Supramolecular system of podophyllotoxin and hydroxypropyl-β-cyclodextrin: Characterization, inclusion mode, docking calculation, solubilization, stability and cytotoxic activity

Accepted Manuscript Supramolecular system of podophyllotoxin and hydroxypropylβ-cyclodextrin: Characterization, inclusion mode, docking calculation, solubilization, stability and cytotoxic activity

Li-Juan Yang, Shu-Hui Wang, Shu-Ya Zhou, Fang Zhao, Qing Chang, Min-Yan Li, Wen Chen, Xiao-Dong Yang PII: DOI: Reference:

S0928-4931(16)32563-2 doi: 10.1016/j.msec.2017.03.197 MSC 7709

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

7 December 2016 21 March 2017 22 March 2017

Please cite this article as: Li-Juan Yang, Shu-Hui Wang, Shu-Ya Zhou, Fang Zhao, Qing Chang, Min-Yan Li, Wen Chen, Xiao-Dong Yang , Supramolecular system of podophyllotoxin and hydroxypropyl-β-cyclodextrin: Characterization, inclusion mode, docking calculation, solubilization, stability and cytotoxic activity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.197

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ACCEPTED MANUSCRIPT Supramolecular System of Podophyllotoxin and Hydroxypropyl-β-Cyclodextrin: Characterization, Inclusion Mode, Docking Calculation, Solubilization, Stability and

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Cytotoxic Activity

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Li-Juan Yang a,*, Shu-Hui Wang a, Shu-Ya Zhou a, Fang Zhao a, Qing

School of Chemistry & Environment, Engineering Research Center of Biopolymer

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a

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Chang a, Min-Yan Li c, Wen Chen b,*, Xiao-Dong Yang b,*

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Functional Materials of Yunnan, Yunnan Minzu University, Kunming 650500, PR China

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education,

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b

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School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China

Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323,

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c

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United States

* Corresponding authors. Tel./fax: +86 871 65910017. E-mail addresses: [email protected] (L.J. Yang), [email protected] (W. Chen), [email protected] (X.D. Yang). 1

ACCEPTED MANUSCRIPT ABSTRACT The inclusion complexation behavior, characterization, and inclusion mode of podophyllotoxin

(POD)

with

hydroxypropyl-β-cyclodextrin

(HPβCD)

were

investigated in both solution and the solid state by means of XRD, DSC, SEM, 1H and

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2D NMR and UV–vis spectroscopy. The results showed that the water solubility and

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thermal stability of POD were obviously increased in the inclusion complex with

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HPβCD. The cytotoxicity of POD/HPβCD inclusion complex against all the human tumor cell lines investigated still remains. This satisfactory water solubility and high

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thermal stability of the POD/HPβCD complex will be potentially useful for their

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application as herbal medicines or healthcare products.

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Keywords:

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Podophyllotoxin; Cyclodextrin; Inclusion Complex; Characterization; Cytotoxic

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Activity

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ACCEPTED MANUSCRIPT 1. Introduction

Podophyllotoxin (POD, Fig.1), a naturally occurring lignan currently extracted from genera of Podophyllum [1], Dysosma [2], Diphylleia [3] and Juniperus [4], has

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played an important role in the pharmaceutical study for its excellent biological activities. The roots of these herbs are used for the treatment of cancer and rheumatoid

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arthritis in traditional medicine. Lots of researches suggest that POD is an excellent

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potent antitumor agent against a panel of human tumor cell lines in vitro [5-10].

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However, because of its unacceptable gastrointestinal toxicity, initial expectations regarding the clinical utility of POD were tempered. Additionally, the tincture of POD

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is recommended as the drug of choice for the treatment of genital warts by World Health Organization. However, cases of poisoning and even death were frequently

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reported for the narrow therapeutic window [11-15]. The clinical adverse syndromes include abdominal pain, abnormal hepatorenal functions, coma, diarrhea, fainting,

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fever, leukopenia, manifested nausea, memory impairment, thrombocytopenia, and vomiting after using this drug [16-18]. Apart from the toxicity, the use of POD as a

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drug is greatly limited by its low water solubility and bioavailability. Although much effort has been made to improve its water solubility, such as ethosome [19], and nanostructured lipid carrier [20], POD still cannot be sufficiently dissolved in water. Therefore, the investigation for an efficient and nontoxic carrier for POD has become important in order to its further clinical application.

