Crumpled Aluminum Hydroxide Nanostructures as a

Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

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Crumpled Aluminum Hydroxide Nanostructures as a Microenvironment Dysregulation Agent for Cancer Treatment Marat I. Lerner,†,∇ Georgy Mikhaylov,‡,∇ Alexey A. Tsukanov,†,∇ Aleksandr S. Lozhkomoev,† Elazar Gutmanas,§ Irena Gotman,∥ Andreja Bratovs,‡ Vito Turk,‡ Boris Turk,‡,⊥,# Sergey G. Psakhye,*,† and Olga Vasiljeva*,‡,○ †

Institute of Strength Physics and Materials Science, Tomsk 634055, Russia Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana SI-1000, Slovenia § Technion-Israel Institute of Technology, Haifa 3200, Israel ∥ Department of Mechanical Engineering, ORT Braude College, Karmiel 2161002, Israel ⊥ Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana SI-1000, Slovenia # Center of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, SI-1000 Ljubljana, Slovenia

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‡

S Supporting Information *

ABSTRACT: Owing to their unique physicochemical properties, nanomaterials have become a focus of multidisciplinary research efforts including investigations of their interactions with tumor cells and stromal compartment of tumor microenvironment (TME) toward the development of nextgeneration anticancer therapies. Here, we report that agglomerates of radially assembled Al hydroxide crumpled nanosheets exhibit anticancer activity due to their selective adsorption properties and positive charge. This effect was demonstrated in vitro by decreased proliferation and viability of tumor cells, and further confirmed in two murine cancer models. Moreover, Al hydroxide nanosheets almost completely inhibited the growth of murine melanoma in vivo in combination with a minimally effective dose of doxorubicin. Our direct molecular dynamics simulation demonstrated that Al hydroxide nanosheets can cause significant ion imbalance in the living cell perimembranous space through the selective adsorption of extracellular anionic species. This approach to TME dysregulation could lay the foundation for development of novel anticancer therapy strategies. KEYWORDS: Crumpled nanosheets, aluminum hydroxide, tumor microenvironment, ion balance, molecular dynamics simulation, cancer treatment

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especially attractive due to its distinct combination of substantial surface area and positive charge.15 In addition, Al hydroxide is approved for clinical applications and has been used as a human vaccine adjuvant over six decades, with a demonstrated safety profile.16 Here we report that Al hydroxide, a well-studied antacid agent17 and selective adsorbent for many chemicals,18,19 when synthesized in the form of crumpled and radially assembled nanosheets, causes tumor growth inhibition and cell apoptosis due to a significant ion imbalance in the tumor microenvironment. Synthesis of Agglomerated Al Hydroxide-Based Crumpled Nanosheets. In this work, we synthesized Al hydroxide mesoporous nanostructures using a modified

ver the past decade, it became increasingly recognized that the tumor microenvironment (TME) plays an important role in cancer progression and metastasis.1−5 Novel TME-manipulating anticancer therapies have been proposed and developed including immunotherapy,6 antiangiogenic,7,8 and anti-inflammatory agents,9 as well as in targeted delivery systems.10,11 Among different strategies, nanoparticles have been widely exploited in cancer therapy because of their unique chemical and physical properties. One of the newly emerging areas for potential nanoparticle applications in treatment of cancer is based on the notion of the sensitivity of cancer cells to deviations from the equilibrium extracellular and intracellular ion concentration.12,13 Therefore, inorganic nanostructured materials with pronounced sorption properties and/or surface charge that could modulate ion concentration gradient across a cell membrane14 could represent a novel treatment strategy in oncology. In this respect, the nanostructured boehmite form of aluminum hydroxide seems © XXXX American Chemical Society

