Combretastatin A4-camptothecin micelles as combination therapy for effective anticancer activity

Article abstract of DOI:10.1039/C8RA08794F

Cancer is a major worldwide health problem, for which chemotherapy is a common treatment option. However drug toxicity and the development of resistance to chemotherapy are two main challenges associated with the traditional anticancer drugs. Combined pharmacological therapy based on different mechanisms might be an effective strategy in cancer treatment, and could exhibit a synergistic therapeutic efficacy. Herein, we aim to combine combretastatin A4 (CA4) and camptothecin (Cpt) chemically into a codrug through two hydrophilic linkers utilizing click chemistry to improve their water solubility and anticancer activity. The synthesized amphiphilic structure could self-assemble into a micelle structure as confirmed by atomic force microscopy (AFM) and dynamic light scattering (DLS), which showed a high stability and improved water solubility at pH 7.4, with a low critical micelle concentration (CMC) value of 0.9 mM. Moreover, in vitro hydrolysis was observed upon incubation of the hybrid compound with an esterase enzyme, which suggested a complete disassembly into the starting active drugs. Finally, cytotoxicity studies on HeLa cancer cells showed that the codrug demonstrated an enhanced (five fold) cytotoxicity as compared with the free drugs. In addition the combination index (CI) was <1, which suggests a synergistic activity for the codrug. Moreover, the tested concentrations of the codrug were not significantly cytotoxic to a noncancerous fibroblast cell line. The imaging of HeLa cells treated with FITC-loaded micelles showed a rapid internalization. In conclusion, the codrug of CA4 and Cpt might be a potential novel anticancer drug as it demonstrated a synergistic cytotoxic activity that might spare noncancerous cells.

Full text of DOI:10.1039/C8RA08794F

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Combretastatin A4-camptothecin micelles as
combination therapy for effective anticancer
Cite this: RSC Adv., 2019, 9, 1055
activity
a
Naim Kittana,b Sahar Alhaj Qasem,a Raghad Adas,a
*
Mohyeddin Assali,
Doaa Saleh,a Asala Arara and Osayd Zohud
b
Cancer is a major worldwide health problem, for which chemotherapy is a common treatment option. However
drug toxicity and the development of resistance to chemotherapy are two main challenges associated with the
traditional anticancer drugs. Combined pharmacological therapy based on different mechanisms might be an
effective strategy in cancer treatment, and could exhibit a synergistic therapeutic efficacy. Herein, we aim to
combine combretastatin A4 (CA4) and camptothecin (Cpt) chemically into a codrug through two hydrophilic
linkers utilizing click chemistry to improve their water solubility and anticancer activity. The synthesized
amphiphilic structure could self-assemble into a micelle structure as confirmed by atomic force microscopy
(AFM) and dynamic light scattering (DLS), which showed a high stability and improved water solubility at pH
7.4, with a low critical micelle concentration (CMC) value of 0.9 mM. Moreover, in vitro hydrolysis was
observed upon incubation of the hybrid compound with an esterase enzyme, which suggested a complete
disassembly into the starting active drugs. Finally, cytotoxicity studies on HeLa cancer cells showed that the
codrug demonstrated an enhanced (five fold) cytotoxicity as compared with the free drugs. In addition the
combination index (CI) was <1, which suggests a synergistic activity for the codrug. Moreover, the tested
concentrations of the codrug were not significantly cytotoxic to a noncancerous fibroblast cell line. The
imaging of HeLa cells treated with FITC-loaded micelles showed a rapid internalization. In conclusion, the
codrug of CA4 and Cpt might be a potential novel anticancer drug as it demonstrated a synergistic cytotoxic
activity that might spare noncancerous cells.
Received 23rd October 2018
Accepted 23rd December 2018
DOI: 10.1039/c8ra08794f
rsc.li/rsc-advances
the treatment. On the other hand, the development of drug
resistance by tumors might be a reason for the treatment failure
1. Introduction
in many cases. Therefore, there is a great demand to develop
a new strategy to ght this lethal disease.6,7 The combination of
multiple drugs with various mechanisms could be an effective
strategy that might exhibit a synergistic activity.8–13 However, the
proper delivery of the combined therapy has some limitations
and a proper chemical combination is highly desired due to the
various physiological barriers and enzymatic processes in the
body.
Combretastatin A4 (CA4) and camptothecin (Cpt) are among
many clinically used anticancer drugs. CA4 is a natural
compound that is extracted from an African willow tree called
Combretum caffrum. It acts as a vascular disrupting agent (VDA).
