Development of an oral satraplatin pharmaceutical formulation by encapsulation with cyclodextrin

Abstract

A novel water-soluble oral satraplatin/β-cyclodextrin inclusion complex was prepared and characterized with a variety of techniques. Molecular dynamics simulations were performed to clarify its inclusion mechanism. Enabled by encapsulation with cyclodextrin, the water solubility of satraplatin was successfully increased to 7.4 mg mL⁻¹ and significantly improved by phase solubility studies. Meanwhile, the stability of satraplatin in acidic and weak alkaline aqueous solution was also effectively enhanced by forming the inclusion complex. Importantly, in an in vitro cytotoxicity test, the satraplatin encapsulated complex displayed superior cytotoxicity compared to free satraplatin against A549 and MCF-7 cells but was almost non-toxic to Caco-2 cells. In an in vivo antitumor test, this satraplatin encapsulated complex has shown much better activity in repressing lung cancer than free satraplatin but nearly no damage to intestinal mucosa by oral administration evaluated in xenograft mice models. Overall, the development of the current satraplatin/β-cyclodextrin inclusion complex has significantly improved the bioavailability of satraplatin and could benefit further applications in related pharmaceutical formulations.

---

1. Introduction

Over the past 4 decades, platinum-based chemotherapy drugs have been widely investigated in the treatment of cancers, and about 30 of them have been used in clinical trials. However, influenced by the quite low water solubility of platinum complexes, they have only exerted limited effects in applications. Although structural modifications have been employed as an important strategy to increase their hydrophilicity, the resulting drugs (e.g. carboplatin, oxaliplatin) have no obvious advantage over cisplatin (approved by the US FDA in 1978), and have serious side effects, such as kidney damage, neurotoxicity and ototoxicity in the treatment of testicular and ovarian cancers.^1,2

Satraplatin (Fig. 1) is a fourth-generation platinum analogue with high activities against human ovarian, cervical, prostate and lung tumours in preclinical studies,^3–7 and has no nephrotoxic or neurotoxic side effects.^8–10 However, since its chemical structure includes two axial acetate groups and a cyclohexyl group, the lipophilic property is apparently reserved in this compound. To afford satraplatin contained solutions with enough concentrations for cell-based assays and vivo evaluations, organic solvents (e.g. DMSO) are normally employed, which is deviated far from medical conditions. Therefore, the development of a water-soluble satraplatin complex, which could reserve its exceeding bioactivities, is highly attractive from the perspective of medical applications.

Fig. 1 Chemical structure of satraplatin and β-cyclodextrin.

It is well-known that cyclodextrins (CDs) are non-toxic macrocyclic oligosaccharides with a hydrophobic internal cavity and a hydrophilic outer surface. CDs have been effectively used to partly or entirely host inorganic or organic molecules within the cavities, and bring them into new hydrophilic environments.¹¹ Benefitted from these intrinsic properties, the water solubility of hydrophobic molecules could be greatly enhanced via encapsulation with CDs.^12–14 However, CDs have been rarely used in combination with platinum-based complexes to afford chemotherapeutic complexes.¹⁵ Recently, our group has reported that by forming inclusion complexes with CDs, the water solubility and bioavailability of natural products, such as cordycepin,¹⁶ nimbin¹⁷ and artemether,¹⁸ could be effectively enhanced. Considering the important potential usages of satraplatin in treating cancers, we decide to develop an oral satraplatin pharmaceutical formulation with cyclodextrins.

In preliminary studies, complex formations between satraplatin and three different CDs (α-CD, β-CD and γ-CD) were investigated (Fig. S6 and S7†). Their water solubility was compared to that of free satraplatin by means of UV-vis spectroscopy. The results showed that the inclusion of satraplatin in β-CD had the strongest absorption, indicating the highest enhancement of water solubility and cell activity in vitro (Table S1†) was realized by interacting with β-CD. In the following studies, we prepared a water-soluble inclusion complex of satraplatin with β-CD. A variety of characterisations, such as FT-IR, NMR, DSC, powder XRD and SEM, were performed to confirm the newly obtained platinum inclusion complexes. Importantly, enhanced cytotoxic activity was successfully achieved by complexation as demonstrated by experiments in vitro and in vivo. Moreover, no apparent toxic effect was detected for this satraplatin/β-CD complex at the examined concentrations. These resulted demonstrated that current inclusion complex could be a useful candidate for anti-tumour applications.

