Preparation of a Camptothecin-conjugated Molecular Carrier and its Cytotoxic Effect Toward Human Colorectal Carcinoma In Vitro

Article abstract of DOI:10.1002/bkcs.11611

Camptothecin (CPT) and its derivatives are the only chemical class that targets the enzymatic activity of DNA topoisomerase I, which is involved in DNA damage and subsequent cell apoptosis. Despite CPT's advantages, its use has been restricted due to extremely poor solubility, drug resistance by several efflux pumps, short half-life, and poor bioavailability. To overcome these limitations, we designed and synthesized a water-soluble CPT-conjugated molecular transporter that successfully and rapidly delivered CPT into cells. The conjugate had affinity toward mitochondria inside cells and it was distributed mainly to kidney, lung, and spleen. MTT assay confirmed that the CPP-conjugate decreased cell viability of colon cancer cell lines to a much greater extent than the parent drug. This novel approach has the potential of improving CPT efficacy for future in vivo applications, especially for colon cancer therapy.

Full text of DOI:10.1002/bkcs.11611

Article
DOI: 10.1002/bkcs.11611
BULLETIN OF THE
D. Jeong et al.
KOREAN CHEMICAL SOCIETY
Preparation of a Camptothecin-conjugated Molecular Carrier and its
Cytotoxic Effect Toward Human Colorectal Carcinoma In Vitro
Dongjun Jeong,† Tarun Pal,‡ Hyungjoo Kim,§ Tae Wan Kim,§ Goutam Biswas,¶ Daeun Lee,k
Tejinder Singh,k Akula S. N. Murthy,k Wanil Kim,** Kyong-Tai Kim,** and Jungkyun Imk,
*
†Department of Pathology, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea
‡Department of Chemistry, Pohang University of Science and Technology, Pohang, Republic of Korea
§Soonchunhyang Medical Science Research Institute, College of Medicine, Soonchunhyang University,
Cheonan, Republic of Korea
¶Department of Chemistry, Cooch Behar Panchanan Barma University, Cooch Behar, India
kDepartment of Chemical Engineering, Soonchunhyang University, Asan, Republic of Korea.
*E-mail: jkim5279@sch.ac.kr
**Department of Life Science, Division of Molecular and Life Science and Division of Integrative
Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of
Korea
Received August 7, 2018, Accepted September 21, 2018
Camptothecin (CPT) and its derivatives are the only chemical class that targets the enzymatic activity of
DNA topoisomerase I, which is involved in DNA damage and subsequent cell apoptosis. Despite CPT’s
advantages, its use has been restricted due to extremely poor solubility, drug resistance by several efflux
pumps, short half-life, and poor bioavailability. To overcome these limitations, we designed and synthe-
sized a water-soluble CPT-conjugated molecular transporter that successfully and rapidly delivered CPT
into cells. The conjugate had affinity toward mitochondria inside cells and it was distributed mainly to
kidney, lung, and spleen. MTT assay confirmed that the CPP-conjugate decreased cell viability of colon
cancer cell lines to a much greater extent than the parent drug. This novel approach has the potential of
improving CPT efficacy for future in vivo applications, especially for colon cancer therapy.
Keywords: Camptothecin, Colorectal cancer, Drug delivery, Molecular carrier
Introduction
undergoes reversible hydrolysis from a closed lactone form
to a hydroxyl carboxylate structure. Under physiological
pH conditions, the carboxylate CPT is the dominant form
in plasma. It was verified that the lactone-CPT form pro-
vides the therapeutic activities, while the carboxylate CPT
form is more toxic yet less active, providing only ca. 10%
of the pharmacological effect compared to the hydrophobic
lactone form.8 Only the lactone form is capable of crossing
cell membranes.9,10 Furthermore, the ionized CPT exhibits
interactions with abundant blood proteins, displaying
100-fold higher binding affinity toward human serum albu-
min (HSA) than the lactone-CPT.4,11 Thus, the equilibrium
reaction favors the ionized product, converting the more
active lactone-CPT to the inactive form (Figure 1).
Third, CPT is a substrate of several efflux pumps belong-
ing to the ATP-binding cassette (ABC) transporter super-
family present in cell membranes.10 P-glycoprotein (P-gp),
also called ABCB1/MDR1, is the most well-studied ABC
transporter that effluxes neutral and cationic hydrophobic
compounds. It is reported that P-gp is good at blocking
CPT and its derivatives, as well as also other anticancer
drugs such as doxorubicin and paclitaxel.3,12,13 multidrug
resistance protein 1 (MRP1), also called ABCC1, and
Camptothecin (CPT) was first extracted from the Chinese
tree Camptotheca acuminata and was used in traditional
Chinese medicine. It was discovered to be a potent agent
with antitumor and antibiotic activities that acted by inhibit-
ing DNA topoisomerase I (Topo I), an essential enzyme
that can release DNA supercoiling during transcription and
replication.1 CPT binds to the Topo I-DNA intermediate,
holding the single- and double-stranded DNA breaks unli-
gated, and the resulting complex can induce DNA damage
and initiate cellular apoptosis, which is programmed-cell
death.2 In particular, CPT is specific to the S-phase of the
cell cycle; thus, it is highly toxic to rapidly replicating cells
like cancerous cells.3,4
Despite the promising antitumor activity of CPT, the suc-
cess achieved in preclinical studies has not led to clinical
uses of CPT for a variety of reasons. First, CPT is known
to have extremely poor aqueous solubility (2.5 μg/mL),
making it difficult for formulation and administration.5,6
Although CPT derivatives have been prepared in sodium
salt form to enhance water solubility, their efficacy was still
not as good as that of the parent drug.7 Second, CPT
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disulfide bond, and the resulting complex was shown to
penetrate into cells of tumor-bearing mice. However, none
of these approaches has yet reached the clinical therapy
stage.
