Invitro Stability of SBA-15

7/30/2019 Invitro Stability of SBA-15

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In vitro stability of SBA-15 under physiological conditions

I. Izquierdo-Barba, M. Colilla, M. Manzano, M. Vallet-Reg *

Dpto. Qumica Inorgnica y Bioinorgnica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramn y Cajal s/n, 28040 Madrid, Spain

Centro de Investigacin Biomdica en Red. Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain

a r t i c l e i n f o

Article history:

Received 22 January 2010Received in revised form 3 March 2010Accepted 20 March 2010Available online 27 March 2010

Keywords:

SBA-15Silica lixiviationIn vitro stability testFunctionalizationStability

a b s t r a c t

The numerous applications of mesoporous silicas are highly dependent on the stability of these matrices.

Consequently, it is convenient to carry out stability studies of these materials under the conditions inwhich they are going to be employed. Thus, when targeting these matrices for biomedical purposes,in vitro stability tests of mesoporous silica matrices are necessary. In this manuscript, the amount of silicalixiviated from SBA-15 matrices and their stability in aqueous solutions mimicking physiological fluids at37 C have been investigated. The selected aqueous media, which exhibit different ionic composition andpresence of proteins, have been: saline solution, a simulated body fluid and completed Dubeccos mod-ified Eagle medium. These are aqueous solutions widely employed as release media in drug delivery, bio-activity and cell culture assays. The obtained results indicate that SBA-15 degrades in all tested mediaand that there is a partial loss of the mesostructure after 60 days of assay. With the aim of decreasingthe silica lixiviation rate, SBA-15 has been organically modified by using different organic functions: alkylchains (methyl and octyl) and aminopropyl groups. In vitro stability tests of functionalized samples revealthat the amount of SiO2 is reduced compared to unmodified SBA-15 due to the organic coverage that actas a protection barrier for the nucleophilic attack of water to the siloxane (SiOSi) groups of the sil-ica framework that leads to the preservation of the mesostructure arrangement. These preliminary testsindicate that organic functionalization of mesoporous silica is a good strategy to decrease SiO2 lixiviation.

2010 Elsevier Inc. All rights reserved.

1. Introduction

Silica based ordered mesoporous materials have been widelyproposed for different biomedical applications [1]. Their outstand-ing structural and textural properties [2] together with their capa-bility to be functionalized with different organic groups [3] makethem attractive candidates for a wide range of biomedical pur-poses, such as controlled drug delivery [47], bone tissue regener-ation [8,9], gene transfection [10,11], cell tracking [12,13] andimmobilization of proteins or enzymes [1416]. Silica mesoporousmaterials are of particular interest for bone tissue regeneration be-cause they exhibit the added value of being able to act as hostmatrices for a wide range of biologically active guest moleculesto locally promote new bone formation or bone healing [17].

Within the world of biomaterials technology, an adequate bio-compatibility for the designed applications is a mandatory require-ment [18]. Amorphous silica has been generally considered as non-toxic, and it has been reported as biocompatible and degradable inliving tissue [19], which is required for bone, cartilage and connec-

tive tissue formation [20,21]. Up to date, biocompatibility and sta-bility tests of different silica materials have been reported [22,23].However, much research effort has to be committed to evaluate theinherent degradability, cytotoxicity and biocompatibility of meso-porous materials. Previous research works demonstrated that theconcentration and particle size of mesoporous silicas play a keyrole in their in vitro toxicity, showing low toxicity at low concen-trations [10,11,24].

Recently, Hudson et al. have performed in vivo studies using dif-ferent mesoporous materials, concretely, MCM-41, MCM-48 andMCF [25]. Histological studies performed after subcutaneousinjection of such mesoporous materials at the sciatic nerve in ratsevidenced a good biocompatibility at all time points. However, in-tra-peritoneal and intra-venous injections in mice resulted indeath or euthanasia. The role of silica in the etiology of this path-ogenesis is still unclear. Although local tissue reaction of mesopor-ous silicas was benign, they caused somewhat systemic toxicity.Authors suggest that toxicity could be mitigated by modificationof the materials, by means of surface adsorption of proteins, func-tional groups or polymers and so forth.

The above mentioned results clearly evidence that toxicity ofmesoporous silicas highly depends on the dosage and the adminis-tration route to the living body. This is tightly related to theamount of silica that the mesoporous matrix releases to the

1387-1811/$ - see front matter 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.micromeso.2010.03.025

* Corresponding author at: Dpto. Qumica Inorgnica y Bioinorgnica, Facultad deFarmacia, Universidad Complutense de Madrid, Plaza Ramn y Cajal s/n, 28040Madrid, Spain.

E-mail address: [email protected] (M. Vallet-Reg).

Microporous and Mesoporous Materials 132 (2010) 442452

Contents lists available at ScienceDirect

Microporous and Mesoporous Materials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m i c r o m e s o
http://dx.doi.org/10.1016/j.micromeso.2010.03.025mailto:[email protected]://www.sciencedirect.com/science/journal/13871811http://www.elsevier.com/locate/micromesohttp://www.elsevier.com/locate/micromesohttp://www.sciencedirect.com/science/journal/13871811mailto:[email protected]://dx.doi.org/10.1016/j.micromeso.2010.03.025


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physiological fluids, where the normal concentration of SiO2 (inhuman plasma) is ca. 1 mg/L[26]. Considering the viability of mes-oporous matrices as biomaterials for bone tissue regeneration, thefirst step should be the in vitro tests in aqueous medium mimickingphysiological conditions. Such studies would allow knowing thematerials reactivity, including the amount of SiO2 leached to theaqueous medium.

Recently, it has been demonstrated that the incorporation ofseveral heteroatoms into the silica network led to decrease the sil-ica lixiviation rate by increasing the stability of materials [27,28].Another proposed strategy consisted in incorporating osteogenicagents, such as parathyroid hormone related-protein (PTHrP) topromote bone tissue regeneration and accelerate the bone integra-tion process [29].

