Maleimidation of dextran and the application in designing a dextran-camptothecin conjugate

Article abstract of DOI:10.1039/c7ra12954h

Camptothecin analogs, as commonly used chemotherapy drugs, usually have poor water solubility which has limited their use in the clinic. In order to improve the water-solubility of camptothecin, a new dextran derivative Dex-Mal was synthesized and used in

Full text of DOI:10.1039/c7ra12954h

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Maleimidation of dextran and the application in
designing a dextran–camptothecin conjugate†
Cite this: RSC Adv., 2018, 8, 2818
*
*
Qiwen Zhu,‡ Bin Bao,‡ Qiumeng Zhang, Jiahui Yu and Wei Lu
Camptothecin analogs, as commonly used chemotherapy drugs, usually have poor water solubility which
has limited their use in the clinic. In order to improve the water-solubility of camptothecin, a new dextran
derivative Dex-Mal was synthesized and used in designing a dextran–camptothecin conjugate which
contained a CTB-sensitive linker. This conjugate could efficiently release the therapeutic drug SN-38 in
the presence of cathepsin B and the antiproliferative activity of the conjugate was similar to the
approved drug Irinotecan hydrochloride. Furthermore, in the presence of dextran, the conjugate could
self-assemble into nanoparticles in water, which could improve the targeting ability through the EPR
effect. This provides a potential way to formulate a drug delivery system for camptothecin analogs or
other drugs which have poor water solubility.
Received 1st December 2017
Accepted 3rd January 2018
DOI: 10.1039/c7ra12954h
rsc.li/rsc-advances
(HMPA),6 poly-L-glutamic acid (PG),7 polyethylene glycol (PEG),8
styrene maleic anhydride (SMA)9 and dextran.10
Introduction
Dextran, which is synthesized from sucrose by certain lactic-
acid bacteria like Leuconostoc mesenteroides and Streptococcus
mutans, is a natural linear polymer of glucose linked by a 1-6
linked-glucopyranoside.11 The great water solubility and
biocompatibility of dextran makes it approved for clinical
application by FDA. Unlike PEG, which only has modiable
groups at the terminal, dextran is easy to modify due to the large
number of hydroxyl groups which can undergo direct esteri-
cation,12 carbonyldiimidazole activation,13 periodate oxida-
tion,14 cyanogens bromide activation and etherication.15
However, these reactions are oen involved in severe reaction
conditions that are not suitable for many substrates. The thiol–
maleimide ‘Click’ reaction is a popular bioorthogonal reaction
which is commonly applied in the eld of bioconjugation.
Meanwhile, the maleimidation is a common method used for
modication of polymers and antibodies.16,17 Nevertheless, very
few research has been disclosed on the maleimidation of
dextran,18 therefore we developed a method to modify dextran
by maleimidation.
“Smart linkers” are usually activated by the specicity of
tumor tissue such as acidic medium, reduction environment
and some specic enzymes.19 Among these linkers, enzyme-
sensitive linkers are more attractive because of their high
selectivities. Cathepsin B (CTB) is a cysteine protease involved
in numerous pathological processes.20 The expression of CTB
was found to be relatively higher in tumor tissue than in normal
tissue and blood plasma.21 Besides, CTB can selectively recog-
nize several specic dipeptide sequences such valine–citrulline,
phenylalanine–arginine and lysine–lysine.22 With these advan-
tages, CTB-sensitive linkers have been successfully applied in
designing probes,23 polymer–drug conjugates and antibody–
Although great efforts have been made in cancer pharmaco-
therapy in the last few decades, there is still a long way to go to
cure cancer. According to the statistics, there will be more than
1.68 million new cancer cases and 0.60 million cancer deaths in
the United States in 2017.1 Traditional anticancer drugs such as
camptothecin (CPT) and paclitaxel (PTX) analogs are commonly
used in clinical cancer therapy due to their high efficiency and
wide spectrum.2 However, the poor water solubility of CPT and
PTX analogs has limited their clinical application. Moreover,
the nonspecic distribution in the body always leads to severe
side effects. To overcome these obstacles, various drug delivery
systems have been developed, including liposomes,3 micelles4
and polymer–drug conjugates;5 these drug delivery systems can
selectively deliver therapeutic agents into tumor tissues via the
enhanced permeability and retention (EPR) effect, as well
as improving the physicochemical and pharmacokinetic
properties.
