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Lipid bilayer-gated mesoporous silica nanocarriers for tumor-targeted delivery of zoledronic acid in vivo Diti Desai, Jixi Zhang, Jouko Sandholm, Jaakko Lehtimäki, Tove Grönroos, Johanna Tuomela, and Jessica M. Rosenholm Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00519 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Molecular Pharmaceutics

Lipid bilayer-gated mesoporous silica nanocarriers for tumor-targeted delivery of zoledronic acid in vivo Diti Desai1, Jixi Zhang2, Jouko Sandholm3, Jaakko Lehtimäki4, Tove Grönroos5,6, Johanna Tuomela*4, Jessica M. Rosenholm*1 Dr. Diti Desai, Prof. Jessica M. Rosenholm Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Turku 20520, Finland E-mail: [email protected] Dr. Jixi Zhang Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, China. Dr. Jouko Sandholm Cell Imaging Core, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland Jaakko Lehtimäki, Dr. Johanna Tuomela Institute of Biomedicine, University of Turku, Turku 20520, Finland E-mail: [email protected] Dr. Tove Grönroos Medicity Research Laboratory, University of Turku Turku 20520, Finland Turku PET Centre, University of Turku, Turku 20520, Finland

Keywords: tethered lipid bilayers, mesoporous silica nanoparticles, zoledronic acid, breast cancer

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Abstract Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate used for the treatment of bone diseases and calcium metabolism. Anticancer activity of ZOL has been established, but its extraskeletal effects are limited due to its rapid uptake and accumulation to bone hydroxyapatite. In this work, we report on the development of tethered lipid bilayer-gated mesoporous silica nanocarriers (MSNs) for the incorporation, retention and intracellular delivery of ZOL. The in vitro anticancer activity of ZOL-loaded nanocarriers was evaluated by cell viability assay and live-cell imaging. For in vivo delivery, the nanocarriers were tagged with folic acid to boost the affinity for breast cancer cells. Histological examination of the liver revealed no adverse offtarget effects stemming from the nanocarriers. Importantly, non-specific accumulation of ZOL within bone was not observed, which indicated in vivo stability of the tethered lipid bilayers. Further, the intravenously administered ZOL-loaded nanocarriers showed tumor growth suppression in breast cancer xenograft-bearing mice.


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1. Introduction Breast cancer commonly metastasizes to bone; over 80% of women with advanced breast cancer develop bone metastases, which ultimately account significantly for morbidity and reduced quality of life. Zoledronic acid (ZOL) and other nitrogen-containing bisphosphonates are the most potent inhibitors of osteoclast activity, and they are extensively used for the treatment of osteoporosis and bone metastases [1, 2]. These nitrogen-containing bisphosphonates have also been reported to exhibit anti-angiogenic and anti-tumor effects [3-5]. ZOL is a potent inhibitor of farnesyl-pyrophosphate synthase. At nanomolar concentrations it interferes with the mevalonate pathway and related critical processes in cell signaling, cell proliferation, and cytoskeletal organization [6, 7]. Thus, ZOL induces apoptosis of tumor cells and inhibits tumor cell growth in vitro in a variety of cancer cell types, including breast and prostate cancer [8-10]. Although an antitumor activity of ZOL in vitro has been reported, only few clinical studies have demonstrated effective prevention of disease recurrence in premenopausal breast cancer when ZOL is given as the adjuvant therapy [11-13]. The reason for difficulties in demonstrating anti-cancer activity of ZOL in patient cohorts is probably due to its pharmacokinetic profile. After intravenous (i.v.) administration, ZOL is rapidly cleared from the circulation by the kidneys and about 50% of the administered dose accumulates in bone [14]. Therefore, a suitable drug delivery system is required to change the biodistribution and the pharmacokinetic profile of ZOL. The system should reduce its bone accumulation, as well as improve the half-life in blood, which would enable ZOL to exert its apoptotic and anti-proliferative effect. In animal models, the delivery of ZOL has been improved by using liposome or self-assembly nanoparticle-based drug delivery systems [15-20]. However, the loading capacities in these nanocarrier systems were typically relatively low.



