilic adduct that formed liposomes. Liposomes of resveratrol-norcantharidin adduct are capable of forming a tadpole-like nanoparticle and exhibited

Full text of DOI:10.1016/j.bmcl.2015.12.099

Accepted Manuscript
Amphiphilic Nanoparticles of Resveratrol-Norcantharidin to Enhance the Tox-
icity in Zebrafish Embryo
Deyue Yan, Lin-Kai Ni, Ho-Lun Chen, Li-Chou Chen, Yau-Hung Chen, Chien-
Chung Cheng
PII:
S0960-894X(15)30412-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.099
BMCL 23460
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 September 2015
10 December 2015
29 December 2015
Please cite this article as: Yan, D., Ni, L-K., Chen, H-L., Chen, L-C., Chen, Y-H., Cheng, C-C., Amphiphilic
Nanoparticles of Resveratrol-Norcantharidin to Enhance the Toxicity in Zebrafish Embryo, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.099
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Amphiphilic Nanoparticles of Resveratrol-Norcantharidin to Enhance the
Toxicity in Zebrafish Embryo
Deyue Yana,*, Lin-Kai Ni,b Ho-Lun Chen,b Li-Chou Chen,c Yau-Hung Chen,d,* Chien-Chung
Chengb,*
a School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240,
P. R. China
b Department of Applied Chemistry, National Chia-Yi University, Chia-Yi City, Taiwan 60004,
ROC
c Department of Styling, Tzu Hui Institute of Technology, Pingtung, Taiwan 926, ROC
d Department of Chemistry, Tamkang University, New Taipei City, Taiwan 25137, ROC
[*] Corresponding Authors
Prof. Deyue Yan
School of Chemistry and Chemical Technology,
Shanghai Jiao Tong University,
Shanghai 200240, P. R. China
Tel: +021-54742664; E-mail: dyyan@sjtu.edu.cn
Prof. Yau-Hung Chen
Department of Chemistry,
Tamkang University,
New Taipei City, Taiwan 25137
Tel: ++886-2-26215656 ex. 3009; E-mail: yauhung@mail.tku.edu.tw
Prof. Chien-Chung Cheng
Department of Applied Chemistry
National Chia-Yi University
Chia-Yi City, Taiwan 60004, Republic of China
Tel: +886-5-2766084; FAX: +886-5-2717901
E-mail: cccheng@mail.ncyu.edu.tw
1

Abstract
Direct coupling of a hydrophobic drug and a hydrophilic natural product via an ester bond
produced an amphiphilic adduct that formed liposomes. Liposomes of resveratrol-norcantharidin
adduct are capable of forming a tadpole-like nanoparticle and exhibited high toxicity in zebrafish
embryos to give the better transportation and the effective concentration into cells. Using
fluorescent chromophore showed the liposome in the stomach and intestinal villi rather than in the
skin and muscle. This result may provide an insight into the mechanism of action of traditional
Chinese medicines, which often contain a significant amount of flavonoids and polyphenol analogs.
Keywords: liposome, resveratrol, norcantharidin, drug delivery, tadpole structure
2

Liposomes are important carriers for transporting drugs into cells.1-3 Many drugs can be
encapsulated by amphiphilic compounds to increase solubility, effective concentration, and
bioavailability.4 For instance, some anticancer drugs, such as doxorubicin5 and daunorubicin,6 are
administered in liposomes to improve their clinical effect. Moreover, cisplatin-encapsulated
liposomes7 are used to treat pancreatic cancer in infants to increase the bioavailability.8 Liposomes
used to deliver drugs are made of phosphatidylcholines, which contain aliphatic fatty acids to
interact with the lipophilic target drug. The liposomes enter cells by diffusion through the lipid
bilayer membranes. The maximum efficiency for encapsulated drugs is usually around 30%. To
reach an effective concentration,9 it is necessary to use a large quantity of liposomes to deliver the
target drug, and the lipophilic portion of the remaining liposomes may have unwanted effects in
biological systems. To solve these potential problems, beside to use other polymeric carriers,10,11 we
have developed a strategy to prepare a chemical adduct that links a lipophilic drug directly to a
hydrophilic antioxidant through an ester bond to generate a new type of amphiphile.
Figure1. Concept for direct coupling of NCTD with resveratrol
We used an antioxidant as the hydrophilic group because the health benefits of antioxidants
have been widely studied.12 For instance, flavonoids are abundant antioxidants in plants13 and
possess a diverse range of biological activities, including chemical signaling, physiological
regulation, and cell cycle inhibition. Polyphenolic stilbenoids such as resveratrol,14 pinosylvin,15
and piceatannol16 also exhibit antioxidant activity. In particular, resveratrol is an anti-inflammatory,
3

