DOI: 10.1002/asia.201100064

Small Mesoporous Silica Nanoparticles as Carriers for EnhancedPhotodynamic Therapy

Jie Zhu,[a] Huixiang Wang,[a] Lei Liao,[a] Lingzhi Zhao,[a] Liang Zhou,[a] Meihua Yu,[b]

Yunhua Wang,[a] Baohong Liu,*[a] and Chengzhong Yu*[b]

Introduction

Photodynamic therapy (PDT) is an important clinical treat-ment for a range of cancers.[1–3] In the overall reaction ofPDT, reactive oxygen species (ROS), such as singlet oxygen(1O2), are generated by cancer-targeting photosensitizers(PSs) upon irradiation. ROS will oxidize biomolecules,create an oxidative stress, and kill the treated cancer cells.In practical applications, PSs that can be activated with redlight are of great interest because red light has deep tissue-penetration depth and the resulting PDT effect can be tar-geted to the area of interest.[4,5] For hydrophilic PSs, theirselective accumulation in a tumor is not high enough forclinical use, thus most existing PSs are hydrophobic andtend to aggregate inside tumor tissue. However, the bio-availability of hydrophobic PSs is rather low and the result-

ing PDT efficiency is hindered.[6] Therefore, much effort hasbeen paid to incorporating PSs into various biocompatibledelivery platforms, such as microcapsules,[7] liposomes,[8] oli-gopeptides,[9] polymeric micelles,[10,11] and nanoparticles.[12]

Because the efficiency of PDT is dependent on both the ir-radiation time and dosage of PSs,[13] a delivery system thatcan increase the dosage of PSs being delivered into cancercells is expected to have great advantages in PDT.

MSNs are a relatively new and biocompatible deliverysystem, with their small particle sizes affording favorable en-docytosis properties, high surface area, and porosity favoringa high loading capacity for carrying a variety of guest mole-cules including hydrophobic ones.[14] Another advantage ofMSNs compared to dense silica coatings comes from theirporous walls, through which ROS generated by PSs loadedin MSNs can be released out of the silica carriers. PSs suchas protoporphyrin IX and palladium–porphyrin were previ-ously modified in the silica framework,[15] either inside thepores or on the outer surface of MSNs.[13,16] Mesoporous-silica-coated NaYF4@silica nanoparticles with a core/shellstructure were also reported for PDT applications.[17] How-ever, the diameters of these MSNs for PS-encapsulation arebigger than 100 nm. It is well-understood that the cellularuptake of particles is size-dependent[18–20] and the diametersof ideal delivery particles are smaller than 100 nm.[21, 22]

Zhang et al. reported a highly efficient multifunctionalnanocomposite (ca. 50 nm in diameter) that contains a non-porous, dye-doped silica core for fluorescence imaging and amesoporous silica shell containing PSs, and tested the singletoxygen generation efficieny.[23] Lo and co-workers reported

Abstract: Small mesoporous silicananoparticles (MSNs; ca. 37 nm in di-ameter) have a high loading capacityfor a hydrophobic photosensitizer,SiPcCl2 (82.6 % in weight), and excel-lent endocytosis properties. As a result,the amount of SiPcCl2 being deliveredto cancer cells is increased by approxi-

mately two orders of magnitude com-pared to pure SiPcCl2 at the samedosage, and the photodynamic therapy

(PDT) efficiency is enhanced by overfourfold. Our method can be widelyused to increase the dosage of hydro-phobic anti-cancer drugs in cancer cellsand therefore increase the cytotoxicityof the drugs.

Keywords: mesoporous · photody-namic therapy · photosensitizers ·silica nanoparticles

[a] J. Zhu, H. Wang, L. Liao, L. Zhao, L. Zhou, Prof. Y. Wang,Prof. B. LiuDepartment of Chemistry and Institute of Biomedical SciencesFudan University220,Handan Road, Shanghai, (200433) (China)Fax: (86) 216-564-1740E-mail : bhliu@fudan.edu.cn

[b] M. Yu, Prof. C. YuARC Centre of Excellence for Functional Nanomaterials and Aus-tralian Institute for Bioengineering and NanotechnologyThe University of QueenslandBrisbane, QLD 4072 (Australia)E-mail : c.yu@uq.edu.au

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/asia.201100064.

