4052 New J. Chem., 2013, 37, 4052--4060 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

Cite this: NewJ.Chem.,2013,37, 4052

Sepiolite based materials for storage and slow releaseof nitric oxide†

Ana C. Fernandes, Fernando Antunes and Joao Pires*

Nitric oxide (NO) is a small endogenous molecule that has important regulatory roles both in

physiology and in pathology. Because of this multiplicity of roles, the level of NO concentration or,

more precisely, the control of its concentration, is crucial to avoid adverse effects including toxicity.

Nanoporous materials have emerged as potential carriers for NO delivery. In this work nanoporous

materials were prepared from clay based solids, having as main goal the study of their potentialities in

the field of storage and release of nitric oxide for therapeutic applications. Prepared materials were

pillared interlayered clays (PILCs), obtained from natural sepiolite clay. Materials were characterized by

X-ray diffraction and nitrogen adsorption at �196 1C. The kinetic data for the nitric oxide storage and

release, in selected materials, were obtained in gas and liquid phases. Toxicological assays with HeLa

cells have also been done to determine material cytotoxicity. Some of the materials prepared in this

work are able to store an amount of NO with biological significance, show slow release kinetics and

have low cytotoxicity.

1. Introduction

Solid materials have shown great potential in recent years inbiological, medical and pharmaceutical applications, namely indrug delivery.1,2 Advanced nanoparticle drug delivery systemshave important advantages over conventional dosage forms.Conventional drug delivery forms are oral, topical, inhaled orinjections. These delivery systems usually result in a drugconcentration profile that initially peaks above the therapeuticrange. Then, there is a relatively fast decrease in the concentrationuntil it falls below the therapeutic range.1,3–5 Controlled drugdelivery systems have, therefore, attracted wide attention inbiomedical research due to their potential to reduce the sideeffects of therapeutics, while making it possible to control theconcentration and location of active drugs released over longperiods of time.1,5,6

Currently, the research on nanoparticle drug delivery systemsfocuses on: (1) the selectiveness and combination of carriermaterials to obtain suitable drug release speed; (2) the surfacemodification of nanoparticles to improve their targeting ability;and (3) the optimization of the preparation of nanoparticles, toincrease their drug delivery capability.6

Nitric oxide (NO) is one of the smallest endogenous mole-cules but, nevertheless, has an important regulatory role in thebody, despite its toxicological potential. It tends to concentratein lipophilic environments, such as membranes and hydro-phobic domains of proteins.7,8 NO is involved in a number ofkey physiological functions such as blood pressure regulation,immune control against pathogens, neurotransmission, inhibi-tion of platelet adhesion, wound healing and nonspecificimmune response to infection.8,9 Because of this multiplicityof effects, the level of NO concentration or, more precisely, thecontrol of its concentration, is crucial in order not to have anyadverse effects or even toxicity. Many metallo-proteins can reactwith NO and exposure to high concentrations of NO may resultin inhibition of their functions. A significant proportion of theNO therapy market will therefore necessarily involve targeteddelivery of NO to specific areas of the body, which will avoidsystemic effects.

Solid carriers are of potential biomedical interest in thedelivery of exogenous NO for anti-bacterial, anti-thrombic andwound healing applications. Delivery of NO is challengingbecause NO is a gas at normal temperature and pressure unlikeregular drug molecules that are usually in the solid or liquidstate. Homogeneous donors that deliver NO directly fromsolution exist but this approach is limited by the systemicnature of delivery, which can cause unwanted side effects.Alternatively, some organic or inorganic materials such aspolymers, nanoparticle metal–organic frameworks (MOFs)and zeolites have been explored as NO delivery platforms.7,10–13

CQB and Department of Chemistry and Biochemistry, Faculty of Sciences,

Building C8, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal.

E-mail: jpsilva@fc.ul.pt; Fax: +351 217500088; Tel: +351 217500903

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nj00452j

Received (in Montpellier, France)30th April 2013,Accepted 10th September 2013

DOI: 10.1039/c3nj00452j

www.rsc.org/njc

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Biocompatibility is a fundamental requirement for a material tobe used in drug delivery.14 Clays and clay minerals play animportant role in the field of health products. In fact, they arefundamental components in several medical products, being usedas excipients and fulfilling some technological functions. The factthat clays are already in use in several medical products for a longtime may be advantageous for their use in other related fields suchas the NO delivery. In addition, clay minerals may be effectivelyused in the development of new drug delivery systems.14–16

Clay minerals are fine-grained silicates with either lamellaror fibrous morphology. Among these, smectites, such asmontmorillonite and saponite have been more commonly studiedbecause of their higher cation exchange capacity compared tothe pharmaceutical silicates.14 However their adsorption capacity islimited.

