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Colloids and Surfaces B: Biointerfaces 94 (2012) 288– 295

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

j our na l ho me p age: www.elsev ier .com/ locate /co lsur fb

H sensitive silica nanotubes as rationally designed vehicles for NSAIDs delivery

élia T. Sousaa,1, Cláudia Nunesb,1, Mariana P. Proenc aa, Diana C. Leitãoa, José L.F.C. Limab,alette Reisb, João P. Araújoa, Marlene Lúciob,∗

IN and IFIMUP, Institute of Nanoscience and Nanotechnology, Rua do Campo Alegre, 687, 4169-007 Porto, PortugalREQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

r t i c l e i n f o

rticle history:eceived 5 September 2011eceived in revised form 18 January 2012ccepted 2 February 2012vailable online 10 February 2012

eywords:anoporous aluminailica nanotubesurface functionalization

a b s t r a c t

A novel pH-sensitive drug delivery system based on functionalized silica nanotubes was developed forthe incorporation of non-steroidal anti-inflammatory drugs (NSAIDs), aimed at a tailored drug releasein acidic conditions characteristic of inflamed tissues. Silica nanotubes (SNTs) were synthesized by ananoporous alumina template assisted sol–gel method. Inner surfaces were physically and chemicallymodified to improve both the functionalization and subsequent incorporation of the drug. Fourier trans-form infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive spectroscopy(EDS) and transmission electron microscopy (TEM) were used to characterize the designed nanocarriersand their functionalization. To achieve the highest degree of functionalization, three types of aminosi-lanes were tested and calcination conditions were optimized. APTES was shown to be the most effective

rug-delivery systemSAIDs

aminosilane regarding the functionalization of the SNTs’ inner surface and an adequate calcinationtemperature (220 ◦C) was found to attain mechanical stability without compromising functionalizationefficiency. Finally, the incorporation of naproxen into the nanotubes was accessed by fluorescence mea-surements and drug release studies were performed, revealing that the electrostatic linkage ensureseffective release of the drug in the acidic pH typical of inflamed cells, while maintaining the SNT-drugconjugates stable at the typical bloodstream pH.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of theorld’s most prescribed drug groups [1]. Although their major

pplication is in acute inflammatory conditions, they are alsoidely used in chronic inflammatory diseases. However, long-termse of NSAIDs is associated with a high risk of gastrointesti-al bleeding. This constitutes a significant limitation of currentherapies since drugs must frequently be administered at subop-imal doses to reduce cytotoxicity. Numerous strategies have beenmployed over the past 50 years to develop NSAIDs that spare theastrointestinal tract [1] but none have been proven successful.ontrolled release systems may signify the answer to this problem

s they increase the overall therapeutic efficiency of the drug byaintaining its concentration in the body since it is only released

∗ Corresponding author at: REQUIMTE, Departamento de Química, Faculdade dearmácia, Universidade do Porto Rua Aníbal Cunha, 164 4099-030 Porto Portugal.el.: +351 222078966; fax: +351 222004427.

E-mail address: mlucio@ff.up.pt (M. Lúcio).1 These authors have contributed equally.

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2012.02.003

in the injured cells. Thus, the used carrier and its linkage with thedrug are the main keys in this process.

In addition, significant advances made in nanoscience and nano-technology over the last decade have led to the development ofnew platforms where all physical properties such as size, poros-ity, geometry and surface functionalization can be controlled at thenanoscale level. In particular, high aspect ratio inorganic nanopar-ticles have aroused great interest and shown great potential indrug delivery [2], particularly those based on silica which havemany advantages over organic polymeric particles since they areextremely stable and their size, shape and porosity can be con-trolled easily [3]. In addition, there is no enlargement or porositychange upon pH variation and no vulnerability to microbial attackwhile these particles have also been shown to effectively protectentrapped molecules (enzymes, drugs, etc.) against degradation[4]. Silica-based particles are also known for their biocompatibil-ity [5–7] and surface functionalization which allows the hydroxylson the surface to be modified with amines, thiols, carboxyls andmethacrylate, according to the desired application [8]. Among

these, the alkoxysilanes with an amino group represent one of themost interesting functionalization agents from the biological pointof view [9,10]. Amino-functionalized surfaces can be obtained bybinding simple compounds bearing an amine group (for example

C.T. Sousa et al. / Colloids and Surfaces B

Fig. 1. Structure of the different aminosilanes. 3D structures were pre-d ®

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-aminopropylalkoxy-silanes) onto the silica surface. On the otherand, the functionalization can be tailored to obtain a sensitive link-ge between the drug and the nanoparticle. Thus, the detachmentan be easily foreseen and controlled.