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that palladium–porphyrins can be covalently bonded toMSNs (ca. 100 nm in diameter) and form a promising cancertheranostic platform[24] Zhao et al. prepared silica nanoparti-cles with diameters of 25–30 nm using triethoxyvinylsilane(TEVS) as a silica source.[25] A PS, silicon phthalocyanine 4,was encapsulated in the silica and the product showed en-hanced PDT efficiency towards melanoma cells.[25] However,the texture property (e.g. nanoporous or nonporous) of suchsilica nanoparticles is not clear, and the use of relatively ex-pensive organosilica source TEVS may also limit their prac-tical applications.

Herein, we report the photocytotoxicity of silicon phtha-locyanine dichloride (SiPcCl2) is greatly increased by usingMSNs nanocarriers with pure silica composition and smalldiameters (ca. 37 nm, measured by electron microscopy).MSNs used in our study have a uniform mesopore size of2.7 nm, and a high loading capacity for SiPcCl2 (82.6 %),owing to their high surface area and pore volume. By com-paring HeLa cells cultivated with SiPcCl2@MSNs and pureSiPcCl2 in the absence of MSNs at the same dosage, thecells cultivated with SiPcCl2@MSNs showed approximatelytwo orders of magnitude higher fluorescence intensity thanthe cells cultivated with SiPcCl2, thereby suggesting the ex-cellent endocytosis property of MSNs. The photocytotoxicitystudies further show that the use of MSNs as nanocarrierscan dramatically enhance the PDT efficiency (>4 fold in-crease in inhibitor efficiency) and cancer cells can be fullyinhibited upon red-light irradiation using our approach.

Results and Discussion

The MSNs were synthesized according to a literature proce-dure[26] using cetyltrimethylammonium chloride (CTAC) asa template, tetraethoxysilane (TEOS) as a silica source, anddiethanolamine (DEA) as an additive agent. A typical trans-mission electron microscopy (TEM) image of calcinedMSNs is shown in Figure 1 a. The average diameter was de-termined to be approximately 37 nm by measuring 100 indi-vidual MSNs directly from the TEM images (Figure 1 b).The dynamic light scattering (DLS) experiments (Figure 1 c)were also carried out to measure the particle size of MSNsin different mediums (Figure 1 c). In both the phosphatebuffer solution (PBS) and Dulbecco�s modified Eagle�smedium (DMEM), the MSNs exhibited an average diameterof 78.8 nm (Figure 1 c1 and c2), larger than that measuredfrom the TEM analysis. In our experiments, calcination wasperformed at 550 8C for 5 hours to remove any surfactants(see the Experimental Section). The large particle size of

the MSNs measured by DLS should be attributed to the par-tial aggregation of MSNs during the high-temperature calci-nation process. Interestingly, when MSNs are dispersed inDMEM supplemented with 10 % fetal bovine serum (FBS)and pure FBS after 6 hours, the particle size of MSNs in-creases to 141.8 and 220.2 nm (Figure 1 c3 and c4), respec-tively, thus indicating that the pure silica MSNs interact withproteins in FBS and form aggregates. Our observation issimilar to that in a previous literature report, where the au-thors further demonstrated that the stability of MSNs in bio-logical systems could be improved by surface modifica-tion.[27] The peaks observed below 30 nm should be attribut-ed to the bovine serum albumin (BSA) in FBS and their ag-gregates.[28]

The MSNs were further characterized using X-ray diffrac-tion (XRD) and nitrogen sorption. Only one broad Braggpeak is observed at 2q= 1.98 (Figure 2 a), thereby indicatinga disordered mesostructure. The nitrogen adsorption–de-sorption isotherms of calcined MSNs are shown in Fig-ure 2 b. The calcined MSNs exhibit a surface area of631 m2 g�1, a large pore volume of 2.0 cm3 g�1, and a narrowpore-size distribution centered at 2.7 nm (Figure 2 c).

The loading capacity of SiPcCl2 in MSNs was quantitative-ly measured using thermogravimetric (TG) analysis (Fig-ure 2 d). Calculated from the weight loss of SiPcCl2@MSNs(41.5 %), SiPcCl2 (89.3%), and MSNs (2.0 %), the loadingpercentage of SiPcCl2 was 82.6 % (per gram of MSNs).After loading SiPcCl2 into MSNs, the XRD pattern ofSiPcCl2@MSNs shows a diffraction peak at the same posi-tion compared to that of pure MSNs (Figure 2 a); however,

Abstract in Chinese:

Figure 1. a) The TEM image of MSNs, b) Histogram showing the size-dis-tribution of MSNs measured from TEM, c) The size-distributions ofMSNs dispersed in PBS solutions (c1), DMEM (c2), DMEM supplement-ed with 10% FBS (c3), and FBS (c4) measured by Nanosizer ZS-90.