3

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ACCEPTED MANUSCRIPT

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Fig. 1. The structures of Podophyllotoxin and HPβCD.

It is well known that cyclodextrins (CDs) are truncated-cone polysaccharides

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mainly composed of six to eight D-glucose monomers linked by α-1,4-glucosidic

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bonds. They have a hydrophobic central cavity and a hydrophilic outer surface and can encapsulate various inorganic/organic molecules to form host–guest complexes or

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supramolecular species. This usually enhances drug solubility in aqueous solutions

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and affects the chemical characteristics of the encapsulated drug in the pharmaceutical industry [21-23]. This fascinating property enables them to be successfully utilized as

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drug carriers [24-26], separation reagents [27], enzyme mimics [28], and

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photochemical sensors [29], etc. Hydroxypropyl-β-cyclodextrin (HPβCD, Fig. 1) is a hydroxyalkylated β-CD derivative that combines relatively high water solubility with low toxicity and satisfactory inclusion ability [30,31]. Recently, the inclusion complex formation between γ-CD and POD [32], as well as the preparation and solubility of complex of POD with HPβCD [33], have been reported. However, to the best of our knowledge, no scientific study on the complete inclusion complexation behavior, characterization and binding ability of POD/HPβCD complex in both the solution and 4

ACCEPTED MANUSCRIPT solid state by means of XRD, DSC, SEM, 1H and 2D NMR and UV–vis spectroscopy have hitherto been reported. More recently, we reported that the inclusion complexation of CDs with natural medicines such as crassicauline A [34], alpinetin [35], naringenin [36], pinocembrin

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[37], and hesperetin [38] significantly enhanced the water solubility and

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bioavailability of the medicines. As a continuation of our studies on natural

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medicines/cyclodextrin inclusion complex, a supramolecular system of POD with HPβCD was investigated.

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Considering HPβCD is an alternative to α-, β- and γ-cyclodextrin, with improved

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water solubility properties and slightly more toxicologically benign [30, 31], we aim to report the preparation and characterization of the water-soluble inclusion complex

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formed by POD and HPβCD (Fig. 1) in this paper. We were particularly interested in

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exploring the solubilization effect of HPβCD on POD and the binding ability of the resulting inclusion complex, which would provide a useful approach for obtaining

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novel POD-based healthcare products with high water solubility and high

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bioavailability.

2. Materials and methods

2.1. Materials POD (molecular weight = 414.41, PC > 98%) used in this work was purchased from the National Institute for Control of Pharmaceutical and Bioproducts. 5

ACCEPTED MANUSCRIPT 2-Hydroxypropyl-β-cyclodextrin (HPβCD, average substitution degree = 4.3, average molecular weight = 1380) was purchased from ABCR GmbH & Co. KG and used without further purification. Other reagents and chemicals were of analytical reagent

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grade. All experiments were carried out using ultrapure water.

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2.2. Methods

2.2.1. Preparation of POD/HPβCD supramolecular system

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POD (0.03 mM, 9.1 mg) and HPβCD (0.01 mM) were completely dissolved in a

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mixed solution of ethanol and water (ca. 7mL, V:V = 1:5, given the poor water solubility of POD, ethanol was used), and the mixture was stirred for 10 days at room

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temperature. After evaporating the ethanol from the reaction mixture, the

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uncomplexed POD was removed by filtration. The filtrate was evaporated under reduced pressure to remove the solvent and dried under vacuum to produce the POD/

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HPβCD complex (yield 83%).

2.2.2. Job plot

The Job plot was established using the UV Spectra data obtained in a mixed solution of water and ethanol at pH 3.0. The total molar concentration of POD and HPβCD) was maintained at 5.6×10-5 M and the molar fraction of POD ( [POD]/([POD] ＋[HPβCD])) varied from 0.1 to 0.9. The absorbance was recorded at 289 nm in UV at 37 oC. And the absorption of each POD solution in the presence and absence of 6

ACCEPTED MANUSCRIPT HPβCD were also carried out in the same condition.

2.2.3. Determination by UV Spectra Absorption spectra measurements were carried out with an Agilent UV 3600 using

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a conventional 1 cm path (1 cm1 cm4 cm) quartz cell in a thermostated

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compartment, which was kept at 25 °C by a Shimadzu TB-85 Thermo Bath unit.

