Bone-Targeted Mesoporous Silica Nanocarrier Anchored by

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Bone Targeted Mesoporous Silica Nanocarrier Anchored by Zoledronate for Cancer Bone Metastasis Wentong Sun, Yu Han, Zhenhua Li, Kun Ge, and Jinchao Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02228 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 18, 2016

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Bone targeted mesoporous silica nanocarrier anchored by zoledronate for cancer bone metastasis

Wentong Sun†, Yu Han†, Zhenhua Li†, Kun Ge†,§*, Jinchao Zhang†*

† Key Laboratory of Chemical Biology of Hebei Province, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, College of Chemistry & Environmental Science, Hebei University, Baoding 071002, China. § Affiliated Hospital of Hebei University, Baoding 071000, China.

Abstract:

Once the bone metastasis occurres, the chance of survival and quality of the life

for the cancer patients decrease significantly. With the development of nanomedicine, nanocarriers loading bisphosphonates have been build to prevent cancer metastasis according to the enhanced permeability and retention (EPR) effects, however, as a passive mechanism, EPR effects can not apply to the metastatic sites due to their lack of leaky vasculature. In this study, we fabricated 40 nm sized mesoporous silica nanoparticles (MSNs) anchored by zoledronic acid (ZOL) for targeting bone sites, and delivered the anti-tumor drug doxorubicin (DOX) in a spatiotemporally controlled manner. The DOX loading and release behaviors, bone-targeted ability, cellular uptake and mechanisms, subcellular localization, cytotoxicity and anti-migration effect of this drug delivery systems were investigated. The results indicated that MSNs-ZOL had better bone targeting ability compared with non targeted

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MSNs. The max loading capacity of DOX into MSNs and MSNs-ZOL was about 1671 and 1547 mg/g with the loading efficiency of 83.56 and 77.34% respectively. DOX@MSNs-ZOL had an obvious pH sensitive DOX release behavior. DOX@MSNs-ZOL entered into cells through ATP-dependent pathway, and then localized in lysosome to achieve effective intracellular DOX release. The antitumor results indicated that DOX@MSNs-ZOL exhibited the best cytotoxicity against A549 cells and significantly decreased cell migration in vitro. This drug delivery system will be promising for the treatment of cancer bone metastasis in the future.

Keywords:

cancer bone metastasis; mesoporous silica nanoparticles; bone targeting;

bisphosphonates.


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1. Introduction Metastasis is the final stage in cancers which is still incurable1, 2. Bone tissue is one of the favorable sites for cancer metastasis because the bone marrow microenvironment can improve the growth of the cancer cells by supplying nutrients, niche and oxygen3-5. Approximately 70% of early breast or prostate cancer and up to 15 - 30% of patients with carcinomas of lung, colon, stomach, bladder, uterus, rectum, thyroid or kidney have bone metastasis6. Once the bone metastasis occurs, the chance of survival and quality of the life for the patients decrease significantly, with a clinical result including persistent pain, augment of catagma and hypercalcemia7, 8. Significant progress has been made in the medical management of bone metastasis. The combination therapy based on chemotherapeutic drugs and bisphosphonates (BPs) as a basic strategy was often used to enhance the therapeutic efficiency9. However, traditional chemotherapy is ineffective due to the low permeability in the skeleton tumor tissues and poor selectivity to the multiple bone metastatic nodules10. Therefore, the side effect due to the non targeted drug release was still the majority obstacle in cancer therapy. BPs as anti-resorptive agent have been widely used to prevent cancer bone metastasis11. But the efficiency of BPs for inhibiting the viability of cancer cells was limited12, and more, high doses of BPs usually cause osteonecrosis of the jaw in clinic13. So it is very important to develop a new tactics for treating cancer metastasis in bone14. With the development of nanomedicine, nanocarriers loading BPs have been built to prevent cancer bone metastasis according to the enhanced permeability and retention (EPR) effects15. Traditional tactics is used nanoscale delivery systems such as liposome16,

