FULL PAPER DOI: 10.1002/asia.201301333
Effective Cancer Cell Killing by Hydrophobic Nanovoid-Enhanced Cavitation under Safe Low-Energy Ultrasound Yang Zhao,[a] Yingchun Zhu,*[a] Jingke Fu,[a] and Lianzhou Wang[b] Abstract: b-Cyclodextrin (b-CD)capped mesoporous silica nanoparticles with hydrophobic internal nanovoids were prepared and used for effective cancer cell killing in synergistic combination with low-energy ultrasound ( 1.0 W cm2, 1 MHz). The water-dispersible nanoparticles with hydrophobic internal nanovoids can be taken up by cancer cells and subsequently evoke a remarkable cavitation effect under ir-
radiation with mild low-energy ultrasound ( 1.0 W cm2, 1 MHz). A significant cancer cell killing effect was observed in cancer cells and in a mouse xenograft tumor model treated with the nanoagents together with the lowKeywords: cancer · cavitation · low-energy ultrasound · mesoporous materials · nanoparticles
Introduction
pressure (ca. 1000 atm), and ultrahigh temperature (ca. 5000 K) as well as a shockwave and liquid jet in the microenvironment when the irradiation intensity is above the threshold level,[5] which could lead to irreversible cell death through coagulative necrosis.[6] However, the LEUS ( 1.0 W cm2, 1 MHz) used in the present study cannot induce a remarkable cavitation effect and antitumor effect due to the failure to overcome the tensile strength of water to produce a new cavity in the absence of cavitation micronuclei.[7] Much effort has been made to reduce the cavitation threshold and to increase the cavitation effect.[8] Nevertheless, it still remains a great challenge to create a remarkable cavitation effect by LEUS ( 1.0 W cm2, 1 MHz) for effective cancer therapy. In this work, we report an innovative approach for cancer treatment by using water-dispersible nanoparticles with hydrophobic internal nanovoids (nanoagents), which can be taken up by cancer cells. Such nanoagents with internal hydrophobic nanocavities can act as bubble nucleation seeds to drastically amplify the ultrasonic cavitation effect by responding to LEUS. Significant cancer cell killing effects were observed in cancer cells and in a mouse xenograft tumor model treated by the nanoagents together with LEUS. By contrast, antitumor effects were not observed when either LEUS or nanoagents are applied alone.
Safe and effective treatments for cancer have long been a challenging goal for biomedical research.[1] Among various therapies and techniques, low-energy ultrasound (LEUS) defined as therapeutic ultrasound, especially with an intensity below 1 W cm2 and a frequency range of 1–3 MHz, may activate integrin signaling and enhance cellular production of signaling molecules and growth factors via mechanical stimulation of cells (sonostimulation).[2] Thus, LEUS has been widely used in medical imaging, wound healing, and fracture healing.[3] Importantly, the results of some studies suggest that the selectivity of LEUS is higher for malignant cells than for normal cells.[4] Based on the aforementioned safety of LEUS, it is strongly expected as a potential and safe treatment for cancer therapy. In principle, ultrasound with high energy (high intensity and low frequency) can induce a drastic ultrasonic cavitation effect involving a large number of free radicals, extreme [a] Dr. Y. Zhao, Dr. Y. Zhu, Dr. J. Fu The State Key Lab of High Performance Ceramics and Superfine Microstructure Key Laboratory of Inorganic Coating Materials Shanghai Institute of Ceramics, Chinese Academy of Sciences 1295 Dingxi Road, Shanghai, 200050 (P. R. China) Fax: (+ 86) 2152412632 E-mail: [email protected]
Results and Discussion
[b] Dr. L. Wang ARC Centre of Excellence for Functional Nanomaterials School of Chemical Engineering and Australian Institute for Bioengineering and Nanotechnology The University of Queensland QLD 4072 (Australia)
Preparation of b-CD-Capped Hydrophobic Mesoporous Silica Nanoparticles The small-angle X-ray diffraction (SAXRD) pattern and nitrogen adsorption–desorption isotherm show that mesoporous silica nanoparticles (MSNs) and silylanized hydropho-
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301333.
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energy ultrasound, showing a distinct dependence on the concentration of nanoagents and ultrasound intensity. By contrast, an antitumor effect was not observed when either low-energy ultrasound or nanoagents were applied alone. These findings are significant as the technique promises a safe, low-cost, and effective treatment for cancer therapy.
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namic light scattering (DLS) data show that b-CD-capped HMSNs have a narrow size distribution with an overall hydrodynamic diameter of about 220 nm in water (Figure S6, Supporting Information). Moreover, after standing for 24 h and 48 h, their hydrodynamic diameter is about 260 nm and 295 nm, respectively. This result demonstrates that the bCD-capped HMSNs could be stably dispersed in water without aggregate formation.
bic mesoporous silica nanoparticles (HMSNs) have a highly ordered hexagonal structure and classical type-IV N2 adsorption–desorption isotherm (Figures S1 and S2, Supporting Information). The FTIR spectrum and TG-DSC analysis reveal a high surface coverage of the SiMe3 group on surface of the HMSNs (Figures S3 and S4, Supporting Information), and the internal mesoporous surfaces of HMSNs are also silylated by SiMe3 groups to obtain hydrophobic internal nanovoid, as evidenced by the decrease in the BJH pore diameter from 2.5 nm to 2.3 nm by silanization. Previous studies suggest that when two contiguous hydrophobic surfaces are separated, they are connected through gas bridges, and these gas bubbles are stable even up to several micrometers. The interfacial free energy of a hydrophobic surface is lower against gas than against water, so that it is energetically favorable to replace water by gas between the hydrophobic surfaces.[9] The floating of the HMSNs on water indicates their hydrophobic nature due to the absence of water in the hydrophobic mesopores (Figure 1 a). The contact angle between the HMSNs and water in air is about 1408 (Figure 1 b). The HMSNs are re-dispersible in water when being externally capped by b-cyclodextrin (b-CD) (Figure 1 a). b-CD has a hydrophobic cavity (internal diameter of 6.0 and height of 7.9 ), which makes it feasible to cap the hydrophobic SiMe3 groups present on the external surface of HMSNs. Upon binding between the inside hydrophobic cavity of b-CD and the hydrophobic groups of HMSNs, the external hydrophilic groups of b-CD could make HMSNs water-dissolvable (Figure 1 a and 1 f). The highly ordered mesopores remain in the HMSNs and b-CD capped HMSNs (Figure 1 c–e and Supporting Information, Figures S1 and S2). The zeta potential of b-CD-capped HMSNs is 16.8 mV in phosphate-buffered saline (PBS, pH = 7.4; Figure S5 in the Supporting Information). The dy-
Assay of the Intensity of Ultrasound Cavitation It is noted that bubble nucleation is the key process during acoustic cavitation, and in the absence of pre-existing gas cavities, bubble nucleation has to scale a high nucleation energy barrier, which leads to a high cavitation threshold and poor cavitation effect.[8b, 10] The large pore volume (1.275 cm3 g1) of HMSNs with internal hydrophobic channels ensures the entrapment and stability of gas bubbles as nucleation seeds; furthermore, the hydrophobic interface could also reduce the tensile strength of water. As a result, both features could promote the reduction of the cavitation threshold and increase of the cavitation effect.[8a, 9a] The gas bubbles can escape from the hydrophobic pore channels when an external acoustic field is applied. Neglecting vapor pressure, the pressure Pg of gas bubbles entrapped inside the hydrophobic pore channel is given by: Pg ¼ PðtÞ þ 2s cos q=R
ð1Þ
where P(t) is the external pressure, s is the surface tension, q is the contact angle between the liquid and hydrophobic surface, and R is the pore size. The second term accounts for the difference in pressure along the interface (DP). Initially, the liquid–gas interface is concave towards the gas phase and DP is towards the liquid phase to stabilize the gas bubble. Thus, the initial contact angle q is an obtuse angle for this stable state.[10] When an ultrasound wave propagates through the liquid, the total external pressure is given by: PðtÞ ¼ P0 þ PA sinðwtÞ
ð2Þ
where PA ¼
Figure 1. Silica nanoagents with hydrophobic mesopores. a) Conversion of a dispersion of mesoporous silica nanoparticles (MSNs) into b-cyclodextrin (b-CD)-capped HMSNs. b) Contact angle (ca. 1408) between a HMSN and water in air. c–e) TEM images of MSN (c), HMSN (d), and b-cyclodextrin (b-CD)-capped HMSN (e) samples. f) Schematic illustration of b-CD-capped HMSNs; the hydrophobic SiMe3 groups on HMSNs are partially capped by b-CD. g) Fluorescence assay of the cavitation effect in dispersions of MSNs and b-CD-capped HMSNs in PBS after ultrasound irradiation for 2 min (1 MHz, 0.6 W cm2). PBS, control in the absence of nanoparticles.
