zoledronic acid (ZOL) for targeting bone sites, and delivered the anti-tumor drug ... Keywords: cancer bone metastasis; mesoporous silica nanoparticles; bone ...
Article pubs.acs.org/Langmuir
Bone-Targeted Mesoporous Silica Nanocarrier Anchored by Zoledronate for Cancer Bone Metastasis Wentong Sun,† Yu Han,† Zhenhua Li,† Kun Ge,*,†,‡ and 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 S Supporting Information *
ABSTRACT: Once bone metastasis occurs, the chances of survival and quality of life for cancer patients decrease significantly. With the development of nanomedicine, nanocarriers loading bisphosphonates have been built to prevent cancer metastasis based on their enhanced permeability and retention (EPR) effects; however, as a passive mechanism, the EPR effects cannot apply to the metastatic sites because of 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 antitumor drug doxorubicin (DOX) in a spatiotemporally controlled manner. The DOX loading and release behaviors, bone-targeting ability, cellular uptake and its mechanisms, subcellular localization, cytotoxicity, and the antimigration effect of this drug delivery system (DDS) were investigated. The results indicated that MSNs−ZOL had better bone-targeting ability compared with that of the nontargeted MSNs. The maximum loading capacity of DOX into MSNs and MSNs−ZOL was about 1671 and 1547 mg/g, with a loading efficiency of 83.56 and 77.34%, respectively. DOX@MSNs−ZOL had obvious pH-sensitive DOX release behavior. DOX@MSNs−ZOL entered into cells through an ATP-dependent pathway and then localized in the 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 DDS is promising for the treatment of cancer bone metastasis in the future.
1. INTRODUCTION Metastasis is the final stage in cancer, which is still incurable.1,2 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 oxygen.3−5 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 metastasis.6 Once the bone metastasis occurs, the chances of survival and the quality of life for the patients decrease significantly, with a clinical result including persistent pain, augment of catagma, and hypercalcemia.7,8 Significant progress has been made in the medical management of bone metastasis. The combination therapy of chemotherapeutic drugs and bisphosphonates (BPs) as a basic strategy was often used to enhance the therapeutic efficiency.9 However, traditional chemotherapy is ineffective because of the low permeability in the skeleton tumor tissues and poor selectivity to the multiple bone metastatic nodules.10 Therefore, side effects due to the nontargeted drug release are still the major obstacle in cancer therapy. BPs as antiresorptive agents have been widely used to prevent cancer bone metastasis.11 But the efficiency of BPs for inhibiting the viability of cancer cells is limited,12 and © 2016 American Chemical Society
high doses of BPs usually cause osteonecrosis of the jaw in clinic.13 So, it is very important to develop new tactics for treating cancer metastasis in bone.14 With the development of nanomedicine, nanocarriers loading BPs have been built to prevent cancer bone metastasis based on their enhanced permeability and retention (EPR) effects.15 Traditional tactics are used in nanoscale delivery systems such as liposomes16,17 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, the EPR effects cannot apply to the metastatic sites because of their lack of leaky vasculature.19 As reported, BPs such as zoledronic acid (ZOL) have a higher bone-binding affinity, and they can bind to the bone surface and inhibit bone resorption by inducing apoptosis of osteoclasts upon administration.20 It has been proved that ZOL can be used as a ligand for bone targeting in bone metastasis diseases.21 Therefore, BPs can not only be used as a drug for bone disease but also be used as a bone-targeting ligand. Received: June 15, 2016 Revised: August 13, 2016 Published: August 16, 2016 9237
DOI: 10.1021/acs.langmuir.6b02228 Langmuir 2016, 32, 9237−9244
Article
Langmuir
2.5. Bone-Targeting Ability. The bone-targeting ability of MSNs−ZOL was investigated.29 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 and comparing with that of nontargeted MSNs−FITC. In addition, specific binding of MSNs−ZOL to bone slices (femur, cow) compared with that of nontargeted MSNs was also studied using SEM. The bone slices were incubated in a nanoparticles solution (0.1 mg/mL) for 0, 4, 12, and 24 h under physiological conditions, washed three times with phosphate-buffered saline (PBS), dried overnight under vacuum, and coated with gold to visualize under a scanning electron microscope. 2.6. DOX Loading and Release Assays. In a 2 mL aqueous system, 2 mg of DOX was mixed with 2 mg of MSNs or MSNs−ZOL, and the resultant mixture was shaken for 48 h under dark conditions at room temperature to obtain DOX@MSNs and DOX@MSNs−ZOL. The loading efficiency (LE) was evaluated by using the following formula
