Molybdocene dichloride intercalation into zirconium phosphate nanoparticles

Journal of Organometallic Chemistry 791 (2015) 34e40

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Molybdocene dichloride intercalation into zirconium phosphate nanoparticles ~ as-Montes a, Agustín Díaz b, Cindy Barbosa a, Coralis Ramos a, Barbara Casan ndez c, Cle mence Queffelec d, Franck Fayon e, Cindy Collazo a, Enrique Mele b d  n a, * Abraham Clearfield , Bruno Bujoli , Jorge L. Colo a

Department of Chemistry, University of Puerto Rico-Río Piedras Campus, PO Box 70377, San Juan, PR 00936-8377, USA Department of Chemistry, Texas A&M University, PO Box 30012, College Station, TX 77842-3012, USA Department of Chemistry, University of Puerto Rico-Mayagüez Campus, P.O. Box 9019, Mayagüez, PR 00681, USA d Chimie Et Interdisciplinarit e: Synth ese Analyse Mod elisation (CEISAM), Universit e de Nantes, CNRS, UMR 6230, 2, rue de la Houssini ere, BP 92208, 44322 Nantes Cedex 3, France e CEMHTI-CNRS, 1D av. de la Recherche Scientifique, 45071 Orl eans Cedex 2, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2015 Received in revised form 19 May 2015 Accepted 20 May 2015 Available online 27 May 2015

Molybdocene dichloride (Cp2MoCl2), a metallocene dichloride currently being evaluated as potential anti-cancer drug, has been intercalated into zirconium phosphate, an inorganic layered nanomaterial. Hydrolysis of the chloride ligands appears to occur, leading to the formation of the monocation species ([Cp2Mo(H2O)(OH)]þ), which intercalates into the ZrP layers by ion exchange. IR spectroscopy, X-ray powder diffraction (XRPD), UVeVis spectrophotometry, scanning electron microscopy with energy dispersive X-ray spectroscopy, NMR spectroscopy, and thermogravimetric analysis were used to confirm the presence of the metallocene between the layers of zirconium phosphate. The XRPD data indicates that a new intercalated phase with an expanded interlayer distance of 11.0 Å was obtained; thermogravimetric analysis indicates up to 64% loading in the context of molar ratio (0.64 mol per ZrP formula unit) or 32% exchange capacity. Further evidence for intercalation was obtained from 31P MAS NMR experiments. IR spectroscopy confirms the presence of the cyclopentadienyl bearing metallocene in the layers. © 2015 Elsevier B.V. All rights reserved.

Keywords: Molybdenum Molybdocene dichloride Layered materials Zirconium phosphate Nanoparticles

1. Introduction The development of targeted drug delivery systems is currently one of the most important areas of drug research. Presently, many of the drugs designed, discovered or already in use for therapeutics are limited by their poor solubility, high toxicity, high dosage, aggregation, nonspecific delivery, in vivo degradation, and short circulating half-lives [1]. Previous reports have shown that nanoparticles can overcome these limitations, particularly by the enhanced permeability and retention effect shown by cancer cells toward nanoparticles presenting a passive mechanism for selective delivery of drugs to tumor cells [2]. Before such efforts can advance, detailed chemical characterization of the drug-immobilized nanoparticles are needed to better guide the understanding of cell- and in-vivo studies.

* Corresponding author. n). E-mail address: [email protected] (J.L. Colo http://dx.doi.org/10.1016/j.jorganchem.2015.05.031 0022-328X/© 2015 Elsevier B.V. All rights reserved.