2. Results and discussion

2.1 Phase-solubility

A linearly increasing trend of the concentrations between satraplatin and β-CD was detected as illustrated in Fig. 2. It indicated the formation of a soluble complex between satraplatin and β-CD. The regression equation was summarized as follows: [satraplatin] = 0.999[β-CD] + 0.753, R² = 0.992. The phase solubility curve can be assorted as a typical A_L type.¹⁹ Since a slope of approximately 1.0 was observed, it suggested that the solubility of satraplatin increased due to the formation of a 1 : 1 complex with β-CD.¹⁹ The apparent stability constant (K_c = 2765 M⁻¹) was calculated from the slope of the phase-solubility diagram according to eqn (1), in which S₀ was the intrinsic water solubility of satraplatin (S₀ = 0.4 mg mL⁻¹, 0.8 mM).²⁰ The obtained high K_c value demonstrated that this inclusion complex was quite stable. The spontaneity and feasibility of the molecular complexation of satraplatin and β-CD were further evaluated using a thermodynamic approach. According to eqn (2), the change in Gibbs free energy (ΔG⁰) upon transferring satraplatin from aqueous solution to the cavity of β-CD was −19.30 kJ mol⁻¹. The negative value of the energy change indicated the complexation process was energetically favourable.

Fig. 2 (A) Phase solubility diagram of satraplatin in water depending on the β-CD concentration; (B) Job's continuous variation plot of the satraplatin/β-CD complex.

2.2 ¹H and 2D NMR analysis

To study the host–guest chemistry of our satraplatin/β-CD complex and establish inclusion modes, NMR experiments were taken.

First, we compared the ¹H NMR spectra of satraplatin with/without the host β-CD (Fig. 3). Due to its extremely low water solubility, satraplatin was difficult to be detected in D₂O by ¹H NMR. When the satraplatin/β-CD complex was examined, it clearly showed the framework protons of satraplatin, indicating the existence of satraplatin incorporated in β-CD. According to the calculation based on integrating β-CD's protons, an inclusion stoichiometry of 1 : 1 ratio was found (Fig. S3–S5†) and NMR data directly verify the accuracy of the Job's continuous variation plot method.

Fig. 3 ¹H NMR spectra of satraplatin in the absence and presence of β-CD in D₂O at 25 °C, respectively.

Several chemical shifts of β-CD were observed in ¹H NMR, suggesting new interactions existed in the complex. As shown in Table 1, the chemical shift of H-1 and H-6, which representing outside protons of β-CD, had a little variation after forming the complex. In comparison, more changes were exhibited by H-5 and H-3, which represent the internal protons of β-CD. It might due to a deep penetration of the drug into the CDs' cavity. Meanwhile, all spins of satraplatin experienced chemical shift changes upon complexation, indicating that satraplatin was probably encapsulated into β-CD. In this way, the satraplatin was also evaluated by measuring the changes in the chemical shifts (Δδ) of the protons in the complex, relative to the free satraplatin (Table S2†), at a temperature of 25 °C.

Table 1 Chemical shifts of ¹H NMR of β-CD protons in the presence and absence of satraplatin

Protons		Chemical shift (ppm)
		δβ-CD	δsatraplatin/β-CD	Δδ
H-1	d	4.931	4.905	−0.026
H-2	dd	3.520	3.500	−0.020
H-3	t	3.825	3.784	−0.041
H-4	t	3.443	3.425	−0.018
H-5	d	3.745	3.714	−0.031
H-6	m	3.762	3.742	−0.020

---

To further confirm the interaction of the satraplatin/β-CD complex, two-dimensional (2D) NMR was employed to detect the spatial proximity between the host and guest groups.^21,22 The ROESY spectrum of the satraplatin/β-CD complex (Fig. 4(A)) apparently illustrated a correlation between the cyclohexane ring of satraplatin and the H-5 and H-3 protons of β-CD, manifesting that the cyclohexane ring was trapped in the β-CD's cavity as depicted in Fig. 4(B).

Fig. 4 (A) ROESY spectrum of satraplatin/β-CD complex in D₂O. (B) Possible inclusion modes and significant ROESY (↔) correlations of the satraplatin/β-CD complex.

