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Rational Design of Multifunctional Dendritic Mesoporous Silica Nanoparticles to Load Curcumin and Enhance Efficacy for Breast Cancer Therapy Jiao Wang, Yue Wang, Qiang Liu, Linnan Yang, Rongrong Zhu, Chengzhong Yu, and Shilong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08400 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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ACS Applied Materials & Interfaces

Rational Design of Multifunctional Dendritic Mesoporous Silica Nanoparticles to Load Curcumin and Enhance Efficacy for Breast Cancer Therapy Jiao Wang,a Yue Wang,b Qiang Liu,a Linnan Yang,a Rongrong Zhu,a Chengzhong Yu,*,b and Shilong Wang *,a

a

Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, PR China

b

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.

Author Information *Corresponding Author: Chengzhong Yu Address: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia. Tel.: +61 7 334 63283 E-mail address: [email protected] Shilong Wang Address: Research Center for Translational Medicine at East Hospital, School of Life Science and Technology, Tongji University, Shanghai, PR China Tel.: +86 21 65982595 E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract Breast cancer is the primary reason of cancer-related death in women worldwide and the development of new formulations to treat breast cancer patients is crucial. Curcumin (Cur), a natural product, exerts promising anti-cancer activities against various cancer types. However, its therapeutic efficacy is hindered as a result of poor water solubility, instability, and low bioavailability. The aim of this work is to assess the curative effect of a novel nano-formulation, i.e., Cur-loaded and calcium-doped dendritic

mesoporous

silica

nanoparticles

modified

with

folic

acid

(Cur-Ca@DMSNs-FA) for breast cancer therapy. The results manifested that Cur-Ca@DMSNs-FA dispersed very well in aqueous solution, released Cur with a pH-responsible profile, and targeted efficiently to human breast cancer MCF-7 cells. Further investigations indicated that Cur-Ca@DMSNs-FA effectively inhibited cell proliferation, increased intracellular ROS generation, decreased mitochondrial membrane potential, and enhanced cell cycle retardation at G2/M phase, leading to a higher apoptosis rate in MCF-7 compared to free Cur. Moreover, the western blotting analysis demonstrated that Cur-Ca@DMSNs-FA were more active than free Cur through suppression of PI3K/AKT/mTOR and Wnt/β-catenin signaling, and activation of the mitochondria-mediated apoptosis pathway. In addition, hemolysis assay showed that the Ca@DMSNs-FA exhibited good biocompatibility. Last, in vivo studies indicated that when Cur was encapsulated in Ca@DMSNs-FA, the Cur concentration in blood serum and tumor tissues was increased after 1 h intraperitoneal injection. In conclusion, Cur-Ca@DMSNs-FA might be acted as a potential anticancer drug formulation for breast cancer therapy.

Keywords: Breast cancer; Curcumin; Dendritic mesoporous silica nanoparticles; Folic acid; pH-responsive release.

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1. Introduction Breast cancer is known as the most common cancer diagnosed in women.1 Recent statistics estimated 246,600 new cases and 40,450 deaths could occur from breast cancer in the United States in 2016.2 Although current therapies such as surgery, radiotherapy, and chemotherapy have proven to be efficient in cutting down primary tumor, the 5-year survival rate of patients with breast cancer is still poor chiefly because of reoccurrence, resistance, and severe side effects.3-5 Recently, medicine derived from natural plants with anti-cancerous properties and good safety profiles has received increasing attention for the tumor treatment.6,7 Curcumin (Cur) is a natural polyphenolic pigment derived from the rhizome of Curcuma longa, which possesses a wide range of pharmacological activities8-11 including a remarkable therapeutic potential for breast cancer.12-14 Various in vitro and in vivo studies have showed that Cur can effectively promote apoptosis and suppress metastasis, invasion, and angiogenesis through modulation of several intracellular signaling pathways.15-17 Besides, clinical trials have indicated that Cur is non-poisonous to humans when taken at a high dose of 8 g a day for 3 months.18 However, owing to low bioavailability caused by poor water solubility and fast degradation, the clinical application of Cur is limited.19-22 Previously, our study revealed that only a very low concentration of Cur could be detected in the serum of mice at 0.5 h after administration with a dosage of 400 mg per kilogram of body weight.23 Therefore, it is necessary to develop a nanomedcine-based Cur formulation for better t eatment of breast cancer. To date, various nano-delivery systems have been prepared to encapsulate Cur to overcome its drawbacks, such as surfactant complexes, liposomes, polymeric micelles, and inorganic oxides.24-27 Thereinto, mesoporous silica nanoparticles (MSNs) have caused great concern in drug delivery because of their unique mesostructures, big surface areas, high chemical stability, good biocompatibility, and adjustable surface chemistry.28-31 In previous years, a series of MSNs-based formulations have been prepared to improve Cur bioavailability. Earlier, Cur was either loaded into CTAB micelles followed by silica coating,32 immobilised onto the internal surface by 3



