Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide

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Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide with Environment Sensitive Drug Release for Effective Treatment of Triple Negative Breast Cancer Xiaohui Wu, Zheng Han, Rebecca Schur, and Zheng-Rong Lu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00398 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 28, 2016

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ACS Biomaterials Science & Engineering

Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide with Environment Sensitive Drug Release for Effective Treatment of Triple Negative Breast Cancer Xiaohui Wu†, Zheng Han†, Rebecca M. Schur and Zheng-Rong Lu* Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United Sates. †Contribute equally

* To whom correspondence should be addressed: Dr. Zheng-Rong Lu M. Frank and Margaret Domiter Rudy Professor Wickenden 427, Mail Stop 7207 10900 Euclid Avenue Cleveland, OH 44106 Phone: 216-368-0187 Fax: 216-368-4969 Email: [email protected]

Submitted to ACS Biomaterials Science & Engineering

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ABSTRACT In this study, we report a novel modality of using a mesoporous silica nanoparticles (MSNs)based drug delivery system with RGD peptide as a targeting ligand to load arsenic trioxide (ATO) (ATO-MSNs-RGD) for treating MDA-MB-231 breast cancer. The MSNs, ATO-MSNs, and ATO-MSNs-RGD were characterized with X-ray diffraction (XRD), thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and Brunauer-Emmet-Teller (BET) method. The data indicated that the MSNs possessed MCM-41 type mesopores with high surface area of ~1021 m2/g and pore diameter of ~2.2 nm. However, both values dramatically decreased after encapsulated with ATO or modified with RGD. The amount of surface anchored RGD peptide was determined to be 0.20 mmol/g. Glutathione (GSH) greatly enhanced the ATO release from MSNs. Confocal laser microscopic images strongly demonstrated that both ATOMSNs and ATO-MSNs-RGD had good cellular uptake improved by incubation time and nanoparticle concentration and the ATO-MSNs-RGD showed clearly improved cellular uptake compared with ATO-MSNs. The MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO were used to treat MDA-MB-231 breast tumors every 5 days and the findings suggested that ATO-MSNsRGD provided superior therapeutic ability over MSNs, ATO-MSNs, and ATO. Keywords: nanoparticles, arsenic trioxide, targeting, breast cancer


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INTRODUCTION The application of nanotechnology in drug delivery has received increasing attention and brought promising benefits in cancer therapy. Conventional chemotherapeutic agents display unfavourable pharmacokinetics, non-specific biodistribution, affecting both cancerous and normal cells, which in turn causes suboptimal treatment and excessive side effects.1 Nanoparticle-based delivery systems accumulate preferentially in solid tumors due to leaky tumor vasculature.2, 3 Accumulated at the tumor sites, nanoparticles delivering drugs act as a local drug depot and provide sustained drug release. In addition, nanoparticles modified with targeting ligands can improve tumor-targeted drug delivery.4 Generally, targeting ligands can be exemplified as peptides, proteins, antibodies, and small compounds.3,

5

Nanoparticle-based

drug delivery systems for cancer therapeutics include, but not limited to, liposomes, dendrimers, micelles, polymeric nanoparticles, protein nanoparticles, inorganic nanoparticles, nanotubes, and viral nanoparticles.6-10 These nanoparticles have brought new possibilities to this burgeoning area of drug delivery. Arsenic trioxide (ATO) is water soluble without odor and taste, and widely known as the “the king of poison/the poison of kings”.11 Arsenic derivatives have been used in treating human diseases for about 3000 years in ancient China and Greece. For example, arsenic was used as a devitalizing agent prior to dental work and as an escharotic to generate a thick black scab called eschar to treat cancers.12 Low doses of a 1% aqueous solution of ATO or arsenous acid resulted in complete remission of leukemia. The US Food and Drug Administration (FDA) approved the use of ATO for treatment of acute promyelocytic leukemia (APL) in 2000. Although ATO has shown good efficacy in treatment of hematological malignancies, it has limited efficacy in treating solid tumors11, 13, because of rapid renal clearance of arsenic, low tumor targeting efficiency, poor therapeutic efficacy at low doses, and the systemic toxicity associated with the high doses. The design and development of targeted ATO delivery systems