Received: April 19, 2018 Revised: July 24, 2018

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DOI: 10.1021/acs.nanolett.8b01592 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Aloohene synthesis and characterization. (a) TEM images showing the stages of water oxidation of a source Al/AlN nanoparticle (from left to right) into radially assembled crumpled AlOOH nanosheets (Aloohene). (b, c) High-resolution transmission and scanning electron micrographs of Aloohene. Microdiffraction pattern (insert on panel b) shows a low crystallinity of Aloohene. (d) HRTEM image of the Aloohene multidomain structure with a domain size of 4−5 nm. (e) X-ray diffraction pattern of Aloohene. Peaks are indexed according to the reference pattern for boehmite (Supporting Information). The broadening of the diffraction peaks and the shift of the (020) reflection compared to boehmite suggest that the synthesized material exhibits a pseudoboehmite structure.

synthesis method15 that allowed us to produce a very pure material, without any salt or Al3+ cations contaminations. The latter is particularly important, since Al3+ can promote generation of reactive oxygen radicals and subsequent oxidative damage that could lead to toxicity effects.20 To obtain Alhydroxide, Al/AlN nanopowder prepared by electric explosion of an aluminum wire in N2 gas environment was oxidized for 1 h in deionized water at 60 °C and atmospheric pressure. The oxidation of Al/AlN nanoparticles proceeds without an induction time, and the released ammonia causes a complete oxidation of aluminum. Figure 1a demonstrates the formation stages of Al hydroxide mesoporous nanostructures. The initial Al/AlN nanoparticles act as growth centers or seeds for the crumpled Al hydroxide nanosheets, which are agglomerated in radially assembled nanostructures. The lateral size of the crumpled nanosheets was 400 nm or less (Figure 1b,c), and their thickness was in the range of 2−5 nm (Figure S1). Highresolution transmission electron microscopy (HRTEM) showed that the crumpled nanosheets have a disordered

multidomain structure; the lateral size of each domain is about 5 nm (Figure 1d). The multidomain structure suggests the presence of multiple structurally defective areas in the vicinity of domain interfaces, where active adsorption centers are located,21 thus providing the Al hydroxide mesoporous nanostructures with high adsorption efficiency. X-ray diffraction (XRD) analysis revealed that the material is the boehmite form of aluminum hydroxide (AlOOH) (Figure 1e). The synthesized boehmite crystallites exhibit a platelet shape as was previously reported for nanocrystalline and microcrystalline boehmite.22,23 Boehmite is a layered aluminum oxide hydroxide with an ordered laminated structure composed of parallel double chains of AlO6 octahedra held together by the relatively weak hydrogen bonds.22,23 Easy cleavage of the hydrogen bonds between the octahedral lattice layers explains the platelet-like morphology of boehmite crystals. The synthesized agglomerates of radially assembled crumpled AlOOH nanosheets have been termed Aloohene, referring to its chemical formula AlOOH. Aloohene has a high specific B


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Figure 2. Antitumor effect of Aloohene in vitro. (a) Analysis of cellular toxicity of Aloohene. Annexin V and PI staining of HeLa, MCF-7, UMSCC-14C, and MMTV-PyMT cells in culture in the presence or absence of Aloohene (5 mg ml−1 concentration) at 37 °C for 24 h. Fluorescence intensity was measured by flow cytometry, and data were analyzed by the Cell Quest software. (b) Proliferation of HeLa, UM-SCC-14C, and MCF7 cells, as measured by BrDU assay. Cell cultures were incubated with Aloohene (5 mg ml−1) at 37 °C for 24 h. Fluorescence intensity was measured 48 h after BrDU labeling at excitation and emission wavelengths of 370 and 470 nm, respectively. Results are means of 3 independent experiments. (c) Combined effect of Aloohene and doxorubicin on PyMT cancer cells. MMTV-PyMT mouse breast cancer cells were pretreated with Aloohene (0.1 mg ml−1) for 24 h and then incubated with doxorubicin (0.1 μg ml−1) for an additional 24 h. Cytotoxicity was determined with the MTT assay. Cell viability was calculated as a percentage of viable cells. Results are means of 3 independent experiments ***P, 0.001.