In addition it can inhibit the polymerization of tubulin result-
ing in cell death.14–16 However, it has two major disadvantages;
the low water solubility and the short half life.10,17,18 On the other
hand, Cpt is extracted from a Chinese plant called Camptotheca
acuminata.19 It inhibits topoisomerase I enzyme, which is
important in the DNA replication, resulting in cell death.20
Similar to CA4, Cpt also suffers from low water solubility, but in
addition it has low stability and preponderance of a less active
carboxylate form at physiologic pH.21,22
Cancer is considered one of the complicated diseases that
presents as a major leading cause of death worldwide.1 Cancer
chemotherapy is among one of the major treatment options for
cancer, besides surgery and radiotherapy when applicable.
Chemotherapy can be used as a supplemental treatment to
attack micro-metastases following surgery and radiation. It can
also be given prior to the surgical procedure in order to shrink
the tumor.2 Anticancer drugs are normally administered
systemically, so that they distribute throughout the body tissues
and may affect not only the cancerous cells but also many
normal cells leading to various side effects on the heart,
kidneys, bladder, digestive system, bone marrow, lungs and
many others.3–5 Drug toxicity and the development of drug
resistance are among the main challenges associated with the
use of traditional anticancer drugs. Toxicity can cause life-
threatening consequences that may require the cessation of
aDepartment of Pharmacy, Faculty of Medicine and Health Sciences, An Najah
National University, P. O. Box 7, Nablus, Palestine. E-mail: m.d.assali@najah.edu
bDepartment of Biomedical Sciences, Faculty of Medicine & Health Sciences, An Najah
National University, P. O. Box 7, Nablus, Palestine
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Solid tumors unendingly grow in size. In many times this pressure. Nuclear Magnetic Resonance (NMR) spectra were
increase in tumor size is not only associated with an increase in recorded with Bruker Avance 400 and 500 MHz spectrometers.
tumor tissue hypoxia, but also a reduction in tumor blood ow Chemical shis were reported in ppm, and coupling constants
and consequently an impairment in the delivery of the were reported in Hz. Infrared spectra were recorded on FTIR
chemotherapeutic agents.23–25 However, some research studies Spectrometer (Nicolet iS5, Thermo Fisher Scientic Company,
suggested that VDAs could otherwise be superior against such USA). Ultraviolet-visible (UV-Vis) spectra were recorded on a UV/
large tumors, because tumor vasculature could be already vis (7315 Spectrophotometer, Jenway, UK), using quartz
susceptible to occlusion due to elevated interstitial uid pres- cuvettes. Perkin Elmer luminescence spectrometer was used to
sure, which are more associated with advanced tumor masses.26 calculate the critical micelle concentration (CMC). Atomic force
Moreover a recent review study demonstrated that combining microscope (AFM) imaging was performed by depositing the
VDAs with traditional chemotherapeutic agents has greater sample of mica using a tapping mode-AFM (Alquds University)
potential to eradicate tumor cells.27,28
and WSxM soware designed by Nanotec Electronica (Madrid,
Herein we aim to combine the VDA agent (CA4) with the Spain) used for image analysis.29 Particle size analysis was
topoisomerase I inhibitor (Cpt) chemically for the rst time measured by dynamic light scattering (DLS) (NanoBrook Omni,
using a suitable hydrophilic linkers of tetraethylene glycol that Brookhaven Instruments Corporation, USA) with a xed scat-
would permit the formation of an amphiphilic structure that tering angle of 90ꢀ at 25 ꢀC. Microplate reader 6000 (Unilab Inc,
could assemble into micelles, which might in turn improve the USA) was used to read the plate for cell viability test. Olympus
water solubility of the resulting molecules and provide a syner- IX73® inverted microscope operated with Pylon Viewer 64 bit®
gistic effect of the combined therapy (Scheme 1).
soware was used for bright eld and uorescence imaging for
cellular uptake experiment.
2. Experimental
2.1. Reagents and instruments
2.2. Synthesis
The following compounds were synthesized and characterized
according to our previous reported study:10 OH-TEG-OTs (1),
OH-TEG-N3 (2), COOH-TEG-N3 (3), CA4 (4) and CA4-TEG-N3 (5).
