Mesenchymal Stem Cells as a Novel Carrier for Targeted Delivery of

Aug 3, 2012 - The success of gene therapy relies largely on an effective targeted gene delivery system. Till recently, more and more targeted delivery...
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Mesenchymal Stem Cells as a Novel Carrier for Targeted Delivery of Gene in Cancer Therapy Based on Nonviral Transfection Yu-Lan Hu,† Bin Huang,† Tian-Yuan Zhang,† Pei-Hong Miao,‡ Gu-Ping Tang,§ Yasuhiko Tabata,∥ and Jian-Qing Gao*,† †

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People's Republic of China Department of Pharmacy, Zhejiang Provincial Corps Hospital of Chinese People's Armed Police Forces, Jiaxing 314000, People's Republic of China § Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310058, People's Republic of China ∥ Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan ‡

S Supporting Information *

ABSTRACT: The success of gene therapy relies largely on an effective targeted gene delivery system. Till recently, more and more targeted delivery carriers, such as liposome, nanoparticles, microbubbles, etc., have been developed. However, the clinical applications of these systems were limited for their several disadvantages. Therefore, design and development of novel drug/gene delivery vehicles became a hot topic. Cellbased delivery systems are emerging as an alternative for the targeted delivery system as we described previously. Mesenchymal stem cells (MSCs) are an attractive cell therapy carrier for the delivery of therapeutic agents into tumor sites mainly for their tumor-targeting capacities. In the present study, a nonviral vector, PEI600-Cyd, prepared by linking low molecular weight polyethylenimine (PEI) and β-cyclodextrin (βCD), was used to introduce the therapeutical gene, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), to MSCs. Meanwhile, the characterization, transfection efficiency, cytotoxicity, cellular internalization, and its mechanism of this nonviral vector were evaluated. The in vitro expression of TRAIL from MSCs-TRAIL was demonstrated by both enzyme-linked immunosorbent assay and Western blot analysis. The lung tumor homing ability of MSCs was further confirmed by the in vitro and in vivo model. Moreover, the therapeutic effects as well as the safety of MSCs-TRAIL on lung metastases bearing C57BL/6 mice and normal C57BL/6 mice were also demonstrated. Our results supported both the effectiveness of nonviral vectors in transferring the therapeutic gene to MSCs and the feasibility of using MSCs as a targeted gene delivery carrier, indicating that MSCs could be a promising tumor target delivery vehicle in cancer gene therapy based on nonviral gene recombination. KEYWORDS: MSCs, tumor targeting, TRAIL, PEI600-Cyd, gene recombination FasL.5 It is considered an optimal candidate for cancer gene therapy because of its tumor cell specificity and has little effect on most normal cells.6 Although several studies have shown the antitumor activity of recombinant TRAIL, its in vivo use is limited due to short half-life in plasma.7 Additionally, previous studies have largely relied on viral vectors to deliver these therapeutic genes, which are associated with safety concerns8 and limit the clinical application of these cytokines. Therefore, new effective therapeutic tools that specifically target tumor sites are needed for improved efficiency and minimizing systematic toxicity of anticancer agents.

1. INTRODUCTION With the development of molecular biology, cancer gene therapy has become a promising approach for the treatment of cancer and has attracted wide attention in recent years. Several cytokines, such as interleukin-2 (IL-2), interleukin-12 (IL-12), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and interferon-β (IFN-β) etc., were shown to have antitumor effects.1−3 Nevertheless, the therapeutic application of exogenously administered cytokines is limited by their short half-lives and poor accessibility to tumor sites.4 These cytokines exhibit rapid blood clearance and poor retention time in the target sites, which results in the necessity for the frequent administration of such agents. Therefore, the therapeutic utility of these cytokines in vivo is limited by its excessive toxicity when administered systemically at high doses and with high frequency.4 For example, TRAIL was shown to be a promising anticancer death ligand with a sequence homology to TNF and © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2698

May 4, 2012 July 23, 2012 August 3, 2012 August 3, 2012 dx.doi.org/10.1021/mp300254s | Mol. Pharmaceutics 2012, 9, 2698−2709

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complexes escape into the cytosol and are subsequently transported into the nucleus.35 Nevertheless, PEI is associated with high cytotoxicity, especially at a high molecular weight. In the present study, PEI600-Cyd, a kind of cationic polymer prepared by a cationic polymer composed of low molecular weight PEI with a molecular weight of 600 Da cross-linked by β-cyclodextrin (β-CD), was chosen as the nonviral vector. It was reported to have lower cytotoxicity and a high transfection efficiency close to 25 kDa PEI in vitro and in vivo.36 However, the transfection efficiency of this vector in MSCs has not been studied. In the present study, we evaluated the tranfection efficiency, cytotoxicity, and cellular internalization of the nonviral vector, PEI600-Cyd, in MSCs, and this vector was used to transfect the therapeutic gene, TRAIL, to MSCs. It was proved the MSCs-TRAIL could notably decrease the lung metastasis numbers the in the C57BL/6 mice and did not cause toxicity on mice lung and liver tissue. The present study is the first attempt to use a nonviral vector, PEI600-Cyd, in the transfection of MSCs and to prove that MSCs delivery of TRAIL could reach a therapeutic effect in vivo as well as induce migration to tumors, suggesting that the gene recombinant MSCs could be used as a potential tumor-targeted gene delivery system in cancer gene therapy.