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covalent bonding,33 or conjugated with solid silica by using a simple wet chemical method.34 However, the cationic surfactant used is toxic to normal cells,35 and the drug release in the system was not responsive in tumor microenvironment. Recently, MCM-41 and KIT-6 were also used for Cur delivery showing enhanced in vitro toxicity against cancer cells.36,37 However, the small pore size of MCM-41 and poor dispersity of KIT-6 restricted their applications. As a result, challenges still remain in the design of multifunctional MSNs that can carry Cur for better cancer treatment. Nowadays, dendritic mesoporous silica nanoparticles (DMSNs) with center-radial pore channels have attracted considerable interest.38-40 Compared with conventional MSNs, they have open 3D dendritic superstructures that are characterized by large pore sizes and highly accessible internal surface areas.41 Till now, there is no report using DMSNs as Cur nano-carriers for cancer treatment. Meanwhile, we just reported the use of perfluorooctanic anions and cationic surfactants to produce DMSNs with small particle sizes.42 In addition, it is a prerequisite that Cur molecules selectively reach into the site of tumor for high activities.43 Thus, it is hypothesized that DMSNs could deliver Cur molecules to the tumor site and achieve its effective pharmacological functions. In this work, we report a DMSNs-based multifunctional drug delivery system (Scheme 1). DMSNs with the mean size of 63 nm were prepared firstly. Then, to selectively target cancer cells, folic acid (FA) was conjugated to DMSNs. FA is an optimal cancer-targeting ligand which has strong chemical attraction with the folate receptor (Kd = 10-10 M), a glycosylphosphatidyinositol-linked protein over-expressed in more than 40% of human tumors, including breast cancer.44-47 Furthermore, to achieve the stimuli-sensitive release, the nanopores of FA functionalized DMSNs (DMSNs-FA) were immobilized with biocompatible calcium hydroxide and Cur was loaded via chelating with divalent calcium.48-50 Finally, the anti-cancer efficacy of Cur loaded-multifunctional DMSNs (Cur-Ca@DMSNs-FA) was evaluated by a battery of in vitro and in vivo experiments. 2. Experimental Section 2.1. Chemicals 4


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Tetraethyl orthosilicate (TEOS, 98%), 3-aminopropyltriethoxysilane (APTES), cetyltrimethylammonium

bromide

4′,6-diamidino-2-phenylindole

(CTAB), (DAPI),

folic

acid

(FA,

triethanolamine

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

bromide

98%), (TEA),

(MTT),

and

dimethylsulfoxide (DMSO) were bought from Sigma-Aldrich Co. (St Louis, MO, USA). 1-ethyl-3-(3-dimethly-aminopropyl) carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), succinic anhydride (SA), anhydrous calcium chloride (CaCl2), and Cur (purity ≥98%) were bought from Aladdin Chemistry Co. Ltd. (Shanghai, China). Sodium perfluorooctanoate (PFO) was bought from Santa Cruz Biotechnology Co. (Dallas, Texas, USA). Sodium hydroxide (NaOH), toluene, acetone, absolute ethanol (EtOH, >99.9%), Tween 80, and methanol (MeOH) were purchased from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). DMEM (high glucose) medium, penicillin-streptomycin solution, fetal bovine serum (FBS), trypsin, and PBS solution (pH 7.4 and 5.0) were supplied from HyClone (Logan, UT, USA). 2.2. Cell Lines and Culture Conditions The human lung cancer cell line A549 and breast cancer cell line MCF-7 were supplied from Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (high glucose) medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator containing 5% CO2. 2.3. Animals BALB/c nude mice (5–6-weeks old females) were supplied from Shanghai Laboratory Animal Co. (SLAC), Ltd. and raised in SPF animal facility in Tongji University. All animal-related procedures were carried out according to the protocol approved by the Institutional Laboratory Animal Resources at University. 2.4. Synthesis Procedures 2.4.1. Synthesis of DMSNs DMSNs were prepared according to our report with slight modifications.42 Briefly, a mixture containing ddH2O (25 mL), TEA (0.068 g), CTAB (0.384 g), and PFO (0.0682 g) was stirred at 80 °C for 1 h. After that, TEOS (4 mL) was added into above 5



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solution and stirred at 80 °C for another 2 h. Subsequently, the resultant products were collected through centrifugation (20,000 rpm, 15 min), washed twice with ddH2O/EtOH (20 mL, 1:1, v/v), and dried at 50 °C for 48 h. Finally, DMSNs were obtained after calcination at 550 °C for 5 h in air. 2.4.2. Synthesis of Ca@DMSNs-FA First, 0.5 g of DMSNs were mixed in dry toluene (50 mL) in a round bottom flask, then APTES (0.8 mL) was added at once and the reaction mixture was refluxed at 115 °C for 20 h. The particles were centrifuged (20,000 rpm, 15 min), washed twice with EtOH and once with acetone, and dried overnight at 50 °C (denoted as DMSNs-NH2). Next, 0.5 g of DMSNs-NH2 were re-dispersed in 30 mL of DMSO with SA (0.45 g) and TEA (0.45 mL) and stirred at 50 °C for 48 h to yield DMSNs-COOH. Furthermore, DMSNs-COOH (0.2 g) were sufficiently dispersed in DMSO solution (20 mL). Then, EDC·HCl (0.1 g) and NHS (0.05 g) were added and stirred at room temperature for 2 h. Subsequently, FA (0.01 g/mL in DMSO, 8 mL) was dropwise added into above solution upon stirring for another 24 h. The products were harvested via centrifugation, washed thrice with ddH2O, and dried with lyophilization (denoted as DMSNs-FA). Last, 0.1 g of DMSNs-FA and 2 mmol CaCl2 were dispersed in methanol (20 mL). The solvent was evaporated after intensive mixing and then again re-dispersed in MeOH (20 mL), followed by the addition of 4 mmol NaOH that dissolved in MeOH (10 mL) to give calcium hydroxide filled DMSNs-FA precipitate. The final products were harvested via centrifugation, washed twice with MeOH, and dried with lyophilization (denoted as Ca@DMSNs-FA). 2.5. Drug Loading Ca@DMSNs-FA (0.1 g) were dispersed in Cur solution (4 mg/mL in EtOH, 25 mL) by ultrasonication. After stirring in the dark for 24 h, the mixtures were centrifuged at 20,000 rpm for 15 min. Then, the harvested products were washed with EtOH (20 mL) to remove any unbound Cur and dried with lyophilization to obtain Cur-loaded Ca@DMSNs-FA which was denoted as Cur-Ca@DMSNs-FA. The above supernatant and washing liquid were collected and the loading weight ratio (weight of Cur in Ca@DMSNs-FA / weight of Cur-Ca@DMSNs-FA ×100%) was calculated. 6