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can potentially address these limitations and extend its clinical use in treating other cancer types by efficient delivery of ATO into solid tumors at efficacious doses and minimize nonspecific systemic toxic side effects. Mesoporous silica nanoparticles (MSNs) possess several advanced characteristics for drug delivery, including high specific surface area, controllable nanoparticle size, tailorable pore size, and dual-functional surfaces (interior and exterior).2 MSNs demonstrate good biocompatibility both in vitro and in vivo and are suitable vehicles for drug delivery. Their outer surface can be readily modified with targeting agents to achieve tumor specific drug delivery. The MSNs have high interior surface areas and loading capacity, and are suitable delivery vehicles for ATO. ATO can be loaded to MSNs via hydrogen bonding with the hydroxyl groups on the inner surface and/or coordination bonds with arsenic after inner surface modification. The mesoporous structure of MSNs will allows high payloads of ATO to deliver sufficient drug into solid tumors for efficacious treatment. Triple negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that lacks the expression of estrogen receptor, progesterone receptor, and HER2 amplification. Currently, there is still limited progress in developing efficient targeted therapy available for TNBC, even though some nanoparticle have been tested in delivery of ATO for treating tumors14-17. We hypothesize that the targeted delivery of ATO with the MSNs has the potential to minimize its systemic toxicity and enhance its efficacy in treating TNBC. In this study, we aimed to develop targeted MSNs loaded with ATO and examine their effectiveness in treating TNBC in a mouse tumor model. We modified both the inner and outer surfaces of MSNs to introduce thiol groups for ATO loading in the mesopores and conjugating a targeting agent on outer surface for targeted drug delivery. ATO loading via the coordination of arsenic with the thiol groups in the MSNs with give an environmental sensitive drug delivery system of targeted


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delivery of ATO into cancer cells. Cyclic peptide Arg-Gly-Asp-D-Phe-Lys (cRGDfK) targeted MSNs loaded with ATO were prepared, characterized, and examined both in vitro and in vivo.

EXPERIMENTAL SECTION Materials. Fluorescein-5-maleimide was purchased from AAT Bioquest, Inc (Sunnyvale, CA, USA). Cetyltrimethylammonium (CTAB), tetraethoxysilane (TEOS), sodium hydroxide, (3mercaptopropyl)

triethoxysilane

(3MPT),

N,N-Diisopropylethylamine

(DIPEA),

diisopropylcarbodiimide (DIC), HOAc, trifluoroethanol (TFE), trifluoroacetic acid (TFA), triisopropylsilane (TIS), arsenic trioxide (ATO), all amino acids for RGD synthesis, and solvents were purchased from Sigma-Aldrich. MDA-MB-231 cells (#HTB-26) were purchased from ATCC. DMEM/High Glucose media and MTT assay kit were purchased from Life Technologies. Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco. Rhodamine phalloidin (RP) was purchased from Cytoskeleton, Inc. Synthesis of thiol-functionalized MSNs (MSNs-SH). In a typical synthesis route, CTAB (1 g) and sodium hydroxide (0.28 g) were completely dissolved in DI water (480 mL) under vigorous stirring at 80 oC. Then TEOS (5 mL) was added to the solution dropwise in 15 min. The vigorous stirring at 80 oC was maintained overnight and the milk-like solution was centrifuged to collect MSNs, followed by reflux in a mixture of ethanol (500 mL) and hydrochloric acid (37.5%, 5 mL) for 12 h, and this procedure was repeated three times to remove CTAB. Then the final products were dried completely at 120 oC overnight. MSNs (100 mg) were suspended in anhydrous toluene (20 mL) thoroughly and 3MPT (150 µL) was added. The mixture was refluxed overnight. Centrifugation at 4000 rpm for 20 min was used to collect MSNs-SH and the MSNs-SH was washed with DI water and ethanol twice, respectively. The surface thiol groups were quantified to be 1.12 mmol/g by TGA.