2.3 DSC, TG, XRD and FT-IR spectroscopy analysis

The differential scanning calorimeter (DSC) and thermogravimetric curves (TG) of satraplatin, β-CD and the inclusion complex were shown in Fig. S8.† The DSC profile of satraplatin (70.0 °C), β-CD (91.2 °C), satraplatin/β-CD complex (68.6 °C) and its physical mixture (60.8 °C) showed the broad endothermic band corresponding to the release of water molecules from their cavities. And DSC results of satraplatin (Fig. S8(a)†) showed two sharp endothermic peaks (195.1 °C and 243.4 °C), corresponding to the melting and boiling points of satraplatin. The thermal profile of β-CD (Fig. S8(b)†) displayed an endothermic signal close to 307.1 °C corresponding to its melting point. However, the DSC curves of the satraplatin/β-CD complex showed new peaks at about 246.3 and 312.9 °C (Fig. S8(c)†). The disappearance of the melting point peak of satraplatin and shifts of the dehydration peaks of cyclodextrin implied the formation of a new complex. This is indicative that satraplatin was no longer as a crystalline form and it is successfully included inside β-CD cavity.

The TG curves showed the decomposition temperature of satraplatin and β-CD were 175 °C (ca.) and 303 °C (ca.) respectively (Fig. S8†). In contrast, the thermal stability of the inclusion complex was changed to 239 °C (ca.), providing another evidence of forming the satraplatin/β-CD complex.

XRD and FT-IR¹⁶ were also used to confirm the structure of the platinum inclusion complex. For analysis details, please see the ESI (Fig. S1 and S2†).

2.4 SEM analysis

Next, to qualitatively understand the structural aspects of our inclusion complex, SEM experiments were further conducted. The SEM photomicrographs of β-CD, satraplatin, satraplatin/β-CD inclusion complex and their physical mixtures were shown in Fig. 5. β-CD crystallized as irregular particles (Fig. 5(a)) and pure satraplatin existed as irregularly shaped crystals (Fig. 5(b)). The physical mixture of satraplatin/β-CD revealed similarities of both crystalline components (Fig. 5(d)).

Fig. 5 Scanning electron microphotographs: (a) β-CD, (b) satraplatin, (c) satraplatin/β-CD inclusion complex, (d) satraplatin and β-CD physical mix.

In contrast, the satraplatin/β-CD inclusion complex was appeared in plate-like structures with variable sizes (Fig. 5(c)). Additionally, the morphology of the new complex was completely different from satraplatin or β-CD. These changes can be taken as a proof of the formation of a new inclusion complex by molecule encapsulation.

2.5 Solubilisation

The water solubility of the satraplatin/CD complex was determined by preparing a saturated solution. During this experiment, an excess amount of the complex was placed in 2 mL water (ca. pH 7.0), and the mixture was stirred for 1 h by ultrasonic wave at room temperature. After removing the insoluble substances by filtration, the resulted residue was dried and weighted to afford a calculated water solubility, which was approximately 7.4 mg mL⁻¹ (S_{0(satraplatin)}: ca. 0.4 mg mL⁻¹). To confirm this solubility result, the satraplatin/β-CD (24.2 mg) complex was mixed with 1 mL water at room temperature, and a clear solution was successfully afforded. It confirmed the inclusion complex has increased the solubility of satraplatin, which would be advantageous to the medical utilization.

2.6 Molecular modelling studies

Molecular docking and binding energy calculations were conducted to rationalise the geometry of our supramolecular complex of satraplatin and β-CD. In this regard, both head and tail inclusion orientations, from the β-CD secondary rim, were considered. The final relative stability energy values referred to the most stable state of each couple optimised in water. Related structures of two couples are represented in Fig. 6, including the longitudinal section, cross-section and ball-and-stick model of all docking configurations. According to the calculation, coaxially encapsulating the cyclohexane ring of satraplatin into β-CD central cavity was more stable than leaving the ring outside the rim (Goldscore = 35.576, binding energy = −19.999 kJ mol⁻¹ in Fig. 6(a); Goldscore = 29.63, binding energy = −14.342 kJ mol⁻¹ in Fig. 6(b)). The calculating results were in a good agreement with other experimental outcomes, highlighting the structural possibility of this supramolecular aggregate.

Fig. 6 The interaction energy of the most two stable shapes of satraplatin/β-CD complex, including longitudinal section, cross section and ball-and-stick model. (a) The cyclohexane ring of satraplatin inserted into β-CD; (b) the cyclohexane ring of satraplatin is outside of larger rim.