Nevertheless, the modification of the silica surface is nottraightforward and involves all processes that lead to changes inhemical composition of the surface, which can be achieved eithery physical treatment, using for example thermal treatment whichhanges the ratio of silanol and siloxane groups of the silica surface,r by chemical treatment using specific surface reactions to exhibitydroxyl groups. Through this surface modification, the adsorptionroperties are significantly affected.

The most important advantage of nanotubes is that the inneroids can be used to incorporate drug molecules, preventing theirontact with the surrounding environment until they reach theesired local delivery. Moreover, given the innovation of the tem-late assisted synthesis using nanoporous anodic aluminum oxideAAO), it is possible to obtain monodisperse nanotubes of almostny material with the desired length and pore diameter solely byhanging the anodization conditions [11].

In this work, we used an alumina template assisted syn-hesis method to produce silica nanotubes (SNTs), which havei O H chemical bonds at the inner surface, allowing directminosilane functionalization to generate a polycationic sur-ace. The quantity of OH groups was determined as a functionf the calcination temperature. An optimum calcination tem-erature of around 220 ◦C was found, as a good compromiseetween mechanical stability of the nanotube and OH groupvailability. Thereafter, the inner surface of these nanotubesas functionalized with three different aminosilane molecules.minopropyltrimetoxysilane (APTMS), aminopropylethoxysilane

APTES) and aminopropyldimethylethoxysilane (APDMES), whosetructural representation is presented in Fig. 1, were chosen sincehey are the most frequently used organosilane agents to preparehe amino-ended films on oxidized silicon wafers. The characteriza-ion techniques used were Fourier transform infrared spectroscopyFTIR), scanning electron microscopy (SEM) and transmission elec-ron microscopy (TEM). These characterization techniques were

pplied at all steps of the nanocarrier systems preparation processn order to optimize each stage.

To enhance the amount of incorporated drug, a pre-treatmentf the polycationic surface of the nanotubes was optimized to

: Biointerfaces 94 (2012) 288– 295 289

activate the amino groups of the organosilane. Finally, the innervoids of SNTs were used to incorporate the NSAID, naproxen, bymeans of an electrostatic interaction. Naproxen was the chosendrug in this study given its common application in several patho-logical conditions and was used in an attempt to improve itstherapeutic value, preserve its efficiency as an anti-inflammatorydrug and reduce its undesirable gastrointestinal effects. Moreover,naproxen contains a carboxylic group which ionizes at neutral pHand establishes the abovementioned electrostatic interaction withthe polycationic nanotube surface. This electrostatic interactionmay lead to ideal anti-inflammatory drug delivery systems becausethis linkage ensures effective release of the drug in the slightlyacidic inflamed tissues, while maintaining the conjugates stablein the bloodstream (pH 7.4). Therefore, the novel acid-sensitivenanosystems developed were adapted to be employed in inflam-matory conditions and have the potential to be applied to severalother non-steroidal anti-inflammatory drugs (NSAIDs) given thatthe carboxyl group used to incorporate the drug into the devel-oped nanotubes is a common feature of this therapeutic group ofdrugs.

A schematic representation of the main steps involved in thenanodelivery system preparation process is shown in Fig. 2.

The incorporation of naproxen into the nanotubes as well as thedrug release studies were based on fluorescence determinationsand have demonstrated that the nanosystems developed are verypromising candidates for therapeutic applications.

To the best of our knowledge, this is the first report of silica nano-tubes being used as drug delivery vehicles for future applicationsto the treatment of chronic inflammatory conditions. Furthermore,the synthesis and functionalization of the nanotubes as well asdrug loading was successfully achieved and thus opens up furtherexploration of biomedical applications of novel silica nanomate-rials for potential translation to the clinic in the future, since theone-dimensional shape and length of nanotubes easily caters forligands, drugs and multiple molecules to be targeted for synergisticeffects.