2.2.1. Synthesis of OH-TEG-alkyne (6). A solution of NaH
(247 mg, 10.3 mmol) dissolved in 5 ml THF was added dropwise
over 30 min to a solution of tetraethylene glycol (1.0 g, 5.15
mmol) in 10 ml THF and stirred at 0 ꢀC. Then, a solution of
propargyl bromide (858 mg, 7.2 mmol) dissolved in 5 ml THF
was added dropwise to the reaction over 30 min at 0 ꢀC and was
kept at room temp for 24 h. The next day, 5 ml of distilled water
was added to the reaction slowly, then THF was evaporated.
100 ml of DCM was added and treated with 50 ml HCl, organic
layer was collected and dried over Na2SO4 which puried by
ash column chromatography in ethyl acetate to get colorless
liquid with 42% yield (500 mg). Rf: 0.26 (EtOAc). FTIR: 3320,
2130 cmꢁ1. 1H NMR (400 MHz, CDCl3): d 4.24 (d, 2H, J ¼ 2.9 Hz,
CH2C), 3.65–3.58 (m, 14H, 7CH2O), 3.57–3.51 (m, 2H), 2.95 (s,
1H, CH), 1.1 (s, 1H, OH). 13C NMR (100.6 MHz, CDCl3): d 92.6,
78.8, 74.5, 72.3, 70.8, 70.7, 70.7, 70.6, 70.3, 69.1, 61.5, 58.4.
2.2.2. Synthesis of alkyne-TEG-COOH (7). Chromic acid (15
ml) was added to a solution of compound OH-TEG-alkyne 6
(410 mg, 1.76 mmol) dissolved in acetone (15 ml). Aer the
reaction was stirred for 3 h, isopropyl alcohol was added until
the color became green. Then, the reaction solution was l-
trated using Celite® and washed by DCM, then evaporated. A
pale yellow oil was obtained with 97% yield (450 mg). Rf: 0.29
(DCM/MeOH (9 : 1)). FTIR: 2980, 2128, 1716 cmꢁ1. 1H NMR (500
MHz, CDCl3): d 6.55 (bs, 1H, COOH), 4.13 (s, 2H, CH2COOH),
4.08 (s, 2H, CH2C^C), 3.80–3.66 (m, 12H, 6CH2O), 2.46 (s, 1H,
C^CH). 13C NMR (125.7 MHz, CDCl3): d 175.2, 79.1, 78.3, 71.2,
70.9, 70.8, 70.7, 70.6, 70.2, 69.1, 62.1.
All chemical materials that used in the experiments were of
analytical grade and were purchased from commercial
suppliers. Tetraethylene glycol, L-ascorbic acid sodium salt
99%, quinoline, propargyl bromide, succinic anhydride 99%, 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC), 2-(3,4,5-trimethoxyphenyl) acetic acid, 3-hydroxy-4-
methoxybenzaldehyde and camptothecin were purchased
from (Alfa Aesar Company, England). 4-(Dimethylamino) pyri-
dine (DMAP) was purchased from Merck Company. Esterase
from porcine liver, pyrene, acetic anhydride, copper, anhydrous
copper sulfate were purchased from (Sigma-Aldrich, USA).
¨
Sodium azide was purchased from (Riedel-de Haen Company,
Germany). Triethylamine and diethyl ether were purchased
from (SDFCL Company, India). HeLa cells and 3T3 broblasts
cell lines were obtained as a kind gi from Dr Johnny Amer (The
source was the American Type Culture Collection [ATCC],
Manassas, VA, USA). DMEM medium, fetal bovine serum (FBS),
L-glutamine and penicillin/streptomycin were purchased from
Biological Industries, Beit Haemek. CellTiter 96® AQueous One
Solution Cell Proliferation Assay (MTS) was purchased from
Promega (Wisconsin, USA).
For ash chromatography, silica gel (Sigma-Aldrich 230–400
mesh) was used. Columns were eluted with positive air
2.2.3. Synthesis Cpt-TEG-alkyne (8). OH-TEG-alkyne
7
(212.0 mg, 0.86 mmol), Cpt (100 mg, 0.29 mmol), EDC (211 mg,
1.16 mmol) and DMAP (106 mg, 0.86 mmol) in anhydrous DCM
Scheme 1 General scheme of the proposed micelle formation.