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Mesoporous silica nanoparticles (MSNs) have emerged as promising drug delivery carriers [21] due to their biocompatibility, well-defined mesoporosity, easily modifiable surface properties and efficient encapsulation efficiency, which makes them suitable carriers for a wide range of therapeutic agents [22-24]. For hydrophilic drug molecules, owing to their very high solubility in aqueous environment, drug release occurs by diffusion immediately upon immersion into physiological/biological fluids. Therefore, to prevent the immediate drug release in aqueous medium, the pore openings should be gated after drug loading. Lipid bilayers can act as diffusional barriers for the loaded water-soluble drug and prevent unwanted drug leakage [25]. Supported lipid bilayers have been used for the development of biosensors and drug delivery systems. As the structures of lipid bilayers are similar to those of cellular membranes, they retain the fluidity and functionality of a natural membrane, can accommodate ligands and proteins, and provide biomimetic coatings enabling barrier formation towards hydrophilic molecules [26-28]. In MSN context, this combination have been coined “protocell technology” and have recently experienced rapid advancement due to the modularity in design options and wide-spread application potential [29, 30]; but several challenges still exist, not the least on the in vivo drug delivery side with very few successful demonstrations to date.

Recently, we have reported on the novel strategy of creating hybrid tethered lipid bilayer-gated mesoporous silica nanoparticles (tLB@MSNs) for higher loading, prolonged retention of sequestered hydrophilic molecules, and low premature leakage of cargo before cellular internalization [31]. These defect-free and stable bilayers were achieved by using polyethyleneimine (PEI) on the MSN surfaces as a cushioning layer to lift off the anchored LBs from the solid surface; thus mimicking the features of actin filaments in cells. In our previous study, we have used phosphatidylcholine (PC) and phosphatidylglycerol (PG) lipids, as these 4 ACS Paragon Plus Environment

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phospholipids make up a large portion of mammalian cell membranes, and they are also common constituents of the most studied liposomal formulations. In this study, we have further included DOTAP lipid (1,2-Dioleoyloxy-3-trimethylammonium propane chloride), as liposomes containing DOTAP lipid have been widely used as transfection agents for the delivery of negatively charged biomolecules such as DNA, RNA, and oligonucleotides [32].

In this study, we describe the formulation of a new ZOL-carrying, folate receptor-targeted delivery system based on our earlier concept of MSN-supported tethered lipid bilayers. Since the tethered lipid bilayer-gated MSNs provide flexibility for further PEGylation and/or conjugation with targeting moieties in a similar manner to liposomes; to potentially extend the blood circulation time and specific targeting in vivo, the tLB@MSNs were PEGylated and further conjugated with folic acid (FA) for boosting the affinity of the delivery system towards the folate receptor-expressing breast cancer cells [33, 34]. As a cellular model, Toll-like receptor 9 (TLR9) siRNA MDA-MB-231 cells and xenografts were used. TLR9 is an innate immunity receptor, which is also expressed in several cancers. We have previously found a new subgroup of triplenegative breast cancer (TNBC) patients, who express low levels of TLR9 and have poor prognosis compared with TNBC patient with high TLR9 [35]. Even if low-TLR9-TNBC patients have the worst prognosis, our recent data have shown that TLR9 siRNA cells and xenografts are more sensitive to ZOL compared with control cells [4]. The use of ZOL in breast cancer treatment is yet limited due to its high affinity to bone. Consequently, the current formulation was optimized in order to obtain high ZOL-loading and efficient intracellular delivery without premature leakage. The DOPC, DOPG and DOTAP tLB@MSNs were evaluated in vitro for cellular uptake and cargo release by confocal microscopy, cellular uptake routing by flow cytometry and drug delivery efficiency by cell viability assay. Further, the anti-proliferative 5 ACS Paragon Plus Environment


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effect of the selected DOPC-DOPE@MSNs was assessed by kinetic IncuCyte™ ZOOM highcontent microscope. Altered in vivo biodistribution for ZOL/FA-DOPC-DOPE@MSNs has been observed compared to free drug, as evaluated by bone density measurements. Finally, as proofof-concept, the therapeutic efficacy of ZOL encapsulated into the selected formulation after i.v. administration was affirmed on mouse xenografts.