surface area of 284 m2 g−1 with the width of the slit-shape pores between the nanosheets being 4−19 nm. The ζ potential of the synthesized Aloohene, measured at 37 °C in the pH range 3.0−8.0, was above 30 mV throughout. The isoelectric point (pI) of Aloohene was found to be 9.6. In water and in the cellular environment, Aloohene acts as a weak base and preferentially adsorbs anions on its surface. Stability of AlOOH nanosheets was assessed by 72 h immersion of Aloohene powder in PBS and other solutions containing different ionic species, with no reduction of the specific surface area detected. Aloohene is essentially insoluble at a physiological pH and is not expected to release measurable amounts of Al3+ ions implicated in various toxic events. Effect of Aloohene on Tumor Cells Viability and Proliferation in Vitro. The unique properties of the synthesized AlOOH nanomaterial were utilized to inhibit tumor cell growth. Three human cancer cell lines (MCF-7, UM-SCC-14C, and HeLa) and primary mouse mammary

tumor (PyMT) cells were cultured in a medium containing 5 mg ml−1 of Aloohene. After 24 h of incubation, a significant reduction of tumor cell viability was detected by fluorescenceactivated cell sorting (FACS) for all tested cells compared to the corresponding nontreated controls (Figure 2a and Figure S2), whereas no effect on pH or lysosomal integrity was detected in the cell cultures upon treatment with Aloohene (Figures S3 and S4). Next, the BrdU (5-bromo-2′-deoxyuridine) assay was used to investigate the influence of Aloohene on proliferation of tumor cells. We observed a 30−37% decrease in proliferation of all tested cell lines (Figure 2b). Collectively, these findings suggest that Aloohene reduce viability and inhibit proliferation of tumor cells. Therefore, we next tested whether such interaction would sensitize tumor cells to clinically approved cytotoxic agents. For this, PyMT mouse breast cancer cells were pretreated with a suboptimal concentration of Aloohene (0.1 mg ml−1; Figure S5) for 24 h, followed by a 24 h treatment with the standard chemoC


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Figure 3. Antitumor effect of Aloohene in vivo in a murine breast cancer model. (a) The PyMT orthotopic breast cancer model was treated with 10 mg mL−1 of Aloohene, and tumor volumes were measured twice a week. Data are presented as mean tumor volume ± standard errors of mean (n = 8 per treatment group). The statistical significance of differences between the treated and control groups was assessed by Student’s t test. Representative images of tumors are presented on the right side of the graph. (b) Quantification of Ki67-positive cells as a percentage of total cells in primary tumors of nontreated and Aloohene-treated mice. Representative images of cell proliferation in primary tumors by immunodetection of Ki67 (brown staining) are shown at ×20 magnification. (c) The percentage of necrotic cells areas was measured using high-power fields of tumors sections from nontreated (n = 8) and Aloohene-treated (n = 8) mice using the TUNEL assay (brown areas). (d) Caspase-3-positive cells were calculated as a percent of total cells in primary tumors of nontreated and Aloohene-treated mice. Representative images of caspase-3-positive staining in primary tumors of nontreated and Aloohene-treated mice are shown underneath. Percentages of proliferative, necrotic, and apoptotic cells are presented as means and standard errors, n = 8. Representative images are shown for control mice and mice treated with Aloohene. The scale bar corresponds to 200 μm. Student’s t test was used to calculate the statistical significance of differences between the groups. *P, 0.05; ***P, 0.001, compared with the control group.

therapeutic efficacy of doxorubicin, which may allow to decrease its effective dose and consequently to reduce its toxicity. Evaluation of Antitumor Activity of Aloohene in Mouse Models of Cancer. We next investigated whether the in vitro cancer cell growth inhibition effect of Aloohene would translate into antitumor efficacy in vivo using a murine model of breast cancer. First, in vivo safety of Aloohene was evaluated in an acute toxicity study using healthy FVB mice treated with 500 or 1000 mg kg−1 of Aloohene. No adverse effects or changes in blood biochemistry profile and histopathology of collected tissues were observed 14 days after administration of Aloohene in treated mice as compared to the nontreated group of animals (Table S1; Figures S8 and S9). Next, the ortothopic

