Acid-controlled release complexes of podophyllotoxin and etoposide with acyclic cucurbit[n]urils for low cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2018.12.035

The targeted or responsive systems are appealing therapeutic platforms for the development of next-generation precision medications. So, we design and prepare acid-controlled release complexes of podophyllotoxin (POD) and etoposide (VP-16) with pH-labile

Full text of DOI:10.1016/j.bmc.2018.12.035

Bioorganic&MedicinalChemistry27(2019)525–532
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acid-controlled release complexes of podophyllotoxin and etoposide with
acyclic cucurbit[n]urils for low cytotoxicity
⁎
Fanjie Lia,c, Dan Liua,c, Xiali Liaoa, Yulin Zhaob, Rongtao Lia, Bo Yanga,
a Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China
b Faculty of Chemical Engineering, Kunming University of Science and Technology, Kunming 650500, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The targeted or responsive systems are appealing therapeutic platforms for the development of next-generation
precision medications. So, we design and prepare acid-controlled release complexes of podophyllotoxin (POD)
and etoposide (VP-16) with pH-labile acyclic cucurbit[n]urils, and their characteristics and inclusion com-
plexation behaviors were investigated via fluorescence spectroscopy, nuclear magnetic resonance and X-ray
power diffraction. Cells incubated with complexes have been analyzed by high-content analysis (HCA), and
cytotoxicity tests have been completed by MTT assay. The results showed that complexes with different binding
constants can release the drug substance in the physiological pH environment of cancer cells, maintain good
anticancer activity, and have low cytotoxicity. This provides a strategy about targeted and responsive systems of
POD and VP-16 for clinical application.
Acid-controlled release
Acyclic cucurbit[n]uril
Podophyllotoxin and etoposide
Inclusion complex
Low cytotoxicity
1. Introduction
in the therapy of tumors and relevant diseases.4,5
Podophyllotoxin (POD, Fig. 1), a natural compound derived from
Discussions on increasing anticancer activity and reducing side ef-
fects of normal cells of anticancer agents have existed in recent years.
Molecular containers as potential drug carriers attracted a lot of at-
tention. The prevailing molecular container compounds include crown
ethers, cryptands, carcerands, calixarenes, cyclophanes, cyclodextrins,
cucurbituril, and complexes self-assembled by metal-ligand and H-
bonding interactions and reversible covalent bonds.1 In general, su-
pramolecular containers have a hollow three-dimensional structure
with a hydrophobic internal cavity and hydrophilic external surface.2
They can form host-guest inclusion complexes with molecules having
suitable size by self-assembly. In this investigation, we aim to study
host–guest interactions between acyclic cucurbit[n]uril with pH sti-
mulus-responsive function and anti-cancer drugs. Therefore, we dis-
close the synthesis of stimulus-response acyclic cucurbit[n]uril and
used to increase drug solubility and targeted drug delivery (Fig. 1).3
From Fig. 1, under mildly acidic conditions (pH 5.5–6.5), host-1 can
degrade into the host-2 and release encapsulated cargoes at an ac-
celerated rate due to the decrease in binding capacity.3 In other words,
when host-1 is delivered onto the surface of tumor cells, anticancer
drugs will be released to play an active role due to mildly acidic con-
ditions of tumor tissues. As a result, smart stimuli-responsive supra-
molecular inclusion complexes have a promising application potential
the roots of Podophyllum pleianthum,6 contains four consecutive chiral
centers and four nearly planar fused rings. This compound has tradi-
tionally and commonly been isolated from podophyllin, resin of Po-
dophyllum rhizome.7,8 It is also an important natural product in
lignan.9,10 POD is widely studied and applied due to its excellent bio-
logical activity such as effective inhibition of herpes virus,11–13 therapy
of toxic sexually transmitted diseases,14–16 treatment of condyloma
acuminate,17–19 etc. The most important thing is that POD has also been
proved to be effective in cancer therapy.8,20 Many studies have shown
that it had good antitumor activity against different tumor cell lines
(such as P-388 murine leukemia, A-549 human lung carcinoma, HT-29
human colon carcinoma and MEL-28 human melanoma).21 What the
mechanism of its antineoplastic and antiviral properties is that it can
lead to the inhibition of tubulin polymerization and the arrest of the cell
cycle in the metaphase.7,22 Unfortunately, it is also lethal to normal
cells and it has a very low water solubility, which prevent it from being
used in clinical research.23 To overcome these shortcomings, podo-
phyllotoxin derivatives have been extensively studied.24–28 Lots of POD
derivatives, such as etoposide, teniposide, and etopophos, have been
developed for achieving higher antitumor activity, better water-solu-
bility, and few side effects. Although great efforts were made to im-
prove its water solubility and cytotoxicity, it’s still worthy of further
⁎ Corresponding author.