17

or

metal-organic framework (MOF)18 to encapsulate BPs, which can increase the cytotoxicity against cancer cells and decrease the viability of osteoclasts. However, as a passive mechanism, EPR effects can not apply to the metastatic sites due to their lack of leaky vasculature19. As reported, BPs such as zoledronic acid (ZOL) have higher bone-binding


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affinity, can bind to bone surface and inhibit bone resorption by inducing apoptosis of osteoclasts upon administration20. It has been proved that ZOL can be used as a ligand for bone targeting in bone metastasis diseases21. Therefore, BPs can not only be used as a drug for bone disease but also used as a bone targeting ligand. However, to our knowledge, there has been no report for the combination of these two properties to treat cancer bone metastasis. Mesoporous silica nanoparticles (MSNs) have great potential as drug delivery system (DDS) for their unique advantages22-28. Inspired by this, we fabricated 40 nm sized MSNs anchored by ZOL for targeting bone sites, and delivered the anti-tumor drug doxorubicin (DOX) in a spatiotemporally controlled manner. The bone targeting ability, DOX loading and release behaviors, cellular uptake, cytotoxicity and anti-migration effect of the DDS were investigated.

2. Experimental Section 2.1. Materials Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), ethanol, and methanol were obtained from Sigma Chemical. Hexadecyl trimethyl ammonium chloride (CTAC), 3-aminopropyltriethoxysilane

(APTES),

fluorescein

isothiocyanate

(FITC),

ZOL,

N,N’-carbonyldiimidazole (CDI), MitoTracker Red (MTR), and LysoTracker Red (LTR) were purchased from Sigma Aldrich. DOX and dimethyl sulfoxide (DMSO) were provided by Sun Pharma Advanced Research Centre (SPARC). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin and fetal bovine serum (FBS) were purchased from Gibco. IL-8 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Boster. Cell counting kit-8 (CCK-8) was obtained from BestBio. Deionized water was used in all experiments. All chemicals were used without further purification.

2.2. Preparation of MSNs and MSNs-NH2 For the synthesis of the MSNs, a procedure was used with some modifications26. CTAC


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(2g) and TEA (0.08g) were dissolved in water (20mL) at 75℃ under stirring. After 2 h, 1.5mL of TEOS was added dropwise, the mixture was stirred for 3 h. The products were separated by centrifugation (11000 rpm, 8 min), washed for several times by ethanol. In order to remove the template, the products were extracted for 3 h with a 1wt% solution of NaCl in methanol. Green fluorescence-labeled MSNs (MSNs-FITC) were obtained by the addition of FITC-APTES, followed by TEOS by a co-condensation route26. The surface of MSNs was functionalized with amine group by APTES26. In brief, MSNs (50 mg) were first dispersed in 40 mL of ethanol, and then the solution was refluxed for 24 h, followed by the addition of 100 µL APTES. After being washed by water and DMSO, the obtained MSNs-NH2 was redispersed in 50 mL of DMSO for further use.

2.3. Preparation of MSNs-ZOL Conjugation of ZOL with MSNs-NH2 was prepared by CDI21. ZOL (100 mg) was dissolved in 50 mL DMF with 2 mL triethylamine. CDI (90 mg) was added to vessel under nitrogen blanket for 24 h at 60 ℃, then triethylamine was evaporated and precipitates were washed three times with acetonitrile. MSNs-NH2 (50mg) and activated ZOL (22.6 mg) were dissolved in DMSO with 2 mL triethylamine in vessel under nitrogen blanket for 12 h. Green fluorescence-labeled MSNs-ZOL (MSNs-ZOL-FITC) were obtained by the addition of FITC-APTES, followed by MSNs-NH2 dispersed in 20 mL of ethanol.

2.4. Characterizations Scanning electron microscopy (SEM) was investigated by a cold field-emission scanning electron microscope JSM-7500F (JEOL). Transmission electron microscopy (TEM) was obtained by a Tecnai G2 F20 S-Twin transmission electron microscope (FEI). Zeta potential was determined using Nano-ZS (Malvern Instruments) in disposable cuvettes. The N2 adsorption/desorption isotherms, BET surface area, and pore volume were obtained on a micromeritics ASAP 2010 M instrument. FT-IR spectra were obtained by PerkinElmer 580B


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(KBr pellet). The loading amount of DOX was determined by UV-vis spectra. The photoluminescence (PL) spectra were measured on an F-7000 spectrophotometer.

