Letter pubs.acs.org/NanoLett
Nanoghosts as a Novel Natural Nonviral Gene Delivery Platform Safely Targeting Multiple Cancers Limor Kaneti, Tomer Bronshtein, Natali Malkah Dayan, Inna Kovregina, Nitzan Letko Khait, Yael Lupu-Haber, Miguel Fliman, Beth W. Schoen, Galoz Kaneti, and Marcelle Machluf* Faculty of Biotechnology and Food Engineering, TechnionIsrael Institute of Technology, Haifa 3200003, Israel S Supporting Information *
ABSTRACT: Nanoghosts derived from mesenchymal stem cells and retaining their unique surface-associated tumortargeting capabilities were redesigned as a selective and safe universal nonviral gene-therapy platform. pDNA-loaded nanoghosts efficiently targeted and transfected diverse cancer cells, in vitro and in vivo, in subcutaneous and metastatic orthotopic tumor models, leading to no adverse effects. Nanoghosts loaded with pDNA encoding for a cancer-toxic gene inhibited the growth of metastatic orthotopic lung cancer and subcutaneous prostate cancer models and dramatically prolonged the animals’ survival. KEYWORDS: mesenchymal stem cells, nanoghosts, nonviral gene therapy, targeted cancer therapy
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more pharmaceutically applicable; however, such vesicles are not equipped with any inherent active-targeting capabilities.4 We have developed membrane vesicles that both have inherent targeting capabilities and can be produced using a technologically scalable and pharmaceutically applicable process. The concept of using such vesicles for targeted drug delivery to various pathologies including HIV/AIDS and cancer was reported by us before.5,6 This platform was termed nanoghosts (NGs) in order to distinguish it from other cell-derived vesicular delivery systems such as exosomes or membranefunctionalized particles, which vary from our NGs in their production, mechanism, applications, and prospects. In our recently published work, we demonstrated that PEGylated NGs, made from mesenchymal stem cells (MSC-NG), can be used to target and treat cancer. 6 Such vesicles were demonstrated to be biocompatible and preserved the cells’ unique surface-associated targeting capabilities toward a variety of tumor cells. Remarkably, a single systemic administration of MSC-NGs loaded with a model drug achieved almost complete tumor inhibition, for at least two weeks, in a prostate cancer xenograf t model.6 The unique tropism of MSCs toward inflammation, which underlie the targeting capabilities of our NGs, along with MSCs’ immunomodulatory capacity and immune evasiveness made them the most popular choice for cell-based therapies for treating solid, metastatic and hematological malignancies, degenerative diseases, and immune disorders.7−12 Nonetheless
he clinical success of gene therapy is still overshadowed by considerable delivery and safety concerns that are mostly associated with the vectors used to induce transgene transfection.1−3 Besides being safe, an optimal vector for gene therapy should selectively target the specific subset of cells requiring genetic intervention. Selective targeting may improve transfection efficiencies, minimize off-target transfection, and reduce genotoxicity, which have long been recognized as the major obstacle of gene therapy.2,3 Unfortunately, designing targeted vectors, either pseudotyped viral vectors or nonviral particulate gene-carriers that are conjugated with active targeting ligands, has proven extremely challenging.2 To guarantee clinical success, vectors should also meet the following criteria: efficient loading with a variety of genetic constructs of different sizes; efficiently introduce the transgenes into the target cells; be nonimmunogenic and nonimmunotoxic; and be produced using a scalable and cost-effective process.1−3 Most of these traits, however, usually come one at the expense of the other. Though needed, it has so far been impossible to combine all these traits into one clinically relevant therapeutic solution. The fate of gene therapy depends, therefore, upon the development of novel systems that can overcome these conflicting prerequisites for the ideal genedelivery vector. Natural membrane-derived vesicles, such as exosomes, represent a new and promising approach for targeted genedelivery.1 Nonetheless, there are currently no satisfactory scalable procedures for exosomes’ biological production and loading with different therapeutics, which hinder their pharmaceutical development.1 Other membrane vesicles, technologically reconstructed from red blood cells, may be © XXXX American Chemical Society
Received: October 18, 2015 Revised: January 28, 2016
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DOI: 10.1021/acs.nanolett.5b04237 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
