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A General Strategy for Dual-Triggered Combined Tumor Therapy Based on Template Semi-Graphitized Mesoporous Silica Nanoparticles

Yi Wang , Kaiyuan Wang , Xueying Yan , and Rongqin Huang *

Y. Wang Center of Analysis and Measurement Fudan University Shanghai 200433, China K. Y. Wang, Prof. X. Y. YanSchool of PharmacyHeilongjiang University of Chinese Medicine Harbin 150040, China Prof. R. Q. HuangDepartment of Pharmaceutics School of PharmacyKey Laboratory of Smart Drug DeliveryMinistry of Education Fudan University Shanghai 201203, ChinaE-mail: rqhuang@fudan.edu.cn

DOI: 10.1002/adhm.201300324

Treating cancer by combining the controllable chemotherapy and near-infrared light (NIR)-mediated photothermal therapy has now become a hot topic due to the potential such as over-coming or reducing multi-drug resistance, improving the anti-cancer therapeutic effect, and minimizing the invasive damage to normal tissues. [ 1 ] Many novel nanomaterials based on noble metal (e.g., gold nanoparticles, [ 2 ] Pd nanosheets, [ 3 ] and silver nanostructures) [ 4 ] with localized surface plasmon resonance (LSPR) or carbon nanomaterials (e.g., carbon nanotubes, [ 5 ] gra-phene, [ 6–8 ] and graphitic shell) [ 9 ] with NIR absorbance were developed for the chemo-photothermal combined therapy. How-ever, in order to confi ne the LSPR absorbance of noble metal in the desirable NIR window for the deep-tissue pen etration and avoid the hydrophobicity of pristine carbon nanomaterials, as well as increase specifi c surface area for effi cient drug loading, labor-intensive preparations and complex modifi cations were needed. [ 10–12 ] Furthermore, in most of these chemo-photothermal nanoplatforms, the hotspots were often centralized in the modifi -cators (core–shell structure) such as mesoporous silica. [ 13–15 ] This core–shell structure may depress the photothermal effect and the NIR-mediated drug release.

As a widely used drug delivery vector, mesoporous silica nanoparticles (MSN) had been always pursued due to its fan-tastic properties such as high surface area, large pore volume with tunable pore size, and hydrophilicity. [ 16–18 ] Moreover, the rich silanol groups on MSN surface make it easy to combine with various functional triggers for controllable drug delivery. [ 19,20 ] However, the residual surfactant during the preparation of MSN was still toxic for the biomedical application even after uneco-nomic and time-consuming removing process. [ 21,22 ] Therefore, utilizing the fantastic properties of MSN but escaping from the toxicity of residual surfactant, at the same time, exploiting the

new functions such as controllable chemo-photothermal com-bined therapy, would be of considerable signifi cance.

Herein, a general strategy was developed for synergistic chemo- and photothermal tumor therapy by using template semi-graphitized mesoporous silica nanoparticles (TsGMSN) as a carrier and photothermal agent. The unique structure of TsGMSN, including the semi-graphitized carbon (sGC) as hot-spots on the pore wall, makes water-insoluble anti-cancer drug doxorubicin (DOX) directly contact the hotspots, which simul-taneously achieves effi cient pH- and NIR-triggered release. The strategy was illustrated in Scheme 1 .

TsGMSN was synthesized by utilizing surfactant (cetyltri-methylammonium bromide, CTAB) and micellar swelling agent (1,3,5-trimethylbenzene, 1,3,5-TMB) as templates via base-catalyzed co-condensation of tetraethyl orthosilicate (TEOS), and then in situ graphitizing the template under nitrogen tempera-ture programming. To well study the properties of TsGMSN, template semi-graphitized mesoporous silica nanoparticles synthesized without 1,3,5-TMB (TsG L MSN) and common MSN were prepared as controls (see the Supporting Informa-tion). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that TsGMSN were homo-geneously dispersed spheres with a diameter of 115 ± 20 nm ( Figure 1 A). TEM in Figure 1 B showed that ordered pores were fi tly distributed in TsGMSN and the pore size was measured to be 1.9 nm as shown in high-resolution TEM (HRTEM) (inset in Figure 1 C). X-ray diffraction (XRD) pattern of TsGMSN exhib-ited three resolved diffraction peaks at 2 θ values of 2.73°, 4.65°, and 5.40°, respectively (Figure 1 C), which can be indexed as the typical (100), (110), and (200) refl ections of a highly ordered hexagonal P6mm mesostructure with unit cell parameter ( a 0 ) of 3.73 nm. [ 23 ] This is in good agreement with the HRTEM result (inset in Figure 1 C). N 2 adsoption–desorption isotherm and corresponding pore diameter distribution curve revealed that TsGMSN had a Brunauer–Emmett–Teller (BET) surface area of 769 m 2 g −1 , a large pore volume of 0.78 cm 3 g −1 , and a narrow pore size distribution centered at about 1.9 nm (in consistence with the TEM result) (Figure 1 D and Table S1, Supporting Information). Interestingly, the TsGMSN contained sGC, which was verifi ed by the Raman spectrum and themogravimetric analysis (TGA). As shown in Figure 1 E, Raman spectrum gave obvious signals similar to the symmetry A1g mode and the E2g mode of sp 2 carbon atoms at 1330 cm −1 and 1580 cm −1 (D- and G-band), respectively. [ 24 ] And the TGA result in Figure 1 F proved that approximately 7.2% weight loss (the content of sGC in TsGMSN) happened at the similar temperature range with that of the decomposition of defective graphene. [ 25 ] The thicker pore wall of TsGMSN (1.83 nm) than that of MSN (1.48 nm)