E-mail address: yangbo6910@sina.com (B. Yang).
c These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2018.12.035
Received 9 November 2018; Received in revised form 14 December 2018; Accepted 27 December 2018
Availableonline28December2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.

F. Li et al.
Bioorganic&MedicinalChemistry27(2019)525–532
Fig. 1. The structures of Podophyllotoxin, Etoposide, Host-2 and Host-1.
research. Therefore, it is important to develop delivery systems for the
wide clinical application of podophyllotoxin and its derivatives. In
order to solve the above problems, we investigated the interaction of
POD with the host-1. In addition, etoposide (VP-16, Fig. 1), a derivative
of podophyllotoxin synthesized in the 1960s, is a topoisomerase II in-
hibitor, which can prohibit the function of topoisomerase II-DNA and
stop the cell cycle at the late S and G2 phases.29 Since VP-16 was dis-
covered, its application in the treatment of human malignancies has
continued to increase, and it has become an essential antitumor drug.
Moreover, etoposide, as a part of an important second-line drug, can
treat a variety of malignancies including small cell lung cancer,30 non-
hodgkin’s lymphoma,31 hodgkin’s disease,32 non-lymphocytic leu-
kaemia,29 ovarian,32 and kaposi’s sarcoma.33 In addition, etoposide is
part of an important second-line drug or remedy for several cancer
treatments.29 However, low water-solubility and bioavailability limit its
application in cancer treatment. Therefore, the construction of efficient
and nontoxic carriers for the delivery of VP-16 has become an im-
portant step in its further clinical application. Our group has reported
the use of cyclodextrin to form inclusion complexes with POD by host-
guest chemistry to increase its water solubility.34 However, this merely
solves its water solubility and the side effects which restrict its clinical
application not resolved.
in this work was purchased from the National Institute for Control of
Pharmaceutical. Other reagents were of analytical grade. All experi-
ments were carried out by using ultra-pure water.
2.2. Synthesis of host-1
Host-2 was synthesized according to the previous literature35 (see
supporting information). The procedure for the preparation of host-1
was given in the following text as a typical example. Host-2 (1.48 g,
1.15 mmol) was dissolved in ultra-pure water (18 ml). Triethylamine
was added and it was stirred at room temperature for 10 min. Subse-
quently, maleic anhydride (3.58 g, 35.45 mmol) was added and dis-
solved in acetonitrile (0.6 ml). The mixture was stirred at room tem-
perature for 8 h. The reaction solution was adjusted to pH 6.0 with HCl
(1 M) solution. The precipitate was collected by centrifugation and
washed with acetone (75 ml × 2). The obtained white solid was dis-
solved in water, and the pH was adjusted to 7.4 with a NaOH (1 M)
solution. The solvent was removed on a rotary evaporator to give a
host-1 as a white solid (1.68 g, 1.04 mmol, 87%). Proton nuclear
magnetic resonance (Supporting information, Fig. S5) (1H NMR,
600 MHz, D2O): 6.56 (s, 4H), 6.21 (d, 4H, J = 12.4 Hz), 5.90 (d, 4H,
J = 12.4 Hz), 5.51 (d, 2H, J = 15.4 Hz), 5.45 (d, 4H, J = 16.2 Hz), 5.31
(d, 2H, J = 9.2 Hz), 5.24 (d, 2H, J = 9.2 Hz), 5.18 Hz (d, 4H,
J = 15.8 Hz), 4.16 (d, 4H, J = 15.8 Hz), 4.11 (d, 4H, J = 16.2 Hz), 3.97
(d, 2H, J = 15.4 Hz), 3.85 (m, 4H), 3.64 (m, 4H), 3.33 (m, 8H), 1.68 (s,
6H), 1.66 (s, 6H).