In recent years, more and more drug/gene targeted delivery carriers, such as magnetic nanoparticles,9 ligand-conjugated nanoparticles,10 stealth liposome,11 and ultrasound microbubbles,12 have been developed and under investigation for their tumor targeting efficiency and effectiveness for cancer treatment.13 However, these drug/gene delivery vehicles are limited in use because of their several disadvantages. For example, the rapid recognition and clearance of liposome by the reticuloendothelial system (RES) from the bloodstream hindered their further applications as drug carriers. Also, magnetic nanoparticles have the problems of low drug-loading capacities and nonuniform particle size distribution and are prone to form agglomerates that may lead to an occlusion of capillaries.4 Till now, several reports used the nanoscale or microscale vehicles to deliver TRAIL or recombinant TRAIL for the treatment of tumor. Guo et al. prepared TRAIL liposome, and it was used for the combination therapeutic study with doxorubicin liposome to treat glioblastoma.14 Others prepared the TRAIL nanoparticles using a low molecular weight polyethylenimine (PEI) modified with myristic acid.15 Also, there is a report using a tumor-targeting carrier, peptide HAIYPRH (T7)-conjugated polyethylene glycol-modified polyamidoamine dendrimer, to codeliver of TRAIL and doxorubicin.16 The microspheres17 or micelles18,19 were also reported to be explored as delivery vehicles for TRAIL. Recently, cell-based therapy was proposed to be a promising alternative therapeutic option for cancer treatment. However, the clinical application of differentiated cells is hindered by the difficulty in obtaining a large number of cells, their lack of ability to expand in vitro, and their poor engraftment efficiency to targeted tumor sites. In 1987, Friedenstein et al.20 found that the bone marrow (BM) single cell can differentiate into bone, cartilage-forming, adipocyte cells under certain conditions. These cells retain the ability of forming bone and cartilage after being transplanted in diffusion chambers after 20−30 cell doublings in vitro and are called mesenchymal stem cells (MSCs) or BM stromal cells. MSCs have emerged as attractive cell therapy vehicles for the delivery of therapeutic agents into tumor cells because of their capability of self-renewal, relative ease of isolation, expansion in vitro, and the homing capacity that allows them to migrate toward and engraft into the sites of tumors.21−23 To develop MSCs as therapeutic agents, efficient gene transfer to the cells is required. The strategies for gene delivery into MSCs include using viral vectors and nonviral vectors. Although viral vectors are widely used for gene delivery,24−26 their immunogenicity, insertional mutagenesism, and oncogenicity have given rise to a lot of problems27 and may cause some adverse effects on MSCs, as these cells are primary cultured and sensitive to the culture conditions. Nonviral vectors, which have several advantages such as the ease of synthesis, low immune response, and low cost in production and which are relatively easy to make some chemical modifications to improve biocompatibility and target specificity, can be a good alternative for gene transfection.28,29 So far, a variety of nonviral delivery approaches have been developed, including calcium phosphate, cationized liposomes, noisomes, and cationic polymers.30−33 A number of cationic polymers have been demonstrated to display considerable transfection efficiency in the past decade. Among those cationic polymers, PEI has emerged as a promising delivery reagent, mainly due to its excellent transfection efficiency in a wide range of cell types.34 Its high proton-buffering capacity results in rapid osmolysis of the endosomes, whereas the PEI/DNA

2. MATERIALS AND METHODS 2.1. Materials. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, and trypsin were obtained from Gibco BRL (Gaithersberg, MD). Plasmid DNA (pGL3 and pEGFP-N1) was kindly provided by the Institute of Infectious Diseases, Zhejiang University. Plasmid DNA coding for the TRAIL gene was kindly provided by Dr. Yagita Hideo (Juntendo University School of Medicine, Japan). The plasmid DNAs were amplified using Escherichia coli DH5α and purified using AxyPrep Maxi Plasmid Kit (Axygene Biotechnology Limited, Hangzhou, China). The purity of the plasmids consisting of supercoiled and open circular forms was checked by electrophoresis on a 1.0% agarose gel, and the concentration of DNA was determined by measuring the UV absorbance at 260 and 280 nm. The luciferase assay and BCA Protein Assay Kit were purchased from Beyontime Co. (Beyontime, China). A TRAIL ELISA Kit was purchased from Boster Company (Wuhan, China). All other chemicals were of analytical grade. 1,1Carbonyldiimidazole (CDI) was from Pierce (Rockford, IL). PEI (PEI25 kDa, MW 25000 Da; PEI600, MW 600 Da), β-CD (Cyd, MW 1135), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from SigmaAldrich (St. Louis, MO). 2.2. Animals. Three weeks old SD (Sprague−Dawley) male rats (50−60 g) and female C57BL/6 mice (6−8 weeks old) were supplied by Zhejiang University Experimental Animal Center, China. All animals were maintained under constant conditions (temperature, 25 ± 1 °C), on a 12 h light/dark cycle with free access to food and water. All of the experimental procedures were in accordance with the Zhejiang University guidelines for the welfare of experimental animals. 2.3. Isolation and Culture of MSCs. MSCs were isolated from the rat BM cells obtained from the hind femurs of 3 weeks old SD rats. Briefly, the bones were aseptically removed, dissected clean of attached muscles, and flushed with 1 mL of DMEM supplemented with 10% FBS, L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin penicillin. The cell suspension was placed into a 100 mm dish and cultured at 37 2699