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(Table S1, Supporting Information) proved that the sGC in TsGMSN by the in situ template carbonization was mainly dec-orated on the inner wall of mesopores and its thickness was estimated to be 0.35 nm.

Notably, 1,3,5-TMB used for preparation of TsGMSN played two roles while did not apparently change the morphology (Figure S1, Supporting Information). First, it was used as a pore swelling agent to expand the mesopore size. [ 26 ] As shown in Figure S1,S2 and Table S1 (Supporting Information), the pore size of pure MSN (about 1.7 nm) was smaller than reported 2–4 nm, [ 26,27 ] due to the shrinkage of unit cell under high tem-perature treatment. [ 28 ] However, the pore size of TsGMSN can reach about 1.9 nm even after decoration with sGC in the inner wall, demonstrating the size expansion function. This was benefi cial for high loading of guest molecules. Second, 1,3,5-TMB acted as the carbon source to increase the content of sGC (Figure S3, Supporting Information, 5.5% in TsG L MSN).

As a result that the hydrophobic sGC was decorated on inner pore wall of the hydrophilic mesoporous silica, TsGMSN was expected to be a good therapeutic vector. First, TsGMSN exhibited a mean dynamic light scattering (DLS) diameter of 122.4 nm (Figure S4, Supporting Information), which was close to the size of single particle in TEM result (Figure 1 ). Second, TsGMSN had a zeta potential of −44.6 mV, which would lead to large repulsion force between particles. [ 26 ] Third, TsGMSN did not apparently deposit until 12 h especially at the low con-centration (20 μ g mL −1 ) (Figure S5, Supporting Information). Fourth, hemolytic test (Figure S6, Supporting Information) demonstrated that TsGMSN showed less than 5% hemolysis even at the high concentration (500 μ g mL −1 ). These results suggested that TsGMSN possesses preliminary water disper-sity and biocompatibility. [ 26,29 ] However, to further improve the water dispersity and increase in vivo circulation time, it would be necessary for the surface functionazation such as PEGyla-tion [ 30 ] or phosphonate modifi cation. [ 31 ] More importantly, TsGMSN had higher NIR absorbance as compared with MSN and TsG L MSN at 808 nm ( Figure 2 A) and allowed for more effective photothermal heating of solutions (Figure 2 B). Since the main difference of these nanoparticles was the content of sGC, it can be concluded that sGC was the hotspots for pho-tothermal effect. Although the hotspots within TsGMSN were

not in high content (only 7.2%) and high graphitization extent, their regular distribution in dispersed nanoparticle assigns TsGMSN superior photothermal effi ciency. For example, at a concentration above 50 μ g mL −1 , rapid photothermal heating to the cell photoablation limit of 50 °C occurred within 2 min irra-diated using an 808-nm laser at 6 W cm − 2 (Figure 2 C). More-over, the photothermal heating effect of TsGMSN exhibited concentration-dependent (Figure 2 C) and laser power intensity-dependent (Figure S7, Supporting Information) manner. These effects will benefi t for controllable photothermal therapy.

Additionally, TsGMSN can stably load large amount of DOX, resulting TsGMSN-DOX drug delivery system (TsGMSND). Around pH 9.0 with initial DOX concentration of 0.25 mg mL −1 , a DOX loading capacity of 3.53 ± 0.01 μ g μ g −1 (DOX/TsGMSN weight ratio) was achieved (Figure S8, Supporting Informa-tion). The high loading capacity can be mainly attributed to the adsorption of graphitic mesopores and the supramolecular π – π stacking interaction of sGC. Some other mechanisms such as the close packing of DOX or clustering between DOX and TsGMSN might also be included. [ 32 ] As shown in Figure 3 , TsGMSND exhibited pH-dependent and NIR-mediated release behavior, which can be attributed to pH-triggered reduction and heat-stimulative dissociation of the strong interaction between DOX and sGC. Because DOX directly contacted with the hot-spots in TsGMSN, the obvious photothermal promoted release was simply realized by NIR irradiation (see the Supporting Information). Meanwhile, the pH- and NIR-triggered release demonstrated synergistic effect. For example, the cumulative release at 120 h enhanced by NIR irradiation was 2.7%, 4.0%, and 11.3%, respectively, at pH 7.4, 6.0, and 5.0. Furthermore, the cumulative release was less than 30% even at 120 h, which achieved great sustained release propertie. These features would be appreciated for in vivo application. First, along with the high DOX-loading capacity, the sustained release might be benefi cial for reducing the dosage of TsGMSND in vivo when maintaining the pharmacodynamic effect. As known, the in vivo accumulation of inorganic materials is still a problem due to their non-biodegradability. [ 33 ] Second, the pH- and NIR-triggered release would be accomplished due to acidic tumor/cell microenvironment [ 34 ] and controllable NIR irradiation, for reducing unwanted side effects to normal tissues.

