Nanosizing Noncrystalline and Porous Silica MaterialâNaturally

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Biological and Medical Applications of Materials and Interfaces

Nanosizing Non-Crystalline and Porous Silica Material—Naturally Occurring Opal Shale for Systemic Tumor Targeting Drug Delivery Qian Guo, Zengyan Chang, Naveed Ullah Khan, Tongtong Miao, Xiufeng Ju, Huaxiang Feng, Lei Zhang, Zhenglong Sun, Hui Li, and Liang Han ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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ACS Applied Materials & Interfaces

Nanosizing Non-Crystalline and Porous Silica Material—Naturally Occurring Opal Shale for Systemic Tumor Targeting Drug Delivery

Qian Guo†, Zengyan Chang†, Naveed Ullah Khan†, Tongtong Miao†, Xiufeng Ju†, Huaxiang Feng†, Lei Zhang†, Zhenglong Sun‡, Hui Li‡, Liang Han*,†,§,¶

†

Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow

University, Suzhou 215123, P. R. China ‡

Suzhou Institute of Biomedical Engineering and Technology, China Academy of

Science, Suzhou 215163, P. R. China §

Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education,

Shanghai 201203, P. R. China ¶

Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical

Sciences, Soochow University, Suzhou 215123, P. R. China

*Corresponding author. Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, E01-1325, 199 Ren’ai Road, Suzhou, 215123, China Tel: (86) 512-65882089. Fax: (86) 512-65882089. E-mail: [email protected]

Keywords drug delivery; nanoparticle; non-crystalline porous silica material; opal shale; tumor passive targeting

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Abstract Opal shale, as a naturally occurring and non-crystalline silica material with porous structure, has the potential to be a drug delivery carrier. In this study, we obtained opal shale nanoparticles (OS NPs) through the techniques of ultrasonic emulsion and differential centrifugation. The OS NPs exhibited markedly lower cytotoxicity than crystalline mesoporous silica nanoparticles. The highly porous structure and the strong adsorbability endowed OS NPs with the ability of loading and sustained release of doxorubicin (DOX). DOX-loaded OS NPs improved tumor cellular uptake and anti-proliferation compared with free drug. Interestingly, OS NPs possessed strong binding with the nuclear envelope, which can be beneficial to the nucleus localization and apoptosis inducing of loaded DOX. We further demonstrated the tumor passive targeting ability, prolonged blood circulation, and enhanced anti-tumor effect with limited in vivo toxicity. Our results suggest OS NPs can be applied for tumor targeting drug delivery, which may have a significant influence on the development of silica-based drug delivery system.

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1. Introduction The rocketing development of drug delivery systems (DDS) increasingly relies on the exploitation of new materials and technologies. Mesoporous materials have been extensively researched for drug delivery. With many unprecedented advantages including mesoporous structure, large pore volume, and tailored surface chemistry, these materials can load, deliver and responsively release drugs with high efficiency.1-3 Mesoporous silica nanoparticles (MSNs) are one representative DDS of this kind, which also display high stability and non-toxic nature. Silica is “generally recognized as safe” by the United States Food and Drug Administration (https://www.fda.gov/food/ingredientspackaginglabeling/gras/scogs/ucm260849.htm). In recent years, most studies focused on synthetic MSNs by purely chemical methods.1-3 However, the application of synthetic MSNs in drug delivery encountered various problems, such as poor biocompatibility, which were partly associated with the crystalline state of MSNs.4,5 Amorphous pozzolana opal shale (OS) is a kind of naturally occurring, hydrous, and non-crystalline silica material (SiO2*nH2O), with various degrees of structural disorder.6,7 OS has many outstanding properties including amorphous state, highly porous structure with nanopore, high surface area, chemical stability, low density and relatively low price.6-11 These unique characteristics endow OS with strong hygroscopicity and adsorbability.6-11 Therefore, OS can efficiently adsorb various substances or elements, for example, rhodamine-B, water, chloride, fluoride, heavy metals, and gas.11-14 In the present work, we hypothesized that the amorphous state of OS would lead to lower cytotoxicity and improved biocompatibility compared with crystalline MSNs. Inspired by that rhodamine-B was used to investigate the adsorption capacity of OS,11 3



we also hypothesized that the mesoporous structure of OS can be used to load small molecule drugs, and the strong adsorbability of OS can give a sustained release of carried drugs. Through this work, we expanded the application of OS to drug delivery. To our best knowledge, no studies concerning the use of OS in construction of DDS have been reported so far. Small molecule based chemotherapy is the most common treatment modality for cancer. On the first hand, the therapeutic efficiency of chemotherapeutic drugs largely depends on the local drug concentration, making drug enrichment a prerequisite for effective therapy. On the other hand, the penetration into normal tissues of these small molecule drugs results in serious side effects. Hence, tumor targeted drug delivery is essential for safe and efficient tumor therapy. Many highly efficient strategies was reported for achieving tumor targeted drug delivery.15-17 Among all the strategies, passive targeting, based on the more permeable leaky vascular structure of solid tumors than healthy tissues, allows nanoparticles (NPs) to specifically accumulate in tumor. The underlying mechanism is well known as the enhanced penetration and retention (EPR) effect of solid tumors.18-20 In this study, we hypothesized that OS should possess nanosized close packed silica spheres according to previous published reports,21-23 and the ultrasonic emulsion technique can further break bigger particles into NPs.24-26 We supposed that these NPs in suspension could be stabilized in the presence of the emulsifying agent and could be collected through differential centrifugation.27-29 Here, we proposed an innovative nanoscale tumor passive targeting OS NPs. We successfully prepared the OS NPs through the extraction process of OS via the ultrasonic emulsion solvent evaporation techniques. Using doxorubicin (DOX) as the model drug, we evaluated the drug loading, release, cytotoxicity, cellular uptake, and 4


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intracellular behavior. We finally investigated the tumor targeting efficiency, pharmacokinetics and pharmacodynamics. Our results suggested that the resulting DOX-loaded OS NPs allowed for targeted drug delivery to tumor cells based on the EPR effect and prolonged blood circulation, efficient uptake by tumor cells and transport into nuclei, strong apoptosis-inducing effect, and enhanced anti-tumor effect with limited in vivo toxicity. Our study reveals a novel DDS for tumor targeting chemotherapy, which may have a notable effect on the future silica-based DDS development.

2. Experimental section 2.1 Preparation of OS NPs Blank OS NPs were prepared through the ultrasonic emulsion solvent evaporation procedures. In a typical process, 100 mg ultrafine powder of OS suspended in 2 mL ethyl ether was stirred at room temperature for about 6 h to disperse OS particles. Then the suspension was added dropwise to 4 mL 2.5% polyvinyl alcohol (PVA403, Kuraray, Tokyo, Japan) under vortex and sonicated to form an oil/water emulsion. The emulsion was poured into a beaker containing 0.3% PVA (100 mL) and stirred for 5 h to evaporate the organic solvent. The suspension was centrifuged at 3000 rpm for 10 min to remove bigger particles. Then the supernatant was centrifuged at 13000 rpm for 30 min to remove the surfactant PVA and collect the OS NPs. Freshly prepared NPs by resuspending the precipitate in PBS 7.4 or H2O were used in the following experiments. The OS NPs resuspended in H2O was lyophilized to calculate the yield. Yield % =

Weight of dried blank OS NPs Weight of all added OS

×100%

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[1]


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For preparation of DOX-loaded OS NPs, 5 mg freshly-prepared blank OS NPs in 2 mL PBS 7.4 were stirred with DOXHCl (0.5 mg) in the presence of triethylamine (3 µL) at room temperature for overnight. Then the mixture was centrifuged at 13000 rpm for 20 min to remove free DOX and collect the DOX-loaded OS NPs. The carried DOX was extracted with dimethyl sulfoxide (DMSO) and measured to calculate the entrapment efficiency and DOX loading using the following formulas. Drug loading %

=

Entrapment efficiency % =

Determined DOX amount in NPs Weight of NPs Determined DOX amount in NPs All DOX added

×100%

[2]

×100%

[3]

For preparation of coumarin-6 or IR780 loaded OS NPs, 10 mg freshly-prepared blank OS NPs in 1.4 mL PBS 7.4 were stirred with coumarin-6 or IR780 (0.2 mg) dissolved in a mixed solution of ethanol and polyethoxylated castor oil (1:1 by volume) at room temperature for 4 h.

