Porous clay heterostructures: A new inorganic host for 5-fluorouracil encapsulation

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IJP 14923 1–11 International Journal of Pharmaceutics xxx (2015) xxx–xxx

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Porous clay heterostructures: A new inorganic host for 5-fluorouracil encapsulation S.A. Gârea a, * , A.I. Mihai a , A. Ghebaur a , C. Nistor b , A. Sârbu b a University POLITEHNICA of Bucharest, Faculty of Applied Chemistry and Materials Science, Advanced Polymer Materials Group, Gh. Polizu 1-7, Bucharest, Romania b National Institute for Research and Development in Chemistry and Petrochemistry. Bucharest, Romania

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 February 2015 Received in revised form 19 May 2015 Accepted 20 May 2015 Available online xxx

This study proposed a new inorganic host for drug encapsulation. Porous clay heterostructure (PCH), synthesized using modified montmorillonite with dodecylamine, was used as host material and 5fluorouracil (5-FU) as guest drug. Drug encapsulation within PCH in different conditions (soaking time, temperature and pH value) was investigated. Possible interactions of 5-FU with PCH were pointed out using different characterization methods like spectroscopic techniques (FT-IR, UV–vis, XPS), thermogravimetrical and BET analysis. The obtained results suggested that PCH host exhibits a high drug encapsulation efficiency which was influenced by factors like soaking time and pH value. PCH zeta potential value was strongly influenced by pH value. The PCH zeta potential significantly varies at acid pH, while a pH value higher than 7 provides a less variation. UV–vis analysis showed that after 30 min PCH host registered a maximum encapsulation efficiency value (44%) at room temperature using an incubation solution with a pH of 11. The soaking temperature does not substantially affect the loading of drug in PCH host. Thermogravimetrical analysis highlighted that drug encapsulation efficiency of PCH was mainly influenced by pH values. BET results confirmed the PCH synthesis and drug loading capacity. ã2015 Published by Elsevier B.V.

Keywords: Host–guest system Porous clay heterostructures Drug 5-Fluorouracil

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1. Introduction Porous clay heterostructures (PCH) are porous materials characterized by attractive properties of micro- and mesoporous structures (Galarneau et al., 1995; Kooli et al., 2006). PCH can be classified between layered silicates (montmorillonite), pillared clays and mesoporous silica. PCH possess a layered structure (smectites), like montmorillonite, pillars between adjacent layers, like pillared clays, and a high surface area, porosity and tunable pore diameters like mesoporous silica. In comparison with classical montmorillonite, characterized by a lower surface area (40–70 m2/g) and porosity (0.006–0.010 cm3/g), PCH show higher value for these two features (Kooli, 2014). In addition, PCH are characterized by the presence of micropores specific for zeolites and mesopores like mesoporous silica (Pires et al., 2008).

* Corresponding author. E-mail address: [email protected] (S.A. Gârea).

Similar to pillared clays, the PCH are synthesized by a cationic exchange and subsequent intercalation which involves the silica precursors polymerization between layers of clay pretreated with organic cation and neutral amines as co-surfactants (Chmielarz et al., 2009a,b,c; Cecilia et al., 2013; Manova et al., 2010). Like in case of synthetic clays (pillared clays, mesoporous silica), the PCH properties (pores dimension, BET surface area, adsorption capacity) depend on the synthesis conditions (surfactant type, surfactant concentration, co-surfactant type, ratio between starting layered silicate: silica precursor: co-surfactant) (Santos et al., 2010; Zapata et al., 2013; Nunes et al., 2008; Chmielarz et al.,  2009a,b,c; Betega de Paiva et al., 2008; Capková et al., 2006; Hedley et al., 2007; Zhou et al., 2004). The advantageous properties of natural and synthetic clays recommend these materials in a wide range of applications, like heterogeneous catalysts, molecular sieves, adsorbents, decontamination agents and drug delivery systems (Qu et al., 2009; Chmielarz et al., 2009a,b,c; Arellano-Cárdenas et al., 2010; Pires et al., 2008; Ku zniarska-Biernacka et al., 2011; Nguyen-Thanh et al., 2006; Apps et al., 2014).

