Enhanced anti-cancer efficacy to cancer cells by doxorubicin loaded water-soluble amino acid-modified β-cyclodextrin platinum complexes

Article abstract of DOI:10.1016/j.jinorgbio.2014.03.012

The effective targeted delivery of insoluble anticancer drugs to increase the intracellular drug concentration has become a focus in cancer therapy. In this system, two water-soluble amino acid-modified β-cyclodextrin (β-CD) platinum complexes were report

Full text of DOI:10.1016/j.jinorgbio.2014.03.012

Enhanced anti-cancer efficacy to cancer cells by doxorubicin loaded water-
soluble amino acids-modified β-cyclodextrin platinum complexes
Mei-Xia Zhao, Meng Zhao, Er-Zao Zeng, Yang Li, Jin-Ming Li, Qian
Cao, Cai-Ping Tan, Liang-Nian Ji, Zong-Wan Mao
PII:
DOI:
Reference:
S0162-0134(14)00089-0
doi: 10.1016/j.jinorgbio.2014.03.012
JIB 9494
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
16 December 2013
20 March 2014
21 March 2014
Please cite this article as: Mei-Xia Zhao, Meng Zhao, Er-Zao Zeng, Yang Li, Jin-
Ming Li, Qian Cao, Cai-Ping Tan, Liang-Nian Ji, Zong-Wan Mao, Enhanced anti-
cancer efficacy to cancer cells by doxorubicin loaded water-soluble amino acids-modified
β-cyclodextrin platinum complexes, Journal of Inorganic Biochemistry (2014), doi:
10.1016/j.jinorgbio.2014.03.012
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Author contributions
M. X. Zhao, Y. Li and E.-Z. Zeng performed the experiments. M. X. Zhao, M. Zhao,
E.-Z. Zeng, Y. Li, J.-M. Li, Q. Cao, C.-P. Tan, Z. W. Mao and L. N. Ji reviewed,
analyzed and interpreted the data. M. X. Zhao and Z. W. Mao wrote the paper. All
authors discussed the results and commented on the manuscript.
a
Key Laboratory of Natural Medicine and Immune Engineering, Henan University,
Kaifeng 475004, China
b
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275,
China.
* Corresponding author. Tel: +86 20 84113788; Fax: +86 20 84112245; E-mail:
cesmzw@mail.susy.edu.cn (Z.-W. Mao).
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Abstract: The effectively targeted delivery of insoluble anticancer drugs to increase
the intracellular drug concentration has become a focus in cancer therapy. In this
system, two water-soluble amino acids-modified -cyclodextrin (-CD) platinum
complexes were reported. They showed preferable binding ability to DNA and
effective inhibition to cancer cells. And they could bind and unwind pBR322 DNA in
a manner, which was similar to cisplatin. Besides, our platinum complexes could
effectively deliver the anticancer drug doxorubicin (Dox) into cells and had higher
cell inhibition ratio, but less toxicity on the normal cells, compared with cancer cells.
In this combination system, Dox was encapsulated into the hydrophobic cavities of
-CD at the optimum molar ratio of 1:1, which were validated by UV-visible
(UV-vis) absorption spectroscopy, fluorescence spectroscopy and MTT experiments.
Moreover, the combination system had higher cell inhibition ratio than free Dox and
amino acids-modified -CD platinum complexes. And the results of high content
screening (HCS) showed that Dox-loaded amino acids-modified -CD platinum
complexes could permeate the cell membrane and enter cells, suggesting the efficient
transport of Dox across the membranes with the aid of the -CD. We expect that the
amino acids-modified -CD platinum complexes will deliver antitumor drug Dox to
enhance intracellular drug accumulation and such combination system showed great
potential as antitumor drug.
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Keywords: Cyclodextrin, Platinum complexes, Doxorubicin, Drug delivery,
Combination system
1. Introduction
The development of platinum complexes as antitumor drug is an area of
considerable current interest, and platinum-based anticancer drugs have been
constantly advanced and modified [1-6]. To achieve more effective antitumor activity,
platinum complexes should be transported across the cell membrane and undergo
hydrolysis [7-9]. Thus, solubility should be taken into consideration in the design of
new active platinum complexes [10, 11]. At present, the popular approach to improve
the bioavailability of insoluble anticancer drugs is carriers-based delivery and
modified by biomolecules, but the application of some delivery systems have been
limited by their complicated construction and poor performance in vivo due to uptake
in non-specific tissues [12, 13]. The platinum complexes with better activity in
tumour cells and less toxicity in normal cells could be obtained through linking
bioactive groups to platinum compounds, e.g., amino acids, peptides, nucleosides, and
nucleobases, etc. [14-17]. The platinum complexes with bioactive groups are revealed
to be good DNA intercalators [18-20]. For example, Farrell et al. studied the
mechanism of cytotoxic action of naphthoquinone-platinum(II) complexes [21]. The
results showed that coordination and stabilization of the quinone structure could
substantially affect the chemical and biophysical properties of the pro-ligands, thus
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improving their DNA binding properties and generating compounds capable of
cleaving DNA and catalytically inhibiting Topo I [21].