Our current interest is in using the tetravalent metal phosphate, zirconium phosphate (zirconium bis(monohydrogen orthophosphate) monohydrate (Zr(HPO4)2$H2O, a-ZrP) as drug carrier for potential anticancer agents such as metallocene dichlorides, in particular molybdocene dichloride (MDC). ZrP has been extensively studied and reported as an inorganic layered nanomaterial (ILN) [3]. The use of ILNs as potential drug carriers has been growing since the discovery of their ability to encapsulate bioactive compounds and control their release via a chemical switch [3e5]. a-ZrP is one of the best characterized ILNs with an interlayer distance of 7.6 Å and a layer thickness of 6.6 Å (Fig. 1) [6]. In a-ZrP the zirconium atoms in each layer nearly align in a plane with bridging phosphate groups located alternately above and below the metal atom plane. Six oxygen atoms from different phosphate groups octahedrally coordinate the Zr atoms while each phosphate group is bonded to three different Zr atoms. The fourth oxygen atom of the phosphate group is protonated and it is

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Fig. 1. A portion of the Zr(HPO4)2$H2O, a-ZrP structure showing the relationship between adjacent layers. A cavity is shown in heavy outline and the negatively marked circles are oxygens bonded to exchangeable protons. Adapted from Ref. [6].

pointing towards the interlayer space and can be deprotonated via an ion exchange reaction [6]. This arrangement creates a zeolitic cavity per formula unit encapsulating a water molecule stabilized by hydrogen bonding with the hydroxy groups of the phosphates inside the cage. These zeolitic cavities interconnect to each other by entrances with a diameter of 2.61 Å. Due to this narrow space just a few cations can diffuse through, limiting the direct ion-exchange capacity of this material [6]. Our laboratory developed an approach that overcomes the difficulty of direct intercalation in a-ZrP [7]. In this approach a highly hydrated phase of ZrP (containing six molecules of water per formula unit, known as the q phase) is used to achieve direct intercalation of large species without the need of a preintercalation process (Fig. 2A). The highly hydrated phase of the layered ZrP is an acidic ion exchanger that has been used for the immobilization of several photo- [7], bio- [5] and redox-active compounds [8]. The applications for these materials can range from being used as catalysts [9], electron-transfer systems [7,8], drug carriers [5], and modified electrodes [8]. In addition, ZrP can be modified by manipulating certain variables in its synthesis to obtain a material with nanoscale dimensions [10], suitable for additional applications in the areas of nanotechnology and drug delivery. q-ZrP was chosen to intercalate MDC (Fig. 2B) to demonstrate its use of these nanoparticles as anticancer drug delivery agents. Since the discovery of titanocene dichloride's antitumor properties in 1979 [11], investigations of other related potential

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metallocene-based drugs begun to emerge due to the lower toxicity exhibited in comparison with the known anticancer drug cisplatin (cis-Pt(NH3)2Cl2). The metallocenes of general formula Cp2MX2 (Cp ¼ cyclopentadienyl; M ¼ Ti, V, Nb, and Mo; X ¼ halides and pseudohalides) have shown antitumor activity against a wide variety of tumor cells [12e14]. Contrary to titanocene dichloride which is unstable in water and looses its Cp ligands to produce insoluble species at physiological pH, MDC maintains its Cp ligands in water and accumulates in significant amounts in the cellular nuclei [15], which provoked our interest in the intercalation of MDC in ZrP. The mechanism of action of MDC is not yet clear in terms of the cellular uptake, distribution, and damage in the tumor cells [12]. The Mo metal in MDC, being a softer metal than Ti, prefers coordination to softer ligands, such as thiols, over phosphate, amino, and carboxylate groups. Therefore, this compound would be more attracted to thiol-containing biomolecules as a cellular target. In addition, MDC has a poor aqueous solubility that limits its utility as an antitumor drug. To overcome the drawbacks of low solubility in water, instability, and non-localized release of MDC we decided to study the direct intercalation of this anticancer drug into q-ZrP. Direct intercalation of metallocenes into q-ZrP has not been previously reported in the literature with exception of the direct intercalation of ferrocene for use as a redox agent in our own effort [8]. Here, we report the successful direct intercalation of MDC into ZrP producing an expanded intercalated phase. In addition, we present an extensive characterization of the drug-immobilized nanomaterial that indicates that the chemical integrity of the cyclopentadienyl rings of MDC are preserved in the MDCintercalated material and that MDC can be released with a pH stimulus. 2. Experimental section 2.1. Materials Zirconyl chloride octahydrate (ZrOCl2$8H2O, 98%), was obtained from Aldrich and used without further purification. Phosphoric acid (H3PO4, 85% v/v) and bis(cyclopentadienyl) molybdocene dichloride (99%) were obtained from Fisher Co. Nanopure water was obtained using a Barnstead purification train (18 MU$cm). All other reagents were of spectroscopic grade and were used without further purification.