In order to improve the water-solubility, absolute bioavailability
and tumor targeting performance of POD and VP-16, we investigated
the interaction of POD and VP-16 with host-1 in aqueous solution.
Binding behaviors of native host-1 with POD and VP-16 and the solu-
bilization effect of host-1 toward POD and VP-16 were also explored,
which provided a highly effective approach to construct novel POD and
VP-16 formulations with high bioavailability. Moreover, mildly acidic
environments in tumor tissues could cause the degradation of host-1
and the release of cargoes. As a result, inclusion complexes of host-1
with POD and VP-16 can precisely deliver drugs to tumor cells to
achieve targeted drug delivery, which has potential application value
and significance in tumor treatment.
2.3. Preparation of POD/host-1 and VP-16/host-1 supramolecular system
The POD/host-1, VP-16/host-1 inclusion complex were prepared in
aqueous solution. Host-1 (200 mg) was dissolved in 10 ml ultra-pure
water and the required amount of POD (or VP-16) was added to obtain
a 1:3 mol ratio. The mixture was stirred for 7 days at room temperature
in the dark, and the undissolved POD or VP-16 were removed by micro
porous filter membrane (0.22 μm). The filtrate was evaporated under
reduced pressure and dried under vacuum to give the POD/host-1, VP-
16/host-1 inclusion complexes.
2. Experimental
2.1. Reagents and materials
2.4. Preparation of physical mixtures
All the reagents were purchased from commercial sources and used
without further purification. POD (molecular weight = 414.41,
PC > 98%) and VP-16 (molecular weight = 588.56, PC > 99%) used
A physical mixture of Host-1 and POD, VP-16 was prepared. The
molar ratio of host-1 to guest molecule was 1:1. After grinding in a
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Bioorganic&MedicinalChemistry27(2019)525–532
mortar for 10 min, the physical mixture of host-1 and guest molecule
was obtained.
humidified atmosphere of 5% CO2 in air. Afterward, cells were seeded
into 96-well microculture plates. Culture for 24 h, drugs, host-1 and
host-1/drugs inclusion complexes was then added, respectively. After
48 h exposure to the compounds, cells viability was determined by the
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT)
cytotoxicity assay by measuring the absorbance at 490 nm with a mi-
croplate spectrophotometer. Each test was performed in triplicate.
Studying on the release of cargo at different pH values, we used free
rhodamine B or host-1/rhodamine B inclusion complexes incubated
with HepG2 cells at 37 °C 1 h in difference pH value. The medium was
adjusted to pH 6.4 and 5.5 with dilute hydrochloric acid. Subsequently,
cells were washed five times with PBS to remove surface-bound rho-
damine B molecules. Cells were imaged by high content analysis with
high throughput cell analyzer (Thermo Scientific Array Scan XTI).
2.5. 1H NMR analysis
NMR experiments were carried out in CDCl3 or D2O.
Tetramethylsilane (TMS) was used as a reference in D2O. The samples
were dissolved in 99.98% D2O or 99.98% CDCl3 and filtered before use.