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Meanwhile, to calculate the percentage of Ca(OH)2 in Cur-Ca@DMSNs-FA, a given amount of Cur-Ca@DMSNs-FA was added to a 6 M HCl solution followed by ultrasonic treatment. The concentration of Ca in collected supernatant was determined by ICP-MS. 2.6. Characterization Dynamic light scattering (DLS) experiments were performed with a Zetasizer NanoZS Instruments. The samples were dispersed in ddH2O under unltrasonication for 5 min, and then measured at 25 °C. TEM images were obtained with a JEOL 1010 operated at 100 kV. The samples were dispersed in ddH2O and then dropped on a carbon film supported on a copper grid. SEM images were obtained using a JEOL 7800 SEM operated at 3 kV. The samples were prepared by dispersing the nanoparticles in EtOH, then dropped onto the aluminum foil pieces attached on conductive carbon tape on SEM mounts. Zeta potential of samples were measured by using a Malvern NanoZS Zetasizer after dispersing them in PBS (pH 7.4) under gentle vortex. Nitrogen (N2) adsorption-desorption isotherms was measured at 77 K by using a Micromeritcs Tristar II system. Before measuring, all samples were degassed at the corresponding temperature under vacuum overnight. The pore size distributions were calculated by the Barrett–Joyner–Halenda (BJH) method. Fourier transform infrared (FTIR) spectra were scanned on Thermo Nicolet Nexus 6700 FTIR spectrometer in the range of 400-4000 cm−1. Thermogravimetric (TGA) measurements were performed by a Setaram TG92 instrument at a heating rate of 2 °C/min in airflow. UV-vis spectra were recorded on a Cary 50 spectrophotometer. 2.7. Dispersity and Drug Release An equivalent quantity of free Cur or Cur-Ca@DMSNs-FA was dispersed in PBS solution (pH 7.4) to investigate their aqueous solubility / dispersity. Furthermore, the release profile of Cur-Ca@DMSNs-FA was studied in PBS (pH 5.0 and 7.4) solution containing Tween-80 (10%, v/v). Briefly, 36 mg of Cur-Ca@DMSNs-FA well suspended in 27 mL of solution was divided equally into 18 tubes, each experiment was performed in triplicates. All tubes were shaken in an incubator at 100 rpm under 37 °C. At different time points, the solution was centrifuged and the supernatant was 7



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removed. The released drug amount was quantified by UV-vis measurement. 2.8. Cell Viability Assay The cytotoxicity of DMSNs, Ca@DMSNs-FA, free Cur, and Cur-Ca@DMSNs-FA was determined by MTT assay.51 Briefly, A549 and MCF-7 cells were seeded into 96-well plates (6×103 per well), respectively. After cell attachment, DMSNs, Ca@DMSNs-FA, free Cur, or Cur-Ca@DMSNs-FA in medium at different concentrations were added into wells and incubated for 24 h. Next, MTT (5 mg/mL in PBS, 20 µL) was added and the plates were further incubated for 4 h. Then, the medium was removed and 150 µL of DMSO was added. Finally, the absorbance at 490 nm was measured by a microplate reader (ELX800 UV, BIO-TEK, USA). 2.9. Intracellular Uptake and Release Study Both intracellular uptake and Cur release behaviors of Cur-Ca@DMSNs-FA were investigated by using a confocal laser scanning microscope (CLSM). For uptake study, the A549 cells and MCF-7 cells were seeded into glass bottom dishes (1.5×105 per dish) and cultivated for attachment. After that, free Cur and Cur-Ca@DMSNs-FA at the same Cur concentration (10 µg/mL) were added and incubated for 1 h. Meanwhile, to examine the active targeting abilities, other two groups of cells were pre-incubated with 2 mM of folic acid for 1 h before Cur-Ca@DMSNs-FA treatment. After that, the medium was sucked out and the cells were rinsed carefully with PBS for three times. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with DAPI at room temperature. Finally, the cells were observed with a confocal laser scanning microscope imaging system (Leica TCS SP5 II). For Cur release, after treating with Cur-Ca@DMSNs-FA for 1 h, the MCF-7 cells were washed with PBS for three times and added into medium for 4 h and 12 h cultivation, respectively. Subsequently, the experiment was processed with the same way as the uptake study. 2.10. Cell Apoptosis Primarily, the apoptosis of Cur-Ca@DMSNs-FA against MCF-7 cells was assessed by Hoechst staining assay. Typically, cells were seeded into glass bottom dishes (1.5×105 per dish) and cultivated for attachment. After that, free Cur and Cur-Ca@DMSNs-FA at an equivalent Cur concentration of 10 µM were added and 8


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incubated for 48 h. Then, the medium was sucked out and the cells were rinsed two times with PBS. Subsequently, the cells were stained with the Hoechst 33342 for 20 min. Finally, the cells were viewed as above. The apoptosis effect of Cur-Ca@DMSNs-FA was also evaluated by Annexin V-APC/PI apoptosis detection kit (KeyGen Biotech, China). MCF-7 cells were seeded into 6-well plates (1.5×105 per dish) and cultivated for attachment. After that, the cells were treated as above. Subsequently, the cells were harvested and resuspended in 500 µL binding buffer containing both Annexin V-FITC (5 µL) and PI (5 µL). Finally, the cells were examined by a flow cytometer (Becton, Dickinson and Company, USA). 2.11. Cell Cycle Assay The effect of Cur-Ca@DMSNs-FA on cell cycle distribution was examined by using the cell cycle detection kit (KeyGen Biotech, China). MCF-7 cells were seeded into 6-well plates (1.5×105 per dish) and cultivated for attachment. After that, free Cur and Cur-Ca@DMSNs-FA at an equivalent Cur concentration of 10 µM were added and incubated for 48 h. Then, cells were harvested and fixed in 70% ethanol (ice-cold) at 4°C overnight. Subsequently, the cells were resuspended in 500 µL of PBS (pH 7.4), incubated with RNase A (40 µg/mL), and stained with PI (10 µg/mL) in the dark. Finally, the cells were analyzed by the Becton–Dickinson flow cytometer. 2.12. ROS Detection The effect of Cur-Ca@DMSNs-FA on intracellular reactive oxygen species (ROS) generation was examined by using the ROS assay kit (Beyotime Biotech, China). MCF-7 cells were handled as depicted in the cell cycle assay. After that, the cells were processed according to the product description. Finally, the cells were analyzed using the Becton–Dickinson flow cytometer. 2.13. Mitochondrial Membrane Potential (∆ψm) Measurement Changes of ∆ψm were determined with mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotech, China). MCF-7 cells were handled as depicted in the cell cycle assay. After that, the cells were processed according to the product description. Finally, the cells were analyzed using the Becton–Dickinson flow cytometer. 9