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Given the poor water solubility of POD, a water/ethanol (V:V = 4:1) solution was used in the spectral measurements. The concentration of POD was held constant at

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0.16 mM. Then, an appropriate amount of HPβCD was added, and the final

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concentrations varied from 0 to 4 mM (HPβCD: 0, 0.2306, 0.3294, 0.4706, 0.6723, 0.9604, 1.3710, 1.9600, 2.800 mM at pH 3.0; 0, 0.1614, 0.3294, 0.4706, 0.6723,

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0.9604, 1.9600, 4.0000 mM at pH 10.5). The absorption spectra measurements were

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taken after 1 h. The measurements were done in the 250–400 nm spectral range. All

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experiments were carried out in triplicate.

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2.2.4. Standard curve of POD A series of POD ethanol solutions with their concentrations ranging from 0.08 to 0.272 mM (0.08, 0.096, 0.112, 0.128, 0.144, 0.156, 0.176, 0.192, 0.208, 0.224, 0.254, 0.256, 0.272 mM) were configured. The absorbance was recorded at 289 nm in UV at 37 oC in order to make a standard curve using concentrations (C, mM) as x-coordinate and absorbance (A) as y-coordinate. We found the standard curve of POD could be depicted by the equation: A = 3.88395C + 0.01901 (R = 0.9991). 7

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2.2.5. Phase-solubility diagram The phase-solubility diagram was carried out according to the method developed by Higuchi and Connors [39]. A series of HPβCD solutions were prepared with

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increasing concentrations: 0 – 12.0 mM (0, 1.5, 3.0, 4.5, 6.0, 9.0, 12.0 mM). An

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excessive of POD was added to each solution and the suspensions stirred for 48 h in

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the dark. Following this, all suspensions were centrifuged and the supernatants were

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experiments were carried out in triplicate.

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filtered over 0.45 μm Millipore membranes and analyzed by UV-vis spectra. All

2.2.6. 1H and 2D NMR

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All NMR experiments were carried out in D2O. Tetramethylsilane was used as a

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reference. The samples were dissolved in 99.98% D2O and filtered before use. 1H NMR spectra were acquired on a Bruker Avance DRX spectrometer at 500 MHz and

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298 K. The one-dimensional spectra of both solutions were run with FID resolution of

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0.18 Hz/point. The residual HDO line had a line width at a half-height of 2.59 Hz. Two-dimensional (2D) ROESY spectra were acquired at 298 K with presaturation of the residual water resonance and a mixing (spin-lock) time of 350 ms at a field of ~2 kHz, using the TPPI method, with a 1024 K time domain in F2 (FID resolution 5.87 Hz) and 460 experiments in F1. Processing was carried out with zero-filling to 2K in both dimensions using sine (F2) and qsine (F1) window functions, respectively.

8

ACCEPTED MANUSCRIPT 2.2.7. Powder X-ray diffraction (XRD) The XRD patterns were obtained using a D/Max-3B diffractometer 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°

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between 2θ = 3° and 50°.

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2.2.8. Thermal analyses

Differential scanning calorimetry (DSC) measurements were performed with a

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NETZSCH STA 449F3, respectively, at the a heating rate of 10 ºC/min from room

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temperature to 500 ºC in a dynamic nitrogen atmosphere (flow rate = 70 mL/min).

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2.2.9. Scanning electron microphotographs (SEM)

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SEM photographs were determined on a FEI QUANTA 200. The powders were previously fixed on a brass stub using double-sided adhesive tape and then were made

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electrically conductive by coating, in a vacuum with a thin layer of gold

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(approximately 300 Å) for 30 s and at 30 W. The pictures were taken at an excitation voltage of 15, 20 or 30 kV and a magnification of 1080, 1200, 1400 or 2000×.