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(8 ml) were stirred for 24 h at room temperature under argon. of each CA4 and Cpt was prepared (conc. 0.5 mg mlꢁ1), then, it
On the next day, 50 ml of 1 M HCl was added to the reaction and was diluted to a series of concentrations and the absorbance
extracted with 100 ml DCM then the aqueous layer was washed was measured by spectrophotometer at l max 290 nm for CA4,
with 50 ml DCM. Aer that, the organic layer was collected, 365 nm for Cpt and 420 nm for codrug 9.
dried over Na2SO4 and evaporated. The crude product was
2.3.2. Preparation of buffer solutions. Acetate buffer solu-
puried by column chromatography with a mobile phase of tion pH 4.5 : 14 ml of 2 M acetic acid was added to 2.99 g of
DCM/MeOH (20 : 1) to provide pale yellow oil with 78% yield. Rf: sodium acetate dissolved in water and diluted to 1000 ml with
0.32 (DCM/MeOH (20 : 1)). 1H NMR (500 MHz, CDCl3): d 8.33 (s, water.
1H, H7), 8.19 (d, 1H, J ¼ 8.3 Hz, H12), 7.87 (d, 1H, J ¼ 8.3 Hz,
Phosphate buffer solution pH 7.4 : 250.0 ml of 0.2 M potas-
H9), 7.76 (t, 1H, J ¼ 6.9 Hz, H11), 7.66 (t, 1H, J ¼ 7.9 Hz, H10), sium dihydrogen phosphate was added to 393.4 ml of 0.1 M
7.21 (s, 1H, H14), 5.70–5.66 (dd, 2H, J ¼ 17.2 Hz, J ¼ 17.2 Hz, sodium hydroxide.
H17), 5.27 (d, 2H, J ¼ 2.8 Hz, H5), 4.31 (d, 2H, J ¼ 7.9 Hz,
Carbonate buffer solution pH 9.2 : 0.1 M sodium carbonate
COCH2O), 4.14 (s, 2H, CH2C^C), 3.66–3.49 (m, 12H, 6CH2O), was prepared by dissolving 1.06 g anhydrous Na2CO3 in water
2.36 (s, 1H, C^CH), 2.32–2.15 (m, 2H, 2H19), 0.99 (t, 1H, J ¼ and diluted to 100 ml and 0.1 M sodium bicarbonate was
7.7 Hz, H18). 13C NMR (125.7 MHz, CDCl3): d 169.7 (CO), 167.2 prepared by dissolving 0.84 g NaHCO3 in water and diluted to
(C21), 157.3 (C16a), 152.2, 148.8, 146.4, 145.4, 131.2, 130.7, 100 ml. Then, 90 ml of diluted sodium bicarbonate were added
129.6, 128.4, 128.2, 128.1, 120.3, 95.9 (C2, C3, C6–C16), 78.2, to 10 ml of diluted sodium carbonate in to 100 ml volumetric
76.4 (C20), 76.2, 71.0, 70.7, 70.6, 70.5, 70.0, 60.5 (CH2C^C), 68.1 ask.
(COCH2O), 67.2 (C17), 49.9 (C5), 31.8 (C19), 7.6 (C18).
2.2.4. Synthesis of Cpt-TEG-trizole-TEG-CA4 9 (codrug 9). codrug 9 was determined by using shake-ask technique as
CPT-TEG-alkyne 8 (100 mg, 0.17 mmol) and CA4-TEG-N3
reported.30 Briey, compound 9 was added in excess to 1.5 ml
2.3.3. Solubility study. The solubility of the synthesized
5
(83 mg, 0.16 mmol) were dissolved in 3 ml DCM. Then a solu- Eppendorf tube containing 500 ml of phosphate buffer (pH 7.4).
tion of sodium ascorbate (31.2 mg, 0.16 mmol) and CuSO4 The tube was placed onto a plate shaker at 300 rpm, 37 ꢀC.
(25.2 mg, 0.16 mmol) in 3 ml H2O was added to the reaction and Sampling was performed aer 24, 48 and 72 h. The excess solid
stirred over a night at room temperature. Aer 24 h, 50 ml water was separated from the solution by centrifugation at 15 000 rpm
was added to the reaction and extracted with 100 ml DCM. for 10 min. Aer that, exactly 100 ml of the supernatant was
Then, the aqueous layer was washed with DCM (50 ml ꢂ 2) and taken and diluted. Then, the absorbance was measured at lmax
the organic layer was collected, dried over Na2SO4 and evapo- 420 nm and concentrations were obtained using calibration
rated. The crude product was puried by ash column chro- curve with correlation coefficient more than 0.99. The intrinsic
matography in DCM/MeOH (20 : 1) and a yellow oil product was solubility represented by steady-state concentrations.