-Cyclodextrin (-CD) is a type of cyclic oligosaccharides consisting of seven
glucose units with lipophilic central cavities and a hydrophilic outer surface. It can be
used as a pharmaceutical excipient to improve the solubility, stability, and
bioavailability of hydrophobic drug molecules [22-27]. There have been many reports
about cyclodextrins as new drug carriers. For instance, Yao et al. synthesized glycol
chitosan-carboxymethyl -CD and studied their inclusion capability of delivering
different hydrophobic anticancer drugs (5-fluorouracil, doxorubicin (Dox), and
vinblastine), which could be successfully loaded into the cavities of the -CD [28].
Gourevich and co-workers also reported the potential of a cyclodextrin-based
nanocarrier as a drug delivery vehicle, and investigated the cell uptake routes of Dox
encapsulated in the drug delivery vehicle [29]. The results showed that the uptake of
encapsulated Dox into cancer cells was increased in amount by a factor of up to 5.5
when ultrasound was applied. Thus, -CD derivatives are attractive carriers since they
possess the hydrophobic cavities and they are highly desirable on the way to improve
the bioavailability of insoluble anticancer drugs via anticancer drug carriers.
In the present work, we designed and synthesized two Dox loaded water-soluble
cyclodextrin platinum complexes. In this system, amino acid ligands are attached to
-CD in order to evaluate whether the presence of these bioactive ligands can
modulate the DNA binding ability and potential cytotoxic activity of the
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platinumcomplexes (Scheme 1). The amino acids-modified -cyclodextrin could
improve the solubility and stability of the platinum complexes, and the application of
L-Arg with positive charge and L-Phe with neutral charge could be preferably bound
to platinum. The water-soluble -CD platinum complexes with amino acid showed
preferable ability to bind DNA and they bound and unwound pBR322 DNA in a
manner similar to that of cisplatin. Besides, the platinum complexes could effectively
deliver the anticancer drug Dox into cells and had less toxicity in the normal cells than
in cancer cells (Scheme 1). The optimum molar ratio of Dox encapsulated into the
hydrophobic cavities of -CD was validated by UV-visible (UV-vis) absorption
spectroscopy,
fluorescence
spectroscopy
and
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) experiments. In
addition, the positive charge of L-Arg led to a considerable increase in the
intracellular delivery of Dox. Moreover, we further investigated intracellular uptake
of the combination system of amino acids-modified -CD platinum complexes loaded
with Dox by a high content screening (HCS) technique.
2. Experimental Section
2.1. Chemicals and reagents
-CD, p-toluenesulfonyl chloride, triethanolamine, L-Arg, L-Phe, and K2PtCl4
were obtained from Shanghai Chemical Factory, China. -CD was recrystallized
twice from twice-distilled water and dried under vacuum for 12 h at 95 °C before use.
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Other organic solvents were purchased from EM Sciences. Fetal bovine serum (FBS)
and Dulbecco minimum essential medium (DMEM) were from Invitrogen
Corporation. MTT and Dox were from Sigma.
2.2. Preparation of the ligands L-amino acid-modified -cyclodextrin
L-Amino acid-modified -CD was synthesized based on reported method with
slight
modification
[30,
31].
Briefly,
the
mono[6-O-(p-toluene-sulfonyl)]--cyclodextrin and amino acid (L-Arg and L-Phe) at
molar ratio of 1:3 were dissolved in water solution containing triethanolamine at
85 °C for 24 h under N2. Most of the solvent was evaporated under reduced pressure,
and the resulting solution was poured into vigorously stirred 500 mL ethanol, then the
resultant mixture was stored in a 4 °C refrigerator for 18 h to produce a pale yellow
precipitate. The precipitate then was purified by chromatography with deionized water
as eluent, followed by repeated recrystallization from water and ethanol, to gain pale
yellow product in 65.2% yield for L-Arg--CD and in 47.3% yield for L-Phe--CD.