Chem. Asian J. 2011, 6, 2332 – 2338 � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org 2333

the intensity of the peak is weaker, consistent with the load-ing of SiPcCl2 into the mesopores and accordingly the de-creased contrast between pore and pore walls. ForSiPcCl2@MSNs, the BET surface area is decreased to213 m2 g�1, the pore volume is reduced to 0.7 cm3 g�1 (Fig-ure 2 b), and the pore size shrinks from 2.7 nm (MSNs) to2.4 nm (Figure 2 c), which can be attributed to the loading ofSiPcCl2 inside the mesopores of MSNs.

The Fourier transform infrared (FTIR) spectra of SiPcCl2

and SiPcCl2@MSNs (Figure 3 a) both show the typical ab-sorption bands of phthalocyanine rings at 1334, 1080, 910,760, and 730 cm�1[29] , thus indicating the stability of SiPcCl2

in the drug-loading process. The SiPcCl2@MSNs also showthe same absorption bands as pure MSNs at 1100 cm�1,which is assigned to the siloxane bond. Owing to the verylow solubility of SiPcCl2 in water, it is difficult to measureits UV/Vis and fluorescence spectra in PBS and DMEM(data not shown). Therefore, toluene was used as the solventto measure the optical properties. The UV/Vis spectrum ofSiPcCl2@MSNs resembles that of pure SiPcCl2; however,the peaks are slightly blue-shifted (Figure 3 b). The absorp-tion spectrum of SiPcCl2@MSNs shows a B-band at 356 nm,an intense and sharp Q-band at 672 nm, together with twovibronic bands at 605 and 642 nm, which are indicative ofnon-aggregated phthalocyanine molecules.[30] For compari-son, the Q-band of SiPcCl2 appears at 694 nm. The fluores-cent spectra of both SiPcCl2 and SiPcCl2@MSNs are alsosimilar to one another (Figure 3 c); however, the main emis-sion peak red-shifts from 678 nm (SiPcCl2) to 683 nm(SiPcCl2@MSNs). The shift in peak positions is thought tobe associated with the change in polarity after SiPcCl2 isloaded into the nanopores of MSNs.

In a PDT process, the generation of 1O2 is mainly respon-sible for the cell death; thus, the 1O2 generation efficiency of

SiPcCl2 and SiPcCl2@MSNs in PBS with the same SiPcCl2

concentration of 40 mg mL�1 was compared using fluores-ceinyl cypridina luciferin analog (FCLA) as a probe mole-cule.[31] Before irradiation with the red light (ca. 5 mw cm�2),the fluorescence intensities of FCLA mixed with SiPcCl2

and SiPcCl2@MSNs were 21 and 23, respectively (Figure 4).After irradiation for 26 minutes, the corresponding fluores-cence intensities increased to 36 and 56 (Figure 4 a and 4 b),respectively. The above results revealed that the efficiencyof photo-oxidation towards FCLA in the case ofSiPcCl2@MSNs is about twice that of SiPcCl2 at the samedosage, thus indicating that SiPcCl2 loaded in MSN candouble the efficiency of 1O2 generation. Our observation isconsistent with a previous report, which suggested thatMSNs act not only as carriers for the PSs, but also as nano-reactors to facilitate the photo-oxidation reaction.[23]

The cytotoxicities and photocytotoxicities of MSNs,SiPcCl2, and SiPcCl2@MSNs toward a human cervical cancerHeLa cell-line have been evaluated using the standard MTTassay. In the dark (Figure 5 a), MSNs exhibit almost no cyto-toxicity to HeLa cells at three concentrations under testing(7.3, 73, 730 mg mL�1; also see the Supporting Information,Table S1), thus showing excellent biocompatibility. BothSiPcCl2 and SiPcCl2@MSNs show very low cytotoxicitytoward HeLa cells at relatively low concentrations (C1, C2;see the Supporting Information, Tables S1 and S2). Evenwhen the concentration of used or loaded SiPcCl2 is as highas 600 mg mL�1 (C3, ca. 100 mm), SiPcCl2 and SiPcCl2@MSNsonly achieve inhibition ratios of 6.3 % and 22.8 % (see theSupporting Information, Table S2), respectively.

Figure 2. a) XRD patterns; b) nitrogen adsorption/desorption isotherms;c) pore-distribution curve determined by the BJH method from the ad-sorption branch data of MSNs (A) and SiPcCl2@MSNs (C); d) TG curvesof MSNs (A), SiPcCl2 (B), and SiPcCl2@MSNs (C).