2.2 Transmission electron microscope (TEM) Images for examining the size and morphology of the OS NPs and MSNs was obtained on a TEM (HT7700, HITACHI, Japan) with samples prepared by dropping 10 µL of OS NPs (0.75 or 5 mg/mL) or MSNs (0.75 mg/mL) suspension on the copper grid.

2.3 Particle size analysis and zeta potential The hydrodynamic diameter of OS NPs was measured using dynamic light scattering (DLS). A transparent cuvette was filled with 1 mL of 0.75 or 5 mg/mL NPs in HPLC-grade water. The capped cuvette was placed in a Zetasizer (Malvern) and DLS data were read. Zeta potential was also measured using the Zetasizer. 6


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2.4 In vitro drug release In vitro DOX release was performed using the dialysis method. A 1.9 mL DOX-loaded OS NPs suspension (4.75 mg OS NPs and 0.285 mg DOX) or free DOXHCl (0.285 mg DOX) was placed in the dialysis bag (MWCO 3500 Da), hermetically clamped, and immediately suspended in 100 mL PBS 5.5, 6.8, and 7.4. At predetermined time intervals, 1 mL releasing medium was withdrawn, followed by replacing with 1 mL of fresh PBS. Detection of DOX was conducted using the fluorescence reading methods at 478/594 nm (Perkin-Elmer).

2.5 Cell lines Human breast cancer (MCF-7) cells were purchased from Cell Bank, Chinese Academy of Sciences (Shanghai, China). Mouse brain microvascular endothelial cell line bEND.3 was purchased from ATCC. Cells were maintained in DMEM supplemented with 10% FBS, 100 UmL-1 penicillin, and 100 µgmL-1 streptomycin and cultured at 37 °C under a humidified atmosphere containing 5% CO2.

2.6 In vitro cytotoxicity evaluation For cytotoxicity of blank OS NPs on MCF-7 cells, MCF-7 cells were seeded at a density of 7.5 × 103 cells/well in 96-well plates for 24 h before treatment. Cells were incubated with blank OS NPs for 24 h. Cells treated with MSNs in the same concentration to that of blank OS NPs were used as a control. Cell viability was then quantified using the Cell Counting Kit-8 (CCK-8). Briefly, after NPs or MSNs treatment, 10 µL of the CCK-8 solution was added to each well of the plate, which 7



were incubated for an additional 3.5 h at 37 ºC. The absorption was measured at 450 nm using a BioTek Instrument ELx800 microplate reader. The percentage cell viability of each sample was determined relative to the control (untreated) cells. For cytotoxicity of DOX-loaded OS NPs on MCF-7 cells, MCF-7 cells were seeded at a density of 4 × 103 cells/well in 96-well plates for 24 h before treatment. To compare the in vitro cytotoxicity against tumor cells with that against normal cells, bEND.3 cells were seeded at a density of 4 × 103 cells/well in 96-well plates for 24 h before treatment. Then DOX-loaded OS NPs were added to cells and incubated with cells for 48 h. The effect on cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8) and compared with blank OS NPs and free DOX according to the NPs concentration and DOX concentration, respectively.

2.7 In vitro cellular uptake MCF-7 cells were seeded in 33-mm φ20mm glass bottom cell culture dishes and incubated for 48 h and checked under the microscope for confluency and morphology. Then cells were incubated with free DOX or DOX-loaded OS NPs at the concentration of 12 µg DOX/mL for 2 h at 37 °C. After treatment, cells were washed with Hank’s saline then incubated with Hoechst 33342 to label the nucleus for 30 min. After nucleus labeling, cells were washed three times with Hank’s saline and examined using a confocal microscope. For qualitative evaluation, cells were seeded at a density of 1 × 105 cells/well in 6-well plates (Corning-Coaster, Tokyo, Japan). The cells were incubated for 48 h and 8


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checked under the microscope for confluency and morphology. Then cells were incubated with free DOX, or DOX-loaded OS NPs at the concentration of 12 µg DOX/mL for 2 h at 37 °C. After treatment, cells were trypsinized and centrifuged at 1600 rpm for 5 min to obtain a cell pellet, which was subsequently resuspended in PBS and analyzed using a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA) equipped with an argon ion laser (488 nm) as the excitation source. The fluorescence of DOX was collected at 575 nm (FL2). For each sample, 10,000 events were collected and data were analyzed with CellQuest Pro software. MCF-7 cells cultured under normal conditions served as the control. To avoid the saturated uptake due to high dose incubation and compare the in vitro cellular uptake against tumor cells with that against normal cells, MCF-7 cells and bEND.3 cells in 24-well plates were incubated with free DOX or DOX-loaded OS NPs at the concentration of 3 µg DOX/mL for 2 h at 37 °C. After treatment, cells were washed with Hank’s saline then incubated with Hoechst 33342 to label the nucleus for 30 min. After nucleus labeling, cells were washed three times with Hank’s saline and examined using a fluorescence microscope. For the effect of OS NPs on the inhibition of efflux of DOX, the MCF-7 cells were grown in 24-well plates and incubated in fresh medium containing 3 µg/mL free DOX or DOX-loaded OS NPs for 2 h. Then drug-free fresh medium was used to replace free DOX or DOX-loaded OS NPs to further incubate the cells. After 0, 2, 4, 6, or 8 hours’ further treatment, the cells were imaged by fluorescence microscope to detect the remaining intracellular drug to compare the DOX efflux. 9



2.8 In vitro intracellular behavior MCF-7 cells were seeded in 33-mm φ20mm glass bottom cell culture dishes and incubated for 48 h and checked under the microscope for confluency and morphology. To demonstrate the cellular uptake of DOX from DOX-loaded OS NPs was not by passive diffusion, MCF-7 cells were stained with DiD to label the cell membrane and Hoechst 33342 to label the nucleus. After membrane labeling, the cells were incubated with DOX-loaded OS NPs at 12 µg DOX/mL for 2 h at 37 °C. Then cells were washed three times with Hank’s saline and examined using a confocal microscope. For further analyzing the intracellular distribution of DOX-loaded OS NPs, MCF-7 cells in 33-mm φ20mm glass bottom cell culture dishes were incubated with DOX-loaded OS NPs at the concentration of 12 µg DOX/mL for 2 h at 37 °C. After treatment, cells were washed with Hank’s saline then incubated with LysoTracker Green DND-26, Mito-Tracker Green, and Hoechst 33342, to label the intracellular acidic lysosomes, mitochondria, and nuclei, respectively. Then cells were washed three times with Hank’s saline and examined using a confocal microscope.

2.9 Animals Female Balb/c nude mice (Laboratory Animal Center of Soochow University, Suzhou, China), 16-18 gram each, were maintained under standard housing conditions. For subcutaneous xenograft model, 3 × 106 MCF-7 cells in 100 µL PBS 7.4 were inoculated subcutaneously in the proximal femur region of the mouse. The in vivo 10


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animal experiments were carried out in accordance with guidelines evaluated and approved by the Institutional Animal Care and Utilization Committee (IACUC) of Soochow University.