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complexes were diluted in 500 μL of Opti-MEM medium. After 4 h of incubation at 37 °C, the Opti-MEM medium was replaced with DMEM medium containing 10% FBS. After 24 h of incubation, the green fluorescence was observed under fluorescent micrographs, and the transfection efficiency was also quantified by flow cytometry (FACS Calibur, BD Biosciences, San Jose, CA). 2.8. Cell Viability Assays. The PEI600-Cyd and PEI25 kDa solutions were filtered through 0.22 μm aseptic filter membranes before there were incubated with MSCs. For the cell viability assay, MSCs were seeded into 96-well plates at a density of 7 × 10 3 cells/well. After 24 h, different concentrations of PEI600-Cyd/DNA and PEI/DNA were added with 100 μL of Opti-MEM media to replace the culture media, and cells were incubated at 37 °C in a humidified 5% CO2-containing atmosphere for 6 h. Then, it was replaced by fresh DMEM medium with 10% FBS. After the cells were incubated for 48 h, 20 μL of MTT solution (5 mg/mL) was added to each well, and the cells were incubated for 4 h. The unreacted dyes were removed, the violet crystals in each well were dissolved in 100 μL of DMSO, and the optical density (OD) was measured by a microplate reader at the wavelength of 570 nm. The cell viability was calculated by OD570 (sample)/ OD570 (control) × 100. All of the experiments were carried out in triplicate to ascertain the reproducibility. 2.9. In Vitro Cellular Uptake. The MSCs were plated on glass coverslips in 24-well plates at 5 × 104 cells/well and were incubated for 18 h. Then, the media were replaced with OptiMEM containing polymer/DNA-FITC (1 μg) complex. After incubation for 0.5, 2, 4, and 6 h, cells were washed in PBS and fixed with 4% paraform. The nucleus was stained with propidium iodide (PI) for 3 min in room temperature and washed with PBS three times. The coverslips were mounted on microslides. Cells were observed with a confocal laser scanning microscope (MRC-1024, Bio-Rad, United Kingdom). 2.10. Treatment with Endocytosis Inhibitors. To evaluate the effect of endocytosis on the transfection efficiency, cells were pretreated with 5 μg/mL of chlorpromazine (CPZ) for 0.5 h to inhibit the clathrin-mediated endocytosis pathway,38,39 or 10 mM methyl-β-CD (MβCD, Sigma) for 0.5 h to inhibit caveolae-mediated endocytosis, or 20 mM amiloride for 15 min to inhibit macropinocytosisi in the culture medium pathway.40,41 The cells were then incubated at 37 °C with the complex for 4 h in the presence of the inhibitors. Subsequently, the medium was refreshed with DMEM supplemented with 10% FBS, L-glutamine, penicillin (50 U/ mL), and streptomycin (50 U/mL). The luciferase assay was performed as described above. Results were reported as relative transfection as compared to that without endocytic inhibitors. 2.11. In Vitro TRAIL Transfection and Enzyme-Linked Immunosorbent Assay (ELISA). For the transfection of TRAIL, MSCs were seeded in A 24-well plate at a density of 7000 cells/well and cultured for 24 h. Before transfection, the culture medium was removed, and the cells were rinsed once with PBS. Each well received 1 μg of TRAIL plasmid, in exclusion from the vector. The PEI600-Cyd/TRAIL complexes were diluted in 500 μL of Opti-MEM medium. After 4 h of incubation at 37 °C, DMEM medium with 10% FBS was added to the plate. Transfection with PEI600-Cyd/EGFP complexes was performed as a negative control. After 1 and 7 days of incubation, the cell culture supernatant and TRAIL protein concentrations in the supernatant were measured using an

°C in a humidified atmosphere containing 95% air and 5% CO2. The medium was changed on day 4 of culture and every 3 days thereafter. When the cells of the first passage became subconfluent, usually 7−10 days after seeding, the cells were detached from the flask using treatment for 5 min at 37 °C with PBS solution containing 0.25 wt % trypsin and 0.02 wt % ethylenediaminetetraacetic acids. Second-passage cells at subconfluence were used for all experiments, and MSCs were identified by flow cytometric analysis to demonstrate the expression of distinct MSC antigens (CD73 and CD90), and the absence of hematopoietic and endothelial antigens (CD45 and CD34) according to the method previously reported.37 2.4. Synthesis and Characterization of the Nonviral Vector PEI600-Cyd. The nonviral vector PEI600-Cyd was prepared according to the method described previously.36 Briefly, β-CD and CDI were dissolved together in N,Ndimethylformamide (DMF), and the mixture was stirred for 1 h under nitrogen and then precipitated in cold diethyl ether. The resulting compound, CyD-CDI, was filtered and dissolved in dimethyl sulfoxide (DMSO) and was added dropwise to the PEI solution with stirring, followed by another reaction for 5 h. Finally, the mixture was dialyzed in water for 2 days and freezedried. The composition of the prepared PEI600-Cyd copolymer was estimated by measuring 1H nuclear magnetic resonance (1H NMR) (AvanceTM 600, Bruker, Germany). 2.5. Characterization of PEI600-Cyd/DNA Complexes. The morphological examination of PEI600-Cyd/DNA complexes was performed by a transmission electron microscope (TEM). The complexes were stained with 2% (w/v) phosphotungstic acid solution for 10 s, immobilized on copper grids, and dried overnight before microscopy. The particle sizes and surface charge (represented by the surface ζ-potential) of the synthesized PEI600-Cyd/DNA complexes with a w/w ratio = 20 were measured using a laser diffraction spectrometry (Malvern Zetasizer 3000HS, Malvern, United Kingdom). The volume of the samples was 1 mL containing a final DNA concentration of 50 μg/mL. 2.6. Assay of Luciferase Activity. The plasmid PGL3 was used to examine the transfection efficiency of both the PEI25 kDa and the PEI600-Cyd vectors on MSCs. MSCs were seeded in a 24-well plate at a density of 1 × 105 cells/well and cultured for 24 h before transfection. PEI600-Cyd/DNA complexes were prepared by adding a copolymer solution to equal volumes of pGL3 solution with gentle vortex mixing and were incubated at room temperature for 20 min. The PEI/DNA complex (N/P = 10, weight per 1 μg of DNA) was also prepared by the same method. The original cell culture media were replaced with the complex solution containing the complexes and an additional 500 μL of Opti-MEM in each well. After incubation for 4 h at 37 °C, the transfection medium was replaced with fresh growth medium containing FBS, and then, the cells were incubated for 24 h. The luciferase assay was carried out according to the manufacturer's instruction (Promega, United States). Light units (LUs) due to luciferase activity were measured with a chemiluminometer (Autolumat LB953, EG&G Derthold, Germany). All of the experiments were carried out in triplicate to ascertain the reproducibility. 2.7. EGFP Expression. For the transfection of EGFP, MSCs were seeded in a 24-well plate at a density of 1 × 105 cells/well and cultured for 24 h before transfection. The culture medium was removed just before transfection, and the cells were rinsed once with PBS. Each well received 1 μg of EGFP plasmid, in exclusion from the vector. The PEI600-Cyd/EGFP 2700