replaced with high-energy probe sonication, introducing the payload into the sonication buffer. Due to the high shear force applied during sonication, which fragments the naked pDNA, loading by sonication required pDNA complexation with polycations (e.g., polyethylenimine, PEI) that compress and protect the DNA from degradation. Over 30% reduction in the viability of target PC3 cells was achieved by NGs’ loaded by sonication with PEI:pPEX complexes (Figure 1A). More
and aside from the common risks associated with the administration of living proliferating cells, owing to their natural role in supporting angiogenesis and immunosuppression, MSCs may also contribute to the pathogenesis of various diseases including cancer.10,11,13 In contrast to the common belief, recent studies have shown that direct surface interactions are at least as important, if not more so, than the soluble ones in governing both MSC targeting capabilities9,13−17 and immunological properties (Figure S1).12,17−20 MSC-NGs, which benefit from MSCs’ surface-associated targeting capabilities and immune evasiveness but lack their cytoplasmatic machinery represent a much safer platform for selective delivery of not only cell-made products and transfection of the target cells themselves. Furthermore, the patients’ biological milieu, which was suggested to drastically limit the long-term effect of whole MSCs (as cell carriers or cell-based therapies), is not expected to interfere with the membrane-associated capabilities of the innate NGs.21 The relatively simple derivation of inherently targeted MSC-NGs presents additional advantages over pseudotyped lentiviral vectors or synthetic vectors conjugated with active targeting moieties, which were largely hindered from clinical applications due to the complexity of their production. Moreover and unlike other actively targeted vectors that are restricted to a specific stage of the pathology, MSC-NGs, harnessing the wide natural capabilities of MSCs, may facilitate the targeting of various diseases and stages involving marked temporal and spatial changes. One of our most surprising discoveries to date was that MSC-NGs accumulate not only in the cytoplasm but were also detected in the nucleus of the targeted tumor cells. Therefore, we hypothesized that our MSC-NGs may also be tailored as a safe targeted nonviral gene delivery platform for various MSCtargetable pathologies.6−8,22 Nonetheless, because of their anionic surface charge, NG loading with negatively charged plasmid cDNA (pDNA) required the development of alternative production and loading methods to the ones we have previously used and published for therapeutic proteins.6 In the current work we provide, for the first time, evidence of the safe and effective transfection ability of MSC-NGs for cancer gene therapy, which accounts for more than half of all gene therapy applications in clinical trials.23 Given the fact that shortterm expression of therapeutic proteins by tumor cells can affect both cancer cells and their supportive environment, genetic therapy presents the opportunity to eradicate tumors while overcoming the toxicities and emergence of drugresistance associated with oncological drugs.23,24 To demonstrate their applicability for gene therapy, MSC-NGs were loaded with plasmid cDNA encoding for the C-terminal fragment of the human matrix metalloprotease-2, also known as the hemopexin-like domain (PEX), which was previously studied for its dual inhibitory effect on both cancer cells and vasculature.25,26 The targeting capabilities and efficacy of this system were demonstrated in two xenograft tumor models orthotopic metastatic pulmonary nonsmall cell lung carcinoma (NSCLC) and subcutaneous prostate cancerproviding overwhelming evidence of its clinical potential for treating various cancers and, possibly, other MSC-targetable pathologies. NG Loading and Characterization. Initial attempts to load the NGs with pDNA during the ghosts’ extrusion into NGs have failed, probably due to losses of pDNA adsorbed to the glass and metal vessels and the polycarbonate membranes used in the process. To avoid such losses, the extrusion was
Figure 1. MSC-NG loading and characterization. (A) Viability of PC3 cells 72 h post-transfection with MSC-NGs carrying pPEX (NGpPEX), pGFP (NG-pGFP), or PEI-complexed pPEX (NG-PEI:pPEX), loaded by sonication or electroporation. (B) pPEX loading efficiency of MSC-NGs electroporated at various cDNA to lipids ratios. (C) FACS analyses of typical MSC surface markers on electroporated NG-pPEX. (D) Cryo-TEM imaging of empty NGs and NGs electroporated with gold-labeled pPEX.