1H NMR spectra were acquired on a Bruker Avance III HD spectrometer
(600 MHz) at 25 °C. The one-dimensional spectra of both solutions were
run with FID resolution of 0.18 Hz/point. The residual HDO line had a
line width at a half-height of 2.59 Hz.
2.6. Preparation of samples for Job’s plots and complex constant
The stoichiometric relationship between host-1 and guest was de-
termined by Job’s curve method.36 We used fluorescence spectroscopy
to monitor the continuously changing data. The total molar con-
centration of host-1 and guest remains unchanged. And the molar
fraction of the guest varies from 0.1 to 0.9. The fluorescence intensity of
the solution at different mole fractions was monitored separately.
3. Results and discussion
3.1. 1H NMR analysis
1H NMR spectroscopy studies were used to give a deep insight into
the host-guest interactions. The addition of 3 eq. of POD (or VP-16) was
added to a solution of host-1 in D2O (6.2 × 10−3 M). POD and VP-16 is
transparent to 1H NMR under most conditions when D2O is used as a
solvent, owing to its poor water solubility. Assessment of the POD
complex by 1H NMR clearly demonstrated the presence of the frame-
work protons of the POD molecule, which was consistent with the
significant solubilization (Fig. 2). Comparing Fig. 2b with Fig. 2c, we
could clearly see that some of the protons of podophyllotoxin have been
shifted, due to the shielding effect of the electronrich host-1 cavity. The
proton resonances of host-1 also displayed slight changes in their che-
mical shifts as well as significant signal broadening because of com-
plexation dynamics between host-1 and POD. The changes in the
1HNMR spectra indicate that POD was completely encapsulated within
the host-1 cavity. Like Podophyllotoxin, VP-16 complex by 1H NMR
also clearly demonstrated the presence of the framework protons of the
VP-16 molecule, which was consistent with the significant solubiliza-
tion (Fig. 3). Comparing Fig. 3, we could find that the protons of the VP-
16 and host-1 have been shifted. From the 1H NMR changes, we can
assure that VP-16 has entered the cavity of the host-1.
2.7. Fluorescence spectroscopy
Fluorescence absorption spectroscopy measurements were per-
formed by using a Shimadzu RF-5301 pc in a constant temperature
compartment using a conventional 1 cm path (1 cm × 1 cm × 4 cm)
quartz cell maintained at 25 °C by Shimadzu TB-85 Thermo. Spectral
titration was performed by the following steps: POD (1.0 × 10−6 M)
and host-1 (1.0 × 10−4 M) solution were provided in a buffer solution
(KH2PO4-NaOH, pH7.4). The concentration of POD was held constant
at 1.0 × 10−6 M. Then, an appropriate amount of host-1 was added,
and the final concentrations varied from 0 to 0.0297 mM (host-1:
0.0000, 0.0017, 0.0033, 0.0050, 0.0083, 0.0115, 0.0147, 0.0208,
0.0297 mM at pH 7.4. The absorption spectra measurements were taken
after 1 h. Spectral titration was performed by the following steps: VP-16
(1.0 × 10−7 M) and host-1 (1.0 × 10−5 M) solution were provided in a
buffer solution (KH2PO4-NaOH, pH7.4). The concentration of VP-16
was held constant at 1.0 × 10−7 M. Then, an appropriate amount of
host-1 was added, and the final concentrations varied from 0 to
0.0297 mM (host-1: 0.0000, 0.0001, 0.0007, 0.0014, 0.0041, 0.0078,
0.0144, 0.0248, 0.0446, 0.0632 mM at pH 7.4). The absorption spectra
measurements were taken after 1 h. Measurements were made in the
220–700 nm spectral range. All experiments were carried out in tripli-
cate.