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pffiffiffiffiffiffiffiffiffiffi 21cI
ð3Þ
Accordingly, the pressure Pg of gas entrapped inside hydrophobic pores is given by: Pg ¼ P0 þ
pffiffiffiffiffiffiffiffiffiffi 21cI sinðwtÞ
þ2s cos q=R
ð4Þ
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where P0 is the static pressure, 1 is the density of the liquid, c is the velocity of sound in the liquid, I is the intensity of ultrasound, PA is the maximum amplitude of the acoustic pressure, and w is the angular frequency of the acoustic wave. The liberation of gas as cavitation nuclei accompanies the change of the contact angle q and movement of the liquid– gas interface. Equation (4) indicates that the contact angle q changes with the time varying of the ultrasound wave. During the negative pressure cycle of ultrasound (P(t) < 0), the pressure Pg inside the gas-filled hydrophobic core is greater than the external pressure. To balance the pressure between the inside and the outside areas, DP changes to point to the gas phase, which causes the liquid–gas interface to become convex towards the liquid phase and the contact angle q to become an acute angle. A further increase in ultrasound intensity could cause the interface to move away from the hydrophobic channels and gas bubbles to be liberated. It is worth pointing out that Equation (4) indicates that a reduction in the surface tension s resulting from the hydrophobic interface and an increase in the ultrasonic intensity would increase the response sensitivity and variation amplitude of the contact angle q when irradiated by ultrasound, which can promote the liberation of free gas bubbles as cavitation nuclei during the negative pressure cycle and sharply enhance the cavitation effect as a response from LEUS. The occurrence of ultrasonic cavitation under LEUS was detected by using fluorescence spectroscopy (Figure 1 g and Figure S7, Supporting Information). Ultrasonic cavitation consequently creates OHC radicals and HC atoms via the dissociation of water;[6b, 11] these radicals can be trapped with non-fluorescent para-phthalic acid to form highly fluorescent hydroxyl(p-phthalic acid).[12] The fluorescence intensity of a PBS solution containing b-CD-capped HMSNs was about 6 times higher than that of PBS solutions with or without MSNs when exposed to ultrasound (1 MHz) with an intensity of 0.6 W cm2 for two minutes (Figure 1 g and Figure S7 a,c, Supporting Information). By contrast, the introduction of either b-CD or hydrophilic MSNs did not lead to an increase in fluorescence intensity of PBS solutions when exposed to ultrasound (Figure S7 b,d, Supporting Information). The results indicate that a remarkable enhancement of the cavitation intensity is caused by silica nanoparticles with hydrophobic mesopores in response to LEUS.
Figure 2. In vitro cellular uptake of b-CD-capped HMSNs (25 mg mL1) in ZR75-30 cells. a) Blue fluorescence image of cell nuclei stained with DAPI. b) Green fluorescence image of FITC-labeled, b-CD-capped HMSNs in cells. c) Overlay image.
The results illustrate a high efficient uptake of b-CD-capped HMSNs by ZR75-30 cells. The in vitro therapeutic effect against ZR75-30 breast cancer cells was evaluated by using the MTT assay. To evaluate the influence of LEUS without nanoagents on the viability of cancer cells, ZR75-30 cells were exposed to ultrasound irradiation with different intensities (1 MHz at continuous cycles and focal distance d = 20 mm). More than 90 % of the cells were viable when the ultrasonic intensity was below 0.8 W cm2, and more than 86 % of the cells were still viable under all test conditions (Figures S8 and S9, Supporting Information). These results show that LEUS ( 1.0 W cm2, 1 MHz) has little effect on the viability of ZR75-30 cells in vitro.[13] Next, in vitro cytotoxicity tests of MSNs and the b-CDcapped HMSNs against ZR75-30 breast cancer cells were performed without ultrasound irradiation. The cell viability was more > 92 % for ZR75-30 cells incubated with MSNs, even at 50 mg mL1 (Figure S10, Supporting Information). The cell viability was > 95 % when the cells were incubated with b-CD-capped HMSNs of 25 mg mL1, and the cell viability was > 92 % when the concentration of the b-CDcapped HMSNs was 50 mg mL1 (Figure 3). Most of the cells were still attached to the plate with a normal morphology in both cases (Figure S11, Supporting Information). Mesoporous silica nanoparticles as a promising drug-delivery carrier have recently been extensively investigated, and most of the obtained data are in line with a negligible cytotoxicity,[14] which is consistent with the findings of this work. In vitro assays of LEUS together with b-CD-capped HMSNs to kill cancer cells were then systematically performed in terms of particle concentration, ultrasound intensity, and irradiation duration (Figure 3). When being irradiated for 20 seconds (1 MHz, continuous cycles and d = 20 mm), the viability of cells cultured with 5 mg mL1 nanoagents decreased from about 98 % to 83 %, 79 %, 58 % and 50 %, respectively, with increasing the irradiation intensity from 0 to 0.4, 0.6, 0.8, and 1.0 W cm2 (Figure 3 a). The cell viability decreased to about 46 %, 35 %, and 10 %, respectively, when the concentration of the nanoagent was increased to 10, 25, and 50 mg mL1 under 1.0 W cm2 irradiation. Obviously, the cell viability moderately decreased with increasing irradiation intensity, and significantly decreased with increasing nanoagent concentration, which is consistent with the measurements of ultrasonic cavitation by fluores-
In Vitro Assay of the Therapeutic Effect and Cytotoxicity To examine the cell-uptake property of the silica nanoagents, b-CD-capped HMSNs were grafted with fluorescein isothiocyanate (FITC) for fluorescence labeling. After treatment with b-CD-capped FITC-HMSNs, ZR75-30 cells were fixed and the nuclei stained with DAPI. The distribution of b-CD-capped FITC-HMSNs in the cancer cells was examined by confocal laser scanning microscopy (Figure 2). After incubation for 12 hours, the b-CD-capped HMSNs were mainly located in the cytoplasm and the perinuclear region.
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Figure 4. Images of ZR75-30 cancer cells exposed to ultrasound from 0 to 1.0 W cm2 for 80 s. a) Cells cultured without nanoparticles (medium). b) Cells cultured with MSNs (50 mg mL1). c) Cells cultured with b-CDcapped HMSNs (50 mg mL1).