However, to our knowledge, there has been no report on the combination of these two properties to treat cancer bone metastasis. Mesoporous silica nanoparticles (MSNs) have great potential as drug delivery systems (DDSs) owing to their unique advantages.22−28 Inspired by this, we fabricated 40 nmsized MSNs anchored by ZOL for targeting bone sites, and delivered the antitumor drug doxorubicin (DOX) in a spatiotemporally controlled manner. The bone-targeting ability, DOX loading and release behaviors, cellular uptake, cytotoxicity, and the antimigration effect of this 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. The Interleukin (IL)-8 enzymelinked 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. MSNs were synthesized using Pan et al.’s26 procedure with some modifications. CTAC (2 g) and TEA (0.08 g) were dissolved in water (20 mL) at 75 °C under stirring. After 2 h, 1.5 mL of TEOS was added dropwise and the mixture was stirred for 3 h. The products were separated by centrifugation (11 000 rpm, 8 min) and washed several times with ethanol. To remove the template, the products were extracted for 3 h with a 1 wt % solution of NaCl in methanol. Green fluorescencelabeled MSNs (MSNs−FITC) were obtained by the addition of FITC−APTES, followed by TEOS by a cocondensation route.26 The surface of the MSNs was functionalized with an amine group using APTES.26 In brief, the 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 of APTES. After being washed with water and DMSO, the obtained MSNs−NH2 were redispersed in 50 mL of DMSO for further use. 2.3. Preparation of MSNs−ZOL. Conjugation of ZOL with MSNs−NH2 was aided by CDI.21 ZOL (100 mg) was dissolved in 50 mL of dimethylformamide (DMF) with 2 mL of triethylamine. CDI (90 mg) was added to the vessel under a nitrogen blanket for 24 h at 60 °C; then, triethylamine was evaporated, and the precipitates were washed three times with acetonitrile. MSNs−NH2 (50 mg) and activated ZOL (22.6 mg) were dissolved in DMSO with 2 mL of triethylamine in a vessel under a 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. Characterization. Scanning electron microscopy (SEM) was performed using a cold field-emission scanning electron microscope (JSM-7500F, JEOL). Transmission electron microscopy (TEM) was performed using a Tecnai G2 F20 S-Twin transmission electron microscope (FEI). Zeta potential was determined using a Nano-ZS (Malvern Instruments) system in disposable cuvettes. The N2 adsorption/desorption isotherms, the Brunauer−Emmett−Teller (BET) surface area, and the pore volume were obtained using a micromeritics ASAP 2010 M instrument. The Fourier transform infrared (FT-IR) spectra were obtained using a PerkinElmer 580B spectrophotometer (KBr pellet). The loading amount of DOX was determined by ultraviolet−visible (UV−vis) spectroscopy. The photoluminescence (PL) spectra were measured on an F-7000 spectrophotometer.
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 using the semipermeable dialysis bag diffusion technique.22 The parallel DOX@MSNs and DOX@MSNs−ZOL were separately 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 °C under shaking. At tested time intervals, 9 mL of acetate or PBS buffer was taken out and the amount of DOX released was measured using fluorescence spectroscopy. An equal volume of fresh acetate buffer or PBS was 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 of penicillin, and 100 U/mL of streptomycin. The cells were kept at 37 °C 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 using the flow cytometry method (FCM).30 A549 cells were seeded in 6-well culture plates (2 × 105 cells/well) overnight. MSNs−ZOL−FITC were added to the wells at a final concentration of 20 μg/mL. The wells containing only cells were used as control. After 1, 4, 8, 12, and 24 h treatment, the cells were collected, centrifuged, and resuspended in a PBS solution. The uptake of particles was analyzed using a flow cytometer (FACS Calibur, BD). The cellular uptake was also measured using a confocal laser microscope (Olympus Fluoview IX81).22 A549 cells were plated on 13 mm coverslips at 5 × 104 per well overnight. The cells were treated with 20 μg/mL of MSNs−ZOL−FITC at 1, 4, 12, and 24 h. Cell imaging was performed using a confocal laser microscope.31 Subcellular localization was measured using a confocal laser microscope.22 A549 cells were plated on coverslips as above. Subsequently, they were treated with MSNs−ZOL−FITC at 37 °C, washed with PBS three times, stained with 100 nM LTR or MTR for 30 min, and observed using a confocal laser microscope. The endocytosis mechanism of MSNs−ZOL−FITC was studied using adenosine triphosphate (ATP)-depleted environment and lowtemperature assays.21 A549 cells were seeded in 6-well culture plates (2 × 105 cells/well) overnight. Then, the cells were incubated with NaN3 for 1 h at 37 °C and MSNs−ZOL−FITC were added to the wells at 20 μg/mL for 1 h. The uptake of particles was analyzed using a flow cytometer. 