2.2.10. Docking Calculation POD was docked into HPβCD using AutoDock (Version 4.2). A grid of 0.8, 0.8, and 0.8 points in the x, y, and z directions was constructed centered on -0.323, 0.059, 9

ACCEPTED MANUSCRIPT and 0.081. We used a grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant for the energetic map calculation. Docking simulation of the compounds were carried out using the Lamarckian genetic algorithm and through a protocol with an initial population of 150 randomly placed individuals, a maximum

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number of 250 million energy evaluations, a mutation rate of 0.02, a crossover rate of

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0.8, and an elitism value of 1. Fifty independent docking runs were carried out for

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each compound, and the resulting conformations that differed by 1.2 Å in positional root-mean-square deviation (rmsd) were clustered together. Cluster analysis was

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the one endowed with the best energy.

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performed by selecting the most populated cluster, which in all cases coincided with

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2.2.11. Solubilization test

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An excess amount of the complex was placed in 2 mL of water (ca. pH 7.0) under nitrogen, sheltered from light, and the mixture was stirred for 24 h at 20 ± 2 ºC. The

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solution was then filtered on a 0.45 μm cellulose acetate membrane. The filtrate was

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evaporated under reduced pressure to dryness and the residue was dosed by the weighing method.

2.2.12. Cytotoxicity assay The assay was in five kinds of cell lines (HL-60, SMMC-7721, A549, MCF-7 and SW480). Cells were cultured at 37 oC under a humidified atmosphere of 5% CO2 in RPMI 1640 medium supplemented with 10% fetal serum and dispersed in replicate 10

ACCEPTED MANUSCRIPT 96-well plates. POD/HPβCD supramolecular complex and POD were then added, respectively. After 48 h exposure to the compounds, cells viability were determined by

the

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide]

(MTT)

cytotoxicity assay by measuring the absorbance at 570 nm with a microplate

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spectrophotometer. Each test was performed in triplicate.

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3. Results and discussion

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3.1. Stoichiometry

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The Job plot is a classic and effective method to calculate the stoichiometry in host-guest chemistry. In this work, a Job plot was employed to obtain the

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stoichiometry of POD and HPβCD via the UV–vis spectroscopy. In Fig. 2,

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x-coordinate is the mole fraction of POD, and y-coordinate is the relative of POD absorbance intensity in the presence and absence of HPβCD. The value of the max

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mole fraction of POD in this work is 0.5 (Fig. 2), which strongly proves the formation

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of the POD/HPβCD inclusion complex at a stoichiometry of 1:1.

11

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ACCEPTED MANUSCRIPT

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289 nm in a water/ethanol (V:V = 4:1, pH = 3.0).

0.00 0.0

0.30

0.5

1.0

1.5

2.0

Absorbance

a

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0.02

0.45

Exp Calcd

2.5

[HPCD]/mM

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0.15 0.00 250

nm

350

0.02

Esp Calcd

0.01

a 0.00 0

0.30

1

2

3

4

[HPCD]/mM

0.15 0.00 250

400

300

nm

350

400

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300

0.45

0.03

A-A0

0.04

B

pH = 10.5 0.60 h

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pH = 3.0 i

Absorbance

0.75

0.06

A0-A

0.60

A

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3.2. Spectral titration

0.75

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Fig. 2. The Job plot for the POD/HPβCD system ([POD]＋[HPβCD]＝5.6×10-5 M) at

Fig. 3. UV–vis spectral changes in POD (0.16 mM) upon addition of HPβCD (A: 0-2.8 mM, from a to i, B: 0-4.0 mM, from a to g) in a water/ethanol (V:V = 4:1, A: pH = 3.0, B: pH = 10.5.) mixed solution, and the nonlinear least-squares analysis (inset) of the differential intensity (ΔA at 289 nm) to calculate the complex stability constant (Ks).

12

ACCEPTED MANUSCRIPT KS 

POD / HPCD  A /  PODHPCD POD0  A /  HPCD0  A /  

Equation 2

 POD0  HPCD0  1 / K s    2 POD0  HPCD0  1 / K s   4 2 POD0 HPCD0 2

A 

2 Equation 3

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A quantitative investigation of the inclusion complexation behavior of HPβCD with

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POD was carried out in a water/ethanol (V : V = 4:1) solution at pH 3.0 using a

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spectrophotometric titration method owing to the rather low water solubility of POD. As illustrated in Fig. 3, the absorbance intensity of POD gradually increased with the

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stepwise addition of HPβCD. The pH of the solution did not change appreciably

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during any of the experimental procedures. As the size-fit, shape-fit, and charge-fit effects are the dominant controlling factors on the formation of inclusion complexes

