Selective binding and controlled release of anticancer drugs by polyanionic cyclodextrins

Article abstract of DOI:10.1016/j.bmc.2018.03.013

The binding stoichiometry, binding constants, and inclusion mode of some water-soluble negatively charged cyclodextrin derivatives, i.e. heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin(H1), heptakis-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin(H2), mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin (H3) and mono-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin (H4), with three anticancer drugs, i.e. irinotecan hydrochloride; topotecan hydrochloride; doxorubicin hydrochloride, were investigated by means of ¹H NMR, UV–Vis spectroscopy, mass spectra and 2D NMR. Polyanionic cyclodextrins H1-H2 showed the significantly high binding abilities of up to 2.6 × 10⁴–2.0 × 10⁵ M^?1 towards the selected anticancer drugs, which were nearly 50–1000 times higher than the corresponding Ks values of native β-cyclodextrin. In addition, these polyanionic cyclodextrins also showed the pH-controlled release behaviors. That is, the anticancer drugs could be efficiently encapsulated in the cyclodextrin cavity at a pH value similar to that of serum but sufficiently released at an endosomal pH value of a cancer cell, which would make these cyclodextrin derivatives the potential carriers for anticancer drugs.

Full text of DOI:10.1016/j.bmc.2018.03.013

Accepted Manuscript
Selective binding and controlled release of anticancer drugs by polyanionic cy-
clodextrins
Jian-Guang Cheng, Hua-Jiang Yu, Yong Chen, Yu Liu
PII:
S0968-0896(18)30242-6
https://doi.org/10.1016/j.bmc.2018.03.013
BMC 14251
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 February 2018
8 March 2018
8 March 2018
Please cite this article as: Cheng, J-G., Yu, H-J., Chen, Y., Liu, Y., Selective binding and controlled release of
anticancer drugs by polyanionic cyclodextrins, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
1
0.1016/j.bmc.2018.03.013
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Selective binding and controlled release of
anticancer drugs by polyanionic
cyclodextrins
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a
a
a
a,b
Jian-Guang Cheng, Hua-Jiang Yu, Yong Chen, Yu Liu*,
a
College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University,
b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P.R. China;