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Synthesis of Maleimido-RGD. cRGDfK was synthesized via a standard Fmoc solid phase peptide synthetic chemistry. Briefly, 2-chlorotrityl chloride resin (2 g, 2.4 mmol) was suspended in anhydrous DCM for 1 h at room temperature with shaking. Fmoc-Arg(Boc)-OH (R, 973 mg, 1.5 mmol) was dissolved in anhydrous DMF (20 mL) containing DIPEA (1000 µL) and this mixture was added to the swelled resin. The reaction continued for 50 min. Then the deprotection and coupling for Fmoc-Gly-OH (G, 892 mg, 3 mmol), Fmoc-Asp(Pbf)-OH (D, 1234 mg, 3 mmol), Fmoc-D-phenylalanine-OH (f, 1162 mg, 3 mmol), and Fmoc-lys(Boc)-OH (K, 1406 mg, 3 mmol) were performed following the similar procedure. Subsequently, cleavage of protected linear peptide from resin was performed with a mild HOAc/trifluoroethanol (TFE)/DCM (1:1:3) mixture without affecting the side chain protecting groups after the deprotection with piperidine/DMF (1:4, v/v). Head-to-tail cyclization was carried out overnight in DCM with DIC (3 eq), 1-hydroxybenzotriazole hydrate (3 eq), and DIPEA (3 eq). Then the crude peptide was precipitated in water and deprotected with a mixture of TFA/water/TIS (95:2.5:2.5) at room temperature for 2 h. The crude peptide was precipitated in cold ether and washed with ether twice to yield cRGDfK-NH2 (add mass here.). The maleimido-RGD conjugate was obtained through the reaction of cRGDfK-NH2 and Maleimido-propionic-PFP (1.5 eq) with DIPEA (2 eq) in DCM for 15 min. Maleimido-propionicPFP was synthesized according to the previous report.18 The maleimido-RGD conjugate was purified by precipitation in cold ethyl ether and dried under reduced pressure overnight. The maleimido-RGD conjugate was characterized by MALDI-TOF (m/z, [M+H]+): 754.876 (obsd.); 754.80 (calcd). Synthesis of MSNs-RGD. The MSN-SH (1 g) was thoroughly suspended in DMF (30 mL) using ultrasound for 30 min and maleimido-RGD conjugate (754 mg, 1.0 mmol) was added to the MSN-SH/DMF mixture under vigorous stirring for 2 h. Then the MSN-RGD was collected by


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centrifugation at 4000 rpm for 20 min, washed with ethanol twice, and dried at 30 oC under reduced pressure overnight. Synthesis of fluorescein labelled MSNs and MSNs-RGD. Fluorescein-5-maleimide (AAT Bioquest, USA) (10 mg, 0.02 mmol) was dissolved in DMSO completely and then mixed with MSN-SH (1 g) or MSNs-RGD (1.75 g), which was presuspended in DMSO using ultrasound for 20 min. The mixture was stirred for 1 h at room temperature and the fluorescent MSNs (FMSNs) or fluorescent MSNs-RGD (F-MSNs-RGD) was collected by centrifugation at 4000 rpm for 30 min, followed by washing with ethanol twice. The F-MSNs or F-MSNs-RGD was thoroughly dried at room temperature under reduced pressure overnight. ATO loading and release. ATO (0.35 g) was dissolved in PBS solution (35 mL) completely. MSNs-RGD (0.2 g) was suspended in the ATO solution and shaken at 37 oC overnight. The MSNs-RGD was collected using centrifugation and washed with PBS three times, followed by drying under reduced pressure at 50 oC. The PBS solution and PBS washings were collected and the concentration of ATO was measured with ICP-AES to calculate ATO loading in MSNsRGD. The ATO in vitro release was studied by suspending above ATO-loaded MSNs-RGD in PBS solution or glutathione (GSH) containing PBS solution (50 µM or 10 mM, 30 mL) under shaking at 37 oC. The supernatant (2 mL) was collected at each release time point (1 h, 4 h, 12 h, day 1, day 2, day 4, and day 10) and the concentration was detected with ICP-AES to study release kinetics. Characterizations. The cRGDfK and maleimido-RGD peptides were characterized by matrix-assisted laser desorption/ionization time-flight (MALDI-TOF) mass spectrometry, which were acquired on a Bruker Autoflex III MALDI-TOF MS using 2,5-dihydroxybenzoic acid (2,5DHB) as a matrix. The morphology of silica nanoparticles were observed using a transmission electron microscopy (TEM, FEI Tecnai F30, USA). The porosity parameters and specific surface area were measured using a Brunauer-Emmet-Teller (BET) method, which was based on the