http://dx.doi.org/10.1016/j.ijpharm.2015.05.053 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Gârea, S.A., et al., Porous clay heterostructures: A new inorganic host for 5-fluorouracil encapsulation. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.053

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The classical pharmaceutical formulations based on antitumor drug used in cancer therapy have many disadvantages like low light stability, low water solubility, modest stability to pH variation, high toxicity (haematological, gastrointestinal and neurological toxicity), fast release of active pharmaceutical ingredients. (Plumb et al., 2012; Specenier et al., 2009). For example, in case of platinum-based anticancer drug (cisplatin), the highest drug concentration after administration was achieved in a very short time period (shorter than 5 min). For these reasons, a new concept of pharmaceutically formulations based on host–guest systems was introduced. Hybrid materials based on natural and synthetic clays were proposed as hosts for drug encapsulation. These host–guest systems were characterized by advantageous features such as high drug encapsulation efficiency, controlled/slow release of active pharmaceutical ingredient, high drug protection against pH variations, etc. (Joshi et al., 2009; Ha and Xanthos, 2011; Kong et al., 2010; Szegedi et al., 2011). The most used natural or synthetic clays as drug delivery vehicles include cationic (montmorillonite, halloysite) and anionic (layered double hydroxide (LDH)) layered silicates, zeolites and mesoporous silica (Datt, 2012; Rimoli et al., 2008; Khodaverdi et al., 2014; Amorim et al., 2012; Vilaça et al., 2013). In the present work, we proposed and investigated a new host– guest system based on PCH (as host) and 5-fluorouracil (a chemotherapeutic drug characterized by high toxicity). Factors, including soaking time, pH value and temperature, which affect the PCH capacity for drug loading, were studied using various characterization methods like BET, FTIR, UV–vis, TGA, XPS and zeta potential measurement.

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2. Experimental

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2.1. Raw materials

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Nanofil 116 (a natural montmorillonite (MMT)), with a cationic exchange capacity (CEC) of 116 mEq/100 g clay, was provided from Southern Clay Products. Hexadecyltrimethylammonium bromide (HDTMA), tetraethyl orthosilicate (TEOS), dodecylamine (DDA) and 5-fluorouracil (5-FU) drug were supplied from Sigma and used as received. The chemical structures of the raw materials are shown in Fig. 1.

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2.2. Synthesis of porous clay heterostructures (PCH)

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PCH host was synthesized using a method described in our previous paper (Gârea et al., 2014). The PCH synthesis involved three main steps: (1) HDTMA intercalation between montmorillonite layers, (2) TEOS polymerization between montmorillonite layers, in the presence of HDTMA as surfactant and DDA as cosurfactant and finally (3) thermal treatment of PCH to remove the organic templates. Following these steps, MMT (10 g) was swelled in deionized water (900 ml) for 1 h at 50
C under mechanically stirring. After the MMT swelling step, a cation exchange reaction occurred due to the presence of HDTMA (6 g). The obtained suspension was maintained for 5 h at 50
C and finally the modified clay was filtered and washed with water. The modified MMT (HDTMA– MMT) was air dried for 24 h and then was treated with a precise amount of DDA and TEOS in the presence of water. For PCH synthesis, an organoclay/amine/TEOS molar ratio of 1:20:120 was used. The PCH precursor was calcined at 650
C, for 6 h.

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83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

Fig. 1. Chemical structure of raw materials.