Herein we propose the organic functionalization of mesoporousmaterials as a straightforward approach to decrease the silica lixiv-iation. The aim of the present work is to evaluate the degradationin different aqueous media of SBA-15 mesoporous material. In thiswork the different selected media have been: saline solution (0.9%NaCl saline solution buffered at pH 7.4), a simulated body fluid(SBF) (an acellular aqueous solution with inorganic ion composi-tion similar to that of human plasma) [30] and completed Dub-eccos modified Eagle medium (DMEM). All of them are aqueoussolutions widely employed as release media in drug delivery, bio-activity and cell culture assays. In the present work the silica deg-radation kinetics from SBA-15 matrix is monitored as a function ofthe degree of ionic complexity of the media and the presence or ab-sence of serum proteins.

The functionalization of ordered mesoporous silica is a wellknown procedure employed to chemically modify the surface ofmesoporous matrices. In the approach here presented, SBA-15has been modified with two different alkyl groups methyl (C1)and octyl (C8) chains, and aminopropyl groups in an attempt tomake the surface more hydrophobic and hence more stable inaqueous media. After verifying the proper functionalization ofSBA-15 with the different organic moieties, the stability of the

resulting samples has been investigated in the same way asunmodified SBA-15. The differences observed in the silica degrada-tion rates have been determined by colorimetrically measuring thespecies of soluble silica leached to the tested media. Moreover, thetextural and structural features of the different mesoporous mate-rials after the in vitro tests have been also carried out.

The chemical strategies and research methodologies carried outin this work have been focused to biotechnological purposes. How-ever, they can be extended to other applications to evaluate thestability of mesoporous materials.

2. Experimental

2.1. Synthesis of mesoporous silica

Pure-silica SBA-15 ordered mesoporous material was synthe-sized according to the method previously reported by Zhao et al.[31]. In a typical synthesis, 4 g of triblock copolymer Pluronic

P123 (poly(ethyleneoxide)poly(propyleneoxide)poly(ethylene-oxide)), PEO20PPO70PEO20, Mavg = 5750) kindly supplied by BASFCo., used as structure directing agent, were dissolved in a mixtureof 104 mL of deionized water and 20 mL of reagent-grade HCl(37 wt.%, Aldrich) under magnetic stirring at 35 C. Once the sur-factant was completely dissolved, 9.15 mL of tetraethyl orthosili-cate (TEOS 98%, Aldrich) were added as silica source to yield amolar composition of 1.00 Si(OEt)4:0.02 P123:5.89 HCl:161.23H2O. The resulting mixture was kept under magnetic stirring dur-

ing 12 h at 35 C in a sealed Teflon

container and subsequentlyaged at 100 C for 24 h in a Teflon-lined stainless steel reactor. So-

lid silica particles were then collected by filtration, gently washedwith deionized water and dried in air at 60 C for 24 h. Finally,dried powders were thermally treated to remove the surfactantat 550 C under N2 flow for 2 h and then under atmospheric airfor further 3 h.

2.2. Functionalization of mesoporous silica

Functionalized materials were produced by organically modify-ing SBA-15 using the post-grafting method and following the pro-cedure described in previous publications [3234]. Thus, 1 g ofcalcined SBA-15, previously dried at 65 C for 24 h and kept underargon flow for 4 h, was reacted with 10 mmol of the correspondingfunctionalizing reactant, see below, in 10 mL of toluene (Aldrich)during 16 h under reflux. The final samples were filtered, washedwith a (1:1)-mixture of dichloromethane (CH2Cl2, Aldrich) anddiethyl ether (Et2O, Aldrich) and left to dry at 60 C for 24 h. Thefunctionalization reactants were 3-aminopropyltriethoxysilane(APTES, 97 wt.%), methyltriethoxysilane (MTES, 98 wt.%) and n-octyltrimethoxysilane (OTMS, 97 wt.%), all purchased from ABCRGmbH, Germany, yielding the mesoporous functionalized samplesdenoted as SBA15NH2, SBA15C1 and SBA15C8, respectively, referredto the silylation with PrNH2, C1 and C8 organic groups.

2.3. Characterization of materials

The presence of functional groups on the pore wall surface wasconfirmed by elemental microanalysis (PerkinElmer 2400 CHNthermo analyzer) and by Fourier transformed infrared (FTIR) spec-troscopy (Thermo Nicolet Nexus spectrometer equipped with aGoldengate attenuated total reflectance (ATR) device).

The structural and textural characterization was performed bypowder X-ray diffraction (XRD) in a Phillips XPert diffractometerequipped with a Cu Ka radiation (wavelength 1.5418 ). XRD pat-terns were collected over the range of 0.6 and 8 of 2h using with astep size of 0.02 and counting time of 4 s per step. Transmission

electron microscopy (TEM) was carried out with a JEOL 3000 FEGelectron microscope fitted with a double tilting goniometer stage(45) and with an Oxford LINK energy dispersive X-ray spectros-copy (EDS) analyzer. TEM images were recorded using a CCD cam-era (MultiScan model 794, Gatan, 1024 1024 pixels, size24 lm 24 lm) using low-dose condition. FT patterns were con-ducted using Digital Micrograph (Gatan). The textural propertiesof materials were determined by N2 adsorption/desorptionanalyses at 196 C on a Micromeritics ASAP 2010 instrument(Micromeritics Co., Norcross, USA). In all cases, 5070 mg of thefunctionalized samples or samples after in vitro tests were out-gassed under vacuum lower than 105 bar during 48 h at 60 Cprior to the measurements. In the case of pure-silica SBA-15 mate-rials before the in vitro assays the outgassing condition consisted in