importantly, changing from extrusion to sonication was shown to increase the NGs’ phospholipids yield by more than 10-fold (Figure S2A) and their purity (as lipids-to-proteins ratio) by almost 2-fold (Figure S2B). Sonication was, therefore, selected for the following experiments as the preferred ghosts’ downsizing method, regardless of the loading method. Sonicated NGs, loaded by postpreparation electroporation with naked pPEX, led to the highest reduction in PC3 viability (>60% compared to untreated cells), which was similar to the reduction achieved using the same amount of pPEX transfected with Lipofectamine (Figure 1A). Electroporation that required no DNA complexation and allowed postpreparation loading, therefore, was selected for the continuation of our studies. NGs loaded via electroporation with pGFP as a nontherapeutic gene control led to no cytotoxic effect. Over 30% loading was achieved when the NGs were electroporated with pPEX at concentrations greater than 1 μg of pDNA/μg of lipids (Figure 1B). Electroporation and pDNA loading had no significant effect (p < 0.05) on the NGs’ size (204 ± 17 nm) or charge (−16 ± 2 mv), compared to nonelectroporated NGs (188 ± 35 nm, −17 ± 2 mv), or their retention of typical MSC surface markers (Figure 1C). Electroporation and loading did not affect the NGs’ unilamellar morphology; however, the content of NGs loaded with gold labeled cDNA appears to be more granulated than that of empty ones (Figure 1D). In Vitro Bioactivity. Confocal imaging of PC3 cells incubated with fluorescently labeled NGs for up to 3 h revealed time-dependent accumulation in the cytoplasm and nucleus of the target cells (Figure 2A). PEX DNA and mRNA copy numbers were 40 and 51 times higher, respectively, inside cells transfected with NG-pPEX compared to cells incubated B
DOI: 10.1021/acs.nanolett.5b04237 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters
Figure 2. In vitro NG-pPEX bioactivity. (A) Representative confocal imaging, out of at least three independent experiments, of DiI-labeled MSCNGs (red) incubated for up to 3 h with PC3 cells stained with phalloidin-FITC (green−actin fibers) and Hoechst (blue−nuclei). (B) pPEX DNA and mRNA levels (copy numbers) in PC3 cells post-transfection with NG-pPEX relative to cells incubated with the same amount of naked pPEX. (C) PEX protein level in PC3 cells 72 h post-transfection with NG-pPEX normalized to the cells’ viability and relative to cells incubated with naked pPEX. The (D) viability, (E) proliferation, and (F) apoptosis of PC3 cells measured up to 7 days post-transfection with NG-pPEX or incubation with empty NGs or naked pPEX, relative to the viability of the untreated cells at each day. (G) Viability of prostate (PC3), lung (A549), and breast cancer (MCF7) cells measured up to 7 days post-transfection with NG-pPEX relative to untreated cells. (H) Migration rate and (I) proliferation of endothelial cells (HUVEC) incubated for up to 24 h with conditioned-media (CM) derived from transfected or untreated PC3 cells or PC3 cells (containing cellular products) incubated with empty NGs or naked pPEX.