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2.14. Western Blot Analysis MCF-7 cells were handled as depicted in the cell cycle assay. After that, the cells were harvested and total cellular protein lysates were extracted by a total protein extraction kit (KenGen Biotech, China). Meanwhile, the cytosolic and nuclear fractions were extracted using a nucleoprotein and cytoplasmic protein extraction kit (KenGen Biotech, China). The protein content was measured with BCA protein assay kit (KenGen Biotech, China). An aliquot of 20 µg extract was fractionated by electrophoresis through SDS-PAGE gel, and transferred to PVDF membrane. After being blocked by 5% BSA at room temperature for 1 h, the protein blots were probed with primary rabbit antibodies against PI3K, p-AKT, p-mTOR, β-catenin, p53, Bcl-2, cytochrome c, caspase 9, caspase 3, PARP, NF-κB p65, IκBα, β-actin, and Lamin A (Cell Signaling Technology, USA) at 4 °C overnight. Then, the membrane was incubated with secondary antibody (KenGen Biotech, China) to detect the target protein. Finally, the proteins were visualized by ImageQuant LAS4000 mini (GE Healthcare Life Science, USA). 2.15. Hemolysis Test Hemolytic activities of DMSNs and Ca@DMSNs-FA were evaluated by detecting the hemoglobin release from mice blood cells. Fresh mice blood was kindly supplied by the Shanghai Research Centre for Model Organisms. Firstly, the blood was centrifuged at 10250 g at 4 °C for 5 min and washed four times with PBS solution so as to obtain Red blood cells (RBCs). The RBCs was then diluted to a concentration of 10% (v/v) with PBS (7.4). Furthermore, the 0.8 mL of the PBS solutions of DMSNs and Ca@DMSNs-FA were added into 0.2 mL of diluted RBCs suspension, respectively, and the final concentrations of the nanoparticles were 5, 10, 20, 40, 80, 160, and 320 µg/mL. Herein, RBCs incubated PBS and ddH2O were served as negative and positive controls, respectively. The mixed solutions were then vortexed and kept in static conditions at room temperature for 2 h. Subsequently, all the samples were centrifuged at 12,000 g at 4 °C for 5 min, and the absorbance at 541 nm were determined using a UV-vis spectrophotometer. The hemolytic degree was expressed by the hemolytic ratio in each sample according to the following formula: 10


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hemolytic ratio = (OD test

OD negative control) / (OD positive control

OD negative control)

×100%. 2.16. Pharmacokinetics and Tumor Distribution BALB/c nude mice (5–6-weeks old females) were given with free Cur and Cur-Ca@DMSNs-FA intraperitoneally (ip) at 30 mg/kg equivalent Cur, respectively. After 1 h, mice were sacrificed and the plasma was obtained through centrifuging the collected blood samples. Finally, Cur was extracted by adding acetonitrile and drug levels were analyzed by HPLC. The xenograft tumor models of MCF-7 breast cancer were established and given with free Cur or Cur-Ca@DMSNs-FA at 30 mg/kg equivalent Cur via ip. 1 h later, mice were sacrificed and tumor tissues were collected. Subsequently, all tissues were homogenized and added acetonitrile. Then, Cur levels in each tissue were determined with HPLC. Finally, the tumor tissue distribution was expressed as µg drug per g proteins. 2.17. Statistical Analyses All data were displayed as mean ± standard deviation (SD). The statistical significance of the differences between the groups was determined using one-way analysis of variance (ANOVA). For all statistical analyzes, P < 0.05 or 0.01 were considered significant.

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Scheme 1. A schematic illustration of the fabrication of Cur-Ca@DMSNs-FA and the pH stimuli responsive release in tumor.

3. Results and Discussion 3.1. Synthesis and Characterization of Multifunctional DMSNs DMSNs were synthesized with a facile approach.42 DLS results showed DMSNs had a mean diameter ~ 63 nm and presented a narrow monodispersed unimodal size distribution mode (PDI = 0.141) (Figure 1A). TEM and SEM images (Figure 1B and Figure 1C) revealed that DMSNs displayed homogeneous spherical morphology and uniform open 3D dendritic and center-radial pore structures. The fabricated and working principle of multifunctional DMSNs used as the carrier to deliver Cur is depicted in Scheme 1. As shown in Figure 1D-1F, after multi-steps of modification, including amino, carboxyl, and folic acid conjugation, the morphology as well as the size of the particles did not change obviously, just the pores turned a little blurred. However, after Ca(OH)2 loading, the pore channels of particles 12


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(Ca@DMSNs-FA) were nearly filled up and became very blurred (Figure 1G). Followed by Cur loading, the pore channels of particles (Cur-Ca@DMSNs-FA) can be hardly distinguished (Figure 1H). When Cur-Ca@DMSNs-FA were dispersed into a mildly lower pH (5.0) solution for a while, the pore structures were observed again, indicating the embedded Ca(OH)2 was dissolved in a slightly acidic environment (Figure 1I). Various DMSNs were studied by zeta potential analysis. As depicted in Figure S1, the zeta potential of DMSNs, DMSNs-NH2, DMSNs-COOH, DMSNs-FA, Ca@DMSNs-FA, and Cur-Ca@DMSNs-FA is -23.3, +3.31, -41.2, -25.1, -16.9, and -15.4 mV, respectively. The changes of zeta potential in each step suggested the successful functionalization.