2.5. Bone-targeted ability The bone-targeted ability of MSNs-ZOL was investigated29. MSNs-FITC and MSNs-ZOL-FITC were incubated with bone slices in 12 well plates for 0, 4, 12, and 24 h. The binding affinity of MSNs-ZOL was determined by detecting the fluorescence of MSNs-ZOL-FITC in the supernatant compared with that of non targeted MSNs-FITC. In addition, specific binding of MSNs-ZOL to bone slices (femur, cow) compared with that of non targeted MSNs was also study by SEM. Bone slices were incubated by nanoparticles solution (0.1 mg/mL) for 0, 4, 12, and 24 h in physiological conditions, and washed three times by phosphate buffer saline (PBS), dried over night under vacuum, and coated with gold to visualize under SEM.

2.6. DOX loading and release assays In a 2 mL aqueous system, 2 mg of DOX were mixed with 2 mg MSNs or MSNs-ZOL respectively, and shaken for 48 h under dark condition at room temperature to obtain DOX@MSNs and DOX@MSNs-ZOL. The loading efficiency (LE) was evaluated by the following formula: LE (%) = [m(total DOX) - m(DOX in supernatant)]/[m(loaded DOX) + m(carrier)] × 100. The DOX released from DOX@MSNs or DOX@MSNs-ZOL was measured by a semipermeable dialysis bag diffusion technique22. The parallel DOX@MSNs and DOX@MSNs-ZOL were dispersed in 1 mL of acetate buffer (pH 5.0) or 1 mL of PBS (pH 7.4), transferred to semipermeable dialysis bags (MWCO = 3500), and then immersed in 9 mL of acetate or PBS at 37 ℃ under shaking, respectively. At tested time intervals, 9 mL of acetate or PBS buffer were taken out and the amount of released DOX was measured by fluorescence spectroscopy, and then an equal volume of fresh acetate buffer or PBS was


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added to the release system.

2.7. Cell Culture Human lung adenocarcinoma cells (A549 cells) were cultured in DMEM, containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. The cells were kept at 37 ℃ in a humidified 5 % CO2 atmosphere.

2.8. Cellular uptake, subcellular localization and mechanisms of cellular uptake The cellular uptake of MSNs-ZOL-FITC was monitored by flow cytometry method (FCM)30. A549 cells were seeded in 6 well culture plates (2×105 cells/well) over night. MSNs-ZOL-FITC was added to the wells at final concentration of 20 µg/mL. The wells only containing cells were used as control. After 1, 4, 8, 12, and 24 h treatment, cells were collected, centrifuged, and resuspended in PBS solution. The uptake of particles was analyzed by flow cytometer (FACS Calibur, BD). The cellular uptake was also measured by a confocal laser microscope (Olympus Fluoview IX81)22. A549 cells were plated on 13 mm coverslips at 5×104/well over night. The cells were treated by 20 µg/mL MSNs-ZOL-FITC at 1, 4, 12, and 24 h. The cell imaging was performed by a confocal laser microscope31. Subcellular localization was measured by a confocal laser microscope22. A549 cells were planted on coverslips as above. Subsequently, A549 cells were treated by MSNs-ZOL-FITC at 37 ℃. Cells were washed by PBS for three times, stained with 100 nM LTR or MTR, for 30 min, and observed by a confocal laser microscope. The endocytosis mechanism of MSNs-ZOL-FITC was studied by adenosine triphosphate (ATP)-depleted environment and low temperature assays21. A549 cells were plated in 6 well culture plates (2×105 cells/well) over night. After that, the cells were incubated with NaN3 for 1h at 37 ℃. And then, MSNs-ZOL-FITC was added to the wells at 20 µg/mL for 1 h. The uptake of particles was analyzed by flow cytometer.