Sepiolite belongs to the phyllosilicate group of clay mineralswith a 2 : 1 ribbon structure, having an ideal unit cell formula ofSi12O30Mg8(OH)4(OH2)4�8H2O.17–20 The general structure ofsepiolite is formed by alternation of blocks and tunnels. Theblocks are composed of two tetrahedral silica sheets and acentral octahedral sheet containing magnesium. The periodicinversion of the tetrahedral silica sheets gives rise to thestructural channels.17–19,21 These channels have dimensionsof 1.1 � 0.4 nm that are accessible to small molecules.22 Thedimensions of the sepiolite crystals vary between 0.2 and 4 mmin length, 10 and 30 nm in width and 5 and 10 nm inthickness.18,20,21 Sepiolite can have high specific surface areavalues up to 200–500 m2 g�1 not only as a result of the smallsize of its particles, but also because of its morphology andintra-crystalline tunnels.18,20 Sepiolite has been commonly usedin several technological and industrial applications. Many ofthese applications are based on the good adsorptive, rheologicaland catalytic properties.17,18 Although it is not generally regardedas an expandable mineral, such as montmorillonite, there arestudies that report the possibility of expanding its basal spacingby intercalation with other species.20

Pillared interlayered clays (PILCs) are obtained by exchangingthe interlayered cations of layered clays with bulky inorganicspecies. The intercalated cations increase the basal space of theclays, and after heating, they are converted into the metal oxideclusters by dehydration and dehydroxylation.23 Materialsobtained by this method have a permanent porosity consistingmainly of micropores (pores having a width less than 20 Å),although materials can also be obtained with mesoporosity(width between 20 and 500 Å).24 The most studied intercalatingspecies, which by calcination form the pillars, are oligomericaluminium cations.23 In addition to the Al species, oligomericspecies based on titanium and cobalt and zinc ion compoundshave also been studied. After pillaring, the possibility exists thatCo and Ti intercalated species in the material can developspecific interactions with the NO molecule, and change itsadsorption–desorption behaviour. Both titanium and cobalthave low toxicity and are therefore compatible with biologicalorganisms.25,26

Here we address NO storage and release by employing, to ourknowledge for the first time, solid carriers based on modified

sepiolite clay. We show that not only these materials are able tostore amounts of NO with biological significance but also thatthey present slow release kinetics and low cytotoxicity.

2. Experimental2.1 Materials

As raw material, a natural clay of sepiolite type, from TolsaGroup, Spain, was used. This clay has the following characteristics:specific area 141 m2 g�1, total porous volume = 0.398 cm3 g�1; basaldistance = 12.19 Å. To obtain aluminium pillared clay, a pillaringoligocation of aluminium ions was prepared from 0.2 M of AlCl3�6H2O (Fluka, >98%) solution and 0.5 M of NaOH (BDH ChemicalsLtd, 98%) with an OH/Al ratio of 2 in a procedure previouslyoptimized.27 Sodium hydroxide was added to the aluminiumchloride solution, which was maintained under stirring at 60 1C.The oligocation was then added to a suspension of 5% clay indeionized water (20 meq of aluminium per gram of clay). Thesample was washed in a dialysis membrane, dried and calcinated at400 1C for 2 h. The resulting material was denoted AlS-P.

Metal ion-exchanged sepiolite (with cobalt and zinc ions)was also prepared. Typically, the clay (5 g) was placed in a0.05 M solution of metal acetate (400 mL, distilled water),(C2H3O2)Co�4H2O and (C4H6O4)Zn�2H2O (both reagent gradefrom Sigma-Aldrich), and stirred for 24 h.28 The materials wererecovered by centrifugation, washed with distilled water severaltimes and dried at 100 1C overnight. The sepiolite ionexchanged materials with cobalt and zinc were designated asCoS and ZnS, respectively.

Titanium pillared materials were prepared, from CoS andZnS, using Ti(OEt)4 (Aldrich, p.a.) as a starting material with thefinal materials labeled as TiCoS-P and TiZnS-P, respectively. Toprepare these materials, Ti(OEt)4 was added to a 5 M HClsolution (H+/Ti4+ = 2.5). The pillaring oligocation was thenincorporated in a suspension of 5% of the exchanged materialclay (CoS and ZnS) in deionized water. Both samples werecalcinated for 2 h at 400 1C, this temperature being selectedaccording to literature information for the decomposition ofcobalt and zinc acetates as well as for Ti-PILCs formation.29–31