2. Material and methods

2.1. Material

The nanoporous alumina templates were fabricated by anodiza-tion of high purity aluminum foils (99.997%).

The precursor for sol–gel (TEOS) and all amino-alkoxysilanesAPTMS, APTES and APDMES were purchased from Aldrich.

Naproxen was obtained from Sigma–Aldrich and was used with-out further purification. Solutions were prepared with HEPES buffer(10 mM, 0.1 M NaCl, pH 7.4).

The release studies were performed in acetate buffer (pH 5.0).All reagents used were the highest purity available commer-

cially. All solutions were prepared with water from a Milli-Q plussystem with specific conductivity less than 0.1 �S cm−1.

3. Methods

3.1. Nanoporous alumina templates

Nanoporous alumina templates were produced through anelectrochemical process in a home-made anodization set-up. Theapparatus used consisted of an electrolyte container made of Teflonwith a stirrer and platinum mesh attached to its lid. The stirrer

was connected to a motor which permitted precise control overthe stirring, thereby maintaining the solution homogeneity andremoving the hydrogen bubbles formed at the anode. The platinummesh represents the cathode in the electrochemical process and the

290 C.T. Sousa et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 288– 295

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ig. 2. Schematic representation of: (a) formation of silica nanotubes (SNTs) insidec) drug loading in the inner voids of silica nanotubes.

ample, the anode where oxidation occurs. A copper cap functionss an electrical contact, being also used to cool the sample throughirculation of a refrigerant. This is needed since lower tempera-ures provide better organization of the alumina pore structure.

oreover, to further improve the structure and organization ofanopores in alumina, the aluminum surface was treated prior tohe anodization process with electropolishing in a HClO4:C2H5OHolution (ratio 1:4) at 10 ◦C and by applying 20 V potential over

min [12]. To obtain nanoporous templates with large areas ofrganized pores, a two-step anodization process was applied [13].he first anodization was carried out for 40 h at 1 ◦C in 0.3 M H2SO4,pplying 25 V. After the first anodization process, a chemical disso-ution of the resulting nanoporous alumina template was achievedy submerging the sample in an aqueous solution of H3PO4 (0.4 M)nd H2Cr2O7 (0.2 M) at 70 ◦C for about 12 h. The second anodizationas then performed using the same conditions described for therst but with different time courses depending on the template’sesired thickness (2.5 �m/h).

.2. Preparation of silica nanotubes by sol–gel process

The silica nanotubes were produced using a template assistedethod and the templates used were obtained with pore diame-

ers of 35 nm. Following their production, the nanoporous alumina

orous alumina templates; (b) silica nanotubes inner surface functionalization and

templates were detached from the aluminum foil placed under-neath using a solution of CuCl2 (II) (0.5 M) and HCl (1 M).

The polymeric silica sol was prepared under acidic conditionsby mixing TEOS, H2O (pH 2, adjusted with HCl) and ethanol at a1:3:1 molar ratio. Further stirring and heating at 70 ◦C for 5 minpromoted silica polymerization and the formation of a sol–gel.

The alumina templates were immersed in the sol–gel for 2 h atroom temperature to promote the adsorption of the silica polymerto the walls of the pores, after which the samples were placed undervacuum conditions for 3 h and then cured at 120 ◦C for 3 h, to evapo-rate the incorporated solution. It is possible that the sol–gel solutionmay become partially attached to the alumina surface, originatinga silica layer in the alumina surface. To remove this, the templateswere washed in ethanol and dried in vacuum after which the tem-plates were further polished with alumina fine powder (averagegrain size of 0.06 �m).

3.3. Surface modifications

The samples (alumina templates containing silica nanotubes)were thermally treated at several temperatures (120, 220, 350, and

500 ◦C) for 5 h, to remove the organic remains and reinforce themechanical structure of the nanotubes.