Mass Spectrometry (MS) (Electrospray ionization (ESI), H2O, m/z) L-Arg--CD:
1
1291.7 (M+H); H NMR (DMSO, Tetramethylsilane (TMS)): 2.98-3.64 (m, 45H),
4.53-4.63 (m, 7H), 4.64-4.82 (m, 7H), 5.58-5.86 (m, 14H), 7.34-8.67 (m, 5H), 12.34
(s, 1H); Anal. Calcd for L-Arg--CD complex (C48H82N4O3610H2O): C 39.18; H 6.99,
N 3.81; found: C 39.32, H 6.65, N 3.81.
1
MS (ESI, H2O, m/z) L-Phe--CD: 1282.3 (M+H); H NMR (DMSO, TMS):
2.78-3.34 (m, 50H), 4.47-4.82 (m, 7H), 5.60-5.71 (m, 14H), 7.20-7.21 (m, 5H); Anal.
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Calcd for L-Phe--CD complex (C51H79NO368H2O): C 42.95, H 6.71, N 0.98; found:
C 42.92, H 6.81, N 0.77.
2.3. Synthesis of L-amino acid-modified -cyclodextrin platinum complexes
A total of 0.5 mmol of the L-amino acid-modified -CD and 0.5 mmol of
K2PtCl4 was suspended in 50 mL of H2O, and the mixture was stirred at 60 °C for 48
h under argon in dark. After that, the reaction mixture was cooled to room
temperature. The solids were then filtered out. And the raw products were separated
by acetone precipitation followed by centrifugation. The solid products were collected
by centrifugation and then purified by chromatography on a Sephadex G-25 column
with deionized water as eluent, to give pale yellow product in suitable yield.
1
[Pt(L-Arg--CD)Cl2]⋅10H2O. Yield as light yellow powder (53.5%). H NMR
([D6]DMSO, TMS): δ = 1.58-1.72 (m, 4H), 2.08 (m, 1H), 3.12-3.15 (m, 2H),
3.28-3.64 (m, 45H), 4.18-4.63 (m, 7H), 4.82-4.91 (m, 7H), 5.67-6.15 (m, 14H),
7.34-8.67 (m, 2H); elemental analysis calcd (%) for Anal. Calcd for
C48H81N4O36PtCl210H2O: C 34.27, H 5.69, N 3.33; Found: C 34.15, H 5.89, N 3.16.
1
[Pt(L-Phe--CD)Cl2]⋅8H2O. Yield as light yellow powder (63.6%). H NMR
([D6] DMSO, TMS): δ = 2.72-2.88 (m, 2H), 3.12-3.15 (m, 2H), 3.26-3.62 (m, 45H),
4.35-4.54 (m, 7H), 4.82-4.96 (m, 7H), 5.52-5.95 (m, 14H), 7.204-7.315 (m, 4H), 8.29
(m, 1H); elemental analysis calcd (%) for C51H78Cl2NO36Pt⋅8H2O: C 36.23, H 5.61, N
0.83; found: C 36.29, H 5.58, N 0.85.
2.4. Spectral measurements
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UV-vis absorption spectra were recorded in a quartz cuvette with a 1 cm path
length using a Perkin-Elmer Lambda-850 UV-vis spectrometer (Perkin-Elmer, USA).
3 mL of Tris buffer solution was added to the reference pool with the sample cell as
the baseline correction. 3 mL of 100 M calf thymus DNA (CT-DNA) solution was
added to sample cell, then same volume (6 L) of 10 mM sample was added to the
sample pool, so that the concentration ratio of the complexes and the DNA increased.
Mixed for 24 h everytime. The ultraviolet-visible spectral changes upon the addition
of the complexwere detected in the range of 180-500 nm.
2.5. Preparation of Dox-loaded -CD platinum complexes
A general protocol for the Dox-loaded amino acids-modified -CD platinum
complexes formation was as follows. Dox and amino acids-modified -CD platinum
complexes were dissolved in phosphate-buffered saline (PBS) at various molar ratios
(nDox : nplatinum = 1:0.5, 1:1, 1:1.5, 1:2, 1:4), then sonicated for 4 h. Afterwards, the
optimum molar ratio of Dox and Dox-loaded amino acids-modified -CD platinum
complexes were validated by UV-vis absorption and fluorescence emission
spectroscopy, respectively.
The UV-vis absorption spectra of free Dox and Dox-loaded amino
acids-modified -CD platinum complexes were recorded using a Perkin-Elmer
Lambda-850 UV-vis spectrometer (Perkin-Elmer, USA). The fluorescence emission
spectra from 500 nm to 800 nm were obtained using a Perkin-Elmer Ls55
luminescence spectrometer (Amer-ican, Perkin-Elmer Co.).