2.9. Cellular DOX Release. The DOX released from DOX@ MSNs or DOX@MSNs−ZOL in vitro was monitored using the FCM.32,33 The detailed procedure is demonstrated as follows: A549 cells were seeded in 6-well plates (1 × 105 cells/well) overnight. Free DOX, DOX@MSNs, and DOX@MSNs−ZOL were added to the plates respectively. The wells containing only cells were used as control. After 1, 4, 8, and 12 h treatment, the cells were collected, 9238
DOI: 10.1021/acs.langmuir.6b02228 Langmuir 2016, 32, 9237−9244
Article
Langmuir
Figure 1. SEM images of MSNs (A), the scale bar is 100 nm. TEM images of MSNs (B), the 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 °C. centrifuged, washed, and resuspended in the PBS solution. The cellular DOX release was analyzed using a flow cytometer. 2.10. Cytotoxicity. The cytotoxicity was assessed using a CCK-8 kit assay.28 A549 cells were seeded in a 96-well plate (3 × 103 cells/ well) overnight. Thereafter, the cells were treated with free ZOL, free DOX, MSNs, MSNs−ZOL, DOX@MSNs, and DOX@MSNs−ZOL for 24 h. CCK-8 solution (10 μL) was added to the wells, and the cells were incubated for 4 h. The absorbance was measured using a microplate reader (Molecular Devices SpectraMax M4) at 450 nm. Cytotoxicity was expressed as the percentage of cell viability compared with the control cells. 2.11. Wound-Healing Assay. The migration of A549 cells was detected by a wound-healing assay.32,33 Briefly, when cells grew to 90% confluency in 6-well plates, a monolayer of cells was scratched by a sterile pipette tip to form a bidirectional wound. Free ZOL, free DOX, MSNs, MSNs−ZOL, DOX@MSNs, and DOX@MSNs−ZOL were added to the culture medium immediately. The wound width was observed using a microscope after 24 h treatment. The migration ratio (%) was calculated as follows
3. RESULTS AND DISCUSSION 3.1. Characterization of MSNs−ZOL. It has been reported that rigid nanoparticles with a long circulation halflife can accumulate in the spleen at a high percentage.35 Nanoparticles of intermediate size (20−100 nm) have the highest potential for in vivo applications because of their ability to circulate in the blood for long periods of time; these nanoparticles are able to avoid renal or lymphatic clearance and have the capability to avoid opsonization. 36 Moreover, nanoparticles within the size range of 20−100 nm are believed to be internalized easily by cells in comparison to smaller or larger particles.36 The SEM images (Figure 1A) and TEM images (Figure 1B) of the particles demonstrated that monodisperse MSNs with an average diameter of 40 nm were synthesized (Figure S1). The N2 adsorption−desorption isotherms (Figure 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 using UV− vis (Figure 1D) and FT-IR (Figure 1E) spectroscopy. The UV−vis spectra of MSNs−ZOL showed an absorbance peak at 210 nm, which indicates the presence of ZOL. As shown in Figure 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 the MSNs was calculated from the standard curve of ZOL. The loading capacity of ZOL onto the MSNs was about 166.8 mg/g. After ZOL bound to the MSNs, the zeta potential changed from −36.7 to −16 mV in water (Figure S2). These results indicate that the surface of MSNs has been successfully modified by ZOL.
migration ratio(%) original wound width − remaining wound width = × 100 original wound width 2.12. IL-8 Detection. A549 cells were seeded at 5 × 105 cells per 30 mm dish. Free ZOL, free DOX, MSNs, MSNs−ZOL, DOX@ MSNs, and DOX@MSNs−ZOL were added to the dish. The medium was immediately replaced by a serum-free medium, the supernatants were collected 30 h later, centrifuged, and stored at −80 °C.34 The IL8 level was quantified using the IL-8 ELISA kit according to the manufacturer’s protocol. 2.13. Statistical Analysis. Data were presented as mean ± standard deviation (SD) and were collected from three separate experiments. The comparison between two groups was made using the two-tailed Student’s t-test, and the significant difference was defined as P value