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surfaces were photographed and counted, and the lung weight was measured. The lung from MSCs-TRAIL and MSCs-GFPtreated group was taken out and underwent paraffin sectioning, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using a TUNEL assay kit (Roche) according to the manufacturer's instructions. 2.17. Toxicity Evaluation of MSCs-TRAIL. As TRAIL exposure may be associated with side effects,42,43 an in vivo toxicology approach was established to assess the effects of MSCs-TRAIL. On the basis of the reported TRAIL liver toxicity,42 C57BL/6 mice were injected with 1 × 106 MSCsTRAIL or with PBS and 1 × 106 MSCs-GFP as controls by tail vein, and the serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for both groups was measured 1, 7, 14, and 30 days after injection. Meanwhile, the lung and liver from the MSCs-TRAIL group were taken out, embedded in OCT compound, rapidly frozen in liquid nitrogen, and cryosectioned. Lung and liver sections (5 μm) were then stained with hematoxylin and eosin (H&E) for microscopic observation. 2.18. Statistics. Data are expressed as the mean ± standard deviation (SD). Statistical evaluation of differences between experimental group means was done by analysis of variance and multiple Student's t tests. A value of P < 0.05 was considered significant. Data points were from at least three independent experiments.

ELISA kit (Boster Biosciences Co., Wuhan, China) according to the manufacturer's instructions. 2.12. Western Blot Analysis. MSCs-TRAIL or MSCsEGFP was lysed in a cell lysis buffer (Beyontime, China). Proteins were separated on a 10% sodium dodecyl sulfate− polyacrylamide gel electrophoresis and transferred onto a nitrocellulose transfer membrane (Whatman). The membranes were first incubated with primary antibodies (anti-TRAIL 1:1000) and then incubated with horseradish peroxidaselabeled secondary antibodies. The peroxidase activity was visualized with the enhanced chemiluminescence kit (Amersham Biosciences) according to the manufacturer's instructions. 2.13. In Vitro Antitumor Effects of MSCs-TRAIL. To examine the in vitro antitumor effects of MSCs-TRAIL, B16F10 cells (2 × 104) were seeded in the lower well of the Transwell plates (Corning Costar). MSCs-TRAIL or MSCsGFP cells of different concentrations (1 × 104, 2.5 × 104, and 5 × 104 per well) were plated in the Transwell inserts containing 0.4 μm pores (Corning Costar). After 5 days, the viability of B16F10 in the lower well was analyzed by MTT assay. All experiments were conducted in triplicate. 2.14. In Vitro Migration of MSCs. The migratory ability of MSCs was determined using Transwell plates (Corning Costar) that were 6.5 mm in diameter with 8 μm pore membranes. MSCs were added to the upper chamber at 4 × 105 cells/mL. DMEM with 0.1% FBS, DMEM with 10% FBS, or B16F10 cells were added to the lower well of the Transwell plate. After they were incubated at 37 °C overnight, cells on the upper side of the membrane were wiped off, and the migrated cells were stained by crystal violet staining. Migration was quantified by counting the cells that passed through the filter. Stained cells from a minimum of five fields of view (200×) for three replicates were counted, and the data were expressed as the average number of migrated cells. Experiments were done in triplicate. 2.15. In Vivo Migration of MSCs. The MSCs were rinsed twice with PBS and incubated in a 20 mM CM-Dil [3,38dioactadecyl-5,58-di(4-sulfophenyl)-oxacarbocyanine, sodium salt, SP-DIOC18, Molecular Probes] solution for 5 min at 37 °C and for 15 min at 4 °C according to the manufacturer's protocol. The labeled cells were then rinsed twice with PBS, collected by centrifuging for 10 min at 1000 rpm, and diluted to 106 cells/200 μL in PBS. The cells were injected into the mice by tail vein. At 1, 7, or 14 days after MSCs inoculation, the lung was taken out and embedded in OCT compound, rapidly frozen in liquid nitrogen, and cryosectioned. The migration toward the lung sties of MSCs was assessed by visualization using a fluorescence microscope. To further explore the location of MSCs at the lung sites, the tumor lungs were imaged at different time points postinjection using the Maestro in vivo imaging system (CRI, Inc., Woburn, MA). 2.16. Gene-Engineered MSCs for Cancer Therapy. B16F10 cells (1 × 105 cells in 0.2 mL of PBS per mouse) were injected into C57BL/6 mice via the tail vein. Following the B16F10 cells injection, mice were randomly divided into different groups. Ten days after the establishment of lung metastases in C57BL/6 mice, intravenous inoculation of 0.2 mL of TRAIL-engineered MSCs (5 × 106/mL), MSCs-GFP (5 × 106/mL), PEI600-Cyd/TRAIL (125 μg/mL TRAIL DNA), or PBS (6 animals/group) was performed via the lateral tail vein. All mice were sacrificed at 21 days after tumor cell injection. Lungs were removed from the mice and the metastastic foci, whose sizes ranged between 0.1 and 0.5 mm in diameter, lung

3. RESULTS 3.1. Synthesis of PEI600-Cyd. PEI600-Cyd was synthesized by a reaction between PEI600 and Cyd in the presence of CDI. Cyd was activated by CDI, and then, the Cyd-CDI was conjugated to PEI to obtain the PEI600-Cyd copolymer via a polycondensation reaction. 1H NMR spectrometry in D2O was used to analyze the molar ratio of PEI600-Cyd. The peaks at δ 2.3−2.8 were assigned to protons of −CH2−CH2−NH− from PEI600, while the peak at δ 4.8 was assigned to the C1 hydrogen of Cyd. Stoichiometry calculated integral values of PEI600 (−CH2−CH2−NH−), and the C1 hydrogen of Cyd (1:8.52) suggested that the molar ratio between PEI and CyD was 1:1 (Figure S1 in the Supporting Information), which was also confirmed using the approach described in the previous report.36 3.2. Characterization, Transfection Efficiency, and Cytotoxicity of the Nonviral Vector. On the basis of our previous report,36 the PEI600-Cyd of N/P = 20 was used for further evaluation. The particle size measured was around 180 ± 13 nm (n = 6). The ζ-potential of the particles at an N/P ratio of 20 was around 29 mV. As shown in Figure S2 in the Supporting Information, the complexes maintained a round shape with an almost homogeneous structure. For the transfection efficiencies in MSCs, the PEI600-Cyd polymer was comparable to those provided by PEI25 kDa (Figure 1Aa). We also investigated the potential cytotoxic effect of both PEI25 kDa and PEI600-Cyd in MSCs, and the cell viability of MSCs was determined by the MTT assay. Figure 1B shows the cell viability of MSCs incubated with various concentrations of PEI25 kDa and PEI600-Cyd. It was shown that PEI25 kDa was considerably more toxic than PEI600-CyD (Figure 1Ab). To further study the gene delivery ability of the nonviral vector, PEI600-Cyd, direct visualization of gene expression of GFP was also performed under fluorescence microscopy. The plasmid pEGFP-N1 encoding GFP was delivered by the vector to 2701