However, in the group treated with LEUS and MSNs, the cells retained a normal morphology (Figure 4). Morphological evaluation of ZR75-30 cancer cells exposed to LEUS was also performed by scanning electron microscopy (Figure 5). Compared with untreated cells, most of cells treated with LEUS (1 MHz, 0.6 W cm2 for 20 s) together with b-CD-capped HMSNs (25 mg mL1) detached from the plate, and changes of the surface structure such as the destruction of microvilli and the formation of dimples or pores were found, which indicate a damage of the cell membrane. However, no noticeable morphological change of cells was observed at treatment with LEUS alone or the combination of LEUS and hydrophilic MSNs. From the results of the in vitro killing assay, a mechanism for the synergetic effect of LEUS and b-CD-capped HMSNs with hydrophobic internal nanovoids to cause cell apoptosis is proposed. Upon incubation of ZR75-30 cancer cells with b-CD-capped HMSNs, the nanoparticles are easily enclosed by the cell membrane to form vesicles and are then internal-
Figure 3. In vitro assay of the killing efficacy of low-energy ultrasound together with b-CD-capped HMSNs against ZR75-30 cells. The killing efficacy was systematically determined with regard to the concentration of nanoagents (5–50 mg mL1), ultrasound intensity (0–1.0 W cm2), and irradiation time (a, 20 s; b, 40 s; c, 60 s; d, 80 s). Each column represents the average plus standard deviation of six independent experiments. Significant differences were calculated by comparing treatment groups with the corresponding group treated with nanoagents alone at the same concentration; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
cence spectroscopy (Figure 1 g and Figure S7, Supporting Information). When the irradiation time was prolonged to 40 s, the viability of cells cultured with 50 mg mL1 nanoagents decreased to about 26 %, 14 %, and 6 % under 0.6, 0.8, and 1.0 W cm2 irradiation, respectively (Figure 3 b). The cell viability decreased to approximately 5 % under both 0.8 and 1.0 W cm2 irradiation for 60 seconds when being cultured with 50 mg mL1 nanoagents (Figure 3 c), thus indicating that a prolonged irradiation time together with a higher nanoagent concentration apparently decreased the cell viability. When the irradiation time was further prolonged to 80 seconds, the survival ratio of ZR75-30 cells cultured with 50 mg mL1 nanoagents was below 4 %, even under 0.8 W cm2 irradiation, while that of the group with 25 mg mL1 nanoagents was about 12 % under 0.8 W cm2 irradiation (Figure 3 d and Figure 4). The killing efficacy of LEUS together with the pristine MSNs (Figure 1 c) was examined as a control. The survival ratios of the cells were maintained above 70 % under the entire test conditions, and no noticeable dose dependence was observed (Figure 4 and Figure S10, Supporting Information). These results indicate that the pristine MSNs without hydrophobic internal channels failed to effectively kill cancer cells when LEUS ( 1.0 W cm2, 1 MHz) is applied. The morphology of cancer cells treated was observed by microscopy. For the cells treated with LEUS and the b-CDcapped HMSNs, most of cells were detached from the plate.
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Figure 5. Morphological evaluation of the surface of ZR75-30 cells after treatment with low-energy ultrasound (1 MHz, 0.6 W cm2) for 20 s. a) Untreated ZR75-30 cells cultured without nanoparticles. b) Treated ZR75-30 cells cultured without nanoparticles. c) Treated ZR75-30 cells cultured with hydrophilic MSNs (25 mg mL1). d) Treated ZR75-30 cells cultured with b-CD-capped HMSNs (25 mg mL1) with hydrophobic internal nanovoids.
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ized by the cells through endocytosis. Upon subsequent treatment with LEUS, bubbles could be released in the cells from the hydrophobic internal nanovoids of the b-CDcapped HMSNs and grow during the rarefaction phase of the sound wave; then, in the compression phase, the bubbles are compressed, which induces a cavitation effect accompanied by physical and chemical processes.[15] On the one hand, the sudden collapse of bubbles inside cells generates momentary a high temperature of several thousand degrees (ca. 5000 K) and a pressure of hundreds of atmospheres in the bubble core. The hot bubble leads to the formation of hydroxyl radicals and hydrogen atoms in the cytosol due to the thermal decomposition of water molecules, which will induce oxidative damage to DNA, proteins, and lipids by direct oxidation and thus leads to cell death. On the other hand, physical processes arising from the collapse of the gas nuclei can cause cell death. The sudden collapse of cavitation bubbles leads to the formation of shock waves and high-velocity liquid microjets near the bubble surface, which could generate secondary stress waves in the cancer cells and mechanically disrupt the cellular membrane. Such processes will result in irreversible cell death without leaving behind any toxic agents.[13c, 16]
Figure 6. In vivo antitumor activity of low-energy ultrasound combined with b-CD-capped HMSNs in a breast cancer xenograft model on nude mice. a) Plot of the relative tumor volume of the control group and treatment groups (b-CD-capped HMSNs, 25 and 50 mg mL1) as a function of treatment time. Each data point represents the average plus standard deviation of six independent excperiments. b) Photographs of tumors from the mice sacrificed at 13 days of treatment. c–e) Histological sections of tumors stained with H&E. c) Tumor of the control group treated with PBS, followed by ultrasound irradiation (1 MHz, 0.8 W cm2). d, e) Tumors of the treatment groups (b-CD-capped HMSNs; d, 25 mg mL1; e, 50 mg mL1), followed by ultrasound irradiation (1 MHz, 0.8 W cm2).
In Vivo Assay of the Therapeutic Effect In the next set of experiments, the in vivo therapy against breast cancer by the LEUS–nanoagent (b-CD-capped HMSNs) system was assessed by using a xenograft tumor model. During these experiments, the mice were comfortably relaxed; the weight of all mice used was quite stable and no abnormality was observed. The size of the tumors was measured every two days. The plot of the relative tumor volume as a function of treatment time shows that the growth rate of the tumor is visibly slower in the treated groups than in the control group (Figure 6 a), and the tumor growth rates are 60 % and 45 % for the 25 mg mL1 group and 50 mg mL1 group, respectively. The tumor sizes of the 25 mg mL1 group and 50 mg mL1 group are visibly smaller than those of the control group (Figure 6 b). Moreover, the inhibition rate is higher in the 50 mg mL1 group than in the 25 mg mL1 group (Figure 6 a–b). Next, histopathological examination was performed on tumor specimens from mice of the three groups. The control group shows typical pathological features of the tumor, that is, a large number of cells with irregular shape and with enlarged and deeply stained nuclei (Figure 6 c). Nevertheless, all of the cancer cells show evident karyopyknosis in the group treated with 25 mg mL1 b-CD-capped HMSNs (Figure 6 d), which indicates the irreversible condensation of chromatin in the nucleus of a cell undergoing apoptosis. Importantly, Figure 6 e shows that a high percentage of hematoxylin-stained chromatin is irregularly distributed throughout the cytoplasm, which obviously suggests that the synergetic treatment by LEUS and b-CD-capped HMSNs (50 mg mL1) induces destructive karyorrhexis of the nucleus and cell necrosis.
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Conclusions In summary, our study shows that irradiation with lowenergy ultrasound can effectively kill cancer cells in vitro and in vivo due to the amplified acoustic cavitation boosted by HMSNs with hydrophobic nanovoids. If combined with targeting agents, such nanoagents may be interventionally or intravenously injected, followed by selective low-energy ultrasound irradiation on the targeted cancer area. The key advantage of this method lies in the synergetic combination of low-cost mild ultrasound and nontoxic targeting nanoagents (when combined with targeting agents) for cancer killing without leaving behind any toxic agents, and thus may open up important application opportunities for in vivo tumor treatment.
Experimental Section Chemicals and Materials MTT was purchased from Sigma–Aldrich. Hexamethyl disilylamine (HMDS), beta-cyclodextrin (b-CD), cetyltrimethylammonium bromide (CTAB), tetraethylorthosilicate (TEOS), para-phthalic acid (PTA), (3aminopropyl)triethoxysilane (APTES), and fluorescein isothiocyanate (FITC) were obtained from Aladdin Chemistry Co., Ltd. 4’,6-Diamidino2-phenylindole (DAPI) was purchased from Nanjing KeyGen Biotech Co., Ltd.
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Preparation of b-CD-Capped HMSNs
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ferent power inputs was evaluated by MTT assay using a microplate reader (Bio-Tek ELx800). For every experimental condition, six repeated experiments were performed (n = 6). The ZR75-30 cells (0.8 105 cells ml1, 150 mL) were seeded in 96-well plates and cultured for 24 h. The medium solution containing MSNs or the b-CD-capped HMSNs (0, 5, 10, 25, and 50 mg mL1) were prepared and added to the cells. After incubation for another 12 h, the culture medium was removed. The cells were washed twice with PBS to remove residual nanoparticles and then fresh medium was added.