From this step forward, samples suffered different treatmentsin order to be functionalized with the aminosilanes. The samples

C.T. Sousa et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 288– 295 291

f the a

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ere placed in a solution of 10% aminosilane in ethanol (v/v) andeft overnight to react on a rocking shaker. Then the samples were

ashed with ethanol and bi-deionized water and dried at 120 ◦Cor 3 h.

.4. Characterization of the nanotubes

The size and morphology of the nanotubes were evaluatedy scanning electron microscopy (SEM) analysis in a FEI Quanta00 FEG SEM and by transmission electron microscopy (TEM)

n a LEO 906E Leica. Energy dispersive spectrometer (EDS) wassed to analyse the composition of the prepared samples. In ordero visualize by SEM the nanotubes formed within the pores, the

lumina template was partially dissolved using NaOH (1 M). Thereparation of the samples for TEM measurements included alu-ina dissolution with H3PO4 (0.4 M), centrifugation at 35,000 × g

or 25 min, followed by thoroughly washing with bi-deionized

lumina template (A and B) and respective EDS spectrum (C).

water (conductivity less than 0.1 �S cm−1) and final resuspensionin 0.5 mL of ethanol.

IR spectra were recorded on an ATI Mattson Genesis series FTIR(software: WinFirst v.2.10) spectrophotometer. The samples werecrushed and mixed with KBr of spectroscopic grade making a pellet.All samples had the same weight.

3.5. Incorporation of naproxen into the nanotubes

After dissolving the alumina template by the same method pre-viously described, the nanotubes were resuspended in a solutionof KOH (0.1 M) for 10 min, centrifuged at 35,000 × g for 25 min andwashed with bi-deionized water. The centrifugation and washing

process was repeated three times to make sure that there were noresidues of KOH left in the nanotubes. Then the nanotubes wereresuspended in a Hepes buffered solution (pH 7.4) of naproxen(10 �M) and incubated in the dark for 2 h with continuous agitation.

2 aces B: Biointerfaces 94 (2012) 288– 295

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n order to take of the excess of naproxen that was not incorporated,he suspension was washed again with bi-deionized water and cen-rifuged three times. Finally the nanotubes were suspended in 1 mLf Hepes buffer.

.6. Drug incorporation and drug release studies

Once naproxen is a fluorescent compound, the incorporationnd release studies were performed by fluorescence analysis. Alluorescence measurements were conducted in a Perkin Elmer LS-0 B spectrofluorimeter equipped with a constant temperature cellolder. All data were recorded at a controlled physiological temper-ture (37.0 ± 0.1 ◦C) in a 1 cm path length cuvette. Excitation andmission wavelength were set to 271 nm and 356 nm, respectively,orresponding to the maximum excitation and emission peaks ofaproxen. For each measurement, fluorescence emission was auto-atically acquired during 50 s.In order to measure the drug released from the nanosystems,

he total concentration of naproxen encapsulated in the nanotubesas in compliance with the sink conditions. The suspension of the

unctionalized nanotubes loaded with the drug was washed, cen-rifuged and resuspended in acetate buffer (pH 5.0) or Hepes bufferpH 7.4). The drug release from the nanotubes was monitored for

h with integration time measurements of 10 s each 10 min.

. Results and discussion

This work describes a detailed and straightforward procedureor the preparation of silica nanotubes combining both anodizationnd sol–gel techniques. By using such template-assisted methods,ilica nanotubes can grow inside the alumina templates by fill-ng the pores with a sol–gel solution. Furthermore, the methodresented here caters for an accurate tailoring of the nanotubes,egarding their required dimensions, size homogeneity and dis-inct functionalization of both inner and outer walls. Concerninghe dimensions of the nanotubes, the pore diameter can vary from0 nm to 200 nm while length can vary from a few nanometerso several micrometers by simply controlling the applied potentialnd anodization time, respectively. In this work, the optimizationf the inner surface functionalization was performed in silica nano-ubes of 35 nm diameter and 100 �m length. The increased length ofhe nanotubes used was aimed at signal enhancement in the char-cterization techniques which included, as previously mentioned,EM, EDS and FTIR analysis.

Both the SEM images and EDS spectrum presented in Fig. 3 asell as the TEM image presented in Fig. 4 clearly show the forma-

ion of the silica nanotubes inside the nanopores of the aluminaemplates. Fig 3A and B shows a SEM image of silica nanotubes

ig. 4. TEM image of a silica nanotube after total removal of the alumina template.