therapeutic drug doxorubicin at the minimal effective concentration (0.1 μg mL−1) determined by dose titration (Figure S6). The results of the MTT cell viability assay demonstrate that pretreatment of PyMT cells with Aloohene significantly enhanced the cytotoxicity of the drug (Figure 2c). The combined effect of doxorubicin and Aloohene was evaluated using the method of isoboles, a graphical method for assessing synergism or subadditivity for agonist drug combinations based on the concept of dose equivalence.24 Isobologram analysis of the dose-dependent cytotoxicity of the PyMT cells monotreatment with doxorubicin and Aloohene (Figures S5 and S6) and a combination of both (Figure 2c) indicates their synergistic effect in vitro (Figure S7). Taken together, this data suggests that Aloohene could potentiate the D


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Figure 4. Antitumor effect of Aloohene in vivo in murine skin melanoma model. (a) Effect of treatment with Aloohene alone and in combination with doxorubicin. Mice were treated with 10 mg ml−1 of Aloohene, 10 mg kg−1 of doxorubicin, and their combination, and tumor volumes were measured twice a week. Data are presented as mean tumor volume ± standard errors of mean (n = 10 per treatment group). The statistical significance of differences between the groups was assessed by Student’s t test. (b) Cell proliferation in primary melanomas determined by Ki67 staining in nontreated mice and mice treated with doxorubicin, Aloohene, or their combination. Ki67-positive cells calculated as percentage of total cells and the corresponding illustrative images are shown. (c) The percentage of necrotic cells areas was measured on high-power fields of primary melanomas of nontreated mice and mice treated with doxorubicin, Aloohene, or their combination using the TUNEL assay (brown areas). The percentages of proliferative, necrotic, and apoptotic cells are presented as means and standard errors, n = 10. Representative images are shown for control mice and mice treated with Aloohene. The scale bar corresponds to 200 μm. Student’s t test was used to calculate the statistical significance of differences between the groups. ***P, 0.001 compared with the control group.

intravenously applied drugs.28,29 Weekly intratumoral injections of a water suspension of as-synthesized Aloohene (100− 500 nm agglomerates) at a concentration of 10 mg ml−1 demonstrated significant tumor growth reduction as compared to the vehicle control (P < 0.01) (Figure 3a and Figure S10). Furthermore, to address the mechanism of tumor growth inhibition by Aloohene, we measured markers of tumor proliferation, cell death, and vascularization. Whereas distribution of the endothelial cell marker CD31 in the analyzed tumor sections indicated that Aloohene treatment had no effect on tumor vascularization (Figure S11), a significant decrease in the proliferation rate of tumors treated with Aloohene as compared to the control group was detected by immunohistochemical (IHC) staining for the proliferation

mouse breast cancer model was developed by the orthotopic implantation of tumor cells, isolated from primary tumors of MMTV-PyVT transgenic mouse mammary tumor model, into the mammary gland of syngeneic immunocompetent recipient mice (FVB/N mouse strain) as previously described.25 The intratumoral route, which was also used for Aloohene administration in this study, has been extensively evaluated in the past few decades in preclinical studies and was also approved for the clinical applications.26,27 It was shown that, compared to systemic drug administration, both the tumor-toorgan ratios and the tumor concentrations of injected compounds improved substantially.28 Furthermore, intratumoral injection results in a major increase of the antitumor efficacy and an improved therapeutic index as compared to the E


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Figure 5. A molecular dynamics simulation of ion redistribution induced by Aloohene nanosheet. (a) Spatial distribution of cations and anions in the extracellular area near the cell membrane in the presence of the Aloohene domain. (b) Ion concentration Ci(z) of cations (red curve) and anions (blue curve) relative to the unperturbed level for same ion C0i (z) at a distance z from the cell membrane. (c) The insert demonstrates an enlarged fragment of the Aloohene domain and the cell membrane. Membrane colors: POPC lipid tails = white, POPC phosphate groups = orange, choline groups = green, POPG lipids = yellow, POPG phosphate groups = red. Aloohene colors: OH groups = white, bridging oxygen = light gray, aluminum = dark gray. Water molecules and lipid hydrogen atoms are not shown.