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2.6. Circular dichroism (CD) measurements
CD measurements were performed on a Jasco J-810 CD spectropo-larimeter at
37 ◦C in a rang of 200-400 nm and the cell length was 1 cm. Titrations were
performed by incrementally adding aliquots of each complex to 25 mM CT-DNA in
10 mM Tris-HCl buffer with pH valueadjusted to 7.4. The concentration of each
complex ranged from 0 to 20 mM. CD spectra were baseline-corrected for signal
contributions due to the buffer. The CD spectrum of CT-DNA alone (25 mM) was
recorded as control. After each addition of complex, the reaction was stirred and
allowed to equilibrate for at least 24 h and a CD spectrum was collected with at least
five scans. Final analysis of the data was carried out using Origin 7.5.
2.7. DNA binding study
Linear DNA fragments were isolated from pBR322 DNA cleavage products by
platinum complexes and purified using a DNA gel extraction kit [32, 33]. Specifically,
pBR322 DNA was incubated with Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2 in 20
mM HEPES buffer, pH 7.20, for a certain period of time (24 h), then 4 L of loading
buffer (0.05% bromophenol blue, 50% glycerol, and 2 mM Na2H2EDTA) was added
to the mixture. Control reactions without platinum complexes were carried out under
the same conditions. Then the samples were loaded directly onto a 0.9% agarose gel
and electrophoresed in TBE buffer (89 mM tris-borate, 2 mM Na2H2EDTA) at a
constant voltage of 80 mV for 100 min. Then the gels were placed in the ethidium
bromide (EB) dilute solution and tained five minutes. The gels were visualized in the
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electrophoresis documentation and analysis system 120. Densitometric calculations
were performed using Image Tools 3.00. The intensities of supercoiled pBR322 DNA
were corrected by a factor of 1.22 as a result of its lower staining capacity by EB [34].
2.8. Cell culture
K562, HepG2 and 7701 (normal liver cells) cells were cultivated in DMEM
medium supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v)
penicillin-streptomycin. These cells were allowed to grow in monolayer in a tissue
culture flask incubated at 37 °C in a 95% humidified incubator with 5% CO2. When
the cells were maintained in an exponential growth phase by periodic subcultivation,
the cells were counted by hemocytometer and seeded in 96-well microplate at a
density of 5104 cells/well.
2.9. Cell viability assay
The optimal molar ratio of Dox and amino acids-modified -CD platinum
complexes was performed using the MTT assay. Dox (5 M) and amino
acids-modified -CD platinum complexes were dissolved in DMEM medium at
various molar ratios (nDox : nplatinum = 1:0.5, 1:1, 1:1.5, 1:2, 1:4). After being sonicated
for 4 h, Dox and Dox-loaded amino acids-modified -CD platinum complexes
incubated with cells for 48 h to determine the optimal molar ratio at maximal cell
inhibition rate.
In vitro cytotoxicity of the samples was determined by a modified MTT
assay[35]. Briefly, the cells (5104 cells/well) were seeded in 96-well microplate for
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24 h, then the different concentrations of samples in DMEM medium (1-200 M)
were added to the wells in triplicates and incubated for 48 h. After the incubation
period, 20 L of MTT solution (2.5 mg mL-1 in PBS) was added to each well and the
plates were incubated in the dark for another 4 h. Then the media were removed and
the formazan crystals were dissolved in 100 L of DMSO. Then the absorbance of
each well at 570 nm was read on the Tecan Infinite F200/M200 multimode Plate
readers. The percentage of cell viability was calculated using the equation: (mean OD
of treated cells/mean OD of control cells) × 100%. Cells treated with vehicle (1%
DMSO) were used as controls. Data were averages of three independent experiments
± standard deviations.
2.10. Cellular uptake of Dox-loaded amino acids-modified -CD platinum complexes
in live cells
HepG2 cells in DMEM medium containing FBS (10 %) were seeded in 96-well
microplate for 24 h. The Dox-loaded amino acids-modified -CD platinum complexes
containing medium were added to the cells at a concentration of 5 M. After
incubation for 6 h, the cells were thoroughly washed with PBS buffer to remove
samples that were not permeated. After washing, the cell nuclei were stained with
Hoechst 33342 solution. Then the cells were analysed by a high content screening
(HCS) technique immediately under excitation at 488 nm.
3. Results and discussion
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3.1. Synthesis and characterization of the platinum complexes
The complexes Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2 were prepared via
the reaction of K2PtCl4 with ligands L-Arg--CD and L-Phe--CD in suitable yields.