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2.9. Cellular DOX release The DOX released from DOX@MSNs or DOX@MSNs-ZOL in vitro was monitored by FCM32, 33. The detailed procedure was demonstrated as follows: A549 cells were seeded in 6 well plates (1×105 cells/well) over night. Free DOX, DOX@MSNs and DOX@MSNs-ZOL were added to the plates respectively. The wells only containing cells were used as control. After 1, 4, 8, and 12 h treatment, the cells were collected, centrifuged, washed and resuspended in PBS solution. The cellular DOX release was analyzed by flow cytometer.

2.10. Cytotoxicity The cytotoxicity was assessed by CCK-8 kit assay28. A549 cells were seeded in a 96 well plate (3×103 cells/well) over night. Thereafter, the cells were treated with free ZOL, free DOX, MSNs, MSNs-ZOL, DOX@MSNs, and DOX@MSNs-ZOL for 24 h. 10 µL of CCK-8 solution was added to the wells and incubated for 4 h. The absorbance was measured by a microplate reader (Molecular Devices SpectraMax M4) at 450 nm. The cytotoxicity was expressed as the percentage of cell viability compared to control cells.

2.11. Wound healing assay The migration of A549 cells was detected by wound healing assay32, 33. Briefly, when cells grew to 90% confluency in 6 well plates, the monolayer of cells was scratched by a sterile pipette tip in order to form a bidirectional wound. Free ZOL, free DOX, MSNs, MSNs-ZOL, DOX@MSNs, and DOX@MSNs-ZOL were added to culture medium immediately. The wound width was observed by a microscope after 24 h treatment. The migration ratio (%) was calculated as follows: Migration ratio (%) = (original wound width – remaining wound width) / original wound width × 100

2.12. IL-8 detection A549 cells were seeded at 5×105 cells per 30-mm dish. Free ZOL, free DOX, MSNs,


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MSNs-ZOL, DOX@MSNs, and DOX@MSNs-ZOL were added to the dish. The medium was replaced by serum-free medium, after 30 h treatment, the supernatants were collected, centrifuged, and stored at -80 ℃34. IL-8 level was quantified by the IL-8 ELISA kit according to the manufacturer’s protocol.

2.13. Statistical analysis Data were presented as mean ± standard deviation (SD), which were collected from three separate experiments. Two group’s comparison was determined by two-tail Student’s t test, and the significant difference was defined as P value < 0.05 at the 95% confidence interval.

3. Results and Discussion 3.1. Characterization of MSNs-ZOL It has been reported that rigid nanoparticles with long circulation half-life can accumulate in the spleen at a high percentage35. Nanoparticles with intermediate size (20-100 nm) have the highest potential for in vivo applications due to their ability to circulate in the blood for long periods of time, these nanoparticles are able to avoid renal or lymphatic clearance and have capability to avoid opsonization36. What’s more, nanoparticles within size range of 20-100 nm are believed to be internalized easily by cells in comparison to smaller or larger particles36. The SEM images (Fig. 1A) and the TEM images (Fig. 1B) of the particles demonstrated that the monodisperse MSNs with average diameter of 40 nm were synthesized (Fig. S1 in Supporting Information). N2 adsorption-desorption isotherms (Fig. 1C) showed a typical type-IV curve with an average pore diameter of 2.09 nm and a narrow pore distribution, which are consistent with the TEM results. The presence of ZOL was confirmed by UV-Visible spectroscopy (Fig. 1D) and FT-IR (Fig. 1E). The UV-Visible spectra of MSNs-ZOL showed absorbance peak at 210 nm which indicated the presence of ZOL. As


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shown in Fig. 1E, the band at 1230 cm-1 can also indicate the presence of ZOL in MSNs-ZOL which belongs to the asymmetric phosphate stretching mode. The grafting density of ZOL on the surface of MSNs was calculated from the standard curve of ZOL. The loading capacity of ZOL onto MSNs is about 166.8 mg/g. After zoledronic acid was bound to MSNs, the zeta potential changed from -36.7 to -16 mV in water (Fig. S2 in Supporting Information). The above results indicated that the surface of MSNs has been successfully modificated by zoledronic acid.