Fig. 2. Structure of (A) Zr(HPO4)2$6H2O, q-ZrP and (B) molybdocene dichloride (MDC).

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2.2. Characterization of molybdocene dichloride-intercalated ZrP materials X-ray powder diffraction (XRPD) measurements were performed from 2 to 40 (in the 2q axis) using a Bruker D8 Advance Xray diffractometer with Cu Ka radiation (l ¼ 1.5406 Å) with BraggBrentano assembly. Bragg's law (nl ¼ 2dhkl sin q) was used for the determination of the interlayer distance in the ZrP layers from the first order diffraction peak, where l is the wavelength of the X-ray source, dhkl is the interlaminar distance between planes in the unit cell and q is the diffraction angle. Vibrational spectroscopy data were obtained using a Bruker-Tensor 27 FT-IR spectrometer with the OPUS Data Collection Program for the analysis. UVevis absorption spectra were obtained using 2501PC Shimadzu spectrophotometer using water as a solvent for MDC. Thermogravimetric experiments were carried out on a NETZSCH TGA/DSC model: STA 449 Jupiter F3 instrument. The temperature was ramped at 5  C min1 under a flow of air up to 1000  C. Transmission electron microscopy of the sample was performed on a TEM Hitachi HF2000 microscope (accelerating voltage of 300 kV, Scherzer resolution of 0.18 nm). The sample was added in ethanol and dispersed ultrasonically. A drop of the suspension was deposited on a copper grid previously covered with a thin holey carbon film. Qualitative elemental analysis by energy dispersive X-ray spectroscopy by means of SEM (SEM-EDX) was performed using a scanning electron microscope JEOL JSM 5800 LV with SDD energy dispersive spectrometer SAMx instrument. Solid-state magic angle spinning (MAS) NMR experiments were performed on a Bruker spectrometer, operating at 7.0 T (1H and 31P Larmor frequencies of 300 and 121.5 MHz, respectively) using a 4 mm double-resonance MAS probe and a spinning frequency of 14 kHz. Quantitative 31P single pulse MAS spectra were acquired using a 20 flip angle (pulse duration of 1 ms) and a recycle delay of 40 s. The 31P-{1H} cross-polarization (CP) MAS spectra were recorded using a ramped cross-polarization [16] with a contact time of 1 ms and a recycle delay of 1 s. For all 31P 1D experiments, 1 H decoupling was achieved using the SPINAL64 sequence [17] with a radio-frequency field strength of 64 kHz. 31P 2D homonuclear dipolar double quantum e single quantum (DQ-SQ) MAS correlation spectra were recorded using the BaBa-xy16 recoupling sequence [18]. The DQ excitation and reconversion periods were set to 1.143 ms (corresponding to 16 rotor periods). 1H SIPNAL64 decoupling with an rf-field of 64 kHz was applied during DQ recoupling periods, t1 evolution, and signal acquisition. Quadrature detection in the indirect dimension was achieved using the States method [19]. The chemical shifts were measured relative to 85 wt% H3PO4. 2.3. Synthesis of the q-ZrP phase A volume of 120 mL of 0.05 M ZrOCl2$8H2O was mixed with 85 mL of 6 M H3PO4 with constant stirring at 94  C for 48 h [20]. The precipitated solid was filtered and washed three times with nanopure water. The wet precipitate was characterized using X-ray powder diffraction (XRPD). The first diffraction peak at 2q ¼ 8.6 corresponds to an interlayer distance of 10.3 Å for this phase using Bragg's law. 2.4. Intercalation of molybdocene dichloride