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nitrogen gas adsorption (ASAP2010, USA). Thermogravimetric analysis (TGA) was conducted on a Q500 thermal analyst in nitrogen at a heating rate of 20 oC min-1 from room temperature to 800 oC. The mesostructure was characterized by small-angle X-ray diffraction (SAXRD, Scintag X-1) using Cu Kα radiation (40 kV and 40 mA) at a scanning rate of 0.02º/min over the range of 1.5-6.5º with a step width of 0.02º. Cell

culture.

MDA-MB-231 cells

were cultured in DMEM/High Glucose media,

supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were incubated on tissue culture plates with 5% CO2 and 95% relative humidity at 37 oC. The culture media was refreshed every other day and the cells were split upon ~80% confluence. Fluorescence confocal microscopy. To visually observe the endocytosis of F-MSNs and F-MSNs-RGD, the cells at a density of 5×104 cells/mL were cultured in 6-well plates for 24 h prior to the treatment with a concentration of 100 µg/mL F-MSNs or F-MSNs-RGD suspended in cell culture media. After the cells were cultured for another 4 h, the culture media was extracted and the cells were thoroughly washed with PBS to remove nanoparticles, not internalized into cells. Afterwards, the cells were fixed with 4% PFA solution for 20 min, washed with PBS, permeabilized with 0.2% Triton X-100, and stained with rhodamine phalloidin (RP) for 1 h in cell incubator.

The cells were imaged using a Olympus FV1000 confocal laser microscopy

(Olympus, Center valley, PA) after stained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI). Cell viability. The cells at a density of 1×105 cells/mL were seeded in 12-well tissue culture plates for 24 h. After the removal of culture media, MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO suspensions at various concentrations (equivalent ATO for ATO-MSNs, ATO-MSNs-RGD, and ATO at each concentration, equivalent total weight for MSNs and ATO-MSNs at each concentration) in culture media were used to culture cells for another 24 h. Then the cell


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number was quantified with an MTT assay (Life Technologies) according to the manufacturer’s manual. Animal tumor model. An animal study was performed according to a protocol approved by the Case Western Reserve University Animal Care Committees. Female athymic nu/nu mice (46 weeks) were purchased from the Athymic Animal & Xenograft Core at Case Comprehensive Cancer Center. The mice were implanted with 1×106 MDA-MB-231 cells in a mixture of PBS (50 µL) and Matrigel (50 µL) in flanks. The in vivo treatment with drug was initiated when the tumor size was ~5 mm in diameter. In vivo tumor treatment. MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO solutions were prepared at predetermined concentrations. The tumor-bearing mice (n=5) were i.v. injected with MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO solutions at an ATO dosage of 0.75 mg/kg every 5 days over the course of 20 days. MSNs were used as a non-therapeutic control and were injected at a dose of 5 mg/kg MSN. Tumor volume was measured at each injection with a caliper and calculated based on the following equation: tumor volume = (x2)(y2)(0.5). On the day 20, the tumors in control group (MSNs) were too large to continue the experiment, and the mice in all treatment groups were sacrificed on the day 20. Tumor tissues were collected, weighed, embedded in optimal cutting temperature compound (OCT), and cryosectioned into 5 µm slices for H&E staining. Stained slices were imaged at 10× with Virtual Slide Microscope VS120 (Olympus, Melville, NY). Statistical analysis. ICP-AES measurements, cellular uptake, and cell viability were performed in quadruplicates for each group at each time point. The statistically significant difference (p < 0.05 or 0.01) between groups was analyzed with the student’s t-test. RESULTS AND DISCUSSION Synthesis and characterizations. The MSNs were synthesized using a sol-gel process using CTAB as a template, which was removed with a mixed solution of ethanol and