Fig. 5. FTIR spectra of the sol–gel precursor solution (a) and the alumina templatewithout (b) and with silica nanotubes (c). Dashed lines indicate the most relevantpeaks.

prepared in alumina templates with a pore diameter of 35 nm andpore depth of 100 �m. The samples show tubular morphologies inhomogenous large areas.

Chemical composition of silica nanotubes analysis was done viaEDS. As expected, since the samples are formed on the aluminatemplate, the EDS image (Fig. 3C) shows high aluminum (Al), oxy-gen (O) and silicon (Si) content. Carbon (C), sodium (Na) and sulfur(S) were also found and correspond respectively, to the carbon gluestrip used to fix the samples for SEM analysis, the sodium hydrox-ide used to partially remove the alumina template and the sulfateion adsorbed to the alumina surface.

Silica nanotubes were also characterized by FTIR and their spec-trum is presented in Fig. 5. Vibrational modes characteristic of silicawere observed, namely the stretching mode at 850–1300 cm−1

(Si O) [14]. The highest peak at about 3600–3200 cm−1 corre-sponds to the stretching mode of the OH groups [14,15]. Peakscorresponding to the vibration modes of oxygen atoms in silicacompounds (Si O Si) [14,16] were also identified: torsional (in-plane bending at about 810–780 cm−1) and stretching (symmetricat about 460–400 cm−1 and asymmetric at about 1200–1100 cm−1).

The condensation reaction used to produce the silica nano-tubes generates water and ethanol and can continuously proceedwith the resulting production of silicon containing alkoxyl andhydroxyl groups [6]. To remove the alkoxyl groups derived fromthe polymerization process, the silica nanotubes were calcined.The temperature of the calcination step had however to be pre-viously tested. Indeed, submitting the SNTs to high temperatures(500 ◦C during the calcination process) could lead to the completeremoval of the OH groups, thereby preventing future functional-ization. Consequently, several temperatures were used to establishthe adequate temperature at which to induce calcination withoutcompromising the number of OH groups available. A sequence oftransmission FTIR spectra of these samples is shown in Fig. 6.

Some changes in surface chemistry were observed as a conse-quence of the heat treatments. At lower temperatures from 120to 220 ◦C, physically adsorbed water molecules desorb from theporous silica surface (dehydration). After dehydration, the silicasurface is covered with vicinal and isolated silanols. Additionaltemperature increments will give rise to dehydroxylation. Firstly,

the vicinal hydroxyls react and form isolated silanols and silox-ane bridges on the surface. Most of such silanols react within aquite narrow temperature region of 200–400 ◦C [17]. However, thedehydroxylation reaction of vicinal silanols may begin at 110 ◦C

C.T. Sousa et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 288– 295 293

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Fig. 8. Normalized fluorescence spectra of (a) naproxen solution; (b) supernatantresulting from thoroughly washing the nanotubes (with naproxen incorporated)with Hepes buffer (pH 7); (c) suspension of silica nanotubes functionalized with

ig. 6. FTIR spectra of the alumina template with silica nanotubes, submitted toifferent temperatures: 120 ◦C (a), 220 ◦C (b), 350 ◦C (c) and 500 ◦C (d). Dashed lines

ndicate the most relevant peaks.

nd continue up to 600 ◦C [17]. The decrease in number of stronglynd weakly hydrogen-bonded hydroxyl groups on the silica surfaceuring heating is clearly observed in the region of 3670–3200 cm−1

hich corresponds to the stretching mode of the OH groups (Fig. 6).he intensity of bands for strongly hydrogen-bonded and weaklyydrogen-bonded (internal) OH groups decreases with tempera-ure enhancement from 120 to 350 ◦C, above which is about theame. Therefore, the ideal temperature to treat the samples wasonsidered to be around 220 ◦C as it does not compromise theumber of OH groups on the surface and still confers mechanicaltrength.

After optimising the calcination procedure, the modificationf the silica nanotubes with the aminosilanes was made whilehe nanotubes were still embedded in the alumina template. This

eans that only the inner walls of the nanotubes were accessibleor chemical modification. Surface modifications were performedsing silanization chemistry.