q-ZrP has 6 water molecules per formula unit whereas a-ZrP has only one. Upon drying, the q-phase rapidly dehydrates and converts to the a-phase of ZrP [7]. Therefore, to maintain the q-phase requires its storage as a suspension in water. Prior to use, the q-phase was quickly filtered (ca. 3 min) and placed in the reaction solvent

while still wet. At the same time, a similar amount of q-ZrP was allowed to dry in an oven at 75  C for 24 h (final product ¼ a-ZrP), in order to determine the exact content of ZrP in the sample and subsequently adjust the desired metallocene dichloride to ZrP molar ratio. The direct intercalation reaction was conducted by suspending q-ZrP in an aqueous solution of molybdocene dichloride at a 1:1 MDC:ZrP concentration ratio at constant stirring and at room temperature for five days. Subsequently, the reaction mixture was filtered using 0.22 mm filters (Millipore), washed with water and the solid dried overnight at room temperature. In a typical experiment, using a mass of q-ZrP equivalent to 95 mg of a-ZrP led to isolation of 130 mg of dry intercalation product. 3. Results and discussion 3.1. Direct intercalation and characterization The direct intercalation of MDC was achieved by the batch method, where the metallocene dichloride was added to a suspension of q-ZrP. After the intercalation reaction was stopped, the resulting solid was filtered, rinsed with water, dried, and pulverized prior to XRPD analysis. Fig. 3 shows the XRPD patterns for MDCintercalated ZrP (MDC@ZrP) and a-ZrP. If pure q-ZrP, being the highly hydrated phase of ZrP, is dried, dehydration converts it to aZrP [7]. This means that for dry materials after the intercalation reaction, what will be observed in the XRPD patterns are either (i) only the peaks corresponding to the 7.6 Å phase (a-ZrP) if no intercalation occurs (in this case, if the sample incorporated any intercalant molecule, it is only surface bound) [21], (ii) a peak corresponding to a larger interlayer distance than 7.6 Å with no 7.6 Å peak present if a pure intercalated product is obtained, or (iii) a peak corresponding to an interlayer distance larger than 7.6 Å accompanied by a 7.6 Å peak if the intercalation product is a mixed phase with a-phase present. Therefore, the presence in the XRPD pattern of the resulting product of a diffraction peak at lower diffraction angle than that of the 002 reflection of a-ZrP (corresponding to its 7.6 Å interlayer distance) proves that an expanded intercalated phase has been produced with an interlayer distance larger than 7.6 Å. The diffraction pattern of MDC@ZrP (Fig. 3) shows than an intercalated phase was obtained when water was used as the intercalation reaction solvent. Since the 7.6 Å peak of a-ZrP is absent and instead a diffraction peak is observed at 11.0 Å, the intercalation reaction was successful and produced a pure expanded phase [7]. The Cl ligands of MDC are known to hydrolyze in aqueous solution to produce the monocation [Cp2Mo(H2O)(OH)]þ [12,13,22]. Using the molecular modeling program Spartan P-Chem we calculated the minimum distance that [Cp2Mo(H2O)(OH)]þ can separate the ZrP layers due to the monocation minimum molecular dimension. That minimum distance is 4.38 Å when the Cp rings are perpendicular, and the Cp-Mo-Cp angle is parallel, to the ZrP layers. We have previously reported that another metallocene, ferrocene, also intercalates with the Cp rings perpendicular to the ZrP layers [8]. Adding the thickness of a layer of ZrP (6.6 Å) [6,23,24] we would have expected an interlayer distance of 10.98 Å, which is consistent with the 11.0 Å obtained from the XRPD experimental results. Experimental confirmation of the loss of the chloride ligands of MDC upon intercalation to form the monocation and how the dimensions of the monocation limit the loading level are discussed further below. In the diffraction pattern of MDC@ZrP it can be observed that the first order diffraction peak corresponding to an interlayer distance of 11.0 Å is followed by the second order diffraction peak at 5.5 Å. Furthermore, the diffraction peaks for the (020) and ð312Þ reflections that confirm that the ZrP layers remained intact and a-