2.2 Characterization methods

High angle powder X-ray diffraction patterns were recordedwith a Philips PW 1730 diffractometer with automatic dataacquisition (APD Phillips (v3.6B) software using a Cu anode(l = 1.5406 Å)). The low angle powder X-ray diffraction patternswere recorded with a Philips Analytical PW 3050/60 X’Pert PROequipped with an X’Celerator detector and with automatic dataacquisition (X’Pert Data Collector v2.0b software) using amonochromatized CuKa radiation (l = 1.5406 Å). Nitrogenadsorption–desorption isotherms were recorded at �196 1Cusing a volumetric automatic apparatus (Micromeritics, ASAP2010). Prior to the measurements the samples were outgassedat 300 1C for 2 h. Specific surface area measured was based onBET formalism and micropore volume was evaluated usingthe t-method.32 Thermogravimetry with differential scanningcalorimetry (TG/DSC) experiments were carried out in an apparatus

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from Setaram (TG-DSC 111), with 0.001 mg and 0.05 mW ofprecision, between 25 and 600 1C, under dry air flux. Thechemical analysis for titanium, cobalt and zinc content wasmade by ICP ‘‘Laboratory of Analysis of IST’’.

2.3 Nitric oxide adsorption and release

Studies of adsorption of NO were made in gaseous phase anddesorption (that is, release) studies were made in both gas andliquid phases. The adsorption–desorption profiles in the gasphase were determined using a microbalance (C.I. Electronics,Disbal) suited for vacuum connected to a high vacuum pumpsystem composed of a turbomolecular pump and a diaphragmpump (Pfeiffer Vacuum). Samples were outgassed in vacuumbetter than 10�2 Pa, for 2 h, at 250 1C. The sample was thenthermostatized at 25 1C and NO was then introduced in themicrobalance until a pressure of 80 kPa. The microbalance wasconnected to a computer and the weight increase was recordedat fixed time intervals of 72 h. After this time, the desorption ofNO started, by creating vacuum in the cell, and the weightdecrease was then recorded for 48 h or until it reached aconstant value.

Release studies in the liquid phase were performed with anoxyhemoglobin assay as described in detail in the literature.33

To avoid the problem of dispersion of the sample in the liquidphase, samples were ground with poly(tetrafluoroethylene)(PTFE, 25 m particle size powder, from Aldrich) (75 sample :25 PTFE wt%).28 The mixture was then pressed into disks(5 mm diameter) under 8 tons for 30 s. Then, 6 mg of the diskswere introduced in small glass in a glass container with a PTFEvacuum valve. The container was connected to a vacuum line,and samples were outgassed for 2 h at 250 1C. After the materialreturned to room temperature, NO was introduced, the valvewas closed, and left equilibrating for 3 days. After evacuationfor 1 minute to remove the NO in gas phase, the container wasfilled with helium until the atmospheric pressure was reached.For the NO release, the samples were added to 3 mL ofoxyhemoglobin solution (1 mM) in a quartz container, whichwas vigorously shaken. Briefly, oxyhemoglobin solution wasprepared by dissolving 20 mg of lyophilized human hemoglobinin 1 mL of 0.1 M phosphate buffer (pH = 7.4). Purification anddesalting were performed by passing the resulting oxyhemoglobinsolution over a column of Sephadex G-25.33 The kinetic experi-ments were performed in a 0.1 M phosphate buffer (pH = 7.4) atambient temperature and spectra were taken at 10 minuteintervals for 2 h, using a UV-Vis spectrophotometer (Genesys10S UV-Vis Spectrophotometer from Thermo Scientific). Initialand final spectra of some samples can be found in the ESI†(Fig. S1). Blank experiments were also conducted on the sampleswithout NO, following the same procedure.

2.4 Cell culture

HeLa cells were cultured in complete medium (RPMI-1640medium supplemented with 25 mM of Hepes and L-glutamine,and antibiotics from Life Technologies). Cells were incubatedat 37 1C in humidified air with 5% carbon dioxide and kept inthe logarithmic phase by routine passage every 2 to 3 days.

Before the day of the experiment cells were seeded at a densityof 7000 or 2500 cells per well for the 24 or 72 hours experiments,respectively. On the day of the experiment, cells were washedand fresh medium was added 1 hour before the experiment.

2.5 Toxicological assays

The compounds to be tested, at a final 450 mg mL�1 concentration,were added to 96-well plates containing the cell culture (10 mLsample/100 mL cell culture media). Assays were performed after 24 hand 72 h, 10 mL alamarBlues was added directly to each well, theplates were incubated at 37 1C for 4 h and the fluorescence signalwas measured on a Spectra Max Gemini EM from MolecularDevices, with SoftMax Pro software. Cell viability was calculatedusing the following equation:

Cell viability = (Fsample/Fcontrol)

where Fsample is the fluorescence of the cells incubated withsamples and Fcontrol is the absorbance of the cells incubatedwithout the sample. All the results were statistically treated, byANOVA tests. Data shown are the average � standard deviationfor 24 measurements. For a selected material (sepiolite) aconcentration study was made. In this case, the concentrationof the material varied between 100 and 1800 mg mL�1. Survivalwas evaluated, after 72 h, as described above. In preliminaryexperiments, it was observed that our test compounds did notaffect alamarBlues fluorescence.