Bioorganic & Medicinal Chemistry
Selective binding and controlled release of anticancer drugs by polyanionic
cyclodextrins
a
a
a
a,b
Jian-Guang Cheng, Hua-Jiang Yu, Yong Chen, Yu Liu*,
a
College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P.R. China
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The binding stoichiometry, binding constants, and inclusion mode of some water-soluble
negatively charged cyclodextrin derivatives, i.e. heptakis-[6-deoxy-6-(3-sulfanylpropanoic
acid)]-β-cyclodextrin(H1), heptakis-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin(H2),
mono-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin (H3) and mono-[6-deoxy-6-(3-
sulfanylpropanoic acid)]-β-cyclodextrin (H4), with three anticancer drugs, i.e. irinotecan
hydrochloride; topotecan hydrochloride; doxorubicin hydrochloride, were investigated by means
Available online
1
of H NMR, UV-Vis spectroscopy, mass spectra and 2D NMR. Polyanionic cyclodextrins H1-
4
5
-1
H2 showed the significantly high binding abilities of up to 2.6  10 2.0  10 M towards the
selected anticancer drugs, which were nearly 50-1000 times higher than the corresponding Ks
values of native β-cyclodextrin. In addition, these polyanionic cyclodextrins also showed the
pH-controlled release behaviors. That is, the anticancer drugs could be efficiently encapsulated
in the cyclodextrin cavity at a pH value similar to that of serum but sufficiently released at an
endosomal pH value of a cancer cell, which would make these cyclodextrin derivatives the
potential carriers for anticancer drugs.
Keywords:
Polyanionic cyclodextrin
Selective binding
Controlled release
Anticancer drugs
Supramolecular chemistry
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
respectively, which greatly restricts the application of β-CD as
carriers of anticancer drugs. Recently, the negatively charged CD
derivatives have attracted more attention because of their
potential applications in drug delivery. For example, Zhang et al.
reported a negatively charged CD named ORG25969 as a good
acceptor to give an extraordinarily high binding affinity towards
Recently, numerous effective anticancer drugs have been used
for the treatment of various human and animal cancers. Among
them, irinotecan hydrochloride (CPT-11), topotecan
hydrochloride (TPT) and doxorubicin hydrochloride (DOX) are
three prominent leader compounds. CPT-11 and TPT are both
water-soluble semi-synthetic derivatives of the alkaloid
camptothecin. CPT-11 exhibits remarkable antitumor activity
in clinical trials against a variety of human tumors, including
colorectal cancer, lung cancer and malignant lymphoma. TPT
is used clinically in the treatment of relapsed ovarian, lung
cancer, and cervical cancer. DOX is a chemotherapeutic agent
used for the treatment of a wide variety of human malignancies
with an anthracycline structure, which consists of an aglycon,
adriamycinone, combined with an amino sugar, daunosamine.
On the other hand, cyclodextrins (CDs), a class of cyclic
oligosaccharides linked by 1,4-glucose bonds, are water-soluble,
nontoxic, compounds commercially available at low price,
and their torus-shaped cavity can bind various
inorganic/organic/biological molecules. This excellent property
enables the wide application of CDs in fields of molecular
recognition and molecular assembly. Among the CD family, the
7
1
rocuronium bromide (Ks up to10 M ), and thus can be clinically
30
used as a reversal agent in the post-operative recovery. Wenz
1,2
and Apostolakis et al. synthesized a series of negatively charged
CDs and researched their binding behaviors with camptothecin.
The result showed that the stabilities of camptothecin complexes
obtained from solubility measurements of negatively charged CD
3-5
6-8
9-12
derivatives were significantly higher than those of other reported
31,32
CD derivatives.
Herein, we selected four negatively charged
CD derivatives, i.e. heptakis-[6-deoxy-6-(3-sulfanylpropanoic
acid)]-β-CD (H1), heptakis-[6-deoxy-6- (2-sulfanylacetic acid)]-
β-CD (H2), mono-[6-deoxy-6-(2-sulfanyl acetic acid)]-β-CD
1
3-16
(
H3) and mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-CD
1
7-23
33
(H4), and investigated their selective binding and controlled
release behaviors towards anticancer drugs CPT-11, TPT and
DOX. Significantly, with binding abilities much stronger than
those of most previously reported CD derivates, these
polyanionic CDs exhibited the pH-responsive release of drug in a
cancer cell environment. That is, the polyanionic CD/anticancer
drug complex was stable in a biological environment such as
serum (pH 7.2), but efficiently released the encapsulated
anticancer drug at pH 5.7 (endosomal pH values of a cancer cell).
24-28
most used one is β-CD that contains 7 glucose units.
Nevertheless, the complex stability constants (Ks) between native
β-CD and anticancer drugs (CPT-11, TPT and DOX) are very
limited, i.e. 2.6  10 M for β-CD/CPT-11 pair, 8.8  10 M
for β-CD/TPT pair, and 2.1  10 M for β-CD/DOX pair
29
2
−1
3
−1
2
−1