obtained with 58% yield. Rf: 0.15 (DCM/MeOH (9 : 1)). 1H NMR
2.3.4. CMC determination. The critical micelle concentra-
(500 MHz, CDCl3): d 8.41 (s, 1H, H7-Cpt), 8.21 (d, 1H, J ¼ 8.5 Hz, tion (CMC) of codrug 9 was determined by using pyrene as
H12-Cpt), 7.95 (d, 1H, J ¼ 7.9 Hz, H9-Cpt), 7.84 (t, 1H, J ¼ 7.4 Hz, uorescent probe.31 Briey, different concentrations ranging
H11-Cpt), 7.76 (bs, 1H, H-triazol), 7.67 (t, 1H, J ¼ 7.4 Hz, H10- from 0.05 to 5 ꢂ 10ꢁ6 mg mlꢁ1 of codrug 9 were added to
Cpt), 7.19 (s, 1H, H14-Cpt), 7.10 (d, 2H, J ¼ 6.9 Hz, 2CH, Ar- a saturated solution of pyrene (6 ꢂ 10ꢁ7 M). The asks were
CA4), 6.98 (s, 1H, CH, Ar-CA4), 6.82 (d, 2H, J ¼ 8.3 Hz, 2CH, then heated for 2 h at 60 ꢀC with shaking to equilibrate the
Ar-CA4), 6.44 (d, 2H, J ¼ 10.1 Hz, CH]CH), 5.42–5.38 (dd, 2H, J pyrene and the micelles. Subsequently the asks were allowed
¼ 17.2 Hz, J ¼ 17.2 Hz, H17-Cpt), 5.22 (d, 2H, J ¼ 2.8 Hz, H5- to cool down over night to room temperature. The uorescence
Cpt), 4.65 (s, 2H, CH2COO), 4.49 (s, 2H, CH2COO), 4.35 (s, 2H, excitation spectra were obtained with an emission wavelength
triazol CH2), 3.87–3.83 (m, 5H, CH2 triazol, OCH3), 3.79 (s, 3H, of 390 nm. The CMC was determined as the intersection when
OCH3), 3.75 (s, 6H, 2OCH3), 3.70–3.55 (m, 20H, 10CH2O), 1.22 extrapolating the intensity ratios of I336/I333 for low and high
(m, 2H, 2H19-Cpt), 0.85 (t, 1H, J ¼ 7.6 Hz, H18-Cpt). 13C NMR concentration regions.
(125.7 MHz, CDCl3): d 173.2 (CO), 170.1 (CO), 169.4 (CO), 166.2
2.3.5. Stability study. The stability study was determined
(C21), 158.3 (C16a), 155.2, 155.0, 154.3, 154.0, 153.2, 148.6, on three different pHs (4.5, 7.4, 9.2). Three stocks of concen-
146.5, 145.2, 131.5, 130.8, 129.5, 128.6, 128.5, 128.4, 128.2, tration 1.0 mg mlꢁ1 were prepared in the three different
127.4, 123.3, 121.3, 120.1, 111.2, 104.4, 96.8, 76.8 (C20), 71.2, prepared buffers and aliquots were withdrawn during one week
70.6, 70.5, 70.4, 69.3, 68.1 (COCH2O), 67.2 (C17), 60.3, 56.2, 55.3, and analyzed at UV spectrophotometer.
50.5 (CH2-triazol), 50.2 (C5), 32.4 (C19), 7.7 (C18).
2.3. Solubility, critical micelle concentration and stability
studies
2.4. Hydrolysis study by esterase enzyme
In order to study the hydrolysis of the drug by esterase enzyme
Calibration curves of CPT, CA4 and codrug 9 were constructed was conducted as documented previously.32 Briey, 1 mg of
as follow:
codrug 9 and 1 mg of esterase (10 U mlꢁ1) were dissolved in 5 ml
2.3.1. Calibration curve of combretastatin A4, camptothe- of phosphate buffer (pH 4.7) and gently stirred at 37 ꢀC. An
cin and codrug 9. A calibration curve was developed by plotting aliquot was withdrawn every 10 min and was replaced with
absorbance vs. concentration. At the beginning, a stock solution equal volume of fresh medium to mimic the sink conditions.
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The absorbance of the collected samples was measured by UV reaction with CA4 to obtain CA4-TEG-N3 5 as shown in
spectrophotometer.