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Fig. 3. (A) XRPD patterns of MDC@ZrP and a-ZrP. (B) TEM image of an MDC@ZrP nano-platelet.

like are present in the diffraction pattern of the intercalated product at 33.8 and 34.2 (2q) [5]. In addition, the sharp peaks in the XRPD pattern indicate significant crystallinity of the ZrP nanoparticles intercalated with MDC. The crystallinity of the MDC@ZrP can be described as high, with a second and even a third order diffraction peak visible. Transmission electron microscopy (TEM) images of the MDC@ZrP intercalation particles show that their size is in the range of 75e200 nm in diameter with the characteristic platelet-like hexagonal shape of ZrP nanoparticles (Fig. 3B) [5,21]. Fig. 4 shows the IR spectra of ZrP, MDC, and MDC@ZrP. The IR spectrum of the MDC@ZrP material should be similar to the sum of the spectra of the ZrP material and MDC if MDC or ZrP does not undergo any chemical change after intercalation. In contrast, the appearance of new peaks or large displacements of the existing ones would indicate changes in the structure of the intercalated MDC molecules or the layered material. The IR spectrum of the MDC@ZrP sample (Fig. 4) shows a decrease in intensity of the ZrP lattice water bands (from water molecules held in the crystal lattice) that appear in the 3580e3200 cm1 and 1630e1600 cm1 regions in the unintercalated ZrP IR spectrum [25]. A decreased intensity of the lattice water vibrational bands is characteristic of intercalated materials, resulting here by the displacement of interlayer water by the metallocene derivative upon intercalation [25]. The characteristic peaks of the metallocene Cp rings (expected ca. 3100 cm1 for the nCeH bands in the Cp rings, 1440 cm1 for the nC]C bands, and 1370 cm1 for the nCeC bands) [26] in the spectrum

of the pure MDC compound are also observed in the MDC@ZrP IR spectrum. In addition, the peak at ca. 820 cm1 characteristic of the vibrational band of the CeH bond in the Cp rings [26] is also observed in the MDC@ZrP IR spectrum. The IR spectroscopy results strongly suggest that the intercalation of the metallocene dichloride was successfully achieved without any significant chemical change of the Cp rings in the intercalated molecules. 31 P MAS NMR experiments were also performed to corroborate intercalation (see Fig. 5). The characteristic a-ZrP 31P signal with a chemical shift of 19.0 ppm corresponds to the orthophosphate group of ZrP, where the phosphorus atom is bonded to three Zr atoms through three oxygen atoms and to an eOH group pointing into the interlayer space when no intercalant is present [21]. The two weak intensity peaks at ca. 17.3 and 20.8 ppm, which are assigned to HPO4 groups with different hydrogen bonds to water molecules, correspond to structural defects. Instead, the 31P MAS NMR spectra of MDC@ZrP show two main signals at ca. 21.8 and 14.6 ppm giving evidence for structural modifications related to MDC intercalation in the interlayer. The intense resonance at 21.8 ppm is attributed to HPO4 groups in the chemical environment caused by the dehydration of ZrP due to the displacement of part of the lattice water by the MDC complex [21]. The intercalation of MDC in between the layers of ZrP will disturb the interaction of the H-bond between the protonated oxygen of the phosphate and the water, therefore producing an upfield shift. The peak at 14.6 ppm is attributed to the deshielding of the

Fig. 4. FT-IR spectra of ZrP, MDC, and MDC@ZrP. The rectangles highlight the ZrP lattice water bands that appear in the 3580e3200 cm1 region and the peaks of the metallocene Cp rings that appear at the 3100, 1440, 1370, and 820 cm1 regions.