with naked pPEX for 6 h (Figure 2B). PEX protein levels, normalized to cell viability, were more than seven times higher in transfected cells compared to cells incubated with pPEX alone (Figure 2C). Six hours of transfection with NG-pPEX led to a substantial time-dependent reduction in PC3 cells’ viability (p < 0.0001, Figure 2D) and proliferation (p < 0.001, Figure 2E), and increased their apoptosis (p < 0.001, Figure 2F) for at least 1 week. Naked pPEX or empty NGs had no significant effect on PC3 cells’ viability, proliferation, and apoptosis. Similar to their effect on PC3 cells, NG-pPEX also decreased the viability of lung (A549) and breast (MCF7) cancer cells (Figure 2G). Conditioned-media derived from transfected PC3 cells reduced (p < 0.01) the migration rate (Figure 2H and Figure S3) and proliferation of human umbilical vein endothelial cells (HUVEC, Figure 2I) compared to no significant effect of media derived from untreated PC3 cells or cells treated with empty NGs or naked pPEX. In Vivo Safety Studies. GFP expression levels measured in dissociated blood-filtering organs harvested from C57BL mice 1 and 7 days postadministration (Figure 3A and B, respectively) of NGs loaded with a reporter genepGFP (NG-pGFP) were similar to those measured in animals administered with PBS or naked pGFP, as controls. Empty NGs administered in escalating doses, up to a dose at least ten times higher than the
therapeutic one, led to no changes in animals’ weights measured 1 week postadministration (Figure 3C). Blind pathological analyses (H&E) revealed that with the possible but unlikely exception of mild vacuolation in the liver, there is no evidence of toxic effects or necrosis in blood-filtering organs harvested from mice administered with up to 5 mg/kg of empty NGs (data not shown). A slight increase (p < 0.05) in the animals’ white blood cells (WBC) count (albeit within the normal range)27,28 was measured 1 day postadministration of empty NG (3−6 mg/kg) with no changes in the lymphocytes, neutrophils, or monocytes levels (Figure 3D, upper row). Other changes, still within the norm, were measured in the WBC (p < 0.001) and lymphocyte counts (p < 0.01) 1 week postadministration of more than 1.5 mg/kg of empty NGs; with no changes in neutrophils and monocytes levels (Figure 3D, lower row). To assess the safety of multiple administrations, C57BL mice were once (Day 7) or twice (Days 0 and 7) administered with empty NGs (0.5 mg/kg). Blood counts taken 1 (Figure 3E, upper row) and 7 days (Figure 3E, lower row) after the last administration revealed no significant changes in the number of WBC, lymphocytes or neutrophils; monocytes were undetectable in all groups. No increase was measured in the lymph nodes’ mRNA levels of IL-1β and TNFα, either 1 (Figure 3F, upper chart) or 7 days (Figure 3F, lower C
DOI: 10.1021/acs.nanolett.5b04237 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Vehicle safety studies in C57BL mice. Off-target GFP expression by cells dissociated from major blood filtering organs of mice administered with NG-pGFP, naked pGFP, or PBS (control) harvested (A) 1 day or (B) 1 week postadministration. (C) Relative change in the animal’s weights 1 week postadministration of empty NGs in escalating doses. (D) Major leukocyte (WBC, lymphocytes, neutrophils, and monocytes) counts 1 day (upper row) or 1 week (lower row) postadministration of empty NGs in escalating doses. (E) Major leukocyte counts taken 1 day (Day 8, upper row) and 1 week (Day 14, lower row) after single (Day 7) and multiple (Days 0 and 7) administrations of empty NGs. (F) Lymph nodes’ mRNA levels of TNF-α and IL-1β 1 day (Day 8, upper chart) and 1 week (Day 14, lower chart) after single (Day 7) and multiple (Days 0 and 7) administrations of empty NGs.