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2B). The cells treated with PEI600-Cyd/DNA complexes without adding any inhibitors were used as a control. As shown in Figure 3C, no obvious difference was observed between the transfection results in the presence of CPZ or amiloride and the control group. The transfection efficiency was decreased in the presence of MβCD as compared with the control group, indicating that PEI600-Cyd/DNA complexes were mainly endocytosed via a caveolae-mediated mechanism. 3.4. In Vitro Secretion of TRAIL and Antitumor Effects Induced by MSCs-TRAIL. MSCs were transduced by plasmid encoding for full-length TRAIL or with control plasmid encoding for EGFP based on nonviral transfection. As TRAIL in nature can be released as a soluble ligand,5 we used an ELISA assay to detect the expression of TRAIL in vitro. It was found that MSCs-GFP does not express TRAIL, while gene modification of MSCs with TRAIL-encoding plasmid allows a relatively high protein expression at 1 and 7 days, indicating that MSCs can be genetically modified to express high levels of TRAIL by nonviral transfection (Figure 3Aa). To assess whether MSCs-TRAIL showed antitumor effects in vitro, we used Transwell plates containing semiporous membranes to separate MSCs-TRAIL and B16F10. It was found that there was a dose-dependent inhibition of B16F10 cell growth in the MSCs-TRAIL group (Figure 3Ab). As it was reported that MSCs could express TRAIL either as membrane-bound (MB) protein or as soluble ligand, both of which can rapidly induce apoptosis in a variety of cancers cells,47 we used Western blot to confirm the expression of TRAIL in MSCs-TRAIL cell lysates. The Western blot analysis showed that there is high expression of TRAIL in MSCs-TRAIL cell lysates (Figure 3B). Therefore, the results of both ELISA measurement and Western blot analysis confirm the success in the transfection of MSCs with TRAIL using the nonviral vector. 3.5. In Vitro and in Vivo Migration of MSCs. As demonstrated in the in vitro migration study, there was a dramatic increase in the migration of MSCs incubated with B16F10 cells. In contrast, few MSCs were detectable in the control group (Figure 4Aa). It was counted that in the B16F10 group, there were 150 ± 12.3 MSCs migrated, while in the control group, only 25 ± 9.2 MSCs migrated (Figure 4Ab). The results suggested that the tumor cells could secret chemokines or cytokines to attract MSCs. To track the homing of transplanted MSCs to tumor cells in the lung metastasis model, MSCs were labeled with the cell tracker dye CM-DiI before in vivo administration. It was shown that the intravenous injection of MSCs migrated toward the lung 1 day after MSC inoculation. After 7 days, the MSCs could also be detected at the lung area (Figure 4B). To further detect the location of MSCs, the in vivo imaging system was used to detect the MSCs at the lung site. After comparing the location of the injected MSCs and the tumor nodules in Figure 4C, we found out that most of MSCs are located at the tumor nodules, indicating that MSCs can localize to tumor mass. This result showed that MSCs possess an excellent migratory and tumor tropism capacity in vivo. 3.6. Therapeutic Effect of MSCs-TRAIL on Mice Lung Metastases Tumor. In the present study, we transferred TRAIL to MSCs using the novel nonviral vectors PEI600-Cyd. Intravenously injected B16F10 cells were used to produce lung metastases in C57BL/6 mice. MSC-TRAIL was then used as an intravenous combined cellular and gene therapy, which was implanted 10 days after tumor cell injection from mice tail vein. At day 21 after B16F10 tumor cell inoculation, the animals

Figure 1. Characterization, transfectionm and cytotoxicvity of nonviral vectors in MSCs. (A) Transfection efficiency and cytotoxicity of PEI25 kDa and PEI600-Cyd. The transfection efficiency of PEI25 kDa and PEI600-Cyd was measured by luciferase activities (a). Cytotoxicity of PEI25 kDa and PEI600-Cyd using MTT assay (b). (B) EGFP transfection by PEI600-Cyd observed under a fluorescence microscope.

examine the GFP expression in MSCs. In MSCs, PEI600-Cyd/ pEGFP showed a relatively stronger fluorescence signal. These results confirmed the transfection efficiency of PEI600-Cyd (Figure 2C). 3.3. Cellular Uptake of PEI600-Cyd/DNA Complex. The cellular uptake of the PEI600-Cyd/DNA complex was observed under confocal laser scanning microscopy. Figure 3A showed the cellular internalization procedure of the PEI600-Cyd/DNA complex. It was shown that the complexes were internalized in 0.5 h, and they were distributed mostly into the endochylema. The complexes entered into the nucleus in about 4 h, and more DNA could be detected in the nucleus after 6 h (Figure 2A). In contrast, PEI25KD could escape from the endosome and enter the nucleus more quickly as it was reported.44 For efficient gene delivery, it is also important to analyze the mechanisms of cellular uptake and trafficking pathways of gene vectors.45 The transfection of the PEI600-Cyd/DNA complexes was examined in the presence of different endocytic inhibitors, including MβCD, to inhibit caveolae-mediated endocytosis, CPZ to inhibit clathrin-mediated endocytosis, and amiloride to inhibit macropinocytosisi.46 The cell viability with PEI600-Cyd/DNA complexes and the inhibitors was found to be above 80%, indicating that these inhibitors are not toxic to MSCs (Figure 2702