3.2. Stoichiometry
The stoichiometry between the host and guest can be effectively
measured by the Job’s plots. In this work, Job’s plot was employed to
obtain the stoichiometry of guest and host-1 via the fluorescence
spectrometer. The concentration of guest solution (1 × 10−6 M) was
kept constant, and it changed the concentration of the host-1 so that the
mole fraction of guest (guest/[guest + host − 1]) varies between 0.1
and 0.9. In the concentration range, the Job’s plot showed a maximum
at a molar fraction of 0.5 (Fig. 4), proving the 1:1 inclusion com-
plexation between POD and host-1. Similarly, VP-16 and host-1 Job’s
plot showed a maximum at a molar fraction of 0.5 (Fig. 4), proving the
1:1 inclusion complexation between VP-16 and host-1.
2.8. Powder X-ray diffraction (XRD)
The XRD patterns were obtained by using
a D/Max-3B dif-
fractometer with Cu Kα radiation (40 kV, 100 mA), at a scanning rate of
5°/min. Powder samples were mounted on a vitreous sample holder and
scanned with a step size of 2θ = 0.02° between 2θ = 5° and 60°.
2.9. Host-1 degradation
Use 1H NMR to monitor the degradation of the host under acidic
conditions. We incubated host-1 at 37 °C and pD 5.0 for 3 h, 6 h and 3 d,
respectively. Then we performed 1H NMR studies on the incubated
product.
2.10. Cell culture study
Here we have selected four human cells (HCT116, HepG2, SY5Y,
293T). Doxorubicin and cisplatin were used as the positive control.
Cells were suspended in RPMI 1640 (Hyclone Corp. Utah, USA) sup-
plemented with 10% fetal bovine serum (Hyclone) at 37 °C in a
Fig. 2. 1H NMR spectra of (a) host-1 in D2O, (b) host-1/POD inclusion complex
in D2O, (c) POD in CDCl3 at 25 °C.
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Bioorganic&MedicinalChemistry27(2019)525–532
Table 1
Complex stability constants (Ks) and Gibbs free energy change −ΔG (KJ mol−1
)
for 1:1 inclusion complexes of guest artesunate with host in PBS (pH 7.4) at
25 °C.
Ks (M−1
)
logKs
−ΔG (KJ mol−1
)
Host-1/POD
Host-1/VP-16
Host-2/POD
Host-2/vp-16
(5.09
(2.22
0
0.12) × 104
4.7
26.85
24.79
N/A
0.08) × 104
4.3
N/A
N/A
Fig. 3. 1H NMR spectra of (a) host-1 in D2O, (b) host-1/VP-16 inclusion com-
0
N/A
plex in D2O, (c) VP-16 in CDCl3 at 25 °C.
illustrates a typical curve-fitting plot for the titration of VP-16 with
host-1, which shows the excellent fit between the experimental and
calculated data and the 1:1 stoichiometry of the VP-16/host-1 inclusion
3.3. Spectral titration
Ks
complex.
The
binding
constant
of
the
complex
was
(1)
H+ G
H·G
(2.22 × 0.08) × 104 M−1 (Table 1).
KS = [G/H]/([G][H]) = (
/
)/{([G]0
/
A
)([H]0
A/ )}
On the other hand, inclusion complex between POD and VP-16 was
not formed from NMR analysis. So, we could think that the binding
constants of host-2 and POD (or VP-16) were 0 respectively. Thus, we
suspect that during the mildly acid conditions, host-1 will be slowly
released during the process of cleavage of host-1 into host-2. This is
similar to the mildly acidity of the microenvironment of the tumor.
Then we could design a targeted drug delivery system for tumor cells.
We suspect that the drugs release was shown in Fig. 5. The following
experiment will further demonstrate that the drugs in the inclusion
compound will slowly release under mildly acidic conditions.
(2)
A
=
·{1/2[H]0 + [G]0 + 1/KS
A
1/4([H]0 + [G]0 + 1/KS) [H]0·[G]0 }
(3)
[H]0: initial host concentration; [G]0: initial guest concentration;
A: change of absorbance; ε: change of coefficiency; Ks: binding con-
stant.