corresponding Ks values of native β-CD. A possible reason may
be that the seven anionic side arms on H1 or H2 (either of H1 or
H2 possesses 7 negative charges) gave the greatly strengthened
electrostatic interactions with the cationic guest. Moreover, the
extended cavity formed by seven side arms may also provide the
additional van der Waals and hydrophobic interactions towards
the accommodated drug. As a result, host H1 exhibited the fairly
high encapsulation efficiency (> 75%) and loading efficiency (>
1
8%) towards the selected anticancer drugs when the
concentrations of anticancer drugs and hosts were fixed at
.1mM, which enables it as a good candidate of anticancer drug
carriers. The anticancer drugs encapsulation efficiency and
0
35
loading efficiency was calculated by the following formulas:
Scheme 1. Chemical structures of host and guest.
Where mloaded is the mass of anticancer drugs that formed
inclusion complex with hosts, m is the mass of anticancer drugs
added, mcd is the mass of hosts, [G] is the initial concentration of
anticancer drugs added, [H] is the initial concentration of
hosts，and M , v and Ks are molecular weight of anticancer
drugs, volume of the solution, and the stability constants of the
inclusion complex, respectively. In addition, the solubility and
stability of inclusion complexes in 10% serum solution were also
investigated. The results showed that the solubility could reach
D
0
2
. Results and discussion
0
D
2
.1. Job plots and binding constants of H1-H4 and anticancer
drugs
UV−vis spectroscopy was employed to determine the host-
guest binding stoichiometry. As shown in Figure 1, the Job plot
of H1/CPT-11 in water gave a maximum at molar fraction of
.5, indicating that H1 formed stoichiometric 1:1 inclusion
0.5 mmol/mL, and the complex could keep stable for at least 24
h.
0
complex with CPT-11. Moreover, the mass spectrum
measurements (Figures S31-S33) also demonstrated the
formation of 1:1 inclusion complexes between cyclodextrin hosts
and anticancer drugs. The quantitative investigation on the
molecular binding behavior of H1 with CPT-11 was examined
by means of UV−vis spectral titration, wherein the UV-vis
spectra of a series of solutions containing the same amounts of
CPT-11 and different amounts of H1 were measured to
determine the binding constant between CPT-11 and H1. As can
be seen from Figure S20, with the addition of H1, the absorbance
maximum of CPT-11 slightly decreased, accompanied by the
appreciable red shift of maximum wavelength. By using the
34
nonlinear least-squares method, the stability constants (Ks)
5
-1
values could be calculated as (1.7  0.2)  10 M according to
the sequential changes of absorbance intensity of CPT-11 with
the different concentrations of H1. Similar 1:1 binding
stoichiometry was also found in the association of hosts H1-H4
with anticancer drugs CPT-11, TPT and DOX, and the
corresponding stability constants (Ks) were listed in Table 1.
Moreover, we also tried to use isothermal titration calorimetry to
determine the binding constants. However, the isothermal
titration calorimetry experiment required the higher
concentrations, and the inclusion complex formed precipitate
under such a concentration. Accordingly, the encapsulation and
loading efficiency of anticancer drugs by hosts were calculated
and listed in Table 1. As seen in Table 1, the native β-CD only
showed very poor binding ability towards the selected anticancer
drugs. Possessing an anionic side arm on the β-CD rim, host H3
Figure 1. Job plot for the binding of H1 with CPT-11 in water at 25 °C,
indicating a 1:1 stoichiometry. The changes of absorbance were measured at
3
69 nm, and the total concentration was maintained at 0.1 mM.
3
4
or H4 showed the moderate binding ability (1.02  10  1.7  10
-1
M ) towards anticancer drugs owing to the electrostatic
interactions between the anionic side arm of host and the cationic
guest. However, host H1 or H2 showed a significantly increased
4
binding ability towards anticancer drugs up to 2.6  10  2.0 
5
-1
1
0 M , which was nearly 50-1000 times higher than the