2.10 In vivo imaging of NPs in subcutaneous tumor-bearing mice Free IR780 and IR780-loaded OS NPs at the dose of 10 µg IR780/mouse were injected into the tail vein of nude mice bearing subcutaneous xenograft, respectively. At 24 h, the nude mice were anesthetized and visualized using IVIS in vivo imaging system (Caliper, CA). Then, mice were sacrificed and organs were excised for further imaging. Fluorescence intensity in each organ was quantified using Living Image 3.0 (Caliper, CA).

2.11 Pharmacokinetics and biodistribution of NPs in normal mice For pharmacokinetics study, 16 ICR mice were randomly selected into two groups. Group 1 received an intravenous injection of free IR780, while group 2 received IR780-loaded OS NPs at an equivalent IR780 dose of 0.75 mg/kg. Blood samples (20 µL) were obtained at 1 min, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h through the tail vein and then mixed with 180 µL DMSO to extract IR780. The whole mixture was transferred to 96-well plate for near-infrared fluorescence imaging and the fluorescence signal intensity was quantified using Living Image 3.0 and compared with standard samples for blood concentration-time data analysis. Free IR780 and IR780-loaed OS NPs were injected into the tail vein of ICR mice at the dose of 0.75 mg IR780/kg, respectively. After 24 h, mice were sacrificed and minced tissues (20 mg) were infiltrated in 180 µL DMSO to extract IR780. Detection

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of IR780 was conducted using the fluorescence reading methods at 780/817 nm (TECAN).

2.12 In vivo antitumor efficiency and toxicity To evaluate the antitumor efficiency, PBS, free DOXHCl, blank OS NPs, and DOX-loaded OS NPs (2 mg DOX/kg) were intravenously administered into the mice. The tumor volume of each group was estimated every 2 days after being injected with various formulations according to the calculation formula: V = A × B2/2 (A: maximum diameter of the tumor; B: minimum diameter of the tumor). We recorded weight changes of each group at the same time interval within 21 days. Blank OS NPs and DOX-loaded OS NPs (2 mg DOX/kg) were administered through tail vein injection into female ICR mice (n = 6 per group). Blood was collected 1 day post-injection via submandibular bleeding. The blood samples were centrifuged at 3000 rpm for 10 minutes to separate the serum, which was then subjected to alanine amino transferase (ALT) and aspartate amino transferase (AST) testing using commercial kits (Sigma MAK055 and MAK052). PBS-treated mice were used as controls.

2.13 Statistical analysis All data were collected in triplicate and reported as mean and standard deviation. Comparison of two conditions was evaluated by a paired Student’s t-test. One-way ANOVA analysis was performed to determine the statistical significance of treatment related changes in survival. A p ≤ 0.05 was considered to indicate a statistically significant difference.

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3. Results and discussion 3.1 Detecting and Analysis of OS The elemental and compound compositions were obtained using X-ray fluorescence spectrometer by national engineering research center of ultrafine powder (Shanghai, China). As revealed in Figure 1 and Figure S1, silicon (59.6 wt%) and oxygen (34.2 wt%) were the two most abundant elements and silica dioxide (92.8 wt%) was the most abundant compound in OS. Automatic surface area and porosity analyzer (TriStar 3000) was used to analyze the mesoporous structure of OS. The data for the BJH parameters were shown in Table 1. Table 1. The BJH parameters of raw OS, OS NPs, and DOX-loaded OS NPs

Figure 1. The elemental (a) and compound (b) compositions of OS were obtained in the form of mass percentage using X-ray fluorescence spectrometer. 13



3.2 Preparation and characterization of blank OS NPs It was reported that OS is a natural nanostructure model, which possesses nanosized close packed silica spheres.21-23 The schematic diagram of preparation of OS NPs was illustrated in Figure 2a. Blank OS NPs were extracted and produced using OS through the ultrasonic emulsion and solvent evaporation technique. Ultrasonication produces cavitation through the release of high energy. The collapse of the cavitation leads to shear forces.24,25 Therefore, ultrasonication during emulsion may smash bigger particles. Ultrasonic lithotripsy is based on this mechanism for clinical treatment of lithiasis.26 It was reported that the addition of suitable surfactants or emulsifying agents during the preparation of NPs could decrease the surface tension to stabilize the NPs in suspension.27,28 And, differential centrifugation can collect NPs with specified diameter according to the centrifugal force.29 We evaluated a series of initial lower centrifugal speeds (3000 rpm to 8000 rpm) and kept the higher centrifugal speed for collecting NPs at 13000 rpm. The size of collected NPs gradually decreased (243 nm to 193 nm) as the initial centrifugal speed heightened (Figure S2). The size data were appraised as good by the Zetasizer. All polydispersity indices (PDI) at all initial centrifugal speeds were less than 0.3 (Figure S2), but higher than 0.2, which suggested moderate homogeneity of OS NPs. Initial centrifugal speed at 3000 rpm was chosen for high yield of OS NPs (4.94% ± 0.83%). Compared with raw OS, OS NPs showed altered BJH parameters including increased cumulative volume of pores and average pore diameter. However, there was no notable difference for cumulative surface area of pores between OS and OS NPs (Table 1). TEM image displayed ~50 nm NPs with irregularly spherical shape and mesoporous morphology (0.75 mg/mL, Figure 2b, Table 2). The average hydrodynamic diameter measured by DLS was 146.0 nm (0.75 mg/mL, Figure 2c) 14


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and 245.7 nm (5 mg/mL, Figure S3). At 0.75 mg/mL, the size discrepancy between data of TEM and DLS may be due to the formation of the hydration layer from the attraction of water molecules by the mesoporous properties of OS NPs. At 5 mg/mL, some aggregated OS NPs were shown besides unaggregated NPs by TEM (Figure S3). So the size discrepancy of hydrodynamic particle size at different concentrations can be attributed to particle aggregation. The aggregation of MSNs was also previously reported,30 where MSNs with size of 66.9 nm (detected by TEM) showed increased hydrodynamic particle size of ~350 nm. The OS NPs had negative zeta potentials at -22.6 ± 5.3 mV (Table 2). All results confirmed the successful preparation of the OS NPs, which were applicable for EPR effect-based tumor passive targeting. Considering that 92.8% of OS was silica dioxide (Figure 1b), we synthesized MSNs with similar size and zeta potential to be compared with OS NPs for cytotoxicity. TEM data revealed 50 nm MSNs with spherical shape and mesoporous morphology (Figure 2d). The MSNs had negative zeta potentials at -27.5 ± 5.5 mV. The cytotoxicity of OS NPs and MSNs was evaluated on MCF-7 cells. As displayed in Figure 2e, the OS NPs evinced no remarkable cytotoxicity (>85% viability) at concentrations less than 62.5 µg/mL. On the contrary, MSNs exhibited significant toxicity to MCF-7 cells with 25% viability at 62.5 µg/mL. This result suggested that the OS NPs may have good biocompatibility to be used as a drug delivery carrier. The cytotoxicity of MSNs was largely associated with the crystallinity of NPs.4,5 Numerous reports showed that crystalline silica can produce great cytotoxicity and damage cells by acting on DNA or producing oxidative stress.31,32 On the contrary, amorphous silica exhibits minimal cytotoxicity and causes no morphologic diversification.31 We performed the small angel X-ray diffraction (XRD) pattern study to compare the crystalline difference between OS NPs and MSNs (Figure S4). The 15



XRD data suggested the amorphous mesoporous structure with a high degree of structural disorder for OS NPs. While the crystalline structure of mesoporous ordering for MSNs was evident by the apparent diffraction peak (Figure S4). This finding, to some extent, explained the better biocompatibility of OS NPs and the toxicity of MSNs.