The inclusion complexation of host-1 with guest was quantitatively
studied in buffer solution (KH2PO4-NaOH, pH7.4) by fluorescence
spectrophotometry. From the change in the intensity of the fluorescence
induced by the addition of the host molecule, we can determine the
binding constants (Ks) of the complexes. From the Job curve experi-
ment, we can know that the host-1 and the guest molecule POD (or VP-
16) inclusion ratio is 1:1, so we can use Eq. (1) to determine their
binding constants. The complex binding constants (Ks) were calculated
for each host–guest combination from the nonlinear squares fit to Eq.
(2). Where host-1 and guest refer to the total concentrations of the guest
and host-1, respectively, the Eq. (2) on the basis of the completion of
the Eq. (3). Finally, the Ks was obtained from the analysis of the se-
quential changes of the fluorescence intensity (Δintensity) at various
host-1 concentrations, with a nonlinear least squares method according
to the curve-fitting Eq. (3). As showed in Fig. S8, the fluorescence in-
tensity of guest increases with the solubility of the host-1 and remained
constant when the host-1 and the guest were saturated. Using a non-
linear least squares curve-fitting method, we obtained the binding
constants for the host-1-guest inclusion complex. Fig. S8b illustrates a
typical curve-fitting plot for the titration of POD with host-1, which
shows the excellent fit between the experimental and calculated data
and the 1:1 stoichiometry of the POD/host-1 inclusion complex. The
binding constant of the complex was (5.09 × 0.12) × 104 M−1. Fig. S9
3.4. XRD analysis
X-ray power diffraction (XRD) was used to examine the crystalline
state of host-1, host-1/guest physical mixture, host-1/guest inclusion
complex and guest. As shown in Fig. 6, host-1 (Fig. 6a), POD (Fig. 6b) is
in crystalline form. The XRD pattern of the physical mixture confirmed
the presence of both species as isolated solids, as the diffractogram
showed both POD peaks and host-1 peaks (Fig. 6c). The inclusion
complexes noted an amorphous halo pattern from the diffractogram, in
which the sharp diffraction peaks of POD completely disappeared
(Fig. 6d). These results further proved that POD had been incorporated
into the cavity of host-1 and presented as the amorphous or disordered
structure. As shown in Fig. S7, free VP-16 is a crystalline solid
(Supporting information, Fig. S10b). The XRD pattern of the physical
mixture confirmed the presence of both species as isolated solids, as the
diffractogram showed both VP-16 peaks and host-1 peaks (Supporting
information, Fig. S10c). The lyophilized inclusion complex has an
amorphous structure (Supporting information, Fig. S10d), probably due
to both the structure of host-1 and the lyophilization process; this is
evidence of the absence of VP-16 crystalline particles.
Fig. 4. Job’s plots by fluorescence spectrometer for the binding stoichiometry of host-1 with POD (a), VP-16 (b).
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Bioorganic&MedicinalChemistry27(2019)525–532
Fig. 5. Host-1 and drug combination and in vivo release schematic diagram.
to host-2 (Fig. 7d). This observation confirms that the degradation
under acidic condition and “charge conversion” from anionic container
to cationic container lead to the guest dissociation.37 When the host-1
was lysed, the drugs entrapped inside were released and the targeted
delivery of the drug was achieved.
We used 1H NMR to demonstrate that host-1 degrade under acidic
conditions and releases drug molecules. From Fig. 8, as the incubation
time of the VP-16/host-1 inclusion complex under acidic conditions was
gradually increased, the drug content gradually decreased. When the
host-1 was completely cleaved, the hydrogen proton signal of the drug
molecule was not visible from the NMR spectra. Host-1 can be cleaved
to host-2 under acidic conditions and that host-2 cannot continue to
wrap the drug to form a drug release.