Scheme 2.
Once the rst moiety has successfully synthesized, the
second linker was prepared by reacting tetraeythylene glycol
selectively with propargyl bromide in the presence of sodium
hydride in THF. Followed by oxidation reaction to obtain
compound 7 that reacts with Cpt through esterication reaction
using EDC as a coupling agent and DMAP to obtain Cpt-TEG
alkyne 8 as shown in Scheme 3.
As the two moieties were successfully synthesized CA4-TEG-
N3 5 and Cpt-TEG-alkyne 8, they were connected through click
reaction, which is occurred in one pot and produced a high yield
reaction with a minimum byproduct.36,37 Herein we used
anhydrous CuSO4 in the presence of ascorbic acid dissolved in
a mixture DCM : H2O to synthesized CA4-TEG-triazole-TEG-Cpt
9 as shown in the Scheme 4. The connection between the two
anticancer drugs was through triazole group that are highly
enzymatic and chemically stable.38 Moreover, the direct linkage
between the CA4, Cpt and linkers was through an ester bond
that can be hydrolyzed easily by esterase enzyme that is usually
2.5. Cytotoxicity study
2.5.1. Cell lines. The cytotoxic activity of the test
compounds was studied on HeLa cancer cells and 3T3
broblasts.
2.5.2. Cell culture. HeLa cells and 3T3 broblasts were
cultured in 15 cm2 plastic culture plate in culture growth
medium (CGM) which consists of DMEM medium supple-
mented with 10% fetal bovine serum (FBS), L-glutamine and
penicillin/streptomycin. Cells were maintained in the above
medium at 37 ꢀC in a humidied atmosphere containing 5%
CO2.
2.5.3. Cell viability study. HeLa cells and 3T3 broblasts
were cultured in 96-well plates. They were incubated with 100 ml
per well CGM supplemented with different concentrations of
the test compounds for 48 h. Then, they were incubated for 1 h
at 37 ꢀC and 5% CO2 with 20 ml per well of MTS reagent. Finally,
the absorbance was measured by a plate reader at a wavelength
of 490 nm. In order to conrm the synergism, Chou and Tala-
lay's method was followed33 using CompuSyn soware to
calculate the LD50, combination index (CI) and the dose
reduction index (DRI), so that the CI ¼ 1 indicates additive
effect, while CI < 1 indicates synergistic activity and CI > 1
indicates antagonism activity.
2.5.4. Cellular uptake study. The cellular uptake of codrug
9 was studied by loading the uorescent dye uorescein iso-
thiocyanate (FITC) into the micelles of codrug 9. 1 mg of FITC
was added to a stock solution of codrug 9 that was previously
prepared as 0.5 mg mlꢁ1 of DMEM medium. The solution was
then sonicated for 30 min. Aer that it was centrifuged at
5000 rpm for 10 min in order to remove the unloaded FITC.
Subsequently 100 ml aliquots of the resulting FITC-loaded
micelles were diluted with 500 ml DMEM and were then added
to HeLa cells cultured in 24-well plates. The cells were imaged
by uorescent microscope (Olympus IX73®) aer 1 and 2 h of
incubation; before imaging the medium was discarded, and the
cells were washed several times with PBS.
Scheme 2 Synthetic scheme of CA4-TEG-N3 5.
3. Results and discussion
3.1. Synthesis
Scheme 3 Synthetic scheme of Cpt-TEG-Alkyne 8.
The synthesis was achieved by using the adequate linkers to
provide the correct hydrophobic/hydrophilic balance in order to
improve their water solubility, therefore, we utilized two deriv-
atives of tetraethylene glycol one for the Cpt and other for the
CA4 since the biocompatible polyethylene glycol (PEG) chain
has shown effective improvement in the solubility of hydro-
phobic anticancer drugs conjugate and the prolong circulation
of the formed micelles.34,35
The rst linker was synthesized through selective mono-
tosylation of tetraethylene glycol followed by nucleophilic
substitution with sodium azide to obtain compound 2 and
terminated by oxidation reaction utilizing Jones reagent to
Scheme
4 Synthetic scheme of CA4-TEG-triazole-TEG-Cpt 9
obtain the bifunctional linker 3. In a nal step, an esterication (codrug 9).
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overexpressed in cancer cells with a high intracellular concen-
tration39,40 in order to give a rapid release prole of the anti-
cancer drugs.