Being different from liposomes and micelles, polymer–drug
conjugates are more stable as the presence of the covalent
attachment. These conjugates are usually obtained by coupling
the drugs to polymers through cleavable linkers which are
sensitive to pH, temperature, reduction, oxidation, enzymes
and so on. There are many kinds polymers used for drug
conjugation, such as N-(2-hydroxypropyl) methacrylamide
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, 3663 North Zhongshan Road, Shanghai 200062, P. R. China. E-mail:
wlu@chem.ecnu.edu.cn; qiumeng-zhang@foxmail.com
† Electronic supplementary information (ESI) available: Spectrum of the
corresponding compounds. See DOI: 10.1039/c7ra12954h
‡ These authors contributed equally.
2818 | RSC Adv., 2018, 8, 2818–2823
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drug conjugates.24–26 In previous studies we have disclosed 19.7 mmol) was added and then was stirred overnight. The
a CTB-activated nanoprodrug which had great water solubility solution was poured into water and extracted with ethyl acetate.
and in vivo anti-tumor activity.24 Herein we introduce a dextran– The organic layer was washed with water followed by brine and
camptothecin conjugate based on our earlier report.
dried over Na2SO4. The nal product was puried by silicagel
column to afford a white solid (5.0 g, 49%), mp 156–158 ꢀC. 1H-
NMR (400 MHz, DMSO-d6) d 11.93 (s, 1H), 8.05 (d, J ¼ 8.3 Hz,
1H), 7.89 (s, 1H), 7.41–7.14 (m, 15H), 4.31 (d, J ¼ 7.5 Hz, 1H),
3.11–2.85 (m, 2H), 2.37–2.21 (m, 2H), 2.14 (t, J ¼ 7.4 Hz, 2H),
1.81 (s, 3H), 1.49–1.30 (m, 4H), 1.28–1.15 (m, 2H); 13C-NMR (100
MHz, DMSO-d6) d 174.3, 169.5, 168.9, 144.3, 129.0, 128.0, 126.7,
65.8, 51.5, 38.3, 34.1, 33.6, 28.5, 25.8, 24.1, 22.4. MS (ESI) m/z ¼
541.3 [M + Na]+
Experimental
Materials and methods
1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DRX-400 MHz spectrometer (400 and 100
MHz, respectively) using CDCl3, D2O, or DMSO-d6 as solvents
with TMS as an internal standard. Chemical shis were re-
ported as d (ppm) and spin–spin coupling constants as J (Hz)
values. The mass spectra (MS) were recorded on a Finnigan
MAT-95 mass spectrometer. Melting points were taken on
a SGW X-4 melting point apparatus, uncorrected and reported
in degrees centigrade. Column chromatography was performed
with silica gel (200–300 mesh). UV-vis absorption spectra were
recorded on a Varian Cary 100 spectrophotometer.
Maleic anhydride, beta-alanine, acepramin and N-acetyl-L-
cysteine were purchased from Energy Chemical Reagent Co.
(Shanghai, China). HATU was purchased from Highne Biotech
Co., Ltd (Suzhou, China). Dextran (20 kDa) was purchased from
Pharmacosmos. Compound 8 was synthesized before and other
chemicals were purchased from Sinopharm Chemical Reagent
Co., Ltd. Human colon cancer cell line HCT-116, human
hepatoma cell line HepG2 and human cervical cancer cell line
HeLa were purchased from the Shanghai Institute of
Biochemistry and Cell Biology (Shanghai, China). The cell
culture uid and FBS were purchased from Thermo Fisher
Scientic.
Compound 8. Compound 8 was synthesized as described in
the ref. 24.
Compound 9. Compound 8 (800 mg, 0.74 mmol) was dis-
solved in dichloromethane and TFA was added. The mixture
was stirred at room temperature for 2 h and then the solvent
was removed under vacuum to give the crude product directly
for the next step. Compound 7 (577 mg, 1.04 mmol) was dis-
solved in anhydrous DMF and HATU (395.2 mg, 1.04 mmol),
DIEA (0.24 mL, 1.4 mmol) was added, then the previous crude
product was dissolved in DMF and added to the solution.
Subsequently the mixture was stirred at room temperature
overnight and the solvent was removed under vacuum to give
the residue. Then it was puried by column chromatography to
afford the target compound (550 mg, 54%). 1H NMR (400 MHz,
DMSO-d6) d 8.30–8.10 (m, 2H), 8.02–7.84 (m, 2H), 7.62 (dt, J ¼
26.2, 16.0 Hz, 3H), 7.46–7.26 (m, 15H), 5.58–5.32 (m, 4H), 5.21–
5.06 (m, 2H), 4.70–4.54 (m, 2H), 4.36 (d, J ¼ 24.2 Hz, 2H), 4.21 (t,
J ¼ 18.7 Hz, 3H), 3.56 (s, 2H), 3.45 (d, J ¼ 9.8 Hz, 2H), 3.27–2.88
(m, 11H), 2.43–2.29 (m, 3H), 2.18 (dt, J ¼ 15.2, 7.8 Hz, 2H), 2.08–
1.91 (m, 3H), 1.88 (d, J ¼ 7.3 Hz, 3H), 1.81–1.58 (m, 3H), 1.57–
1.20 (m, 12H), 0.92 (dt, J ¼ 16.4, 7.0 Hz, 8H). HR-MS m/z:
1480.7810, (calculated for C80H94N11O15S, 1480.6652).