Recently, our group has designed and developed a series
of advanced molecular carriers that contain carbohydrates
as a scaffold. For example, monosaccharides (e.g., glucose,
mannose, allose, and galactose), disaccharides (e.g., lactose
and trehalose), stereoisomers of inositols, and sorbitol were
utilized as scaffolds.24–28 Similar to CPP, multiple guani-
dine units were attached to the carbohydrate scaffold via
linkers to facilitate cell membrane penetration. Furthermore,
these synthetic carbohydrate-based molecular carriers dem-
onstrated their greatest utility in delivering diverse cargoes
into mammalian tissues and organs. To date, small mole-
cules such as paclitaxel, ibuprofen, and AZT, as well as
larger sized substances such as proteins and nanoparticles
were readily delivered to targets by these molecular
carriers.29–34 Therefore, to make CPT a more potent drug
and to treat colon cancer efficiently with lower doses of
CPT, the need for an efficient drug delivery vector is
straightforward. We herein synthesized a CPT-conjugated
molecular transporter, studied its cell membrane permeabil-
ity, and measured its anticancer activity.
Figure 1. The equilibrium reaction of CPT and the chemical struc-
tures of lactone-CPT and carboxylate-CPT generated by reversible
hydrolysis.
MRP2, also called ABCC2, transport anionic substrates and
have been shown to bind and pump CPT and its derivatives
out of the cell.10,14,15 When a potent inhibitor of MRP2
was administered to MRP2 overexpressed cell lines, the
efflux of CPT was completely inhibited, suggesting the crit-
ical role of CPT resistance by the transporter.11 Thus, mul-
tiple drug resistance in chemotherapy with CPT is mostly
attributed to overexpression of P-gp and MRPs. In addition,
breast cancer resistant protein (BRCP), also called ABCG2,
was identified to confer strong resistance to many CPT
derivatives, such as topotecan and 9-aminocamptothecin,
suggesting that this efflux pump can also associate with
CPT.15,16 Because these ABC transporters are overex-
pressed in tumor cells, their presence can confer resistance
to CPT and finally reduce the intracellular accumulation
of CPT.
Experimental
Due to the above-mentioned issues, CPT trials in clinical
studies have disappointed, engendering many attempts to
meet these challenges. Over the last few decades, various
CPT derivatives with moderate aqueous solubility were dis-
covered or developed; among them, irinotecan (trade name:
Camptosar) and topotecan (Hycamtin) have been approved
for clinical use by the US FDA.2 Currently, irinotecan and
topotecan are mainly used for the clinical treatment of colo-
rectal and ovarian cancers, respectively. Unfortunately,
derivatives have shown a dramatic drop in anticancer effi-
cacy compared to the parent drug, necessitating the devel-
opment of alternative routes for CPT delivery.15,17,18
In one effort for the efficient delivery of CPT, liposome
formulation has been of interest for last few years. CPT can
be dissolved in the lipid bilayer of a liposome, and the pH
can be maintained inside the liposome to favor the equilib-
rium to the lactone-CPT form.19 Similarly, CPT has been
encapsulated in several supramolecules, such as cyclodex-
trin, cucurbituril, and dendrimers, for a safe delivery.20–22
These formulations commonly provide a hydrophobic cav-
ity that can stabilize CPT by weak non-covalent interac-
tions, while the inclusion complex maintains good aqueous
solubility. Also, cell-penetrating peptides (CPP) have been
employed to improve cellular internalization of CPT.23 In
CPP, lysine or arginine rich sequences, which are known as
protein transduction domains (PTD), can associate with
negatively charged molecules over the cellular membrane
and promote drug penetration into the cytoplasm. For
instance, CPT was covalently attached to the CPP via a
Chemicals and Methods of Synthesis. S-(+)-Camptothe-
cin, fluorescein-5-isothiocyanate (FITC-I), triethylamine
(Et3N),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC), 4-dimethylaminopyridine (DMAP), diisopropy-
lethylamine (DIPEA), and trifluoroacetic acid (TFA) were
purchased from Sigma-Aldrich Korea (Yongin, Korea). All
solvents were purified and dried by standard methods prior
to use. NMR structural analyses were performed with a
Bruker ASPECT 300 instrument (Ettlingen, Germany)
operating at 300 MHz for 1H and 75 MHz for
13C. Chemical shifts were recorded in parts per million
(ppm) relative to tetramethylsilane (TMS) or deuterium
1
oxide (D2O), which were used as internal standards for H
NMR. Mass spectra were obtained using the fast atom
bombardment (FAB) method. In analysis of high molecular
weight compounds, matrix assisted laser desorption/ioniza-
tion (MALDI-TOF) data was obtained by Micromass
M@DI at the Biomolecular Diversity Core Facility
(POSTECH, Pohang, Korea). Medium pressure liquid chro-
matography (MPLC) was performed by CombiFlash RF+
Lumen UV with Fluka 100 C8-reversed phase silica gel
(St. Gallen, Switzerland). Analytical Reverse Phase-High
Performance Liquid Chromatography (RP-HPLC) and pre-
parative RP-HPLC were performed on an Agilent 1220
Infinity LC Chemstation (Santa Clara, CA, USA).