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hydrochloric acid.19, 20 The thiol groups were introduced to the MSN inner and outer surfaces to enhance ATO loading and conjugate the targeting group (Figure 1a). The morphology and mesostructure of MSNs were characterized and confirmed using TEM (Figure 1b-d). The MSNs had a size of 100 ± 30 nm in diameter as measured using ImageJ software (NIH, Bethesda, MD). Well-ordered mesopores of MSNs with uniform size were observed, consistent with previous reports.19, 20 The mesostructures of MSNs-SH and MSNs-RGD were also visualized with TEM and both had morphological features identical to MSNs (Figure 1b-c), indicating that modification with (3-mercaptopropyl)triethoxysilane and maleimido-RGD did not affect the mesostructure of MSNs. However, the loading of ATO into MSNs-RGD prominently changed the morphological features (Figure 1d) with the disappearance of porous structure, demonstrating that the particles were loaded with ATO in the pores, through both physical adsorption by pores and chemical complexation between thiol groups on MSNs and arsenic. The surface properties of the drug loading nanoparticles were also determined with BET nitrogen adsorption (Table 1). The BET surface areas of MSNs and MSN-SH were as large as ~1021 m2/g and 958 m2/g and the BJH pore sizes were measured to be ~2.2 in diameter, respectively, which agreed with previous reports.19 After modification with RGD, both the BET surface area and BET pore volume of MSNs-RGD decreased substantially to ~779 m2/g and 0.467 cm3/g, respectively. This change was more significant for ATO-MSNs and ATO-MSNsRGD due to the adsorption of ATO into mesopores. The XRD patterns of both MSNs and MSNs-SH (Figure 2a) clearly showed typical MCM-41 type ordered frameworks, in which three distinct diffraction peaks at ~2.2o, 3.9o, and 4.5o (2θ) were indexed to the reflection planes of (100), (110), and (200), respectively.19 However, all reflection planes of hexagonal mesoporous structure faded a little after the modification with RGD (MSNs-RGD). Consistent with TEM observation, the reflection planes of (110) and (200) completely disappeared when the MSNs-RGD was loaded with ATO (ATO-MSNs-RGD),


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indicating that the ATO was absorbed into the pores. The nanoparticle composition was also analyzed using TGA measurement (Figure 2b). The water residue in the nanoparticle was removed at a temperature of >100oC and the surface -OH groups, -SH groups, and RGD peptide degraded at > 300oC. The ATO with a boiling point of 460 oC started to show fast weight loss around 460 oC. The weight losses of MSNs, MSNs-SH, MSNs-RGD, and ATO-MSNs-RGD were measured to be 15%, 25%, 40%, and 52%, respectively. The amounts of surface anchored -SH groups, RGD peptide, and loaded ATO were determined to be 1.12 mmol/g, 0.20 mmol/g, and 0.61 mmol/g, respectively. In vitro ATO release. The release of ATO from ATO-MSNs-RGD was investigated in PBS, 20 µM glutathione (reduced form, GSH), and 10 mM GSH PBS solution. Two different GSH concentrations were used to mimick the extracellular and intracellular environments21. ATO was gradually released from the nanoparticles under all tested conditions (Figure 1 and Figure 3). ATO release was slightly faster in 20 µM GSH/PBS solution, a condition similar to extracellular environment, than in PBS solution, but difference was no significant (p > 0.05). The ATO release rate was significantly accelerated in 10 mM GSH/PBS solution, a condition similar to intracellular environment, and reached almost 100% at 50 h. The presence of high concentration GSH triggered the dissociation of ATO from the thiolated MSNs and greatly facilitated the intracellular drug release. The results suggested that ATO-MSNs-RGD had a controlled drug release function with slow a drug release during systemic circulation and fast drug release once internalized in the targeted cells. In vitro cellular uptake. Drug action of ATO requires cancer cell specific internalization of the nanoparticles and drug release to induce apoptosis.22 Cellular uptake was investigated with fluorescein labelled nanoparticles, as shown in Figure 4a. Both F-MSNs and F-MSNs-RGD can be internalized into cells. The cells presented prominently higher uptake of F-MSNs-RGD than F-MSNs, indicated by the higher intensity of green color. The MSNs modified with RGD (F-