Figure 2. Preparation and characterization of OS NPs. (a) Schematic diagram for the preparation of OS NPs through the ultrasonic emulsion solvent evaporation techniques and the collection through differential centrifugation. (b) TEM image of OS NPs. (c) The hydrodynamic size distribution of OS NPs by DLS. (d) TEM image of MSNs. (e) In vitro cytotoxicity of OS NPs (blue) and MSNs (black) in MCF-7 breast cancer cells after incubation for 24 h (n = 6). **P < 0.01. 16


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Table 2. Properties of OS NPs and DOX-loaded OS NPs

3.3 Characterization of DOX-loaded OS NPs

The DOX-loaded OS NPs were prepared by stirring the mixture of blank OS NPs, DOXHCl, and triethylamine in PBS 7.4 for overnight (Table 2). With the feed mass ratio of blank OS NPs to DOX at 10:1, the entrapment efficiency and DOX loading were 46.3% and 4.4%, respectively. However, DOXHCl loading without triethylamine, compared with DOX loading, decreased by 18.2% to 3.6%. Triethylamine modifies DOXHCl’s physicochemical properties (e.g. enhanced hydrophobicity and lowered water solubility), which influence its loading by NPs. Therefore, the drug loading mechanism may be the hydrophobic interaction between DOX and OS NPs. Many papers reported the use of synthetic MSNs as efficient drug delivery systems. A theranostic nanoplatform based on synthetic MSNs with surface area of 302.6 m2/g displayed DOX loading capacity of 5.17% (close to OS NPs).33 In another paper, MSNs with surface area of 446 m2/g were reported to be able to load 8.9% DOX,34 which was two times that of OS NPs. These reports together with our results suggest that drug loading capacity of MSNs may be not completely dependent on the surface area. OS NPs may have stronger adsorbability for higher drug loading per unit surface area. In addition, we tried using ultrasonication to obtain OS NPs with higher surface area and DOX loading capacity can be further increased to 8.6%.

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Upon carrying, DOX distributed preferentially inside the nanopores, with intense drug-associated density increase and all OS NPs showed DOX loading under TEM (Figure 3a, Table 2). Compared with OS NPs, DOX-loaded OS NPs showed lowered cumulative volume of pores, average pore diameter, and cumulative surface area of pores (Table 1), which further demonstrated DOX loaded inside OS NPs. Afterward, the release manner of DOX from OS NPs was monitored at different pH (Figure 3b). While free DOX was completely released within very short time (95.6%, 6 h), the hydrophobic interaction between DOX and the interior nanopore of OS NPs resulted in a stable DDS and a slow release. At physiological pH 7.4, a small amount of DOX (17.50%) was released from OS NPs within the first 4 h, after which 26.26% of DOX was leaked out in 5 days. However, at acidic pH, especially pH 5.5, DOX release accelerated with 25.27% DOX release in the first 4 h. The reason for the faster release rate under acidic condition may be that DOX was diffused much quicker at pH 5.5 than at pH 7.4. This result was consistent with a previous report, where the pH-dependent DOX release from MSNs was attributed to the improved solubility of DOX.35

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Figure 3. Preparation and characterization of DOX-loaded OS NPs. (a) Schematic diagram for drug loading by OS NPs and TEM images of DOX-loaded OS NPs. (b) Drug release kinetics of free DOX at PBS 7.4 and DOX-loaded OS NPs at PBS 7.4, 6.8, and 5.5 (n = 3).

3.4 In vitro cytotoxicity and cellular uptake Breast cancer is the most common invasive cancer in women and affects ~12% of women worldwide.36 Nearly 30% of women diagnosed with early-stage breast cancer will develop metastatic disease. There is a clear need to optimize and develop new therapies for the treatment of breast cancers and related metastatic tumors. MCF-7 is a classic breast cancer cell line and the source of much of current knowledge about breast cancer. Therefore, MCF-7 cells were selected to investigate drug delivery efficiency of OS NPs. DOX-loaded OS NPs manifested significantly higher cytotoxicity than blank OS NPs and free DOX (Figure 4a). The IC50 of DOX-loaded OS NPs on MCF-7 cells was 0.346 µg DOX/mL (~0.596 µM), which was obviously lower than that for free DOX (0.620 µg/mL, 1.028 µM). The IC50 of free DOX on MCF-7 cells was consistent with reported values (0.32-0.65 µg/mL).37,38 In addition, consistent with Figure 2e, the blank OS NPs had minimal cytotoxicity to MCF-7 cells (Figure 4a). As shown in Figure S5, the cytotoxicity of both DOX-loaded OS NPs (0.817 µg/mL) and free DOX (1.074 µg/mL) on bEND.3 normal microvascular endothelial cells was lower than that on MCF-7 tumor cells. Because the bEND.3 cells used in this work are immortalized and actually in vivo most microvascular endothelial cells don’t proliferate, the cytotoxicity difference between tumor cells and normal cells may be bigger. We studied the cellular uptake to explain the higher cytotoxicity of DOX-loaded OS NPs than free DOX. As evident in Figure 4b, both free DOX and DOX-loaded OS NPs led to almost 100% positive cellular uptake for two hours’ incubation at a 19



concentration of 12 µg DOX/mL. However, the mean fluorescence intensity of DOX-loaded OS NPs treated cells was slightly stronger than that of DOX-treated cells (86.8 versus 78.9). Under the confocal microscope, cells treated with DOX-loaded OS NPs possessed stronger red fluorescence than cells treated with free DOX (Figure 4c). The results were consistent with the quantitative data (Figure 4b). Both data verified that the slightly higher cytotoxicity to MCF-7 cells of DOX-loaded OS NPs might be due to the higher cellular uptake. At DOX concentration of 3 µg/mL, the uptake of DOX-loaded OS NPs by MCF-7 cells was significantly higher than that of free DOX (Figure S5), which was different to the uptake data at DOX concentration of 12 µg/mL (Figure 4c). This result further demonstrated the higher cytotoxicity of DOX-loaded OS NPs than free DOX. However, the uptake of DOX-loaded OS NPs in bEND.3 cells was notably lower than that in MCF-7 cells (Figure S5), which demonstrated a lower uptake rate of bEND.3 cells and the biosafety of intravenously injected DOX-loaded OS NPs. As a substrate of different ATP-binding cassette membrane pumps, such as P-glycoprotein, DOX can be actively pumped out from cancer cells, reducing its anti-tumor effects.39 For free DOX-treated MCF-7 cells, further incubation with drug-free medium obviously reduced intracellular drug concentration (Figure S6). In contrast, for DOX-loaded OS NPs treated cells, intracellular DOX signal maintained even after 6 hours’ further incubation. This result proved that OS NPs can inhibit DOX efflux by MCF-7 cells.

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Figure 4. In vitro cytotoxicity and cellular uptake of DOX-loaded OS NPs were studied on MCF-7 breast cancer cells. (a) In vitro cytotoxicity of empty OS NPs, free DOX and DOX-loaded OS NPs after 48 h incubation (n = 3). (b) Flow cytometric analysis of MCF-7 cells after treatment with free DOX (green) or DOX-loaded OS NPs (red) at a DOX concentration of 12 µg/mL for 2 h (n = 3). (c) Confocal fluorescence microscope images of MCF-7 cells after treatment with free DOX or DOX-loaded OS NPs at a DOX concentration of 12 µg/mL for 2 h. Scale bar 100 µm. 21



We also studied the cellular uptake of coumarin-6-loaded OS NPs. The strong hydrophobicity of coumarin-6 made its release almost negligible (0.08%, 14 d, Figure S7). The signal represented dye-loaded OS NPs rather than the released coumarin-6. The remarkable cellular uptake of NPs was shown for cells treated with NPs at 0.5 and 1 µg NPs/mL for 2 h (Figure S7).

