Microporous and Mesoporous Materials 147 (2012) 200–204

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Microporous and Mesoporous Materials

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A biocompatible drug delivery nanovalve system on the surfaceof mesoporous nanoparticles

Li Du, Huiyu Song ⇑, Shijun LiaoSchool of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e i n f o

Article history:Received 11 May 2011Received in revised form 12 June 2011Accepted 20 June 2011Available online 30 June 2011

Keywords:Mesoporous nanoparticlesNanovalveBio-stabilityDrug delivery

1387-1811/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.micromeso.2011.06.020

⇑ Corresponding author. Tel.: +86 20 87113586; faxE-mail addresses: hysong@scut.edu.cn (H. Song), c

a b s t r a c t

A biocompatible nanovalve attached to the surface of MCM-41 mesoporous nanoparticles is designed torelease encapsulated guest molecules controllably under pH activation. This nanovalve system is com-prised of a-cyclodextrin (a-CD) rings that encircle p-anisidino linkers, and can be tuned to respond underspecific pH conditions through chemical modification of the linkers. One of the distinctive features of thisfunctional nanovalve system lies in its excellent bio-stability and durability in cell culture medium solu-tion, the binding between the a-CD and p-anisidino groups was not interrupted or disintegrated by theproteins in the DMEM solution without adjusting the pH value. Luminescence spectroscopy demonstratesthat the on-command pH-activated system displays very good bio-stability—no drug leakage at pH �7.4and excellent drug release performance not only in H2O but also in cell culture medium at pH �5.5.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Mesoporous silica nanoparticles have attracted increasingattention due to their potential applications in adsorption, separa-tion, catalysis, drug delivery, sensors, photonics, machines, andnanovalves [1]. Recently, some reports have demonstrated thatboth molecular and supramolecular valves can regulate the releaseof cargo molecules from mesoporous nanoparticles using redoxchemistry [2–4], pH [5–8], competitive binding [9], and light[10–12] as actuators. These systems rely on the switching of com-ponents on the nanoparticle surfaces that have been modified suchthat the entranceways to the nanopores can be opened and gatedon demand [13,14]. Although these nanovalve systems functionexceptionally well, some of them still exhibit a degree of cargoleakage or must be used in organic solvents. Therefore, we hopeto construct a nanovalve with biocompatible components thatcan be operated in solvents compatible with physiological condi-tions, while simultaneously allowing controlled drug release,which is crucial for therapeutic applications.

The ability to use mechanised mesoporous nanoparticles asdrug delivery carriers is due not only to their porous performancefor drug storage, but also to their uniform small size that allows foreasy uptake by lysosomes in cancer cells (the size of lysosomesvaries from 0.1 to 1.2 lm) [15]. It should be noted that themechanised nanoparticles are transmitted within the blood orextracellular fluid before their uptake by cancer cells. As is well

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: +86 20 87113586 808.hsjliao@scut.edu.cn (S. Liao).

known, the pH within blood and extracellular fluid isapproximately 7.4 [16], and the human cytosolic pH range is 7.0–7.4 [17], which necessitates that the nanoparticles not releasethe drugs at pH �7.4. Interestingly, mechanised nanoparticles car-rying drugs can first be phagocytosed by lysosomes after penetrat-ing the cell membrane of the cancer cell. However, the pH withinlysosomes is 4.8–6.4, substantially less than that of cytosol (pH�7.4), which may be very favorable to drug release from a nanov-alve system. Hence, a pH-responsive drug delivery nanovalve sys-tem is urgently needed for therapeutic applications, which requiresnanoparticles to easily release their drugs within the lysosomesunder slightly acidic conditions, after which the drugs can continueto disperse in the cytosol and carry on attacking the cancer cell.

Previously, we reported a rotaxane-based ‘nanovalve’ to controlthe escape of drugs from the pores of hollow silica nanoparticles[18]. Construction of that pH-responsive ‘nanovalve’ relies on thehydrogen bonding interaction between a-CD and a linker moleculecontaining an aniline group. Herein, based on our previous work,we mainly investigate the bio-stability and capability of a biocom-patible pH-responsive nanovalve using MCM-41 mesoporousnanoparticles as the supporting framework, propidium iodide (PI)as the cargo, and a-CD as the capping agent. Luminescence studieshave demonstrated that the release of cargo molecules occurswhen the pH in the external medium is tuned down from neutralto acidic, as p-anisidino nitrogen (on the linker) is being proton-ated. This nanovalve system has been tested and proven functionalin water as well as in cell culture medium. Adding potential bio-applicabilities to the list of key attributes—commercial availability,versatility, functionality in mild pH conditions, and simplicity of

L. Du et al. / Microporous and Mesoporous Materials 147 (2012) 200–204 201

design—this new pH-responsive nanovalve paves the way forfuture therapeutic applications.