3. Results and discussion3.1 Characterization of the samples

For the ion exchanged materials both Co and Zn contents werenear 2 wt% in each sample. After the introduction of titaniumpillars, the content in Co and Zn decreased for 1 and 0.1 wt%respectively. The titanium content introduced was 8 wt% inTiCoS-P and 7 wt% in TiZnS-P. The TG-DSC curves for theinitial sepiolite and for the uncalcined aluminium pillaredmaterial (AlS-P), Co and Zn exchanged materials (CoS andZnS) and titanium pillared materials from CoS and ZnS(TiCoS-P and TiZnS-O) are depicted in Fig. 1. When the sepioliteis heated, it undergoes structural changes due to the dehydrationprocess which takes place during the heating. The loss of wateroccurs in several stages: the loss of surface-adsorbed and zeoliticwater, that is, the water bound to the exchange cations; the loss ofthe first molecules of water coordinate to the octahedral sheet;and loss of the rest of the coordinated water, and dehydroxylationof the tetrahedral sheet.34–36 The main weight loss occurredbetween 50–125 1C for all samples, 5–17 wt% for sepiolite,AlS-P and TiCoS-P, 23 wt% for TiZnS-P, attributable to the lossof physically adsorbed water. In the case of the Co and Znexchanged materials, this weight loss was lower (near 8 wt%).For sepiolite, a second stage of mass loss, 3 wt%, occurredbetween 250–300 1C, which can be assigned to the removal ofthe zeolitic water on the structural pores and a last stage wasnoticed, until 600 1C, with mass loss of 4 wt% due to thedesorption of coordinated water from sepiolite.34

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For AlS-P, the DSC peak around 100 1C is sharper comparedto the sepiolite case, additionally, a mass loss of 6 wt% wasobserved between 250–300 1C, associated with an endothermicpeak in the DSC curve. This peak was attributed before in theliterature27 to the transformation of the intercalated oligomericspecies in the respective oxide pillars.

For the ion-exchanged samples, and respective pillared materials,the TG/DSC curves are similar to that above. TiCoS-P and ZnCoS-P

present a slightly endothermic peak more intense than CoS andZnS, near 100 1C. For the titanium pillared materials a smallpeak between 250–300 1C was observed with mass loss between4 and 6 wt%, which, as in the case of the AlS-P sample can berelated to the formation of the oxide pillars. Further, massdecreased (2–8 wt%) up to 600 1C, attributable to the continuousdehydroxylation.

Fig. 2(a) presents the X-ray diffraction patterns of the startingclay and materials derived from it. Sepiolite diffractogram ischaracterized by a strong and sharp reflection at about 71 2y(d001), corresponding to a basal spacing of approximately 12 Å(check also Fig. S2 (ESI†) where data are compared with the ciffile for sepiolite) and several other moderate to weak reflections(at 19, 21, 23, 34, 37 and 401 2y) characteristic of sepiolite.37 Afterion-exchange, CoS still retains a well-defined d001 peak butfor ZnS the structure is now less ordered. This reduction ofstructural order is also noticed in titanium oxide pillared materials.Conversely, for AlS-P the d001 reflection is clearly noticed near 71 2y.The effect of pillaring can be better noticed in Fig. 2(b) wherelow-angle XRD is shown, a type of data that, to our bestknowledge, has not been used before in the literature in thecontext of pillaring the sepiolite material. This figure displays a

Fig. 1 TG (solid lines) and DSC (broken lines) curves for sepiolite and (a) AlS-P;(b) CoS and TiCoS-P; (c) ZnS and TiZnS-P.

Fig. 2 High angle (a) and low angle (b) X-ray diffraction.

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new and broad peak (absent for the initial sepiolite material)that can be tentatively ascribed to the pillar formation.

Fig. 3 shows the nitrogen adsorption–desorption isothermsof sepiolite and the pillared clays. Nitrogen adsorption curvesare similar for all materials being of type IIb.32 Below 0.4 p/p0,that is, a relative pressure range where adsorption in micro-pores (pore width less than 20 Å) is completed but capillarycondensation in mesopores (pore width between 20 and 500 Å)has not started, AlS-P is the material which adsorbs the most,presenting the higher ABET and as a consequence the increasein the microporous volume due to the pillaring process. Theextension of the hysteresis loops is similar for the variousmaterials but is slightly wider in the pillared samples (AlS-P,TiCoS-P and TiZnS-P). In general, the pillaring process did notgenerate a large increase of the specific surface area (Table 1) ormicropore volumes, the highest values being achieved for theAlS-P sample.