2. Experimental Section Synthesis of MSNs: The starting particles (MSN-NH2) were prepared by co-condensation procedure using TEOS and APTES as silica sources according to previously reported recipe by us [31]. At first, a mixed solution was prepared by dissolving and heating CTAB (0.45 g) in a mixture of water (150 mL) and ethylene glycol (30 mL) at 70 ⁰C in a round bottom flask. Once a clear solution was obtained, decane (2.1 mL) was subsequently added to the system. Further, 1,3,5-trimethylbenzene (TMB, 0.51 mL) was added into the mixture after 0.5 h and stirring continued for another 1.5 h to homogenize the solution. Then, ammonium hydroxide (30 wt%, 2.5 mL) was introduced to the system as catalyst, and TEOS (1.5 mL) and APTES (0.3 mL) was added consecutively to initiate the reaction. The molar ratio used in the synthesis was 1 TEOS: 0.19 APTES: 0.18 CTAB: 0.55 TMB: 1.6 decane: 5.9 NH3: 88.5 ethylene glycol: 1249 H2O. To obtain fluorescence labeled MSNs tetramethylrhodamine (TRITC, 1mg in 1 mL ethanol) has been pre-reacted with 10 µL APTES at room temperature for 2 hours, and added to the reaction mixture. The reaction was allowed to proceed for 3 h at 70ºC. Then, the heating was stopped and the as-synthesized colloidal suspension was then aged at 70ºC without stirring for 24 h. After the suspension was cooled to room temperature, the suspension was separated by centrifugation. Ethanol was used to wash the centrifuged particle. The template removal was performed by an ion-exchange method. The purified nanoparticles were redispersed in a solution containing 60 mg 6 ACS Paragon Plus Environment

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ammonium nitrate in 20 mL of ethanol, and then the mixture was stirred at 60ºC for 30 min. The procedure was repeated three times to completely remove the surfactants. The final product was suspended in ethanol for further use.

PEI functionalized MSNs were synthesized by acid-catalyzed hyperbranching surface polymerization of aziridine according to the previous protocols [52, 53]. A typical synthesis was performed with toluene as a solvent. MSN-NH2 particles obtained from the previous synthesis step were centrifuged, washed with toluene two times. Then the toluene suspension (20 mL) was subsequently subjected to argon atmosphere. Catalytic amount of acetic acid was added under stirring, after which aziridine was added in an amount of 0.5 mL per gram of particles. The suspension was stirred under argon atmosphere overnight at 70⁰C, centrifuged, washed with toluene, and dispersed in ethanol for further use.

Characterization of particles: Full redispersibility of particles was confirmed by redispersion of the dry particles in 25 mM HEPES buffer at pH 7.2 and Milli-Q water, and subsequent dynamic light scattering (DLS) measurements (Malvern ZetaSizer NanoZS). Efficient surface modifications of MSNs with PEI and different lipid layers were evaluated by zeta potential measurements (Malvern ZetaSizer NanoZS, Malvern Instruments Ltd., Worcestershire, UK). The size, monodispersity, morphology and non-agglomerated state of the nanoparticles were further confirmed by scanning electron microscopy (SEM; Jeol JSM-6335 F, JEOL Ltd., Tokyo, Japan). The mesoscopic ordering of the nanoparticles was confirmed by transmission electron microscopy (TEM; JEM 1400-Plus, JEOL Ltd., Tokyo, Japan) operating at 120 kV with a LaB6 filament and a 2k × 2 k CCD camera).



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Zoledronic acid loading into MSNs: Loading of ZOL into MSN-PEI was performed by soaking particles in a solution of ZOL in MES buffer (pH 5.0). For ZOL adsorption isotherm measurements, MSN-PEI particles (2 mg) were suspended in water (2 mL) with different initial concentrations of ZOL (0.1-0.4 mg mL-1). Subsequently, the mixture was homogenized by continuous rotation at room temperature for 4 hours. Then, the ZOL-loaded MSN-PEI particles were separated by centrifugation and the supernatant liquid was collected. The amount of ZOL adsorbed by MSN-PEI was calculated using the UV absorbance difference in the supernatant before and after the adsorption at a wavelength of 208 nm using a UV-Vis Spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific Inc., USA). Lipid bilayer coating on ZOL loaded PEI-MSNs were then preceded similarly as empty PEI-MSNs.