2. Experimental section

2.1. Materials

Analytical and reagent grade materials were used as purchased:tetraethoxysilane (TEOS) (98%, Aldrich), cetyltrimethyl-ammo-nium bromide (CTAB) (P98%, Aldrich), toluene (PhMe) (P99.5%,EMD), methanol (MeOH) (200 proof, pharmaco-AAPER), 3-iodopro-pyltrimethoxysiliane (IPTMS) (P95%, Gelest), p-anisidine (99%, Al-drich), propidium iodide (PI) (P95%, Aldrich), and a-cyclodextrin(a-CD) (P98%, Aldrich). All reagents were used without furtherpurification.

2.2. Instrumentation

Transmission electron microscope (TEM) images were collectedon a JEM1200-EX (JEOL) machine provided by the California Nano-System Institute (CNSI). X-ray diffraction (XRD) patterns were col-lected using a Philips X’Pert Pro diffractometer equipped withCuKa radiation. All solid-state NMR experiments were performedon dry powder samples using a Bruker DSX-300 MHz spectrometerand with a 4 mm double-resonance Bruker probe head. Zirconiumoxide 4 mm rotors were used with Kel-F caps for all measure-ments. The controlled release profiles were obtained via lumines-cence spectroscopy using an Acton SpectraPro 2300i CCD and acoherent Argon Innova 90C-5-excitation laser.

2.3. Preparation of MCM-41 nanoparticles

The nanoparticles templated by cetyltrimethylammonium bro-mide (CTAB) were synthesized according to a procedure publishedelsewhere [19]. Through solvent extraction using concentrated HClsolution (12 M, 9 mL) and refluxing under heat for 24 h, CTAB wasremoved from silica particles (1.5 g) that were suspended in MeOH(160 mL). The precipitate was collected using vacuum filtrationthen washed with additional MeOH, and the final product was ob-tained after drying in a vacuum.

2.4. Construction of supramolecular nanovalves

In order to achieve a p-anisidino modified silica surface, meso-porous nanoparticles (100 mg of MCM-41) were first treated withlinker molecules (0.1 mmol of IPTMS) in dried toluene (10 mL) andallowed to reflux under heat for 24 h. Then p-anisidine (1.0 mmol)and triethylamine (3.0 mmol, as catalyst) were added to the IPTMSfunctionalized particles in toluene solution, and the reaction mix-ture was heated under N2 at reflux for another 2 days. Lastly, thep-anisidino functionalized material was then collected using vac-uum filtration, followed by additional washing with toluene andMeOH, prior to drying in a vacuum. Cargo molecules—propidiumiodide (1 mM aqueous solution)—were loaded into the nanoporesby diffusion for 24 h in RT. Finally, the PI-loaded, p-anisidino func-tionalized nanoparticles were capped with a-CD (0.2 g). The result-ing mixture was stirred for 1 day in RT prior to further washing,filtering, and drying.

2.5. Controlled release experiments

A sample of PI-loaded, a-CD capped nanovalve nanoparticles(4 mg) were placed in the corner of a cuvette. Distilled H2O(7 mL) or cell culture medium (DMEM, pH �7.4, 7 mL) was care-fully added into the cuvette so as not to agitate or disarrange the

nanoparticles. The emission of PI in the solution above the particleswas measured as a function of time by using a 514 nm excitationbeam (10 mW) to excite the drug molecules as they were releasedfrom the nanopores. The PI emission spectrum was recorded as afunction of time at 1-s intervals. Release profiles were obtainedby plotting the luminescence intensities of PI at the emission max-imum (650 nm) as a function of time. Activation of the nanovalveswas accomplished by adjusting the pH value of the solution to �5.5with the addition of HCl solution (0.1 or 1 M). The solution wasgently stirred in the cuvette throughout all controlled releaseexperiments.