24 h at 200 C. The BrunauerEmmettTeller (BET) equation wasused to calculate the surface area (SBET) from adsorption data ob-tained at P/P0 between 0.05 and 0.2. The total volume of pores,VP, was calculated from the amount of the N2 adsorbed at P/P0 = 0.97, assuming that adsorption on the external surface wasnegligible compared to adsorption in pores. The average mesoporesize (DP) was obtained from the maximum of the pore size distri-bution calculated from the adsorption branch of the isothermby means of the (BJH) BarrettJoynerHalenda method [35]. Thewall thickness (twall) was calculated using from N2 adsorptionand XRD data as previously reported [36]. To assess the possibleexistence of micropores (pore diameter < 2 nm) in samples, thet-plot method was employed, which allowed the estimation ofthe microporous fraction contribution (VlP) to the total pore vol-

ume. The amount of SiOH groups in all materials before and afterin vitro assays was determined by thermogravimetric analysis

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(TGA) in a PerkinElmer Diamond analyzer. To determine the SiOHconcentration, the samples were first heated from 20 to 130 C at arate of 5 C/min; then they were kept at this temperature for 1 hand finally they were heated at a rate of 5 C/min from 130 to1100 C. All measurements were performed under air stream. Thehydroxyl concentration in the mesostructured silica was calculatedfrom the weight loss after the complete removal of physisorbedwater and specific surface area. In the case of functionalized sam-ples, the organic content was also taken into account to performthe corresponding calculations.

2.4. In vitro stability assays

To carry out the in vitro stability tests, three different aqueousmedia were selected. The silica degradation rate in those mediaas function of ionic content and complexity of each medium (pres-ence or absence of the serum proteins) was determined. The firstmedium consisted in 0.9% NaCl saline solution (SS) buffered atpH 7.4. The second medium was a simulated body fluid (SBF)[30], an acellular aqueous solution with inorganic ion compositionsimilar to that of human plasma. The third medium was Dubeccosmodified Eagle medium (DMEM, Sigma) completed with 2 mMglutamine, 100 U/mL penicillin, 100 g/mL streptomycin and 10% fe-tal calf serum (FCS). To avoid microorganism contamination allmedia were filtered with a 0.22 lm Millipore System and all oper-ation/manipulation of the pieces and solutions were performed ina laminar flux cabinet Telstar AV-100.

The in vitro stability tests of samples were performed by soak-ing disk shaped pieces of material into 100 mL of the differenttested media at 37 C under orbital stirring (at 100 rpm) in a HTEcotron. Beforehand, portions of 100 mg of powder each samplewere compacted at 0.5 MPa uniaxial and 1 MPa isostatic pressuresat room temperature (13 mm in diameter and 2 mm in height) toobtain the disk shaped pieces. Three pieces of each material weresoaked into the three media to determine the reproducibility of

the tests. Each piece was mounted vertically using platinum wirein order to avoid non desirable precipitations on the disk surface.The amount of SiO2 leached to the different delivery media versustime was determined throughout colorimetrical analysis by usingan equivalent method to US EPA 370.1 [37]. This method is basedin the formation of a greenish-yellow complex proportional tothe dissolved silica concentration upon addition of the molybdateion in acidic solution. The molybdate complex is measured spec-trophotometrically at 660 nm in the 0.110 lg/mL range. Briefly,four reagents were used. The reagent #1 corresponds to a 2 MHCl solution, the reagent #2 to 10% (w/v) solution of ammoniummolybdate tetrahydrate solution ((NH4)6Mo7O244H2O, SigmaAl-drich) at pH of 78 by NaOH addition, the reagent #3 to 10% (w/v) solution of oxalic acid (H2C2O42H2O, SigmaAldrich) and the re-

agent #4 to 5% (w/v) solution of ascorbic acid (C6H8O6, SigmaAl-drich). To carry out the measurement a SmartChem140 automaticdiscrete analyzer (Alliance Instruments, AMS France) was em-ployed, including a software that allows to set up different param-eters such as the amount of each reagent as well as the delay timebetween their additions. In this particular case, for 350 lL of sam-ple, were consecutively added 7, 15, 11 and 11 lL of reagents #1,#2, #3 and #4, respectively with a delay time of 36, 36, 144 and206 s, respectively.

To prepare the silica patron solution, 4.73 g of sodium metasil-icate nonahydrated (Na2SiO39H2O, SigmaAldrich) were dissolvedin silicon-free MilliQ water until adjust the concentration to1000 mg/L SiO2 by gravimetric technique. All reagents and patronsolution were storage in plastic bottles. Four samples were used

for each measurement and the results are show as mean standarddeviation.

3. Results and discussion

3.1. Characterization of materials

The functional groups content in functionalized samples wasdetermined by means of CHN elemental analysis by determiningthe percentage of carbon present in each sample. In the case ofthe SBA-15 functionalized with PrNH

2groups, the amount of

nitrogen was also used to determine the functionalization degreeand the obtained result was in good agreement with that derivedfrom carbon content. The resulting functionalization degrees were:2.02 0.22 mmol APTES/g SiO2 (19.7 2.2 wt.% of APTES) forSBA15NH2, 2.58 0.25 mmol C1/g SiO2 (14.7 1.4 wt.% of C1) forSBA15C1 and 0.497 0.026 mmol C8/g SiO2 (8.1 0.43 wt.% of C8)for SBA15C8 (see Table 1).