Characterization of Synthetic Compounds
20-O-[N-(tert-butoxycarbonyl)-6-aminohexanoyl] camp-
tothecin (1). To a solution of (S)-(+)-camptothecin (40 mg,
0.11 mmol) in dry DMF were added 6-tert-butoxycabonyl-
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Preparation of a Camptothecin-conjugated Molecular Carrier
KOREAN CHEMICAL SOCIETY
aminohexanoic acid (51 mg, 0.22 mmol), EDC (46 mg,
0.24 mmol), and DMAP (7 mg, 0.055 mmol) at rt. under
an argon atmosphere, and the solution was stirred over-
night. The reaction mixture was diluted with water (20 mL)
and then extracted with ethyl acetate (20 mL) three times.
The combined extracts were washed sequentially with
0.1 M hydrochloric acid (30 mL) and distilled water
(30 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude residue was
purified by SiO2 column chromatography (CH2Cl2:
MeOH = 25:1) to afford compound 1 (63 mg, 98%) as a
Rf
0.36
(CH2Cl2:MeOH = 10:1),
1H
NMR
(CDCl3 + CD3OD): δ 1.0 (t, J = 7.5 Hz, 3H), 1.38–1.54
(m, 4H), 1.62–1.71 (m, 2H), 2.11–2.28 (m, 2H), 2.37–2.59
(m, 6H), 3.13 (t, J = 6.3 Hz, 2H), 5.31 (s, 2H), 5.40 (d,
J = 17.1 Hz, 1H), 5.63 (d, J = 17.1, 1H), 7.33 (s, 1H),
7.66–7.71 (m, 1H), 7.82–7.87 (m, 1H), 7.99–8.01 (m, 1H),
8.15–8.18 (m, 1H), 8.54 (s, 1H). 13C NMR (d6-DMSO): δ
7.5, 24.1, 25.7, 28.7, 30.2, 31.8, 32.2, 33.1, 38.1, 50.2,
75.6, 94.6, 118.8, 127.7, 128.5, 128.9, 129.7, 130.4, 131.6,
145.4, 146.0, 147.8, 152.2, 152.3, 156.5, 162.3, 167.2,
171.9, 172.0; MS (FAB) m/z calcd. for C30H32N3O8
562.22, found 562.37 [M + H]+.
ꢀ
pale yellow solid: mp 154 C, Rf 0.38 (CH2Cl2:MeOH =
20:1); 1H NMR (CDCl3): δ 0.91 (t, J = 7.4 Hz, 3H),
1.38–1.49 (m, 13H), 1.64–1.68 (m, 2H), 2.10–2.36 (m,
2H), 2.49 (td, J = 2.5, 7.3 Hz, 2H), 3.04–3.08 (m, 2H),
4.75 (br s, 1H, NH), 5.26 (s, 2H), 5.39 (d, J = 17.2 Hz,
1H), 5.65 (d, J = 17.2 Hz), 7.22 (s, 1H), 7.65–7.68 (m,
1H), 7.80–7.83 (m, 1H), 7.92 (d, J = 8.1 Hz, 1H), 8.20 (d,
J = 8.4 Hz, 1H), 8.39 (s, 1H); 13C NMR (CDCl3): δ 7.7,
24.4, 26.2, 28.5, 29.8, 31.9, 33.8, 40.4, 50.1, 67.2, 75.9,
96.3, 120.5, 128.2, 128.3, 128.4, 12.5, 130.9, 131.6, 146.1,
146.2, 148.8, 152.4, 156.1, 157.6, 167.7, 172.7; MS (FAB)
20-O-[6-(fluoresceinyl-5-thioureido)-hexanoyl)
camp-
tothecin (4). To a solution of 3 (15.6 mg, 0.027 mmol) in
DMF (1 mL) at rt. were added Et3N (0.17 mL, 0.90 mmol)
and FITC-I (17.60 mg, 0.041 mmol). After stirring for 12 h
in the dark, the reaction mixture was azeotroped with tolu-
ene five times at 45 ꢀC. The concentrated solution was
purified by SiO2 column chromatography and then by pre-
parative TLC to yield 4 (19 mg, 84%) as a sticky yellow
oil: Rf 0.42 (CH2Cl2:MeOH = 10:1 + AcOH (0.1%)); 1H
NMR (CDCl3 + MeOD): δ 0.88 (t, J = 6.6 Hz, 3H),
1.15–1.92 (m, 6H), 2.05–2.38 (m, 2H), 2.39–2.63 (m, 2H),
3.51–3.60 (m, 2H), 5.30 (s, 2H), 5.53 (dd, J = 60, 17.1 Hz,
2H), 6.43–6.84 (m, 6H), 7.10 (d, J = 9 Hz, 1H), 7.27 (s,
1H), 7.70 (t, J = 7.2 Hz, 1H), 7.83 (t, J = 7.2 Hz, 1H),
7.85 (s, 2H), 7.86–8.00 (m, 1H), 8.20 (d, J = 8.4 Hz, 1H),
8.48 (s, 1H); HR-MS (FAB) m/z calcd. for C47H39N4O10S
851.2387, found 851.2382 [M + H]+.
m/z
562.37 [M + H]+.