ESI-MS spectrometry is an important method for the investigation of the formation of
platinum complexes. Fig. S1 and S2 in the Supporting Information showed the MS
spectra of the Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2. The ESI-MS spectrum in
Fig. S1 in the Supporting Information showed the molecular ion fragment peaks at
m/z 1579.1 and m/z 798.0 which was assigned to the complexes
[Pt(L-Arg--CD)Cl2+Na]+ and [Pt(L-Arg--CD)Cl2+H2O]2+, respectively. Moreover,
the ESI-MS spectrum in Fig. S2 in the Supporting Information showed the species of
m/z 1546.3 which was assigned to the complex [Pt(L-Phe--CD)Cl2]-. These results
provided strong evidence for the presence of the corresponding platinum complexes.
1
Fig. S3 and S4 in the Supporting Information showed the H NMR spectra of
Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2 complexes. The disappearance of
carboxylic acid signal and the shift of amido proton are both indicative of the
coordination of platinum as shown in Scheme 1.
3.2. DNA binding properties of the platinum complexes
The DNA binding properties of the complexes to CT-DNA were investigated by
various spectral techniques. CD is a useful technique to assess whether nucleic acids
undergo conformational changes as a result of complex formation or environmental
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changes. Fig. 1 showed the CD spectral change upon the titration of
Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2 in Tris-HCl buffer at pH 7.4, respectively.
In Fig. 1a, the presence of Pt(L-Arg--CD)Cl2 induced increase in intensity at ~220
and ~245 nm but decrease at ~277 nm, indicating that there was perhaps non-covalent
interaction between Pt(L-Arg--CD)Cl2 and DNA [36]. In Fig. 1b, the presence of
Pt(L-Phe--CD)Cl2 displayed negaligible effect on the CD spectrum, indicating that
perhaps the complexes could not bind to DNA [37]. This may be due to the disruption
of base stacking, whichis required to optimize the binding interactions [38]. More
significantly, in Fig. 1a a strong negative band at 295 nm was observed, suggesting
the presence of Pt(L-Arg--CD)Cl2 induced conformational change of the CT-DNA
from B to Z-configuration [37]. These may reflect local untwisting of the CT-DNA
helical backbone and changes in relative orientation of the bases to accommodate the
probes within a particular base pair [39, 40]. The results indicated the different
interaction modes or strength between the binding of the two complexes to CT-DNA.
UV-vis spectroscopy were also employed to investigate DNA binding ability of
Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2. As shown in Fig. 2, the UV-vis
absorption spectra both showed hyperchromic effect with the addition of
Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2, and the hyperchromic effect became
stronger with the increased complexes concentration. It was also noted that the
UV-vis absorption at 256 nm split into two bands (231 nm and 272 nm) in the case of
Pt(L-Arg--CD)Cl2 (Fig. 2a). The hyperchromic effect might be ascribed to
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electrostatic binding or partial uncoiling of the helix structure of DNA, exposing more
bases of the DNA [41]. These results confirmed that the complexes could bind to
DNA, in accordance with the results of CD, and further implied that the intercalation
between the base pairs was not prevalent in these cases.
The binding abilities of cisplatin, ligands and complexes to pBR322 DNA were
evaluated by gel electrophoresis. As shown in Fig. 3, the mobility of DNA decreased,
after incubated with complex Pt(L-Arg--CD)Cl2 and cisplatin for 24 h, whereas the
ligands L-Arg--CD, L-Phe--CD and complex Pt(L-Phe--CD)Cl2 had little effect
on the mobility of DNA. It is obvious that the complex Pt(L-Arg--CD)Cl2 could bind
to pBR322 DNA in a similar way as cisplatin. The increaseed mobility indicates that
the binding of the platinum complexes changes the sign of the writhe of DNA to a
more positive value [42, 43]. It also indicates that complex Pt(L-Arg--CD)Cl2 is able
to bind to plasmid DNA more efficiently than ligands L-Arg--CD, L-Phe--CD and
complex Pt(L-Phe--CD)Cl2, which suggests a plausible synergistic effect between
the Pt(II) ion and the positively- charged arginine residue.