2.3. The influence of soaking parameters (time, temperature and pH value) on drug encapsulation

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The adsorption experiments were performed at different soaking times, temperature and pH values, in order to reach the optimum parameters for a maximum drug loading into PCH. pH effect on drug loading within PCH was studied by maintaining 0.05 g of PCH with 0.01 g of 5-FU for 1 h, at room temperature, in incubation solutions with different pH value (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The samples were abbreviated as follow: PCH5-FU-pH 2, PCH-5-FU-pH 3, PCH-5-FU-pH 4, PCH-5-FU-pH 5, PCH5-FU-pH 6, PCH-5-FU-pH 7, PCH-5-FU-pH 8, PCH-5-FU-pH 9, PCH5-FU-pH 10 and PCH-5-FU-pH 11. The influence of soaking time was performed by mixing 0.01 g of 5-FU with 0.05 g PCH at pH 9 and 11 for 5, 10, 30, 60, 120, 180, 240 and 360 min. The samples were abbreviated as follow: PCH-5FU-5 min, PCH-5-FU-10 min, PCH-5-FU-30 min, PCH-5-FU-60 min, PCH-5-FU-120 min, PCH-5-FU-180 min, PCH-5-FU-240 min, PCH5-FU-360 min. The same quantities of drug and PCH were used to study the influence of temperature on the drug encapsulation. In this case, the mixtures based PCH and drug dissolved in 10 ml of incubation solution were stirred for 1 h at different temperature (20
C, 40
C and 60
C). The samples were abbreviated as follow: PCH-5-FU20
C, PCH-5-FU-40
C and PCH-5-FU-60
C. The final samples were
centrifuged and freeze dried at -50 C for 3 h.

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2.4. Characterization techniques

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UV–vis spectra were recorded on UV 3600 Shimadzu equipment provided with a quartz cell having a light path of 10 mm. The UV spectra were measured at l = 266 nm.

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FTIR spectra were recorded in the 400–4000 cm1 wavenumbers range, on a Bruker VERTEX 70 spectrometer using 32 scans with a 4 cm1 resolution. Thermogravimetrical analysis (TGA) was done on Q500 TA instrument. The samples were heated up to 900
C using a heating rate of 10
C/min, under constant nitrogen flow rate. X-Ray photoelectron spectroscopy (XPS) spectra were registered on a Thermo Scientific K-Alpha equipment, fully integrated with an aluminium anode monochromatic source. The C1s band, at 284.8 eV, was taken as internal standard to correct possible deviations. X-ray diffraction (XRD) spectra were recorded on Panalytical X’Pert Pro MPD equipment, with a Cu-Ka radiation. Transmission electronic microscopy (TEM) was performed on a TECNAI F30 G2 HRTEM equipment. For TEM investigation the powder samples were embedded in epoxy resin and cut with a microtome (Leica EM UC6) into ultrathin sections. Zeta potential measurements of PCH suspensions were registered by a light scattering technique using a Malvern Zeta Sizer. Each zeta potential measurement was repeated 3 times to determine reproducibility.

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Textural parameters of starting clay (MMT) and PCH samples were determined by nitrogen adsorption–desorption isotherms at 196
C, using a NOVA 2200e Automated Gas Sorption instrument. Samples were pre-treated (degassed) under vacuum at 40
C, for 5 h. The surface areas of MMT and PCH samples were calculated by BET method, while the total pore volume was measured in the highest point of the isotherm (at p/po = 0.99). The pore-size distributions were determined from the desorption branch of the isotherms with BJH model.

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3. Results and discussion

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3.1. PCH host characterization

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In the first step, the inorganic host (PCH) was characterized using the following methods: XRD, TEM, FTIR and TGA.

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3.1.1. XRD and TEM characterization Valuable informations about PCH structure and morphology were obtained using XRD and TEM analyses (Fig. 2).

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Fig. 2. X-ray diffractograms and TEM image of PCH.