FTIR spectra also confirmed the effective functionalization ofthe materials through the presence of the vibration bands ascribedto the different functional groups. Fig. 1 displays the FTIR spectraof SBA-15 before and after being functionalized with the differentorganic groups. FTIR spectrum of SBA-15 is characteristic of apure-silica material, in which the stretching vibration modes ofsurface silanol groups at ca. 37003200 cm1 are clearly observed.The width of this band together with the presence of anotherbroad vibration in the 16501600 cm1 range, ascribed to the dHOH(H2O) points to the existence of adsorbed water on the materialsurface. Moreover, in the 12501000 cm1 range, high-intensitybands attributed to mSiO of siloxane groups (SiOSi) are observed.Asymmetric stretching vibrations of SiOSi (mSiO) at ca.1100 cm1, symmetric stretching vibrations from SiO bonds(mSiO) at ca. 800 cm

1 and bending vibrations from SiOSi (dSiOSi)a 456 cm1 are clearly observed. The presence of free silanolgroups (SiOH) is evidenced by the presence of a acute signal at3745 cm1 (mOH) together with a weak band at ca. 980 cm

1

(mSiO). The chemical bond between SiOH groups and alkoxysilanegroups of modifying agents induces a decrease in the relativeintensity of both signals.

When aminopropyl modification was carried out absorptionsbands appearing at 3369 cm1 (mNH) and at 1150 cm

1 (dHNH) con-firm the presence of amine groups in SBA15NH2. Moreover, the or-ganic modification of SBA-15 is evidenced by the appearance ofnew bands at 2980 cm1, attributed to the CH stretching vibra-tion modes of residual ethoxy groups [38], and bands at 2937and 2874 cm1 assigned to CH2 stretching due to the alkyl groupsintroduced during silylation [39]. Moreover, a new absorptionband assigned to the deformation dHNH of NH2 group at ca.1540 cm1 is observed.

The successful functionalization of SBA-15 with C1 and C8groups was confirmed by the presence of different signals in the28003000 cm1 range belonging to the stretching vibrations(mCH) of the alkyl chains.

The structural characterization of SBA-15 before and after PrNH2, C1 and C8 functionalization has been performed bypowder XRD and TEM. Fig. 2A displays the XRD patterns at smallangles corresponding to SBA-15, SBA15NH2, SBA15C1 and SBA15C8samples. In all cases three well-defined diffractions maxima areobserved, which could be indexed as (10), (11) and (20)reflections based in a 2D-hexagonal mesoporous arrangement(p6mm plain group) [40]. Such results show that the mesoporousarrangement is preserved even after functionalization, being theunit cell parameter (a0), calculated from the d10 reflection,around 11 nm in all cases. TEM images corresponding toSBA-15, SBA15NH2, SBA15C1 and SBA15C8 materials (Fig. 3) arein very good agreement with the observations derived fromXRD patterns. TEM images show very good ordered arrange-

ment in the 2D-hexagonal system (p6mm plane group) in allcases.

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Fig. 2B shows the N2 adsorption isotherms for SBA-15 beforeand after being modified with the different organic groups. Allcurves can be identified as type IV isotherms in the BDDT classifi-cation, which are typical from mesoporous solids [41]. The pres-ence of H1 type hysteresis loops in the mesopore range indicatesthe existence of open-ended cylindrical mesopores with narrowpore size distributions, which are characteristic of SBA-15 meso-porous matrices. The main textural features derived from theappropriate treatment of N2 adsorption and XRD data are summa-rized in Table 1. It can be observed that there is a general decreasein the textural properties of SBA-15, i.e. BET surface area (SBET),pore volume (VP), micropore volume (VlP) and pore diameter

(DP), after being organically modified, which confirms the effectivefunctionalization of mesoporous silica walls.

In SBA15NH2, the decrease in the SBET and VP values com-pared to pure SBA-15 is more noticeable than in SBA15C1 andSBA15C8 samples. These results agree with those derived fromelemental analyses, in which the functionalization degree ofSBA15NH2 (19.7 wt.% of APTES) is greater than that of SBA15C1(14.7 wt.% of C1) and SBA15C8 (8.1 wt.% of C8). This notablereduction after functionalization with APTES moieties is inagreement with previous results.[5,42] Regarding to the VlP, itis observed the total loss of the VlP for the sample SBA15NH2and a decrease in this value of around 40% and 70% for SBA15C1and SBA15C8 samples, respectively. Concerning the pore size(DP), there is a decrease from 8.1 nm in unmodified SBA-15 to

5.4 and 6.1 nm in SBA15NH2 and SBA15C1 samples, respectively.This would also confirm functionalization of the inner surface of

Wavenumber (cm-1)

2800320036004000

O-HO-H(Si-OH)

(H2O, Si-OH)

N-H

(-NH2) C-H

C-H

C-H

800120016002000 400

SiO(Si-O-Si)

SiO

SiOSi

(Si-O-Si)

SiO(Si-OH)HOH

(H2O)

HNH

(-NH2)

SBA15C8

SBA15C1

SBA15NH2

SBA-15

Fig. 1. FTIR spectra of SBA-15, SBA15NH2, SBA15C1 and SBA15C8 samples.

Table 1

Textural and structural parameters obtained from N2 adsorption and XRD for the different mesoporous materials before and after 10 days of in vitro degradation tests in different

aqueous media. The values of %C and %N of all materials derived from chemical analyses are also displayed.a

Sample SBET (m2/g) VP (cm

3/g) VlP (cm3/g) DP (nm) twall (nm) a0 (nm) %C (wt.) %N (wt.)