3.2. DOX loading and release properties As an efficient DDS, the high drug-loading capability is necessary. The max loading capacity of DOX into MSNs and MSNs-ZOL is about 1671 and 1547 mg/g with the loading efficiency of 83.56 and 77.34% respectively, which exhibits the high drug loading performance. The UV-vis and FT-IR spectra of DOX, MSNs, DOX@MSNs, and DOX@MSNs-ZOL are shown in Fig. 1D and 1E. For the UV-vis spectra, free DOX has highest absorbance at 482 nm, and MSNs have no obvious absorbance from 340 to 900 nm. DOX@MSNs and DOX@MSNs-ZOL have the highest absorbance at 484 nm, which indicates that DOX has been successfully loaded into MSNs37. For the FT-IR spectra of MSNs, it shows characteristic groups of Si-O-Si (υs: 1098 cm-1, υas: 814 cm-1), Si-OH (υs: 965 cm-1), and Si-O (δ: 469 cm-1)23. For DOX@MSNs and DOX@MSNs-ZOL, the FT-IR spectra include all the absorption bands of bare DOX and MSNs, and no new absorption band can be observed, indicating that the DOX has been absorbed on MSNs successfully. In vitro release profiles of DOX from DDS were measured in PBS buffer at pH 7.4 and acetate buffer at pH 5.0. Fig. 1F showed that the amount of cumulative released drug from DOX@MSNs-ZOL and DOX@MSNs is almost the same (about 10%) at pH 7.4. When the medium pH value was reduced to 5.0 (pH value of lysosomes was about 5.0), the accumulated released drug from DOX@MSNs-ZOL and DOX@MSNs quickly improved to


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38 and 25%. The results clearly demonstrated that DOX@MSNs-ZOL have an obvious pH sensitive DOX release behavior. This could be the reason that the protons can easily penetrate the mesoporous in acidic buffer solution to protonate the amino group of DOX, thus accelerate the drug release and increase cytotoxicity38. It was worth noting that DOX@MSNs-ZOL showed the faster drug release rate than DOX@MSNs in acid buffer. The reason is that when MSNs-ZOL has not enough hydroxyl to create a hydrogen bond with the amino group of DOX, which decreased the interaction between DOX molecular and MSNs-ZOL, and further promoted the drug release. Moreover, the DDS showed an initial burst release within 10 h, which may be assigned to the weak adsorption of the DOX on the surface of MSNs-ZOL, then followed by a slow sustained release of DOX molecules which can be assigned to the strong interaction between MSNs-ZOL and DOX23. The drug release process for the DOX@MSNs-ZOL system can sustain for more than 100 h. Such gradual and prolonged release behavior is preferable for the enhanced efficiency of cancer therapy, because continually sustained drug release can effectively prevent cancer growth in long-term treatment39.

3.3. Bone targeting ability Dominating obstacle in cancer therapy is non-specific action which will lead to unexpected side effects in normal sites40. Hydroxyapatite is rich in bone microenvironment, especially in the sites of lesions occurred, where the bone turnover is upgrade. ZOL have higher binding affinity with bone slices even though they were used as surface ligands with nanoparticles, and so they can bind to bone surface and prevent bone resorption20. The bone targeting ability of the MSNs-ZOL was confirmed by SEM (Fig. 2A). MSNs-ZOL binds to the bone slices immediately. The fluorescence of supernatants of MSNs-ZOL obviously decreased (Fig. 2B). These results demonstrated that MSNs-ZOL had better bone targeting ability compared with non targeted MSNs.