Figure 2. UV-vis spectral titration of TPT upon addition of H1 in H
5 C. The nonlinear least-squares analysis (inset) of the differential
absorbance to calculate the complex stability constant. The changes of
2
O at
absorbance were measured at 381 nm. ([TPT] = 0.05 mM, H1 = 0.00, 0.01,
0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22 mM from a to l)
2
Table 1. Stability constants (Ks), encapsulation efficiency and loading efficiency of anticancer drugs by negatively charged
cyclodextrins in water at 25 °C
-
1
host
guest
Ks / M
encapsulation efficiency/%
loading efficiency/%
2
3
2
36
37
38
β-CD
CPT-11
TPT
2.6  10
8.8  10
2.1  10
2.29
36.0
2.02
1.26
14.5
DOX
1.03
25.7
5
5
H1
H2
H3
H4
CPT-11
TPT
(1.7  0.2)  10
(1.5  0.2)  10
(2.0  0.31)  10
(2.6  0.2)  10
(4.4  0.4)  10
(2.6  0.2)  10
(5.1  0.5)  10
(1.7  0.2)  10
(2.3  0.1)  10
(4.1  0.1)  10
(9.7  0.8)  10
(1.02  0.07)  10
7
7
8
5
6
5
2
4
1
2
3
8
8.5
7.3
0.0
4.3
2.4
4.3
7.1
7.3
6.2
3.8
7.7
.53
18.6
24.4
18.7
15.8
17.4
13.6
17.4
7.53
12.0
14.0
4.02
5
DOX
4
CPT-11
TPT
4
4
DOX
3
CPT-11
TPT
4
3
DOX
3
CPT-11
TPT
3
3
DOX
2
.2. Binding mode of H1 with anticancer drugs
by fluorescence spectra or naked eyes. For example, the
fluorescence intensity of DOX showed the obvious decrease, and
its color turned dark, after the association with H1 (Figure 4). In
the control experiment, the pH dependence of DOX fluorescence
changes in the absence of -CD hosts was relatively limited.
Therefore, the fluorescence spectra were used to investigate the
controlled release behaviors of polyanionic CD/anticancer drug
systems at different physiological pH values. As shown in Figure
2D NMR spectroscopy is an essential method to investigate
binding mode between host and guest. As shown in Figure 3, we
could see NOE correlations between protons of CPT-11 and
interior protons of H1 from the ROESY spectrum of an
equimolar mixture of H1 with CPT-11. The cross peak A was
assigned to NOE correlations between H18/H23 protons of CPT-
11 and H3 protons of H1, and the cross peak B was assigned to
5, a very slow release of DOX was observed at pH 7.2, indicating
NOE correlations between H28 protons of CPT-11 and H3/H5
protons of H1, and H5 protons gave stronger NOE correlations
than H3 protons. Moreover, the cross peak C was assigned to
NOE correlations between H11/H12/H13/H14 protons of CPT-
that H1/DOX was stable in a biological environment such as
serum. However, the release rate was significantly enhanced
when the polyanionic CD/anticancer drug system was placed in
an acidic solution. That is, > 70% was released at pH 5.7
11 and H3 protons of H1. Therefore, we deduced that the CPT-
(endosomal pH values of a cancer cell) within 140s. The
11 guest entered the H1 cavity from the wide side. Based on the
binding/release of DOX at different pH was mainly owing to the
different binding abilities of H1 towards DOX at different pH.
That is, the binding ability of H1 towards DOX was much
stronger at pH 7.2, which led to the lower release level at pH 7.2.
In addition, the UV-vis spectroscopy experiment demonstrated
that the drug binding/release could be reversible for several
times. Other polyanionic CD/anticancer drug systems also
showed the similar controlled release behaviors of their drugs.
This pH-responsive release of drug in a cancer cell environment
will not only improve its cytotoxic efficacy against tumor cells
but also reduce the toxicity of drug to normal tissues.
ROESY and molecular simulation experiments, the geometry of
inclusion complex of H3 with CPT-11 was proposed where the
CPT-11 guest entered the H1 cavity from the wide side.
Figure 3. 2D ROESY spectra of H1•CPT-11 complex in D
2
O. The cross
peaks indicate intermolecular interactions between CPT-11 and H1
2.3. Controlled release of anticancer drugs
In addition to the UV-vis spectral changes, the association with
polyanionic CDs also led to the obvious fluorescence and color
changes of anticancer drugs, which could be readily monitored

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2
3
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5
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reach 10
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Supplementary Information
Selective Binding and Controlled Release of Anticancer Drugs by
Polyanionic Cyclodextrins
a
a
a
,a,b
Jian-Guang Cheng, Hua-Jiang Yu, Yong Chen, Yu Liu*
a
College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University,
b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P.R.
China;
yuliu@nankai.edu.cn