SBA-15 899 1.03 0.046 8.1 2.7 10.8 0.19 0.05SBA-15-SS 434 0.891 0.022 9.8 1.1 10.9 0.35 0.07SBA-15-SBF 312 0.757 0.017 10.0 1.4 11.4 0.93 0.24SBA-15-DMEM 314 0.603 0.001 9.5 1.6 11.1 1.91 0.61

SBA15NH2 381 0.663 $0 5.4 5.0 10.4 7.16 2.25SBA15NH2-SS 328 0.637 $0 8.8 2.5 11.3 4.27 1.58SBA15NH2-SBF 339 0.750 $0 7.0 3.6 10.6 4.31 1.61SBA15NH2-DMEM 251 0.450 $0 8.1 2.2 10.3 10.69 3.10

SBA15C1 688 0.913 0.028 6.1 5.0 11.1 2.83 0.05SBA15C1-SS 391 0.877 0.014 10.0 1.4 11.4 1.12 0.10SBA15C1-SBF 335 0.909 0.010 7.9 3.2 11.1 1.63 0.32SBA15C1-DMEM 230 0.524 $0 9.3 1.9 11.2 5.7 1.74

SBA15C8 683 0.851 0.015 7.8 2.8 10.6 4.85 0.08SBA15C8-SS 354 0.753 0.008 9.6 1.7 11.3 3.56 0.13SBA15C8-SBF 364 0.905 0.005 8.0 3.0 11.0 4.09 0.22SBA15C8-DMEM 291 0.645 $0 7.7 3.5 11.2 9.55 1.85

a SBET is the surface area determined by using the BET method between the relative pressures (P/P0) 0.050.25. VP and VlP are respectively, the total pore volume andmicropore volume obtained using the t-plot method. The total pore volume was estimated from the amount adsorbed at a relative pressure of 0.97. DP is the pore diametercalculated by means of the BJH method from the adsorption branch of the isotherm. a0 is the unit cell parameter calculated by XRD, being a0 2=

ffiffiffi

3p

d10. twall is the wall

thickness calculated using the equation twall = a0 DP [36]. %C and %N are respectively, the percentages of carbon and nitrogen in samples calculated from chemical analyses.



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mesopores, which leads to a pore diameter reduction and to anincrease in the mesoporous wall thickness (twall) which is corre-

lated with the upholstered of silica wall with organic moieties[5,6].

3.2. In vitro stability tests

In vitro stability tests of SBA-15 before and after being function-alized with different alkyl chains (C1 and C8) and PrNH2 were

0 1 2 3 4 5 6 7 8

2/degree

Intensity

(a.u

)

0.0 0.2 0.4 0.6 0.8 1.0

0

200

400

600

800

1000

1200

1400

1600

1800

SBA15C8

SBA15C1

SBA15NH2

Volumeadsorbed/cm3.g

-1(STP)

P/P0

SBA-15

SBA-15

SBA15NH2

SBA15C1

SBA15C8

(A) (B)

(10)

(11)

(20)

0 1 2 3 4 5 6 7 8

2/degree

Intensity

(a.u

)

0.0 0.2 0.4 0.6 0.8 1.0

0

200

400

600

800

1000

1200

1400

1600

1800

SBA15C8

SBA15C1

SBA15NH2

Volumeadsorbed/cm3.g

-1(STP)

P/P0

SBA-15

SBA-15

SBA15NH2

SBA15C1

SBA15C8

(10)

(11)

(20)

Fig. 2. (A) XRD patters and (B) N2 adsorption isotherms of SBA-15 mesoporous materials before and after being functionalized with different organic groups. The isothermsfor samples SBA15NH2, SBA15C1 and SBA15C8 were vertically offset by 500, 800 and 1200 cm

3/g, respectively.

Fig. 3. TEM images corresponding to SBA-15, SBA15NH2 , SBA15C1 and SBA15C8 samples. In all cases, TEM images have been taken with the electron beam perpendicular to themesoporous channels of a 2D-hexagonal structure (p6mm plane group).



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carried out in three different aqueous media, SS, SBF and DMEM, ascommented in Section 2. The amount of soluble silica species wascolorimetrically determined and the resulting degradation pat-terns are plotted in Fig. 4. The SiO2 release profiles correspondingto unmodified and functionalized SBA-15 matrices are noticeablydifferent. The highest amount of lixiviated SiO2 was that from pureSBA-15 independently of the tested media. In all cases, the curvesare characterized by exhibiting a relatively fast dissolution rate upto ca. 250 h followed by plateau in which the measured SiO 2 re-mains constant until the end of the in vitro assay, which indicatesthat the maximum level of saturation has been reached. In this

case, the saturation level for unmodified SBA-15 is 120 mg SiO2/g, whereas such value does not exceed 80 mg SiO2/g in the restof materials independently of the tested media. This is a clear indi-cation that functionalizing the mesoporous matrices, less dissolu-tion is observed, so higher stability is reached. The saturationphenomenon was predictable considering the static standardin vitro conditions here selected. Focusing on the degradationbehaviour before saturation, i.e. between 0 and 250 h (right sideof Fig. 4), clear differences can be observed among functionalizedsamples regarding the degradation kinetics and the maximumamount of lixiviated SiO2. The kinetic constant values of lixiviated

0 200 400 600 800 1000

0

20

40

60

80

100

120

140

160SS

Time (h)

mgS

iO2

/gmaterial

0 200 400 600 800 1000

0

20

40

6080

100

120

140

160

Time (h)

SBF

mgSiO2

/g

material

0 200 400 600 800 1000

0

20

40

60

80

100

120

140

160

Time (h)

DMEM

mgSiO2

/gmaterial

SBA-15 SBA15NH2 SBA15C1 SBA15C8

0 50 100 150 200 250

0

20

40

60

80

100

120

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Time (h)

mg

SiO

2/gmaterial

0 50 100 150 200 250

0

20

40

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160

mgSiO

2/gm

aterial

Time (h)

SBF

0 50 100 150 200 250

0

20

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60

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100120

140

160

mgSiO

2/gmaterial

Time (h)

DMEM

SS

0 200 400 600 800 1000

0

20

40

60

80

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160SS

Time (h)

mgS

iO2

/gmaterial

0 200 400 600 800 1000

0

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6080

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Time (h)

SBF

mgSiO2

/g

material

0 200 400 600 800 1000

0

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Time (h)

DMEM

mgSiO2

/gmaterial

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0 50 100 150 200 250

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Fig. 4. Silica release profiles (mg of SiO2 lixiviated per 1 g of material) from SBA-15, SBA15NH2, SBA15C1, SBA15C8 materials in the different media tested in this work: NaCl0.9 wt.% pH 7.4 (SS), simulate body fluid (SBF) and completed Dubeccos modified Eagle medium (DMEM).