The concentration effect was also evaluated for Pt(L-Arg--CD)Cl2,
Pt(L-Phe--CD)Cl2 and cisplatin. As shown in Fig. 4, the mobility of DNA decreased
with the increase of concentrations of Pt(L-Arg--CD)Cl2 and cisplatin. However,
such mobility disappeared when the concentration of Pt(L-Phe--CD)Cl2 reaches 180
M (Fig. 4b). Such difference suggested that Pt(L-Arg--CD)Cl2 exhibit more
effective binding activity with DNA compared to Pt(L-Phe--CD)Cl2, and the binding
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mode to DNA might be in a non-intrusive way similar to cisplatin [44]. A possible
explanation is that the complex Pt(L-Arg--CD)Cl2 has more positive charges and
less steric than complex Pt(L-Phe--CD)Cl2, which benefits the covalent interaction
of Pt(L-Arg--CD)Cl2 with DNA. On the other hand, the hydrophobic phenyl group
in complex Pt(L-Phe--CD)Cl2 may be embedded in the hydrophobic cavity of the
β-CD, which increases the steric hindrance and hampers its interaction with DNA
[45].
3.3 Drug delivery ability of platinum complexes
To evaluate the drug delivery ability of amino acids-modified -CD platinum
complexes as hydrophobic anticancer drugs, a strategy via host-guest interactions was
proposed in Scheme 1. The two Dox-loaded amino acids-modified -CD platinum
complexes were prepared with the ultrasonic method at room temperature and
protected from light. Dox was loaded into lipophilic central cavities of -CD by
hydrophobic interaction. UV-vis spectroscopy was used to prove the synthetic product
and the optimum molar ratio of Dox-loaded amino acids-modified -CD platinum
complexes. The UV-vis spectra of Dox-loaded platinum complexes at different molar
ratios of Dox to amino acids-modified -CD platinum complexes are shown in Fig. 5.
Dox had a maximum absorption peak at 498 nm, and this absorption increased upon
the addition of two amino acids-modified -CD platinum complexes, indicating that
the -CD formed inclusion complexes with drug Dox [46, 47]. Moreover, when the
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molar ratio between Dox and amino acids-modified -CD platinum complexes was
1:1, the absorption reached the maximum, suggesting that the inclusion complex was
most stable at the ratio of 1:1 for Dox-loaded amino acids-modified -CD platinum
complexes, thus 1:1 was the matching molar ratio.
In order to further clarify the optimum molar ratio for producing the most stable
complexes and forming inclusion complexes, the Dox-loaded amino acids-modified
-CD platinum complexes were characterized by fluorescence spectra. The
fluorescence spectra of Dox-loaded amino acids-modified -CD platinum complexes
formed at different molar ratios of Dox to amino acids-modified -CD platinum
complexes were shown in Fig. 6. After adding amino acids-modified -CD platinum
complexes, the fluorescence intensity of the Dox-loaded amino acids-modified -CD
platinum complexes decreased sharply compared with free Dox, suggesting that both
Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2 complexes had the ability to encapsulate
Dox in PBS. Besides, when the molar ratio between Dox and both the two amino
acids-modified -CD platinum complexes was 1:1, the emission spectra reached the
minimum, producing the most stable complexes.
In addition, MTT experiments were also carried out to validate the optimum
molar ratio of the Dox to amino acids-modified -CD platinum complexes. As shown
in Fig. 7, the Dox-loaded amino acids-modified -CD platinum complexes presented
higher cell inhibition ratio compared to free Dox, confirming that Dox was
encapsulated within lipophilic central cavities of -CD. Cell inhibition ratio reached
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maximum at the molar ratio of 1:1, which was consistent with the results of UV-vis
and fluorescence spectroscopy. In other words, the intracellular drug concentration of
the more stable Dox-loaded amino acids-modified -CD platinum complexes was
higher, leading to higher cell inhibition ratio of the drug combination system-treated
cells compared to free Dox-treated (or platinum complex-treated) cells at the same
concentration (5 M).
The above results proved that amino acids-modified -CD platinum complexes
could solubilize hydrophobic anticancer drugs and form stable combination system of
drug and amino acids-modified -CD platinum complexes. Therefore, based on the
combined results of UV-vis and fluorescence experiments, it can be summarized that
amino acids-modified -CD platinum complexes can encapsulate hydrophobic
anticancer drugs into the cavities of -CD to obtain drug combination system, thereby
possessing very good loading ability for anticancer drugs.