chart) after single or multiple administrations of empty NGs. Blood chemistry tests, drawn 1 and 7 days after the last administration, revealed no changes in the blood levels of urea, albumin, alkaline phosphatase, AST, or ALT in any of the groups compared to the control animals that were twice administered with PBS (Figure S4). In Vivo Efficacy in a Subcutaneous Prostate Cancer Model. To assess the NGs’ tumor targeting potential, subcutaneous tumors were explanted and blood-filtering organs were harvested 14 days postadministration, dissociated into single cells, and analyzed by FACS for NG uptake. Substantial NG uptake by tumors (38%) was measured, whereas all bloodfiltering organs were NG-free (Figure 4A). A single dose of NG-pPEX resulted in 76% tumor growth inhibition (%TGI), as measured 2 weeks postadministration compared to untreated animals (Figure 4B), and no effect by equivalent doses of naked pPEX or empty NGs (Figure 4C). As seen in Figure 4D, repeated NG-pPEX administrations (Days 0, 7, and 14) maintained a TGI of 77%, for at least 25 days, compared to 54% TGI in animals administered only once (Day 0). Animal survival studies, presented in Figure 4E, reveal that multiple administrations dramatically increased the time-tofirst-event (TFE) and half-life to 50 and 58 days, respectively, compared to untreated animals (26 and 28 days) and those treated only once (32 and 48 days). Histological (H&E) examination and immunohistochemical (IHC) analyses were carried out on tumors harvested from singly administered mice
to determine the scope of the NG-pPEX anticancer effect (Figure S5). Image analyses of IHC micrographs revealed significant and substantial reductions in tumor vascularization (CD31, p < 0.001, Figure 4F) and proliferation (Ki67, p < 0.01, Figure 4G), as well as increase in tumor apoptosis (Caspase 3, p < 0.001, Figure 4H). Empty NGs had no significant effect compared to animals treated with naked pPEX (p > 0.05). Nonetheless, compared to the untreated animals, empty NGs had a significant yet much smaller effect than NG-pPEX on the vascularization (p < 0.05), proliferation (p < 0.0001), and apoptosis (p < 0.01) indices presented in Figure 4F, G, and H, respectively. Wide expression of PEX was found throughout the entire bulk of tumors harvested 14 days post-NG-pPEX administration, analyzed by immunofluorescence (Figure 4I) and compared to control animals injected with PBS (Figure 4J). In Vivo Efficacy in an Orthotopic Metastatic Pulmonary NSCLC Model. The metastatic progression following iv inoculation of A549 cells was followed by MRI revealing the development of pulmonary metastases (Figure 5A). To assess the NGs’ tumor targeting potential, the lungs and other bloodfiltering organs of both tumor-bearing and non-tumor-bearing animals, either treated (50 days postinoculation) or untreated, were harvested 7 days postadministration, dissociated into single cells, and analyzed by FACS for NG uptake. Substantial NG accumulation in the tumor-bearing mice was accounted only in their lungs (Figure 5B), whereas in the non-tumorbearing animals the NGs have accumulated only in the spleen D
DOI: 10.1021/acs.nanolett.5b04237 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 4. NG-pPEX in vivo efficacy in a subcutaneous prostate cancer model. (A) Representative FACS histograms (n = 5 animals per group) of human CD90 expression in dissociated tumors and blood-filtering organs harvested from tumor-bearing mice 14 days postadministration of NGpPEX (0.5 mg/kg, purple curves) compared to untreated animals (black curves). Tumor size progression in mice administered with (B) NG-pPEX or (C) empty NGs or naked pPEX, compared to tumor-bearing untreated animals. (D) Tumor size progression and (E) survival of tumor-bearing mice singly (Day 0) or multiply (Days 0, 7, and 14) administered with NG-pPEX, compared to untreated animals. Tumor sizes are presented as mean ± SE (n = 8 animals per group). (F) Vascularization (CD31, microvessel density), (G) proliferation (Ki67), and (H) apoptosis indices (Caspase 3) calculated by image analyses of IHC micrographs of tumors harvested 14 days after a single administration of naked pPEX, empty NGs, and NG-pPEX (n = 5 animals/group). Representative immunofluorescent micrographs (n = 5 animals/group) of tumors harvested from (I) mice treated with NG-pPEX and (J) untreated mice, sectioned and stained for PEX (red) and cell nuclei (blue).
(Figure 5C). To assess their therapeutic efficacy, NG-pPEX were administered once a week, starting 50 days post tumor inoculation. Weight analysis of lungs harvested 7 days after the last administration from treated and untreated mice and nontumor-bearing animals (Figure 5D) demonstrated over 50% TGI (p < 0.05). A closer look at the individual lung weights (Figure 5E) reveals that treatment has also narrowed the weight distribution in the responsive part of the treated group (12 out of 15 animals) to less than 30% (coefficient of variance), compared to over 60% for the untreated group (n = 19) and less than 15% for normal lungs harvested from non-tumorbearing mice at the same age (n = 6). Histopathological (H&E) examination of lungs harvested from untreated animals, revealed a large number of neoplastic nodules covering the entire lungs (>50) that vary in size from small groups of cells around 0.1 mm in diameter to coalescing irregular long and oval 2.5 mm structures (Figure 1F, left panel). In the treated animals, the number of metastatic nodules (