3.2 Nitric oxide adsorption and release

Data for adsorption and release, in the gas phase, of nitric oxide areshown in Fig. 4(a) and (b), respectively. NO adsorbed amounts arebetween 0.8 and 5.9% in mass (see Table 1). AlS-P material was the

one which adsorbed the most leading to an increase of 5.9% inmass, a value similar to what was found for instance for type-Azeolites.28 The sepiolite ion exchanged with zinc presented lessNO adsorbed amounts. Cobalt ions have a better affinity for NOthan zinc ions, since CoS and TiCoS-P adsorb more NO,although the introduction of titanium oxide pillars leads to adecrease in NO adsorption. The initial sepiolite clay alsoadsorbs NO. From the sequence of the NO adsorbed amountsin Fig. 4(a) and the sequence of the specific surface area values

Fig. 3 Adsorption–desorption nitrogen isotherms at �196 1C for the varioussamples.

Table 1 Textural parameters of the samples: specific surface area (ABET),microporous (Vm) and mesoporous volumes (Vm). NO adsorption and release

MaterialABET

(m2 g�1)Vm

(cm3 g�1)Vm

(cm3 g�1)NOAds

a

(%) NOAdsb

NOreleasedc [NO]L

d

Sepiolite 141 0.007 0.391 1.2 12.2 8.4 1.29AlS-P 206 0.039 0.193 5.9 59.0 26.4 2.10CoS 161 0.004 0.454 5.4 53.6 39.2 1.58e

TiCoS-P 185 0.007 0.442 3.2 31.9 12.8 0.72ZnS 134 0.006 0.378 0.8 7.7 8.0 0.43TiZnS-P 160 0.008 0.286 2.3 23.5 15.4 0.27

a Maximum NO adsorbed in the gas phase. b Maximum NO adsorbedin the gas phase (mgNO per gsolid). c Mass of NO released in 1 h inthe gas phase (mgNO per gsolid). d Concentration of NO released in 2 h inthe liquid phase (nM). e Maximum release after 10 min.

Fig. 4 Gas phase results for: (a) NO adsorption and (b) NO release.

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in Table 1, and with the exception of the TiCoS-P sample, it canbe observed that the sequence of NO adsorbed amounts is inline with the specific surface areas. This is indicative that themechanism of NO adsorption in the studied material will beessentially related to physical adsorption with no clear evidenceof significant specific interactions. Concerning nitric oxiderelease, as can be observed in Fig. (4b), ZnS has a fast kineticrelease, releasing the entire NO amount in the first hour. Thepillared materials AlS-P and TiCoS-P presented a similar releasebehavior, the release is slow but, with final values retained,after 20 h, being still high when compared with other solids.

Nitric oxide release data were analyzed according to Higushi,M/M0 = kt1/2, (1) Korsmeyer et al., M/M0 = ktn (2) and Peppas andSahlin, M/M0 = kdtm + krt

2m (3) equations,38–41 where M/M0 is thereleased drug fraction at time t, k is the release rate constantcharacteristic of the NO–clay system, n is the diffusionalexponent for drug release that depends on the release mechanismand the shape of the matrix tested, kd and kr are the diffusionand relaxation rate constants, respectively, m is the purelyFickian diffusion exponent for a device of any geometrical shapewhich exhibits controlled release.38,39 Regression parameterswere determined using OriginPro 8.5 software. Since modelsfrom eqn (1) to (3) have a different number of adjustableparameters Akaike’s Information Criterion (AIC)42 instead ofthe regression coefficient (R2) was used to determine the bestfitting model. According to the AIC method the equation to beused both for releasing in the gas or liquid phase is theKorsmeyer equation (eqn (2)), evidencing the prevalence of aFickian diffusional release that occurs by the usual moleculardiffusion due to a chemical potential gradient.41

Fig. 5 illustrates the fractional NO release in gas phase vs.time fitted with eqn (2) and fitting parameters are presented inTable 2. As for Fig. 5 and Table 2, it can be observed that therelease kinetics in the gas phase (except for ZnS) are notmarkedly different among the various solids. Nevertheless,(Table 2) the material pillared with aluminium oxide (AlS-P)presents the slowest kinetics and the initial sepiolite materialthe fastest. Data of release kinetics in the ZnS sample could notbe fitted by any of eqn (1) to (3).