Synthetic procedures
Compound 2. Maleic anhydride (10.0 g, 0.102 mol) and b-
Dextran–camptothecin conjugate. Compound 9 (100 mg,
alanine (9.90 g, 0.112 mol) were dissolved in glacial acetic acid 0.068 mmol) was suspended in dichloromethane and TFA was
(150 mL) and the solution was heated to reux for 8 h then added to form a yellow solution. Triethyl silicane (0.05 mL, 0.31
acetic acid was removed under vacuum to give the crude mmol) was added to the solution and it turned light kelly. Aer
product which was recrystallized in diethyl ether to afford stirring for 2 h, NaHCO3 (120 mg, 1.5 mmol) in 10 mL water was
a white solid (9.50 g, 55%). Mp 105–107 ꢀC. 1H-NMR (400 MHz, added and the mixture turned colourless and Dex-Mal (120 mg,
DMSO-d6) d 12.4 (s, 1H), 7.02 (d, J ¼ 4.4 Hz, 2H), 3.62 (t, J ¼ 0.069 mmol maleimide unit) was added. The solution was
7.3 Hz, 2H), 2.53–2.45 (m, 2H).
Compound 4. Compound 4 was synthesized as described in water for 24 h in a dialysis tube (MWCO: 10 000). Finally, the
the ref. 28.
solution in the dialysis tube was lyophilized to give the target
Dex-Mal. Compound 4 (calculated for 5.91 mmol) was added product as a white solid.
stirred for another 4 h and then dialyzed against deionized
to a solution of dextran (T20, 11.8 mmol glucose unit) in
anhydrous DMSO. The reaction mixture was heated to 45 ꢀC
overnight and dialyzed against deionized water for 24 h in
a dialysis tube (MWCO: 10 000). Finally, the solution in the
General HPLC method
dialysis tube was lyophilized and washed with cold ethanol to HPLC analysis was performed at room temperature using
give Dex-Mal as a white occulent solid.
a Diamonsil C18 (250 mm ꢁ 4.6 mm) and a mobile phase
Compound 6. Compound 6 was synthesized as described in gradient from 10% CH3CN/buffer (0.1% TFA/H2O) to 30%
the ref. 27.
CH3CN/buffer (0.1% TFA/H2O) for 3 min, 30% CH3CN/buffer
Compound 7. To a solution of compound 6 (8.0 g, 19.7 (0.1% TFA/H2O) to 90% CH3CN/buffer (0.1% TFA/H2O) for
mmol) in anhydrous DMF was added DIEA (6.85 mL, 39.4 12 min, 90% CH3CN/buffer (0.1% TFA/H2O) for 5 min, a ow
mmol) and HATU (7.49 g, 19.7 mmol) in batches. The mixture rate of 1.0 mL minꢂ1, and plotted at 373 nm. This method was
was stirred at room temperature for 2 h and acepramin (2.58 g, used in stability studies.
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Calculation of drug loading
Cell viability assay
Compound 9 was dissolved in DMSO and diluted to different HCT-116, HepG2 and Hela cells were cultured in McCoy's 5A (for
molar concentrations and the dextran–camptothecin conjugate HCT-116) or MEM (for HepG2), or DMEM (for Hela) medium,
was diluted to a given mass concentration as well. The UV-vis supplemented with 10% heat-inactivated FBS and 1% penicillin/
absorption spectrum was recorded on a Varian Cary 100 spec- streptomycin (Thermo Fisher Scientic). All cell lines were
trophotometer. The calibration curve was obtained and the drug maintained at 37 ꢀC with 5% CO2. Cell viability was evaluated by
loading was calculated according to the absorption at 365 nm.
the MTT assay. Cells were seeded in 96-well plates and incubated
for 12 h. A range of concentrations of the test compounds were
added and the plates were incubated for 72 h. Before the addition
of 10 mL 5 mg mLꢂ1 MTT, 100 mL of medium was removed in each
well. Aer 4 h of incubation, 50 mL of lysis solution (10% SDS, 5%
isobutanol and 0.012 mol Lꢂ1 HCl) was added to each well and
incubated for another 12 h. The absorbance was measured using
Water solubility of the dextran–camptothecin conjugate and
Irinotecan hydrochloride
Excess amount of Irinotecan hydrochloride and the dextran–
camptothecin conjugate was added into 0.1 mL water respec-
tively and dissolved thoroughly. The undissolved substances
were ltered by a 0.22 mm lter, and the ltrate was diluted with
DMSO, and the UV-vis absorption spectrum of the dilution was
recorded on a Varian Carry 100 Spectrophotometer.