2.7 Stability in aqueous buffer solutions

In order to evaluate the stability of the satraplatin/β-CD complex, we tracked the absorbance changes of satraplatin and satraplatin/β-CD in different aqueous buffer solutions, including a simulated acidic body fluid (ca. pH 2.2, citric acid/Na₂HPO₃/water buffer system) and a simulated alkaline body fluid (ca. pH 7.3, NaH₂PO₃/Na₂HPO₃/water buffer system). The absorbance of related solutions of satraplatin and satraplatin/β-CD was analyzed at 208.5 nm by UV/vis spectra every 25 min at room temperature. Fig. S9† illustrated the trend of the relative absorbance A/A₀ (A was the absorbance at the recording time and A₀ was the original absorbance) of different solutions. As shown in Fig. S9(A),† when pH = 2.2, the A/A₀ value of satraplatin changed faster than the complex before 225 min. After 24 h, the relative absorbance of both solutions reached equilibrium, and the changing ranges were about 96% for satraplatin and 41% for satraplatin/β-CD complex. When pH = 7.3, the A/A₀ value followed a similar trend to that of acidic conditions. As shown in Fig. S9(B),† satraplatin kept a faster change rate of the relative absorbance than the inclusion complex at the beginning. After 24 h, the changing ranges for satraplatin and the satraplatin/β-CD complex were about 76% and 19% respectively. These results suggested that the satraplatin/β-CD complex reserved much better stabilities than free satraplatin under either aqueous acidic or alkaline conditions.

2.8 In vitro cytotoxicity

The cytotoxicity of free satraplatin and the satraplatin/β-CD inclusion complex against A549 and MCF-7 cells was individually evaluated using the MTT assay.^23,24 For A549 cells, the IC₅₀ values of satraplatin (Table S1†) and the inclusion complex were 10.12 mmol mL⁻¹ and 6.09 mmol mL⁻¹ respectively, which corresponded to relative standard deviations (RSDs) of 6.45% for satraplatin and 5.34% for the inclusion complex. For MCF-7 cells, the IC₅₀ values were 0.25 mmol mL⁻¹ and 0.14 mmol mL⁻¹ respectively, which represented a RSD value of 7.25% for satraplatin and 6.12% for the complex. In a blank control experiment, β-CD did not show any inhibitory activity against both cells. Therefore, the satraplatin/β-CD complex displayed superior cytotoxicity compared to free satraplatin against A549 and MCF-7 cells, demonstrating that enhanced anticancer activities were obtained by encapsulating free satraplatin with cyclodextrin.

The cytotoxicity of satraplatin and the satraplatin/β-CD inclusion complex against Caco-2 cells was evaluated in a concentration range from 0.3125 mmol mL⁻¹ to 10 mmol mL⁻¹, using MTT test (Fig. S10†). The satraplatin/β-CD complex displayed less cytotoxicity compared to free satraplatin on same concentration, demonstrating that the satraplatin/β-CD complex was almost non-toxic to human epithelial colorectal adenocarcinoma Caco-2 cells.

2.9 In vivo antitumor potency

The effects of the satraplatin, β-CD and the satraplatin/β-CD inclusion complex on inhibiting tumour growth were carefully evaluated in xenograft models. Based on the in vitro biological experiments, the in vivo antitumor activities of satraplatin, β-CD and the satraplatin/β-CD complex were determined in the human lung cancer A549 xenograft model. As shown in Fig. 9, when treated with the inclusion complex, remarkable antitumor effects were observed for mice. In these experiments, the intragastrical method was applied for five consecutive days. At a dose of 100 mg kg⁻¹ for the satraplatin/β-CD complex (equivalent to 30 mg kg⁻¹ satraplatin) and 30 mg kg⁻¹ for satraplatin, the body weight and tumour volume of the mice were monitored during the entire period (Fig. 9(A)). Feedings between the treated groups and the control group were the same.

As shown in Fig. 7 and 8, no tumour metastasis was found in the liver, lungs, heart, kidneys and intestine evaluated using histopathological analysis stained with H&E under optical microscope.

Fig. 7 Representative organs pictures in tumour-bearing nude mice.

Fig. 8 Histopathological analysis of representative organs in tumour-bearing nude mice. Pathologic section was stained with H&E (×400 magnifications).