20-O-(6-aminohexanoyl)camptothecin
calcd.
for
C31H36N3O7
562.25,
found
trifluoroacetic
acid salt (2). A solution of 1 (61 mg, 0.108 mmol) in 20%
TFA-CH2Cl2 (2 mL) was stirred at rt. for 1 h. After evapora-
tion of the solvents under reduced pressure, the residue was
triturated with diethyl ether (20 mL). The obtained solid
was filtered, washed with CH2Cl2 (20 mL), and dried under
vacuum to provide the TFA salt of 2 (49 mg, 79%), which
1-O-trityl-2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-
butoxycarbonyl)-guanidino)-propyl]}-6-aminohexanoyl)-
D-sorbitol (5). Compound 5 was prepared according to the
previously published procedure.24 In brief, α-D-glucose, the
starting material, was selectively tritylated by using TrCl in
pyridine. The product 6-O-tritylated glucose was reduced
by using NaBH4 in MeOH which afforded monotritylsorbi-
tol as white solid. The other secondary hydroxyl group was
protected by tert-butylchlorodiphenylsilyl group (TBDPS)
by using TBDPSCl in DMF. The protected-sorbitol deriva-
tive was fully acylated to afford Boc-protected octaguani-
dines. To remove the TBDPS group from one terminal of
the sorbitol scaffold, the compound was treated with tetra-
butylammonium fluoride solution 1.0 M in THF to provide
the product 5.
1
ꢀ
did not require further purification: mp 214 C, H NMR
(CD3OD): δ 1.04 (t, J = 7.4 Hz, 3H), 1.42–1.52 (m, 2H),
1.62–1.78 (m, 4H), 2.14–2.28 (m, 2H), 2.59–2.65 (m, 2H),
2.90 (t, J = 7.6 Hz, 2H), 5.32 (s, 2H), 5.47 (d,
J = 16.8 Hz, 1H), 5.61 (d, J = 16.8 Hz), 7.36 (s, 1H),
7.68–7.74 (m, 1H), 7.85–7.90 (m, 1H), 8.07 (d,
J = 8.2 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.64 (s, 1H); 13C
NMR (CD3OD): δ 8.0, 25.1, 26.6, 28.2, 32.0, 34.2, 40.5, 51.4,
67.7, 77.5, 97.7, 120.5, 129.1, 129.6, 129.8, 130.7, 131.9, 133.3,
147.4, 148.4, 149.4, 153.3, 158.9, 169.6, 173.8; MS (FAB) m/z
calcd. for C26H28N3O5 462.20, found 462.36 [M + H]+.
20-O-(N-succinyl-6-aminohexanoyl)camptothecin (3). To
a solution of compound 2 (30 mg, 0.052 mmol) in dry
DMF (2 mL) were added succinic anhydride (7.8 mg,
0.078 mmol), DMAP (3.1 mg, 0.026 mmol), and pyridine
(12.7 mL, 0.156 mmol). The solution was stirred at rt. under
an argon atmosphere for 12 h. The reaction mixture was
diluted with distilled water (20 mL) and extracted with
ethyl acetate (20 mL) three times. The combined extracts
were washed sequentially with 0.1 M hydrochloric acid
(30 mL) and distilled water (30 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated in
vacuo. The crude residue was purified by SiO2 column
chromatography (CH2Cl2:MeOH = 10:1) to afford com-
1-O-[N-(tert-butoxycarbonyl)-6-aminohexanoyl]-
2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-butoxycarbo-
nyl)-guanidino)-propyl]}-6-aminohexanoyl)-6-O-trityl-D-
sorbitol (6). Compound 5 (250 mg, 0.076 mmol) was dis-
solved in dry CH2Cl2 (20 mL) along with 6-tert-butoxyca-
bonyl-aminohexanoic acid (35.4 mg, 0.152 mmol), EDC
(29 mg, 0.152 mmol), and DMAP (2 mg, 0.015 mmol).
The reaction mixture was stirred under a N2 atmosphere at
rt. for 2 days. After addition of distilled water to quench
the reaction, the solution was extracted with CH2Cl2. The
organic extract was first washed with saturated NaHCO3
solution and then brine and dried over anhydrous Na2SO4.
ꢀ
pound 3 (28 mg, 95%) as a pale yellow solid: mp 162 C,
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Preparation of a Camptothecin-conjugated Molecular Carrier
KOREAN CHEMICAL SOCIETY
The organic phase was concentrated in vacuo, and the resi-
due was purified by SiO2 column chromatography to afford
compound 6 (250 mg, 93%): 1H NMR (CDCl3): δ
1.51–1.77 (m, 191H), 2.28–2.35 (m, 10H), 2.54–2.61 (m,
24H), 3.08–3.11 (m, 2H), 3.43 (brs, 16H), 3.60–4.41 (m,
4H), 4.81–5.62 (m, 4H), 7.24–7.42 (m, 15H), 8.51 (s, 8H),
11.5 (s, 8H); MALDI-TOF-MS: m/z calcd. for
C172H292N29O45 3484.14, found 3484.23 [M + H]+.
(6-aminohexanoyl)-D-sorbitolꢁ9HCl
(9).