a platelet aggregation inhibitor, and prevents coronary heart disease in addition to its anti-oxidant
properties.14 Moreover, phenol-containing compounds complexed with many metal ions exhibit
various catalytic activities and potential medical applications.17 It has recently been reported that
resveratrol-modified stearate (RMS) liposomes form nanoparticles capable of releasing an
encapsulated drug through a spontaneous structural transformation.18 Therefore, we used resveratrol
as the hydrophilic moiety in our amphiphilic compound. For the lipophilic moiety, the bicyclic
compound norcantharidin (NCTD)19 was used instead of stearic acid. NCTD retains its potential
antitumor activity and apoptotic properties, and exhibits reduced inflammation and nephrotoxicity
compared with cantharidin20,21 as shown in Figure 1. However, because NCTD is a smaller
molecule than cantharidin, it has a shorter retention time and a lower effective concentration in the
body.
O
O
OH
OH
OH
O
OH
OH
+
O
O
HO
O
O
O
1
2
3
O
O
O
O
O
O
O
O
O
HO
N
resvertrol
Na2CO3
OMe
MeOH
heat at 60oC
O
OMe
OH
OMe
O
OH
OH
O
O
O
O
O
O
O
N
DCC/DME
rt, 5h
THF; rt; 18 h
O
1
4
5
6
Scheme 1. Synthesis of Res-NCTD.
The size of NCTD can be increased by altering its structure to extend its retention time and
accumulate a sufficiently high effective concentration. However, modifying NCTD may involve
tedious synthetic procedures and affect its biological activity. In this work, we prepared a functional
amphiphile to link NCTD directly to resveratrol and create a molecule with hydrophilic and
hydrophobic moieties to increase the bioavailability. The monomeric resveratrol-norcantharidin
(RES-NCTD, 6) was less toxic than RES-NCTD liposomes in zebrafish embryos. The experimental
procedure is performed in a similar manner as previously reported.22 Furthermore, fluorescent
4

NCTD was prepared and confirmed that the uptake pathway was through the stomach in zebrafish.
Our results show that generating liposomes by combining two compounds directly with no linker to
form an amphiphile is a promising strategy for delivering small molecule drug candidates.
Target compound 6 was prepared as follows. Initially, we attempted to react the anhydride
group of NCTD with resveratrol, although this approach failed, even in the presence of Lewis acids
or NaH under reflux conditions. In general, anhydrides readily undergo ring opening with a
nucleophile; however, steric hindrance may have prevented the reaction from occurring in NCTD.
The anhydride moiety underwent ring opening in the presence of aqueous sodium hydroxide in
methanol under heating. Therefore, this also suggested that steric hindrance prevented the ring
opening of NCTD with a bulky nucleophile. The product of the ring-opening reaction was
confirmed by 1H-NMR. The bridged protons of NCTD were shifted from 5.1 to 4.96 and 4.89 ppm.
The carboxylic acid moiety of compound 4 was reacted with N-hydroxyl succinimide in the
presence of a coupling reagent to generate active ester 5. The final adduct was obtained by reacting
resveratrol with 5 in the presence of Na2CO3 at room temperature for 18 h. Purification by silica gel
column chromatography gave RES-NCTD (6) in yields of 10%–24%. The NMR spectrum showed a
doublet at 7.60 ppm, which was shifted by 0.23 ppm from the signal at 7.37 ppm observed in
resveratrol, indicating the formation of a 4-styryl-substituted resveratrol derivative.18 The aromatic
proton signals at 6.41 and 6.14 ppm of the dihydroxyphenyl group showed a negligible shift (<0.02
ppm) compared with resveratrol. The esterification of resveratrol only occurred at the
4-hydroxystyryl position owing to the resonance effect between the 4-hydroxy group and the
styrene moiety.23 RES-NCTD was also characterized by electrospray ionization-tandem mass
spectrometry, and an [M+H+] molecular ion peak was observed at m/z 410.14.
5