3.2. Solubility, stability and micelle formation
In order to investigate whether the conjugation of the two low-
water-soluble drugs with the hydrophilic linkers could improve
the overall aqueous solubility, the shake-ask method was
implemented to study the solubility of codrug 9 at 37 ꢀC in
sodium phosphate buffer (pH 7.4). The test was performed
based on calibration curves constructed by UV-Vis spec-
trophotometery. The solubility test was performed in triplicates.
The results showed an improvement in the total solubility of
codrug 9 under the specied conditions with a concentration of
1.8 mM, whereas the solubilities of Cpt and CA4 were 5 mM and
17.2 mM respectively. This enhancement of solubility could be
related to the introduced hydrophilic chains that connect
between the two anticancer drugs. More interestingly, as the
synthesized codrug 9 consists of hydrophobic molecules and
hydrophilic chains (amphiphilic structure), this organized
structure can self-assembly into diverse supramolecular struc-
ture. Moreover, the 1,2,3-traizole group has the potential to
enhance the construction of various supramolecular systems as
the triazole rings facilitate the p–p interactions and has
a polarized C–H bond, which could be used as a H-bond
donor.21,41,42 In this direction, we studied the capability of our
compound to self-assemble into micelle structure through
atomic force microscopy (AFM) and dynamic light scattering
(DLS). The image of AFM resembles the formation of micelle
supramolecular structure with a spherical shape as shown in
Fig. 1A. In addition, the DLS results showed good dispersibility
with a PDI of 0.2 and a hydrodynamic diameter of 146 nm as
shown in Fig. 1B. This diameter range is optimum to permit
a preferential internalization to cancer cells over the normal
cells as previously reported.43,44 Moreover, the CMC was esti-
mated using pyrene as a uorescent probe.31 As the concentra-
tion of the compound 9 increased, a red shi of pyrene
absorbance was noticed in the excitation spectrum (Fig. 1C). In
Fig. 2 (A) Percentage of hydrolysis at different pHs. (B) Percentage of
hydrolysis in the presence of esterase enzyme.
particular, the absorbance band of pyrene in water was at
333 nm, and it shied to 336 nm when pyrene was embedded in
micelles as the pyrene transfer from the water environment to
the hydrophobic core of the micelle. Fig. 1C shows the plot of
the intensity ration I336/I333 versus concentration. The CMC
value was measured to be 1 ꢂ 10ꢁ3 mg mlꢁ1 (0.9 mM) as the
intersection of the two tangent lines. This value is considered
thermodynamically stable enough as a drug delivery vehicle.45
A stability study was also conducted for one week on the
synthesized codrug 9 at three various pHs (4.5, 7.4 and 9.2). The
percentage of conversion of the co-drug to their parent drugs
were measured for one week based on the prepared calibration
curves. The obtain results indicated that the synthesized codrug
9 is totally stable at pH 7.4 during the whole week as shown in
Fig. 1D. Regarding pH 4.5, a hydrolysis was observed with 20%
of the sample aer 24 h and maintained this percentage during
the whole week. In the case of pH 9.2, a higher hydrolysis was
observed with 50% aer 24 h and also maintained this
percentage during the whole week as shown in Fig. 2A.
Moreover, the complete hydrolysis of the co-drug was also
studied in the presence of esterase enzyme. An incubation study
was conducted with porcine liver esterase. It was observed
a complete hydrolysis of the co-drug aer 50 min of incubation
with esterase enzyme, which conrms the total conversion of
the co-drug to the two active anticancer drugs aer 50 min as
shown in Fig. 2B.
3.3. MTS study
The cytotoxicity of codrug 9 was investigated on HeLa cells and
was compared with the free CA4 and Cpt alone. The effect of
Fig. 3 % Viability of HeLa cancer cells and 3T3 fibroblasts determined
by MTS assay after 48 h of treatment. *p # 0.05 compared to 0 nM, $p
Fig. 1 (A) AFM image; (B) DLS result; (C) CMC result of codrug 9 and # 0.05 compared to corresponding CA4 concentration, #p # 0.05
(D) vial photograph demonstrates the stability of codrug 9 after 1 week. compared to corresponding CPT concentration, N ¼ 3.