The FTIR spectra of the resulting nanotubes are shown in Fig. 7.omparison between the structures and volume of the three com-ounds (Fig. 1) suggest that the attachment to a silica surface

ig. 7. FTIR spectra of the bare silica nanotubes (a), and silica nanotubes modifiedith APTMS (b), APDMS (c) and APTES (d). Arrows indicate the most relevant peaks.

APTES and without naproxen; (d) silica nanotubes with naproxen incorporatedwithout the pre-treatment with KOH; (e) silica nanotubes with naproxen incor-porated after pre-treatment with KOH. All samples were prepared in Hepes buffer.

should be similar since there are no significant structural changes.However, APTES presented the most enhanced signals for thepresence of the amino groups. In fact, according to the litera-ture APTES is deposited only as multilayers while APTMS andAPDMS form mono or submonolayers under organic solvent reac-tion conditions [18]. This type of deposition can occur since thetrialkoxysilane compound naturally has a greater probability offorming a three-dimensional network leading to the formation ofthick films during self-assembly [19]. Notwithstanding this, APTESalso has the highest number of molecules per nm2 which in itself,would account for the enhanced signal obtained [19].

Therefore, following functionalization of the nanotubes withAPTES, new peaks characteristic of the amino groups appearedat 1029, 1130 cm−1 and 2372 cm−1, corresponding to the stretch-ing vibration mode of the C N bonds and N H bonds. Moreover,the peak at about 3600–3200 cm−1 which corresponds to thestretching mode of the OH groups is sharper for this compound,which coincides with a decrease in the hydroxyl groups due to theattachment of the aminosilane. Although these described changeswere more evident for APTES functionalization, for all three com-pounds used, it is still possible to observe a shoulder at 3270 cm−1

which corresponds to a stretching mode of vibration of protonatedamino groups, indicating a successful functionalization with all theaminopropylalkoxy-silanes tested.

The incorporation of naproxen in the nanotubes was performedby means of an electrostatic bond. At pH 7.4 (physiological pH ofthe blood stream and healthy cells), the amino group of APTES ispositively charged and the carboxylic group of naproxen is nega-tively charged. Consequently, an electrostatic interaction will beestablished between these groups, allowing the drug molecules tobe incorporated in the inner voids of the nanotubes.

Prior to incorporation of naproxen within the nanotubes, theaminosilane linkers must initially be chemically activated, whichwas achieved by KOH pre-treatment. This pre-treatment stage isnecessary since the terminal amino groups of the functionalizednanotubes (when in contact with air) strongly react with CO2 [20].In fact, CO2 reacts with the terminal NH2 and forms NH2CO3, pre-

venting an efficient coupling between the amino groups from thenanotubes and the carboxyl group from the drug.

Having optimized all conditions, several fluorescence measure-ments were performed with a view to accessing the degree of

294 C.T. Sousa et al. / Colloids and Surfaces B:

Fig. 9. Release profile of naproxen from the inner voids of silica nanotubes at pH 5.0(a

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Araújo also thanks the Fundacão Gulbenkian for its financial sup-

a) and at pH 7.4 (b), assessed by fluorescence measurements. Results are reporteds mean ± SD (n = 3).

aproxen incorporation. Fig. 8 presents the emission spectrum ofaproxen solution, before (Fig. 8a) and after being incorporated intohe nanotubes (Fig. 8d and e). This figure also includes the spectrumf silica nanotubes without drug incorporated (Fig 8c) and the spec-rum of supernatant resulting from washing the nanotubes withrug loaded with a buffer solution of pH 7.4 (Fig. 8b).

The fluorescence spectra confirm incorporation of naproxennto the silica nanotubes. Comparison between the spectrum ofhe naproxen solution before (Fig. 8a) and after (Fig. 8d and e)ncorporation into the nanotubes reveals a significant decrease inuorescence intensity which implies a successful incorporationither into the nanotubes pre-treated with KOH (Fig. 8e) or intohe non-treated nanotubes (Fig. 8d). However, it could be con-luded that pre-treatment significantly increased the incorporationf the drug. Indeed, with pre-treatment, drug incorporation stood atpproximately 40% which corresponds to 165 �g of naproxen whileor the non-treated nanotubes, drug incorporation was significantlyess (20%). This confirms that pre-treatment is an essential step inreparation of the nanosystems.

