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RECONSTRUCTED STEM CELL NANO-GHOSTS: A NATURAL TUMOR TARGETING PLATFORM Naama Ester Toledano Furman, Yael Lupu Haber, Tomer Bronshtein, Limor Kaneti, Nitzan Letko, Eyal Weinstein, Limor Baruch, and Marcelle Machluf Nano Lett., Just Accepted Manuscript • Publication Date (Web): 20 Jun 2013 Downloaded from http://pubs.acs.org on June 21, 2013
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RECONSTRUCTED STEM CELL NANO-GHOSTS: A NATURAL TUMOR TARGETING PLATFORM Naama E. Toledano Furman†, Yael Lupu-Haber†, Tomer Bronshtein†, Limor Kaneti†, Nitzan Letko, Eyal Weinstein, Limor Baruch, and Marcelle Machluf*
Faculty of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel
† These authors have equally contributed to this work. Keywords: Nano-ghosts, Drug delivery, Mesenchymal stem cells, Cancer targeted therapy.
ABSTRACT The ultimate goal in cancer therapy is achieving selective targeting of cancer cells. We report a novel delivery platform, based on nano-ghosts (NGs) produced from the membranes of mesenchymal stem cells (MSCs). Encompassing MSC surface molecules, the MSC-NGs retained MSC-specific in vitro and in vivo tumor targeting capabilities and were cleared from blood-filtering organs. MSC-NGs were found to be biocompatible. Systemic administration of drug loaded MSC-NGs demonstrated 80% inhibition of human prostate cancer.
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The ultimate goal in cancer therapy is a ‘magic bullet’ that allows selective targeting of cancer cells1, 2. Three main considerations must be addressed when designing any such delivery system: biocompatibility, long circulation time, and selectivity1. In cancer therapy, passively-targeted drug-carrying particles are still the predominantly used drug-delivery platform1. Based on their nano-size and physical properties, such systems were shown to accumulate in the tumor surroundings—owing to the enhanced permeability and retention (EPR) effect of tumor vasculature and microenvironment. Passive targeting, however, is still limited due to varying degrees of tumor vascularization and permeability affected by the tumor type and stage3. To overcome these limitations, active cancer targeting moieties, such as antibodies, have been incorporated into polymeric drug-carriers made from nanoparticles, micelles or liposomes4-6. However, the relatively short circulation times4 and the complexity of producing such actively-targeted carriers7 hinder their clinical applications. Here we report on a novel targeted delivery platform, based on nano-ghosts (NGs) that are reconstructed from the whole cell membrane of mesenchymal stem cells (MSCs). To assure targeting, the lineage integrity of the MSCs from which the NGs were produced was continuously validated using flow cytometry for typical MSC markers (Fig. S1). In contrast to exosomes or other extracellular vesicles that are shed or bud from cells, MSC-NGs are manufactured in a reproducible process by isolating intact MSC cell membranes (ghost cells), and homogenizing them into nano-sized vesicles (nano-ghosts) while entrapping a therapeutic of choice. This approach, presenting a new paradigm for active cancer-targeted drug-delivery, is supported by our previous publication demonstrating the in-vitro targeting of HIV-infected cells by NGs expressing the receptor for a viral ligand found on infected cells8, 9. The reasoning for choosing MSCs as a source to produce cancer-targeting NGs lies in their hypo-immunogenicity and ability to target many kinds of cancers at different developmental stages10, 11. Such targeting was shown to involve both chemotaxis 12 and
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surface interactions13. Nonetheless, isolated membrane fractions of tumor cells, and not their cytoplasmatic fractions, appear to contain the most potent MSC attractants14, 15. The MSC targeting mechanism is also known to be tumor-specific but not species-specific, allowing the targeting of susceptible tumors by MSCs isolated from different species16, 17. Moreover, MSCs expressing exogenous anti-cancerous proteins, suggested for cell-based cancer therapy due to their homing abilities and hypo-immunogenicity, demonstrate some benefits when administered as whole cells into animal models18. Therefore, using MSCderived NGs (MSC-NGs), a variety of tumors requiring MSC support10, 19 may be targeted by their own invitation, extended to these Trojan horses. Most importantly, this targeting system does not entail the elaborate production of targeting molecules and their incorporation into passive vehicles, constituting a simpler and more clinically relevant approach than existing particulate drug-delivery vehicles. Unlike exosomes, shed-vesicles or cell-based delivery systems, which are predominantly intended for the delivery of products manufactured by the cells themselves, MSC-NGs can be made in different sizes and loaded with a variety of therapeutics, not only cell-made ones, through a reproducible and clinically relevant technological process. In the reported study, the efficacy of our MSC-derived NGs against cancer was demonstrated using a prostate cancer model and by encapsulating the biologic model drug sTRAIL—the soluble form of TNF-related apoptosis-inducing ligand. sTRAIL, applied through cell-based therapy, was shown to have considerable anti-cancerous impact when secreted into the tumor environment by transfected tumor cells 20 or targeted MSCs21. sTRAIL was selected due to its short biological half-life and hepatotoxicity, limiting its clinical use despite its apparent selectivity and potency. Moreover, sTRAIL administered in the form of controlled–release formulation showed no effect when administered without additional drugs; further emphasizing the shortcomings of previously reported delivery platforms22.