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Fig. 2 shows X-ray diffractograms recorded for MMT and PCH. As one may notice, the starting clay (Nanofil 116) sample is mainly constituted by montmorillonite and small amounts of feldspar and quartz (Siebel et al., 2010). For PCH, the diffraction peak (0 0 1), assigned to the ordering of the clay layers, was detected at lower values of 2u (6.9
), this fact suggested an increase of basal spacing (d0 0 1) with 1.9 nm than for MMT. This increase was attributed to pillaring process (Chmielarz et al., 2011; Cecilia et al., 2015). In addition, the intensity of this basal reflection was very low, and therefore we may concluded that the structure of this material was mainly exfoliated (delaminated). The two-dimensional (non-basal) hk diffraction peaks, characteristic (0 2 0, 11 0, 2 0 0, 0 6 0) for layered clay, were detected in the PCH diffractogram, but also a significant decrease of its intensity was recorded, and therefore a structural change due to the formation of pillars in the interlayer space of MMT was confirmed (Cecilia et al., 2015). The broad band in the 15–32
range is assigned to amorphous silica that constitutes pore walls (Pinto et al., 2014). This type of disordered layered structure was reported by Chmielarz for Ti-PCH. The XRD result was confirmed by TEM analysis. TEM image suggested that PCH sample exhibits an exfoliated structure, in which the structure of individual tactoids are affected due to the pillaring process. Only some few stacked layers tactoids with parallel orientation were observed. These could be responsible for the small shoulder diffraction peak (0 0 1) which appeared in XRD analysis. Similar results, regarding the exfoliated PCH structure, were reported by Pálková et al. for Laponite derived porous clay heterostructures (Pálková et al., 2009). 3.1.2. FTIR characterization The FTIR analysis confirmed the PCH structure formation by the presence of several characteristic peaks of amorphous silica (Fig. 3). The peaks of PCH at 3738 cm1 (nSi–OH), 3441 cm1 (OH stretching vibrations of water molecules adsorbed on PCH), 1631 cm1 (bending vibration of adsorbed water molecules), 1083 cm1 (vibrations of three dimensional silica network), 807 cm1 (symmetric stretching of the SiO Si or Si OAl),

Fig. 4. TGA curves of 1-MMT and 2-PCH.

575 cm1 (Al O bending vibrations) and 458 cm1 (Si O bending vibrations) were observed (Pálková et al., 2009; Madejova et al., 2009; Zhao et al., 1997; Cecilia et al., 2013).

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3.1.3. TGA tests Fig. 4 showed thermogravimetrical curves of starting clay (MMT) and PCH sample. In case of montmorillonite sample (Nanofil 116), thermogravimetrical curve highlighted two main weight loss as follows: a weight loss, at low temperature, attributed to the water adsorbed at the MMT external surface and a weight loss related to the dehydroxylation process of MMT, detected between 400 and 700
C. Similar thermogravimetric profile was reported by Cecilia et al., for starting clay used for Zr-PCH sample synthesis (Cecilia et al., 2013). Regarding of PCH sample, TG test highlighted a small weight loss, at lower temperature, assigned to the loss of physisorbed water at the external PCH surface. Moreover, between 300 and 800
C temperature range, TG curve of PCH showed a slow and continuous weight loss assigned to the dehydroxylation process. Similar TG curves for PCH was reported by Cecilia et al. (2013). In case of PCH, dehydroxylation process occurred in two steps, a first

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Fig. 3. FTIR spectrum of PCH.

Please cite this article in press as: Gârea, S.A., et al., Porous clay heterostructures: A new inorganic host for 5-fluorouracil encapsulation. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.05.053

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pH value

Zeta potential (mV)

2 3 4 5 6 7 8 9 10 11

+3 3 23 29 39 39 43 44 44 44




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step (300–500 C) related to loss of hydroxyl groups located in the pillars and second step situated between 500 and 800
C related to the dehydroxylation of hydroxyl groups located between the tetrahedral sheets of MMT.