Further proof of drug incorporation into the nanotubes is pro-ided by the shift observed in maximum emission wavelength. Inact, by observing the spectrum of naproxen incorporated into theilica nanotubes (Fig. 8d and e) the existence of a well-defined peaklightly off (362 nm) the maximum emission of naproxen (356 nmig. 8a) can be seen. This red shift can be attributed to changes inhe chemical environment surrounding the drug molecule due tohe location of this drug inside the nanotube void.

It is also possible to verify that all spectra corresponding to nano-ubes have an increased baseline (Fig. 8c–e). This is due to the lightcattered by the suspension of the nanotubes.

The chemical functionalization of the nanotubes’ inner surfacensures that naproxen remains within the nanotubes, even afterhese nanotubes have been washed several times with a bufferolution of pH 7.4 (which is the physiological pH of the blood-tream and healthy cells). Indeed, the supernatant resulting fromhe washing procedure indicated that no naproxen was releasedFig. 8b).

Having achieved successful incorporation of drug, the release of

he drug within the nanotubes was tested at pH 7.4 and pH 5.0,hich is the pH of inflamed cells. Fig. 9 summarizes these results.

At pH 5.0, the release profile indicates a burst release up to aaximum of 35% naproxen within 60 min (Fig. 9 a). The major

Biointerfaces 94 (2012) 288– 295

factor affecting release ratio is ionic interaction. The strength ofelectrostatic interactions between the drug molecules and theamino groups on the inner surface of nanotubes is mainly deter-mined by the pKa value of the drug, although several other factorsshould be considered such as hydrogen bonding, hydrophobicinteraction and solubility. The pH of the solutions used for drugrelease experiments at pH 5.0 was controlled by an acetate buffersolution and the amine group of APTES was used as counterpart ofionic interaction with drug molecules. Naproxen (pKa 4.2) is onlypartially ionized at pH 5.0 and the much weaker Coulomb inter-action between the negatively charged naproxen and protonatedamino groups on the interior of SNTs contributes considerably tothe partial drug release. Nevertheless, provided the release is opti-mized to occur only in inflamed cells whose pH is 5, the release per-centage of 35% (which corresponds to 57 �g of released naproxen)is significantly higher than that actually needed for an effectivetherapeutic action [21]. This represents no limitation to a thera-peutic application of the nanotubes. Furthermore, the controlledrelease experiments made at pH 7.4 indicate, as expected, thatnaproxen is not released at this pH (Fig. 9b) suggesting that whilein blood circulation the drug will remain inside the nanotubes.

5. Conclusions

In summary, we have successfully synthesized silica nanotubeswith internal diameters as small as 35 nm using a template-assistedmethod combined with sol–gel chemistry. The nanotubes areuniform and present a very high aspect ratio. The temperature-dependence of the density of hydroxyl groups on the surface ofthe silica nanotubes was studied and found to have a significantinfluence on the attachment of other molecules to the surface.

Surface functionalization with the amino-silanes was achievedby a preparation method that was shown to be very effective andsimple. APTES was shown to exhibit the most enhanced signal in theFTIR spectra and was therefore considered to have the most effec-tive bonding to the surface. Hence, the method for fabricating thenanotubes was optimized and the method presented herein pro-vides a detailed description for those conducting research withinthe nanotechnology field.

Incorporation of naproxen within the inner voids of the nano-tubes was also achieved as was its controlled release, by solelychanging the pH conditions of the surrounding medium. Thelinkage between nanotubes and drug was based on electrostaticinteraction which ensures effective release of the drug in theslightly acidic conditions typical of inflamed tissues (pH 5), whilemaintaining the conjugates stable in the bloodstream (pH 7.4).This makes these optimized silica nanotubes an exquisite pH-sensitive delivery system for inflammatory diseases which canlargely diminish the adverse side effects associated with continu-ous use of the NSAIDs. External functionalization of the nanotubeswith folic acid as a vector to inflamed tissues and cellular tox-icity tests will be further required to optimize the nanosystemsdeveloped.