Nitric oxide release studies were also made in the liquidphase using the oxyhemoglobin assay33 as described in theExperimental section. Selected examples of the initial and final(after 120 minutes) UV-Vis spectra are given in ESI† (Fig. S1).In Fig. 6 the results in the form of NO concentration versustime are presented. AlS-P was the material which released thehighest amount of NO, reaching a concentration of 2.10 nM,followed by the initial sepiolite clay. The CoS sample releasedNO in a very short time, which made the data interpretationvery limited for this material.

As a general observation from Fig. 5 and 7, we can see thatNO release kinetics is slower in the liquid phase. Eqn (1) to (3)were also tested for the results in the liquid phase. BothKorsmeyer and Pepas & Sahlin equations presented good fittingresults although eqn (2), as seen above, represents the mostadequate method to be used. The release rate constant (k) andthe diffusional exponent (n) are more uniform for the gas phase

studies than for the liquid phase release. The values of n(Table 2) are in general higher for the liquid phase than forthe gas phase which can be due to different mechanisms ofdiffusion in gas and liquid phases.41 In fact higher values of ncan be interpreted as a result of a mechanism that includesrelaxational release due to, for instance, the swelling of thematerial,41 as a consequence of interaction with the releasemedium. These interactions with the release medium are morelikely to occur in the liquid than in the gas phase, and canjustify the larger differences in the k and n values foundamongst the materials in the case of the studies in liquidphase. It should be emphasized (Fig. 7) that sepiolite andTiCoS-P present release profiles in the liquid phase thatapproach linearity. This is the most favorable situation sincea direct relationship between the time and the amount releasedallows easier control of the drug concentration.

3.3 Toxicological assays

Taking into account the obtained results, namely, NO amountsadsorbed/released as well as the release kinetics parameter,toxicological assays were carried out with the samples sepiolite,AlS-P and TiCoS-P. The results after 24 and 72 h are displayed in

Fig. 5 NO fractional release (M/M0) in the gas phase. Experimental points andfitting to eqn (2) (continuous line) are shown.

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Fig. 8(a), where a material concentration of 450 mg mL�1 wasused. This concentration was selected because it is close to thesuperior limit that is normally used in the literature for evaluatingcytotoxicity of silicas and other porous materials.43–46 After 24 h,cell survival is above 80% for all materials and after a 72 h assay,the % survival suffered a slight decrease although it remainedabove 70%. As the sepiolite sample, being a natural clay, andconsidering cytotoxicity, release kinetics, the final concentration in

the liquid phase and its linearity with time, presented mostfavorable results from the studied materials, it was selected for astudy of the survival rate where the concentration of the solidwas changed (Fig. 8(b)). As can be seen from Fig. 8(b), the % cellsurvival tends to decrease with the increase of sepiolite concen-tration, falling down to nearly 50% for concentrations around1000 mg mL�1. It should be emphasized that the survival rate inFig. 8(b) is high when compared with the literature and, inclusively,the concentration of 450 mg mL�1 (Fig. 8(a)) is also amongst thehighest that was studied for other porous materials such as silicas,modified clays or metal organic frameworks.43–47

Table 2 Korsmeyer equation fitting to NO release in gas and liquid phases

Material

Gas phase Liquid phase

k n k n

Sepiolite 0.477 � 0.0108 0.162 � 0.0055 0.040 � 0.6778 0.678 � 0.0187AlS-P 0.389 � 0.0082 0.200 � 0.0051 0.100 � 0.0153 0.492 � 0.0346CoS 0.401 � 0.0152 0.200 � 0.0091 1.400 � 0.0598 �0.065 � 0.0107TiCoS-P 0.433 � 0.0091 0.178 � 0.0051 0.009 � 0.0025 1.002 � 0.0644TiZnS-P 0.413 � 0.0181 0.192 � 0.0105 0.152 � 0.0257 0.398 � 0.0392

k, Korsmeyer kinetic constant; n, release exponent. The fitting was not possible for the ZnS sample.

Fig. 6 NO release in the liquid phase.

Fig. 7 NO fractional release (M/M0) in the liquid phase. Experimental pointsand fitting to eqn (2) (continuous line) are shown.

Fig. 8 Cell survival (a) after 24 and 72 h with samples solution at 450 mg mL�1

and (b) as a function of sepiolite concentration after 72 h.

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According to the amounts of used material (as described inthe Experimental section), the concentrations of NO released inthe liquid phase from Table 2, and taking for instance the caseof the AlS-P sample, the concentration released in the cellviability studies was 0.64 nM for this material. It should benoticed that signal transduction is operating at sub-nanomolarconcentrations of NO,48 and it can be calculated that endothelialcells donate only 20–100 pM NO to the underlying smoothmuscle target cells.49 Higher concentrations, in the 50–300 nMrange, inhibit the mitochondrial respiration chain,50 which workalso as concentration thresholds for lethal cellular effects.51

Thus, our compounds are capable of delivering NO at biologically-relevant non-toxic concentrations.