Fig. 5. 31P CP-MAS NMR spectra of (a) a-ZrP and (b) MDC@ZrP. (c) MAS spectrum of MDC@ZrP.

31

P quantitative

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phosphorus of the phosphate caused by the unprotonated oxygen (i.e., PO4 units), due to an ion exchange with MDC. The line widths of these resonances (~2 ppm) are two times larger than that of aZrP indicating a more pronounced degree of local disorder in the MCD@ZrP structure. The observation of a very weak intensity signal at 19.0 ppm (about 2% of the total intensity in the 31P quantitative MAS spectrum) indicates that only a small amount of an unmodified ZrP phase remains in the sample. Therefore, 31P MAS NMR results corroborate the successful intercalation of MDC in ZrP. As mentioned previously MDC has poor aqueous solubility; nevertheless, the aqueous chemistry of MDC has been well characterized [13]. For MDC the Cp ligands are reported to be very stable, but the hydrolysis of the chloride ligands is very fast, with more than half the chloride atoms being displaced upon dissolution and at pH 7.4 the chloride hydrolysis is too rapid to be measured [13]. Previous potentiometric titrations of aqueous solutions of MDC by Manohari et al. showed two deprotonations with pKa (1) ¼ 5.5 and pKa (2) ¼ 8.5. Therefore, it was concluded that at physiological conditions the monocation ([Cp2Mo(H2O)(OH)]þ) is the predominant species present; we expected that this species would intercalate in the ZrP layers by ion exchange [13,22]. To corroborate this, we performed qualitative elemental analysis by SEM-EDX of the intercalated material. Fig. 6 shows that the EDX spectrum for the MDC@ZrP intercalated product includes the characteristic peaks indicating the presence of Mo, Zr, P, O, and C, but no Cl peak was observed. The absence of the chloride peak in the EDX spectrum is consistent with the hypothesis that the monocation ([Cp2Mo(H2O)(OH)]þ) is the species present in the intercalated material. In order to assess the loading percent of the metallocene dichloride inside the layered ZrP, thermogravimetric analysis (TGA) for ZrP and MDC@ZrP was performed (Fig. 7). The TGA thermogram obtained in air for a-ZrP shows two major weight losses (centered around 110 and 500  C, respectively), with a total loss of ca. 12%. As reported by Mosby et al. these weight losses will lead to the complete dehydration and condensation of the phosphate groups producing zirconium pyrophosphate as the final product [21]. For the MDC@ZrP intercalation product, the weight loss occurs in three main steps, corresponding to a ca. 45% total weight loss significantly higher than ZrP, which clearly confirms the presence of metallocene species intercalated in the layered ZrP. The first weight loss below ca. 150  C (ca. 9%) is assigned to be the loss of the remaining interlayer water. The second weight loss in the 250e500  C range (ca. 15%) may be assigned to the decomposition of the Cp rings of the intercalated metallocene species and condensation of the phosphate groups. The final degradation step