A mixture of CTAB (1.0 g), NaOH (2 m, 3.5 mL), and deionized H2O (480 mL) was stirred for 30 min at 80 8C. TEOS (5 mL) was then added dropwise, and the mixture was vigorously stirred for 3 h at 80 8C. The white precipitate was filtered off, washed with ethanol, and dried at 80 8C. The soft template CTAB was then removed completely through washing twice in 100 mL of ethanol containing 10 mL of HCl (1 m) at 60 8C for 8 h. The MSNs were obtained after centrifugation at 14800 g for 10 min and a freeze drying process. Hydrophobic MSNs with hydrophobic internal nanovoids (HMSNs) were prepared by adding HMDS (1.3 mL) to a suspension of MSNs (4 mg mL1, 25 mL) in n-hexane. After stirring for 24 h at 25 8C, the resulting products were centrifuged at 14800 g for 10 min, washed thoroughly with n-hexane, and dried at 40 8C in vacuum. Subsequently, HMSNs (100 mg) were added to a solution of b-CD (0.5 g) in deionized water (100 mL). After vigorous stirring for 24 h at 25 8C, a milk-white solution suspension was obtained, and white products were suspended at the surface of the solution after stewing for a few hours. b-CD-capped HMSNs were collected by discarding the clarified liquid at the bottom and were then redispersed (2 mg mL1) in 50 mL of PBS (0.01 m, pH = 7.4).
For the irradiation procedure, the culture plate was placed on the ultrasound probe with a water layer (ca. 20 mm, 37 8C) as intermediate to avoid rapid temperature changes of cells. To further avoid any increase in the temperature of the medium, ultrasound irradiation was not continuous. For every experimental condition six duplicate experiments were performed (six wells), and every well was cyclically exposed to ultrasound irradiation for 2 s, followed by a 10 s pause. After incubation for another 12 h, the viability of cells treated under different ultrasound intensities (from 0.4 to 1.0 W cm2) and accumulative irradiation times (20 s, 40 s, 60 s, and 80 s; that is, the total irradiation time in the six repeated experiments is 2 min, 4 min, 6 min, and 8 min, respectively) was determined by MTT assay, and the morphology of cells was observed by optical microscopy. Viability was quantified as the fraction of surviving cells relative to the untreated cells as control group. Statistical analysis was performed using the Students two-tailed t test. All data are expressed as means standard deviation (n = 6).
Ultrasound Apparatus and Treatment An ultrasonic therapeutic apparatus (Metron Accusonic, Australia) with a resonant frequency of 1.0 MHz at a continuous cycle was used in all experiments. The irradiation was conducted at intensities in the range from 0.2 to 1.0 W cm2.
For morphological observations via SEM, cells were cultured for 24 h in cover glass bottom dishes and then treated with nanoparticles (25 mg mL1). After incubation for another 12 h, the medium was removed. The cells were washed twice with PBS to remove residual nanoparticles and then fresh medium was added. Based on the abovementioned procedure, the cells were then exposed to ultrasound irradiation for 20 s (1 MHz, 0.6 W cm2). The cover glass was then removed to rinse twice with PBS (pH 7.4) for the removal of unattached cells. The cells were then fixed with 3 % glutaraldehyde solution in a sodium cacodylate buffer (pH 7.4, Invitrogen) for 30 min. Prior to SEM observations, the specimens were dehydrated sequentially in a series of ethanol solutions (30, 50, 75, 90, 95, and 100 %, v/v) for 10 min each, with the final dehydration conducted twice in absolute ethanol followed by drying in in a series of ethanol solutions containing HMDS.
Assay of the Intensity of Ultrasound Cavitation by Fluorescence Spectrometry A PTA dosimeter (5 mm PTA, 10 mm NaOH) was routinely prepared in PBS (pH 7.4) as a substrate solution. The preparation was stored in amber bottles at 4 8C. Prior to use, the solution was warmed to room temperature. MSNs and b-CD-capped HMSNs, respectively, were dispersed in the PTA solution at the same concentration (20 mL, 1 mg mL1). After exposure to ultrasound irradiation for 2 min with intensities in the range from 0.2 to 0.8 W cm2, the PL spectra of the two solutions and of PBS without MSNs were obtained under excitation with UV light (310 nm). Cell Culture
Mouse Tumor Models and Assay of the In Vivo Therapeutic Effect
Breast cancer ZR75-30 cells were cultured and maintained in RPMI 1640 medium supplemented with 2.0 g L1 sodium bicarbonate, 4.5 g L1 glucose, 0.11 g L1 sodium pyruvate, 2.383 g L1 HEPES, and 10 % fetal bovine serum. All cells were maintained at 37 8C in a humidified incubator under 5 % CO2. In all experiments, cells were harvested by the use of 0.25 % trypsin (Sigma) in PBS solution (pH = 7.4) and resuspended in fresh medium before plating.
FVB/Neu mice were obtained from the Shanghai Laboratory Animal Center and were housed under specific pathogen-free conditions. All experimental procedures and euthanasia were done painlessly in compliance with the Guide for the Care and Use of Laboratory Animals. Under sterile conditions, well-developed tumors were cut into 1 mm3 fragments and transplanted into mammary fat pad of nude mice using a trocar. When the volume of tumors reached around 100 to 200 mm3, eighteen tumor-bearing FVB/Neu mice were divided into three groups: 1) a control PBS group, 2) a 25 mg mL1 b-CD-capped HMSNs group, and 3) a 50 mg mL1 b-CD-capped HMSNs group. The b-CD-capped HMSNs were directly injected into tumors at a dose of 10 mL every two days. Two hours after each injection, mice were subjected to ultrasound (0.8 W cm1, 1 MHz at continuous cycle) through a water bag (ca. 20 mm) for 3 min plus 2 min with a pause of 1 min to prevent any increase in temperature. The size of the tumors was measured every two days using microcalipers. Mice were sacrificed at 13 days after injection and tumors were collected for histological analysis. The tumor volume (V) was calculated as follows: V = (length width2)/2. The therapeutic effect was assessed using the individual relative tumor volume (RTV), which was calculated as follows: RTV = Vt/V0, where Vt is the measured volume every two days, and V0 is the initial volume at the beginning of the treatment.
Cellular Uptake of Fluorescently Labeled b-CD-capped HMSNs Fluorescein-labeled HMSNs (denoted as FITC-HMSNs) were obtained through the successive addition of FITC-APTES (0.67 mL) and TEOS (5 mL) by a co-condensation route as reported previously.[17] FITCAPTES was prepared by the addition reaction between FITC (10 mg) and APTES (24 mL) in methanol (4 mL) for 24 h under light-sealed and dry conditions at room temperature. For confocal laser scanning microscopy (Fluo View FV1000, Olympus) observations, ZR75-30 cells (0.6 105 cells per dish) were seeded in 35 mm petri dishes and then treated with b-CD-capped FITC-HMSNs (25 mg mL1). After incubation for 12 h, the medium was removed, and the cells were then washed twice with PBS to remove residual nanoparticles. Subsequently, a solution of DAPI (0.5 mL) in methanol (10 %) was added, and the cells were incubated for 15 min to stain the nuclei and to fix the cells. The cells were then gently washed twice with methanol to remove excessive DAPI. At last, 1 mL of PBS was added and the cells were visualized by CLSM.
Characterization In Vitro Assay of the Therapeutic Effect and Cytotoxicity
TEM images were obtained on a JEM-2010 microscope with an accelerating voltage of 200 kV. Small-angle X-ray diffraction (SAXD) data were recorded on a Rigaku D/Max-2550V diffractometer using CuKa radiation
The cell viability of ZR75-30 cells at incubation with MSNs or b-CDcapped HMSNs with or without low-energy ultrasound irradiation at dif-
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(40 kV and 40 mA) at a scanning rate of 0.58 min1. Nitrogen adsorption– desorption experiments were carried out on a Micromeritics Tristar 3000 analyzer at 77 K. Thermogravimetry/differential scanning calorimetry (TG/DSC) was carried out on a TA thermal analyzer at a heating rate of 10 8C min1 in air. FTIR analysis was carried out using KBr discs on an IR Prestige-21 (Shimadzu) spectrophotometer. The mean diameter and size distribution of samples were measured by dynamic light scattering using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA). Photoluminescence (PL) spectra were recorded on a FluoroMax-4 fluorescence spectrometer. SEM images were obtained by field emission scanning electron microscopy (S-4800, Hitachi). The size distribution and zeta potential of samples were measured by dynamic light scattering using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA).
[6]
[7] [8]
[9]
Acknowledgements
[10]
The authors thank Wenfu Tan for help with animal experiments. The authors acknowledge financial support from the Natural Science Foundation of China (51072217), the Science and Technology Commission of Shanghai (11XD1405600), the National High Technology Research and Development Program of China (2008AAO3Z303), and the State Key Lab of High Performance Ceramics and Superfine Microstructure.