3. Results and discussion

The size, dispersion and pore structure of MCM-41 nanoparti-cles were evaluated using transmission electron microscopy(TEM), as shown in Fig. 1. TEM images (Fig. 1A–C) shows that bothunfunctionalized and functionalized nanoparticles have very goodmonodispersion and homogeneous particle size, even though theyare p-anisidino functionalized, PI loaded and a-CD capped. Fur-thermore, the size of all the nanoparticles is around 100 nm, whichis obviously smaller than lysosomes and some cancer cells. There-fore, the nanoparticles are extremely likely to be taken up by thecells. The ordered hexagonal pore structure of MCM-41 nanoparti-cles can be seen in Fig. 1D. It should be noted that the p-anisidinofunctionalized nanoparticles also possess ordered hexagonal porestructure (see Fig. 1E), it means that the pore structure of the nano-particles could not be changed by linkers molecules functionalized.However, the nanoparticles after PI loaded and a-CD capped exhi-bit a less than clear pore structure because they contain PI, a-CD,and linker molecules (see Fig. 1F).

Fig. 2 shows the small-angle XRD patterns of pure MCM-41nanoparticles, p-anisidino functionalized nanoparticles and a-CDcapped nanoparticles. Clearly, there exist four distinct diffractionpeaks at 2h � 2.45�, 4.05�, 4.66�, and 6.05� indexed to (1 0 0),(1 1 0), (2 0 0) and (2 1 0) planes, respectively, indicate that purenanoparticles have a highly ordered 2D hexagonal symmetry (seecurve A in the Fig. 2). Compared with pure nanoparticles, the inten-sity of the diffraction peaks appears to be a little weaker after p-anisidino functionalized. Furthermore, the nanoparticles stillmaintain characteristic reflection of d100 (see curve B in theFig. 2). This implies that p-anisidino functionalized nanoparticlesstill retain an ordered 2D hexagonal structure, coincident withthe ordered pore structure of TEM images. However, the intensityof the diffraction peaks decreases sharply after a-CD capped withp-anisidino functionalized nanoparticles. Up to now, we have notthought out a reasonable explanation for this phenomenon, andwe will try to get it in our future work.

To check their functionalized state, the nanoparticles’ 13C CP/MAS SSNMR was verified using SSNMR. The 13C CP/MAS SSNMRvalues of pure particles and of MCM-41 nanoparticles functional-ized by different linkers were obtained (see Fig. 3). The unfunction-alized nanoparticles have three peaks in the aliphatic region, whichare attributable to the reaction of free alcohol (MeOH and EtOH)—formed by the condensation reaction—with surface silanol groups(Fig. 3A) [20]. The p-anisidino linker functionalized nanoparticles(for chemical structure, see Fig. 3C) were gained by using a two-step synthesis method. First, the pure silica was modified by 3-iodopropyltrimethoxysiliane (IPTMS). The 13C CP/MAS SSNMRspectrum of IPTMS functionalized nanoparticles has three peaksin the aliphatic region, from the propyl chain (Fig. 3B). To achievethe p-anisidino linker functionalized nanoparticles, the IPTMSfunctionalized nanoparticles were treated with p-anisidine. The13C CP/MAS SSNMR spectrum for the p-anisidino linker functional-ized nanoparticles shows four peaks in the aliphatic region from

1 2 3 4 5 6 7 8 9 10

C

210200110

100

BA

Inte

nsity

(a.u

.)

2 theta (deg)

Fig. 2. The low angle XRD patterns of unfunctionalized nanoparticles (A), p-anisidino functionalized nanoparticles (B) and a-CD capped nanoparticles (C).

Fig. 1. TEM images of MCM-41 nanoparticles. Unfunctionalized nanoparticles are shown in A and D, p-anisidino functionalized nanoparticles are shown in B and E, whilenanoparticles loaded with PI and capped by a-CD are shown in C and F.

202 L. Du et al. / Microporous and Mesoporous Materials 147 (2012) 200–204

the propyl and methoxy groups, and four benzyl peaks in the aro-matic region (Fig. 3C). Hence, this demonstrates that the linkermolecules are successfully tethered to the nanoparticles.

Prior to doing any cell culture studies using this nanovalve sys-tem, we wished to establish the bio-stability of this system in a cellculture medium. The bio-stability of the new nanovalve system un-der biological conditions was verified in three experiments. Thefirst experiment involved leakage monitoring using luminescencespectroscopy on PI-loaded nanoparticles suspended in both H2Oand cell culture medium (DMEM) over a 500-min period. As is evi-dent in Fig. 4, virtually no release of PI was observed over thecourse of the experiment when particles were simply suspendedin either H2O (pH �7.0) or cell culture medium (pH �7.4). Remark-ably, a-CD with �1.5 nm ring could tightly encapsulate thep-anisidino region of the linker, thereby obstructing any departure

of loaded PI (�1.3 nm size) molecules from the �2.5 nm nanoporesof the silica nanoparticles. This experiment demonstrates the bio-stability and durability of the nanovalves system in both H2Oand DMEM solution. Furthermore, these results also imply thatthe binding between the a-CD and p-anisidino groups was notinterrupted or disintegrated by the proteins in the DMEM solution.