3.6. In vitro cytotoxicity studies
Fig. 6. Powder X-ray diffractograms for: (a) host-1, (b) POD, (c) host-1/POD
The IC50 values, which represented the concentration of a drug
required for 50% reduction of cellular growth, had been calculated and
showed in Table 2. The evaluation was performed by MTT assay using
doxorubicin and cisplatin as the positive control. It could be seen from
the table that the inclusion complexes exhibited good cytotoxicity and
were less toxic than the drug alone. Consequently, the inclusion com-
plex could reduce the toxicity of the drugs to the cells, and the inclusion
complexes were not toxic to 293T. The inclusion complex has low
toxicity to normal cells and maintains good activity against tumor cells.
In order to study host-1 and to achieve in vitro release of anti-tumor
drugs, we designed an in vitro uptake assay for HepG2 cells. Rhodamine
B is used as a model drug, because it is the most commonly used as
fluorescent substance in fluorescence analysis. To mimic the normal
1:1 (mol proportion) physical mixture, (d) host-1/POD inclusion complex.
3.5. Host-1 degrade and drug release
As showed in Fig. 7, a small portion of the product was decomposed
at 3 h incubation. From this 1H NMR, it was observed that both the
product and the raw material exist (Fig. 7b). After degradation for 6 h, it
was manifestly seen that most of the host-1 was degraded. Meanwhile,
it could be seen from the 1H NMR that the content of the host-1 was
significantly less than that of the incubation for 3 h (Fig. 7c). After
degradation for 3 d, we observed a near complete conversion of host-1
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Fig. 7. 1H NMR spectra of host-1 in D2O for 0 h (a), after incubation in pD 5.0 buffer for 3 h (b), after incubation in pD 5.0 buffer for 6 h (c), and after incubation in pD
5.0 buffer for 3 d (d). and host-2 in pD 7.2 buffer (e). The shadow region indicates the complete degradation of host-1 and host-2.
Table 2
Cytotoxic activities of drugs and inclusion complex in vitro (IC50, µMa).
HCT116
HepG2
SY5Y
293T
POD
0.031
1.255
> 100
8.617
5.931
0.26
0.187
16.88
> 100
3.519
19.05
6.35
0.043
2.45
1.225
16.34
> 100
> 100
> 100
1.81
VP-16
host-1
> 100
7.53
POD/host-1 inclusion complex
VP-16/host-1 inclusion complex
Doxorubicin
13.24
5.45
Cisplatin
8.13
27.2
13.38
2.17
a
The concentrations of free drugs and inclusion complexes mentioned in this
table are as per mole of drugs.
respectively. We then washed the cells with PBS solution in order to
clean the dyes that were not taken up by the cells for subsequent ex-
perimental measurements. We used high-throughput cell analyzers to
observe cells and measure the uptake of Rhodamine B by HepG2 cells
under different conditions. As shown in Fig. 9, at pH 7.4, free Rhoda-
mine B cell uptake is more effective than host-1/Rhodamine B inclusion
complex, because host-1 can encapsulate drugs and reduce their in-
tracellular differentiation efficiency. In contrast, the uptake of fluor-
escent substances by the cells at pH 6.4 and 5.5 was significantly higher
than pH 7.4. This is because host-1 rapidly cleaves and releases cargo in
Fig. 8. 1H NMR spectra of POD/host-1 inclusion complex in pD 5.0 buffer for
1 h (a), after incubation in pD 5.0 buffer for 3 h (b), after incubation in pD 5.0
buffer for 6 h (c), and after incubation in pD 5.0 buffer for 3 d (d).
physiological environment and the extracellular environment of the
acidic tumor, we incubated HepG2 cells with free rhodamine B or host-
1/rhodamine B inclusion complex for 1 h at pH 7.4, 6.4 or 5.5,
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Acknowledgments
This work was supported by Yunnan Applied Basic Research
Projects (No. 2018FA047 and 2018FB018), and the National Natural
Science Foundation of China (NNSFC), (No. 21362016, 21642001,
21361014 and 21302074), which are gratefully acknowledged.
Conflict of interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2018.12.035.
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