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Effective Cancer Cell Killing by Hydrophobic Nanovoid-Enhanced Cavitation under Safe Low-Energy Ultrasound Yang Zhao,[a] Yingchun Zhu,*[a] Jingke Fu,[a] and Lianzhou Wang[b] Abstract: b-Cyclodextrin (b-CD)capped mesoporous silica nanoparticles with hydrophobic internal nanovoids were prepared and used for effective cancer cell killing in synergistic combination with low-energy ultrasound ( 1.0 W cm2, 1 MHz). The water-dispersible nanoparticles with hydrophobic internal nanovoids can be taken up by cancer cells and subsequently evoke a remarkable cavitation effect under ir-
radiation with mild low-energy ultrasound ( 1.0 W cm2, 1 MHz). A significant cancer cell killing effect was observed in cancer cells and in a mouse xenograft tumor model treated with the nanoagents together with the lowKeywords: cancer · cavitation · low-energy ultrasound · mesoporous materials · nanoparticles
Introduction
pressure (ca. 1000 atm), and ultrahigh temperature (ca. 5000 K) as well as a shockwave and liquid jet in the microenvironment when the irradiation intensity is above the threshold level,[5] which could lead to irreversible cell death through coagulative necrosis.[6] However, the LEUS ( 1.0 W cm2, 1 MHz) used in the present study cannot induce a remarkable cavitation effect and antitumor effect due to the failure to overcome the tensile strength of water to produce a new cavity in the absence of cavitation micronuclei.[7] Much effort has been made to reduce the cavitation threshold and to increase the cavitation effect.[8] Nevertheless, it still remains a great challenge to create a remarkable cavitation effect by LEUS ( 1.0 W cm2, 1 MHz) for effective cancer therapy. In this work, we report an innovative approach for cancer treatment by using water-dispersible nanoparticles with hydrophobic internal nanovoids (nanoagents), which can be taken up by cancer cells. Such nanoagents with internal hydrophobic nanocavities can act as bubble nucleation seeds to drastically amplify the ultrasonic cavitation effect by responding to LEUS. Significant cancer cell killing effects were observed in cancer cells and in a mouse xenograft tumor model treated by the nanoagents together with LEUS. By contrast, antitumor effects were not observed when either LEUS or nanoagents are applied alone.
Safe and effective treatments for cancer have long been a challenging goal for biomedical research.[1] Among various therapies and techniques, low-energy ultrasound (LEUS) defined as therapeutic ultrasound, especially with an intensity below 1 W cm2 and a frequency range of 1–3 MHz, may activate integrin signaling and enhance cellular production of signaling molecules and growth factors via mechanical stimulation of cells (sonostimulation).[2] Thus, LEUS has been widely used in medical imaging, wound healing, and fracture healing.[3] Importantly, the results of some studies suggest that the selectivity of LEUS is higher for malignant cells than for normal cells.[4] Based on the aforementioned safety of LEUS, it is strongly expected as a potential and safe treatment for cancer therapy. In principle, ultrasound with high energy (high intensity and low frequency) can induce a drastic ultrasonic cavitation effect involving a large number of free radicals, extreme [a] Dr. Y. Zhao, Dr. Y. Zhu, Dr. J. Fu The State Key Lab of High Performance Ceramics and Superfine Microstructure Key Laboratory of Inorganic Coating Materials Shanghai Institute of Ceramics, Chinese Academy of Sciences 1295 Dingxi Road, Shanghai, 200050 (P. R. China) Fax: (+ 86) 2152412632 E-mail: [email protected]
Results and Discussion
[b] Dr. L. Wang ARC Centre of Excellence for Functional Nanomaterials School of Chemical Engineering and Australian Institute for Bioengineering and Nanotechnology The University of Queensland QLD 4072 (Australia)
Preparation of b-CD-Capped Hydrophobic Mesoporous Silica Nanoparticles The small-angle X-ray diffraction (SAXRD) pattern and nitrogen adsorption–desorption isotherm show that mesoporous silica nanoparticles (MSNs) and silylanized hydropho-
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301333.
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energy ultrasound, showing a distinct dependence on the concentration of nanoagents and ultrasound intensity. By contrast, an antitumor effect was not observed when either low-energy ultrasound or nanoagents were applied alone. These findings are significant as the technique promises a safe, low-cost, and effective treatment for cancer therapy.
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namic light scattering (DLS) data show that b-CD-capped HMSNs have a narrow size distribution with an overall hydrodynamic diameter of about 220 nm in water (Figure S6, Supporting Information). Moreover, after standing for 24 h and 48 h, their hydrodynamic diameter is about 260 nm and 295 nm, respectively. This result demonstrates that the bCD-capped HMSNs could be stably dispersed in water without aggregate formation.
bic mesoporous silica nanoparticles (HMSNs) have a highly ordered hexagonal structure and classical type-IV N2 adsorption–desorption isotherm (Figures S1 and S2, Supporting Information). The FTIR spectrum and TG-DSC analysis reveal a high surface coverage of the SiMe3 group on surface of the HMSNs (Figures S3 and S4, Supporting Information), and the internal mesoporous surfaces of HMSNs are also silylated by SiMe3 groups to obtain hydrophobic internal nanovoid, as evidenced by the decrease in the BJH pore diameter from 2.5 nm to 2.3 nm by silanization. Previous studies suggest that when two contiguous hydrophobic surfaces are separated, they are connected through gas bridges, and these gas bubbles are stable even up to several micrometers. The interfacial free energy of a hydrophobic surface is lower against gas than against water, so that it is energetically favorable to replace water by gas between the hydrophobic surfaces.[9] The floating of the HMSNs on water indicates their hydrophobic nature due to the absence of water in the hydrophobic mesopores (Figure 1 a). The contact angle between the HMSNs and water in air is about 1408 (Figure 1 b). The HMSNs are re-dispersible in water when being externally capped by b-cyclodextrin (b-CD) (Figure 1 a). b-CD has a hydrophobic cavity (internal diameter of 6.0 and height of 7.9 ), which makes it feasible to cap the hydrophobic SiMe3 groups present on the external surface of HMSNs. Upon binding between the inside hydrophobic cavity of b-CD and the hydrophobic groups of HMSNs, the external hydrophilic groups of b-CD could make HMSNs water-dissolvable (Figure 1 a and 1 f). The highly ordered mesopores remain in the HMSNs and b-CD capped HMSNs (Figure 1 c–e and Supporting Information, Figures S1 and S2). The zeta potential of b-CD-capped HMSNs is 16.8 mV in phosphate-buffered saline (PBS, pH = 7.4; Figure S5 in the Supporting Information). The dy-
Assay of the Intensity of Ultrasound Cavitation It is noted that bubble nucleation is the key process during acoustic cavitation, and in the absence of pre-existing gas cavities, bubble nucleation has to scale a high nucleation energy barrier, which leads to a high cavitation threshold and poor cavitation effect.[8b, 10] The large pore volume (1.275 cm3 g1) of HMSNs with internal hydrophobic channels ensures the entrapment and stability of gas bubbles as nucleation seeds; furthermore, the hydrophobic interface could also reduce the tensile strength of water. As a result, both features could promote the reduction of the cavitation threshold and increase of the cavitation effect.[8a, 9a] The gas bubbles can escape from the hydrophobic pore channels when an external acoustic field is applied. Neglecting vapor pressure, the pressure Pg of gas bubbles entrapped inside the hydrophobic pore channel is given by: Pg ¼ PðtÞ þ 2s cos q=R
ð1Þ
where P(t) is the external pressure, s is the surface tension, q is the contact angle between the liquid and hydrophobic surface, and R is the pore size. The second term accounts for the difference in pressure along the interface (DP). Initially, the liquid–gas interface is concave towards the gas phase and DP is towards the liquid phase to stabilize the gas bubble. Thus, the initial contact angle q is an obtuse angle for this stable state.[10] When an ultrasound wave propagates through the liquid, the total external pressure is given by: PðtÞ ¼ P0 þ PA sinðwtÞ
ð2Þ
where PA ¼
Figure 1. Silica nanoagents with hydrophobic mesopores. a) Conversion of a dispersion of mesoporous silica nanoparticles (MSNs) into b-cyclodextrin (b-CD)-capped HMSNs. b) Contact angle (ca. 1408) between a HMSN and water in air. c–e) TEM images of MSN (c), HMSN (d), and b-cyclodextrin (b-CD)-capped HMSN (e) samples. f) Schematic illustration of b-CD-capped HMSNs; the hydrophobic SiMe3 groups on HMSNs are partially capped by b-CD. g) Fluorescence assay of the cavitation effect in dispersions of MSNs and b-CD-capped HMSNs in PBS after ultrasound irradiation for 2 min (1 MHz, 0.6 W cm2). PBS, control in the absence of nanoparticles.