Compound
8 (60 mg, 0.016 mmol) was stirred with HCl (g) saturated
ethyl acetate (10 mL) at rt. for 2 days. The solvent was then
removed under vacuum at rt. The resulting precipitate was
washed with hexane at least three times and dried under
vacuum. The product was dissolved in distilled water, fil-
tered through a polytetrafluoroethylene (PTFE) syringe fil-
ter, and lyophilized to give the crude product, which was
purified by preparative RP-HPLC (GRACEVYDAC,
Leicestershire, UK, C-18), (2.0 mL/min, 10:90 = CH3CN:
H2O, 220 nm) to yield 9 (35 mg, 92%) as a white foamy
solid (HCl salt): 1H NMR (D2O + MeOD): δ 1.08 (t,
J = 7.2 Hz, 3H), 1.27–1.42 (m, 12H), 1.67–1.69 (m, 22H),
2.05–2.08 (m, 14H), 2.26–2.54 (m, 14H), 2.72 (m, 2H),
3.01 (m, 2H), 3.25–3.33 (m, 46H), 3.57 (m, 2H), 4.01–4.52
(m, 4H), 4.53–5.70 (m, 8H), 6.75–8.41 (m, 6H); 13C NMR
(D2O + MeOD): δ 8.5, 23.9, 24.4, 25.1, 26.7, 27.1, 27.9,
29.5, 30.7, 31.5, 32.1, 33.3, 34.7, 39.7, 40.5, 40.7, 51.7,
54.5, 55.4, 68.2, 77.9, 99.0, 120.1, 129.1, 129.6, 132.7,
134.1, 149.1, 152.5, 154.5, 158.3, 159.2, 171.6, 176.1;
MALDI-TOF-MS: m/z calcd. for C98H171N32O18 2084.34,
1-O-[N-(tert-butoxycarbonyl)-6-aminohexanoyl]-
2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-butoxycarbo-
nyl)-guanidino)-propyl]}-6-aminohexanoyl)-D-sorbitol
(7). A column of flash silica gel was packed first in hexane
with 1% Et3N at the bottom and then in hexane with 1%
TFA; sea sand layer was placed in between. Compound
6 (240 mg, 0.068 mmol) was dissolved in CH2Cl2. The
solution was loaded onto the column and eluted with
increasing concentrations of MeOH in CH2Cl2 to give com-
1
pound 7 as a colorless, foamy solid (120 mg, 54%): H
NMR (CDCl3): δ 1.26–1.69 (m, 175H), 1.91–2.04 (m,
16H), 2.33 (brs, 10H), 2.86–3.22 (m, 26H), 3.47 (brs,
16H), 3.70–4.51 (m, 4H), 4.65–5.53 (m, 4H), 8.51 (s, 8H),
11.45 (s, 8H); 13C NMR (CDCl3): δ 24.4, 26.2, 27.9, 28.2,
33.8, 38.5, 40.3, 50.4, 53.1, 79.2, 83.2, 153.0, 156.5,
163.3, 170.5, 172.5, 172.7; MALDI-TOF-MS: m/z calcd.
For C153H278N29O45 3242.03, found 3242.15 [M + H]+.
found
2084.39
[M + H]+;
analytical
HPLC
(GRACEVYDAC, C-18): tR = 2.72 min (flow rate = 1
mL/min, UV 220 nm, CH3CN:H2O = 70:30).
1-O-[20-O-(N-succinyl-6-aminohexanoyl)camptothecin]-
2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-butoxycarbo-
nyl)-guanidino)-propyl]}-6-aminohexanoyl)-6-O-
1-O-[20-O-(N-succinyl-6-aminohexanoyl)camptothecin]-
2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-butoxycarbo-
nyl)-guanidino)-propyl]}-6-aminohexanoyl)-6-O-[N-(tert-
[6-(fluoresceinyl-5-thioureido)-hexanoyl]-D-sorbitolꢁ8HCl
(10). To a solution of compound 9 (10 mg, 0.004 mmol) in
DMF (2 mL) at rt. were added diisopropylethylamine
(0.015 mL, 0.083 mmol) and fluorescein-5-isothiocyanate
(2 mg, 0.005 mmol). After stirring for 24 h in the dark, the
solution was repeatedly concentrated at 45 ꢀC under
reduced pressure by azeotropic removal with toluene. The
residue was dissolved in MeOH, filtered through a polyte-
trafluoroethylene (PTFE) syringe filter, and the resulting
solution was evaporated. The crude product was sequen-
tially purified using MPLC (C8 column) and preparative
butoxycarbonyl)-6-aminohexanoyl]-D-sorbitol
(8).
Compound 7 (100 mg, 0.030 mmol) was dissolved in dry
CH2Cl2 along with compound 3 (34 mg, 0.060 mmol),
EDC (11.5 mg, 0.060 mmol), and DMAP (1.0 mg,
0.006 mmol). The reaction mixture was stirred under a N2
atmosphere at rt. for 2 days. After addition of distilled
water to the reaction mixture, the resulting solution was
extracted with CH2Cl2. The organic extract was first
washed with saturated NaHCO3 and then brine and was
dried over anhydrous Na2SO4. The organic layer was con-
centrated in vacuo, and the residue was purified by
SiO2 column chromatography to afford compound
RP-HPLC
(GRACEVYDAC,
MONOMER
C-18),
(2.0 mL/min, 10% CH3CN in H2O, 220 nm) to give com-
pound 10 (6 mg, 52%) as a sticky orange solid: H NMR
1
1
8 (60 mg, 52%): H NMR (CDCl3): δ 0.98 (t, J = 7.2 Hz,
3H), 1.25–1.77 (m, 197H), 2.00–2.84 (m, 42H), 3.08–3.20
(m, 4H), 3.44–3.46 (m, 16H), 3.65–4.5 (m, 4H), 4.87–5.70
(m, 8H), 7.21 (s, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.84 (t, J =
7.8 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz,
1H), 8.42 (s, 1H), 8.50 (s, 8H), 11.47 (s, 8H); 13C NMR
(CDCl3): δ 8.7, 21.3, 24.7, 25.9, 27.1, 28.2, 28.5, 31.9,
33.7, 34.0, 39.3, 45.9, 50.1, 51.4, 52.9, 53.6, 53.7, 67.2,
79.3, 83.1, 96.1, 128.2, 128.4, 128.7, 129.6, 130.9, 131.5,
146.1, 146.4, 149.0, 152.5, 153.2, 156.3, 157.5, 163.7,
172.8; MALDI-TOF-MS: m/z calcd. for C183H307N32O52
3785.23, found 3785.29 [M + H]+.