Figure 3. a) The infrared spectra of SiPcCl2, MSNs, and SiPcCl2@MSNs,b) UV/Vis spectra, and c) fluorescence spectra of SiPcCl2 andSIPcCl2@MSNs in toluene.

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The photocytotoxic activities of MSNs, SiPcCl2, andSiPcCl2@MSNs on HeLa cells after exposure to red-light ir-radiation were studied further (Figure 5 b). The inhibitionratios for pure MSNs are very low in all experiments and nosignificant changes at different concentrations are observed,thus suggesting that MSNs show no photocytotoxicity. Foreach group of SiPcCl2 or SiPcCl2@MSNs, the inhibitionratios increase as a function of the concentration. Moreover,

at a given concentration, the inhibition ratio is significantlyhigher in the presence of red-light irradiation compared tothat in dark (see details in the Supporting Information,Table S3). Importantly, by comparing the photocytotoxicitiesof SiPcCl2@MSNs to SiPcCl2 at the same drug dosage, ap-proximately 6.3 and 7.0 fold enhancements in inhibitory effi-ciencies are observed (C2 and C3, respectively), thus show-ing that the use of MSNs as nanocarriers can dramaticallyenhance the PDT efficiency. At the highest concentration inour experiments, the inhibition ratio in the case of SiPcCl2 isonly 15.0 % at 600 mg mL�1, whilst the use of SiPcCl2@MSNsat 1330 mg mL�1 (loaded SiPcCl2 is also 600 mg mL�1) leads toa much-enhanced inhibition ratio of 104.3 %, thus indicatingthat all of the cancer cells are damaged at this dosage ofphotosensitizer and red-light irradiation.

The efficiency of PDT is dependent on not only a chosenPS and its dosage, but also the intensity of red light and theexposure time. Previously, it was shown that the PDT couldkill cancer cells with high efficiency by using a high-powerlaser.[13,16] However, it has been reported previously that theuse of high-power laser light (130 mw cm�2) will damageblood cells.[32] Other groups used light with a comparativelylower strength. For example, Brevet et al. used a monopho-tonic irradiation (ca. 6 mw cm�2) to stimulate one water-soluble PS,[33] and Xue et al reported a study based on aphthalocyanine photosensitizer irradiated by a light-emittingdiode array with a power density of 6~7 mw cm�2.[34] Never-theless, red light at this level is still difficult to obtain indaily life, thus additional equipment should be applied inPDT. It is noted that in our study, a red light with lowpower (600~710 nm, 0.8 mw cm�2) was chosen. For compari-sion, the power density of red light (600~710 nm) from sun-light was measured to be 3.9 and 0.2 mw cm�2 under directsunshine and under the tree at 14 o’clock in Shanghai inautumn, respectively. The excellent phototoxicity achievedin our study under low fluency (similar to that of moderateheliotherapy) shows not only the advantages of MSNs as thedelivery system, but also the convenience of the PSs/MSNsfor future practical applications of PDT.

To understand how the MSNs promote the PDT efficiencyof SiPcCl2, flow cytometric analysis (FCM) was used tostudy the uptake of SiPcCl2 by HeLa cells in two systems(SiPcCl2@MSNs and pure SiPcCl2). The results of FCManalysis (Figure 6) show that the fluorescence intensity fromHeLa cells cultivated with SiPcCl2@MSNs for 2 hours is ap-proximately two orders of magnitude higher than that culti-vated with SiPcCl2 at the same dosage, which can be attrib-uted to the excellent endocytosis properties and high drug-loading capacity of MSNs, owing to the small particle sizeand large pore volume of MSNs used in our study.

The confocal microscopy images further reveal that thefluorescence intensity from HeLa cells cultivated withSiPcCl2@MSNs is much-higher than that with SiPcCl2

(Figure 7), consistent with the FCM results. As shown inFigure 7 a, the red fluorescence is observed in the blue re-gions more or less in each cell, thereby indicating thatSiPcCl2 has been transported into the nucleus of HeLa cells.

Figure 4. The fluorescence spectra of FCLA in PBS mixed witha) SiPcCl2 and b) SiPcCl2@MSNs after irradiation by red light at differenttimes (1, 6, 11, 16, 21, 26 min, from the bottom to the top).