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of CDs [21], these results indicate that the binding behavior is mainly dependent on

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the individual structural features of the host and guest. Assuming a 1:1 stoichiometry for the POD/CD inclusion complex, the inclusion complexation of POD with HPβCD

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could be expressed by Equation 1, and the stability constant (Ks) could be calculated

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from Equation 2, where [POD/HPβCD], [POD], [HPβCD], [POD]0 and [HPβCD]0 refer to the equilibrium concentration of the POD/HPβCD inclusion complex, the equilibrium concentration of POD, the equilibrium concentration of HPβCD, the original concentration of POD, and the original concentration of HPβCD, respectively, and Δε is the differential molar extinction coefficient of POD in the absence and presence of HPβCD. According to Lambert–Beer Law, it was found that the concentration of the POD/HPβCD complex was equal to ΔA/Δε (Equation 2). We 13

ACCEPTED MANUSCRIPT then derived Equation 3 from Equation 2. Finally, the Ks was obtained from the analysis of the sequential changes of absorption (ΔA) at various HPβCD concentrations, with a nonlinear least squares method according to the curve-fitting Equation 3.

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Using a nonlinear least squares curve-fitting method [40], we obtained the

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complex stability constant for the host–guest combination. Fig. 3 (inset) illustrates a

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typical curve-fitting plot for the titration of POD with HPβCD, which shows the excellent fit between the experimental and calculated data and the 1:1 stoichiometry

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of the POD/HPβCD inclusion complex. In the repeated measurements, the Ks values

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were reproducible within an error of ±5%. The stability constant (Ks) and Gibbs free energy change (−ΔGº) for the inclusion complexation of HPβCD with POD are listed

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in Table 1.

Table 1 The stability constant (Ks and logKs) and Gibbs free energy change (−ΔG0)

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for the inclusion complexation of HPβCD with POD guest in a water/alcohol (v/v

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=4:1, ca. pH 3.0, 7.0 or 10.5) mixed solution Ks/M-1

logKs

699

2.844

16.235

3.0

2.857

16.300

7.0

2.9263

16.706

10.5

719a 845

-△Go(KJ/moL)

a

pH

The stability constant was obtained from the phase-solubility diagram (see 3.3. Phase-solubility)

3.3. Phase-solubility 14

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2.4

[POD]=0.17253[HPCD] + 0.00634 R=0.99195

[POD](mM)

2.0 1.6 1.2 0.8

0.0

0

2

4

6

8

[HPCD](mM)

10

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0.4

12

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Fig. 4. Phase-solubility diagram for the POD/ HPβCD system at 25 oC.

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The phase-solubility diagram of the POD/HPβCD system (Fig. 4) showed POD solubility increased linearly with increasing HPβCD concentration. This diagram can

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be classified as AL type according to the model established by Higuchi and Connors [39]. Therefore, it can be related to the formation of a soluble inclusion complex at a

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stoichiometry of 1:1. The apparent stability constant (Ks), was calculated from the linear fit of the curve according to the following equation: Ks = Slope/[S0(1-Slope)],

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where Slope is the value found in the linear regression (Slope = 0.17253) and S0 is the aqueous solubility of POD at pH 7 (S0 = 120 mg/L= 2.90×10-4 M) in the absence of

o

C.

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HPβCD, determined using the UV–vis spectroscopy. This gave a Ks of 719 M-1 at 25

3.4. 1H and 2D NMR analysis

15

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

ppm

ACCEPTED MANUSCRIPT 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

ppm

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

ppm

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

ppm

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7.5

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Fig. 5. 1H NMR spectra of HPβCD in the absence and presence of POD in D2O at 25

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ºC. (a) HPβCD, (b) POD/HPβCD complex (asterisk highlights the water peak).

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In order to explore the possible inclusion mode of the POD/HPβCD complex, we

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compared the 1H NMR spectra of HPβCD in the absence and presence of POD (Fig. 5). Owing to its poor water solubility, POD is transparent to 1H NMR under most

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conditions when D2O is used as a solvent. Assessment of the POD complex by 1H

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NMR clearly demonstrated the presence of the framework protons of the POD molecule, consistent with the significant solubilization. As illustrated in Fig. 5, the

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majority of POD protons displayed chemical shifts at δ 7.5–5.5 ppm, which were

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distinct from the HPβCD protons (δ 1.0–5.5 ppm). By comparing the integration area of these protons with that of the HPβCD’s H-1 protons, we calculated the inclusion stoichiometry of the POD/HPβCD complex, that is, 1:1 for the POD/HPβCD.