Particle size distribution of the dextran–camptothecin
conjugate in water
The Dex-Mal and dextran–camptothecin conjugate was dis-
solved in water with a suitable concentration and the size
distribution was measured on a dynamic light scattering (DLS)
(ZetasizerNano ZS, Malvern Instruments, UK).
Enzyme-sensitivity of the dextran–camptothecin conjugate
Fig. 1 The 1H-NMR of the Dex-Mal.
CTB (10 units) from bovine spleen was dissolved in 1 mL buffer
containing 25 mM sodium acetate (pH ¼ 5.0) and 1 mM EDTA at
ꢀ
ꢂ80 C before use. The above stock solution (60 mL) was acti-
vated with 120 mL buffer containing 30 mM dithiothreitol (DTT)
and 15 mM EDTA for 15 min at 37 ꢀC. The activated CTB
solution was diluted with 0.82 mL buffer containing 25 mM
acetate (pH ¼ 5.0) and 1 mM EDTA. The nal concentration of
CTB in this solution was 0.6 unit per mL. Then, the dextran–
camptothecin conjugate in 10 mL DMSO was added to CTB
solution and incubated at 37 ꢀC. The sample (70 mL) was
collected and quenched by the addition of 70 mL acetonitrile.
The concentration of drug released was determined by HPLC
using external standard method.
Scheme 1 Synthesis of Dex-Mal, reagents and conditions: (a) beta-
alanine, glacial acetic acid, reflux; (b) anhydrous acetone, TEA, methyl
chloroformate, NaN3; (c) toluene, reflux; (d) dextran (T-20), DBTDL,
DMSO.
Scheme
2 Synthesis of the dextran–camptothecin conjugate.
Reagents and conditions: (a) TrtCl, DMF; (b) acepramin, HATU, DIEA,
DMF; (c) DMF, HATU, DIEA; (d) TFA, DCM; (e) TFA, DCM, NaHCO3, H2O.
2820 | RSC Adv., 2018, 8, 2818–2823
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Results and discussion
Design of the dextran–camptothecin conjugate
Dex-Mal was synthesized by the reaction of isocyanate and
dextran, which is catalyzed by DBTDL (Scheme 1). The gra
ratio of maleimide which was dened as the number of mal-
eimide per D-glucose units was calculated by the proton peak
areas of maleimide group (f, at about 6.7 ppm) and the proton
peak areas of 1-H in the glucose unit (a) of dextran (at about 4.9
ppm) in the 1H-NMR spectrum of Dex-Mal (Fig. 1). From Fig. 1,
we were noticed that the gra ratios of maleimide of Dex-Mal
were 10%, which means that there was a maleimide group
every ten glucose unit in the Dex-Mal. As mentioned in our
previous paper,24 compound 8 had been successfully synthe-
sized, a spacer with a sulfydryl terminal was need to attach
compound 8 to the polymer. Amidation on the N-terminal of the
dipeptide (Val-Cit) is a common strategy and acepramin is
a frequently used spacer. Cysteine is an endogenous amino
acid, so it was employed as the sulfydryl terminal spacer of
compound 7 (Scheme 2). The Boc group of compound 8 was
removed successfully but the product was unstable, therefore it
was directly treated with the activated intermediate of
compound 7 to afford compound 9 which was conrmed by 1H-
NMR and HR-MS. Compound 9 was treated with TFA and
triethyl silicane to remove the protecting group but it was easily
oxidized so it was directly attached to Dex-Mal at the presence of
PBS (pH ¼ 7.4, 0.1 N). The nal product was puried by dialysis
and analyzed by the UV-vis absorption spectrum. Through the
UV-vis absorption spectrum, we calculated the gra ratio of SN-
38 to the dextran was about 8.3%, and the drug loading rate was
Fig. 2 The UV-vis absorption spectrum of Irinotecan hydrochloride
and dextran–camptothecin conjugate solution.
Fig. 3 The size distribution of dextran–camptothecin conjugate.
a SpectraMax M5 microplate reader at 570 nm. The concentration
of drug inhibiting 50% of cells (IC50) was calculated using the
soware of Graphpad Prism 5.
Fig. 4 The release mechanism of SN-38 from the dextran–camptothecin conjugate.
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