of 5 × 104 B16F10 cells into C56BL/6J mice. After 2 weeks of weekly intratumoral administration of Aloohene, significantly decreased tumor growth was observed compared to the vehicle control animals (Figure 4a). A combination therapy of the standard-of-care anticancer drug doxorubicin with Aloohene was also investigated in vivo. Mice were pretreated with an intratumoral injection of 10 mg ml−1 of Aloohene followed by systemic administration of doxorubicin at a single dose of 10 mg kg−1. Although the treatment with doxorubicin alone resulted in tumor growth inhibition, the combination treatment with Aloohene and doxorubicin showed a much stronger suppressive effect on tumor growth (Figure 4a and Figure

marker Ki67 (Figure 3b). Furthermore, large areas of dead cells were detected by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) method in tumors treated with Aloohene, unlike in the control group (P < 0.05) (Figure 3c). The latter observation in combination with the results of IHC staining for the apoptosis marker caspase-3, suggests a possible role of Aloohene in the induction of tumor cell apoptosis in vivo (Figure 3d). To evaluate whether these findings have a broad applicability, the antitumor effect of Aloohene was additionally tested in a melanoma mouse cancer model. A highly malignant mouse skin melanoma was developed by intradermal injection F


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nanomaterials in the development of more effective anticancer strategies. Methods. Aloohene Synthesis. Al/AlN nanoparticles produced by the electrical explosion wire technique (Advanced Powder Technologies, Russia) were used to synthesize Aloohene employing a water oxidation method.15 A suspension of 1 g of Al/AlN nanoparticles in 100 mL of room-temperature deionized water was placed on a hot plate at 85 °C and stirred using a magnetic stirrer for 1 h. The suspension was filtered, and the solid residue dried at 120 °C for 2 h. Characterization of Aloohene. The crystalline structure of Aloohene was characterized by X-ray diffraction analysis (XRD) using an XRD 6000 diffractometer (Shimadzu, Japan) operating with cobalt Kα radiation at 40 kV and 30 mA and scanning between 10° (2θ) and 95° (2θ). The peaks were identified using PDF-2 Release (2014 database). Microstructure characterization was performed using transmission electron microscopes Titan 80−300 keV S/TEM (FEI, USA) and JEM-2100 (JEOL, Japan) and the scanning electron microscope Zeiss Ultra-Plus FEG-SEM (Zeiss, Germany). Texture characteristics of Aloohene were calculated using the low-temperature adsorption/desorption N2 isotherms obtained by the BET (Brunauer−Emmett−Teller) and BJH (Barrett− Joyner−Halenda) methods. Data were processed by use of the TriStar 3020 (Micromeritics, USA) analyzer software. The ζ potential was measured in deionized water at 37 °C and pH 7.4 with Zetasizer Nano ZSP (Malvern Instruments, UK). Cell Cultures. MMTV-PyMT primary mouse mammary cancer cells were prepared and maintained as previously described.25 MCF-7 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Sigma), 2 mM L-glutamine, 100 μg mL−1 of streptomycin, and 100 units of penicillin (Invitrogen). The squamous carcinoma cell line UM-SCC-14C was provided by Cell Line Service GmbH (Germany) and cultured in DMEM and Ham’s F12 (1:1, v/v) medium containing 10% fetal bovine serum and 2 mM L-glutamine. Cell cultures were maintained in a 5% CO2 humidified atmosphere at 37 °C. In Vitro Toxicity and Proliferation Assay. MCF-7, UMSCC-14C, PyMT, and HeLa cells were maintained in DMEM supplemented medium as described above. For the in vitro toxicity assay, cells were incubated in a medium containing 5 mg mL−1 of Aloohene in a phosphate buffer, pH 7.4, for 24 h at 37 °C. Cells cultivated in the Aloohene-free medium were used as controls. DNA fragmentation was labeled by Annexin V-PE in the presence of propidium iodide (PI) and analyzed by the flow cytometry performed with the FACScalibur (Becton Dickinson, USA) using the CellQuest software. For the in vitro proliferation assay, MCF-7, UM-SCC-14C, PyMT, and HeLa cells were incubated in a medium containing 5 mg mL−1 of Aloohene in phosphate buffer, pH 7.4, for 12 h at 37 °C. Cells cultivated in the Aloohene-free medium were used as controls. Proliferation of cells was detected by incorporation of 5-bromo-2′-deoxyuridine (BrdU), a synthetic analogue of thymidine, during DNA replication. Fluorescence of BrdU was examined in a 96-well Safire plate reader (TECAN, Austria) at excitation and emission wavelengths of 370 and 470 nm, respectively. In Vitro Cell-Based Cytotoxicity Assay with Aloohene and Doxorubicin. MMTV-PyMT murine mammary cancer cells were seeded (5 × 103 cells/well) in 96-well plates and cultured for 24 h in a supplemented medium. Cells were first treated with 0.1 mg mL−1 of Aloohene for 24 h followed with 0.1 μg