4. Conclusions

A set of clay-based materials was prepared by ion-exchange andby pillaring a natural sepiolite with various types of oligocations.The materials were characterized concerning their structure andporosity. Adsorption and release of nitric oxide, an endogenousmolecule with an important biological action, was studied in theprepared materials, both in the gas and in the liquid phase. Theadsorbed amounts of NO in the gas phase, as well as the releasepatterns, with the exception of one material (TiCoS-P), were inline with textural properties such as the specific surface area.The adsorption (storing) of NO in the studied materials occursessentially by physical adsorption. The released amounts of NOin the liquid phase were within biologically meaningful amountsand, for some materials, a slow release kinetics was obtainedwith, inclusively, an almost direct relationship between thereleased fraction and time. The toxicological studies made withHeLa cells showed a high survival rate, even for materialconcentrations higher than 450 mg mL�1, a fact that, in ourview, can be related to the use of materials that are preparedfrom a naturally occurring clay. The obtained results showedthat pillared clay materials, particularly those prepared bysimple methodologies as was the case of the AlS-P sample aswell as the unmodified sepiolite clay, can be considered in thecontext of the storage/release of NO for therapeutic purposessince they are capable of delivering NO at biologically-relevantnon-toxic concentrations.

Acknowledgements

Thanks to the Foundation for Science and Technology (FCT)for funding the Strategic project Pest-OE/QUI/UI0612/2013(CQB/FC/UL) and for the grant SFRH/BD/72058/2010(ACF).

References

1 M. F. Donatella Paolino, P. Sinna and M. Ferrari, in Encyclopediaof Medical Devices and Instrumentation, ed. J. G. Ebster,John Wiley & Sons, Inc., 2006.

2 Z. Liu, Y. Jiao, Y. Wang, C. Zhou and Z. Zhang, Adv. DrugDelivery Rev., 2008, 60, 1650–1662.

3 A. C. R. John, T. Santini Jr., R. Scheidt, M. J. Cima andR. Langer, Controled Drug Delivery, Wiley-VCH2000,pp. 2396–2407.

4 B. S. John, Pharmaceutical Manufacturing and Packing Sourcer,Ballantyne Ross Ltd International Media, London, 2000.

5 S. Wang, Microporous Mesoporous Mater., 2009, 117, 1–9.6 J. Yang, Adv. Drug Delivery Rev., 2012, 64, 965–966.7 P. Sonveaux, B. F. Jordan, B. Gallez and O. Feron, Eur. J.

Cancer, 2009, 45, 1352–1369.8 A. Ghaffari, D. H. Neil, A. Ardakani, J. Road, A. Ghahary and

C. C. Miller, Nitric Oxide, 2005, 12, 129–140.9 E. Karpuzoglu and S. A. Ahmed, Nitric Oxide, 2006, 15, 177–186.

10 Z. Ma and B. Moulton, Coord. Chem. Rev., 2011, 255, 1623–1641.11 M. L. Pinto, J. Rocha, J. R. B. Gomes and J. Pires, J. Am.

Chem. Soc., 2011, 133, 6396–6402.12 R. E. Morris, N. J. Hinks, A. C. McKinlay, B. Xiao and

P. S. Wheatley, Microporous Mesoporous Mater., 2010, 129,330–334.

13 R. E. Morris and P. S. Wheatley, Angew. Chem., Int. Ed., 2008,47, 4966–4981.

14 V. Anand, R. Kandarapu and S. Garg, Drug Discovery Today,2001, 6, 905–914.

15 P. C. C. Viseras, R. Sanchez, I. Salcedo and C. Aguzzi, Appl.Clay Sci., 2010, 48, 291–295.

16 A. C. Fernandes, M. L. Pinto, F. Antunes and J. Pires,J. Mater. Chem. B, 2013, 1, 3287–3294.

17 S. Tunç, O. Duman and A. çetinkaya, Colloids Surf., A, 2011,377, 123–129.

18 Y. Yu, S. Qi, J. Zhan, Z. Wu, X. Yang and D. Wu, Mater. Res.Bull., 2011, 46, 1593–1599.

19 S. Akyuz, T. Akyuz and A. E. Yakar, J. Mol. Struct., 2001,565–566, 487–491.

20 S. Argast, Clays Clay Miner., 1989, 37, 371–376.21 M. Suarez and E. Garcıa-Romero, Appl. Clay Sci., 2012,

67–68, 72–82.22 B. Wicklein, M. Darder, P. Aranda and E. Ruiz-Hitzky,

Langmuir, 2010, 26, 5217–5225.23 A. G. Gil, L. M. Gandia and M. Vicente, Catal. Rev.: Sci. Eng,