(MeOD): δ 1.04 (t, J = 7.2 Hz), 1.38–1.42 (m, 12H), 1.69
(m, 22H), 2.01–2.04 (m, 14H), 2.39–2.69 (m, 16H),
3.07–3.13 (m, 50H), 3.72–4.51 (m, 4H), 4.54–5.65 (m,
8H), 6.61–6.65 (m, 2H), 6.70 (s, 2H), 6.83–6.87 (m, 2H),
7.20 (d, J = 7.2 Hz, 1H), 7.38 (s, 1H), 7.71–7.76 (m, 2H),
7.87 (t, J = 8.1 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 8.18 (d,
J = 9.0 Hz, 1H), 8.32 (m, 1H), 8.69 (s, 1H); 13C NMR
(MeOD): δ 8.6, 25.6, 25.9, 27.8, 30.0, 30.4, 32.2, 32.6,
35.1, 37.2, 40.4, 44.0, 52.2, 54.9, 68.3, 98.3, 104.2, 120.1,
129.7, 130.3, 131.4, 132.5, 149.3, 159.2, 174.2; MALDI-
TOF-MS: m/z calcd. For C119H181N33O23S 2472.37, found
2472.51 [M]+; analytical HPLC (GRACEVYDAC C-18):
tR = 2.30 min (flow rate = 1 mL/min, UV 220 nm,
CH3CN:H2O = 70:30), purity 98+%.
1-O-[20-O-(N-succinyl-6-aminohexanoyl)camptothecin]-
2,3,4,5-tetra-O-(N-{bis-[3-(N0,N00-bis-(tert-butoxycarbo-
nyl)-guanidino)-propyl]}-6-aminohexanoyl)-6-O-
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Biological Assays
recent studies in CPT conjugation have focused on the car-
bon 20 position, which has a secondary alcohol group.38–40
Thus, in an attempt at efficient conjugation of CPT to the
sterically crowded scaffold (sorbitol), a CPT derivative with
Materials. High glucose Dulbecco’s modified Eagle’s
medium (DMEM), Roswell Park Memorial Institute (RPMI)
1640, Dulbecco’s phosphate buffered saline (DPBS, pH 7.4),
fetal bovine serum (FBS), and trypsin/EDTA were obtained
from Gibco™, Thermo Fischer Scientific (Loughborough,
UK). Mitotracker and Lysotracker were obtained from Invi-
trogen Inc. (Grand Island, NY, USA). 3-(4,5-Dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
was purchased from Sigma-Aldrich Korea. Milli-Q purified
water (18.2 MΩ) was used to prepare all solutions.
a
flexible linker was synthesized (Scheme 1). The
C20-hydroxyl group of CPT was first coupled to Boc-
aminohexanoic acid in the presence of EDC and DMAP to
afford compound (1) in excellent yield. The Boc protecting
group was removed with 10% TFA in CH2Cl2 to give 2 in
good yield, which did not require further purification. Inter-
mediate 2 was treated with succinic anhydride, DMAP, and
pyridine in DMF to afford 3, which was used as a precursor
for the later CPT conjugation. The acid still had poor aque-
ous solubility, but good solubility in organic solvents such
as DMSO and DMF.
For the purpose of comparing delivery efficiency
between the CPT-conjugated molecular carrier and the
blank compound (i.e., CPT without molecular carrier),
compound 2 was treated with FITC-I and Et3N in DMF to
form 4, which incorporated fluorescein via an isothiocya-
nate bond (Scheme 2).
ꢀ
Cell Culture. HeLa cells were incubated at 37 C in a
humidified 5% CO2 environment in DMEM and 10% (v/v)
FBS with penicillin. In cases of SW480 and HT-29 cells,
RMPI 1640 was used as the medium.
Cellular Uptake Experiment by Confocal Microscopy.
HeLa cells (1 × 105 cells per well) were seeded onto a
35-mm cover glass bottomed dish (SPL Ltd., Pocheon,
Korea) and cultured for 24 h. The cells were washed with
PBS (×1) and then treated with 2 mL of serum free DMEM
(1% v/v DMSO) containing compound 4 or 10 (10 μM
each). After incubation for 30 min at 37 ꢀC, cells were
washed with cold PBS three times to remove non-
internalized samples. The live HeLa cells were investigated
by confocal laser scanning microscopy (Olympus Fluoview
FV1000; Tokyo, Japan) equipped with an oil immersion
lens (NA 1.30, 40×). FITC was excited at 488 nm with an
argon laser, and its fluorescence was observed by emission
band filter 500–530 nm. Mitotracker and Lysotracker were
excited at 543 nm by HeNe laser, and their fluorescence
was observed by 600–700 nm emission filter. Each analysis
was performed in triplicate.