S12). For all treatment regimes, a significant effect was detected on cell proliferation and death rates, but not on tumor vascularization (Figure 4b,c and Figure S13). Taken together, these results demonstrate a significant antitumor effect of Aloohene and its potential to evolve into a novel strategy of cancer treatment, possibly in combination with established chemotherapy drugs, such as doxorubicin. In addition, systemic administration of Aloohene with a longer treatment course could possibly lead to a more homogeneous distribution and a better efficacy of the compound, which should be evaluated in the future studies. Molecular Dynamics Study of a Possible Mechanism of Tumor Inhibition by Aloohene. Due to the prevalence of base surface sites in its structure, AlOOH could affect the ion balance in the perimembranous space of tumor cells. To evaluate this hypothesis, we employed a direct molecular dynamics (MD) simulation, which is a powerful computational tool for analyzing the interaction of nano-objects with biological structures.30,31 In this study, Aloohene was represented by a single domain of an Al hydroxide nanosheet (Figure 1d), and the ionic composition of the modeled medium was chosen to be similar to the biological extracellular environment. Ion density profiles in the extracellular fluid near a negatively charged mixed POPC/POPG (1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylglycerol) lipid bilayer were evaluated in the presence of a single Aloohene domain and in its absence (unperturbed state) (Figure 5). The interaction of Aloohene with the extracellular anionic species (e.g., Cl−, HCO3−) leads to their redistribution in the extracellular fluid (Figure 5b). The anions are localized close to the Aloohene domain surface and form an anionic “cloud” around the domain. The concentration of Cl− and HCO3− anions in the space distant from the membrane decreases to 0.7−0.8 of the unperturbed value, whereas their concentration in the anionic cloud increases more than twice (Figure 5b, blue curve). It has been previously reported32,33 that a reduction of extracellular Cl− concentration can inhibit cell growth, e.g., by regulating the activity of ion channels and/or transporters, thereby suggesting that this unique mechanism can be also applied to explain the antiproliferative properties of Aloohene nanoparticles. Collectively, our molecular dynamics simulations confirm that Aloohene can alter ion concentration in the perimembranous space and thus affect ion transport, tumor cell nutrition, and vital cellular functions, thus providing a plausible explanation for the anticancer effect of this type of nanomaterials. In summary, the present study demonstrates that nanoparticle-based systems capable of disturbing extracellular TME ion balance are a novel class of inorganic materials that exhibit a strong antitumor effect. Here we report that Al hydroxide nanostructures in the form of agglomerates of crumpled and radially assembled nanosheets (Aloohene) trigger cancer cell death in vitro and inhibit tumor growth in vivo. The disturbing effect of Aloohene on ion balance in the TME is supported by our direct molecular dynamics simulation. Furthermore, our results show that Aloohene nanostructures can potentiate the anticancer action of the cytotoxic agent doxorubicin and thus could improve the efficacy of state-of-the-art chemotherapy when used in combination. The findings of the present research underscore the important role of this novel class of TME-dysregulating G