As illustrated in Fig. 9(B), the average tumour volumes of two satraplatin-treated groups (free satraplatin and satraplatin/β-CD complex) were consistently smaller than those of the control groups (olive oil, β-CD). Also, growth rates of tumours treated with free satraplatin or the inclusion complex were much slower than control groups. The tumour weight was significantly reduced compared to the control groups in the presence of satraplatin or satraplatin/β-CD (Fig. 9(C)) after two weeks of treatment. Notably, mice treated with the satraplatin/β-CD complex had significantly smaller tumours than those treated with free satraplatin (p = 0.0357). In addition, there was no obvious difference in body weight or change in movement. No salivation, vomiting or oedema was detected. As also shown in Fig. 8, almost normal intestinal mucosa can be seen. This information showed that the satraplatin/β-CD complex was almost non-toxic.

Fig. 9 Evaluation of tumour growth inhibition of β-CD, satraplatin and satraplatin/β-CD complex (30 mg kg⁻¹ satraplatin equivalent) on established A549 tumour-xenografts in athymic mice. Mice were randomly separated into four groups (n = 6) and were administered with 0.2 mL olive oil, β-CD, satraplatin or satraplatin/β-CD complex by IG everyday for five consecutive days. (A) Body weight; (B) tumour volume curve; (C) tumour weight; (D) image of solid tumours. The results are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, compare with the β-CD group at day 24.

2.10 Histopathological analysis of tumour tissue

Next, tumour tissue samples came from mice treated with β-CD, satraplatin and the satraplatin/β-CD inclusion complex were evaluated using histopathological analysis (Fig. 10). Under optical microscope (×200, ×400), cells of the tumour tissue treated with β-CD, satraplatin and the satraplatin/β-CD inclusion complex groups were distributed focally or clustered, and an adenoid structure was observed. The nuclei were large, deeply stained, and pathologic karyokinesis was observed. Necrosis and degeneration of the tumour tissue was obviously observed in addition, an increase in interstitial infiltration was found. These results indicated that xenograft model was established successfully.

Fig. 10 Histological analysis of different subgroups. Sections of tumour tissues recovered from nude mice in the β-CD, satraplatin and satraplatin/β-CD complex groups were stained with H&E (top ×200, bottom ×400 magnifications).

3. Materials and methods

3.1 Materials

Pure satraplatin (FW = 500.29) was purchased from Kunming Guiyan Pharmaceutical Co., Ltd. (China). β-CD (FW = 1135) was purchased from ABCR GmbH & Co. KG and used without further purification. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), phosphate buffered saline (PBS), D₂O (D 99.9%) and dimethyl sulfoxide-d₆ (DMSO-d₆, D, 99.9% + 1% v/v TMS) were obtained from Sigma-Aldrich. Other reagents and chemicals were of pharmaceutical grade and/or approved for human use. All experiments were carried out using ultrapure water.

3.2 Preparation of satraplatin/β-CD

A pre-mixed solution of ethanol and water was used to dissolve satraplatin (0.02 mM, 10.0 mg) and β-CD (0.01 mM) (ca. 10 mL, v/v = 1 : 4) for 7 days at room temperature. After filtering the resulted solution through a 0.22 μm membrane, lyophilization with a freeze dryer (Alpha2-4, Christ) was employed to obtain the solid complex (yield 92%): ¹H NMR (500 MHz, D₂O, TMS): δ 4.91 (d, 7H, H-1 of β-CD), 3.79 (t, 7H, H-3 of β-CD), 3.65–3.74 (m, 14H, H-6 and H-5 of β-CD), 3.47–3.50 (dd, 7H, H-2 of β-CD), 3.42 (t, 7H, H-4 of β-CD), 2.84 (s, –CH– of the cyclohexane ring protons for satraplatin), 1.88 (m, 4H, H-1 and H-5 of the cyclohexane ring protons for satraplatin), 1.80 (s, 6H, CH₃– protons for satraplatin), 1.65 (m, 4H, H-2 and H-4 of the cyclohexane ring protons for satraplatin) and 1.14 (m, 3H of the cyclohexane ring protons for satraplatin). A fine powder of the physical mixture of satraplatin with β-CD in a 1 : 1 molar ratio was prepared by simply stirring and filtering through a #100 sieve.