Synthesis of Sorbitol-based Molecular Carrier with
Camptothecin Conjugate. The molecular carrier was
designed to have multiple guanidines at its terminus via
linkers, a fluorescent probe at the one end for microscopy
analysis, and CPT as a cargo at the other end. Following
several steps according to a previous publication, α-D-
glucose was reduced to a linear structure, which is sorbi-
tol.24 As previously described, compound (5) was obtained
by tritylation and acylation to sorbitol. Compound (5) has
four branched-side chains with Boc-protected bis-guani-
dines, to give a total of eight Boc-protected guanidines,
Tissue biodistribution study was carried out as previ-
ously described.35
Cytotoxicity Measurement by MTT Assay. SW480 and
HT-29 cells (4.5 × 103 cells per well) were seeded into
96-well plates. In 24 h, the media was changed to non-
serum RMPI 1640, and after a further 24 h, the cells were
treated with 9 or CPT at different concentrations (1.28, 6.4,
32 nM, 0.16, 0.8, 4, 20, 100 μM). The treated cells were
incubated for 48 h, washed with cold PBS, and then
exposed to MTT with media for 4 h. The media was chan-
ged to DMSO, and the dissolved formazan dye was quanti-
fied by measuring the absorbance at 500 nm. As a control,
non-treated cells were examined in the same manner.
Results and Discussion
Synthesis of Camptothecin with Linker or Fluorescein.
CPT consists of five rings, including one lactone with a sin-
gle chiral center at carbon 20. Since it has been reported
that the 20-(S) isomer is between 10 and 100-fold more
active than the 20-(R) isomer of CPT in the inhibition of
Topo-I, (S)-(+)-CPT was used for the synthesis.36,37 Most
Scheme 1. (a) HOOC(CH2)5NHBoc, EDC, DMAP, DMAP, DMF,
rt., 12 h; (b) TFA, CH2Cl2, rt., 1 h; (c) succinic anhydride, DMAP,
pyridine, DMF, rt., 12 h; (d) FITC-I, Et3N, DMF, rt., 12 h.
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Scheme 2. (a) EDC, DMAP, HOOC(CH2)5NHBoc, rt., 2 days; (b) SiO2 flash column with hexane-1% TFA and hexane-1% Et3N; (c) 3,
EDC, DMAP, CH2Cl2, rt., 2 days; (d) EtOAc/HCl, rt., 2 days; (e) FITC-I, DMF, DIPEA, rt., 2 days.
which were expected to allow efficient cell penetration.
It has been previously confirmed that more than five or six
guanidines is required to gain reasonable cellular uptake.41
In order to attach a fluorescent probe, Boc-aminohexanoic
acid as a linker was first coupled to 5 by stirring with EDC
and DMAP in DMF to form 6. The trityl group in 6 was
selectively removed in the presence of multiple Boc groups
with an eluting gradient of CH2Cl2/MeOH mixture through
a flash SiO2 column packed with hexane containing 1%
Et3N on the bottom and in hexane containing 1% TFA at
the top. The resulting compound (7) was coupled with the
CPT derivative (3) by stirring with EDC and DMAP in
DMF to obtain the product (8) in moderate yield. The drug
CPT and linker were covalently bonded to the molecular
carrier via ester bonds, which can be hydrolyzed after cellu-
lar uptake to release the payload. All of the Boc groups in
8 were gently removed by treating the compound with ethyl
acetate saturated with HCl to produce 9, which was purified
by prep HPLC. Thus, all of the guanidine moieties were
converted to guanidinium chloride salts, which permit high
aqueous solubility. Compound (9), the CPT-conjugated
molecular carrier, was used as a model substrate for our
later bioassays. Additionally, for attachment of a fluores-
cent probe, compound (9) was treated with FITC-I and
DIPEA in DMF to afford 10. The crude product was puri-
fied by MPLC with a C-8 reverse phase column. This prod-
uct was re-purified by prep HPLC to give 10 in a purity of
98+%.
Cellular Uptake Study of the CPT-conjugated Molecu-
lar Carrier. The synthetic conjugate (10) was investigated
by means of confocal microscopy (Olympus Fluoview
FV1000, NA 1.30, 40×) to determine if it could cross the
cell membrane and translocate into cells. Live HeLa cells
were treated with the conjugate (10) dissolved in serum free
media (10 μM) and then further incubated at 37 ꢀC for
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KOREAN CHEMICAL SOCIETY
30 min. After twice washing the cells with cold PBS, the
cellular uptake in live cells was visually examined by the
fluorescence of the labeled conjugate. Strong green fluores-
cence appeared mainly in the cytoplasm, indicating 10 was
able to penetrate the cell membrane and diffuse into the
cytoplasm. Unlike the conjugate, however, when HeLa
cells were incubated in the same manner with the CPT con-
jugated only with FITC (4), there was no fluorescence
inside the cells. These observations indicate that the molec-
ular carrier successfully delivered CPT, and that multiple
guanidines in the molecular carrier were essential for
imparting cell membrane permeability. Although the mech-
anism of cellular uptake is quite dependent on the charac-
teristics of the delivery vector, cell line, cargo, and other
factors, it has recently been suggested that there are strong
binding events through electrostatic forces and hydrogen
bonding between guanidiniums and cell surface groups
such as phosphates, carboxylates, and sulfates.42 As a con-
sequence, the molecular carrier might enter the cell by
either passive diffusion or endocytosis (Figure 2).
Figure 3. The intracellular localization images by confocal
microscopy. Compound (10) (green, 10 μM, 1 h incubation) was
coincubated with subcellular markers (red, 30 min incubation).
(a) 10 and Mitotracker (100 nM), (c) 10 and Lysotracker
(200 nM). The relative intensity of each fluorescence along the
arrow is shown in (b) and (d).
to internalize into cells by endocytosis, the CPT-conjugate
may enter the cell by a different process from those sug-
gested for common CPPs.43,44 Also, because the CPT is
conjugated by an ester bond via a linker, it is expected the
conjugate could be easily hydrolyzed inside the cell to
release the CPT which can finally reach the cell nucleus.