Figure 1. Particle size distribution determined by DLS (A), TEM image (B), and SEM image (C) of DMSNs. TEM images of (D) DMSNs-NH2, (E) DMSNs-COOH, (F) DMSNs-FA, (G) Ca@DMSNs-FA, and (H) Cur-Ca@DMSNs-FA. (I) TEM image of the Cur-Ca@DMSNs-FA obtained after incubation in weakly acidic solutions.

Nitrogen sorption analysis was performed to describe the porous nature. The 13



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nitrogen adsorption and desorption isotherms (Figure S2A) indicated that DMSNs displayed a type-IV isotherm curve with a major capillary condensation step at a high relative pressure (P/P0) of ~ 0.9. Besides, DMSNs exhibited a BET surface area of 422 m2/g, a total pore volume of 1.10 cm3/g, and a relatively broad pore size distribution in the range of 3-9 nm computed using Barrett-Joyner-Halenda (BJH) model from the adsorption branch (Figure S2B). After functionalization with -NH2, -COOH, and FA, the surface areas decreased in general. After incorporation of Ca(OH)2, small micropores at 0.8 nm were observed. Meanwhile, the BET surface area and pore volume changed to 116 m2/g and 0.87 cm3/g, respectively. This phenomenon may be explained by the deposition of Ca(OH)2. After Cur loading, the pore size cannot be detected and the surface area together with pore volume decreased (Table S1). The whole synthetic processes were also explored by infrared spectroscopic (IR) analysis (Figure S3). DMSNs displayed two absorption peaks at 806 and 1068 cm−1, which were assigned to flexible vibration of Si–O and stretching vibration of Si−O−Si, respectively. The amino modification led to new peaks at 698, 1500, and 2928 cm−1, which stand for vibration of N-H, symmetric vibration of -NH3+, and asymmetric vibration of C-H. The carboxyl modification resulted in the peak at 1704 and 1413 cm−1, which can be ascribed to asymmetric and symmetric vibrations of COO-. For free FA, three bands at 1691, 1604, and 1481 cm−1 correspond to benzene (conjugated double bonds) and C=O vibration, ester bond, and hetero-ring (conjugated double bonds), respectively.47 The DMSNs-FA exhibit the characteristic peaks of FA at 1649, 1562, and 1409 cm−1 corresponding to benzene (conjugated double bonds) and C=O vibration, hetero-ring (conjugated double bonds), and symmetric COO-, respectively. The shift from 1691 to 1649 cm-1 as well as 1604 to 1562 cm-1 is due to the conjugation site near the amine group of FA. Ca@DMSNs-FA showed decreased intensity of absorption and new peak at 3650 cm-1, which were attributed to the filling of Ca(OH)2. Typically, the two C=O stretching bands at 1427 and 1506 cm−1 are the characteristic peaks of Cur. However, after loading into Ca@DMSNs-FA, these bonds were shifted to lower wavenumbers (1419 and 1498 cm−1), manifesting the 14


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participation of carbonyl of the Cur in the process of chelation with divalent calcium.49,50 Besides, the emerge of extra bands such as 1218 and 1317 cm−1 further confirmed that the Cur was loaded in Ca@DMSNs-FA successfully. To further confirm the drug carrying in Ca@DMSNs-FA via Ca2+-Cur coordinate bonding, UV−vis absorption spectroscopy was carried out. As shown in Figure S4, free Cur has a distinctly high absorbance peak at 425 nm when dissolved in an ethanolic solution. However, after loading into Ca@DMSNs-FA, the characteristic peak was turned to around 448 nm, demonstrating the involvement of carbonyl of Cur in metal complexation. The composition of our formulation was investigated using TGA measurement (Figure S5). The water residue was removed at a temperature of > 100 °C and the – NH2 groups, -COOH groups, and FA molecule degraded at > 300 °C. Meanwhile, the Ca(OH)2 decomposed at > 580 °C. The weight losses of DMSNs, DMSNs-NH2, DMSNs-COOH, DMSNs-FA, and Ca@DMSNs-FA were measured to be 3.4, 17.2, 23, 27.9, and 37.7%, respectively. In the case of Cur-Ca@DMSNs-FA, the TGA analysis displayed the total weight loss about 37.5%, which was ascribed to the removal of -NH2, -COOH, FA, Ca(OH)2, and Cur. Based on these results, the Cur amount loaded in Ca@DMSNs-FA was found to be ~ 8%, which was consistent with the value measured by the supernatant collection method depicted in Materials and Methods (2.5). Besides, the ICP results demonstrated the percentage of Ca(OH)2 in the Cur-Ca@DMSNs-FA is around 41.54%. Collectively, the results discussed above confirmed the successful fabrication of multifunctional DMSNs and loading of Cur. 3.2. In Vitro Dispersity and Drug Release To date, poor aqueous solubility has restricted the clinical applications of Cur.19-22 Herein, the multifunctional DMSNs were synthesized and used as reservoirs for delivering Cur. To confirm our formation was able to improve its solubility, equal amounts of free Cur and Cur-Ca@DMSNs-FA were suspended in an equal volume of PBS (pH 7.4). As depicted in Figure 2A, it was observed that free Cur is easily soluble in EtOH, while hardly in aqueous media with visible precipitation. In contrast, Cur-Ca@DMSNs-FA were well dispersed in aqueous solutions and presented a dark 15



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red color. When the pH was lowered to 5.0, the color of dispersion turned into light yellow. The pH-responsive drug release behavior was measured quantitatively that was described in Figure 2B. In neutral PBS solution, the cumulative Cur release was just around 35% at 12 h. In contrast, the Cur-Ca@DMSNs-FA showed remarkable increased release rates, which can reach to 80% in acidic PBS solution (pH 5.0) just for 0.5 h. All the results discussed above demonstrated that the complexation between the Cur and the Ca2+ in Cur-Ca@DMSNs-FA could be dissociated under acidic environment and resultantly led to Cur release. This observation is important for cancer therapies because of mildly acidic microenvironment in cancer tissues.