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pffiffiffiffiffiffiffiffiffiffi 21cI
ð3Þ
Accordingly, the pressure Pg of gas entrapped inside hydrophobic pores is given by: Pg ¼ P0 þ
pffiffiffiffiffiffiffiffiffiffi 21cI sinðwtÞ
þ2s cos q=R
ð4Þ
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where P0 is the static pressure, 1 is the density of the liquid, c is the velocity of sound in the liquid, I is the intensity of ultrasound, PA is the maximum amplitude of the acoustic pressure, and w is the angular frequency of the acoustic wave. The liberation of gas as cavitation nuclei accompanies the change of the contact angle q and movement of the liquid– gas interface. Equation (4) indicates that the contact angle q changes with the time varying of the ultrasound wave. During the negative pressure cycle of ultrasound (P(t) < 0), the pressure Pg inside the gas-filled hydrophobic core is greater than the external pressure. To balance the pressure between the inside and the outside areas, DP changes to point to the gas phase, which causes the liquid–gas interface to become convex towards the liquid phase and the contact angle q to become an acute angle. A further increase in ultrasound intensity could cause the interface to move away from the hydrophobic channels and gas bubbles to be liberated. It is worth pointing out that Equation (4) indicates that a reduction in the surface tension s resulting from the hydrophobic interface and an increase in the ultrasonic intensity would increase the response sensitivity and variation amplitude of the contact angle q when irradiated by ultrasound, which can promote the liberation of free gas bubbles as cavitation nuclei during the negative pressure cycle and sharply enhance the cavitation effect as a response from LEUS. The occurrence of ultrasonic cavitation under LEUS was detected by using fluorescence spectroscopy (Figure 1 g and Figure S7, Supporting Information). Ultrasonic cavitation consequently creates OHC radicals and HC atoms via the dissociation of water;[6b, 11] these radicals can be trapped with non-fluorescent para-phthalic acid to form highly fluorescent hydroxyl(p-phthalic acid).[12] The fluorescence intensity of a PBS solution containing b-CD-capped HMSNs was about 6 times higher than that of PBS solutions with or without MSNs when exposed to ultrasound (1 MHz) with an intensity of 0.6 W cm2 for two minutes (Figure 1 g and Figure S7 a,c, Supporting Information). By contrast, the introduction of either b-CD or hydrophilic MSNs did not lead to an increase in fluorescence intensity of PBS solutions when exposed to ultrasound (Figure S7 b,d, Supporting Information). The results indicate that a remarkable enhancement of the cavitation intensity is caused by silica nanoparticles with hydrophobic mesopores in response to LEUS.
Figure 2. In vitro cellular uptake of b-CD-capped HMSNs (25 mg mL1) in ZR75-30 cells. a) Blue fluorescence image of cell nuclei stained with DAPI. b) Green fluorescence image of FITC-labeled, b-CD-capped HMSNs in cells. c) Overlay image.
The results illustrate a high efficient uptake of b-CD-capped HMSNs by ZR75-30 cells. The in vitro therapeutic effect against ZR75-30 breast cancer cells was evaluated by using the MTT assay. To evaluate the influence of LEUS without nanoagents on the viability of cancer cells, ZR75-30 cells were exposed to ultrasound irradiation with different intensities (1 MHz at continuous cycles and focal distance d = 20 mm). More than 90 % of the cells were viable when the ultrasonic intensity was below 0.8 W cm2, and more than 86 % of the cells were still viable under all test conditions (Figures S8 and S9, Supporting Information). These results show that LEUS ( 1.0 W cm2, 1 MHz) has little effect on the viability of ZR75-30 cells in vitro.[13] Next, in vitro cytotoxicity tests of MSNs and the b-CDcapped HMSNs against ZR75-30 breast cancer cells were performed without ultrasound irradiation. The cell viability was more > 92 % for ZR75-30 cells incubated with MSNs, even at 50 mg mL1 (Figure S10, Supporting Information). The cell viability was > 95 % when the cells were incubated with b-CD-capped HMSNs of 25 mg mL1, and the cell viability was > 92 % when the concentration of the b-CDcapped HMSNs was 50 mg mL1 (Figure 3). Most of the cells were still attached to the plate with a normal morphology in both cases (Figure S11, Supporting Information). Mesoporous silica nanoparticles as a promising drug-delivery carrier have recently been extensively investigated, and most of the obtained data are in line with a negligible cytotoxicity,[14] which is consistent with the findings of this work. In vitro assays of LEUS together with b-CD-capped HMSNs to kill cancer cells were then systematically performed in terms of particle concentration, ultrasound intensity, and irradiation duration (Figure 3). When being irradiated for 20 seconds (1 MHz, continuous cycles and d = 20 mm), the viability of cells cultured with 5 mg mL1 nanoagents decreased from about 98 % to 83 %, 79 %, 58 % and 50 %, respectively, with increasing the irradiation intensity from 0 to 0.4, 0.6, 0.8, and 1.0 W cm2 (Figure 3 a). The cell viability decreased to about 46 %, 35 %, and 10 %, respectively, when the concentration of the nanoagent was increased to 10, 25, and 50 mg mL1 under 1.0 W cm2 irradiation. Obviously, the cell viability moderately decreased with increasing irradiation intensity, and significantly decreased with increasing nanoagent concentration, which is consistent with the measurements of ultrasonic cavitation by fluores-
In Vitro Assay of the Therapeutic Effect and Cytotoxicity To examine the cell-uptake property of the silica nanoagents, b-CD-capped HMSNs were grafted with fluorescein isothiocyanate (FITC) for fluorescence labeling. After treatment with b-CD-capped FITC-HMSNs, ZR75-30 cells were fixed and the nuclei stained with DAPI. The distribution of b-CD-capped FITC-HMSNs in the cancer cells was examined by confocal laser scanning microscopy (Figure 2). After incubation for 12 hours, the b-CD-capped HMSNs were mainly located in the cytoplasm and the perinuclear region.
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Figure 4. Images of ZR75-30 cancer cells exposed to ultrasound from 0 to 1.0 W cm2 for 80 s. a) Cells cultured without nanoparticles (medium). b) Cells cultured with MSNs (50 mg mL1). c) Cells cultured with b-CDcapped HMSNs (50 mg mL1).