3.5 Intracellular behavior To investigate the uptake pathway of DOX-loaded OS NPs, MCF-7 cells were first pre-stained with cell membrane tracker DiD and then incubated with NPs. As shown in Figure 5a (first row), the outstanding DOX-associated fluorescence inside the cell revealed the cellular uptake of OS NPs. Notably, partial green signal (membrane) was entrapped and overlapped with red signal in the cell. The yellow signal in merged image indicated binding of OS NPs with the cell membrane based on the strong adsorbability of OS NPs. Red signal appeared as separate dots, suggesting that DOX-loaded OS NPs might be absorbed via the absorption endocytosis pathway rather than simple diffusion by the released DOX. To further corroborate the absorption endocytosis pathway, the intracellular lysosomes were labeled. Red signal from DOX-loaded OS NPs was almost completely colocalized with green signal from labeled lysosomes, proving OS NPs’ absorption endocytosis pathway (Figure 5a, second row). In addition, the red signal shown in the merged image may indicate that some OS NPs had escaped from endosomes. We also evaluated if DOX-loaded OS NPs can migrate to the mitochondria (Figure 5a, last row). No obvious overlap revealed that OS NPs could not be used for mitochondria targeting. 22


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Figure 5. Intracellular behavior of DOX-loaded OS NPs. (a) Confocal fluorescence microscope images of MCF-7 cells after treatment DOX-loaded OS NPs at a DOX concentration of 12 µg/mL for 2 h. Cytomembrane, lysosomes and mitochondria were stained by DiD, LysoTracker Green and Mito-Tracker Green respectively and shown in green. (b) The nuclear envelope binding and nuclear localization of OS NPs were evaluated using the confocal fluorescence microscope. Original and magnified images of MCF-7 cells after treatment DOX-loaded OS NPs at a DOX concentration of 12 µg/mL for 30, 60, and 120 min were acquired. Scale bar 25 µm.

23



We further studied the intracellular behavior of DOX-loaded OS NPs after different incubation time to investigate the nucleus targeting efficiency of OS NPs because DOX needs to enter the nucleus to induce the tumor cell apoptosis. In confocal experiments, all tumor cells manifested cell membrane binding (30 min) and uptake (60 min) of DOX-loaded OS NPs (Figure 5b). In the magnified image of cells treated for 30 min, endocytic vesicles closed to the cell membrane were lightened with bright red fluorescence. After 60 min incubation, most DOX signal was in the cytoplasm, or more deeply in endosome and lysosome, because of absorption endocytosis mediated cellular uptake of DOX-loaded OS NPs (supported by the 2nd row in Figure 5a). DOX has improved solubility in acidic pH than neutral pH, which led to the faster DOX release from OS NPs at endosome-lysosome pH 5.5 (Figure 3b). Therefore, DOX signal inside cells incubated with DOX-loaded OS NPs for 60 min, can be divided into released DOX and loaded DOX according to the release profiles shown in Figure 3b. Free DOX can spontaneously move to the nucleus and bind with DNA inside the nucleus, which was supported by the significant DOX signal in the nucleus at 120 min. Interestingly, for cells after 60 min and 120 min incubation, obvious red signal was also found around the nucleus in almost all cells, even depicted the karyotheca. This signal may originate from the loaded DOX rather than free DOX. This result indicated strong binding of OS NPs with the karyotheca (Figure 5b), which may be beneficial to the nuclear localization of loaded DOX that didn’t release inside endosome-lysosome. According to Figure 4 and Figure 5, we speculated that the uptake disparity was due to the distinct uptake mechanisms. Small molecule drug enters cells mainly through 24


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simple diffusion.40 In this study, DOX-loaded OS NPs entered tumor cells primarily via the absorption endocytosis pathway (Figure 5a). The strong adsorbability resulting from mesoporous structure and high surface area of the OS may mediate highly effective interactions between OS NPs and cell membrane, which can promote the following efficacious cellular uptake.41-43 The strong adsorbability may also contribute to the strong nuclear envelope binding and nuclear localization (Figure 5), which may be beneficial to the cell inhibiting effect of DOX. The enhanced cellular uptake and nuclear distribution of DOX-loaded OS NPs were consistent with the higher tumor cell killing efficiency of DOX-loaded OS NPs than free drug (Figure 4a).

3.6 Fluorescent imaging of OS NPs’ targeted delivery to tumor in vivo DOX was used as the model drug in this study. Considering its non-negligible release (Figure 3b), DOX imaging may reflect the biodistribution of loaded DOX rather than OS NPs. However, imaging of OS NPs should be beneficial to expand the application of OS NPs as drug delivery system to the delivery of many other drugs. In addition, it is hard for in vivo fluorescence imaging system to distinguish DOX (emission at 560-660 nm) from mouse autofluorescence. IR780, a near-infrared fluorescent probe, allows for efficient detection using the IVIS imaging system. Considering its negligible release,17 IR780 was used for characterizing in vivo biodistribution and pharmacokinetics of OS NPs. Under the imaging system (Figure 6a), the fluorescence signal of IR780 was shown in suspension of IR780-loaded OS NPs (2) and resuspension of centrifugal precipitation of IR780-loaded OS NPs (4). However, the signal was absent in empty OS NPs (1) and the supernatant of 25



IR780-loaded OS NPs after centrifugation (3). This result proved the successful loading of IR780 in the OS NPs. Imaging of mice with subcutaneous MCF-7 tumor at 24 h after intravenous injection of IR780-loaded OS NPs was studied to assess the tumor passive targeting efficiency of OS NPs (Figure 6b-d). It was reported that it was very hard for IR780 to be released from solid terpolymeric NPs due to the strong hydrophobic interaction with NPs.17 So IR780 can be used to image the in vivo behavior of OS NPs. Free IR780 was used as a control. As shown in Figure 6b, tumor was highlighted remarkably by IR780-loaded OS NPs. The fluorescence signal in tumor from mouse treated with IR780-loaded OS NPs was slightly stronger than that in tumor from mouse treated with free IR780. The fluorescence signal in excised organs was further imaged and quantified (Figure 6c,d). For mouse treated with IR780-loaded OS NPs, fluorescence signal was obviously accumulated in tumor (Figure 6c). The fluorescence intensity in tumor was 2.22-fold and 2.54-fold higher than that in liver and kidney, respectively. While for mouse treated with free IR780, the fluorescence intensity in tumor was 75.2% and 118% of that in liver and kidney, respectively. Free IR780 by itself was reported to have the ability of efficient tumor targeting.44 The obviously higher accumulation ratio of tumor to liver or kidney for IR780-loaded OS NPs than free IR780 demonstrated efficient tumor targeting ability of IR780-loaded OS NPs. Considering IR780’s encapsulation inside OS NPs and negligible release,17 the tumor targeting mechanism of IR780-loaded OS NPs was speculated to be from OS NPs rather than loaded IR780. These results suggested that OS NPs were capable of enriching in tumor with high efficiency. Considering the size of OS NPs (Figure 2b,c), the tumor targeting mechanism was speculated to be the passive targeting based

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on the EPR effect of solid tumors for NPs. This finding was consistent with reports published before, which used MSNs as tumor targeting DDS.45,46

Figure 6. In vivo tumor targeting efficiency of IR780-loaded OS NPs. (a) The loading of IR780 by OS NPs was demonstrated by the IVIS in vivo imaging system (Caliper, CA). (b) In vivo fluorescence imaging of the mice with subcutaneous MCF-7 xenografts at 24 h after intravenous injection of free IR780 or IR780-loaded OS NPs via the tail vein. (c) Ex vivo fluorescence imaging of excised organs at 24 h postinjection. From top to bottom: brain, heart, liver, spleen, lung, kidney and tumor. (d) Quantitative analysis of fluorescence intensities of excised organs at 24 h postinjection (n = 3).