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3.2. PCH host for drug encapsulation

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The influence of pH value, temperature and soaking time on drug–PCH interactions was followed using different characterization methods (FT-IR, TGA, UV–vis, XPS and BET analyses). 3.2.1. The influence of pH value on 5-fluorouracil encapsulation within PCH 3.2.1.1. Zeta potential measurements. The first step, in this study, was to determine the zeta potential of PCH dispersions characterized by different pH values. Zeta potential measurement offers valuable informations about clay dispersion stability and also about adsorption of organic cations and polycations on clay (montmorillonite) (Zadaka et al., 2010). In our case, zeta potential results were useful to guide the choice of pH value for the drug encapsulation within PCH. The change of PCH zeta potential as a function of pH value was monitored. The measured zeta potentials of PCH dispersed in different pH solutions are summarized in Table 1. The zeta potential of PCH was shifted from +3 mV (at pH 2) to 44 mV at a basic pH > 9. A significant variation of PCH zeta

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potential was observed in the 2–6 pH range, followed by a stabilization registered at pH value above 8. The high value of negative zeta potential (44 mV), in the basic pH value, implies that the PCH is well dispersed and stable against aggregation in the aqueous solution. The zeta pontential results showed that the PCH particles are stabilized in aqueous solution by adjusting the pH value to a high value (above 9). In this range the PCH particles exhibit a high enough surface charge. The pH value exhibits a significant influence on both PCH surface charge and drug solubility. Regarding the 5-FU solubility in water, it is known that this drug type is a weak acid with pKa about 7.6 and therefore a basic medium favors a high solubility of drug in the aqueous solution (Li et al., 2008; Faisant et al., 2006). Considering this aspect, we may concluded that a high pH value (pH > 9) of dispersing medium favored drug solubility and PCH dispersion stability.

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3.2.1.2. FTIR characterization. The influence of pH value on PCH drug encapsulation capacity was also pointed out using FTIR Spectroscopy (Fig. 5). The appearance of new peaks at 1689–1701 cm1 (assigned to C¼C stretching vibrations) was a confirmation of drug presence within PCH's surface or/and pores. In addition, the FTIR analysis highlighted a dependence of encapsulated drug concentration on the pH value of incubation solution. The peak assigned to C¼C stretching vibrations showed a higher intensity in case of PCH-5FU systems synthesized at high pH value (pH > 8). The peaks assigned to C H asymmetric and symmetric stretching vibrations (2970, 2921 cm1) were detected for host–guest system synthesized at pH of 11. FTIR results are in agreement with Zeta potential measurements, regarding the effect of high pH value on drug entrapment within PCH, due to the PCH surface activation and enhancement of drug solubility.

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3.2.1.3. UV–vis analysis. The UV–vis data confirmed the FTIR results regarding the influence of pH value on the drug encapsulation efficiency (EE) of PCH (Fig. 6).

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Fig. 5. FTIR spectra of 1-drug (5-FU), 2-PCH host and PCH-5-FU host–guest systems synthesized at different pH values: 3-pH 2, 4-pH 5, 5-pH 7, 6-pH 11.

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Fig. 6. Drug encapsulation efficiency against pH value.

Fig. 7. TGA curves of 1-PCH and PCH-5FU host–guest systems synthesized at different pH values: 2-pH 2; 3-pH 7; 4-pH 9; 5-pH 11. 286 287 288 289 290

The highest drug EE (44%) was recorded at pH 11. This behavior may be assigned to the synergetic effect of PCH surface activation and higher 5-FU solubility in the basic medium. 3.2.1.4. Thermogravimetrical analysis. TGA tests confirmed the presence of drug molecules within inorganic host and also the

existence of a drug encapsulation efficiency dependence against pH value (Fig. 7). As one may observe, all the host–guest systems exhibit a higher weight loss than PCH, which was assigned to the degradation of drug loaded within inorganic host. The highest weight loss (10%) was recorded for host–guest system synthesized at pH 11. From TGA results we can conclude that a high pH value favored an increase of drug concentration loaded within PCH.