Scheme 1. The general strategy for pH- and NIR-triggered, synergistic chemo-photothermal therapy based on TsGMSND.

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In virtue of fascinating structure and properties, TsGMSND can be exploited for tumor chemo-photothermal therapy. In this study, the combined therapeutic effect of TsGMSND was evalu-ated using glioma U251 cells. First the qualitative cytotoxicity with different treatments was determined by confocal micros-copy. As shown in Figure 4 , at all tested concentrations, chemo-photothermal therapy resulted in the highest cytotoxicity com-

pared to single chemotherapy or photothermal therapy, whereas TsGMSN exhibited no obvious toxicity to glioma cells. And the cytotoxicity increased with the dose of TsGMSN. However, the effect for lethal attack of glioma cells by the photothermal treatment, as deduced from the red fl uorescence signals, was much better than that by chemotherapy, suggesting the lower drug tolerance for photothermal therapy than chemotherapy.

Figure 1. A) SEM image, TEM image, and corresponding particle size distribution curve (insets) of TsGMSN. B) TEM images of multi- and single (inset) TsGMSN. C) XRD pattern and HRTEM image (inset) of TsGMSN. D) N 2 adsorption–desorption isotherm and pore size distribution curve (inset) of TsGMSN. E) Raman spectrum of TsGMSN. F) TGA curve of TsGMSN.

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These facts were also proved by the quantitative cell viability assay as revealed in Figure 5 . The IC 50 of each treatment was shown in Table S2 (Supporting Information), and the combi-nation index (CI) of chemotherapy and photothermal therapy

Figure 4. Confocal microscopy of glioma cells via LIVE–DEAD staining under different treatments. For each treatment, the left panels represent the dead cell images, and the right panels indicate corresponding live–dead cell images. The concentration was quantifi ed with the mass of TsGMSN. Bar = 75 μ m.

Figure 2. A)UV–vis absorption curves and B)Photothermal heating curves (6 W cm − 2 NIR at 808 nm) of different vectors at 50 μ g mL −1 . C) Photo-thermal heating curves (6 W cm − 2 NIR at 808 nm) of TsGMSN solution with different concentrations. Data were expressed as mean ± S.E.M. ( n = 3).

Figure 3. Cumulative release profi les of DOX from TsGMSN at different pH with or without 6 W cm − 2 NIR irradiation. Data were expressed as mean ± S.E.M. ( n = 3).

based on TsGMSND was 0.589 (<1), demonstrating the syner-gistic therapeutic effect. [ 35 ] Notably, the prepared TsG L MSND by the general strategy also has the function of dual-triggered combined tumor therapy, but the effect was lower than that of TsGMSND (Figure S9,S10, Supporting Information), which might be attributed to the relatively smaller pore size and lower content of sGC.

In summary, TsGMSN was synthesized and developed as the platform for combined glioma therapy. This is a general strategy which can realize many merits: 1) preliminary water dispersity and nontoxicity due to the biocompatible mesoporous silica surface and the effective elimination of toxic surfactant (CTAB), 2) high loading capacity (at high pH value) and sus-tained release (at low pH value) of DOX achieved mainly by the adsorption of mesopores (hydrophobic interaction) and the π – π stacking interaction with semi-graphitized pore wall, 3) high photothermal heating effect resulting from the regular distribu-tion of the hotspots in TsGMSN particles, and 4) sensitive pH- and NIR-triggered release due to the direct contact of DOX with the hotspots. In addition, the method was very simple and the prepared TsGMSN can facilitate further functionalization for targeted drug delivery or imaging. Conclusively, this work pro-vides a simple strategy for effective synthesis of a robust bio-compatible platform for chemo-photothermal tumor therapy.

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Most charmingly, this strategy is general and can be extended for other MSN-based materials.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was supported by the grants from National Key Basic Research Program (2013CB932502) of China (973 Program) and “Zhuo Xue” Talent Plan of Fudan University.

Received: August 2, 2013 Published online: September 1, 2013

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