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Figure 2. Cellular internalization of PEI600-Cyd/DNA complexes and the mechanisms. (A) Intracellular distribution of PEI600-Cyd/FITC-plasmid complexes in MSCs at different time points was observed by a confocal fluorescence microscope. (B) Cell viability in the presence of different inhibitors was determined by the MTT assay (a); in vitro transfection efficiency of PEI600-Cyd/DNA complexes in MSCs in the presence of caveolae (MβCD, 10 mM), macropinocytosis (amiloride, 20 mM), and clathrin (CPZ, 5 μg/mL) inhibitors (b).

showed that there is no evidence of abnormal and inflammatory cell infiltration in both lung and liver sections (Figure 6B), indicating that MSCs-TRAIL do not cause toxicity on normal lung and liver of the mice.

were sacrificed. The pulmonary metastases were counted by visual inspection. The data demonstrated that treatment with MSCs-TRAIL lead to a significant reduction of lung metastases (Figure 5A, P < 0.01). Also, the lung weight of the control group (0.38 ± 0.07 g) was much higher than the group treated by MSCs-IL12 (0.20 ± 0.04 g). Meanwhile, to evaluate the apoptosis-inducing ability of TRAIL secreted from MSCs in vivo, the paraffin section of lung from MSCs-TRAIL and MSCs-GFP-treated group were stained by TUNEL. Apoptosis was detected at the MSCs-TRAIL treat group, while in the MSCs-GFP group, the apoptotic activity was negligible, indicating that MSCs-TRAIL could induce tumor cell death in vivo (Figure 5B). 3.7. In Vivo Toxicology Evaluation of MSCs-TRAIL. To evaluate the toxicity of TRAIL engineered MSCs, we injected the MSCs-TRAIL, MSCs-GFP, or PBS to C57BL/6 mice by tail vein. It was shown that in MSCs-TRAIL-treated mice, liver enzyme levels are normal, and liver histology does not provide evidence of abnormal features. As seen in Figure 6A, at 1, 7, 14, and 30 days, there are no differences in both AST and ALT levels in PBS, MSC-GFP, and MSC-TRAIL groups. The H&E staining for the lung and liver section from MSCs-TRAIL group

4. DISCUSSION Recently, genes delivered by MSCs have emerged as a strategy to improve the efficacy and minimize the toxicity of current gene therapy approaches. Several properties of MSCs favor the development of engineered MSCs as a vehicle to deliver therapeutic genes for cancer therapy. For example, MSCs are easily obtained and easy to expand in vitro, and they have a low immunogenicity and intrinsic mutation rate. Also, they could be genetically modified to express the therapeutic proteins and secrete these proteins into the tumor microenvironment. Most importantly, MSCs have high migration potential to the injury or tumor sites.48 It appears that MSC-derived cells are present in increased numbers in wounded or regenerating tissues. Tumor microenvironments are pathologically altered tissues that resemble unresolved wounds,49 which favor the homing of MSCs to the tumor sites. As compared with the tumor-targeted nanocarrier systems, which simply involve the ligand−receptor 2703

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Figure 3. In vitro secretion of TRAIL and antitumor effects of MSCs-TRAIL. (A) MSCs were transduced with TRAIL by PEI600-Cyd. At days 1 and 7 after infection, the concentration of secreted TRAIL in culture supernatant was analyzed by ELISA (a); the antitumor study was performed using a transwell plate and analyzed by MTT assay (b). (B) Western blot analysis of TRAIL from MSCs cell lysates at days 1 and 7 after MSCs were transduced with TRAIL by PEI600-Cyd.

cultured neurons, utilization of this vector for gene transfection in MSCs has not been reported yet. From our studies, it was shown that the vector has a high transfection efficiency close to that offered by PEI25 kDa but has a lower toxicity in MSCs than PEI25 kDa. The cytotoxicity of PEI is probably caused by polymer aggregation on cell surfaces, which impairs cell membrane functions.54 It was reported that high molecular weight PEI is significantly more toxic than low molecular weight PEI,55,56 and the primary amine was reported to disrupt PKC function through disturbance of protein kinase activity.57,58 Therefore, the decreased cytotoxicity by incorporating PEI600 with Cyd might be caused by lower molecular weight PEI55 and the lower amine content. Moreover, it was supposed that the ability and the time for the nonviral vectors to escape from the endosome and enter the nucleus play an important role for their transfection efficiency and the cytotoxicity. From the cellular uptake studies, it was shown that the PEI600-Cyd/DNA complex could escape from endosomes at a proper rate so that the transfection efficiency could be increased, while the cytoxicity was not that high. Hence, this vector was used for further evaluation by transfection of the therapeutic gene, TRAIL, to MSCs. TRAIL is known to selectively target tumor cells while remaining harmless to most normal cells.47 It signals via two pro-apoptotic death receptors (DR4 and DR5), inducing a caspase-8-dependent apoptotic cascade in tumor cells.59 Recently, it was introduced as an attractive candidate for cancer treatment.60 However, because of its short pharmacokinetic half-life,7 the recombinant TRAIL should be administrated at high frequency and doses to produce the desired effect, which limits its application. To improve the efficiency of cytokine delivery, use of genetically engineered cells expressing cytokines against tumors is considered to be a promising approach. As compared with systemic delivery of therapeutic

interaction, more factors were implicated in the homing of MSCs to tumor sites; therefore, a higher tumor target efficiency of MSCs would be expected. However, the processes and factors underlying the migration of MSC to tumors sites have not been well characterized. Two possible mechanisms, the secretion of chemokines/cytokines from tumors tissues50 and the interaction of the cytokines or chemokines with its corresponding receptors secreted by MSCs,51 may contribute to the tumor tropism of MSCs. Nevertheless, the evidence that the tumor microenvironment favors the homing of exogenous MSCs supports the rationale for developing engineered MSCs as a tool to track tumor sites and to deliver anticancer agents within these areas. To develop MSCs as vehicles for delivery therapeutic agents, efficient gene recombination is a prerequisite. Development of an efficient and safe gene delivery vector into cells is one of the important subjects in the field of gene therapy. Till recently, more and more nonviral vectors were used for transfection for their ease of synthesis, less immunogenicity, and lower cost.28,52 Although the nonviral vectors hold promise in delivering therapeutic genes to MSCs, most current studies on these vectors are still limited to the in vitro evaluation for their transfection efficiency.53 In the present study, we report a cationic polymer, which was synthesized by linking low molecular weight PEI with difunctionalized cyclodextrin, as a nonviral vector that is able to mediate gene transfection in MSCs. This vector was reported to be degradable in the physiological condition.36 The degradation products of the polymer are a low MW PEI and cyclodextrin, both of which are presumably relatively easy to be excluded from the cells and then be eliminated from the body. Even though the low MW PEIs still remain inside the cell, they should have less pronounced effects on cell functions as compared with PEI25 kDa.36 Although the transfection of this vector was reported in 2704