3.3 Phase solubility analysis

Phase solubility studies were performed according to the method of Higuchi and Connors.¹⁹ Satraplatin was added into solutions of β-CD from 0.44 to 2.2 mM. Resulted mixtures were stirred for 48 h shaded from light at room temperature. After reaching equilibrium, the solutions were first filtered through a 0.45 μm membrane. Then, a UV spectrophotometer at 208.5 nm was used to detect the obtained solutions. By comparing with a standard curve of pure satraplatin contained solution, the concentrations of satraplatin in these new solutions were determined. The apparent stability constant (K_c) of the satraplatin/β-CD complex was calculated according to the following equation:^19,25

In eqn (1), S₀ meant the solubility of satraplatin in the absence of β-CD, slope meant the corresponding slope of the phase solubility diagram.

The change in the Gibbs free energy (ΔG⁰) upon transfer of satraplatin from aqueous solution to the cavity of CDs was calculated with the following equation:²⁵

ΔG⁰ = −2.303RT log K_c (2)

The stoichiometry of the complex was determined using the continuous variation of Job's method. The total molar concentration of the satraplatin and β-CD aqueous mixture was kept constant at 8 × 10⁻⁵ M. The molar ratio was varied from 0 to 1. The absorbance was recorded at the different molar ratios by a UV spectrophotometer.

3.4 Characterisation of the satraplatin/β-CD complex

NMR spectra were obtained using D₂O as a deuterium solvent (Bruker 500 MHz). 2D-ROESY spectra were obtained at 298 K using the TPPI method at a field of ∼2 kHz. Sine (F₂) and qsine (F₁) window functions were used, and the processing was carried out with zero-filling to 2 K.

For the FT-IR experiments, β-CD, satraplatin, their physical mixture and the inclusion complex were separately mixed with spectro grade KBr powder, and results were collected from 4000–400 cm⁻¹ on a Nicolet 550-H FT-IR spectrophotometer (Nicolet, USA). FT-IR spectra were analysed by the spectrophotometer software OMNIC 5.2.

The powder X-ray was carried out with a Rigaku TTRIII instrument using Cu Kα radiation (40 kV, 100 mA) at 5° min⁻¹. The samples were mounted and scanned with a step size of 2θ = 0.02°.

TG and DSC measurements were conducted on a NETZSCH STA 449F3 and a 2960 SDT V3.0F instrument, respectively.

For the SEM experiments, all samples were prepared by coating with a thin layer of gold (approximately 300 Å) in a vacuum for 30 s and at 30 W, before detecting with a FEI QUANTA 200 scanning electron microscope operated at an excitation voltage of 15, 20 or 30 kV.

3.5 Molecular modelling^26,27

Molecular modelling was carried out based on an analysis of the NMR results. The initial structures of satraplatin and β-CD were constructed using Chembio3D ultra (Version 10.0, Cambridge Soft com., USA), and were individually optimized using the PM3 method by Gaussian 03 (ref. 28) (Gaussian Inc., Wallingford, USA). After energy minimisations of satraplatin and β-CD, a molecular dynamics simulation of the satraplatin/β-CD complex was conducted based on PM3 optimization in water solvent. For the inclusion process, the glycosidic oxygen atoms of β-CD were placed on the X, Y plane, and the centre was defined as the centre of the coordination system. The guest molecule approached the β-CD cavity along its Z-axis. The 2-OMe and 3-OMe groups in each glucose unit were placed pointing toward the positive Z-axis. The coordination bond toward Pt centre in satraplatin was placed on the Z-axis in the head up orientation during the docking process. Satraplatin approached and passed through the cavity of β-CD along the Z-axis. The relative distance between satraplatin and β-CD ranged from 8 to −8 Å with a step of 0.5 Å. A systematic search of the energy-minimised structure of the satraplatin/β-CD complex was performed at each distance point, set at 298.15 K in water. Finally, the structure with the lowest heat energy at all positions was obtained as the optimal complex structure and further established in PyMol.