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3.2.1.5. Textural parameters characterization. The pH influence on drug entrapment within PCH was also pointed out using BET analysis, textural parameters being determined at two pH values (9 and 11). Textural properties (specific surface area, pore volume, pore diameter and pore-size distribution) of the pristine clay (MMT), modified inorganic host (porous clay heterostructure – PCH) and 5FU loaded PCH samples were determined to prove the conversion of MMT to PCH and also to understand the drug adsorption process within PCH. Nitrogen adsorption–desorption isotherms and pores size distribution of above mentioned samples are shown in Fig. 8, while Table 2 summarizes the calculated textural parameters. As a first observation, BET analysis results confirmed the conversion of MMT to PCH. The starting clay (MMT) exhibits a nitrogen adsorption–desorption isotherm that is classified as type II, with the hysteresis loop corresponding to H3 type. This type of isotherm was related to aggregates of plate-like particles which have no ordered pore structure, indicating the presence of house of cards structure (Cecilia, et al., 2013; Naumov, 2009; ArellanoCardenas et al., 2008). Similar shape for MMT was reported by He et al. (2008). In comparison with the starting clay (MMT), PCH exhibit higher value of SBET (734 m2/g) and a substantially increase of Vt (0.71 cm3/ g). These changes attest the successful formation of PCH (Chmielarz et al., 2014). In case of PCH and 5-FU loaded PCH samples, the BET results highlighted a gradual increase in nitrogen sorption in the entire range of relative pressure values. This change suggested the formation of micropores and small mesopores at low relative pressures and larger mesopores and small macropores at higher to medium relative pressure (Cecilia et al., 2013). The shape of nitrogen adsorption-desorption isotherms of PCH and 5-FU loaded PCH samples correspond to type II isotherm, with a H4 hysteresis loop. After PCH treatment with 5-FU, a significant decrease of textural parameters values (Vp, Dp and SBET) was recorded. These

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Fig. 8. N2 adsorption–desorption isotherms (a) and pore-size distributions (b) of 1-MMT, 2-PCH-5-FU-pH 9, 3-PCH-5-FU-pH 11, 4-PCH.

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Table 2 Textural data for MMT, PCH and 5-FU loaded PCH samples synthesized at different pH values. Samples

a

MMT PCH PCH-5FU-pH 9 PCH-5FU-pH 11

80 734 334 279

a b c

335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361

SBET (m2/g)

b

Dp (nm)

3.86 3.82 3.65 3.46

c

Vt (cm3/g)

0.10 0.71 0.54 0. 40

SBET: specific surface area. Dp: pore diameter. Vt: total pore volume.

change confirmed that 5-FU was adsorbed not only on PCH external surface, but mainly inside the pores and therefore a decrease of available space for nitrogen adsorption occurred. Similar behavior was reported by other authors for modified inorganic hosts (mesoporous silica (MCM-41)) with drug (ibuprofen) or grafting agents (silanization agents): 3-aminopropyltriethoxysilane and 3-propanonitrile triethoxysilane (Arean et al., 2013). The 5-FU loaded PCH samples exhibit narrower pore sizes distribution and smaller pore diameters comparing with the initial support (PCH). This comes to validate our previous statement that the small mesopores of PCH are obstructed by the drug molecules entrapped inside. The BET results are in agreement with previous analyses regarding the drug adsorption dependence on soaking conditions. The PCH-5FU-pH 11 sample exhibits the highest decrease of SBET and Vp values, this fact confirmed that drug adsorption on both pore and PCH surface was favored by a high pH value. The BET results recommend the PCH as a new possible host for adsorption of various drugs. 3.2.2. The influence of soaking temperature on the 5-FU loading within PCH Once, it has been determined the optimum pH value for incubation solution, we performed a study regarding the influence of PCH’s soaking temperature on drug EE. The influence of this parameter was studied using FTIR, UV–vis and XPS characterization methods.

Fig. 10. TGA curves for 1-PCH and different host–guest systems synthesized at various temperatures: 2–40
C; 3–60
C.