2000, 42, 145.24 K. S. W. Sing, D. H. Everett, R. A. W. Haul, L. Moscou,

R. A. Pierotti, J. Rouquerol and T. Siemieniewska, Pure Appl.Chem., 1985, 57, 603–619.

25 G. K. Zhang, X. M. Ding, F. S. He, X. Y. Yu, J. Zhou, Y. J. Huand J. W. Xie, Langmuir, 2008, 24, 1026–1030.

26 K. Bahranowski, J. Janas, T. Machej, E. M. Serwicka andL. A. Vartikian, Clay Miner., 1997, 32, 665–672.

27 J. Pires, M. Brotas de Carvalho and A. P. Carvalho, Zeolites,1997, 19, 107–113.

28 P. S. Wheatley, A. R. Butler, M. S. Crane, S. Fox, B. Xiao,A. G. Rossi, I. L. Megson and R. E. Morris, J. Am. Chem. Soc.,2006, 128, 502–509.

29 A. V. Ghule, K. Ghule, C.-Y. Chen, W.-Y. Chen, S.-H. Tzing,H. Chang and Y.-C. Ling, J. Mass Spectrom., 2004, 39, 1202–1208.

30 J. L. Valverde, P. Sanchez, F. Dorado, C. B. Molina andA. Romero, Microporous Mesoporous Mater., 2002, 54,155–165.

Paper NJC

4060 New J. Chem., 2013, 37, 4052--4060 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

31 T. Wanjun and C. Donghua, Chem. Pap., 2007, 4, 329–332.32 J. R. Françoise Rouquerol and K. Sing, Adorption by powders

porous solids, Academic Press, London, 1999.33 D. K. Martin Feelisch and J. Werringloer, in Methods in

Nitric Oxide Research, ed. M. F. a. J. S. Stamler, John Wiley &Sons Ltd, 1996, p. 732.

34 R. Ruiz, J. C. del Moral, C. Pesquera, I. Benito andF. Gonzalez, Thermochim. Acta, 1996, 279, 103–110.

35 L. Zhou, Z. Jing, Y. Zhang, K. Wu and E. H. Ishida, Appl. ClaySci., 2012, 69, 30–36.

36 J.-e. Zhou, Y. Dong, S. Hampshire and G. Meng, Appl. ClaySci., 2011, 52, 328–332.

37 ICDD-JCPDS, file no. 13-259.38 J. Siepmann and F. Siepmann, J. Controlled Release, 2012,

161, 351–362.39 Y.-S. Lin, PhD thesis, Graduate School of the University

Minnesota, 2012, p. 203.40 C. Ferrero, I. Bravo and M. R. Jimenez-Castellanos,

J. Controlled Release, 2003, 92, 69–82.41 N. A. Peppas and J. J. Sahlin, Int. J. Pharm., 1989, 57, 4.42 A. C. H. J. Motulsky, Fitting Models to Biological Data using

Linear and Nonlinear Regression. A Practical Guide to CurveFitting, GrapfPad Software, Inc, San Diego CA, 2003.

43 A. Bogershausen, S. J. Pas, A. J. Hill and H. Koller, Chem.Mater., 2006, 18, 664–672.

44 F. Y. Qu, G. S. Zhu, S. Y. Huang, S. G. Li, J. Y. Sun,D. L. Zhang and S. L. Qiu, Microporous Mesoporous Mater.,2006, 92, 1–9.

45 S. W. Song, K. Hidajat and S. Kawi, Langmuir, 2005, 21,9568–9575.

46 N. Venkatesan, J. Yoshimitsu, Y. Ito, N. Shibata andK. Takada, Biomaterials, 2005, 26, 7154–7163.

47 B. D. Kevadiya, R. P. Thumbar, M. M. Rajput, S. Rajkumar,H. Brambhatt, G. V. Joshi, G. P. Dangi, H. M. Mody,P. K. Gadhia and H. C. Bajaj, Eur. J. Pharm. Sci., 2012, 47,265–272.

48 A. M. Batchelora, K. Bartusa, C. Reynella, S. Constantinoua,E. J. Halveya, K. F. Heldb, W. R. Dostmannb, J. Vernona andJ. Garthwaite, PNAS, 2010, 107, 22060–22065.

49 K. Chen and A. S. Popel, Free Radical Biol. Med., 2006, 41,668–680.

50 F. Antunes, A. Boveris and E. Cadenas, PNAS, 2004, 101,16774–16779.

51 C.-Q. Li, B. Pang, T. Kiziltepe, L. J. Trudel, B. P. Engelward,P. C. Dedon and G. N. Wogan, Chem. Res. Toxicol., 2006, 19,399–406.

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