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Nano Letters mL−1 of doxorubicin administration and additional 24 h cell incubation at 37 °C. Untreated cells and cells incubated with Aloohene or doxurubicin alone were used as controls. The cell viability was assessed using the colorimetric MTT assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and the absorbance measurement at 570 nm using a Safire plate reader. Acute Toxicity Study. FVB male mice were treated with 500 mg kg−1 (n = 5) and 1000 kg−1 (n = 5) of Aloohene and PBS pH 7.2 (n = 5) as a control. Mice were sacrificed at day 14 after intraperitoneal injection of Aloohene. Blood was collected and serum separation performed by centrifugation in Li-heparin 0.6 mL vials (Fuji Photo Film Co., Ltd. Life Science Products Division). Biochemical parameters were analyzed by the biochemical analyzer Fujifilm DRI CHEM 3500i (Fuji Photo Film Co., Ltd. Life Science Products Division). The kidneys, spleen, liver, heart, lung, and abdominal lymph node tissues were collected and fixed in 10% neutral formalin. Organs were dehydrated and maintained in paraffin blocks. Tissue sections (5 μm) were stained by hematoxylin and eosin for histopathological analysis. Animal Models. Procedures for animal care and use were in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86−23, 1996) and “PHS Policy on Human Care and Use of Laboratory Animals”. Mice were used in accordance with the protocols approved by the Veterinary Administration of the Republic of Slovenia and the Government Animal Ethics Committee. Primary MMTV-PyMT tumor cells obtained from transgenic MMTV-PyMT mice were culture-expanded, suspended in serum-free DMEM medium (Invitrogen), and then 5 × 105 cells were inoculated into the left inguinal mammary gland of the FVB/N recipient mice. Primary B16F10 melanoma cells were obtained from LGC Standards GmbH (Germany), and 5 × 104 cells were administered intradermally to the ventral side of C56BL/6J mice. When the tumor volume reached 125 mm3 in the breast cancer model and 70 mm3 in the melanoma model, respectively, mice were treated with Aloohene at a dose of 10 mg mL−1weekly via two intratumoral injections. In addition, melanoma model was treated with doxorubicin injected intraperitoneally at a single dose of 10 mg kg−1 and in combination with Aloohene at the indicated above dose and regimen. The horizontal and vertical tumor diameters were measured by a digital calliper every second day until the end of treatment, and the volume was calculated using the formula π V = 6 ab2 where a and b are the longer and shorter diameters of the tumor, respectively. One week after the last injection, the mice were sacrificed and the excised tumor volumes calculated. Immunohistochemistry. Proliferation in mammary tumor sections was evaluated by detection the proliferation marker Ki67 using rat anti-Ki67 antibody (Dako, 1:10 dilution) staining. TUNEL assay (ApopTag; Oncor) was used for the in situ measurement of DNA fragmentation of dying cells. Detection of primary antibodies was done by the Vectastain Elite ABC kit (Vector Laboratories, USA) in accordance with the manufacturer’s instructions, followed by incubation with 3,3-DAB (Sigma-Aldrich) and nuclear staining with hematoxylin. Vascularization of cryopreserved MMTV-PyMT tumors was analyzed by immunodetection of endothelial cell with rat anti-CD31 (BD Pharmingen; 1:100 dilution) and secondary donkey antirat Cy3 (Dianova; 1:800 dilution) antibodies. For

