Side-on binding of p-sulphonatocalix[4]arene to the dinuclear platinum complex trans-[{PtCl(NH3)2}2μ-dpzm]2+ and its implications for anticancer drug delivery

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Abstract

The utility of p-sulphonatocalix[4]arene (s-CX[4]) as a drug delivery vehicle for multinuclear platinum anticancer agents, using trans-[{PtCl(NH3)2}2μ-dpzm]2+ (di-Pt; where dpzm = 4,4′-dipyrazolylmethane) as a model complex, has been examined using 1H nuclear magnetic resonance, electrospray ionisation mass spectrometry, molecular modelling and in vitro growth inhibition assays. s-CX[4] binds di-Pt in a side-on fashion in a ratio of 1:1, with the dpzm ligand of the metal complex located within the s-CX[4] cavity with binding further stabilised by ion–ion interactions and hydrogen bonding between the metal complex am(m)ine groups and the s-CX[4] sulphate groups. Partial encapsulation of di-Pt within the cavity does not prevent binding of 5′-guanosine monophosphate to the metal complex. When bound to two individual guanosine molecules, di-Pt also remains partially bound by s-CX[4]. The cytotoxicity of free di-Pt and s-CX[4] and their host guest complex was examined using in vitro growth inhibition assays in the A2780 and A2780cis human ovarian cancer cell lines. Free di-Pt has an IC50 of 100 and 60 μM, respectively, in the cell lines, which is significantly less active than cisplatin (1.9 and 8.1 μM, respectively). s-CX[4] displays no cytotoxicity at concentrations up to 1.5 mM and does not affect the cytotoxicity of di-Pt, probably because its low binding constant to the metal complex (6.8 × 104 M−1) means the host–guest complex is mostly disassociated at biologically relevant concentrations.

Introduction

In the 40 years since the approval of cisplatin as an anticancer agent just two other platinum-based drugs have received world-wide approval [1]. New families of platinum drugs continue to be synthesised and tested, including: platinum(IV) complexes [1], [2], sterically hindered complexes [1], [2], [3], DNA intercalators [4], [5] and multinuclear drugs [6], [7]. We hypothesise, however, that the biggest break-through in the next decade for platinum-based chemotherapy will come from improved chemical delivery of already approved drugs. Better drug delivery can be achieved through the use of encapsulating agents, either using polymers to form liposome/micelle formulations or using macrocycles to encapsulate single drug molecules. The drugs Prolindac and Aroplatin are examples of successful formulations of oxaliplatin using liposomes and micelles [8], [9], [10], [11], [12].

Our group is investigating the targeted delivery of mono- and multinuclear platinum drugs using small macrocycles functionalised with targeting agents. In the first phase of our research we are examining a range of macrocycles that fully or partially encapsulate single molecules of drug, in order to select the best vehicle for further development. Previously we have examined cucurbit[n]urils in detail [5], [13], [14], [15], [16], [17], and cyclodextrins and calix[n]arenes to a lesser extent [18].

Calix[n]arenes are a family of macrocycles made from the hydroxyalkylation of a phenol and an aldehyde and are bowl or cone shaped molecules [19], [20]. p-Sulphonatocalix[4]arene (s-CX[4]; Fig. 1) is a particularly interesting member of the calix[n]arene family and has potential as a drug delivery vehicle. The four sulphate groups impart high water solubility on the molecule, it is able to bind a range of small molecules and proteins with high affinity, it has demonstrated zero haemolytic toxicity in vitro at concentrations up to 5 mM, and it is non-toxic in vivo at doses up to 100 mg/kg [21]. Previously we have examined the encapsulation of a family of platinum(II)-based DNA intercalators by s-CX[4] [18]. The host–guest chemistry of the resultant complexes was unusual, with a 2:2 complex being formed where two intercalator molecules were stacked head-to-tail and capped on either end by s-CX[4] [18].

In this paper we report the side-on binding of s-CX[4] to a dinuclear platinum complex, trans-[{PtCl(NH3)2}2μ-dpzm]2+ (di-Pt; Fig. 1), where dpzm = 4,4′-dipyrazolylmethane, by 1H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionisation mass spectrometry and molecular modelling. The ability of the macrocycle to slow down the metal complex binding to guanosine and the affect of s-CX[4] on the cytotoxicity of di-Pt in the A2780 and A2780cis ovarian cancer cell lines are also reported.

Section snippets

Materials

Di-Pt was made as previously described [22]. p-Sulphonatocalix[4]arene sodium salt and 5′-guanosine monophosphate disodium salt were purchased from Sigma–Aldrich. D2O (99.9%) was purchased from Cambridge Isotope Laboratories. Insulin, hydrocortisone, amphotericin B, gentamicin, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RPMI-1640 medium were purchased from Sigma. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, and l-glutamine were purchased from

Host–guest complex solubility

As the 2+ cation, di-Pt is soluble in water at concentrations up to 13.5 mM. The higher 4- charge of s-CX[4] means it is soluble at concentrations greater than 20 mM. When s-CX[4] and di-Pt are mixed in equimolar concentrations, however, the solubility of the host–guest complex, which has an overall charge of 2-, is around 4.5 mM. This is significantly higher than some cucurbit[n]uril-platinum(II) host–guest complexes which are only soluble at concentrations less than 1 mM [16], [28].

1H NMR

The titration

Conclusions

In this paper we examined the utility of s-CX[4] as a drug delivery vehicle for multinuclear platinum anticancer drugs using the dinuclear complex, di-Pt, as a model. The side-on binding of the macrocycle to the metal complex, its low binding constant, and its inability to provide steric hindrance to attack of the metal complex by guanosine indicates that s-CX[4] may not be a suitable drug delivery vehicle for multinuclear anticancer drugs.

Abbreviations

    di-Pt

    trans-[{PtCl(NH3)2}2μ-dpzm]2+

    DMEM

    dulbecco’s modified eagle’s medium

    Dpzm

    4,4′-dipyrazolylmethane

    ESI-MS

    electrospray ionisation mass spectrometry

    MTT

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

    ROESY

    rotating frame Overhauser effect spectrometry

    s-CX[4]

    p-sulphonatocalix[4]arene

Acknowledgement

This work was supported by a University of Strathclyde Faculty of Science Starter Grant awarded to N.J.W.

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