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Figure 4. In vitro and in vivo migration of MSCs. (A) In vitro migration of MSCs in response to B16F10 cell lines using a Transwell plate (8 μm pores). Representative photomicrographs of stained filters show migrated MSCs. (Magnification, 200×); 0.5% FBS was used as a control (a). Mean migrated MSCs from five random fields are shown; **p < 0.01. (B) MSCs were labeled with the cell tracker dye CM-DiI and injected into the mice with established lung tumor model via tail vein. The lung was taken out at 1 and 7 days, followed by cryosection. In vivo migration of MSCs was observed under fluorescence microscope. (C) Observation of MSCs by the in vivo imaging system.

migrate to the lower well as compared with the control group (data not shown), indicating that SDF-1 may play an important role in the migration of MSCs. For the in vivo migration of MSCs by iv injection, Studeny et al.62 demonstrated that 1 day after iv injection, MSCs were randomly distributed in the lung parenchyma and tumor nodules of mice with established A375SM melanomas. However, after 8 days, MSCs were found mainly in the tumors and had been cleared from normal lungs. It was also proved that during the initial period of iv administration, the majority of MSCs were reported to be filtered by the lung, and only rare MSCs were integrated into the tumor (at least when examined 6−7 days after injection).63 In the present studies, we proved that MSCs were mainly localized to the lung sites after iv injection by tail vein, and the in vivo study demonstrated the tropism of MSCs to tumor cells, supporting the concept that MSCs home to tumors. The other organs, such as liver, spleen, heart, etc., were also taken out for the observations of MSCs. Although some of the MSCs were found in other organs than

agents, MSCs have the advantages of offering a continuous and concentrated targeted delivery of therapeutic cytokines like TRAIL, thus reducing its nonselective targeting and allowing higher therapeutic efficiency and potency for a longer period of time. This was demonstrated by our studies that the MSCsTRAIL showed better antitumor effects than the TRAIL delivered by the nonviral vector. Thus, MSCs could be used for the targeted delivery of tumor therapeutic genes transfected by nonviral vectors for their engraftment efficiency to tumor sites. The in vitro migration of MSCs was stimulated by conditioned medium from cultured tumor cells. This indicates that soluble factors released from tumor cells could induce the migration of MSCs. Therefore, the interaction of the cytokines or chemokines secreted by tumor cells with its corresponding receptors expressed on MSCs would induce the migration of MSCs toward tumor microenvironment.61 To find out if the cytokines that might affect the migration of MSCs, we added the stromal cell-derived factor-1 (SDF-1) to the lower well (containing 0.5% FBS) and found that more MSCs could 2705

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Figure 5. In vivo studies for antitumor activity of MSCs transfected with TRAIL by PEI600-Cyd. MSCs were transfected with TRAIL gene by PEI600Cyd in vitro for 6 h. After 1 day, cells were gathered and were injected into the C57BL/6 mice at 1 × 106 cells vail tail vein. (A) The lung was photographed, and the metastasis numbers were counted and compared; **P < 0.01. (B) TUNEL staining of lung section from the MSCs-TRAILtreated group.

Figure 6. Toxicity evaluation of MSCs-TRAIL. (A) Serum transaminase (AST-ALT) levels in PBS, MSC-GFP, and MSC-TRAIL group. (A) No significant differences in AST and ALT are identified. (B) H&E staining of lung (a) and liver sections (b) from the MSC-TRAIL group (100×).

Targeted delivery of anticancer drugs/genes to tumor site can improve their therapeutic index through minimizing their toxic effects. Currently, a lot of delivery systems have been developed for anticancer agents to enhance their therapeutic values.64 For instance, nanocarrier drug delivery systems emerged as a platform for cancer therapy.13 Also, cell- or tissue-specific receptors can provide a useful target for sitespecific delivery of anticancer drugs. MSCs were proposed as a promising targeted-delivery vehicle in cancer gene therapy as we previously reported.22 Here, our results demonstrated that

lung, the numbers were significantly lower as compared with those at the lung sites (data not shown). Here, we have shown that MSCs can be engineered to express TRAIL in vitro. These cells were able to kill cancer cell lines in vitro, indicating that MSCs-TRAIL exerts a cytotoxic effect on target tumor cell lines. In a systemically delivered in vivo metastasis model, MSCs-TRAIL has significant antitumor effects as compared with nonviral TRAIL gene therapy, indicating that the site-specific delivery of TRAIL could have more pronounced effects. 2706