3.4. Cell inhibition ratio of platinum complexes and Dox-loaded platinum complexes
In order to investigate the cell inhibition ratio of the Dox-loaded amino
acids-modified -CD platinum complexes and amino acids-modified -CD platinum
complexes, cytotoxic activity was examined by incubating the three types of cells
(K562, HepG2 and 7701) with different concentrations of complexes, cisplatin and
Dox, respectively, for 48 h. The IC50 values of the complexes, cisplatin and Dox by
the MTT assay at 48 h were shown in Table 1. And the cell inhibition ratios of
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Dox-loaded platinum complexes, platinum complexes, cisplatin and Dox were shown
in Fig. 8. The cell inhibition ratio of L-Arg--CD and L-Phe--CD in K562, HepG2,
and 7701 cells were all under 35%, which were relatively low even with higher
concentrations, indicating that the ligands amino acids-modified -CD had no
significant growing inhibition to cells. For the same cell, Pt(L-Arg--CD)Cl2 showed
better activity than complex Pt(L-Phe--CD)Cl2, L-Arg--CD and L-Phe--CD but it
was much less active than cisplatin. Perhaps it was the positive charge of the L-Arg
resulting in a considerable increase in the intracellular delivery of Dox. As expected,
the cell growth inhibition efficiency of amino acids-modified -CD platinum
complexes-encapsulated Dox was higher than that of either amino acids-modified
-CD platinum complexes or free Dox. That is to say, encapsulated Dox via amino
acids-modified -CD platinum complexes is able to increase the cell inhibition ratio
versus free Dox by increased accumulation of intracellular drug. The cell inhibition
ratio of the combination system of Dox and amino acids-modified -CD platinum
complexes increased approximately 3 folds compared to free Dox. And the cell
inhibition ratio under our experimental conditions (final concentration ≤ 1% DMSO)
were in the order: Pt(L-Arg--CD)Cl2/Dox > Pt(L-Phe--CD)Cl2/Dox > Dox >
cisplatin  Pt(L-Arg--CD)Cl2  Pt(L-Phe--CD)Cl2  free ligands (L-Arg--CD and
L-Phe--CD). It was also observed that the two types of Dox-loaded platinum
complexes had less toxicity in the normal 7701 cells than other two types of cancer
cells. The synergistic effects of platinum complexes and Dox increased the
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intracellular drug concentration and the percentage of cell death. Thus, the
Dox-loaded platinum complexes had high cell inhibition ratio and certain selectivity
for cancer cells.
3.5. Cellular uptake of Dox-loaded platinum complexes
We further studied the cellular uptake of Dox-loaded platinum complexes by
high content screening (HCS) (Fig. 9). Fig. 9 showed that after 2 h incubation with the
Pt(L-Arg--CD)Cl2/Dox, the red fluorescence from Dox was observed in the
cytoplasm of cells and the intensity was weak, indicating that the Dox was still loaded
by -CD. After 6 h of incubation, the red fluorescence started to aggregate and the
intensity increased, indicating that Dox was released from the combination system
Pt(L-Arg--CD)Cl2/Dox.
Moreover, we compared the fluorescence intensity of free Dox and two Dox-loaded
platinum complexes in cells. Fig. 10 showed that the red fluorescence from Dox was
all observed in the cytoplasm of HepG2 cells after 24 h incubation with the free Dox
and Dox-loaded platinum complexes. We can see that the intensity of the red
fluorescence from free Dox was weak in the cytoplasm in HepG2 cells. However, the
intensity of the red fluorescence displayed elevated increase in HepG2 cells based on
the delivery of Dox by Pt(L-Arg--CD)Cl2 and Pt(L-Phe--CD)Cl2, suggesting the
efficient transport of Dox across the membranes with the aid of the -CD. Comparing
the two combination system of Pt(L-Arg--CD)Cl2/Dox and Pt(L-Phe--CD)Cl2/Dox,
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the red fluorescence signal of Dox delivered by Pt(L-Arg--CD)Cl2 was stronger than
that delivered by the Pt(L-Phe--CD)Cl2, suggesting Pt(L-Arg--CD)Cl2 possessed
the higher Dox transport efficiency. This is probably because the Dox was loaded into
the Pt(L-Arg--CD)Cl2 more easily than the Pt(L-Phe--CD)Cl2 due to the stronger
positive charge of the Pt(L-Arg--CD)Cl2. Thus, the combination system of
Pt(L-Arg--CD)Cl2/Dox and Pt(L-Phe--CD)Cl2/Dox could affect Dox accumulation
and showed great potential as antitumor drugs.
4. Conclusion
In conclusion, we reported two water-soluble amino acids-modified -CD
platinum complexes, which showed preferable binding ability to DNA and effective
inhibition to cancer cells. They bind to and unwind pBR322 DNA in a manner similar
to cisplatin. Besides, the combination system of Dox and amino acids-modified -CD
platinum complexes was synthesized and it could effectively deliver the anticancer
drug Dox into cells and had higher cell inhibition ratio, with less toxicity in the
normal cells than in cancer cells. The combination system of Dox and amino
acids-modified -CD platinum complexes in cells helped to promote the synergistic
effects of the drug Dox and platinum, enhancing intracellular drug accumulation and
potency to kill cancer cells. Hence, the amino acids-modified -CD platinum
complexes is capable of deliver antitumor drug to form the combination system and
show great potential as antitumor drug.