3.2.2.1. FTIR analysis. The FTIR analysis provided only qualitative informations regarding the dependence of drug loading against soaking temperature. As one may observe from FTIR spectra, the temperature value does not substantially affect the drug encapsulation efficiency of PCH (Fig. 9). Regardless of temperature value, in all cases, the peak assigned to C¼C stretching vibrations (1681/1686/1688 cm1), from drug structure, was detected. The intensity of this peak was maintained constant in all cases, this result being a qualitative proof that drug encapsulation efficiency does not depend on temperature of incubation solution. Generally, the loading of drugs poorly soluble in water is strongly influenced by temperature. The temperature can enhance the drug solubility and therefore the ionization of drug increases (Wilson and Crowley, 2011). In our case, 5-FU is a relatively easily ionized drug and therefore it can be explained the slight influence of temperature on drug encapsulation.

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3.2.2.2. TGA tests. Thermogravimetrical results were in agreement with the FTIR analysis conclusions. Fig. 10 shows an example of thermogravimetrical curves for PCH and two host-guest systems obtained at different temperatures of incubation solution.

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Fig. 9. FT-IR spectra of 1-drug, 2-PCH and PCH-5-FU at different soaking temperature: 3–20
C, 4–60
C, 5–40
C.

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Fig. 11. Dependence of drug EE against temperature for different host–guest systems synthesized at various pH value.

TGA tests suggested that the host-guest samples exhibit similar weight loss values (7.3%) which proved that an equal amount of drug retained within PCH samples was subjected to degradation process.

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3.2.2.3. UV–vis analysis. UV–vis analysis results confirmed the conclusions of FTIR and TGA analyses regarding the influence of temperature on drug EE (Fig. 11). This quantitative analysis highlighted a low variation of drug EE against temperature regardless the pH value.

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3.2.2.4. XPS analysis. XPS analysis was used as a qualitative method to point out the presence of 5-FU on PCH surface or inside the micro- and mesoporous framework. XPS analysis confirmed the surface chemical composition of PCH precursor (before thermal treatment) and PCH (after thermal treatment) (Fig. 12a). The XPS spectrum of PCH precursor shows four main signals assigned to the Si2p, O1s, C1s and N1s. The presence of organic fraction (HDTMA or/and DDA) on PCH precursor surface was highlighted by C1s and N1s signals. After calcination step, the XPS analysis highlighted some changes on PCH surface composition. Therefore, the surface

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Fig. 12. XPS spectra of (a) 1-PCH precursor, 2-PCH after calcination, (b) host–guest system synthesized at different temperature: 1–20
C, 2–40
C; 3–60
C and (c) C1s deconvolution of the PCH precursor and PCH after calcination.

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Table 3 Surface elemental composition from XPS analysis. Sample

O1s (atomic %)

C1s (atomic %)

N1s (atomic %)

Si2p (atomic %)

PCH precursor (before calcination) PCH after calcination PCH-5-FU-20
C PCH-5-FU-40
C PCH-5-FU-60
C

46.9 66.8 63.3 63.2 63.1

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2.4 – – – –

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Fig. 13. FTIR spectra of 1-drug, 2-PCH and PCH-5-FU-pH 11 at different soaking times: 3–5 min, 4–10 min, 5–60 min, 6–180 min, 7–300 min. 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430