Detection of ZOL release from MSN: The loaded particles were incubated at 37°C in 20 mM phosphate buffer (pH 7.4) at a concentration of 0.5 mg mL-1. Particles were separated by centrifugation and the amount of ZOL released into the solution was analyzed through the UV absorbance in the supernatant at a wavelength of 208 nm using a UV-Vis Spectrophotometer (NanoDrop 2000c).

Cell line and live cell imaging: MDA-MB-231 cells were from ATCC (USA). The MDA-MB231 TLR9 siRNA cell line is a kind gift from Dr. Katri Selander (University of Alabama at Birmingham) and it has been established and characterized earlier by Tuomela and co-workers [35]. Cells were seeded to 96-well cell culture plates. Plates were imaged with the IncuCyte ZOOM instrument (Essen BioScience). The default software parameters for a 96-well plate (Corning) and a 10x objective were used. The IncuCyte ZOOM 2014A software was used to calculate mean confluencies from phase contrast images for 96 hours. 8 ACS Paragon Plus Environment

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In vivo experiment: Two independent animal experiments were performed. In both, the MDAMB-231 TLR9 siRNA cells (5 x 105 cells in 100 µL PBS, n = 10 in each group), were inoculated into the mammary fat pads of four-week-old, athymic nude mice (Athymic nude/nu Foxn1 mice, Harlan, the Netherlands), as previously described [35]. One week after tumor cell inoculation, the mice were divided into treatment groups. In the first experiment, mice were treated with vehicle, free ZOL, empty FA-DOPC-DOPE@PEI-MSNs or ZOL-loaded FA-DOPC-DOPE@PEI-MSNs once a week 4 times. The drugs were administered intraperitoneally (i.p). In the second experiment, mice were treated with vehicle or with ZOL-loaded FA-DOPC-DOPE@PEI-MSNs at the concentrations equivalent to 0.1 mg ZOL/kg and 0.3 mg ZOL/kg, three times a week. The drugs were administered via tail vein injections. In both experiments, tumor diameters were measured throughout the experiment and tumor volumes were calculated using the formula V = (π/6)(d1 × d2)3/2, where d1 and d2 are the perpendicular tumor diameters. The first experiment was terminated after 5 weeks and second after 4 weeks. The mice were sacrificed and the tumors were dissected and measured. The mice were also weighed throughout the experiment. The animals were maintained under controlled pathogen-free environmental conditions. Animal welfare was monitored daily for clinical signs. The experimental procedures were reviewed and approved by The National Animal Experiment Board in Finland (3257/04.10.07/2014).

pQCT measurement and analysis: Bone mineral density was measured and analyzed from proximal tibia using peripheral quantitative computed tomography (Stratec XCT Research M pQCT device, Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany). For the tibiae, scan lines were placed to 1-4 mm and 7 mm from the proximal end of the tibia, using the scout view



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given by the pQCT device. Calculations of cortical thickness were made using the ring model supplied by the Stratec software.

Histological and immunohistochemical stainings: For the immunohistochemical staining, tumors and livers from each mouse were formalin-fixed, embedded in paraffin and cut into 5 µm-thick sections, mounted on glass and dried overnight at 37°C. All sections were then de-paraffinized in xylene, rehydrated through a graded alcohol series and washed in phosphate-buffered saline (PBS). Tissue sections were heated twice in a microwave oven for 5 min at 700W in citrate buffer (pH 6). The tumor sections were stained with monoclonal rat anti-CD34 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) o/n at +4°C, followed by biotin-labeled anti-rat IgG (DAKO Denmark A/S, Glostrup, Denmark). All samples were processed under the same conditions. Stained tumors were then scanned using Pannoramic slide scanner (3DHistech, Budapest, Hungary). The number of CD34-positive blood vessels was counted from whole tumor area using Pannoramic slide viewer (3DHistech, Budapest, Hungary). The liver sections were stained with hematoxylin & eosin using standard procedures, and were imaged using Pannoramic slide scanner.

Statistics: One-way analysis of variance or Student’s t-test (GraphPad Prism 6 for Windows, GraphPad Software, La Jolla, CA, USA) was used to analyze the data. The data were represented as mean ± standard error of the mean. The level of significance was set at probabilities of *p

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