3.7 Pharmacokinetics and biodistribution of OS NPs in normal mice Blood concentration curves displayed different blood behavior between free IR780 and IR780-loaded OS NPs (Figure 7a). Free IR780 was more quickly eliminated from 27



blood with the concentration of 4.48 µg/mL post injection and 1.07 µg/mL at 48 h. IR780-loaded OS NPs exhibited a relatively sustained clearance with the blood concentration of 4.69 µg/mL post injection and 1.88 µg/mL at 48 h. According to blood concentration-time curves (Figure 7a), pharmacokinetic parameters were calculated (Table 3). The statistical analysis of the AUC0-t (area under the curve from zero to time t, ***P < 0.001), CL (clearance time, ***P < 0.001), MRT0-t (the mean residence time from zero to time t, ***P < 0.001), t1/2α (distribution half-time, *P < 0.05) and t1/2β (elimination half-time, ***P < 0.001) showed remarkable differences between the two groups. The results demonstrated that the IR780-loaded OS NPs prolonged the retention of the IR780 in blood circulation with a 1.90-fold increase in AUC0-t and 1.61-fold increase in MRT0-t. Prolonged blood circulation and the EPR effect are beneficial to the passive tumor targeting accumulation of NPs.47,48 The pharmacokinetic data further proved efficient passive tumor targeting can be accomplished by OS NPs. Quantitative evaluation of NPs’ biodistribution was performed in healthy mice at 24 h after intravenous injection (Figure 7b). In contrast to the findings in mice with subcutaneous MCF-7 tumor (Figure 6d), IR780-loaded OS NPs showed significantly higher drug concentration in liver and spleen than free drug (2.24-fold and 4.42-fold, respectively). However, the drug concentration in kidney for IR780-loaded OS NPs was lower than that for free IR780 in both animal models (47.2% and 53.0% for tumor bearing and tumor free model, respectively). This difference may indicate the sustained renal excretion, which raised the concentration of OS NPs in liver in normal mice and that in tumor in mice with subcutaneous MCF-7 tumor.

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Table 3. Pharmacokinetic parameters after intravenous administration

Figure 7. (a) Time courses of IR780 levels in blood of normal ICR mice following intravenous administration of free IR780 and IR780-loaded OS NPs at a dosage of 0.75 mg IR780/kg. (b) In vivo biodistribution in normal mice at 24 h after intravenous injection of IR780-loaded OS NPs and free IR780, respectively (n = 3). *P < 0.05, **P < 0.01.

For liver and spleen in tumor-bearing mice, drug-loaded OS NPs evinced lower drug concentration than free drug (Figure 6c,d). As opposed to this finding, for liver and spleen in healthy mice without tumor, drug-loaded OS NPs exhibited higher drug concentration than free drug (Figure 7b). These seemingly opposite data might be due to the sustained renal excretion of OS NPs from the body, as evident by the lower kidney drug concentration in both models (Figure 6d, Figure 7b). OS NPs distributed

29



primarily in liver of healthy mice, while mainly accumulated in tumor of tumor-bearing mice owing to the EPR effect. Many papers reported the clearance of MSNs in vivo. Tamanoi and co-workers proved that injected MSNs were mainly excreted in the form of intact MSNs.49 In another report, partially degraded MSNs were found in the urine.50 For MSNs entering into cells, MSNs can be degraded into silicic acid, which was then cleared from human body.51

3.8 Therapeutic efficacy studies We studied the in vivo anti-tumor effect of DOX-loaded OS NPs using the MCF-7 breast tumor model. As depicted in Figure 8a, mice with MCF-7 tumor were injected with different formulations including PBS, empty OS NPs, free DOX and DOX-loaded OS NPs for eight times every other day. The tumor growth was monitored starting from treatment. DOX-loaded OS NPs exhibited outstandingly stronger tumor inhibiting effect than free DOX (Figure 8b). The relative tumor volumes of PBS, blank OS NPs, free DOX, and DOX-loaded OS NPs treated mice after 18 days were 6.14, 6.35, 4.91, and 3.31, respectively. There was significant difference between free DOX and DOX-loaded OS NPs (***P < 0.001). The pharmacodynamics results were in agreement with the data shown in a recently published report, where DOX-loaded synthetic MSNs (DOX@GdMSNs) displayed remarkably strong tumor inhibiting effect with DOX loading capacity of 5.17%.33 Mice body weight change curves (Figure 8c) and blood biochemical indexes for liver function (Figure 8d,e) were used to explore the in vivo toxicity. DOX-loaded OS NPs treatment expressed the stable tendency of body weight (Figure 8c). The liver

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function evaluation by ALT and AST assays suggested that both blank OS NPs and DOX-loaded OS NPs had limited in vivo toxicity (Figure 8d,e).

Figure 8. Antitumor activities of various formulations. (a) Schematic illustration to show the tumor challenge experiment design. (b) Relative tumor volume curves of different groups after various treatments (n = 10). ***P < 0.001. (c) Body weight change of nude mice bearing MCF-7 tumors as a function of days post treatment for various groups (n = 10). In vivo toxicity of blank OS NPs and DOX-loaded OS NPs through evaluating liver function by testing AST (d) and ALT (e). 31



4. Conclusions In summary, we proposed and validated a novel tumor passive targeting OS NPs, based on a naturally occurring and non-crystalline silica material with highly porous and nanosized sphere structure. Our results suggest that OS NPs can be applied for systemic delivery of chemotherapeutic agents to the tumor. Due to the simple construction from abundant materials with minimal toxicity, the precise drug loading and release, the highly efficient tumor cellular uptake and nucleus localization, the prolonged blood circulation, the tumor passive targeting effect, and the enhanced anti-tumor effect with limited in vivo toxicity, we anticipate that OS NPs will serve as a promising direction for the future design and application of DDS. However, it remains to be explored for further functionality. Future work will be aiming at further functionalizing the OS NPs via surface modification for intelligent responsive drug release, engineering OS NPs for being as protein and gene delivery carrier, and systematically researching the biodegradability.

Supporting Information Available Materials. Synthesis of MSNs. The elemental composition of OS except silicon and oxygen and compound composition of OS except silica dioxide (Figure S1). The hydrodynamic size distribution and polydispersity index (PDI) of OS NPs using different initial lower centrifugal speeds (Figure S2). Size analysis of OS NPs at concentration of 5 mg/mL by TEM and DLS (Figure S3). XRD pattern of MSNs and 32


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OS NPs (Figure S4). In vitro cytotoxicity of blank OS NPs, free DOX and DOX-loaded OS NPs on normal endothelial cells and in vitro cellular uptake of free DOX and DOX-loaded OS NPs at DOX concentration of 3 µg/mL by MCF-7 tumor cells and by normal endothelial cells (Figure S5). Fluorescence microscopy images of MCF-7 cells after treatment with free DOX or DOX-loaded OS NPs for 2 h and further incubation with fresh drug-free medium to investigate the drug efflux (Figure S6). Cellular uptake and drug release of coumarin-6-loaded OS NPs (Figure S7).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements This work was supported by the National Natural Science Foundation of China (81703428), the Natural Science Foundation for Colleges and Universities in Jiangsu Province of China (17KJB350010) and the Open Project Program (SDD2016-05) of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China.

References (1) Chen, Y.; Ai, K.; Liu, J.; Sun, G.; Yin, Q.; Lu, L. Multifunctional Envelope-Type Mesoporous Silica Nanoparticles for pH-Responsive Drug Delivery and Magnetic Resonance Imaging. Biomaterials 2015, 60, 111-120.