Figure 2. (A) Solubility of free Cur and Cur-Ca@DMSNs-FA in different solvents. (i) Free Cur (2 mg) added in PBS (pH 7.4) was insoluble, (ii) Free Cur (2 mg) was fully soluble in EtOH, (iii) Cur-Ca@DMSNs-FA (25 mg, equivalent of 2 mg Cur) dispersed in PBS (pH 7.4), and (iv) Cur-Ca@DMSNs-FA (25 mg) in PBS (pH 5.0). (B) Cur release profiles from Cur-Ca@DMSNs-FA in PBS solution (pH 5.0 and 7.4) containing Tween-80 (10%, v/v). 16


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3.3. In Vitro Cytotoxicity and Cellular Uptake In order to measure the toxic effects caused by pure nanoparticles. We first tested the cellular toxicity of DMSNs and Ca@DMSNs-FA at different concentrations on A549 and MCF-7 cells. As depicted in Figure 3A and 3B, the bare DMSNs and Ca@DMSNs-FA had no significant cellular toxicity to both cell lines after 48 h treatment, even the tested concentration reached as high as 320 µg/mL. Subsequently, the cytotoxicity comparison of free Cur and Cur-Ca@DMSNs-FA were evaluated. As shown in Figure 3C and 3D, both free Cur and Cur-Ca@DMSNs-FA showed dose-dependent cytotoxicity on A549 and MCF-7 cells, respectively. Besides, the Cur-Ca@DMSNs-FA showed a higher cytotoxicity than that of free Cur at all the treated concentrations, especially obvious towards MCF-7 cells. In detail, cell viability of MCF-7 cells incubated with free Cur at 5, 10, and 20 µM were 94%, 79%, and 33%, respectively. In contrast, the cell viability of MCF-7 cells incubated with free Cur-Ca@DMSNs-FA at 5, 10, and 20 µM decreased to 71%, 35%, and 9%, respectively. The different effects of Cur-Ca@DMSNs-FA on survival rate of both cancer cells could be attributed to the folate receptor meditated targeting endocytosis.

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Figure 3. Cytotoxicity of DMSNs and Ca@DMSNs-FA against cancer cell lines at different concentrations: (A) A549; (B) MCF-7. Cytotoxicity of free Cur and Cur-Ca@DMSNs-FA against cancer cell lines at different concentrations: (C) A549; (D) MCF-7.

To further corroborate the folate receptor mediated targeted delivery of our formulation, intracellular uptake of free Cur and Cur-Ca@DMSNs-FA on A549 (folate receptor negative cells) and MCF-7 (folate receptor positive cells) was investigated by confocal laser scanning microscope.52,53 Figure 4 illustrated the fluorescence images of A549 and MCF-7 cells incubated with free Cur or Cur-Ca@DMSNs-FA in media at an equivalent Cur concentration of 10 µg/mL for 1 h. It was observed that both A549 and MCF-7 cells treated with free Cur showed no detectable fluorescence intensity. The A549 and MCF-7 cells incubated with Cur-Ca@DMSNs-FA both showed obvious fluorescence signals and the latter is much stronger. Besides, the fluorescence intensity of MCF-7 cells treated with Cur-Ca@DMSNs-FA decreased sharply when the cells was pre-treated with folic acid,

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indicating that the folate receptors on FA positive cells are responsible for the target delivery of nanoparticles. The intracellular release behavior of Cur from Cur-Ca@DMSNs-FA in MCF-7 cells was also monitored. As depicted in Figure S6, it displayed strong fluorescence signals as “big” spots around the MCF-7 cells when treated with Cur-Ca@DMSNs-FA for 1 h. After 1 h incubation, the cell culture medium was removed, a fresh medium was added and the whole fluorescence was detected for the next 4 h and 12 h. It showed that the green fluorescence signals appeared in the cytoplasm gradually and decreased into small "spots" with relatively weak intensity, achieving a more homogenous distribution in the cells. Combined with the in vitro release behavior shown in Figure 2B, it is suggested that Cur-Ca@DMSNs-FA phagocytized by tumor cells gradually release the drugs in the cytosol with a slightly acidic environment.

Figure 4. Fluorescence confocal images of free Cur and Cur-Ca@DMSNs-FA uptake by A549 cells and MCF-7 cells in 1 h. Bar = 50 µm.

3.4. Apoptosis Ability of Cur-Ca@DMSNs-FA The above results have demonstrated that the Cur-Ca@DMSNs-FA could enter into MCF-7 cells through folate-mediated targeting and then released the Cur to enhance its cytotoxicity. To further investigate the mechanism by which Cur-Ca@DMSNs-FA exerted much more cytotoxicity than free Cur to MCF-7 cells, a series of experiments 19



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were performed. The apoptosis-inducing effect of Cur-Ca@DMSNs-FA was firstly evaluated using the Hoechst 33342 staining.54 As depicted in Figure 5A, the nuclei of untreated cells were round and unbroken showing low fluorescence intensity. However, when the cells were incubated with free Cur, the nuclei became lager and condensed with higher fluorescence intensity. The nuclei condensation became more serious and some fragmentation can be observed for the most cells after Cur-Ca@DMSNs-FA treatment. In addition, we quantitatively investigated the apoptosis effects using the Annexin V-APC/PI apoptosis detection kit. As depicted in Figure 5B, Cur-Ca@DMSNs-FA had stronger apoptosis ability compared to free Cur. Specifically, the cells after incubated with free Cur-Ca@DMSNs-FA for 48 h showed the total apoptosis ratio of 25.85%, which was far better than 12.5% of free Cur. Taken together, the results from Figure 5 indicated the stronger cytotoxic effect of Cur-Ca@DMSNs-FA on MCF-7 cancer cells might be attributed to induction of more apoptosis than that by free Cur at the comparable doses.