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MSNs-RGD) significantly enhanced their cellular uptake via receptor mediated endocytosis compared with MSNs, in a good agreement with previous studies.23, 24 Cell viability. Concentration dependent cell viability for MSNs, ATO-MSNs, ATO-MSNsRGD, and ATO was also evaluated using a MTT assay (Figure 4b). Herein, ATO-MSNs, ATOMSNs-RGD, and ATO were assessed based on the ATO equivalent concentrations, Figure 4b. The MSNs concentration was calculated as the equivalent concentration of MSNs as in ATOMSNs. Similar to early reports,2,

25

MSNs showed excellent biocompatibility without inducing

cytotoxicity at any concentration. ATO-MSNs, ATO-MSNs-RGD, and ATO showed dosedependent cytotoxicity with elevated concentration. As expected, ATO-MSNs-RGD showed significantly higher cytotoxicity than ATO-MSNs and ATO when the drug concentration was higher than 20 µg/mL. Free ATO exhibited lower cell viability than ATO-MSNs when the ATO concentration was over 100 µg/mL. High cellular uptake of ATO-MSNs-RGD (Figure 4a) clearly enhanced the cytotoxicity as compared with ATO-MSNs. On the other hand, we found that the cytotoxicity of ATO-MSNs-RGD and free ATO had no significant differences, even though both showed great cytotoxicity, especially when the concentrate increased. It is common that targeted nanoparticle based drug delivery systems to exhibit similar in vitro cytotoxicity to the corresponding free drug because free drugs are generally internalized by the cells by diffusion.26 The advantage of targeted nanosized drug delivery systems is their favourable pharmacokinetics and tumor targeting due to the EPR effect and receptor-mediated endocytosis. These in vitro experiments demonstrated that ATO-MSNs-RGD had several advantageous features, including cancer cell specific cellular uptake, enhanced cytotoxicity, and environment-responsive intracellular drug release. All these features of ATO-MSNs-RGD suggest that the targeted nanoparticles ATO-MSNs-RGD are an effective targeted delivery system of ATO for cancer therapy.


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Anticancer efficacy. The therapeutic efficacy of ATO-MSNs-RGD was demonstrated in mice bearing MDA-MB-231 human TNBC xenografts. The tumor volumes during the treatments and the tumor weights at the end of the treatments were shown in Figure 5a and b. ATO-MSNsRGD resulted in more significant tumor growth regression. The volume of tumors treated with ATO-MSNs-RGD was significantly smaller than other groups at the same time point and the difference was greater as the treatment continued. Tumor weight measurement at the end of the treatments demonstrated that ATO-MSNs-RGD resulted in the smallest tumors. ATO-MSNs and ATO also showed some therapeutic effect as compared with MSNs group. It is interesting to note that the ATO-MSNs resulted in more efficient inhibition of tumor growth than free ATO. Previously, it was demonstrated that MSNs themselves preferentially accumulated in tumors and the camptothecin-loaded MSNs effectively delivered the drug to the tumors and suppressed the growth of human breast tumors.2 In our present study, the enhanced efficacy of ATO-MSNs as compared to ATO might be similarly attributed to the passive accumulation of the ATO loaded nanoparticles into the tumor tissue. Consequently, the ATO-MSNs showed slightly better therapeutic efficacy than ATO. Consistent to the in vitro assessment, ATO-MSNs-RGD demonstrated significantly improved therapeutic efficacy over ATO and ATO-MSNs. Specific binding of ATO-MSNs-RGD to the αvβ3 integrin overexpressed in the tumor

27, 28

certainly enhanced their tumor accumulation, resulting

in more significant inhibition of tumor growth than ATO and ATO-MSNs. In a good agreement, the histological analysis at the end of treatment revealed that the tumor treated with ATOMSNs-RGD showed the reduced cell density as compared to the tumors in other groups, and apoptotic cells that shrank and were separated from neighboring cells with surrounding halo-like clear spaces (Figure 5d). The tumors treated with MSNs and ATO had higher cell density than other groups.