Materials
I
2
, Ph
these were used without further purification.β-Cyclodextrin (β-CD, Kermel, China) was purified twice by
recrystallization from water before use. N,N-Dimethylformamide was stirred overnight with CaH and
distilled under reduced pressure prior to use. irinotecan hydrochloride (CPT-11), topotecan hydrochloride
TPT) and doxorubicin hydrochloride (DOX) were purchased from Knowshine (Shanghai)
Pharmachemicals Inc. (Shanghai, China). heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin
H1), heptakis-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin (H2), mono-[6-deoxy-6- (2-
sulfanylacetic acid)]-β-cyclodextrin (H3) and mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin
H4) were synthesized and purified according to the previous literatures.
3
P was purchased from HEWONS, and 3-sulfanylpropionic acid was purchased from TCI. All of
2
(
(
(
Synthesis of heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin (H1)
Per-6-iodo-β-cyclodextrin (CD-I) was prepared from β-CD according to procedures reported by stoddart
1
and Kaifer et al. Ph
3
P (13.3 g, 48 mmol) was dissolved in anhydrous DMF (30 mL) under stirring, I
2
(13.5
g, 53 mmol) dissolved in anhydrous DMF (30 mL) was added dropwise to the Ph
3
P solution under nitrogen
protection. Heat evolved during the addition and the solution was heated to 80 °C after addition. Anhydrous
β-CD (4.0 g, 3.3 mmol) was then added to this dark brown solution and stirred at 80 °C for 15 h. After that,
the solution was concentrated under vacuum to half of its original volume and the pH was adjusted to 9-10
by the addition of sodium methoxide under cooling with ice-water bath. The reaction mixture was stirred at
ambient temperature for 30 min and was then poured into methanol (500 mL) to form a precipitate, which
was filtered and washed with methanol. The product was purified by Soxhlet extract with methanol for 1
day and recovered as white powder after drying under vacuum. 3-Sulfanylpropionic acid (1 eqv) was
dissolved in dry DMF (45 mL) under N at room temperature. Sodium hydride (2.2 eqv) was added to this
2
solution in three portions and the mixture was stirred for a further 30 min. A solution of 6-per-deoxy-6-per-
iodo-β-cyclodextrin (0.1 eqv) in dry DMF (45 mL) was then added dropwise to this mixture. After addition,
the reaction mixture was heated at 70 °C for 12 h. After cooling, water (10 mL) was added to the mixture
and the volume was reduced to 40 mL in vacuo. Addition of ethanol (250 mL) resulted in precipitation. The

solid precipitate was collected by filtration and dialyzed for 36 h. The volume was then reduced to 20 mL
in vacuo. Ethanol was then added, and the precipitate was collected by filtration and dried to afford H1 as a
1
white solid. H NMR (D
2
O) δ 5.03 (7H, s), 3.88-3.79 (14H, m), 3.50-3.46 (7H, m), 3.10 (7H, d), 2.89-2.78
-
(
21H,m), 2.51(14H, s); MALDI-MS: m/z 1749.44(M-7Na+6H) .
1
Figure S1. H NMR spectrum (400 MHz, D O, 298 K) of
2
Maldi-MS of
Synthesis of heptakis-[6-deoxy-6-(2-sulfanyl acetic acid)]-β-cyclodextrin (H2)
1
H2 was synthesized in a similar way as described above for H1. H NMR (D2O) δ 5.00(7H, s), 3.94(7H, s),
3
+
3
1
.82 (7H, t), 3.61-3.52 (14H, m), 3.07 (7H, d), 2.85 (7H, s); MALDI-MS: m/z 1853.26 (M+2Na+H) ,
-
651.29 (M-Na+6H) .

1
Figure S3. H NMR spectrum (400 MHz, D O, 298 K) of
2
Maldi-MS of
Synthesis of Mono-[6-deoxy-6-(2-sulfanyl acetic acid)]-β-cyclodextrin (H3)
To a suspension of sodium hydride (0.38g) in dry DMF (20mL) was added 3-sulfanylpropanoic acid (0.7
g,4.6 mmol), and the resulting mixture was stirred for 20 min. Mono-[6-O-(p-toluenesulfonyl)]-β-CD (3.2
g, 2.12mmol) was then added to the mixture, the reaction mixture was heated to 100 °C and stirred for 90

min. After the mixture was cooled to room temperature, acetone was added to the solution and the
precipitate was collected by filtration. The residue was dissolved in a minimum of water and precipitated in
1
ethano, and the precipitate was collected by filtration and dried to afford H3 as a white solid. H NMR
(
(
D O) δ 5.01-4.94 (7H, m), 3.84-3.82 (26H, m), 3.54-3.46 (14H, m), 3.04 (1H, d), 2.76-2.7 (3H, m), 2.35
2
+
+
-
2H, t); MALDI-MS: m/z 1245.28 (M+H) , 1267.26 (M+Na) , 1221.45 (M-Na) .
1
Figure S5. H NMR spectrum (400 MHz, D O, 298 K) of
2
Figure S6. Maldi-MS of