Table 2 The chemical shifts (δ) of HPβCD and POD/HPβCD complex δ (ppm)

H-1

d

HP-β-CD

POD/HP-β-CD complex

4.99

4.96 16

ACCEPTED MANUSCRIPT dd

3.54

3.52

H-3

dd

3.87

3.90

H-4

dd

3.49

3.48

H-5

m

3.77

3.76

H-6

dd

3.77

3.75

H-Me

d

1.03

1.04

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H-2

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To further explore the inclusion mode, the chemical shifts of HPβCD protons in the

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absence and presence of POD were listed in Table 2. As can be seen from Table 2,

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inclusion complexation with POD had a negligible effect on the δ values of the H-2, H-4, H-5 and H-6 protons of HPβCD (≤0.02 ppm). In contrast, those values of the

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H-1 and H-3 protons exhibited the significant changes (0.03 ppm). It is fairly noteworthy that the H-3 protons shifted ca. 0.03 ppm, but that the H-5 protons showed

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relatively weak shifts (0.01 ppm) after resulting inclusion complex. Because both H-3 and H-5 protons are located in the interior of HPβCD cavity, and H-3 protons are near

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the wide side of cavity while H-5 protons near the narrow side, this phenomenon may

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indicate that POD should penetrate into the HPβCD cavity from the wide side.

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ppm 0.5 1.0 1.5 2.0 2.5 3.0

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3.5 4.0 4.5

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5.0 5.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

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6.0

4.5

4.0

3.5

3.0

6.5 7.0 7.5

2.5

2.0

1.5

1.0

0.5

8.0 ppm

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Fig. 6. ROESY spectrum of the POD/HPβCD complex in D2O.

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Two-dimensional (2D) NMR spectroscopy provides important information about the spatial proximity between host and guest atoms by observation of intermolecular

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dipolar cross-correlations [41]. Two protons closely located in space can produce a nuclear overhauser effect (NOE) cross-correlation in NOE spectroscopy (NOESY) or

AC

ROESY. The presence of NOE cross-peaks between protons from two species indicates spatial contacts within 0.4 nm [42]. To gain more conformational information, we obtained 2D ROESY of the inclusion complex of POD with HPβCD. The ROESY spectrum of the POD/ HPβCD complex (Fig. 6) showed key correlations of H-5 protons of POD with H3, H-5/H-6 protons of HPβCD (peaks a), as well as an appreciable correlations of H-8 and H-2'/H-6' protons of POD with H3, H-5/H-6 protons of HPβCD (peak b). These results indicate that the D ring of POD was 18

ACCEPTED MANUSCRIPT included in the HPβCD cavity. Based on these observations, together with the 1:1 stoichiometry deduced by NMR spectra and UV-vis spectrophotometric titration, we deduced the possible inclusion

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modes for the POD/HPβCD complex as illustrated in Fig. 7.

Fig. 7. Possible inclusion mode and significant NOESY (↔) correlations of the

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D

POD/HPβCD inclusion complex.

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3.5. Docking calculation

Recently, docking calculation can be used as the theoretical basis to predict

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inclusion mode [43-45]. In order to rationalize the NMR experimental results, POD was docked into HPβCD using AutoDock (Version 4.2). The study revealed a preferred final orientation for the complex. The minimum energy complex obtained for HPβCD and POD under our docking study is shown in Fig. 8. Possible H-bonding interactions are shown by green dotted line. The results are in very good accordance with that obtained by the NMR experiment.

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Fig. 8. The model of POD docked into HPβCD.

4000

a

0

600

b

400

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Intensity [cps]

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2000

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3.6. XRD analysis

200

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c

10

0 10

20

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2[deg.]

40

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Fig. 9. XRD patterns: (a) POD, (b) HPβCD, (c) POD/HPβCD inclusion complex.