(A)
(B)
Figure 2. TEM images showing the structural transformation of RES-NCTD liposomes (A) after
0.3 h, and (B) after 24 h.
The RES-NCTD liposomes were formed by the sonication method.24 The liposomes were
assembled by immediately adding an ethanol solution of the resveratrol derivative to deionized
water at room temperature, followed by sonication (42 W) using a probe (with a size 1.10 × 1.25
cm) for an additional 5 min at 4 oC. The sonication probe with a large surface area produces a better
particle size distribution. Figure 2 shows the transmission electron microscopy (TEM) images of the
morphologies of self-assembled RES-NCTD liposomes. The samples used for TEM images were
prepared according to standard preparation procedures, including drying with 2 % phosphotungstic
acid. The RES-NCTD liposomes were prepared at room temperature by sonication, and then stored
at 8 o C or room temperature. The average particle size of RES-NCTD formed after 20 min was
estimated as 231.96 ± 18.68 nm by dynamic light scattering. The RES-NCTD liposomes
spontaneously transformed to a tadpole-like structure after 24 h (Figure 2b). The tadpole tail
appeared to be a lamellar structure, which is not observed in RMS liposomes. It has been reported
that RMS liposomes spontaneously undergo structural transformation from spherical vesicles to
wire-like linear structures, and finally to inert spherical nanoparticles.18 The resveratrol-NCTD
derivative may form a tadpole-like structure because of the small hydrophobic moiety and bulky
NCTD moiety.
6

Table 1. Survival rates (standard error, SE) of zebrafish after exposure to 10, 25, or 50 ppm of the
compounds and then to stain solution for 80 min. Each (averaged) survival rate and its standard
error are calculated from 4 replicated experiments, and each experiment contains 30 zebrafishes for
scrutiny.
Survival Rate % (SE)
7 dpf Group
10
25
50 (ppm)
Control
100 (0)
NCTD, 1
99.2 (0.83) 96.7 (1.36)
99.2 (0.83) 99.2 (0.83)
90.8 (4.17)
95.8 (1.66)
0 (0)
RES-NCTD, 6 (monomer)
RES-NCTD, 6 (liposome)
98.3(0.96)
100 (0)
Next, we examined the toxicity of the RES-NCTD monomer and liposomes after modification.
NCTD and its derivatives showed negligible inhibition of the growth of Escherichia coli. Although
NCTD has potential antitumor activity in humans, it may not have an effect on prokaryotic cells.
Therefore, the toxicity of RES-NCTD derivatives was evaluated in zebrafish embryos that were
staged according to their age (dpf) with a standard procedure.22 As shown in Table 1, low
RES-NCTD doses of 10 and 25 ppm showed no toxicity for either the monomer or liposomes on 7
dpf zebrafish. However, at a RES-NCTD liposome concentration of 50 ppm, the survival rate of
zebrafish was 0%, whereas the RES-NCTD monomer control showed no change in survival
(95.8%). This suggests that the liposome is crucial for increasing the toxicity of RES-NCTD
through the liposomal transportation of NCTD into cells to exert its biological effect. Moreover, this
result demonstrates that NCTD directly coupled to resveratrol retains its biological activity.
Therefore, RES-NCTD liposomes show the validity of our strategy of improving the bioavailability
of active compounds by coupling the active compounds with a hydrophilic antioxidant to form
liposomes.
+ N
O
N
+N
O
N
+ N
O
N
O
O
NH2
5
H2N
DCI / CH2Cl2
OMe
H
O
O
O
O
H2N
N
HO
THF; rt; 4 h
N
N
H
H
8
9
Rhodamine B, 7
Scheme 2. Synthesis of fluorescent compound by linking rhodamine B with NCTD.
7

Because liposomes can penetrate the cell membrane, it is possible that the toxic effect in
zebrafish may be caused by RES-NCTD liposomes being taken up through the skin. To clarify the
uptake pathway of NCTD derivatives, a rhodamine fluorophore was coupled with NCTD instead of
resveratrol (Scheme 2). Compound 9 was prepared by coupling the external amine of rhodamine
B25 with an activated NCTD ester. Zebrafish treated with fluorescent compound 9 showed the
compound in the stomach and intestinal villi rather than in the skin and muscle. This suggests that
the oral adminstration for zebrafish is still the main uptake pathway for modified NCTD, which
agrees with the reported behaviors of NCTD based on clinical observations.
In summary, direct coupling of a hydrophobic NCTD and a hydrophilic resvertrol via an ester
bond produced an amphiphilic adduct that formed liposomes. The RES-NCTD liposomes showed
high toxicity in zebrafish embryos compared with the monomer, demonstrating that the strategy of
encapsulating active compounds in natural product liposomes improved the transportation and the
effective concentration in vivo. This result may provide an insight into the mechanism of action of
traditional Chinese medicines, which often contain a significant amount of flavonoids and
polyphenol analogs. The biological activity of compounds of traditional Chinese medicines may
arise from the formation of liposomes, nanoparticles, or supramolecules to facilitate transportation
into the cells. We are currently conducting further studies of these liposomes.
Acknowledgements
The authors thank Prof. Ping-Hung Yeh at Tamkang University for kindly providing the TEM
facilities. We gratefully acknowledge the financial support of the Ministry of Science and
Technology in Taiwan.
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