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Physical characterization of NGs NGs were prepared from the cytoplasmatic membranes of human and rat MSCs (hMSC and rMSC, respectively), and human Smooth Muscle Cells (SMC, as a non-mesenchymal control). Briefly, the cells (Fig. 1a) are harvested and hypotonically treated with Tris-Magnesium buffer followed by mild homogenization to allow cytosol removal without substantially disrupting cell membrane. Cells are centrifuged, precipitated and washed several times to remove most nucleic matter, as apparent by staining with 4',6-diamidino-2-phenylindole (DAPI, Fig. 1b). The homogenized cytoplasm-free cells (termed ghost cells or ghosts) are then mildly sonicated and washed again (Fig. 1c). As seen, some membrane fractionation occurs in this step; nonetheless, the product is large enough to be observable by light microscopy and to be separated by low speed centrifugation, allowing further removal of the cytosol and nuclei residues and resulting in much less evidence for DAPI staining. The sonicated ghosts were extruded into NGs in a medium containing sTRAIL and retrieved by ultracentrifugation. The NGs exhibited narrow size distributions with similar average diameters of ~180 nm (Fig. 1d) and Zeta potential of -12 mV (Fig. 1e). sTRAIL encapsulation efficiency was 30% (data not shown) and had no apparent effect on the size or unilamellar morphology of hMSC-NGs imaged with cryo-TEM (Fig. 1f). Out of the sTRAIL released during five days in 37oC (18±4 μg out of 300 μg/sample), about 70% was released during the initial six-hour burst-release, followed by a linear sustained release profile (Fig. 1g). All hMSC surface markers (>50%) were retained on the NGs, as shown by flow cytometric analysis of Dynabeads™ conjugated with hMSC-NGs and immunostained (Fig. 1h). The overall marker retention was slightly reduced compared to MSC-NGs that were not PEGylated (Fig. S2a). Substantial marker retention was also demonstrated by direct flow cytometry of PEGylated MSC-NGs that were not attached to Dynabeads™ (Fig. S2b).
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Figure 1. Physical characterization of NGs. Flourscent micrscopy images of (a) human MSCs counterstained with DAPI before processing into (b) homogenized ghosts (circled by a white dashed line), and (c) sonicated ghosts. Images are representative of at least three indpemedent samples. (d) Size and (e) Zeta-potential of NGs made from hMSC, rMSC and human smooth muscle cells (SMC, n=3). (f) Representative Cryo-TEM images (n>3) of hMSCNGs. (g) Cumulative sTRAIL release from hMSC-NGs; 100% release refers to the total amount of protein released during the time of the assay, which equals 18±4 μg out of 300 μg per sample (n=6). (h) Representative (n=3) FACS histograms of MSC markers on the surface of NGs conjugated with Dynabeads™ to achieve a FACS detectable size (see figure S2 for analyses of non-PEGylated or PEGylated Dynabeads™-free MSC-NGs). Data is represented as mean ±SD.
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In vitro: Targeting, cytotoxicity, and immunogenicity of hMSC-NGs Fluorescence-activated cell sorting (FACS) analyses were used to determine the selectivity of hMSC-NGs binding to specific targets (PC3 and MCF7) in comparison to their binding to two non-specific targets: Baby hamster kidney (BHK) cells and human smooth muscle cells, and quantified as Log Odds Ratio (LOR). The hMSC-NGs exhibited time-dependent selectivity towards PC3 and MCF7 cells, when compared to both non-specific targets (Fig. 2a). PC3 cells were selected for the continuation of our study because the NGs demonstrate lesser selectivity towards them; i.e., they present a bigger challenge than MCF7 cells. hMSC-NGs were found to accumulate inside the cytoplasm and nucleolus of PC3 cells over time, as shown by confocal microscopy (Fig. 2b). When incubated with NGs for more than 12 hrs, PC3 cells became surrounded by large clusters of NGs (Fig. 2c, left panel), also apparent inside the cells, and fused with the cell membrane (Fig. 2c, right panel). Free sTRAIL, and to a larger extent (p