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5. Abbreviations
-CD
Dox
-cyclodextrin
doxorubicin
HCS
high content screening
FBS
Fetal bovine serum
DMEM
MTT
CT-DNA
CD
Dulbecco Minimum Essential Medium
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
calf thymus DNA
Circular dichroism
EB
ethidium bromide
PBS
phosphate-buffered saline
50% inhibitory concentration
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
IC50
HEPES
Acknowledgements
This work was supported by the Project supported by the National Natural
Science Foundation of China (No., 20901005, U1204201), Postdoctoral Science
Foundation Funded Project of China (No. 2012M511570) and Department of
Education Science and Technology Research Key Projects of Henan (13A150063).
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Table legends:
Table 1. Summary of the cytotoxicity activity of complexes.
Samples
IC50 (M)
K562
HepG2
 200
7701
L-Arg--CD
 200
 200
Pt(L-Arg--CD)Cl2
L-Phe--CD
84.22.6
 200
77.53.2
 200
117.53.6
 200
Pt(L-Phe--CD)Cl2
Cisplatin
115.63.5
22.63.4
9.42.8
3.42.6
3.73.3
118.62.4
22.92.2
9.33.1
2.92.1
3.12.7
135.53.3
25.62.7
9.62.6
12.72.2
10.23.2
Dox
Pt(L-Arg--CD)Cl2/Dox
Pt(L-Phe--CD)Cl2/Dox
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Figure and Scheme Legends:
Scheme 1. Synthetic strategy of L-amino acid-modified -CD platinum complexes
and Dox loading into L-amino acid-modified -CD platinum complexes and the
cellular uptake of Dox-loaded amino acids-modified -CD platinum complexes.
Fig. 1. CD spectra of CT-DNA modified by complexes at different
complexes-to-DNA ratios. (a) DNA was titrated with Pt(L-Arg--CD)Cl2 at r of 0, 0.5,
1.0, respectively; (b) DNA was titrated with Pt(L-Phe--CD)Cl2 at r of 0, 0.5, 1.0,
respectively.
Fig. 2. Absorption spectra of complexes (a) Pt(L-Arg--CD)Cl2 and (b)
Pt(L-Phe--CD)Cl2 in Tris-HCl buffer in the presence of CT-DNA with increasing
amounts of complexes. [CT-DNA] = 100 μM, [complexes] = 0-180 M from bottom
to top. Arrows indicate the change in absorbance upon increasing the complexes
concentration.
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Fig. 3. Agarose gel electrophoresis images of pBR322 DNA after incubated with
cisplatin, ligands and complexes in 20 mM HEPES, pH 7.20 at 37 oC for 24 h. Lane
0 (control), contained only 50 M bp DNA ; lane 1, 50 M DNA+50 M of cisplatin,
lane 2, 50 M DNA+200 M of ligand L-Arg--CD, lane 3, 50 M DNA+200 M
of complex Pt(L-Arg--CD)Cl2, lane 4, 50 M DNA+200 M of ligand L-Phe--CD,
lane 5, 50 M DNA+200 M of complex Pt(L-Phe--CD)Cl2.
Fig. 4. The concentration effect of complexes and cisplatin on their DNA (50 M bp)
unwinding activity, (a) Pt(L-Arg--CD)Cl2, (b) Pt(L-Phe--CD)Cl2, and (c) cisplatin,
pH 7.20 at 37 oC for 24 h. Lane 1 for a, b, c: DNA control; lanes 2-10 for a, b: 20 M,
40 M, 60 M, 80 M, 100 M, 120 M, 140 M, 160 M, 180 M; lanes 2-10 for c:
5 M, 10 M, 15 M, 20 M, 25 M, 30 M, 40 M, 50 M, 60 M.
Fig. 5. UV-vis absorption spectra of (a) Pt(L-Arg--CD)Cl2 and (b)
Pt(L-Phe--CD)Cl2 at different molar ratios of Dox to amino acids-modified -CD
platinum complexes with a fixed concentration of Dox (5 M) in PBS solution.
Fig. 6. Fluorescence emission spectra of (a) Pt(L-Arg--CD)Cl2 and (b)
Pt(L-Phe--CD)Cl2 at different molar ratios of Dox to amino acids-modified -CD
29