takes place in the 700e900  C range (~16%) and the weight loss is consistent with volatilization of MoO3 formed after 600  C. Volatilization of MoO3 at those temperatures have been previously observed [27], while Braga et al. have reported thermogravimetric analysis data showing that in an inclusion complex of MDC inside b-cyclodextrin all the Mo is volatilized completely well below 1000  C [28]. An EDX spectrum obtained for a sample of MDC@ZrP heated to 1000  C showed no Mo present (data not shown). In addition, the XRPD pattern of the heated sample was identical to that of Zr pyrophosphate (data not shown), consistent with our hypothesis that the final product from the weight loss of intercalated ZrP in the thermogravimetric analysis is zirconium pyrophosphate [21]. From the TGA analysis we calculated a proposed formula for the intercalated material of Zr(H0.68PO4)2([Cp2Mo(H2O)(OH)]þ)0.64$2.20H2O (i.e., 64% loading). The amount attributed to the Cp2Mo(H2O)(OH)]þ species is in the context of molar ratio (0.64 mol per ZrP formula unit) or 32% exchange capacity. We can calculate the theoretical amount of [Cp2Mo(H2O)(OH)]þ per formula unit of ZrP for a sample with full loading of the complex inside ZrP. Using the dimension of [Cp2Mo(H2O)(OH)]þ mentioned above and considering that is a tetrahedral geometry we can calculate a cross-sectional area of 38.42 Å2 if the Cp ligands are perpendicular to the layers. Using this cross-sectional area and the area of a ZrP formula unit (24 Å), a theoretical amount of 0.72 [Cp2Mo(H2O)(OH)]þ per ZrP formula unit is obtained. The experimental amount obtained from the TGA data of [Cp2Mo(H2O)(OH)]þ was of 0.64 ions per ZrP formula unit, which is near to the capacity limit of the ZrP interlayer space. To further estimate the loading percent of MDC in ZrP, the liquid phase of the reaction medium was analyzed by UVevis spectrophotometry monitoring the peak at 231 nm, the absorption lmax for MDC, before and after the intercalation reaction. By difference, the amount of metallocene species intercalated was estimated to be close to 63%, which is in complete agreement with the TGA result (for more details see page S-2). 3.2. Preliminary drug release experiment A drug-delivery agent must be capable of releasing the drug when the agent has reached its intended target. A preliminary controlled release experiment at pH 7.4 was performed in a Phosphate Buffer Solution (PBS) (m ¼ 0.1) to study the release of MDC from ZrP layers (See Fig. S-2). The quantity of the MDC complex released was monitored with UVevis spectrophotometry by monitoring over time the absorption band at 231 nm (for more details see page S-2). At pH ¼ 7.4, a flash release is observed in the first minute and 88% cumulative release occurs after 2 h. The drug release result shows that a pH stimulus can trigger release in-vitro. We are conducting a complete drug release study in-vitro at different pHs and the results of those investigations as well as cell studies will be presented in a future publication. 4. Conclusion

Fig. 6. SEM-EDX spectrum of MDC@ZrP.

The direct intercalation of molybdocene dichloride in ZrP produced a new material with an interlayer distance of 11.0 Å, as determined by XRPD. The extensive characterization of the intercalated material indicates that upon intercalation the Cp ligands of the metallocene are still present, whereas the chloride ligands are lost, owed to hydrolysis in water during the intercalation reaction. The complex resulting from hydrolysis is a positive charged species that intercalates via ion exchange inside the layers of ZrP. The in vitro release of intercalated MDC at pH 7.4 takes place within the first 2 h up to an 88% cumulative release. Further studies are being conducted to completely characterize the conditions for drug

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Fig. 7. TGA thermograms of a-ZrP and MDC@ZrP.

release and the cytotoxicity of this intercalated compound in comparison with the free drug using cell viability assays. The results of these investigations will have a considerable impact on the development of metallocene dichloride delivery carriers that can overcome the disadvantages of solubility, stability, and nonlocalized release that anti-cancer drugs usually present. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

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Acknowledgments We acknowledge that this work was supported in part by Puerto Rico Louis Stokes Alliance for Minority Participation Grant No. HRD0601843, NSF-GK-12 Grant No. 0841338, The Robert A. Welch Foundation Grant A-673, NIH-RISE Program Grant No. 2R25GM061151-13, NIH-SCORE Grant No. S06 GM008103-37, and a Chateaubriand Fellowship (BCM) provided by the ‘Service pour la Science et la Technologie (SST)’ of the French Embassy in Washington, DC (USA). Appendix A. Supplementary data

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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jorganchem.2015.05.031.

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