3.6 Measurement of cytotoxicity

The cytotoxicity of the samples on tumour cells was measured by MTT method.²⁹ MCF-7 (a human breast adenocarcinoma cell line), A549 (a human lung adenocarcinoma cell line) cells and Caco-2 (human epithelial colorectal adenocarcinoma) were seeded into 96-well microculture plates. In this assay, the increase or decrease in the number of viable cells was linearly correlated with the mitochondrial activity, which was indicated by the conversion of the tetrazolium salt MTT into formazan crystals that can be solubilised and quantified spectrophotometrically. First, cells were grown in 96-well plates at 5000 cells per well in a final volume of 200 μL of culture medium (A549: 10% of RPMI-1640; MCF-7 and Caco-2: 10% of DMEM) per well. Then, the cells were cultured in an incubator (5% CO₂, 37 °C) until the cells reached 70–80% confluence. A549 and MCF-7: satraplatin and its complex were added at a concentration range from 0 mmol mL⁻¹ to 9.6 mmol mL⁻¹ per well; Caco-2: satraplatin and its complex were added at a concentration range from 0.3125 mmol mL⁻¹ to 10 mmol mL⁻¹ per well. After culturing the cells in an incubator for 24–48 h, MTT solution (20 μL) was added to each well and incubated for 2 h. The culture medium was discarded, and DMSO (200 μL) was added to each well. The solution was then swirled gently and left in the dark for 10 min. The absorbance in each well was measured at 570 nm using a microtiter plate reader. The mean IC₅₀ was determined from the results of three independent tests. The inhibition/viability of cell proliferation was calculated by the following formula:

3.7 In vivo tumour growth inhibition and histopathological study

Female BALB/c nude mice at 4–6 weeks of age were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and housed individually in a specific pathogen free facility. The mice were inoculated subcutaneously with A549 cells (2 × 10⁶ suspended in 0.1 mL of PBS for each mouse). After reaching an average tumour volume of 100–150 mm³, the animals were randomised into groups and treated by intragastric administration with 30 mg per kg per day satraplatin, 100 mg per kg per day satraplatin/β-CD complex (compound dissolved in 0.2 mL of olive oil), vehicle control (0.2 mL olive oil) or 70 mg per kg per day β-CD (compound dissolved in 0.2 mL of olive oil), respectively for five consecutive days and monitoring of tumour progression was done every 3 days. The tumour volumes were estimated by measuring two dimensions of the tumour using a digital calliper and calculated by the formula V = a × W² × 0.52, with V being the volume, a being the length, and W being the width of the tumour nodule. At the end of the experiment (day 24), the mice were sacrificed, the solid tumours were obtained and weighted, and the liver, lungs, heart and kidneys were removed. The organs and tumour tissue were gained and fixed in 4% formaldehyde, embedded in paraffin and sectioned at 4 μm for haematoxylin–eosin (H&E) staining for histopathological analysis. H&E staining was performed according to the manufacturer's instructions. All animal experimental protocols were approved by the Administrative Panel on Laboratory Animal Care of Kunming Medical University.

4. Conclusions

In conclusion, we have successfully developed a novel satraplatin/β-CD inclusion complex, providing a hydrophilic antitumor formulation by encapsulating a hydrophobic drug molecule within the cavity of cyclodextrin. A molar ratio of 1 : 1 (satraplatin : β-CD) was determined in this complex by NMR spectrum and phase solubility. After complexation, the changing ranges for satraplatin and the satraplatin/β-CD complex were about 76% and 19% in aqueous buffer solutions by ultraviolet absorption spectrum, respectively. And the results suggested that the satraplatin/β-CD complex reserved much better stabilities than free satraplatin under either aqueous acidic or alkaline conditions. Importantly, in vitro cytotoxicity test, the satraplatin encapsulated complex has displayed superior cytotoxicity compared to free satraplatin against A549 and MCF-7 cells but almost non-toxic to Caco-2 cells. In vivo antitumor test, this satraplatin encapsulated complex has shown much better activity in repressing lung cancer than free satraplatin but nearly no-damage to intestinal mucosa by oral administration evaluated in xenograft mice models. So significant enhancements in the tumour growth inhibition effect were observed both in vitro and in vivo but low toxicity at examined concentrations. These results showed that this satraplatin/β-CD inclusion complex could be used as a novel candidate for applications as an anti-tumour drug.

Acknowledgements

This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13095), the National Natural Science Foundation of China (No. 21262043, U1202221 and 21442006), the Science Research Fund of Yunnan Provincial Education Department (2015J004). The authors thank the High Performance Computing Center at Yunnan University for use of the high performance computing platform.