Figure 5. a) Cytotoxicities of MSNs (A), SiPcCl2 (B), and SiPcCl2@MSNs(C) against HeLa cells in the dark, and b) under red-light (0.8 mw cm�2,8 h) irradiation. C1, C2, and C3 refer to the cytotoxicities measured inthree groups of experiments at different concentrations (for details, seethe Supporting Information, Table S1, S2, and S3).

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Small Mesoporous Silica Nanoparticles as Carriers for Enhanced Photodynamic Therapy

Interestingly, the confocal microscopy Z-scan image seriesof one HeLa cell (Figure 8) clearly revealed that SiPcCl2 hasnot only been transported into the cytoplasma of cells, but

also into the nucleus. On the contrary, this phenomenoncannot be observed in the case of pure SiPcCl2 (Figure 7 b).The subcellular localization of a photosensitizer determinesthe site where the initial photodynamic damage occurs. Thedirect delivery of SiPcCl2 into the nucleus is thought to beanother important factor in explaining the remarkable pho-tocytotoxity of SiPcCl2@MSNs toward HeLa cells. To thebest of our knowledge, SiPc itself cannot enter the nucleus,despite the report that the employment of DNA-targetingplatinum(II) moieties allows SiPc to approach DNA on themolecular scale and execute effective red-light-induced oxi-dative damage to it.[35] Our control experiments at 4 8C (seethe Supporting Information, Figure S2) show no signal forSiPcCl2 in the case of SiPcCl2@MSNs under the same testingparameters, thereby indicating that the endocytosis of MSNsby HeLa cells at 37 8C is mainly through an energy-depen-dent pathway.[36]

Conclusions

In conclusion, we have shown that by loading SiPcCl2 intothe nanopores of MSNs, the 1O2 generation efficiency canbe enhanced. Moreover, MSNs with small particle size andhigh pore volume have high photosensitizer-loading capaci-ty, excellent endocytosis properties, and can deliver photo-sensitizers into cancer cells at a high dosage; thus the PDTefficiency is greatly enhanced. It is expected that further sur-face functionalization of MSNs will allow them to be devel-oped as a targeted drug carrier for efficient cancer therapy.

Experimental Section

Materials

All chemicals were used as received without further purification. SiPcCl2

was purchased from Sigma–Aldrich. Free acid FCLA was purchasedfrom Tokyo Kasei Kogyo Co., Ltd. The HeLa cell-line was provided bythe Institute of Biochemistry and Cell Biology, SIBS, CAS (China).

Figure 6. The fluorescence intensity distributions of HeLa cells (A), andHeLa cells cultivated with SiPcCl2 (B) and SiPcCl2@MSNs (C) after 2 hobtained from flow cytometry Analysis.

Figure 7. Fluorescence confocal micrographs of HeLa cells cultivatedwith a) SiPcCl2@MSNs and b) SiPcCl2 for 3 h at 37 8C. The blue regionsrepresent the signal from the nucleus, the red regions are the signal fromSiPcCl2 and the purple regions are the result of overlapping of the blueand red regions.

Figure 8. The confocal microscopy Z-scan image series of one HeLa cell cultivated with SiPcCl2@MSNs for 3 h at 37 8C.

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CTAC, TEOS, DEA, and ethanol were purchased from Shanghai Chemi-cal Corp.

The Synthesis of Mesoporous Silica Nanospheres

The mesoporous silica nanospheres used in this work were synthesizedaccording to a literature reported previously.[27] Briefly, 7.2 mL of water,0.9 g of ethanol, 0.26 g of CTAC, and 0.02 g of DEA were mixed andstirred in a water bath at 40 8C for 30 min. Then 0.73 mL of TEOS wasadded into the mixture dropwise within 2 min under stirring. The solutionturned white gradually. A further 2 h of stirring was necessary. The milkymixture was allowed to perform dialysis for 48 h. The white powderswere obtained by evaporation of the solution after dialysis. After calcina-tion at 550 8C for 5 h, the final product MSNs were obtained.

The Loading of SiPcCl2

40 mL ethanol was heated to 80 8C. Then 0.1 g of MSNs and 0.1 g ofSiPcCl2 were added to the solution. The mixture was stirred and distilledat 80 8C for 24 h and centrifuged (10 000 rpm) to afford SiPcCl2@MSNs.The obtained SiPcCl2@MSNs were washed with hot ethanol (ca. 80 8C)and centrifuged for several times until the supernatant after centrifuga-tion was almost transparent. Finally, the SiPcCl2@MSNs was dried at100 8C.