The powder X-ray diffraction (XRD) patterns of HPβCD, POD, and their inclusion complex are illustrated in Fig. 9. As indicated in Fig. 9, HPβCD (Fig. 9b) is 20

ACCEPTED MANUSCRIPT amorphous, but POD (Fig. 9a) is in crystalline form. The lyophilized inclusion complex (Fig. 9c) has an amorphous halo pattern, in which any diffraction peak of POD cannot be found from the diffractogram, probably due to both the structure of

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HPβCD and the lyophilization process [46].

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3.7. Thermal analysis

a o

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b

o

83 C

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DSC/(w/g)

185 C

o

exo

360 C

o

c

80

160

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269.8 C

240

o

320

400

Temperature / C

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complex.

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Fig. 10. DSC thermograms: (a) POD, (b) HPβCD, (c) POD/HPβCD inclusion

The differential scanning calorimetry (DSC) diagram of POD, HPβCD and their inclusion complex are presented in Fig. 10. The thermogram of HPβCD shows a very board endothermic band, between 60 and 150 oC, which gains a maximum at 83 oC (Fig. 10b), indicating dehydration process. The trace of POD illustrates a sharp endothermic peak at 185 oC (Fig. 10a). However, in the DSC curves of the lyophilized 21

ACCEPTED MANUSCRIPT POD/HPβCD complex, we can find the endothermic peak at about 270 oC (Fig. 10c), corresponding to the free POD disappears, and the board exothermic peak at about 310 oC, corresponding to the combustion of this complex. The disappearance of the POD fusion peak can be related only to conversion of the crystalline compound to

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amorphous, as shown by XRD. This is perhaps a consequence of inclusion complex

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formation, but also numerous other phenomena can cause the same effect.

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3.8. SEM analysis

Fig. 11. Scanning electron microphotographs: (a) HPβCD, (b) POD, (c) POD and HPβCD physical mixture (1:1 molar ratio), (d) POD/HPβCD inclusion complex.

Scanning electron microscopy (SEM) is a qualitative method used to study the structural aspects of raw materials, i.e., CDs and drugs or the products obtained by 22

ACCEPTED MANUSCRIPT different methods of preparation, such as physical mixing, solution complexation, coevaporation and others [47]. The SEM photographs of HPβCD, POD, their inclusion complex and their physical mixture are shown in Fig. 11. Typical structure of HPβCD appeared as amorphous spherical particles with cavity structures (Fig. 11a)

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and pure POD appears as irregular-shaped particles with small dimensions (Fig. 11b).

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The physical mixture POD/HPβCD reveals some similarities with the crystal of the

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free molecules and shows both crystalline components (Fig. 11c). However, the POD/HPβCD inclusion complex appears as a compact and homogeneous plate-like

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structure crystal and is quite different from the sizes and shapes of POD and HPβCD

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(Fig. 11d), which confirms the formation of the inclusion complex.

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3.9. Solubilization

The water solubility of POD/HPβCD complex is assessed by preparation of its

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saturated solution [48]. An excess amount of complex was placed in 2mL of water (ca.

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pH 7.0), and the mixture was stirred for 24 h at 37 oC. After removing the insoluble substance by filtration, the absorbance of the filtrate was recorded at 289 nm in UV at 37 oC and the residue is dosed by the standard curve of POD. The results show that the water solubility of the POD, compared with that of native POD (ca. 120 μg/mL), was remarkably increased to approximately 3.4 mg/mL by the solubilizing effects of HPβCD. In the control experiment, a clear solution is obtained after dissolving POD/ HPβCD complex (14.7 mg), which is equivalent to 3.4 mg of POD, in 1 mL water at 23

ACCEPTED MANUSCRIPT 37 oC. This subsequently confirmed the reliability of the obtained satisfactory water solubility of POD/HPβCD complex, which will be beneficial to the medical utilization of this compound.

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3.10. Cytotoxic activity

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Table 3 Cytotoxic activities of POD and POD/HPCD inclusion complex in vitrob

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(IC50, mMa) SMMC-7721

A-549

MCF-7

POD

<0.064

<0.064

<0.064

3.60

POD/HPCD complex

<0.064

<0.064

<0.064

<0.064

Cisplatin

1.92

5.31

8.68

13.96

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HL-60

a

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Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay. b Data represent the mean values of three independent determinations.