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The ATO dose used in this study was 0.75 mg/kg for ATO-MSNs and ATO-MSNs-RGD, much lower than the previously reported dose of 2-6.5 mg/kg for treating solid tumors,29 which was considerably higher than the standard dosage of 0.16 mg/kg for treating human patients with APL. Unfortunately, the data suggested that ATO at 2-6.5 mg/kg was highly toxic to the major organs, including the heart, liver, and kidneys, when systemically administered to mice.2931

The dosing frequency used in this study was also much less than the previously reported

frequency for free ATO, every other or 2 days.30, 32, 33 Clearly, ATO-MSNs-RGD were effective in treating TNBC at a low dose and dosing frequency, which could certainly minimize dose related toxic side effects. The targeted ATO-MSNs-RGD was designed to possess the ability for controlled release of ATO in intracellular environment with minimal leakage in the circulation. MSNs encapsulated with ATO exhibited faster drug release intracellularly than in the extracellular environment, indicating better stability in the circulation. Once the targeted ATO-MSNs arrived at tumor sites and were internalized by tumor cells via receptor-mediated endocytosis, the intracellular environment with a high GSH concentration facilitate the ATO release from ATO-MSNs-RGD. Therefore, targeted ATO delivery with ATO-MSNs-RGD minimized drug leakage during the circulation and increased the drug concentration in tumors, resulting in improved therapeutic efficacy at a substantially low dose in treating TNBC in the mouse TNBC tumor model. The reduced dose and dosing frequency with the targeted nanoparticles can also minimize potential dose-dependent toxic side-effect of ATO. These advantageous features of the targeted ATOMSNs-RGD warrant further investigation and development of targeted ATO delivery systems with optimized therapeutic efficacy for safe and efficacious treatment of solid tumors, including triple negative breast cancer.

CONCLUSIONS


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A targeted ATO delivery system ATO-MSNs-RGD with environment sensitive drug release was prepared and explored for treating human triple negative breast cancer. ATO-MSNs-RGD showed clearly higher cellular uptake in cancer cells than non-targeted ATO-MSNs in MDA-MB-231 human TNBC cells. The in vivo study demonstrated that ATO-MSNs-RGD was effective to inhibited tumor growth at a much lower ATO dosage of 0.75 mg/kg and less dosing frequency in a mouse model of human triple negative breast cancer. ATO-MSNs-RGD is a promising therapeutic regimen in treating solid tumors.

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Captions of Table and Figures Figure 1. (a) Synthesis of the ATO-MSNs-RGD. TEM images of (b) MSNs (c) MSNs-RGD, and (d) ATO-MSNs-RGD. Scale bar of 20 nm is applicable to all. Table 1. Surface properties of nanoparticles determined by BET nitrogen adsorption Figure 2. (a) XRD patterns of MSNs, MSNs-SH, MSNs-RGD, and ATO-MSNs-RGD. (b) TGA curves of MSNs, MSNs-SH, and MSNs-RGD. Figure 3. In vitro drug release in PBS, 10 mM GSH/PBS, and 20 µM GSH/PBS at different time points. Error bars indicate SD of the means (n = 4). Figure 4. (a) Fluorescence confocal microscope images of MDA-MB-231 cells. The nuclei were stained with DAPI (blue), the MSNs or MSNs-RGD were labeled with Fluorescein-5-maleimide (green), and the cytoplasma was stained with RP (red). Scale bar of 200 µm is applicable to all. (b) Cell viability normalized to TCP, determined by a MTT assay. *: p < 0.05 relative to MSNs, #: p < 0.01 relative to MSNs, ^: p < 0.01 relative to MSNs. Error bars indicate SD of the means (n = 4). Figure 5. In vivo treatment of MDA-MB-231 breast tumors with MSNs, ATO-MSNs, ATO-MSNsRGD, and ATO every five days. (a) Tumor volume change with treatment days. Error bars indicate SD of the means (n = 5). (b) Tumor weights treated with different drugs at day 20. Error bars indicate SD of the means (n = 5). (c) Digital camera images of tumors treated with different drugs at day 20. (d) Histological view of MDA-MB-231 tumors treated with MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO at day 20 (H&E staining; magnification, ×7.5).