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silica have been calculated using the Higuchi model [43] in the 048 h range. The proposed dissolutiondiffusion model is com-monly employed to describe the whole release process withoutlimitation of different dissolution rate or dissolubility [44]. The dif-fusion coefficient and the dissolution rate constants are evaluatedfrom measured release profiles which are well-fitted to the follow-ing equation (see Table 2):

Wt K t1=2

where Wt is amount of silica lixiviated after t time and K is the re-lease kinetic constant, which indicates the matrix degradation rate.

In the case of functionalized materials, both K and maximumsilica released (Wmax) values are higher than those of unmodifiedSBA-15 in all tested media. Nevertheless, no significant differences

in such values are observed as function of the employed media forSBA-15, being ca. 16 103 mg g1 s1/2 and 120 mg/g, the K andWmax values, respectively. This indicates that the complexity ofthe aqueous medium regarding the ionic composition and thepresence or absence of proteins do not significantly affect theSiO2 lixiviation in the case of SBA-15.

The K decrease after modifying SBA-15 materials more evidentfor SBA15NH2 material in SS and SBF solutions (ca. 6 103 mg g1 s1/2). In fact, K value is around three times smallerfor SBA15NH2 thanforpureSBA-15,whichpointstoahigherstabilityexpectedfortheformermatrix.Moreover, Kvaluesinthesamemed-ia for SBA15C1 and SBAC8 are similar (ca. 11 103 mg g1 s1/2),being around twice than that of SBA15NH2. This last fact could beexplained attending to the highest functionalization degree

observed for SBA15NH2 sample compared to SBA15C1 and SBA15C8,as previously indicated. In this sense, the coverage of the silica sur-facewithorganic moieties confer SBA-15of a certainhydrophobicitydegree that would make difficult the nucleophilic attack of water tothe siloxane bonds (SiOSi) of silica network. This fact resultsinareducedSiO2 lixiviation in SS and SBFsolutions, which is in goodagreement with an increase of hydrophobicity of the sample due tothe incorporation of aminopropyl (PrNH2) and different alkylchains (C1 and C8) [4547].

Likewise, there are significant differences in the lixiviated SiO 2from functionalized samples depending on the presence of pro-teins in the aqueous medium. SBA15C1 and SBA15C8 samples showsmaller SiO2 release rates in DMEM comparing to SS or SBF.Besides, the maximum amount of SiO2 lixiviated to DMEM de-

creases compared to the other aqueous media. Thus, K value de-crease from ca. 11 mg g1 s1/2 for C1 and C8 functionalized

samples in SS and SBF media to ca. 7 mg g1 s1/2 in DMEM med-ium. However, such differences are not found in the in vitro testscarried out with SBA15NH2 material, which exhibit K values of ca.6 103 mg g1 s1/2 in the three tested aqueous media. Moreover,the maximum amount of SiO2 release to DMEM is ca. 60 mg/g,which is 25% less than that released to SS or SBF media.

This is probably due to the different organic adsorption of theglycoproteins from FCS. The adsorption of proteins on the surfaceof mesoporous matrices has been reported to mainly consist inelectrostatic, hydrogen bonding and hydrophobic interactions[4850]. The different protein adsorption observed in the threefunctionalized samples here tested can not be easily explaineddue to the high complexity of the DMEM medium, which containsa wide variety of proteins. Therefore, the driving forces governingproteins adsorption can not be clearly established in this medium.However, it seems clear that proteins adsorption in SBA15C1 andSBA15C8 reduces the silica lixiviation from these matrices.

Up to this point, the effects of materials degradation have beeninvestigated in the media themselves. Moreover, it is also possibleto study the evolution of the materials surfaces during the degra-dation tests. Thus, with the aim of monitoring the evolution ofthe surface of the different SBA-15 materials chemical analysis,FTIR, XRD and TEM measurements of samples were performedafter in vitro assays in the different media.

Chemical analyses (see Table 1) reveal that all functionalizedmaterials exhibit a partial loss of organic groups after 10 days ofassay in SS and SBF. A decrease in C content (in SBA15 C1 andSBA15C8 samples) and in C and N contents in SBA15NH2 sample isobserved. This fact has been previously reported in the literaturefor organically modified mesoporous matrices after being soakedin aqueous media [51]. However, this loss of functional groupswas not total, since FTIR studies revealed that even after 60 daysof assay in all tested media the characteristic vibration bands ofsuch groups are observed. This fact indicates that there are stillfunctional groups remaining in the surfaces of functionalizedmaterials after 2 months of tests (Fig. 6 (upper part)).

Chemical analyses of all functionalized samples after beingsoaked in DMEM medium reveal a significant increase in the %Cand %N contents. Such increase in the organic content was notnoteworthy after the in vitro tests of SBA-15 in DMEM. These datawould be in agreement with the higher protein adsorption fromFCS in the functionalized samples compared to unmodified SBA-15. This would be also in good concordance with the increase inthe hydrophobicity degree of SBA-15 due to the organic modifica-tion of its surface, as previously mentioned [48,49]. Such increasein %N ranges from 1.74 and 1.89 for SBA15C1 and SBA15C8, respec-tively, to 0.85 for SBA15NH2. This higher proteins adsorption degreecould explain the decrease in the amount of SiO2 leached to DMEMcompared to SS and SBF and the subsequent decrease in the K va-lue. FTIR spectra of functionalized samples after being soaked in

DMEM confirm these results (Fig. 6). In such spectra, the character-istic vibration bands at 1635, 1530 and 3305 cm1 correspondingto amide I, amide II band and amide III indicate the presence of gly-coproteins from FCS deposited onto the materials surfaces areclearly observed [52].