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3.4. Cellular uptake, subcellular localization and mechanisms of endocytosis Different kinds of endocytic mechanisms occurred due to the composition, shape and size of vesicles, which can affect the fate of the internalized materials41. The mechanisms of delivery of nanoparticles are very important to determine the destination and residence time of DDS, as well as effectiveness. Endocytosis is known as a general entry mechanism of various extracellular materials and the process is energy dependent, which means it will be hindered when incubation is carried out in an ATP-depleted environment42. The cellular uptake of the nanoparticles was verified by FCM and confocal laser scanning microscopy. As shown in Fig. 3A and 3C, the green fluorescence strengthed with incubation time increase. This suggests that the cellular uptake depends on time. In order to detect subcellular localization, the lysosomes of A549 cells was stained by LTR probe. The co-localization of MSNs-ZOL-FITC with lysosomes produced a yellow fluorescence in merged images (Fig. 3C). We further studied the subcellular localization of MSNs in mitochondria (Fig. S3 in Supporting Information). The results showed the green fluorescence of MSNs-ZOL-FITC did not overlap with fluorescence of MitoTracker Red. This indicated that MSNs-ZOL-FITC was mainly distributed in the lysosomes, but not in mitochondria. The cellular uptake of the MSNs-ZOL-FITC by energy dependent endocytosis was further confirmed by ATP-depleted environment and low temperature assays (Fig. 3B).

3.5. Cellular drug release and cytotoxicity in vitro The therapeutic efficacy of DDS depends on the amount of drug available from the nanocarriers internalized by cells43. The cellular drug release profiles of DOX@MSNs and DOX@MSNs-ZOL were measured by flow cytometer. As shown in Fig. 4A, when A549 cells were incubated with DOX@MSNs, there is a weak fluorescence intensity even after 12 h. However, when A549 cells were incubated with DOX@MSNs-ZOL, the fluorescence was significantly increased with prolonging the incubation time.


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ZOL is a third-generation BPs which contains a nitrogen heterocycles44. It is extensively used to impede cancer bone metastasis45, 46. It was proved that ZOL had anti-cancer activity, it can effectively restrain vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) signal in tumor tissue47, 48. ZOL also shows synergistic effect with chemotherapeutic agents such as DOX in cancer bone metastasis treatment49. The cytotoxicity of the DDS was measured by CCK-8 assay (Fig. 4B). The cell viability of A549 cells remained above 90% when they were treated with MSNs, but less than 50% after treatment with MSNs-ZOL up to 40 µg/mL for 24 h. This is similar to the reported finding that silica is not essentially toxic and ZOL can inhibit cancer cell viability20,50. Next, the anti-cancer efficiency was evaluated. Both free ZOL and DOX can enhance the cytotoxicity. Interestingly, DOX@MSNs-ZOL exhibited significantly better cytotoxicity than free drugs at 24 h. Moreover, we can distinctly find that DOX@MSNs-ZOL exhibites the best anti-cancer efficiency due to the higher cellular release of DOX21.

3.6. Inhibition of cancer cell migration To evaluate the effect of the DDS on inhibiting the cancer invasion process, cell migration was assessed by wound healing assay. As illustrated in Fig. 5A and B, free ZOL and DOX significantly inhibited migration of A549 cells, and ZOL was much more effective in retarding migration of A549 cells. It can be observed that DOX@MSNs-ZOL exhibits the best ability in retarding migration of A549 cells in comparison to DOX@MSNs and MSNs-ZOL. However, we can find that empty MSNs have no effect on the cancer metastasis. Cancer cells can secrete various factors to stimulate bone resorption, the sustained release of these factors promotes cancer cell spreading and bone resorption. Cancer cells can secrete interleukin (IL) -1, -6, -8, transforming growth factor (TGF) –β and tumor necrosis factor (TNF) – α which contribute to bone lesion34. In particular, as a molecular mediator of cancer metastasis, IL-8 is significantly up regulated in cancer tissue51. For example, Wang et


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al. proved that CD147 was a potential therapeutic target for preventing cancer bone metastasis because it plays a key role in decreasing IL-834. IL-8 secreted from A549 cells increases osteoclast formation in vitro52, moreover, sera from lung cancer patients with bone metastasis contains higher level of IL-853, so we chose this molecule as a representative candidate for further investigation. As shown in Fig. 5C, higher level of IL-8 was significantly decreased in A549 cells when cells were treated with DOX@MSNs-ZOL. These data indicate that the DDS can inhibit cancer cells migration due to retarding the expression of IL-8 secreted by A549 cells.