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antimetastatic activity of interleukin 12 against murine tumors. J. Exp. Med. 1993, 178 (4), 1223−1230. (2) Jin, P.; Wang, E.; Provenzano, M.; Stroncek, D.; Marincola, F. M. Gene expression signatures of interleukin-2 in vivo and in vitro and their relation to anticancer therapy. Crit. Rev. Immunol. 2007, 27 (5), 437−448. (3) Chada, S.; Ramesh, R.; Mhashilkar, A. M. Cytokine- and chemokine-based gene therapy for cancer. Curr. Opin. Mol. Ther. 2003, 5 (5), 463−474. (4) Einhorn, S.; Grander, D. Why do so many cancer patients fail to respond to interferon therapy? J. Interferon Cytokine Res. 1996, 16 (4), 275−281. (5) Wiley, S. R.; Schooley, K.; Smolak, P. J.; Din, W. S.; Huang, C. P.; Nicholl, J. K.; Sutherland, G. R.; Smith, T. D.; Rauch, C.; Smith, C. A.; et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995, 3 (6), 673−682. (6) Park, S. J.; Kim, M. J.; Kim, H. B.; Sohn, H. Y.; Bae, J. H.; Kang, C. D.; Kim, S. H. Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells. Exp. Cell Res. 2009, 315 (11), 1809−1818. (7) Ashkenazi, A.; Pai, R. C.; Fong, S.; Leung, S.; Lawrence, D. A.; Marsters, S. A.; Blackie, C.; Chang, L.; McMurtrey, A. E.; Hebert, A.; DeForge, L.; Koumenis, I. L.; Lewis, D.; Harris, L.; Bussiere, J.; Koeppen, H.; Shahrokh, Z.; Schwall, R. H. Safety and antitumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 1999, 104 (2), 155−162. (8) Okada, H.; Pollack, I. F. Cytokine gene therapy for malignant glioma. Expert Opin. Biol. Ther. 2004, 4 (10), 1609−1620. (9) Moritake, S.; Taira, S.; Ichiyanagi, Y.; Morone, N.; Song, S. Y.; Hatanaka, T.; Yuasa, S.; Setou, M. Functionalized nano-magnetic particles for an in vivo delivery system. J. Nanosci. Nanotechnol. 2007, 7 (3), 937−944. (10) Tanaka, T.; Shiramoto, S.; Miyashita, M.; Fujishima, Y.; Kaneo, Y. Tumor targeting based on the effect of enhanced permeability and retention (EPR) and the mechanism of receptor-mediated endocytosis (RME). Int. J. Pharm. 2004, 277 (1−2), 39−61. (11) Li, X.; Ding, L.; Xu, Y.; Wang, Y.; Ping, Q. Targeted delivery of doxorubicin using stealth liposomes modified with transferrin. Int. J. Pharm. 2009, 373 (1−2), 116−123. (12) Liu, Y.; Miyoshi, H.; Nakamura, M. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery. J. Controlled Release 2006, 114 (1), 89−99. (13) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2 (12), 751−760. (14) Guo, L.; Fan, L.; Pang, Z.; Ren, J.; Ren, Y.; Li, J.; Chen, J.; Wen, Z.; Jiang, X. TRAIL and doxorubicin combination enhances antiglioblastoma effect based on passive tumor targeting of liposomes. J. Controlled Release 2011, 154 (1), 93−102. (15) Li, J.; Gu, B.; Meng, Q. G.; Yan, Z. Q.; Gao, H. L.; Chen, X. S.; Yang, X. K.; Lu, W. Y. The use of myristic acid as a ligand of polyethylenimine/DNA nanoparticles for targeted gene therapy of glioblastoma. Nanotechnology 2011, 22 (43), 435101. (16) Han, L.; Huang, R.; Li, J.; Liu, S.; Huang, S.; Jiang, C. Plasmid pORF-hTRAIL and doxorubicin co-delivery targeting to tumor using peptide-conjugated polyamidoamine dendrimer. Biomaterials 2011, 32 (4), 1242−1252. (17) Kim, T. H.; Jiang, H. H.; Park, C. W.; Youn, Y. S.; Lee, S.; Chen, X.; Lee, K. C. PEGylated TNF-related apoptosis-inducing ligand (TRAIL)-loaded sustained release PLGA microspheres for enhanced stability and antitumor activity. J. Controlled Release 2011, 150 (1), 63−69. (18) Lee, A. L.; Wang, Y.; Pervaiz, S.; Fan, W.; Yang, Y. Y. Synergistic anticancer effects achieved by co-delivery of TRAIL and paclitaxel using cationic polymeric micelles. Macromol. Biosci. 2011, 11 (2), 296−307. (19) Lee, A. L.; Dhillon, S. H.; Wang, Y.; Pervaiz, S.; Fan, W.; Yang, Y. Y. Synergistic anti-cancer effects via co-delivery of TNF-related

MSCs transfected with a therapeutic gene, TRAIL, using PEI600-Cyd, can be used as a powerful tool in cancer gene therapy. Although others reported the use of MSCs as gene delivery systems to the delivery of TRAIL for the cancer treatment,65,66 most of these therapies are based on the viral transfection and using intratumoral injections. To our knowledge, our strategy represents the first example of cancer gene therapy based on MSC transfected by the novel nonviral vector, PEI600-Cyd, to reach a therapeutic effect. In a systemically delivered metastasis model, we showed that TRAIL-MSCs reduced metastases in C57BL/6 mice by iv injection and MSCs-TRAIL do not show any evidence in causing toxicity on the lung or liver of normal mice. We believe that this therapy may have an important future therapeutic role in preventing metastasis and the treatment of lung diseases, although further work needs to be carried out in relation to the long-term safety and fate of MSCs after entering the body.

5. CONCLUSION Targeted delivery of anticancer drugs/genes to tumor sites can improve the therapeutic index of a drug/gene by minimizing their toxic effects. The promise of MSCs as a targeted delivery vehicle was demonstrated by the in vitro and in vivo tumor targeting experiment as well as the lung tumor treatment studies. Thus, the homing of MSC to tumor sites and the potential of genetically modified MSCs therapy for antitumor effects suggested that MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites.

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ASSOCIATED CONTENT

S Supporting Information *

Figures of 1H NMR spectra of PEI600-Cyd copolymer in D2O and TEM of PEI600-Cyd/DNA complexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People's Republic of China. Tel/Fax:+86571-88208437. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (30873173 to J.-Q.G. and 81001410 to Y.-L.H.), Zhejiang Provincial Natural Science Foundation of China (R2090176 to J.-Q.G.), Fundamental Research Funds for the Central Universities, and China-Japan Scientific Cooperation Program (81011140077 to J.-Q.G. and Y.T.) supported by both NSFC, China, and JSPS, Japan. We thank Dr. Yagita Hideo (Juntendo University School of Medicine, Japan) for providing plasmid DNA coding for TRAIL gene. We thank Rongrong Tao and Feng Han (Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University) for their technical assistance.

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