Figure 5. (A) Influence of free Cur and Cur-Ca@DMSNs-FA on the nuclear morphology of MCF-7 cells. Bar = 25 µm. (B) Apoptosis effects of free Cur and Cur-Ca@DMSNs-FA on MCF-7 cells. 20


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Cell cycle damage is one of the vital features for detecting apoptotic cells. For example, Leong et al. reported that the SiO2 can induce cell toxicity by affecting cell cycle via disruption of microtubule assembly.55-57 Previous studies also reported that treatment with Cur caused cell cycle arrest in a series of cancer cells.10,58 Figure 6 showed the cell cycle distribution of MCF-7 cells incubated with free Cur and Cur-Ca@DMSNs-FA at identical concentrations (10 µM) for 48 h. It was observed that free Cur and Cur-Ca@DMSNs-FA could both induce G2/M cell cycle arrest, and the effect of latter is more significant. Specifically, untreated cells, cells incubated with free Cur, and cells incubated with Cur-Ca@DMSNs-FA showed 19.42%, 24.54%, and 41.07% of cells in G2/M phase, respectively. Hence, these results suggested that Cur-Ca@DMSNs-FA exerted a higher apoptotic effect on MCF-7 cells via much more G2/M cell cycle arrest compared with free Cur.

Figure 6. Effect of free Cur and Cur-Ca@DMSNs-FA on the cell cycle distribution of MCF-7 cells. *p< 0.05 and **p< 0.01 vs. control.

It is well known that ROS plays a vital role in anticancer activity of many 21



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chemotherapeutic agents. Previous studies demonstrated that Cur can induce cancer cell apoptosis via ROS generation.59 To explore whether ROS was involved in Cur-Ca@DMSNs-FA-induced apoptosis of MCF-7 cells, intracellular ROS generation was measured. As depicted in Figure 7, the peak of cellular fluorescence in cells incubated with Cur-Ca@DMSNs-FA displayed more significant shift compared to cells incubated with free Cur. Specifically, the mean fluorescence intensity of cells incubated with Cur-Ca@DMSNs-FA was 2.5 times more than that of free Cur. These results confirmed that Cur-Ca@DMSNs-FA might suppress cell proliferation and promote apoptosis through boosting intracellular ROS generation in MCF-7 cells.

Figure 7. Effect of free Cur and Cur-Ca@DMSNs-FA on ROS production in MCF-7 cells. *p< 0.05 and **p< 0.01 vs. control.

Mitochondrial membrane potential (∆ψm) is a crucial parameter for evaluating mitochondria function, and the decrease of ∆ψm has been considered as an early event in apoptosis.60 Herein, the ∆ψm of MCF-7 cells after treating with free Cur and Cur-Ca@DMSNs-FA were measured by JC-1 staining under flow cytometry. In intact

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cells, JC-1 is more gathered at mitochondria as J-aggregates emiting red fluorescence. While, in apoptotic cells due to loss of ∆ψm it will transfer into cytosol as green-fluorescent monomers. Accordingly, the specific value between red and green fluorescence can be served as a standard measure of ∆ψm.51,61 As shown in Figure 8, we can observe an increase in percentage of cells that emitted JC-1 green fluorescence from 1.57% in control cells to 6.80% and 47.9% in cells incubated with free Cur and Cur-Ca@DMSNs-FA, respectively. Moreover, in comparison with control, the ∆ψm of cells decreased about 25% after treating with free Cur and greatly dropped to 20% when cells were treated with Cur-Ca@DMSNs-FA. These results indicated that Cur-Ca@DMSNs-FA might induce apoptosis of MCF-7 cells through mitochondria mediated apoptosis pathway.

Figure 8. Effect of free Cur and Cur-Ca@DMSNs-FA on the ∆ψm of MCF-7 cells. *p< 0.05 and **p< 0.01 vs. control.

3.5. Apoptosis Mechanism of Cur-Ca@DMSNs-FA. 23



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To illuminate the molecular mechanism of Cur-Ca@DMSNs-FA induced apoptosis in MCF-7 cells, the alterations of expression levels of proteins associated with apoptosis, including PI3K, p-AKT, p-mTOR, β-catenin, p53, Bcl-2, cytochrome c, caspase 9, caspase 3, PARP, NF-κB p65, and IκBα were determined by western blot analysis. As shown in Figure 9A, the level of proteins including PI3K, p-AKT, p-mTOR, β-catenin, Bcl-2, and NF-κB p65 decreased under the free Cur treatment compared to the control, especially in the Cur-Ca@DMSNs-FA group. Meanwhile, we found significant activation of proteins including p53, cytochrome c, caspase 9, caspase 3, PARP, and IκBα after both free Cur and Cur-Ca@DMSNs-FA treatment, and the latter is much stronger. According to reports, the mitochondria-mediated (intrinsic) pathway plays a crucial role in Cur-induced apoptosis of many cancer cells.62 In detail, DNA damage induced by Cur promoted p53 activation that led to decreased Bcl-2 levels followed by loss of ∆ψm. Subsequently, the cytochrome c release from the mitochondrion into the cytosol was triggered and it will combine with the apoptotic protease-activating factor complex so as to induce the cleavages of molecules such as caspase-7, caspase-3, and PARP, which eventually caused apoptosis. Meanwhile, PI3K/AKT/mTOR is the proverbial main regulatory signaling pathway that modulates cell growth and proliferation in cancer cells. It had been already reported that Cur showed its anti-cancer effect through inhibiting this pathway.63,64 Besides, abnormal activation of the Wnt/β-catenin signaling pathway and succedent upregulation of β-catenin driven downstream targets such as c-Myc and cyclin D1 are related to the development of breast cancer.65 Prasad et al. reported that Cur inhibited cell proliferation through modulation this pathway in MCF-7 cancer cells.66 NF-κB regulates anti-apoptotic gene expressions in cancer cells and is considered to be at “the crossroads of life and death’’. In the rest state, NF-κB exists in the cytoplasm, as an inactive heterodimer composing of p50, p65, and IκBα subunits. While, in the active state, NF-κB p65 will be transferred into nucleus due to the degradation of IκBα.67 Literature reports have shown that Cur can inhibit the activation of NF-κB and its gene products that are participated in cancer cell proliferation and apoptosis.15,17,68 All together, the result of 24