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Figure 1. (a) Synthesis of the ATO-MSNs-RGD and in vitro release of ATO. TEM images of (b) MSNs (c) MSNs-RGD, and (d) ATO-MSNs-RGD. Scale bar of 20 nm is applicable to all.



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Table 1. Surface properties of nanoparticles determined by BET nitrogen adsorption Materials MSNs MSNs-SH MSNs-RGD ATO-MSNs ATO-MSNs-RGD

BET Surface Area 2 (m /g) 1021.334 957.765 779.215 69.762 58.512

BET Pore Volume 3 (cm /g) 0.531 0.520 0.467 0.012 0.013


BJH Pore Diameter (nm) 2.214 2.194 2.187 0.126 0.103

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(a) Intensity (a.u.)

(100)

(110)

MSNs

(200)

MSNs-SH MSNs-RGD ATO-MSNs-RGD

2

3

4

5

6

Degree (2θ)

(b)100 90 Weight (%)

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MSNs

80

MSNs-SH

70 MSNs-RGD

60

ATO-MSNs-RGD

50 40

0

200

400

600

800

o

Temperature ( C)

Figure 2. (a) XRD patterns of MSNs, MSNs-SH, MSNs-RGD, and ATO-MSNs-RGD. (b) TGA curves of MSNs, MSNs-SH, MSNs-RGD, and ATO-MSNs-RGD.



100

Drug release (wt%)

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80 60

PBS 10 mM GSH/PBS 20 µM GSH/PBS

40 20 0 0

100

200

300

400

500

Time (h) Figure 3. In vitro drug release in PBS, 10 mM GSH/PBS, and 20 µM GSH/PBS at different time points. Error bars indicate SD of the means (n = 4).


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Figure 4. (a) Fluorescence confocal microscope images of MDA-MB-231 cells. The nuclei were stained with DAPI (blue), the MSNs or MSNs-RGD were labeled with Fluorescein-5-maleimide (green), and the cytoplasma was stained with RP (red). Scale bar of 200 µm is applicable to all. (b) Cell viability normalized to TCP, determined by a MTT assay. *: p < 0.05 relative to MSNs, #: p < 0.01 relative to MSNs, ^: p < 0.01 relative to MSNs. Error bars indicate SD of the means (n = 4).



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Figure 5. In vivo treatment of MDA-MB-231 breast tumors with MSNs, ATO-MSNs, ATO-MSNsRGD, and ATO every five days. (a) Tumor volume change with treatment days at the ATO dosage of 0.75 mg/kg. Error bars indicate SD of the means (n = 5). (b) Tumor weights treated with different drugs at day 20. Error bars indicate SD of the means (n = 5). (c) Digital camera images of tumors treated with different drugs at day 20. (d) Histological view of MDA-MB-231 tumors treated with MSNs, ATO-MSNs, ATO-MSNs-RGD, and ATO at day 20 (H&E staining; magnification, ×7.5).



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Table of Contents Graphic

Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide with Environment Sensitive Drug Release for Effective Treatment of Triple Negative Breast Cancer Xiaohui Wu, Zheng Han, Rebecca M. Schur and Zheng-Rong Lu

We report a RGD peptide targeted mesoporous silica nanoparticles (MSNs)-based drug delivery system of arsenic trioxide (ATO) (ATO-MSNs-RGD) for treating MDA-MB-231 breast cancer model. Because of the targeting effect, enhanced therapeutic efficacy was achieved with targeted nanoparticles at a low dose due to efficient tumor targeting and less release of arsenic trioxide in extracellular spaces.


Targeted Mesoporous Silica Nanoparticles Delivering Arsenic Trioxide

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