33



(2) Liu, J.; Luo, Z.; Zhang, J.; Luo, T.; Zhou, J.; Zhao, X.; Cai, K. Hollow Mesoporous Silica Nanoparticles Facilitated Drug Delivery via Cascade pH Stimuli in Tumor Microenvironment for Tumor Therapy. Biomaterials 2016, 83, 51-65 (3) He, Q.; Gao, Y.; Zhang, L.; Zhang, Z.; Gao, F.; Ji, X.; Li, Y.; Shi, J. A pH-Responsive Mesoporous Silica Nanoparticles-Based Multi-Drug Delivery System for Overcoming Multi-Drug Resistance. Biomaterials 2011, 32, 7711-7720. (4) Guidi, P.; Nigro, M.; Bernardeschi, M.; Lucchesi, P.; Scarcelli, V.; Frenzilli, G. Does the Crystal Habit Modulate the Genotoxic Potential of Silica Particles? A Cytogenetic Evaluation in Human and Murine Cell Lines. Mutat. Res. Genet. Toxicol. Environ Mutagen. 2015, 792, 46-52. (5) Petushkov, A.; Intra, J.; Graham, J. B.; Larsen, S. C.; Salem, A. K. Effect of Crystal Size and Surface Functionalization on the Cytotoxicity of Silicalite-1 Nanoparticles. Chem. Res. Toxicol. 2009, 22, 1359-1368. (6) Brown, L. D.; Ray, A. S.; Thomas, P. S. 29Si and 27Al NMR Study of Amorphous and Paracrystalline Opals from Australia. J. Non-Cryst. Solids 2003, 332, 242-248. (7) Jia, Y.; Wang, B. M. Mineralogy and Thermal Analysis of Natural Pozzolana Opal Shale with Nano-pores. J. Wuhan Univ. Technol. 2017, 32, 532-537. (8) Sun, Z. M.; Bai, C. H.; Zheng, S. L.; Yang, X. P.; Frost, R. L. A Comparative Study of Different Porous Amorphous Silica Minerals Supported TiO2 Catalysts. Appl. Catal A-Gen. 2013, 458, 103-110. (9) Sun, Z. M.; Kong, W. A.; Zheng, S. L.; Frost, R. L. Study on Preparation and Thermal Energy Storage Properties of Binary Paraffin Blends/Opal Shape-Stabilized Phase Change Materials. Sol. Energy Mater. Sol. Cells 2013, 117, 400-407. 34


Page 34 of 40

Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Channing, A.; Butler, I. B. Cryogenic Opal-A Deposition from Yellowstone Hot Springs. Earth. Planet. Sci. Lett. 2007, 257, 121-131. (11) Yang, D. F.; Wei, C. D.; Ning, W. K.; Xu S. N.; Jiang, Y. S. Structure and Adsorption Properties of Nenjiang Opal Shale. J. Jilin Univ. (Earth Sci. Ed.) 2010, 40, 1061-1065.

(12) Gullick, R. W.; Weber, W. W. Jr. Evaluation of Shale and Organoclays as Sorbent Additives for Low-Permeability Soil Containment Barriers. Environ. Sci. Technol. 2001, 35, 1523-1530. (13) Biswas, G.; Dutta, M.; Dutta, S.; Adhikari, K. A Comparative Study of Removal of Fluoride from Contaminated Water Using Shale Collected from Different Coal Mines in India. Environ. Sci. Pollut. Res. Int. 2016, 23, 9418-9431. (14) Jabłońska, B.; Siedlecka, E. Removing Heavy Metals from Wastewaters with Use of Shales Accompanying the Coal Beds. J. Environ. Manage. 2015, 155, 58-66. (15) Sykes, E. A.; Chen, J.; Zheng, G.; Chan, W. C. Investigating the Impact of Nanoparticle Size on Active and Passive Tumor Targeting Efficiency. ACS Nano 2014,

8, 5696-5706. (16) Huang, X.; Peng, X.; Wang, Y.; Wang, Y.; Shin, D. M.; El-Sayed M. A.; Nie, S. A Reexamination of Active and Passive Tumor Targeting by Using Rod-Shaped Gold Nanocrystals and Covalently Conjugated Peptide Ligands ACS Nano 2010, 4, 5887-5896. (17) Han, L.; Kong, D. K.; Zheng, M. Q.; Murikinati, S.; Ma, C.; Yuan, P.; Li, L.; Tian, D.; Cai, Q.; Ye, C.; Holden, D.; Park, J. H.; Gao, X.; Thomas, J. L.; Grutzendler, 35



Page 36 of 40

J.; Carson, R. E.; Huang, Y.; Piepmeier, J. M.; Zhou, J. Increased Nanoparticle Delivery to Brain Tumors by Autocatalytic Priming for Improved Treatment and Imaging. ACS Nano 2016, 10, 4209-4218. (18) Han, L.; Ma, H.; Guo, Y.; Kuang, Y.; He, X.; Jiang, C. pH-Controlled Delivery of Nanoparticles into Tumor Cells. Adv. Healthc. Mater. 2013, 2, 1435-1439. (19) Ngoune, R.; Peters, A.; von Elverfeldt, D.; Winkler, K.; Pütz, G. Accumulating Nanoparticles by EPR: A Route of No Return. J. Control. Release 2016, 238, 58-70. (20) Perry, J. L.; Reuter, K. G.; Luft, J. C.; Pecot, C.V; Zamboni, W.; DeSimone, J. M. Mediating Passive Tumor Accumulation through Particle Size, Tumor Type, and Location. Nano Lett. 2017, 17, 2879-2886 (21) Gaillou, E.; Fritsch, E.; Aguilar-Reyes, B.; Rondeau, B.; Post, J.; Barreau, A.; Ostroumov, M. Common Gem Opal: an Investigation of Micro- to Nano-Structure. Am. Mineral. 2008, 93, 1865-1873. (22) Pramatarova, L.; Pecheva, E.; Presker, R.; Schwarz, U.; Kniep, R. Natural Opal as a Model System for Studying the Process of Biomineralization. Solid State Phenom. 2014, 106, 75-78. (23) Sodo, A.; Casanova Municchia, A.; Barucca, S.; Bellatreccia, F.; Della Ventura, G.; Butini, F.; Ricci, M. A. Raman, FT-IR and XRD Investigation of Natural Opals. J. Raman Spectrosc. 2016, 47, 1444-1451. (24) Maleki, A.; Aghaei, M. Sonochemical Rate Enhanced by a New Nanomagnetic Embedded

Core/Shell

Nanoparticles

and

Catalytic

Performance

in

the

Multicomponent Synthesis of Pyridoimidazoisoquinolines. Ultrason. Sonochem. 2017, 38, 115-119. 36


Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(25) Chang, Y.; Yan, X.; Wang, Q.; Ren, L.; Tong, J.; Zhou, J. High Efficiency and Low Cost Preparation of Size Controlled Starch Nanoparticles through Ultrasonic Treatment and Precipitation. Food Chem. 2017, 227, 369-375. (26) Harper, J. D.; Sorensen, M. D., Cunitz B. W.; Wang, Y. N.; Simon, J. C.; Starr, F.; Paun, M.; Dunmire, B.; Liggitt, H. D.; Evan, A. P.; McAteer, J. A.; Hsi, R. S.; Bailey, M. R. Focused Ultrasound to Expel Calculi from the Kidney: Safety and Efficacy of a Clinical Prototype Device. J. Urol. 2013, 190, 1090-1095. (27) Keum, C. G.; Noh, Y. W.; Baek, J. S.; Lim, J. H.; Hwang, C. J., Na, Y. G., Shin, S. C.; Cho, C. W. Practical Preparation Procedures for Docetaxel-Loaded Nanoparticles Using Polylactic Acid-coGlycolic Acid. Int. J. Nanomedicine 2011, 6, 2225-2234. (28) Ortega-Oller, I.; Del Castillo-Santaella, T.; Padial-Molina, M.; Galindo-Moreno, P.; Jódar-Reyes, A. B.; Peula-García, J. M. Dual Delivery Nanosystem for Biomolecules. Formulation, Characterization, and In Vitro Release. Colloids Surf. B Biointerfaces 2017, 159, 586-595. (29) Liu, X.; Wu, F.; Tian, Y.; Wu, M.; Zhou, Q.; Jiang, S.; Niu, Z. Size Dependent Cellular Uptake of Rod-like Bionanoparticles with Different Aspect Ratios. Sci. Rep. 2016, 6, 24567. (30) Menon, N.; Leong, D. T. Cytotoxic Effects of Phosphonate-Functionalised Mesoporous Silica Nanoparticles. ACS Appl. Mater. Interfaces 2016, 8, 2416−2422. (31) Yang, H.; Wu, Q. Y.; Lao, C. S.; Li, M. Y.; Gao, Y.; Zheng, Y.; Shi, B. Cytotoxicity and DNA Damage in Mouse Macrophages Exposed to Silica Nanoparticles. Genet. Mol. Res. 2016, 15. (32) Pozzolini, M.; Vergani, L.; Ragazzoni, M.; Delpiano, L.; Grasselli, E.; Voci, A.; Giovine, M.; Scarfì, S. Different Reactivity of Primary Fibroblasts and 37