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The cytotoxic potential of POD, POD/HPβCD inclusion complex was evaluated in vitro against a panel of human tumor cell lines according to procedures described in

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the literature [49,50]. The panel consisted of myeloid leukaemia (HL-60), liver

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carcinoma (SMMC-7721), lung carcinoma (A549), and breast carcinoma (MCF-7). Cisplatin (DDP) was used as the reference drug. The results are summarized in Table 3 (IC50 value, defined as the concentrations corresponding to 50% growth inhibition). The results show that the cytotoxicity of POD/HPβCD inclusion complex against all these human tumor cell lines still remains.

4. Conclusions 24

ACCEPTED MANUSCRIPT In summary, the inclusion complexation behavior, characterization, binding ability, solubilization and stability of POD with HPβCD was investigated. The results showed that HPβCD could enhance not only the water-solubility but also the stability of POD. The cytotoxicity of POD/HPβCD inclusion complex against all the human tumor cell

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lines investigated still remains. Given the shortage of application of POD and the easy

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and environmentally friendly preparation of POD/HPβCD complex, this inclusion

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complexation should be regarded as an important step in the design of a novel

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formulation of POD for the herbal medicine or healthcare products.

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Acknowledgments

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This work was supported by NSFC (21162042, 21562048 and 21662043),

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Innovative Team of Yunnan Minzu University, Key Laboratory of Resource Clean Conversion in Ethnic Regions of Yunnan. We also thank the Program for the

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Changjiang Scholars and Innovative Research Team in University (IRT13095) and

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Excellent Young Talents of Yunnan University for financial support.

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ACCEPTED MANUSCRIPT Figure captions

Fig. 1. The structures of Podophyllotoxin and HPβCD. Fig. 2. The Job plot for the POD/HPβCD system ([POD]＋[HPβCD]＝5.6×10-5 M) at

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289 nm in a water/ethanol (V:V = 4:1, pH = 3.0).

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Fig. 3. UV–vis spectral changes in POD (0.16 mM) upon addition of HPβCD (A:

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0-2.8 mM, from a to i, B: 0-4.0 mM, from a to g) in a water/ethanol (V:V = 4:1, A: pH = 3.0, B: pH = 10.5.) mixed solution, and the nonlinear least-squares analysis

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(inset) of the differential intensity (ΔA at 289 nm) to calculate the complex stability

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constant (Ks).

Fig. 4. Phase-solubility diagram for the POD/ HPβCD system at 25 oC.

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Fig. 5. 1H NMR spectra of POD in the absence and presence of HPβCD in D2O at 25

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ºC. (a) HPβCD, (b) POD/HPβCD complex (asterisk highlights the water peak). Fig. 6. ROESY spectrum of the POD/HPβCD complex in D2O.

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Fig. 7. Possible inclusion mode and significant NOESY (↔) correlations of the

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POD/HPβCD inclusion complex. Fig. 8. The model of POD docked into HPβCD. Fig. 9. XRD patterns: (a) POD, (b) HPβCD, (c) POD/HPβCD inclusion complex. Fig. 10. DSC thermograms: (a) POD, (b) HPβCD, (c) POD/HPβCD inclusion complex. Fig. 11. Scanning electron microphotographs: (a) HPβCD, (b) POD, (c) POD and HPβCD physical mixture (1:1 molar ratio), (d) POD/HPβCD inclusion complex. 30

ACCEPTED MANUSCRIPT Table titles

Table 1

The stability constant (Ks and logKs) and Gibbs free energy change (−ΔG0)

for the inclusion complexation of HPβCD with POD guest in a water/alcohol (v/v

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=4:1, ca. pH 3.0 or 10.5) mixed solution

Cytotoxic activities of POD and POD/HPCD inclusion complex in vitrob

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Table 3

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Table 2 The chemical shifts (δ) of HPβCD and POD/HPβCD complex

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(IC50, mMa)

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Graphical Abstract

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ACCEPTED MANUSCRIPT Highlights

► A novel POD/HPβCD inclusion complex was prepared. ► The inclusion behavior and characterization of POD with HPβCD was

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investigated. ► The water solubility and thermal stability of POD was obviously increased in the

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inclusion complex.

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► The cytotoxicity of POD/HPβCD inclusion complex against all five human tumor

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cell lines still remains.

► The POD/HPβCD inclusion complex will be potentially useful for its medical

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application.

33