Synthesis of Mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin (H4)
1
H4 was synthesized in a similar way as described above for H3 H NMR (D2O) δ 4.92 (7H, m), 3.86-3.77
+
(
26H, m), 3.55-3.50 (14H, m), 3.05 (2H, d), 3.01 (1H, m), 2.74 (1H, m); MALDI-MS: m/z 1231.3 (M+H) ,
+
-
1
253.3 (M+Na) , 1207.37 (M-Na) .
1
Figure S7. H NMR spectrum (400 MHz, D O, 298 K) of
2
Figure S8. Maldi-MS of

Job plots of H1-H4 and anticancer drugs
Figure S9. Job plot for the H1 + TPT system in water at 25 °C, indicating a 1:1 stoichiometry. The changes
of absorbance were measured at 381 nm, and the total concentration was maintained at 0.1 mM.
Figure S10. Job plot for the H1 + DOX system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 482 nm, and the total concentration was maintained at 0.1 mM.
Figure S11. Job plot for the H2 + CPT-11 system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 369 nm, and the total concentration was maintained at 0.1 mM.

Figure S12. Job plot for the H2 + TPT system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 381 nm, and the total concentration was maintained at 0.1 mM.
Figure S13. Job plot for the H2 + DOX system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 482 nm, and the total concentration was maintained at 0.1 mM.
Figure S14. Job plot for the H3 + CPT-11 system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 369 nm, and the total concentration was maintained at 0.1 mM.

Figure S15. Job plot for the H3 + TPT system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 381 nm, and the total concentration was maintained at 0.1 mM.
Figure S16. Job plot for the H3 + DOX system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 482 nm, and the total concentration was maintained at 0.1 mM.
Figure S17. Job plot for the H4 + CPT-11 system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 369 nm, and the total concentration was maintained at 0.1 mM.

Figure S18. Job plot for the H4 + TPT system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 381 nm, and the total concentration was maintained at 0.1 mM.
Figure S19. Job plot for the H4 + DOX system in water at 25 °C, indicating a 1:1 stoichiometry. The
changes of absorbance were measured at 482 nm, and the total concentration was maintained at 0.1 mM.
UV-vis spectral titration of the inclusion complexes
Figure S20. UV-vis spectral titration of CPT-11 upon addition of H1 in H
2
O at 25 C. The nonlinear least-
squares analysis (inset) of the differential absorbance to calculate the complex stability constant, Ks = (1.7

5
-1

0.2)  10 M , (λ = 369 nm，[CPT-11] = 0.05 mM, H1 = 0.00, 0.004, 0.008, 0.012, 0.016, 0.020, 0.024,
0
.028, 0.032, 0.036, 0.04, 0.056, 0.072, 0.094 mM from a to n)
Figure S21. UV-vis spectral titration of DOX upon addition of H1 in H
2
O at 25 C. The nonlinear least-
squares analysis (inset) of the differential absorbance to calculate the complex stability constant, Ks = (2.0
5
-1

0.3)  10 M , (λ = 482 nm, [DOX] = 0.05 mM, H1 = 0.00, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14,
0
.16, 0.18, 0.2, 0.22 mM from a to m).
Figure S22. UV-vis spectral titration of CPT-11 upon addition of H2 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (2.6  0.2)  10 M . (λ = 369 nm,
CPT-11] = 0.05 mM, H2 = 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.23 mM
from a to m)
2
O at 25 C. The nonlinear least-
4
-1
[

Figure S23. UV-vis spectral titration of TPT upon addition of H2 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (4.4  0.4)  10 M . (λ = 381 nm,
TPT] = 0.05 mM, H2 = 0.00, 0.016, 0.024, 0.032, 0.04, 0.048, 0.056, 0.072, 0.088, 0.12, 0.14, 0.16, 0.18,
.20, 0.22, 0.24, 0.26, 0.28 mM from a to r)
2
O at 25 C. The nonlinear least-
4
-1
[
0
Figure S24. UV-vis spectral titration of DOX upon addition of H2 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (2.6  0.2)  10 M . (λ = 482 nm,
DOX] = 0.05 mM, H2 = 0.00, 0.04, 0.08, 0.12, 0.16, 0.20, 0.24, 0. 28, 0. 32, 0.36, 0.40, 0.44, 0.48 mM
from a to m)
2
O at 25 C. The nonlinear least-
4
-1
[