XRD and TEM studies were carried out for all samples in the dif-ferent tested media to study the structural variations as a functionof soaking time in the different media. Whereas XRD do not indi-cate significant variations in the diffraction profiles after thein vitro assays, there is a decrease in the intensity of diffraction pat-ters, which would point to a small loss of structural order. In thissense, an exhaustive study by TEM reveals clear differences inthe evolution of the material as a function of incubation timeand the type of material tested (unmodified and modified SBA-15).

Fig. 5 displays the TEM studies performed for SBA-15 before andafter 10 and 60 days of in vitro assay in SS. TEM images evidence

Table 2

Main parameters resulting from the measurement of the amounts of silica released to

the different release media.a.

Sample K (103)(mg g1 s1/2)

Wmax SiO2 released(mg/g)

R

SBA-15 (SS) 15.80 0.99 123.4 2.6 0.99SBA15NH2 (SS) 5.89 0.37 79.8 1.9 0.90SBA15C1 (SS) 10.75 1.50 83.4 2.1 0.91

SBA15C8 (SS) 10.24 0.90 80.8 1.8 0.90SBA-15 (SBF) 15.51 0.62 119.2 1.9 0.98SBA15NH2 (SBF) 5.51 0.24 78.2 1.3 0.97SBA15C1 (SBF) 12.39 0.69 89.5 1.5 0.97SBA15C8 (SBF) 11.40 0.68 84.6 2.0 0.96

SBA-15 (DMEM) 16.40 2.76 114.2 3.8 0.80SBA15NH2 (DMEM) 7.27 1.25 87.39 2.1 0.92SBA15C1 (DMEM) 8.28 0.41 63.4 2.1 0.98SBA15C8 (DMEM) 6.68 0.56 59.5 2.1 0.93

a Kis the kinetic constant derived from the adjust of the experimental data to theHiguchi equation up to 48 h of experiment: Wt = K t1/2. K is the kinetic constant.Wmax is the maximum amount of SiO2 released to the different delivery media and Ris the regression coefficient.



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that there is an evolution of the mesostructural order during thein vitro assays. In fact, TEM images indicate that after 10 days in

SS the mesoporous arrangement is preserved, as it was observedby XRD. Furthermore, after 60 days in SS, the lower magnificationof TEM image shows the partial loss of ordered mesoporousarrangement on the surface of pure SBA-15. However, some do-mains with 2D-hexagonal mesoporous arrangement are still ob-served. However, the degradation rate is relatively low, sinceTEM studies indicate that after two months of assay there is a par-tial loss of the ordered mesoporous arrangement in SBA-15, butthere are still some domains with 2D-hexagonal structure.

As previously mentioned, Fig. 4 shows that the maximumamount of soluble silica species released from all tested materialsto the different delivery media is reached after ca. 250 h of assay.This could lead to presuppose that there is a stabilization of themesoporous materials in the different media without further deg-

radation. However, the fact of reaching the saturation state doesnot necessary means that the materials do not continue degrad-

ing. In fact, the degradation and the subsequent loss of the mes-ostructural order are clearly evidenced by performing TEM

studies after different times of assay for unmodified SBA-15(Fig. 5).

On the contrary, TEM studies carried out in functionalized sam-ples after in vitro tests indicate that all samples exhibit a well-or-dered mesoporous arrangement in the 2D-hexagonal structureeven after 60 days in SS (Fig. 7). These findings indicate the lowerdegradation rate of functionalized samples due to the organicgroups covering the material surfaces compared to unmodified-SBA-15, as it has been commented above when using other charac-terization techniques.

N2 adsorption measurements allow obtaining information aboutthe evolution of the mesoporous structures after in vitro assays inthe different media. The silica degradation in all samples is evidentsince inall cases a decrease in the maintextural parameters, such as

SBET and VP, is observed independently of the employed medium.Such decrease is more remarkable for unmodified SBA-15, which

Fig. 5. TEM images at magnification 60 K (left) and 150 K (right) corresponding to SBA-15 after different times in SS.



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exhibits a relative SBET loss of 52% and 65% afterbeing soaked during10 days in SS and SBF, respectively. SBA15C1 and SBA15C8 samplesexhibit quite similar behaviours. Thus, the relative SBET decreaseis43% and 51% for SBA15C1 after 10 days in SS and SBF, respectively.Besides, the relative SBET decrease is 48% and 46% for SBA15C8 after10 days in SS and SBF, respectively. Certainly, all these findingsagree with the SiO2 lixiviation profiles observed in Fig. 4 and thekinetics parameters derived from the fit of the experimental datato the Higuchi model (Table 2), that showed that K values wereca. 16 103 mg g1 s1/2 for SBA-15 and ca. 11 mg g1 s1/2 forboth SBA15C1 and SBA15C8, which would induce lower decreasein SBET after the in vitro assays compared to unmodified SBA-15.

However, the high extent of preservation of the textural proper-ties of SBA15NH2 sample after being soaked in SS and SBF should be

highlighted. Accordingly, the relative SBET reduction is only 14%and 11% for SBA15NH2 after 10 days soaked in SS and SBF, respec-tively. This behaviour would be in good agreement with the lowerrelease rate of SiO2 leaching for aminopropyl functionalized SBA-15.

Regarding twall derived from XRD and N2 adsorption measure-ments, it is observed a decrease in such parameter for all samplesafter in vitro assays (see Table 1), which is correlated with the silicaleaching from the mesoporous surface together with the loss of or-ganic moieties in the functionalized matrices. From the variationsobserved in this parameter it is easy to predict that the higher silicalixiviation takes place from unmodified SBA-15.