Notes and references

1. B. Rosenberg, L. Van Camp and T. Krigas, Nature, 1965, 205, 698–699.
2. B. Rosenberg, L. Van Camp, J. E. Trosko and V. H. Mansour, Nature, 1969, 222, 385–386.
3. L. R. Kelland, G. Abel, M. J. McKeage, M. Jones, P. M. Goddard, M. Valenti, B. A. Murrer and K. R. Harrap, Cancer Res., 1993, 53, 2581–2586.
4. K. J. Mellish, L. R. Kelland and K. R. Harrap, Br. J. Cancer, 1993, 68, 240–250.
5. P. R. Twentyman, K. A. Wright, P. Mistry, L. R. Kelland and B. A. Murrer, Cancer Res., 1992, 52, 5674–5680.
6. K. Wosikowski, L. Lamphere, G. Unteregger, V. Jung, F. Kaplan, J. P. Xu, B. Rattel and M. Caligiuri, Cancer Chemother. Pharmacol., 2007, 60, 589–600.
7. K. J. Mellish, C. F. Barnard, B. A. Murrer and L. R. Kelland, Int. J. Cancer, 1995, 62, 717–723.
8. M. J. McKeage, F. E. Boxall, M. Jones and K. R. Harrap, Cancer Res., 1994, 54, 629–631.
9. M. J. McKeage, L. R. Kelland, F. E. Boxall, M. R. Valenti, M. Jones, P. M. Goddard, J. Gwynne and K. R. Harrap, Cancer Res., 1994, 54, 4118–4122.
10. M. J. McKeage, S. E. Morgan, F. E. Boxall, B. A. Murrer, G. C. Hard and K. R. Harrap, Br. J. Cancer, 1993, 67, 996–1000.
11. V. T. Karathanos, I. Mourtzinos, K. Yannakopoulou and N. K. Andrikopoulos, Food Chem., 2007, 101, 652–658.
12. W. Misiuk and M. Zalewska, Carbohydr. Polym., 2009, 77, 482–488.
13. Y. Liu and Y. Chen, Acc. Chem. Res., 2006, 39, 681–691.
14. H. H. Wu, H. Liang, Q. Yuan, T. Wang and X. Yan, Carbohydr. Polym., 2010, 82, 613–617.
15. G. Horvath, T. Premkumar, A. Boztas, E. Lee, S. Jon and K. E. Geckeler, Mol. Pharm., 2008, 5, 358–363.
16. J. Q. Zhang, D. Wu, K. M. Jiang, D. Zhang, X. Zheng, C. P. Wan, H. Y. Zhu, X. G. Xie, Y. Jin and J. Lin, Carbohydr. Res., 2015, 406, 55–64.
17. L. J. Yang, B. Yang, W. Chen, R. Huang, S. J. Yan and J. Lin, J. Agric. Food Chem., 2010, 58, 8545–8552.
18. B. Yang, J. Lin, Y. Chen and Y. Liu, Bioorg. Med. Chem., 2009, 17, 6311–6317.
19. T. Higuchi and K. A. Connors, Adv. Anal. Chem. Instrum., 1965, 4, 117–212.
20. L. Chen, P. F. Lee, J. D. Ranford, J. J. Vittal and S. Y. Wong, J. Chem. Soc., Dalton Trans., 1999, 1209–1212.
21. D. Bardelang, A. Rockenbauer, H. Karoui, J. Finet and P. Tordo, J. Phys. Chem. B, 2005, 109, 10521–10530.
22. M. Kfoury, L. Auezova, H. Greige-Gerges, S. Ruellan and S. Fourmentin, Food Chem., 2014, 164, 454–461.
23. J. Carmichael, W. G. DeGraff, A. F. Gazdar, J. D. Minna and J. B. Mitchell, Cancer Res., 1987, 47, 943–946.
24. T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
25. J. Madan, B. Baruah, M. Nagaraju, M. O. Abdalla, C. Yates, T. Turner, V. Rangari, D. Hamelberg and R. Aneja, Mol. Pharm., 2012, 9, 1470–1480.
26. M. T. Faucci, F. Melani and P. Mura, Chem. Phys. Lett., 2002, 358, 383–390.
27. J. Q. Zhang, K. Li, Y. W. Cong, S. P. Pu, H. Y. Zhu, X. G. Xie, Y. Jin and J. Lin, Carbohydr. Res., 2014, 396, 54–61.
28. Gaussian 03, Revision C.02, ed. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, J. T. Vreven, K. N. Kudin and J. C. Burant, Gaussian, Inc., Wallingford CT, 2004.
29. M. Ferrari, M. C. Fornasiero and A. M. Isetta, J. Immunol. Methods, 1990, 131, 165–172.

---

Footnotes

† Electronic supplementary information (ESI) available: XRD, FTIR, DSC, TG, stability, inhibitory concentration of satraplatin/β-CD. See DOI: 10.1039/c5ra27182g

‡ These authors contributed equally to the work.

---