4. Conclusion In summary, we fabricated the 40 nm sized MSNs anchored with ZOL for targeting bone sites, and delivered the antitumor drug DOX in a spatiotemporally controlled manner. The targeted drug delivery system could enhance the bone homing capacity, and induce higher cellular uptake and drug release in vitro. DOX@MSNs-ZOL entered into cells through ATP-dependent pathway, and then localized in lysosome to achieve effective intracellular DOX release. The antitumor results indicated that DOX@MSNs-ZOL exhibited better cytotoxicity against A549 cells and significantly decreased cancer cell migration in vitro. This kind of bone-targeted nanoparticles may be a promising candidate for the treatment of cancer bone metastasis. Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXX. The particle size distribution of MSNs derived from SEM image; Zeta potential of MSNs and MSNs-ZOL in water; Subcellular location of MSNs-ZOL-FITC in A549 cells for 1, 4, 12, and 24 h at 37 ℃.


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AUTHOR INFORMATION Corresponding Author * J. C. Zhang. E-mail: [email protected]. * K. Ge.

E-mail: [email protected].

Notes The authors declare no competing financial interest.

Acknowledgments Financial support information: This research was supported by the National Natural Science Foundations of China (31470961, 21271059), Key Basic Research Special Foundation of Science Technology Ministry of Hebei Province (14961302D), Hebei Province “Hundred Talents Program” (BR2-202), Hebei Province “Three Three Three Talents Program” (A201401002) and Natural Science Foundation of Hebei Province (B2016201209, B2015201097), Training Program for Innovative Research Team and Leading Talent in Hebei Province University (LJRC024), and Science and Technology Support Program of Baoding (15ZF055).

Reference (1) Wang, J.; Rouse, C.; Jasper, J.S.; Pendergast, A.M. ABL Kinases Promote Breast Cancer Osteolytic Metastasis by Modulating Tumor-Bone Interactions through TAZ and STAT5 Signaling. Sci. Signal. 2016, 9, 475-482. (2) Bednarz-Knoll, N.; Efstathiou, A.; Gotzhein, F.; Wiknnan, H.; Mueller, V.; Kang, Y.; Pante, K. Potential Involvement of Jagged1 in Metastatic Progression of Human Breast Carcinomas. Clin. Chem. 2016, 62, 378-386. (3) Tasnim, A.; Declerck, Y.A. Interleukin-6 in Bone Metastasis and Cancer Progression. Eur.


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FIGURE LEGENDS Fig. 1. SEM images of MSNs (A), Scale bar is 100 nm. TEM images of MSNs (B), Scale bar is 50 nm. (C) N2 sorption isotherms and the corresponding BJH pore size distribution curves (inset) of MSNs. (D) UV-Visible spectra of the MSNs, free ZOL, MSNs-ZOL, free DOX, DOX@MSNs, and DOX@MSNs-ZOL. (E) FT-IR spectra of the MSNs, free ZOL, MSNs-ZOL, free DOX, DOX@MSNs, and DOX@MSNs-ZOL. (F) DOX release profiles of DOX@MSNs or DOX@MSNs-ZOL at pH 5.0 and 7.4 at 37 ℃. Fig. 2. Bone targeting ability of MSNs and MSNs-ZOL. (A) SEM images of MSNs-ZOL and MSNs surface interactions with bone slices (scale bar: 200 µm). (B) The fluorescence intensity of supernatant after MSNs-ZOL-FITC and MSN-FITC were incubated with bone slices for different time at 37℃. (C) Quantification of bone targeting ability of MSNs-FITC and MSNs-ZOL-FITC. Fig. 3. Cellular uptake (A) and mechanisms of cellular uptake (B) of MSNs-ZOL-FITC. (C) Fluorescence images of intracellular distribution of MSNs-ZOL-FITC in A549 cells for 1, 4, 12, and 24 h at 37 ℃. Scale bar is 40 µm. ***p

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