However, in the group treated with LEUS and MSNs, the cells retained a normal morphology (Figure 4). Morphological evaluation of ZR75-30 cancer cells exposed to LEUS was also performed by scanning electron microscopy (Figure 5). Compared with untreated cells, most of cells treated with LEUS (1 MHz, 0.6 W cm2 for 20 s) together with b-CD-capped HMSNs (25 mg mL1) detached from the plate, and changes of the surface structure such as the destruction of microvilli and the formation of dimples or pores were found, which indicate a damage of the cell membrane. However, no noticeable morphological change of cells was observed at treatment with LEUS alone or the combination of LEUS and hydrophilic MSNs. From the results of the in vitro killing assay, a mechanism for the synergetic effect of LEUS and b-CD-capped HMSNs with hydrophobic internal nanovoids to cause cell apoptosis is proposed. Upon incubation of ZR75-30 cancer cells with b-CD-capped HMSNs, the nanoparticles are easily enclosed by the cell membrane to form vesicles and are then internal-
Figure 3. In vitro assay of the killing efficacy of low-energy ultrasound together with b-CD-capped HMSNs against ZR75-30 cells. The killing efficacy was systematically determined with regard to the concentration of nanoagents (5–50 mg mL1), ultrasound intensity (0–1.0 W cm2), and irradiation time (a, 20 s; b, 40 s; c, 60 s; d, 80 s). Each column represents the average plus standard deviation of six independent experiments. Significant differences were calculated by comparing treatment groups with the corresponding group treated with nanoagents alone at the same concentration; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
cence spectroscopy (Figure 1 g and Figure S7, Supporting Information). When the irradiation time was prolonged to 40 s, the viability of cells cultured with 50 mg mL1 nanoagents decreased to about 26 %, 14 %, and 6 % under 0.6, 0.8, and 1.0 W cm2 irradiation, respectively (Figure 3 b). The cell viability decreased to approximately 5 % under both 0.8 and 1.0 W cm2 irradiation for 60 seconds when being cultured with 50 mg mL1 nanoagents (Figure 3 c), thus indicating that a prolonged irradiation time together with a higher nanoagent concentration apparently decreased the cell viability. When the irradiation time was further prolonged to 80 seconds, the survival ratio of ZR75-30 cells cultured with 50 mg mL1 nanoagents was below 4 %, even under 0.8 W cm2 irradiation, while that of the group with 25 mg mL1 nanoagents was about 12 % under 0.8 W cm2 irradiation (Figure 3 d and Figure 4). The killing efficacy of LEUS together with the pristine MSNs (Figure 1 c) was examined as a control. The survival ratios of the cells were maintained above 70 % under the entire test conditions, and no noticeable dose dependence was observed (Figure 4 and Figure S10, Supporting Information). These results indicate that the pristine MSNs without hydrophobic internal channels failed to effectively kill cancer cells when LEUS ( 1.0 W cm2, 1 MHz) is applied. The morphology of cancer cells treated was observed by microscopy. For the cells treated with LEUS and the b-CDcapped HMSNs, most of cells were detached from the plate.
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Figure 5. Morphological evaluation of the surface of ZR75-30 cells after treatment with low-energy ultrasound (1 MHz, 0.6 W cm2) for 20 s. a) Untreated ZR75-30 cells cultured without nanoparticles. b) Treated ZR75-30 cells cultured without nanoparticles. c) Treated ZR75-30 cells cultured with hydrophilic MSNs (25 mg mL1). d) Treated ZR75-30 cells cultured with b-CD-capped HMSNs (25 mg mL1) with hydrophobic internal nanovoids.
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ized by the cells through endocytosis. Upon subsequent treatment with LEUS, bubbles could be released in the cells from the hydrophobic internal nanovoids of the b-CDcapped HMSNs and grow during the rarefaction phase of the sound wave; then, in the compression phase, the bubbles are compressed, which induces a cavitation effect accompanied by physical and chemical processes.[15] On the one hand, the sudden collapse of bubbles inside cells generates momentary a high temperature of several thousand degrees (ca. 5000 K) and a pressure of hundreds of atmospheres in the bubble core. The hot bubble leads to the formation of hydroxyl radicals and hydrogen atoms in the cytosol due to the thermal decomposition of water molecules, which will induce oxidative damage to DNA, proteins, and lipids by direct oxidation and thus leads to cell death. On the other hand, physical processes arising from the collapse of the gas nuclei can cause cell death. The sudden collapse of cavitation bubbles leads to the formation of shock waves and high-velocity liquid microjets near the bubble surface, which could generate secondary stress waves in the cancer cells and mechanically disrupt the cellular membrane. Such processes will result in irreversible cell death without leaving behind any toxic agents.[13c, 16]
Figure 6. In vivo antitumor activity of low-energy ultrasound combined with b-CD-capped HMSNs in a breast cancer xenograft model on nude mice. a) Plot of the relative tumor volume of the control group and treatment groups (b-CD-capped HMSNs, 25 and 50 mg mL1) as a function of treatment time. Each data point represents the average plus standard deviation of six independent excperiments. b) Photographs of tumors from the mice sacrificed at 13 days of treatment. c–e) Histological sections of tumors stained with H&E. c) Tumor of the control group treated with PBS, followed by ultrasound irradiation (1 MHz, 0.8 W cm2). d, e) Tumors of the treatment groups (b-CD-capped HMSNs; d, 25 mg mL1; e, 50 mg mL1), followed by ultrasound irradiation (1 MHz, 0.8 W cm2).
In Vivo Assay of the Therapeutic Effect In the next set of experiments, the in vivo therapy against breast cancer by the LEUS–nanoagent (b-CD-capped HMSNs) system was assessed by using a xenograft tumor model. During these experiments, the mice were comfortably relaxed; the weight of all mice used was quite stable and no abnormality was observed. The size of the tumors was measured every two days. The plot of the relative tumor volume as a function of treatment time shows that the growth rate of the tumor is visibly slower in the treated groups than in the control group (Figure 6 a), and the tumor growth rates are 60 % and 45 % for the 25 mg mL1 group and 50 mg mL1 group, respectively. The tumor sizes of the 25 mg mL1 group and 50 mg mL1 group are visibly smaller than those of the control group (Figure 6 b). Moreover, the inhibition rate is higher in the 50 mg mL1 group than in the 25 mg mL1 group (Figure 6 a–b). Next, histopathological examination was performed on tumor specimens from mice of the three groups. The control group shows typical pathological features of the tumor, that is, a large number of cells with irregular shape and with enlarged and deeply stained nuclei (Figure 6 c). Nevertheless, all of the cancer cells show evident karyopyknosis in the group treated with 25 mg mL1 b-CD-capped HMSNs (Figure 6 d), which indicates the irreversible condensation of chromatin in the nucleus of a cell undergoing apoptosis. Importantly, Figure 6 e shows that a high percentage of hematoxylin-stained chromatin is irregularly distributed throughout the cytoplasm, which obviously suggests that the synergetic treatment by LEUS and b-CD-capped HMSNs (50 mg mL1) induces destructive karyorrhexis of the nucleus and cell necrosis.
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Conclusions In summary, our study shows that irradiation with lowenergy ultrasound can effectively kill cancer cells in vitro and in vivo due to the amplified acoustic cavitation boosted by HMSNs with hydrophobic nanovoids. If combined with targeting agents, such nanoagents may be interventionally or intravenously injected, followed by selective low-energy ultrasound irradiation on the targeted cancer area. The key advantage of this method lies in the synergetic combination of low-cost mild ultrasound and nontoxic targeting nanoagents (when combined with targeting agents) for cancer killing without leaving behind any toxic agents, and thus may open up important application opportunities for in vivo tumor treatment.
Experimental Section Chemicals and Materials MTT was purchased from Sigma–Aldrich. Hexamethyl disilylamine (HMDS), beta-cyclodextrin (b-CD), cetyltrimethylammonium bromide (CTAB), tetraethylorthosilicate (TEOS), para-phthalic acid (PTA), (3aminopropyl)triethoxysilane (APTES), and fluorescein isothiocyanate (FITC) were obtained from Aladdin Chemistry Co., Ltd. 4’,6-Diamidino2-phenylindole (DAPI) was purchased from Nanjing KeyGen Biotech Co., Ltd.
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Preparation of b-CD-Capped HMSNs
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ferent power inputs was evaluated by MTT assay using a microplate reader (Bio-Tek ELx800). For every experimental condition, six repeated experiments were performed (n = 6). The ZR75-30 cells (0.8 105 cells ml1, 150 mL) were seeded in 96-well plates and cultured for 24 h. The medium solution containing MSNs or the b-CD-capped HMSNs (0, 5, 10, 25, and 50 mg mL1) were prepared and added to the cells. After incubation for another 12 h, the culture medium was removed. The cells were washed twice with PBS to remove residual nanoparticles and then fresh medium was added.