Characterization

TEM images were obtained with a JEOL 2011 microscope operated at200 kV. The size-distribution and Zeta potential of particles were mea-sured at 37 8C by Nanosizer ZS-90 (Malvern). Weight changes of theproducts were monitored using a Mettler Toledo TGA-SDTA851 ana-lyzer (Switzerland) from 25 to 900 8C with a heating rate of 10 8C min�1.Nitrogen sorption isotherms were measured at 77 K with a MicromeritcsTristar 3000 analyzer. XRD patterns were recorded on a Bruker D4 X-Ray Diffractometer with Nickel-filtered CuKa radiation (l=1.54056 �) ata voltage of 40 kV and a current of 40 mA. FTIR spectra were measuredwith an Avatar spectrophotometer (FTIR-360) using the standard KBrmethod. UV/Vis spectra were obtained with a UV/Vis Spectrophotome-ter (Agilent, 8453). The fluorescence spectra were measured with a Fluo-rescence Spectrophotomer (Varian, Cary Eclipse). Both the UV/Vis andfluorescence spectra were measured by dispersed 40 mgmL�1 MSNs or18 mgmL�1 SiPcCl2 in toluene.

In the study of the generation efficiency of ROS, SiPcCl2 andSiPcCl2@MSNs were mixed with fresh PBS solutions of FCLA. The finalconcentrations of SiPcCl2 and SiPcCl2@MSNs were 40 mg mL�1 and89 mgmL�1 (containing 40 mg mL�1 of SiPcCl2), respectively. The finalconcentration of FCLA was 14 mmol L�1. The fluorescence of FCLA wasmeasured after irradiation by red light every 5 min. The wavelength ofred light ranged from 600 to 710 nm, and the intensity is ca. 5 mw cm�2.

Photocytotoxicity

Cell-viability following PDT was determined by an MTT assay. Briefly,cells were plated in 96-well flat-bottomed plates at 6� 103 cells per welland allowed to grow overnight prior to exposure to MSNs, SiPcCl2,SiPcCl2@MSNs with different concentrations followed by irradiation withred light. After 24 h of further incubation, the MTT reagent was addedfor 4 h at 37 8C to allow conversion of MTT into a purple formazan prod-uct by active mitochondria. Then the formazan product was dissolved indimethyl sulfoxide (DMSO) and quantified by absorption spectropho-tometry at 490 nm on an enzyme-labeled instrument (SUNOSTIK SPR-960).

Cellular Uptake

To test the uptake of silica nanoparticles, HeLa cells were plated innormal growth medium in 60 mm tissue culture dishes at 5 � 105 cells perdish supplemented with 10% Fetal Bovine Serum (FBS) at 37 8C and5% CO2 and allowed to grow for 24 h. After incubation for 24 h, thecells were washed 3 times with PBS. Then, HeLa cells were separately in-cubated with 40 mgmL�1 MSNs or 18 mgmL�1 SiPcCl2 in a serum-free-medium for 2 h at 37 8C under 5% CO2. Then the cells were harvestedby trypsinization. Flow cytometric analysis was obtained with an EPICS

ALTRA automatic flow cytometry (BECKMAN COULTER). SiPcCl2

was excited by a broadband UV laser (335–365 nm) and fluorescenceemission was collected with a 650 nm long-pass filter.

Subcellular Localization

HeLa cells were grown in Dulbecco�s modified Eagle�s medium(DMEM) supplemented with 10 % Fetal Bovine Serum (FBS) at 37 8Cand 5% CO2. Cells (5 � 108 L) were seeded on 14 mm glass cover slips ina chamber and allowed to adhere for 24 h. After incubation for 24 h, thecells were washed 3 times with PBS. Then, HeLa cells were incubatedwith 40 mgmL�1 MSNs or 18 mgmL�1 SiPcCl2 separately in a serum-free-medium for 3 h at 4 8C or 37 8C under 5% CO2. After incubation, thecells were fixed with 4% paraformaldehyde, and their nuclei werestained with 5 ug ml�1 4’,6’-diamidino-2-phenylindole (DAPI) in 10 %glycerol. Confocal fluorescence imaging was performed with an OlympusFluoView FV1000 confocal laser scanning microscope and a 60 � oil-im-mersion objective lens. A helium/neon laser supplied the 633 nm excita-tion wavelength to excite the SiPcCl2, and a 650 nm long-pass filter wasused to collect the emission.

Acknowledgements

This research was supported by the 973 program (2010CB226901,2007CB714506), the NSFC (20925517), the STCM of Shanghai(10XD1406000,09JC1402600), and the Australian Research Council.

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Received: January 26, 2011Published online: July 8, 2011

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