Cellular Localization of the Conjugate. To get the
insight of the internalization processes, the intracellular
localization was examined more in detail. The HeLa cells
were stained simultaneously with 10 and specific organelle
markers. Live HeLa cells were treated with 10 (10 μm) for
initial 30 min, and then with Mitotracker (100 nM, Invitro-
gen) which is a mitochondria marker, for another 30 min.
Significant colocalization from both fluorescence was
observed (Figure 3(a)), suggesting the conjugate (10) has
high affinity toward mitochondria. In contrast, when 10 and
Lysotracker (200 nM, Invitrogen), which is a lysosome
marker, were treated in a similar way to HeLa cells, coloca-
lization was not observed in both signals (Figure 3(b)).
Intensity profiles of each fluorescence signal along with the
arrow are displayed (Figure 3(b) and (d)), making the
degree of colocalization clear. The result suggests there is
no indication of endocytic process for the cellular uptake.
Because many CPP or CPP-cargo complexes are reported
Tissue Biodistribution of the Conjugate. The tissue dis-
tribution of the conjugate in mice was observed, when the
compound was injected intraperitoneally (ip). Compound
(10) (HCl salt, 93.4 mg/kg) was dissolved in sterile distilled
water and the solution was injected into 8-week-old mice
(C57BL/6). After 30 min, paraformaldehyde (4%) in PBS
(pH 7.4) was perfused into the treated mice, and the major
organs (brain, heart, kidney, liver, lung, and spleen) were
collected and incubated overnight in a solution of sucrose
(0.5 M in PBS). The tissues were frozen in cryoprotectant
and were cut into 15 μm sections. After drying, each
section was transferred to glass slides, and was treated with
Triton X-100 for 15 min. According to the observations
with a fluorescent microscope (Axioplan 2, Carl Zeiss,
Oberkochen, Germany), the conjugate was found mainly in
kidney, lung, spleen, and also to small extent in brain
(Figure 4). These results are different from those of past tis-
sue distribution experiments with other sorbitol-G8 conju-
gates where fluorescence was also seen in the
brain.26,27,29–31,34 However, the CPT-conjugate show much
less distribution in the brain, suggesting the conjugate is
difficult to reach the brain through the Blood Brain Bar-
rier (BBB).
Cytotoxicity Measurement by MTT Assay. To investi-
gate whether the CPT-conjugated molecular carrier had
drug efficacy, the in vitro cytotoxicity of CPT and 9 were
compared. For the assay, compound (9) without FITC was
selected as the conjugate model to prevent any superfluous
effect induced by the chemical fluorescent probe. Because
CPT derivatives are being used clinically in colon cancer
chemotherapy, the tested cell lines were SW480 and HT-
29, which are established from primary adenocarcinoma of
Figure 2. Confocal microscopy images of live HeLa cells incu-
bated with 10 and 4 (10 μM each, 30 min incubation). (a) and
(d) Fluorescence images; (b) and (e) DIC images of 10 and 4,
respectively. (c) Combination of (a) and (b); (f ) combination of
(d) and (e).
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Preparation of a Camptothecin-conjugated Molecular Carrier
KOREAN CHEMICAL SOCIETY
Figure 4. The image of tissue distribution in mice with compound 10 (bottom) and control (top). Exposure times (ms): (a) and (b) 9000,
(c) and (d) 2000, (e) and (f ) 2500, (g) and (h) 2000, (i) and (j) 7000, (k) and (l) 1500.
human colon tissue.45,46 Each cell line was incubated for
48 h in nine concentrations of CPT and 9, and then cell
viabilities were measured by MTT assay (Figure 5). Conju-
gate (9) showed reduced cell viability greater than that of
CPT, demonstrating that 9 provides far better drug efficacy
toward the colon cancer cell lines. The IC50 values of CPT
and 9 in SW480 cells were determined to be 1.53 and
0.54 μM, while those in HT-29 cells were 0.99 and
0.57 μM, respectively. These results indicate that 9 enters
cells more efficiently than CPT alone, thus improving drug
efficacy inside cells.
Conclusions
Although CPT is known to have an anticancer effect, there
are many challenges in using CPT as a chemotherapy drug.
For the purpose of improving drug delivery issue of CPT, we
designed and synthesized a CPT-conjugated molecular trans-
porter that included eight guanidines, which are essential for
cell internalization. A fluorescent probe was also attached to
the conjugate to allow imaging. Using confocal microscopy,
the conjugate was confirmed to internalize rapidly into HeLa
cells and target the mitochondria. The conjugate showed pref-
erence to kidney, lung, and spleen. The cytotoxicity of the
conjugate was quantified by MTT assay, and the resulting
IC50 values of the conjugate in SW480 and HT-29 cells were
much lower than those of CPT. Therefore, we confirmed that
the drug efficacy of the conjugate was well maintained after
internalization. Further studies are underway to exploit the
conjugate in other in vivo experiments and to demonstrate the
potentiating effect of CPT on cancer treatment.
Acknowledgments. I would like to express my gratitude to
Dr. Sung-Kee Chung (Honorary professor at POSTECH) for
his kind advice and interest. This research was supported by
Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry
of
Science,
ICT
&
Future
Planning
(NRF-
2015R1C1A1A01054657) and partly supported by the Indus-
try Technology R&D program of MOTIE/KEIT [10051080,
Development of mechanical UI device core technology for
small and medium-sized flexible display]. This work was also
supported by the Soonchunhyang University.
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