chemical composition of PCH was confirmed by the presence of two main signals attributed to Si 2p and O1s. The residual C1s signal from XPS spectrum of PCH was assigned to carbon dioxide physically adsorbed on mesoporous surface. The C1s spectra of PCH precursor and PCH after calcination were deconvoluted using Gausian–Lorentzian function, centered at 284.8 eV, corresponding to the C C binding energy (Fig. 12c). The deconvolution of C1s spectrum of PCH precursor presents a peak at 285.9 eV assigned to C N+ species. This peak disappear after calcinations, but a new peak attributed to C O species (286.5 eV), that indicate an oxidation reaction, was observed. This type of reaction has been achieved during the calcination step because it was not made under an inert atmosphere. The presence of physically adsorbed CO2 was also highlighted by Chmielarz et al. using thermogravimetrical tests (Chmielarz et al., 2009a,b,c). Also, Wang et al. assigned the C1s weak signal to the CO2 adsorbed on the nanosilica surface (Wang et al., 2008). After the 5-FU loaded within PCH, some changes in the surface atomic percentages of different elements were observed (Fig. 12b, Table 3). The 5-FU loaded PCH samples exhibit an increase of C1s surface atomic percentage due to the drug presence. The signals, attributed for N1s and F1s from drug, was not detected in XPS spectra, probably due to the low percentage which was below to the detection limit of the equipment. The XPS results suggested that the temperature effect on drug loaded within PCH is not significant. In all XPS spectra, the C1s percentage values were similar, regardless temperature value. The PCH surface activation is not affected by temperature variation. The

pH value remains the main factor that influence the activation of PCH surface.

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3.2.3. The influence of soaking time on the drug encapsulation within PCH host The influence of soaking time parameter was studied using a qualitative (FTIR) and quantitative (UV–vis) characterization methods. Experimental tests were done at two pH’s values (pH of 9 and 11).

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3.2.3.1. FTIR analysis. The FTIR spectra of host–guest systems synthesized at different soaking times were registered to confirm the presence of drug within PCH (Fig. 13). As one may observe the drug loaded within PCH reached an equilibrium after 180 min. From this point, we can consider that drug concentration gradient was equal between inside and outside of the inorganic host. The intensity of peak assigned to C¼C stretching vibrations from 5-FU structure remained constant after 180 min. Similar behavior was detected for montmorillonite used as host for 5-FU encapsulation (Lin et al., 2002).

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3.2.3.2. UV–vis analysis. A quantitative evaluation of soaking time influence was obtained by UV–vis characterization (Fig. 14). Drug encapsulation efficiency (EE) of PCH was calculated using the results provided by UV–vis spectra. As one may observe the highest drug EE (44%) was reached after 30 min for a pH of 11. After this time, the drug EE recorded a slight decrease (for 60 and

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Fig. 14. Drug encapsulation efficiency (EE) of PCH against soaking time at different pH value.

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120 min), the equilibrium being reached after 180 min. A decrease of incubation solution pH value leads to lower drug EE. For example, at a pH value of 9, the drug EE reached a maximum value of 23% after 90 min. This variation of drug EE by changing pH value of incubation solution could be occurred due to the different PCH surface charging or increasing of 5-FU solubility. Zeta potential measurements indicated that the value of PCH at pH 9 is equal with those at pH 11 (44 mV) and therefore the 5-FU solubility remains the main factor which influence the EE at different pH values.

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4. Conclusions

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New host–guest systems based on PCH and 5-FU were successfully synthesized. All the results of this study suggested that PCH can be considered a possible host candidate for 5fluorouracil encapsulation. Drug loading capacity of PCH is strongly influenced by the pH value of incubation solution and soaking time. All the results confirmed that the optimal parameters for achieving a maximum drug encapsulation efficiency (EE = 44%) involve drug–PCH interactions at 20
C, 30 min, using an incubation solution characterized by a pH value of 11. These parameters influenced the synergistic effect of PCH surface activation and drug solubility (ionization). BET results confirmed that the drug adsorption on pores and PCH surface was favored by a high pH value.

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5. Uncited references

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Magniez (2015), Szegedi et al. (2012), Tunc and Duman (2010), Volzone et al. (2002) and Zeng et al. (2006).

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Acknowledgements

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Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI) and National Research Council (CNCS) are gratefully acknowledged for the financial support for the PN II research project: DRUG DELIVERY HYBRIDS BASED ON POLYMERS AND POROUS CLAY HETEROSTRUCTURES (DELPOCLAY), No. 154/2012. Authors are thankful to Dr. E. Vasile for TEM and XRD analysis.

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