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western blot analysis indicated that Cur-Ca@DMSNs-FA induced higher apoptosis of MCF-7 cancer cells through inhibiting the PI3K/AKT/mTOR, as well as Wnt/β-catenin signaling pathway and promoting the mitochondria-mediated signaling pathway (Figure 9B).

Figure 9. (A) Influence of free Cur and Cur-Ca@DMSNs-FA on the expression of key proteins participated in apoptosis. (B) The diagram of the mechanisms of Cur-Ca@DMSNs-FA on breast cancer.

3.6. Biocompatibility of the Nanoparticles Hemolysis is the key risk projects connected with the biocompatibility of nanoparticles intended for injection.69 It is well known that the RBCs are broken to release the hemoglobin into solution in the process of hemolysis, and the resulting solution will become visually red. According to the ISO/TR7405 standard, the formulation is determined to possess a risk of hemolysis when the hemolytic ratio exceeds 5%. As shown in Figure 10A, the optical images displayed the direct observation of hemolysis at different concentrations (5, 10, 20, 40, 80, 160, and 320 µg/mL) of DMSNs and Ca@DMSNs-FA, respectively. In terms of DMSNs, it was observed that the color of the resulting solution did not change obviously until the

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concentration reached as high as 160 and 320 µg/mL compared with the negative control. However, no visible color changes can be observed in terms of Ca@DMSNs-FA. To further determine the hemolysis effect, the OD value of the supernatant at 541 nm was determined with UV-vis spectroscopy. As depicted in Figure 10B, when the treating amount of DMSNs reached 160 and 320 µg/mL, the hemolytic ratio was 7.86% and 31.95%, respectively. However, the hemolytic ratio was just 4.38% for Ca@DMSNs-FA at the highest testing concentration (320 µg/mL). The hemolysis of red blood cells when incubated with DMSNs under high concentration may be attributed to the silanol group (Si-OH) on the surfaces that can bind with the phosphatidylcholine-rich RBC membrane. Alltogether, it could be seen from Figure 10 that Ca@DMSNs-FA exhibited better hemocompatibility than DMSNs at high concentrations.

Figure 10. Hemolysis assay of DMSNs and Ca@DMSNs-FA samples: (A) The optical images and (B) Hemolysis percentages.

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3.7. In Vivo Bioavailability The anti-cancer efficiency of Cur is greatly restrained due to the low bioavailability.19,21 To investigate whether our formulation can improve its bioavailability, the plasma concentration as well as tumor distribution was evaluated in BALB/c mice after intraperitoneal injection with free Cur or Cur-Ca@DMSNs-FA at 30 mg/kg for 1 h. As shown in Figure 11A, the plasma level of Cur was about 1.06 µg/mL after treated with free Cur, while it achieved as higher as 3.90 µg/mL after Cur-Ca@DMSNs-FA treatment. Meanwhile, the Cur content found in tumor was 1.09 µg/g protein when mice were injected with Cur-Ca@DMSNs-FA, which was much higher than 0.36 µg/g protein after treated with free Cur (Figure 11B). Thus, the plasma concentration and the accumulation in tumors for Cur can be significantly increased when it was carried by Ca@DMSNs-FA. In summary, our formulation can improve the bioavailability of Cur so that exerted higher anti-cancer effects.

Figure 11. In vivo bioavailability. (A) Plasma concentration of Cur in mice given with free Cur and Cur-Ca@DMSNs-FA in 1 h point. (B) The concentrations of Cur in tumors of mice given with free Cur and Cur-Ca@DMSNs-FA in 1 h point. **Indicates significant difference at p < 0.01.

4. Conclusion In summary, we have successfully developed multifunctional dendritic mesoporous silica nanoparticles (Ca@DMSNs-FA). It was demonstrated that this nano-delivery system promoted intracellular delivery of Cur and enhanced the anticancer effect against MCF-7 breast cancer cells, mainly due to the targeted delivery, pH responsible 27



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drug release, excellent biocompatibility, and higher bioavailability. This work provides a proof-of-concept for the use of this novel nanoparticle-based Cur for effective killing of breast cancer cells. Additional studies, however, are required to further verify the improved therapeutic efficiency of this breast cancer treatment approach.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant No. 81301157, 31570849, 81271694), and the Fundamental Research Funds for the Central Universities. The authors also acknowledge the Australian Research Council, Queensland Cancer Council and Queensland Government for financial supports. We thank the Australian National Fabrication Facility and Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

Associated Content Supporting Information Zeta potential distribution of samples in PBS solution, N2 adsorption/desorption isotherms and pore size distribution curves of samples, FTIR spectra of samples, UV-Vis spectra of Cur and Cur-Ca@DMSNs-FA in aqueous solutions, TGA analyses of samples, Confocal microscopy analysis of Cur release by Cur-Ca@DMSNs-FA at Cur concentration of 10 µg/mL.

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