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Table 1 LD50 and the combination indexes calculated by CompuSyn software
CI value ata
DRI value at
LD50
Drug
LD50 (nM)
LD50
LD75
LD90
LD95
LD75
LD90
LD95
CA4
Cpt
Codrug 9
58.3
50.9
0.29
405
354
912
1199
2054
4064
3567
9322
0.005
0.002
7 ꢂ 10ꢁ4
4 ꢂ 10ꢁ4
a
The combination index (CI) value of <, ¼, > 1 indicate synergism, additive effect and antagonism, respectively.
codrug 9 was also investigated on the noncancerous 3T3 bro- studies.47 Importantly, there was no signicant cytotoxic activity
blasts. The MTS assay was implemented in order to objectively with 3T3 broblasts, which might reect a preferential activity
estimate the cell viability under various concentrations over against cancerous cells.
48 h treatment interval, which was the minimal time period
required for the drug to affect the cells as shown in Fig. 3. The
results demonstrated a concentration dependent cytotoxic 3.4. Cellular uptake study of FITC loaded micelles
activity on HeLa cells under all tested concentrations. Inter-
Cellular uptake of the formed micelles into the cancerous cells
estingly, there was a clear signicant improvement in the
is an essential step to have an effective anticancer activity. Since
anticancer activity of the codrug 9 in comparison to the free
Camptothecin and Combretastatin A4 have low uorescent
drugs over the used concentration range which was up to
intensity, FITC was encapsulated in the self-assembled micelles
approximately ve folds, which might be attributed to a syner-
to obtain a better visualization of the internalization.48 The
cellular uptake was investigated by inverted uorescent micro-
scope. Fig. 4 demonstrated the obtained results aer the treat-
ment of HeLa cells with FITC loaded micelles at two time
gistic activity for the combined drugs as they work in two
different modes of actions, which allowed a concentration as
low as 23.75 nM to exhibit a strong cytotoxic effect. Moreover,
the synergistic effect was conrmed by calculating the combi-
intervals (1 h and 2 h). As shown in the Fig. 4, a bright green
nation index (CI) values at different lethal doses (LD50, LD75,
uorescence was detected inside the HeLa cells, which indicates
LD90, LD95) using CompuSyn soware developed by Chou and
an effective and rapid internalization of the developed micelles.
Together these ndings indicate that the developed micelles
Martin.46 As shown in Table 1, a very low LD50 of the developed
codrug 9 (0.29 nM) was obtained in comparison to the CA4 and
have an effective cytotoxicity with rapid internalization by HeLa
Cpt with LD50 of 58.3 nM and 50.9 nM, respectively. More
cancer cells and a good cytocompatibility on 3T3 non-cancerous
interestingly, the CI values at all doses were less than 0.1 which
cells.
conrm a synergistic effect of the developed co-drug 9. This
synergistic activity not only enhances the therapeutic activity
but also allows the use of lower doses as conrmed by the
calculated dose reduction index (DRI) of each drug as shown in
4. Conclusions
Table 1, which would be expected to decrease the associated
side effects. This synergistic effect coincide with the previously
reported results of the conjugation of camptothecin and cytar-
abine through disulde linkage that showed a rapid cellular
In conclusion, codrug 9 was successfully synthesized with
a strategy to improve the water solubility of the used anticancer
drugs (CA4 and Cpt). The obtained amphiphilic structure has
the capability to self-assemble into micelle structure as
uptake with a synergistic anticancer activity in vitro and in vivo
conrmed by AFM, DLS and CMC determination. The synthe-
sized codrug 9 showed a good stability at the physiological pH,
but could completely hydrolyze into the parent drugs in the
presence of an esterase enzyme. The MTS study showed a great
improvement in the anticancer activity with a strong synergistic
effect as conrmed by the calculated CI without cytotoxic effect
on 3T3 normal cells. Moreover, the cellular uptake study
showed a rapid internalization of the developed micelles within
1 h of the treatment. The developed codrug 9 could provide
a novel therapy for cancer and we recommend further in vivo
studies to determine the pharmacokinetic behaviour and the
anticancer activity in vivo.
Fig. 4 Uptake of FITC-loaded micelles by HeLa cells. The fluorescent Conflicts of interest
FITC signal was detected in HeLa cells treated with FITC-loaded
micelles after 1 h (A–C) or 2 h (D–F).
There are no conicts to declare.
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23 L. Bentzen, S. Keiding, M. R. Horsman, T. Gronroos,
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at An Najah National University under fund identication
number ANNU-1718-Sc-001. We acknowledge Dr Mohammad
Qneibi and Ms Shorooq Sobuh for facilitating cell imaging in
their lab.
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