the staining assessment, 20 randomly selected fields of view per tumor were recorded with Olympus IX 81 microscope (×40 objective). Quantitative analysis of the immunohistochemistry staining was performed using TissueQuest software (TissueGnostics, Austria). Statistical Analysis. Quantitative data are expressed as the means ± standard errors of the means. Student’s t test was used to evaluated the statistical significance of differences between the groups. P values less than 0.05 were regarded as statistically significant. Molecular Dynamics Simulations. Aloohene was modeled as a single AlOOH domain. AlOOH unit cell structure was adopted from ref 34. In the present model the Aloohene domain had a surface charge density of 0.11 C m−2. A model cell membrane was composed of 320 neutral POPC (1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) lipids and 40 negatively charged POPG (1-palmitoyl-2-oleoyl-snglycero-3-phosphatidylglycerol) lipids. To parametrize lipids the CHARMM27 force field35 was utilized. The initial simulation box dimensions were 122 Å × 96 Å × 250 Å. Simulations were performed in the presence and absence of the Aloohene domain. The domain was placed at a distance of about 45 Å from the membrane central plane. The average ion concentrations were 0.147 M for Na+, 6.41 mM for K+, 4.49 mM for Ca2+, 1.92 mM for Mg2+, 0.038 M for free OH− anions, 0.109 M for Cl− and 0.032 M for HCO3−. All simulations used the following parameters: The Lennard-Jones and bond parameters were chosen based on the CLAYFF force field.36 The bicarbonate and free hydroxyl anions were parametrized employing the CHARMM36 force field; the partial atomic charges for the bicarbonate anion were adopted from ref.37 The charges of the hydrogen and oxygen atoms of the free OH− anion were taken as +0.135 e and −1.135 e, respectively. The cutoff distance for nonbonded interaction was 10 Å. The Lennard-Jones potential and the pairwise Coulomb interactions were smoothly shifted to zero between 8 and 10 Å. Long-range electrostatic interactions were calculated using the particle−particle particle-mesh (PPPM) algorithm with an accuracy of 10−4. All simulations were performed in the isothermal−isobaric (NPT) ensemble at human body temperature and pressure (310 K, 1 atm) with a 1 fs integration step. The simulations were performed using the LAMMPS package (Sandia National Laboratory).38 The simulations were carried out on the Lomonosov cluster of the Supercomputing Centre of Moscow State University. Result images were created with the VMD software.39

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b01592. Supplementary figures including HRTEM image of Aloohene, analysis of cellular toxicity of Aloohene, analysis of MMTV-PyMT cells conditioned in medium pH in the presence of Aloohene, analysis of lysosomal integrity in MMTV-PyMT cells treated with Aloohene, dose-dependent cytotoxicity of Aloohene in cancer cells, dose−response modeling of doxorubicin toxicity in cancer cells, the linear isobole with an observed synergistic point, body weight measurements during an acute toxicity study, histopathological analysis of the H


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organs of animals treated with Aloohene, the antitumor effect of Aloohene on breast tumor growth, vascularization of mammary carcinomas and skin melanomas after treatment with Aloohene, the antitumor effect of Aloohene on melanoma tumor growth, and biochemical parameters of FVB mice on day 14 after treatment with Aloohene in the acute toxicity study (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Olga Vasiljeva: 0000-0003-3648-5460 Present Address ○

O.V.: CytomX Therapeutics Inc., South San Francisco, California 94080, USA. Author Contributions ∇

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.I.L., G.M., and A.A.T. contributed equally. Funding

This work was supported by the Russian Science Foundation (grant no. 14-23-00096, M.I.L., G.M., A.A.T., A.S.L., E.G., I.G., V.T., B.T., S.G.P., and O.V.) and by the Slovenian Research Agency (grant no. P1-140, B.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Moscow State University for access to the supercomputer Lomonosov and Tomsk State University for access to the supercomputer SKYF Cyberia. The authors are grateful to Professor Dan Shechtman for fruitful discussions.

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ABBREVIATIONS TME, tumor microenvironment; HRTEM, high-resolution transmission electron microscopy; XRD, X-ray diffraction; FACS, fluorescence-activated cell sorting; IHC, immunohistochemical; MD, molecular dynamics; BET, Brunauer− Emmett−Teller; BJH, Barrett−Joyner−Halenda; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline

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Letter

Nano Letters (38) Plimpton, S. J. Comput. Phys. 1995, 117, 1−19. (39) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33−38.

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Crumpled Aluminum Hydroxide Nanostructures as a

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