A mixture of CTAB (1.0 g), NaOH (2 m, 3.5 mL), and deionized H2O (480 mL) was stirred for 30 min at 80 8C. TEOS (5 mL) was then added dropwise, and the mixture was vigorously stirred for 3 h at 80 8C. The white precipitate was filtered off, washed with ethanol, and dried at 80 8C. The soft template CTAB was then removed completely through washing twice in 100 mL of ethanol containing 10 mL of HCl (1 m) at 60 8C for 8 h. The MSNs were obtained after centrifugation at 14800 g for 10 min and a freeze drying process. Hydrophobic MSNs with hydrophobic internal nanovoids (HMSNs) were prepared by adding HMDS (1.3 mL) to a suspension of MSNs (4 mg mL1, 25 mL) in n-hexane. After stirring for 24 h at 25 8C, the resulting products were centrifuged at 14800 g for 10 min, washed thoroughly with n-hexane, and dried at 40 8C in vacuum. Subsequently, HMSNs (100 mg) were added to a solution of b-CD (0.5 g) in deionized water (100 mL). After vigorous stirring for 24 h at 25 8C, a milk-white solution suspension was obtained, and white products were suspended at the surface of the solution after stewing for a few hours. b-CD-capped HMSNs were collected by discarding the clarified liquid at the bottom and were then redispersed (2 mg mL1) in 50 mL of PBS (0.01 m, pH = 7.4).
For the irradiation procedure, the culture plate was placed on the ultrasound probe with a water layer (ca. 20 mm, 37 8C) as intermediate to avoid rapid temperature changes of cells. To further avoid any increase in the temperature of the medium, ultrasound irradiation was not continuous. For every experimental condition six duplicate experiments were performed (six wells), and every well was cyclically exposed to ultrasound irradiation for 2 s, followed by a 10 s pause. After incubation for another 12 h, the viability of cells treated under different ultrasound intensities (from 0.4 to 1.0 W cm2) and accumulative irradiation times (20 s, 40 s, 60 s, and 80 s; that is, the total irradiation time in the six repeated experiments is 2 min, 4 min, 6 min, and 8 min, respectively) was determined by MTT assay, and the morphology of cells was observed by optical microscopy. Viability was quantified as the fraction of surviving cells relative to the untreated cells as control group. Statistical analysis was performed using the Students two-tailed t test. All data are expressed as means standard deviation (n = 6).
Ultrasound Apparatus and Treatment An ultrasonic therapeutic apparatus (Metron Accusonic, Australia) with a resonant frequency of 1.0 MHz at a continuous cycle was used in all experiments. The irradiation was conducted at intensities in the range from 0.2 to 1.0 W cm2.
For morphological observations via SEM, cells were cultured for 24 h in cover glass bottom dishes and then treated with nanoparticles (25 mg mL1). After incubation for another 12 h, the medium was removed. The cells were washed twice with PBS to remove residual nanoparticles and then fresh medium was added. Based on the abovementioned procedure, the cells were then exposed to ultrasound irradiation for 20 s (1 MHz, 0.6 W cm2). The cover glass was then removed to rinse twice with PBS (pH 7.4) for the removal of unattached cells. The cells were then fixed with 3 % glutaraldehyde solution in a sodium cacodylate buffer (pH 7.4, Invitrogen) for 30 min. Prior to SEM observations, the specimens were dehydrated sequentially in a series of ethanol solutions (30, 50, 75, 90, 95, and 100 %, v/v) for 10 min each, with the final dehydration conducted twice in absolute ethanol followed by drying in in a series of ethanol solutions containing HMDS.
Assay of the Intensity of Ultrasound Cavitation by Fluorescence Spectrometry A PTA dosimeter (5 mm PTA, 10 mm NaOH) was routinely prepared in PBS (pH 7.4) as a substrate solution. The preparation was stored in amber bottles at 4 8C. Prior to use, the solution was warmed to room temperature. MSNs and b-CD-capped HMSNs, respectively, were dispersed in the PTA solution at the same concentration (20 mL, 1 mg mL1). After exposure to ultrasound irradiation for 2 min with intensities in the range from 0.2 to 0.8 W cm2, the PL spectra of the two solutions and of PBS without MSNs were obtained under excitation with UV light (310 nm). Cell Culture
Mouse Tumor Models and Assay of the In Vivo Therapeutic Effect
Breast cancer ZR75-30 cells were cultured and maintained in RPMI 1640 medium supplemented with 2.0 g L1 sodium bicarbonate, 4.5 g L1 glucose, 0.11 g L1 sodium pyruvate, 2.383 g L1 HEPES, and 10 % fetal bovine serum. All cells were maintained at 37 8C in a humidified incubator under 5 % CO2. In all experiments, cells were harvested by the use of 0.25 % trypsin (Sigma) in PBS solution (pH = 7.4) and resuspended in fresh medium before plating.
FVB/Neu mice were obtained from the Shanghai Laboratory Animal Center and were housed under specific pathogen-free conditions. All experimental procedures and euthanasia were done painlessly in compliance with the Guide for the Care and Use of Laboratory Animals. Under sterile conditions, well-developed tumors were cut into 1 mm3 fragments and transplanted into mammary fat pad of nude mice using a trocar. When the volume of tumors reached around 100 to 200 mm3, eighteen tumor-bearing FVB/Neu mice were divided into three groups: 1) a control PBS group, 2) a 25 mg mL1 b-CD-capped HMSNs group, and 3) a 50 mg mL1 b-CD-capped HMSNs group. The b-CD-capped HMSNs were directly injected into tumors at a dose of 10 mL every two days. Two hours after each injection, mice were subjected to ultrasound (0.8 W cm1, 1 MHz at continuous cycle) through a water bag (ca. 20 mm) for 3 min plus 2 min with a pause of 1 min to prevent any increase in temperature. The size of the tumors was measured every two days using microcalipers. Mice were sacrificed at 13 days after injection and tumors were collected for histological analysis. The tumor volume (V) was calculated as follows: V = (length width2)/2. The therapeutic effect was assessed using the individual relative tumor volume (RTV), which was calculated as follows: RTV = Vt/V0, where Vt is the measured volume every two days, and V0 is the initial volume at the beginning of the treatment.
Cellular Uptake of Fluorescently Labeled b-CD-capped HMSNs Fluorescein-labeled HMSNs (denoted as FITC-HMSNs) were obtained through the successive addition of FITC-APTES (0.67 mL) and TEOS (5 mL) by a co-condensation route as reported previously.[17] FITCAPTES was prepared by the addition reaction between FITC (10 mg) and APTES (24 mL) in methanol (4 mL) for 24 h under light-sealed and dry conditions at room temperature. For confocal laser scanning microscopy (Fluo View FV1000, Olympus) observations, ZR75-30 cells (0.6 105 cells per dish) were seeded in 35 mm petri dishes and then treated with b-CD-capped FITC-HMSNs (25 mg mL1). After incubation for 12 h, the medium was removed, and the cells were then washed twice with PBS to remove residual nanoparticles. Subsequently, a solution of DAPI (0.5 mL) in methanol (10 %) was added, and the cells were incubated for 15 min to stain the nuclei and to fix the cells. The cells were then gently washed twice with methanol to remove excessive DAPI. At last, 1 mL of PBS was added and the cells were visualized by CLSM.
Characterization In Vitro Assay of the Therapeutic Effect and Cytotoxicity
TEM images were obtained on a JEM-2010 microscope with an accelerating voltage of 200 kV. Small-angle X-ray diffraction (SAXD) data were recorded on a Rigaku D/Max-2550V diffractometer using CuKa radiation
The cell viability of ZR75-30 cells at incubation with MSNs or b-CDcapped HMSNs with or without low-energy ultrasound irradiation at dif-
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(40 kV and 40 mA) at a scanning rate of 0.58 min1. Nitrogen adsorption– desorption experiments were carried out on a Micromeritics Tristar 3000 analyzer at 77 K. Thermogravimetry/differential scanning calorimetry (TG/DSC) was carried out on a TA thermal analyzer at a heating rate of 10 8C min1 in air. FTIR analysis was carried out using KBr discs on an IR Prestige-21 (Shimadzu) spectrophotometer. The mean diameter and size distribution of samples were measured by dynamic light scattering using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA). Photoluminescence (PL) spectra were recorded on a FluoroMax-4 fluorescence spectrometer. SEM images were obtained by field emission scanning electron microscopy (S-4800, Hitachi). The size distribution and zeta potential of samples were measured by dynamic light scattering using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA).
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The authors thank Wenfu Tan for help with animal experiments. The authors acknowledge financial support from the Natural Science Foundation of China (51072217), the Science and Technology Commission of Shanghai (11XD1405600), the National High Technology Research and Development Program of China (2008AAO3Z303), and the State Key Lab of High Performance Ceramics and Superfine Microstructure.
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