Acknowledgments

The authors are grateful to Prof. Carlos Afonso and Dr. Sara Cravofor help with FTIR measurements. C.N. thanks FCT for the doctoralgrant (SFRH/BD/38445/2007). C.T. Sousa is thankful to FCT for doc-toral grant, SFRH/BD/38290/2007, project FEDER/POCTI n2-155/94and projects POCI/FP/81979/2007, CERN/FP/83643/2008 and. J.P.

port within the “Programa Gulbenkian de Estimulo a InvestigacaoCientifica.” The authors acknowledge funding from FCT through theAssociated Laboratories—IN and REQUIMTE.

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eferences

[1] C. Hawkins, G.W. Hanks, J. Pain Symptom Manage. 20 (2000) 140–151.[2] S.J. Son, X. Bai, S.B. Lee, Drug Discov. Today 12 (2007) 650–656.[3] S.J. Son, X. Bai, A. Nan, H. Ghandehari, S.B. Lee, J. Control. Release 114 (2006)

143–152.[4] R.K. Sharma, S. Das, A. Maitra, J. Colloid Interface Sci. 284 (2005) 358–361.[5] M. Shimada, S. Natsugoe, K. Tokuda, T. Kumanohoso, K. Nakamura, K. Yamada,

T. Aikou, 23rd International Symposium on Controlled Release of BioactiveMaterials 1996 Proceedings, 1996, pp. 387–388.

[6] C. Barbe, J. Bartlett, L.G. Kong, K. Finnie, H.Q. Lin, M. Larkin, S. Calleja, A. Bush,G. Calleja, Adv. Mater. 16 (2004) 1959–1966.

[7] N. Hebalkar, K. Nischala, T.N. Rao, Colloid Surf. B 82 (2011) 203–208.[8] P.K. Jal, S. Patel, B. Mishra, Talanta 62 (2004) 1005–1028.[9] J.M. Qian, X.X. Li, A.L. Suo, Prog. Biochem. Biophys. 29 (2002) 394–397.10] M. Lal, L. Levy, K.S. Kim, G.S. He, X. Wang, Y.H. Min, S. Pakatchi, P.N. Prasad,

Chem. Mater. 12 (2000) 2632–2639.

[[[

: Biointerfaces 94 (2012) 288– 295 295

11] D.T. Mitchell, S.B. Lee, L. Trofin, N.C. Li, T.K. Nevanen, H. Soderlund, C.R. Martin,J. Am. Chem. Soc. 124 (2002) 11864–11865.

12] D.C. Leitao, C.T. Sousa, J. Ventura, F. Carpinteiro, J.G. Correia, M.M. Amado, J.B.Sousa, J.P. Araujo, Phys. Status Solidi C 5 (2008) 3488–3491.

13] H. Masuda, K. Fukuda, Science 268 (1995) 1466–1468.14] E. Pretsch, T. Clerc, J. Seibl, W. Simon, Spring-Verlag (1983), pp. 201–211.15] C. Xu, L.F. Cao, L.F. Zhu, C.Y. Gao, Acta Phys. Chim. Sin. 22 (2006) 451–455.16] J. Serra, P. Gonzalez, S. Liste, C. Serra, S. Chiussi, B. Leon, M. Perez-Amor, H.O.

Ylanen, M. Hupa, J. Non-Cryst. Solids 332 (2003) 20–27.17] G. Fink, B. Steinmetz, J. Zechlin, C. Przybyla, B. Tesche, Chem. Rev. 100 (2000)

1377–1390.18] K.M.R. Kallury, P.M. Macdonald, M. Thompson, Langmuir 10 (1994) 492–

499.19] J.H. Moon, J.W. Shin, S.Y. Kim, J.W. Park, Langmuir 12 (1996) 4621–4624.20] B.A. McCool, W.J. DeSisto, Adv. Funct. Mater. 15 (2005) 1635–1640.21] F. Lapicque, R. Jankowski, P. Netter, B. Bannwarth, C. Guillemin, M.C. Bene, C.

Monot, M. Wayoff, J. Pharm. Sci. 79 (1990) 791–795.