Figure S25. UV-vis spectral titration of CPT-11 upon addition of H3 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (5.1  0.5)  10 M . (λ = 369 nm,
CPT-11] = 0.05 mM, H3= 0.00, 0.04, 0.12, 0.2, 0.28, 0.4, 0.52, 0.68, 0.76, 0.84 mM from a to j)
2
O at 25 C. The nonlinear least-
3
-1
[
Figure S26. UV-vis spectral titration of TPT upon addition of H3 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (1.7  0.2)  10 M . (λ = 381nm,
TPT] = 0.05 mM, H3=0.00, 0.010, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.10, 0.11, 0.12,
.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.10, 0.20, 0.21mM from a to v)
2
O at 25 C. The nonlinear least-
4
-1
[
0

Figure S27. UV-vis spectral titration of DOX upon addition of H3 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (2.3  0.1)  10 M . (λ = 482 nm,
DOX] = 0.05 mM, H3 = 0.00, 0.08, 0.16, 0.32, 0.4, 0.48, 0.56, 0.64, 0.72, 0.8, 0.88, 0.96, 1.04, 1.12, 1.20,
.28 mM from a to q)
2
O at 25 C. The nonlinear least-
3
-1
[
1
Figure S28. UV-vis spectral titration of CPT-11 upon addition of H4 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (4.1  0.1)  10 M . (λ = 369 nm,
CPT-11] = 0.05 mM, H4 = 0.00, 0.03, 0.07, 0.11, 0.15, 0.19, 0.23, 0.27, 0.31, 0.35, 0.39, 0.43, 0.47, 0.51,
.55, 0.59, 0.63, 0.67, 0.69, 0.73 mM from a to t)
2
O at 25 C. The nonlinear least-
3
-1
[
0

Figure S29. UV-vis spectral titration of TPT upon addition of H4 in H
squares analysis (inset) to calculate the complex stability constant, Ks = (9.7  0.8)  10 M . (λ = 381nm,
TPT] = 0.05 mM, H4 = 0.00, 0.02, 0.6, 0.1, 0.14, 0.18, 0.22, 0.26, 0.3, 0.34, 0.38, 0.42, 0.48, 0.52, 0.56,
.6 mM from a to p)
2
O at 25 C. The nonlinear least-
3
-1
[
0
Figure S30. UV-vis spectral titration of DOX upon addition of H4 in H O at 25 C. The nonlinear least-
2
3
-1
squares analysis (inset) to calculate the complex stability constant, Ks = (1.02  0.07)  10 M . (λ = 482
nm, [DOX] = 0.05 mM, H4 = 0.00, 0.08, 0.016, 0.024, 0.032, 0.04, 0.056, 0.072, 0.088, 0.10, 0.12, 0.136,
0
.152, 0.168 mM from a to n)
The mass spectra of the inclusion complexes

3000
2337. 79
2500
2000
1500
1000
2293. 83
500
0
2000
2200
2400
2600
2800
3000
3200
3400
m/ z
Figure S31. Maldi-MS of a solution containing H1+CPT-11, The peak at 2337.79 was assigned to
+
[
H1+CPT-11-7Na-Cl+7H] .
1000
800
2172. 60
600
400
200
0
2
000
2200
2400
2600
2800
3000
3200
3400
m/ z
Figure S32. Maldi-MS of a solution containing H1+TPT, The peak at 2172.60 was assigned to [H1+TPT -
+
7
Na-Cl+7H] .
2294. 82
800
600
400
200
0
2000
2200
2400
2600
2800
3000
3200
3400
m/ z
Figure S33. Maldi-MS of a solution containing H1+DOX, The peak at 2294.82 was assigned to [H1+DOX
+
-
7Na-Cl+7H] .
The fluorescence and color changes of inclusion complex

Figure S34. Fluorescence spectral changes of CPT-11(a) and TPT(b) upon addition of H1. inset:
photographs of fluorescence changes CPT-11(a) and TPT(b) upon addition of H1 in water. (c) Visible
colour changes of CPT-11 solution upon addition of H1 (d) Visible colour change of DOX solution upon
addition of H1
Figure S35. Fluorescence spectral changes of DOX at pH 5.7 and pH 7.2. ([DOX] = 0.05 mM)
Reference:
1
. Rojas, M. T., Köeniger, R., Stoddart, J. F., Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-343.