The second experiment was conducted to check whether thenanovalves were still operable after a 500-min suspension in cellculture medium. As the pKa value of the p-anisidino nitrogen isapproximately 6, when the external pH drops below this value,a-CD will lose its binding affinity to the p-anisidino group, ulti-mately leading to the release of cargo molecules. As shown inFig. 4A, PI was still capable of being released from the nanovalvesystem in H2O at pH �5.5 after the nanoparticles had been soakedin cell culture medium for 500 min.

Finally, we addressed one of the most important questions inthis set of experiments: Does the nanovalve work in DMEM as itdoes in H2O? The answer is yes. Fig. 4B displays the release profileof PI from the nanovalve system in DMEM solution at pH �5.5.According to these results, this pH-responsive system displays verygood bio-stability––no drug leakage at pH �7.4 and excellent drugrelease performance not only in H2O but also in cell culture med-ium at pH �5.5, which is in the range of pH values withinlysosomes.

To certify the controllable function of a-CD, we carried out acontrol experiment. The PI release of the nanovalve system wastested in the absence of a-CD. As shown in Fig. 5, when the PIloaded nanoparticles were put into the H2O solution, a continualrelease could be observed (Curve a), indicating that the PI mole-cules will quickly escape from the nanoparticles without the con-trolling of a-CD. Furthermore, if the PI loaded nanoparticles werewashed thoroughly, almost no PI release could be observed (SeeCurve b in Fig. 5), even though we lowered the pH value at a shorttime of 15 min, there still no obvious PI molecules release could beobserved, implying that the load PI molecules could be totallywashed out without the controlling of a-CD. It is concluded thatthe loaded PI molecules is very easy uncontrollable released fromthe nanoparticles in the absence of the controlling of a-CD.

NH

SiOO

O

OMe

ISiOO

O

Fig. 3. 13C CP/MAS SSNMR spectra of pure silica (A), and functionalized by different linkers (B–C). All of the samples were dried under vacuum.

+N N+

NH2

H2NI- I-

CH3CH3

CH3

0 25 100 200 300 400 500

0

20

40

60

80

100

pH~7.0

pH~5.5

% R

elea

se

Time (mins)

(A)

(B)

H2O

DMEM

0 25 100 200 300 400 500

0

20

40

60

80

100

% R

elea

se

Time (mins)

pH~5.5

pH~7.4

Fig. 4. pH dependence of the release profiles of PI from nanoparticles in differentsolutions: (A) H2O and (B) DMEM. Inset shows the assembly of pH-responsiveMCM-41 nanoparticles with a PI-loaded, a-CD capped nanovalve system.

0 30 60 90 120 150 180 210

b. Washed

a. No wash

Inte

nsity

(a.u

.)

Time (mins)

Fig. 5. The release profiles of PI from nanoparticles without a-CD capping agent inH2O. (a) The nanoparticles without wash after PI loaded and (b) the nanoparticleswere washed thoroughly after PI loaded.

L. Du et al. / Microporous and Mesoporous Materials 147 (2012) 200–204 203

4. Conclusion

This biocompatible pH-responsive nanovalve was designed andsuccessfully synthesized based on MCM-41 mesoporous nanopar-ticles. Luminescence spectroscopy has been used to demonstratethat this nanovalve was able to contain PI molecules inside thenanopores at neutral pH, and release them when an acidic pHwas being adjusted upon the protonation of p-anisidino nitrogenatoms (part of the linker). No leakage was observed in either H2Oor DMEM solution before the pH was tuned. In addition, TEM re-sults have demonstrated that the nanoparticles possess uniformsmall size and good monodispersion—beneficial features for theiruptake by cells. Finally, from a biological standpoint, the applica-bility of this pH-responsive, supramolecular nanovalve is immense,judging from its performance and bio-stability in cell culture med-ium at a mildly acidic pH value. Efforts are now underway to

204 L. Du et al. / Microporous and Mesoporous Materials 147 (2012) 200–204

explore the applications of this nanovalve by testing its deliverycapability in different types of human cancer cells at lysosomalpH levels.

Acknowledgments

This work was supported by the National Scientific Foundationsof China (NSFC, Project No. 20876062) and the Fundamental Re-search Funds for the Central Universities. We thank ProfessorJeffrey I. Zink and the Zink group, who assisted with the TEM andluminescence spectroscopy measurements.

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