Page 38 of 40

Endothelial Cells towards Crystalline Ailica: A Surface Radical Matter. Toxicology 2016, 361-362, 12-23. (33) Sun, Q.; You, Q.; Wang, J.; Liu, L.; Wang, Y.; Song, Y.; Cheng, Y.; Wang, S., Tan, F.; Li, N. Theranostic Nanoplatform: Triple-Modal Imaging-Guided Synergistic Cancer Therapy Based on Liposome-Conjugated Mesoporous Silica Nanoparticles. ACS Appl. Mater. Interfaces 2018, 10, 1963−1975. (34)

Moghaddam,

S.

P.

H.;

Yazdimamaghani,

M.;

Ghandehari,

H.

Glutathione-Sensitive Hollow Mesoporous Silica Nanoparticles for Controlled Drug Delivery. J. Controlled Release 2018, DOI: 10.1016/j.jconrel.2018.04.032. (35) Chen, L.; Zhou, X.; Wei, N.; Zhang, Q.; Wang, W.; Zhang, Y.; He, C. Multifunctional Redox-Responsive Mesoporous Silica Nanoparticles for Efficient Targeting Drug Delivery and Magnetic Resonance Imaging. ACS Appl. Mater. Interfaces 2016, 8, 33829−33841. (36) Mcguire, A.; Brown, J. A.; Malone, C.; Mclaughlin, R.; Kerin, M. J. Effects of Age on the Detection and Management of Breast Cancer. Cancers 2015, 7, 908−929. (37) Goswami, U.; Dutta, A.; Raza, A.; Kandimalla, R.; Kalita, S.; Ghosh, S. S.; Chattopadhyay, A. Transferrin-Copper Nanocluster-Doxorubicin Nanoparticles as Targeted Theranostic Cancer Nanodrug. ACS Appl. Mater. Interfaces 2018, 10, 3282−3294. (38) Hao, T.; Chen, D.; Liu, K.; Qi, Y.; Tian, Y.; Sun, P.; Liu, Y.; Li, Z. Micelles of d-α-Tocopheryl Polyethylene Glycol 2000 Succinate (TPGS 2K) for Doxorubicin Delivery with Reversal of Multidrug Resistance. ACS Appl. Mater. Interfaces 2015, 7, 18064−18075. (39) Kopecka, J.; Campia, I.; Olivero, P.; Pescarmona, G.; Ghigo, D.; Bosia, A.; Riganti, C. A LDL-Masked Liposomal-Doxorubicin Reverses Drug Resistance in Human Cancer Cells. J. Controlled Release 2011, 149, 196-205. (40) Yi, Y.; Kim, J. H.; Kang, H. W.; Oh, H. S.; Kim, S. W.; Seo, M. H. A Polymeric Nanoparticle Consisting of mPEG-PLA-Toco and PLMA-COONa as a Drug Carrier: Improvements in Cellular Uptake and Biodistribution. Pharm. Res. 2005, 22, 200-208. 38


Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(41) Namgung, R.; Zhang, Y.; Fang, Q. L.; Singha, K.; Lee, H. J.; Kwon, I. K.; Jeong, Y. Y.; Park, I. K.; Son, S. J.; Kim, W. J. Multifunctional Silica Nanotubes for Dual-Modality Gene Delivery and MR Imaging. Biomaterials 2011, 32, 3042-3052. (42) Slowing, I.; Trewyn, B. G.; Lin, V. S. Effect of Surface Functionalization of MCM-41- Type Mesoporous Silica Nanoparticles on the Endocytosis by Human Cancer Cells. J. Am. Chem. Soc. 2006, 128, 14792-14793. (43) Xing, X.; He, X.; Peng, J.; Wang, K.; Tan, W. Uptake of Silica-Coated Nanoparticles by HeLa Cells. J. Nanosci. Nanotechno. 2005, 5, 1688-1693. (44) Zhang, C.; Liu, T.; Su, Y.; Luo, S.; Zhu, Y.; Tan, X.; Fan, S.; Zhang, L.; Zhou, Y.; Cheng, T.; Shi, C. A near-Infrared Fluorescent Heptamethine Indocyanine Dye with Preferential Tumor Accumulation for In Vivo Imaging. Biomaterials 2010, 31, 6612-6617. (45)

Xie,

M.;

Xu,

Y.;

Shen,

H.;

Shen,

S.;

Ge,

Y.;

Xie,

J.

Negative-Charge-Functionalized Mesoporous Silica Nanoparticles as Drug Vehicles Targeting Hepatocellular Carcinoma. Int. J. Pharm. 2014, 474, 223-231. (46) Meng, H.; Xue, M.; Xia, T.; Ji, Z.; Tarn, D. Y.; Zink, J. I., Nel, A. E. Use of Size and a Copolymer Design Feature to Improve the Biodistribution and the Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model. ACS Nano 2011, 5, 4131-4144. (47) Chen, Y.; Li, Z.; Wang, H.; Wang, Y.; Han, H.; Jin, Q.; Ji, J. IR-780 Loaded Phospholipid Mimicking Homopolymeric Micelles for Near-IR Imaging and Photothermal Therapy of Pancreatic Cancer. ACS Appl. Mater. Interfaces 2016, 8, 6852−6858. (48) Deng, T.; Peng, Y.; Zhang, R.; Wang, J.; Zhang, J.; Gu, Y.; Huang, D.; Deng, D. Water-solubilizing

Hydrophobic

ZnAgInSe/ZnS

QDs

with

Tumor-targeted

cRGD-Sulfobetaine-PIMA-Histamine Ligands via a Self-assembly Strategy for Bio-Imaging. ACS Appl. Mater. Interfaces 2017, 9, 11405−11414. (49) Lu, J.; Li, Z.; Zink, J. I.; Tamanoi, F. In vivo Tumor Suppression Efficacy of Mesoporous Silica Nanoparticles-Based Drug-Delivery System: Enhanced Efficacy by Folate Modification. Nanomedicine 2012, 8:212-220. 39



Page 40 of 40

(50) Huang, X.; Li, L.; Liu, T.; Hao, N.; Liu, H.; Chen, D.; Tang, F. The Shape Effect of

Mesoporous

Silica

Nanoparticles

on

Biodistribution,

Clearance,

and

Biocompatibility in vivo. ACS Nano 2011, 5:5390-5399. (51) Tolstik, E.; Osminkina, L. A.; Matthäus, C.; Burkhardt, M.; Tsurikov, K. E.; Natashina, U. A.; Timoshenko, V. Y.; Heintzmann, R.; Popp, J.; Sivakov, V. Studies of Silicon Nanoparticles Uptake and Biodegradation in Cancer Cells by Raman Spectroscopy. Nanomedicine 2016, 12:1931-1940.

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Nanosizing Noncrystalline and Porous Silica MaterialâNaturally

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