With the aim of giving some accurate chemical informationthat may support the observed modifications in the variations

SBA15C8

SBA15C1

SBA15NH2

SBA-15

C-H

HOH(H2O)

HNH

(-NH2)

SS

2600300034003800 800120016002000 400

SBA15C8

SBA15C1

SBA15NH2

SBA-15

N-H(Primary amide)

Wavenumbers (cm-1)

C=O(Amide I)

C=O(Amide I)

DMEM

SBA15C8

SBA15C1

SBA15NH2

SBA-15

C-H

HOH(H2O)

HNH

(-NH2)

SS

2600300034003800 800120016002000 400

SBA15C8

SBA15C1

SBA15NH2

SBA-15

N-H(Primary amide)

Wavenumbers (cm-1)

C=O(Amide I)

C=O(Amide I)

DMEM

Fig. 6. FTIR spectra corresponding to SBA-15, SBA15NH2, SBA15C1 and SBA15C8 after 60 days ofin vitro assays in SS and 10 days in DMEM media.



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in the textural properties and the silica release behaviour, the

number of silanol groups (SiOH) was determined for all mesopor-ous materials before and after being soaked during 10 days in SS.It has been previously reported that the aqueous stability of mes-oporous materials can be investigated in terms of the pore wallthickness and the SiOH density [53]. Thus, to determine theamount of SiOH groups a thermogravimetric analysis (TGA) ofthe samples before and after in vitro assays were performed.TGA is a widely recognized efficient method to determine thehydroxyls density in mesostructured silica on the basis of waterloss due to the condensation of silanol groups [54,55]. The SiOHgroups/nm2 values are in the 4.25.3 range for the as-synthesizedSBA-15 and functionalized samples. These values are in agree-ment with those data previously by Zhuravlev reported for differ-ent silica samples [56,57], which are in the 4.25.7 SiOH/nm2

range. Concretely, unmodified SBA-15 exhibits a value of 4.9SiOH/nm2 before the in vitro assay. This value increases a 20%

up to 6.2 SiOH/nm2, after being soaked during 10 days in SS. This

result evidences the destruction of SiOSi linkages fromSBA-15, which are then converted to SiOH bonds, leading tothe rehydroxilation of the surface and eventually to the partiallixiviation of soluble silica species, as confirmed in Fig. 4. Onthe contrary, all functionalized samples undergo a decrease inthe number of silanol groups of ca. 25% after being submittedto the soaking process. Certainly, the un-modification in the num-ber of silanol groups in trimethylsilyl-functionalized samples afterwater treatment has been previously reported [58]. This fact hasbeen attributed to the protection of the SiOSi bonds fromhydrolysis due to the functional groups capping the hydrophilicsilanol groups. In our case, the amount of silanol groups experi-ences a decrease, which could be explained by the further con-densation of SiOH groups to originate SiOSi bonds. The

decrease in the number of silanol groups would originate a stabil-ization of the mesoporous silica framework which would agree

Fig. 7. TEM images at magnification 60 K (left) and 150 K (right) corresponding to SBA15 functionalized with PrNH 2, C1 and C8, respectively after 60 days in SS.



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with the results displayed in Fig. 4, showing a decrease in the sil-ica lixiviation from all functionalized samples compared tounmodified SBA-15.

In the particular case ofin vitro assays in DMEM, the decrease inthe textural parameters is more noticeable in functionalized sam-ples than in unmodified SBA-15 material (see Table 1). This couldbe explained by the high extent of the proteins adsorption in thematerial surfaces. This fact agrees with the results derived fromCHN chemical analyses, in which an increase in the organic contentin all mesoporous samples is observed after the in vitro assays inDMEM. In addition, N2 adsorption results displayed in Table 1 forsamples after 10 days soaked in DMEM, evidence that there is amore noticeable decrease in SBET for SBA15C1 and SBA15C8 samplesthan SBA15NH2, in agreement with the higher protein adsorptiondegree (confirmed by chemical analyses) and the smaller SiO2 re-lease rate obtained for the former during the first days of assay.

4. Conclusions

In vitro stability tests of SBA-15 in solutions mimicking physio-logical fluids using three aqueous media (saline solution, SBF and

DMEM) at 37 C, reveal that SBA-15 is partially soluble in all testedmedia. The complexity of the aqueous medium regarding the ioniccomposition and the presence of proteins does not significant af-fect the silica dissolution from SBA-15. The degradation of SBA-15 produces some loss of its textural and structural properties.However, the degradation rate is relatively low, since TEM studiesindicate that after 60 days of assay there is a partial loss of the or-dered mesoporous arrangement in SBA-15, but there are still somedomains with 2D-hexagonal structure. This is in agreement withthe results derived from XRD measurements.

The reduction in the silica lixiviation rate has been successfullyachieved by organically modifying SBA-15 using different organicgroups: alkyl chains (methyl and octyl) and aminopropyl groups.The in vitro degradation tests indicate that functionalization of

SBA-15 decreases the SiO2 leaching to the three media tested com-paring with to unmodified SBA-15. These results open promisingexpectations for the use of these materials in many biomedicalapplications where low silica release to the physiological mediumis essential.

The decrease in the degradation rate of functionalized samplescompared to SBA-15 is accompanied by a preservation of the mes-ostructure arrangement, as confirmed by TEM and XRD measure-ments after 60 days of assay. This is undoubtedly an added valuewhen long-term applications, such as bone tissue regenerationand other biotechnological applications, are targeted, which re-quire mesoporous materials with stable mesostructural order. Thisstudy, which has been performed for biotechnological purposes,could be suitable for other applications that require and evaluationof the stability of mesoporous matrices.

Acknowledgments

We thank the following for funding this work: the Spanish CI-CYT through projects MAT2008-00736 and the Comunidad Aut-noma de Madrid via the S2009MAT-1472 program grant. We alsothank Fernando Conde (CAI X-ray Diffraction), CAI Elemental Anal-yses, CAI Electron Microscopy of Universidad Complutense de Ma-drid for their valuable technical assistance.

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Invitro Stability of SBA-15

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